The Interactive Fly

Genes involved in tissue and organ development

Development of the Optic Lobe



  • Spatio-temporal pattern of neuronal differentiation in the Drosophila visual system: A user's guide to the dynamic morphology of the developing optic lobe
  • Interaction between EGFR signaling and DE-cadherin during embryonic optic lobe morphogenesis
  • The pan-neural bHLH proteins Deadpan and Asense regulate mitotic activity and cdk inhibitor dacapo expression in the Drosophila larval optic lobes
  • Notch signaling regulates neuroepithelial stem cell maintenance and neuroblast formation in Drosophila optic lobe development
  • Coordinated sequential action of EGFR and Notch signaling pathways regulates proneural wave progression in the Drosophila optic lobe
  • Conserved miR-8/miR-200 defines a glial niche that controls neuroepithelial expansion and neuroblast transition
  • miR-7 buffers differentiation in the developing Drosophila visual system
  • Notch regulates the switch from symmetric to asymmetric neural stem cell division in the Drosophila optic lobe
  • Changes in Notch signaling coordinates maintenance and differentiation of the Drosophila larval optic lobe neuroepithelia
  • Serrate-Notch-Canoe complex mediates glial-neuroepithelial cell interactions essential during Drosophila optic lobe development
  • Fat/Hippo pathway regulates the progress of neural differentiation signaling in the Drosophila optic lobe
  • The tumour suppressor L(3)mbt inhibits neuroepithelial proliferation and acts on insulator elements
  • bantam is required for optic lobe development and glial cell proliferation
  • Temporal patterning of Drosophila medulla neuroblasts controls neural fates
  • A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system
  • Brain-specific-homeobox is required for the specification of neuronal types in the Drosophila optic lobe
  • A temporal mechanism that produces neuronal diversity in the Drosophila visual center
  • Integration of temporal and spatial patterning generates neural diversity
  • Ecdysone-dependent and ecdysone-independent programmed cell death in the developing optic lobe of Drosophila
  • Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper
  • Wnt signaling specifies anteroposterior progenitor zone identity in the Drosophila visual center
  • A unique class of neural progenitors in the Drosophila optic lobe generates both migrating neurons and glia
  • Function of Nerfin-1 in preventing medulla neurons dedifferentiation requires its inhibition of Notch activity
  • Development of the anterior visual input pathway to the Drosophila central complex

    Proteins in establishment of optic lobe circuitry
  • Signals transmitted along retinal axons in Drosophila: Hedgehog signal reception and the cell circuitry of lamina cartridge assembly
  • Neuropil pattern formation and regulation of cell adhesion molecules in Drosophila optic lobe development depend on Synaptobrevin
  • Ig superfamily ligand and receptor pairs expressed in synaptic partners in Drosophila
  • Control of synaptic connectivity by a network of Drosophila IgSF cell surface proteins
  • Dpr-DIP matching expression in Drosophila synaptic pair
  • An axon scaffold induced by retinal axons directs glia to destinations in the Drosophila optic lobe
  • Robo-3-mediated repulsive interactions guide R8 axons during Drosophila visual system development
  • The highly ordered assembly of retinal axons and their synaptic partners is regulated by Hedgehog/Single-minded in the Drosophila visual system
  • Recognition of pre- and postsynaptic neurons via nephrin/NEPH1 homologs is a basis for the formation of the Drosophila retinotopic map
  • Localized netrins act as positional cues to control layer-specific targeting of photoreceptor axons in Drosophila
  • Sequential axon-derived signals couple target survival and layer specificity in the Drosophila visual system
  • Analyzing dendritic morphology in columns and layers
  • Birth order dependent growth cone segregation determines synaptic layer identity in the visual system
  • Visual circuit assembly requires fine tuning of the novel Ig transmembrane protein Borderless
  • Multiple interactions control synaptic layer specificity in the Drosophila visual system
  • The developmental rules of neural superposition in Drosophila
  • Identifying functional connections of the inner photoreceptors in Drosophila using Tango-Trace
  • Mapping chromatic pathways in the Drosophila visual system
  • The developmental rules of neural superposition in Drosophila

    Optic Lobe circuitry and function
  • Cholinergic circuits integrate neighboring visual signals in a Drosophila motion detection pathway
  • The temporal tuning of the Drosophila motion detectors is determined by the dynamics of their input elements
  • The emergence of directional selectivity in the visual motion pathway of Drosophila
  • Transgenic line for the identification of cholinergic release sites in Drosophila melanogaster
  • Orientation selectivity sharpens motion detection in Drosophila
  • Cellular evidence for efference copy in Drosophila visuomotor processing
  • Cross-modal influence of mechanosensory input on gaze responses to visual motion in Drosophila
  • Optogenetic control of fly optomotor responses
  • A directional tuning map of Drosophila elementary motion detectors
  • RNA-seq transcriptome analysis of direction-selective T4/T5 neurons in Drosophila
  • Complementary mechanisms create direction selectivity in the fly
  • Candidate neural substrates for off-edge motion detection in Drosophila
  • Functional specialization of neural input elements to the Drosophila ON motion detector
  • A common evolutionary origin for the ON- and OFF-edge motion detection pathways of the Drosophila visual system
  • Neural mechanisms for Drosophila contrast vision
  • Direction selectivity in Drosophila emerges from preferred-direction enhancement and null-direction suppression
  • In vivo imaging reveals composite coding for diagonal motion in the Drosophila visual system
  • Direct measurement of correlation responses in Drosophila elementary motion detectors reveals fast timescale tuning
  • Multiple redundant medulla projection neurons mediate color vision in Drosophila
  • Processing properties of ON and OFF pathways for Drosophila motion detection
  • Neural circuit to integrate opposing motions in the visual field
  • Nonlinear circuits for naturalistic visual motion estimation
  • The metabolism of histamine in the Drosophila optic lobe involves an ommatidial pathway: β-alanine recycles through the retina
  • Synaptic circuits and their variations within different columns in the visual system of Drosophila
  • Comprehensive characterization of the major presynaptic elements to the Drosophila OFF motion detector
  • Direct neural pathways convey distinct visual information to mushroom bodies
  • Automatic segmentation of Drosophila neural compartments using GAL4 expression data reveals novel visual pathways
  • Subcellular imaging of voltage and calcium signals reveals neural processing in vivo
  • Visual projection neurons in the Drosophila lobula link feature detection to distinct behavioral programs
  • Object-detecting neurons in Drosophila
  • The comprehensive connectome of a neural substrate for 'ON' motion detection in Drosophila

    Spatio-temporal pattern of neuronal differentiation in the Drosophila visual system: A user's guide to the dynamic morphology of the developing optic lobe

    Visual information processing in animals with large image forming eyes is carried out in highly structured retinotopically ordered neuropils. Visual neuropils in Drosophila form the optic lobe, which consists of four serially arranged major subdivisions; the lamina, medulla, lobula and lobula plate; the latter three of these are further subdivided into multiple layers. The visual neuropils are formed by more than 100 different cell types, distributed and interconnected in an invariant highly regular pattern. This pattern relies on a protracted sequence of developmental steps, whereby different cell types are born at specific time points and nerve connections are formed in a tightly controlled sequence that has to be coordinated among the different visual neuropils. The developing fly visual system has become a highly regarded and widely studied paradigm to investigate the genetic mechanisms that control the formation of neural circuits. However, these studies are often made difficult by the complex and shifting patterns in which different types of neurons and their connections are distributed throughout development. This study has reconstructed the three-dimensional architecture of the Drosophila optic lobe from the early larva to the adult. Based on specific markers, it was possible to distinguish the populations of progenitors of the four optic neuropils and map the neurons and their connections. This paper presents sets of annotated confocal z-projections and animated 3D digital models of these structures for representative stages. The data reveal the temporally coordinated growth of the optic neuropils, and clarify how the position and orientation of the neuropils and interconnecting tracts (inner and outer optic chiasm) changes over time. Finally, the emergence of the discrete layers of the medulla and lobula complex were analyzed using the same markers (DN-cadherin, Brp) employed to systematically explore the structure and development of the central brain neuropil. This work will facilitate experimental studies of the molecular mechanisms regulating neuronal fate and connectivity in the fly visual system, which bears many fundamental similarities with the retina of vertebrates (Ngo, 2017).

    Interaction between EGFR signaling and DE-cadherin during embryonic optic lobe morphogenesis

    Dynamically regulated cell adhesion plays an important role during animal morphogenesis. The formation of the visual system in Drosophila embryos has been used as a model system to investigate the function of the Drosophila classic cadherin, DE-cadherin, which is encoded by the shotgun (shg) gene. The visual system is derived from the optic placode, which normally invaginates from the surface ectoderm of the embryo and gives rise to two separate structures, the larval eye (Bolwig's organ) and the optic lobe. The optic placode dissociates and undergoes apoptotic cell death in the absence of Shotgun, whereas overexpression of Shotgun results in the failure of optic placode cells to invaginate and of Bolwig's organ precursors to separate from the placode. These findings indicate that dynamically regulated levels of Shotgun are essential for normal optic placode development. Overexpression of Shotgun can disrupt Wingless signaling through titration of Armadillo out of the cytoplasm to the membrane. However, the observed defects are likely the consequence of altered Shotgun mediated adhesion rather than a result of compromising Wingless signaling, since overexpression of a Shotgun-alpha-catenin fusion protein, which lacks Armadillo binding sites, causes defects similar to Shotgun overexpression. The genetic interaction between Shotgun and the Drosophila EGF receptor homolog, Egfr, was studied. If Egfr function is eliminated, optic placode defects resemble those following Shotgun overexpression, which suggests that loss of Egfr results in an increased adhesion of optic placode cells. An interaction between Egfr and Shotgun is further supported by the finding that expression of a constitutively active Egfr enhances the phenotype of a weak shg mutation, whereas a mutation in rhomboid (rho) (an activator of the Egfr ligand Spitz) partially suppresses the shg mutant phenotype. Finally, Egfr can be co-immunoprecipitated with anti-Shotgun and anti-Armadillo antibodies from embryonic protein extracts. It is proposed that Egfr signaling plays a role in morphogenesis by modulating cell adhesion (Dumstrei, 2002).

    The head ectoderm of early Drosophila embryos is subdivided into several domains that realize different morphogenetic programs. The embryonic eye field is the posterior-medial region of the procephalic neurectoderm that gives rise to the visual system, which includes the larval eye (Bolwig's organ) and adult eye, as well as the optic lobe. Around gastrulation, cells of the eye field undergo a convergent extension directed laterally. Shortly afterwards these cells form two morphologically visible placodes, one on either side of the embryo. These optic placodes sink inside and become the optic lobe primordia, epithelial double layers attached to the posterior surface of the brain. The optic placode of a stage 12-13 embryo is V-shaped, with the anterior leg of the V representing the anterior lip, which later forms the inner anlage of the optic lobe, and the posterior leg forming the posterior lip, later forming the outer anlage. As the invagination deepens and the two lips 'sink' inside the embryo, ectodermal cells that earlier surrounded the perimeter of the optic placode approach each other and eventually form a closed epidermal cover. Abundant cell death accompanies the closing of the head epidermis over the optic lobe anlage, and the subsequent separation of this anlage from the epidermis. A small number of cells that originally formed part of the posterior lip of the optic placode remain integrated in the head epidermis and form the larval eye or Bolwig's organ. As these cells move away from the optic lobe anlage their basal ends become drawn out and form axons that constitute Bolwig's nerve (Dumstrei, 2002).

    Shotgun is expressed throughout the ectoderm including the eye field and its epithelial derivatives. One would expect that normal optic lobe development requires modulation of Shotgun activity to allow, for example, the segregation of the invaginating optic placodes from the surrounding ectoderm. Since cell culture studies have indicated that the mammalian EGF receptor can disrupt cadherin-based adhesion, it was of interest to see whether Drosophila Egfr is expressed in the visual system to allow for such a possibility in Drosophila as well. Egfr is expressed in a complex and dynamic pattern that closely parallels the pattern of double-phosphorylated ERK (dpERK) expression, indicating activation of the MAP kinase signaling pathway. During stage 7 both rho (an activator of Egfr signaling) and dpERK are expressed in two stripes in the head ectoderm. The expression of dpERK in these two stripes is the result of Egfr activity. The anterior stripe corresponds to part of the head midline, while the posterior stripe reaches into the eye field. Distribution of dpERK in the two stripes becomes patchy during stage 10. At the same time, the posterior stripe widens dorsally to overlap with part of the optic lobe placode. Finally, at the late extended germ band stage and during germ band retraction, dpERK becomes restricted to the optic lobe placodes and cells of the dorsal head midline. This expression pattern demonstrates that Egfr activation accompanies the determination, morphogenesis and differentiation of the embryonic visual system (Dumstrei, 2002).

    On the subcellular level, Egfr is expressed diffusely on the membrane of epithelial cells and neuroblasts. Egfr overlaps with Armadillo, the Drosophila ß-catenin homolog, which is an integral component of the cadherin-catenin complex. Like Shotgun, Armadillo is concentrated strongly in the apically located zonula adherens but is also found at lower levels in the entire lateral membranes (Dumstrei, 2002).

    A second type of junction, called a septate junction, develops in Drosophila epithelial cells at a slightly later stage than the zonula adherens. Septate junctions have been implicated in maintaining epithelial stability. The Coracle protein forms part of the septate junctional complex, and an antibody against Coracle serves as a sensitive marker for this junction. Applying this marker to embryos of different stages it was found that all ectodermally derived epithelia express Coracle, except for the optic lobe and the invaginations that form the stomatogastric nervous system. Accordingly, no septate junctions have been reported in previous electron microscopic investigations of these tissues. The reliance on adherens junctions alone may make the optic lobe (and stomatogastric nervous system) susceptible to changes in the stability of these junctions; such changes occur resulting from manipulations of Shotgun and Egfr (Dumstrei, 2002).

    A finely adjusted level of Shotgun is required for optic placode morphogenesis, and ß-catenin, as well as EGFR signaling, is involved in this process. Reduction in Shotgun results in dissociation of the placode around the time when it normally invaginates, suggesting that the forces exerted on the epithelial sheet while folding may disrupt cell contacts. A similar phenotype was described for other epithelial invaginations, including the Malpighian tubules and stomatogastric nervous system. Abolishing Armadillo/ß-catenin function results in a similar, if somewhat weaker phenotype. If Shotgun is overexpressed, invagination is also impaired. Cells stay together in a placode-like formation (as would be expected from 'hyperadhesive' epithelial cells), but do not noticeably constrict apically. It should be noted that the interpretation of this failure of optic placode cells to constrict is complicated by the accompanying increase in cell death in surrounding head epidermal cells. This phenomenon, in addition to a direct effect of an increased amount of Shotgun in the optic placode cells, could be part of the pathology responsible for the non-invagination phenotype. By contrast, the non-disjunction of optic lobe and larval eye is likely to be a rather direct consequence of an increased amount of Shotgun expression. Interestingly, other adhesion systems, notably the Drosophila N-CAM homolog FasII, are also involved in optic lobe-larval eye separation. Thus, the down regulation of FasII by the 'anti-adhesion' molecule Beaten path is also required for normal larval eye morphogenesis (Dumstrei, 2002).

    Overexpression of Shotgun or the DE-cad-alpha-catenin fusion construct causes a dramatic change in optic lobe morphogenesis, without causing much disruption in other epithelia. It is speculated that this enhanced sensitivity of optic lobe cells towards an increased level of Shotgun may be in part due to the fact that adherens junctions form the only means of contact between optic lobe cells. In other epithelia, such as epidermis, trachea and hindgut, septate junctions form by far the more prominent junctional complex. Septate junctions have been implicated in epithelial stability. One could surmise that embryonic epithelia, as they enter the phase of differentiation during mid-embryogenesis, construct septate junctions that add to the adherens junctions developed at an earlier stage. This additional junctional complex makes late epithelia more resistant to changes in cadherins, a notion supported by the finding that blocking cadherins (by applying calcium chelators, or tyrosine kinase inhibitors) in early embryos up to stage 10 leads to a break down of epithelia, whereas it has only a small effect in later stages. The optic lobe, which does not differentiate but gives rise to a population of neuroblasts later dring the larval period, does not form septate junctions, which could account for its strong reliance on normally functioning adherens junctions (Dumstrei, 2002).

    Expression of a fusion construct, DE-cad-alpha-catenin, in which the cytoplasmic domain of Shotgun is replaced by a truncated alpha-catenin, thereby preventing a reduction in the cytoplasmic pool of Arm, results in a similar phenotype as overexpressing full length Shotgun. This finding lends support to the notion that dissociation of the cadherin-catenin complex (CCC) may not occur at the interface between Shotgun and Arm or Arm and alpha-catenin. If one were to assume that dissociation occurred between any components of the CCC, one would expect a stronger phenotype, given that by overexpressing the fusion construct one not only increases the amount of Shotgun molecules interconnecting cells, but also the stability with which they are coupled to the cytoskeleton. Biochemical studies in vertebrates and Drosophila also show that phosphorylation of the CCC does not result in increased dissociation of Arm or alpha-catenin from the CCC, suggesting that the dissociation occurs distal to alpha-catenin (Dumstrei, 2002).

    The strength of the CCC and other structural molecules driving morphogenesis has to be controlled in a complex spatiotemporal pattern. Numerous widely conserved signaling pathways have been implicated in this process. In vertebrate embryos, mutations of the Wnt, Shh and BMP signaling pathways result in impressive examples which tissues and organs show defects in morphogenesis. Furthermore, it became clear that frequently signaling proteins affect fundamental cellular behaviors, such as proliferation, motility, adhesiveness and survival. This prompted the hypothesis that in many developmental scenarios, the 'proximal' effect of receiving a signal could be a change in morphogenetic behavior. The discovery that one of the Wnt signal transducers, ß-catenin, leads a 'double life' as a structural component of the cadherin-catenin complex, fueled the idea that Wnt signal could directly exert an effect on the adhesiveness on the cell, an idea that is supported by cell culture experiments. However, genetic studies have demonstrated that in Drosophila, the roles of ß-catenin as a signaling transducer and a CCC component seem to be quite separate. Although it is clear that the cytoplasmic and membrane bound ß-catenin pools are in a steady state, binding of more ß-catenin to the membrane, by overexpression of Shotgun, reduces the cytoplasmic pool resulting in a wg minus phenotype. However, Wnt/Wg signaling seems to have no effect on the amount of membrane bound ß-catenin. Thus, in Drosophila, it appears that Shotgun mediated adhesion, at least under experimental conditions, interferes with Wnt/Wg signaling by competing for ß-catenin but Wnt/Wg signaling may not have a direct effect on adhesion mediated by the CCC (Dumstrei, 2002).

    The findings suggest that another signaling pathway, the Egfr pathway, is involved in modulating cadherin-mediated adhesion and thereby controls morphogenesis. Egfr, similar to its function in the developing compound eye, is activated in the precursors of the larval eye and adjacent optic lobe at a stage preceding optic lobe invagination and larval eye separation. The ligand for Egfr is Spitz, which is activated by Rhomboid (Dumstrei, 2002).

    In a small subset of larval eye precursors (the 'Bolwig's organ founders') loss of Egfr signaling results primarily in cell death, lending further support to the view that Egfr signaling functions generally in the ectoderm and its derivatives to maintain cell viability. Recent studies in Drosophila indicate that MAPK directly controls the expression and protein stability of the cell death regulator, Hid (W; Wrinkled). If cell death is prohibited by a deficiency of the reaper-complex, cells of the optic placode and most other embryonic cells that undergo apoptosis in Egfr loss-of-function mutants survive. Both optic lobe and Bolwig's organ express several of their normal differentiation markers, but show a characteristic 'hyperadhesive phenotype', consisting in the failure of optic Iobe invagination and Bolwig's organ separation. Based on the similarity of this phenotype to the one resulting from Shotgun overexpression, and the genetic interaction between Egfr and Shotgun mutants in the ventral ectoderm, it is proposed that Egfr activation is required in normal development to phosphorylate the CCC and thereby allows optic lobe invagination and Bolwig's organ separation to occur. This would be in line with experimental results obtained in vertebrate cell culture studies, which have demonstrated that drug- or Egfr-induced phosphorylation of the CCC leads to dissociation between CCC and cytoskeleton. Recent findings have shown that another phosphorylation event, mediated by the rho/rac GTPases, also affects adhesion by dissociating alpha-catenin from the CCC (Dumstrei, 2002).

    Co-IP data indicates that Egfr is linked to the CCC in Drosophila as well. This implies that the effect of Egfr on Shotgun mediated adhesion could be a direct one that occurs at the cell membrane and does not involve MAPK signal transduction to the nucleus. It has been shown in a number of vertebrate cell culture systems that tyrosine phosphorylation of ß-catenin results in a disassembly of the CCC complex and a consecutive loss in cadherin-mediated adhesion. Phenotypically, this results in increased invasiveness of tumor cell lines, neuronal and growth cone motility. Several tyrosine kinases and phosphatases have been identified that can increase or decrease the degree of phosphorylation of the CCC. For example, v-src transfected into cultured cells phosphorylates ß-catenin and causes cells to dissociate, round up, and become more motile. Egfr also phosphorylates the CCC and forms an integral part of this complex. This opens up the possibility that growth factors, with their widespread expression and biological activity in the developing embryo, may exert part of their effect on cell behavior by modulating, in a rather direct way, cell adhesion at the membrane. Such a mechanism would account for the 'rapid mode' of control of adhesion molecules. Systems such as the optic placode of the Drosophila embryo, where matters of different cell fates are decided at the same time when morphogenetic movements change the arrangement and shape of the cells involved, constitute favorable paradigms to address how signaling systems control both processes (Dumstrei, 2002).

    The pan-neural bHLH proteins Deadpan and Asense regulate mitotic activity and cdk inhibitor dacapo expression in the Drosophila larval optic lobes

    Developmental regulators and cell cycle regulators have to interface in order to ensure appropriate cell proliferation during organogenesis. An analysis of the roles of the pan-neural genes deadpan and asense defines critical roles for these genes in regulation of mitotic activities in the larval optic lobes. Loss of deadpan results in reduced cell proliferation, while ectopic deadpan expression causes over-proliferation. In contrast, loss of asense results in increased proliferation, while ectopic asense expression causes reduced proliferation. Consistent with these observations endogenous Deadpan is expressed in mitotic areas of the optic lobes, and endogenous Asense is expressed in cells that will become quiescent. Altered Deadpan or Asense expression results in altered expression of the cyclin dependent kinase inhibitor gene dacapo. Thus, regulation of mitotic activity during optic lobe development may, at least in part, involve deadpan and asense mediated regulation of the cyclin dependent kinase inhibitor gene dacapo (Wallace, 2000).

    Optic lobes begin development during embryogenesis between stages 11 and 12 when a group of 30-40 epidermal cells delaminates and moves from the surface of each brain hemisphere. Once delaminated, these cells remain inactive until the embryo hatches as a first instar larva. This inactive state of the cells is partially mediated by the glycoprotein Anachronism, secreted by glia surrounding the developing optic lobe (Ebens, 1993). In the first instar larva the cells begin to divide, a process requiring the function of the trol gene. These first divisions appear to be synchronous and continue through the beginning of pupal development. A total of approximately 3,000 cells are produced in the mitotically active areas of the optic lobe. During second instar some of the cells of the developing lamina and medulla begin to differentiate into neurons and glia. This differentiation is accompanied by the innervation of the first and second optic lobes by photoreceptor axons. Their arrival and the release of Hedgehog protein in the developing optic lobes begins the differentiation of the lamina cells into neurons and glia. The outer proliferation center (OPC) represents one of the major areas of mitotic activity in the optic lobe. The OPC becomes a distinct structure at late second instar and the cells in the OPC and the inner proliferating center (IPC) continue to divide until all of the photoreceptor axons have innervated the optic lobe and initiated differentiation of the lamina precursors. The lamina furrow spreads outward in a semicircle and passes through the OPC where the cells for the developing lamina originate. As the lamina furrow advances outward, the cells in the passing furrow arrest in G1 phase (Wallace, 2000 and references therein).

    The IPC, which is the second major area of mitotic activity in the optic lobe, forms in a crescent shape at a more interior position of the brain with respect to the OPC. The IPC represents a pool of cells that produces the cells for the medulla and the lobula. The IPC cells, however, do not divide and differentiate in as synchronous an order as the OPC (Wallace, 2000).

    To determine the functional properties of Dpn during larval development the pUAST/Gal4 system was used to test the effects of ectopic dpn expression. The 71B Gal4 driver line was used in this analysis, since it drives the expression of pUAST constructs in most cells of the second and third instar larval optic lobes. In addition, this line also drives strong expression in the wing discs. Bromodeoxyuridine (BrdU) incorporation and histone H1 RNA expression were used as S phase specific markers to detect changes in mitotic activity (Wallace, 2000).

    In the larval central nervous system, ectopic Dpn expression results in a striking increase in the size of the brain lobes as compared to wild-type brains. In brains with ectopic Dpn expression, an increase in the number of mitotically active cells is apparent across the entire surface of the enlarged brain. In addition, a breakdown of the mitotic domain pattern that is present in wild-type third instar optic lobes is also evident. The over-proliferation phenotype that is associated with ectopic Dpn expression is fully penetrant. It can range from >10 times to two- to four-fold the size of a normal wild-type brain lobe, and appears sensitive to the accumulation modifiers in the genome (Wallace, 2000).

    HES proteins can have opposite functions from proteins of the AS-C in neural development. ase, a member of the AS-C, has been reported to be expressed in the developing third instar optic lobes and loss of ase function results in disturbances of the adult optic lobe. It was asked whether the AS-C protein Ase can modulate mitotic activity. To this end, the effects of ectopic ase expression on mitotic activity in the developing larval optic lobe were investigated. As with Dpn, ectopic expression of Ase results in strong expression in most cells of the second and third larval optic lobes. This expression results in breaks in the normally continuous pattern of S phase positive cells in the OPC suggesting that increase and/or ectopic expression of the Ase protein decreases mitotic activity in the optic lobe (Wallace, 2000).

    It was asked whether mitotic activity is altered in third instar larval optic lobes of dpn1 homozygous loss of function mutants. dpn1 is an apparent null allele of the dpn gene. In the OPC of dpn1 homozygous third instar larvae, sporadic breaks are evident in the normally continuous area of S phase positive cells. These breaks can vary in size and location in the OPC, but can be found in the OPC of nearly all homozygous dpn1 mutant larvae. In addition, S phase activity in the developing lamina appears compressed and disorganized (Wallace, 2000).

    To better determine whether reduction in the amount of OPC neuroblasts in dpn1 mutants results in a significant loss of OPC neuroblast progenies, assays were performed for developing lamina cells that represent direct progeny of the OPC cells. If there is a reduction in the amount of cells in the OPC, a subsequent reduction in the amount of developing lamina cells could be expected. Anti-Dachshund antibodies were used to mark the cells of the developing lamina. In dpn1 homozygous mutant third instar larvae, a reduced number of Dachshund (Dac) positive cells is evident as compared to the wild type. Photoreceptor axons that innervate the lamina are responsible for initiating the differentiation of the cells into neurons and glia. It was necessary to determine whether the reduced number of cells is due to an aberrant projection of photoreceptor axons. Anti-HRP antibodies were used to mark the photoreceptor axons in dpn1 homozygous larvae. Overall size and morphology of the eye disc, as well as photoreceptor axon extension and innervation of the lamina in dpn1 larvae appears normal. One difference, however, is that the area of innervation is smaller than wild type. The reduced number of developing lamina cells in dpn1 loss of function larvae indeed may, therefore, be due to a reduced amount of OPC neuroblasts rather than aberrant axon projection (Wallace, 2000).

    To analyze the possible involvement of ase gene function on mitotic activity in the larval optic lobes, the S phase activity in third instar larval brains was determined. In ase1/scb57 mutants, there is an expansion of S phase activity to include the normally mitotic quiescent cells between the OPC and the lamina precursor cells (LPCs), as well as scattered S phase activity in the lamina. ase1 is a deletion of the ase coding region and scB57 is a deletion of the entire AS-C as well as the proximal complementation group EC4 making the larva homozygous mutant for ase and heterozygous for the other members of the AS-C. In contrast, +/scB57 larval optic lobes show normal S phase activity. Thus, ase loss of function mutants show an increase in the S phase activity between the OPCs and the LPCs, and a random pattern of increased S phase activity in the lamina (Wallace, 2000).

    If Dpn is involved in the positive regulation of mitotic activity, as indicated by the dpn loss of function and ectopic expression phenotypes, then Dpn would be expected to be expressed in mitotic active areas. Endogenous dpn protein expression was examined in the wild-type larval CNS; Dpn was found to be expressed in areas of active cell division in the optic lobes. Dpn is expressed in the OPC of the late third instar larva and stops at the edge of the OPC. After cells exit the OPC, S phase activity ceases and the cells subsequently arrest in G1 as they pass through the lamina furrow. Dpn is also expressed in the cells of the IPC. Thus, expression of Dpn in the larval optic lobes is in agreement with a possible role as one positive regulator of the cell cycle (Wallace, 2000).

    If Ase is involved in the termination of mitotic activity in the larval optic lobes, then Ase expression would be expected in or near areas where the cell cycle is arresting. Ase protein expression was examined in the larval optic lobes; Ase was found to be present in a band at the posterior edge of the OPC that partially overlaps with Dpn expression. Ase is expressed just before the cells exit the OPC and cease S phase activity. These cells then arrest in G1 phase as they pass through the lamina furrow. Ase is also expressed in cells of the IPC and at a low level in the lamina furrow. The expression pattern of Ase, which comes to a maximum at the posterior edge of the OPC, is in agreement with a possible role for Ase in aiding cell cycle arrest as cells leave the OPC (Wallace, 2000).

    Cdk inhibitors have been shown to represent key regulators of mitotic activity. In Drosophila a cdk inhibitor gene, dap, has been identified that is transiently expressed during embryogenesis in cells prior to entering their last mitosis and at the onset of terminal differentiation. Ectopic expression of dap results in G1 arrest, while loss of dap function has been shown to cause one extra cell division in embryonic epidermal cells. Dpn appears to promote the continuation of mitotic activity, while Ase has a role in ending cell proliferation in the developing optic lobes. Therefore, it was asked whether altered expression of Dpn and Ase can modulate the expression of the dap. In wild-type third instar larva, optic lobe expression of dap occurs in specific domains. dap is expressed in cells of the lamina furrow and scattered cells of the lamina. There is also strong expression of dap in a subset of cells in the IPC throughout third instar. In contrast, dap expression is virtually absent from the cells of the OPC (Wallace, 2000).

    The effects were determined of the loss of dpn function on the expression of dap. In homozygous dpn1 mutant third instar larva, expression expands into the area of the OPC. Also, cells of the lamina begin to express dap more strongly. In contrast, in larvae with ectopic Dpn expression, dap expression is strongly reduced or absent in the optic lobes of third instar larva. Thus, dpn activity has a negative regulatory effect on the dap RNA level (Wallace, 2000).

    In homozygous ase mutant third instar larvae, there is a strong reduction of dap RNA throughout the entire developing optic lobe while dap expression in the developing eye disk appears normal. The ase loss of function phenotype demonstrates that ase activity is necessary for the expression of dap throughout the developing optic lobe. When Ase is ectopically expressed in third instar optic lobes, ectopic activation of dap expression becomes evident. Therefore, ase activity has a positive regulatory effect on the dap RNA level (Wallace, 2000).

    It was asked whether the phenotypical effects on cell proliferation produced by alterations of Dpn and Ase expression may be caused, at least in part, by changes in the levels of dap transcript. During embryogenesis, alteration in the levels of dap expression through either ectopic expression or by loss of function, result in dramatic changes in mitotic activity. Therefore, the mitotic activity in optic lobes of homozygous dap6 mutant third instar larvae were analyzed. While predominately recessive lethal, a few dap6 homozygous escapees can be viable to adulthood. Therefore, the larval optic lobes of homozygous dap6 mutant third instar larva can be analyzed. In such homozygous dap6 mutant larvae over-proliferation of the cells of the optic lobes is evident. There is a significant increase in the number of mitotically active cells and break down of mitotic domains, as compared to the wild type (Wallace, 2000).

    The over-proliferation phenotype of dap6 null mutants can be compared to the over-proliferation phenotype in larvae with ectopic dpn expression, and the associated suppression of dap expression. Although the over-proliferation in both cases is similar, there are clearly more cells produced in the dpn over-expressing brain lobes. This strongly indicates that other cell cycle regulators are also likely to be affected by the ectopic expression of dpn in the optic lobes (Wallace, 2000).

    A model is proposed for mitotic control in the developing third instar optic lobe in which cell proliferation is modulated by a positive regulator of mitotic activity such as Dpn and a negative regulator of mitotic activity such as Ase. In this model, one role of Dpn and Ase would be to interface with cell cycle regulation through the direct or indirect modulation of dap expression. Mitotic control during optic lobe development may involve the following events. Cells that give rise to the optic lobe delaminate from the neuroectoderm during embryogenesis and remain quiescent until first instar with the help of proteins such as Anachronism. The mitotic activity is then initiated through a process that requires the trol gene product and the developmental regulator and transcription factor Eve to begin the proliferation of neuroblasts to form the OPC. The mitotically active state of OPC cells would be maintained in part by Dpn. In the absence of Dpn, the cells in the OPC have a greater chance of exiting mitosis by allowing Dap to be expressed. As cells arrive at the edge of the OPC, Ase is expressed at high levels, allowing the neuroblasts to become quiescent only after they pass out of the region where Dpn is expressed. Suppression of dap by Dpn in the OPC would allow the neuroblasts to be mitotically active while the increased expression of Ase at the posterior edge of the OPC allows the neuroblasts to exit mitosis and begin differentiation. In addition, the resulting quiescent state needs to be maintained in the lamina; otherwise the cells may reenter mitosis (Wallace, 2000 and references therein).

    Notch signaling regulates neuroepithelial stem cell maintenance and neuroblast formation in Drosophila optic lobe development

    Notch signaling mediates multiple developmental decisions in Drosophila. This study examined the role of Notch signaling in Drosophila larval optic lobe development. Loss of function in Notch or its ligand Delta leads to loss of the lamina and a smaller medulla. The neuroepithelial cells in the optic lobe in Notch or Delta mutant brains do not expand but instead differentiate prematurely into medulla neuroblasts, which lead to premature neurogenesis in the medulla. Clonal analyses of loss-of-function alleles for the pathway components, including N, Dl, Su(H), and E(spl)-C, indicate that the Delta/Notch/Su(H) pathway is required for both maintaining the neuroepithelial stem cells and inhibiting medulla neuroblast formation while E(spl)-C is only required for some aspects of the inhibition of medulla neuroblast formation. Conversely, Notch pathway overactivation promotes neuroepithelial cell expansion while suppressing medulla neuroblast formation and neurogenesis; numb loss of function mimics Notch overactivation, suggesting that Numb may inhibit Notch signaling activity in the optic lobe neuroepithelial cells. Thus, these results show that Notch signaling plays a dual role in optic lobe development, by maintaining the neuroepithelial stem cells and promoting their expansion while inhibiting their differentiation into medulla neuroblasts. These roles of Notch signaling are strikingly similar to those of the JAK/STAT pathway in optic lobe development, raising the possibility that these pathways may collaborate to control neuroepithelial stem cell maintenance and expansion, and their differentiation into the progenitor cells (Wang, 2011).

    This study find that Notch signaling plays an essential role in the maintenance and expansion of neuroepithelial cells in the optic lobe; it also inhibits medulla neuroblast formation. Clonal analyses of several pathway components indicate that this dual function bifurcates downstream of Su(H) with E(spl)-C only partly involved in the inhibition of medulla neuroblast formation but not the maintenance and expansion of neuroepithelial stem cells (Wang, 2011).

    In the optic lobe, Notch signaling plays a role analogous to lateral inhibition during embryonic CNS development. However, the selection of neuroblasts in the OPC neuroepithelium is an all-or-none process rather than selecting individual neuroblasts from the neuroepithelium. Medulla neuroblasts are generated in a wave progressing in a medial to lateral direction in the OPC neuroepithelium with all cells at a particular position along the medial-lateral axis differentiating into neuroblasts. Interestingly, this wave of medulla neuroblast formation coincides with the down-regulation of both Delta and Notch expression in the medial cells in the OPC, which might reduce Notch signaling activity, thereby allowing medulla neuroblasts to form. What factors drive the recession of both Delta and Notch expression in the OPC neuroepithelium along the medial-lateral axis is not known. When Notch signaling is inactivated, neuroepithelial cells in the OPC change cell morphology and differentiate into medulla neuroblasts prematurely. The results indicate that Notch signaling actively controls neuroepithelial integrity, possibly by regulating the adherens junction (AJ), since in Notch pathway mutant mosaic clones in the OPC, the apical determinants PatJ, Crumbs and aPKC are cell autonomously reduced or lost and the mutant cells change to rounded or irregular morphology. Further experiments will be needed to determine how Notch signaling activity affects the maintenance of neuroepithelial integrity, particularly the stability of the adherens junction (Wang, 2011).

    Is neuroblast formation also actively inhibited by Notch signaling or simply a default state of neurogenic epithelial cells? In the latter model, Notch signaling may only maintain neuroepithelial integrity and promote their expansion while medulla neuroblasts form when the neuroepithelial integrity is disrupted. The argument against this model is that changes in neuroepithelial integrity are not always accompanied with cell fate changes. In N, Dl or Su(H) mosaic clones located in the OPC neuroepithelium, it was found that in about 25% of the clones, the mutant cells changed morphology or lost apical marker expression but did not become neuroblasts (Dpn-negative), whereas in E(spl)-C mosaic clones, Dpn+ cells were prematurely induced, which indicate that the cells begin to differentiate into neuroblasts, but these cells still retained columnar epithelial cell morphology and apical marker expression. This suggests that the suppression of neuroblast formation by Notch signaling activity is separable from the maintenance of neuroepithelial integrity and that medulla neuroblast formation is actively suppressed by Notch signaling. A possible scenario is that activation of the Notch pathway turns on the E(spl)-C genes, which in turn suppress proneural gene expression in the optic lobe neuroepithelia. Indeed, at least one member in the E(spl)-C genes, E(spl)m8, appears to be activated in the neuroepithelial cells by the Notch pathway, as the E(spl)m8-lacZ reporter is expressed in a pattern similar to Delta and Notch expression in the OPC and IPC. E(spl)m8 protein and possibly additional members of the E(spl)-C may suppress the expression of proneural genes in the optic lobe. The proneural genes of the achaete-scute complex (as-c) comprise four members, achaete, scute, L'sc, and asense. achaete is not expressed in the optic lobe, but scute is expressed in both the neuroepithelial cells and neuroblasts in the OPC implying that scute expression in the neuroepithelial cells is not suppressed by Notch signaling activity. By contrast, asense is only expressed in the neuroblast and GMCs and L'sc is transiently detected in an advancing stripe of neuroepithelial cells of 1-2 cells wide that are just ahead of newly formed medulla neuroblasts. Thus, E(spl)-C proteins may suppress L'sc and/or ase expression, the release of this suppression may allow the neuroepithelial cells to begin to differentiate into medulla neuroblasts. It should be noted, however, that the removal of the E(spl)-C activity does not seem to be sufficient to allow full differentiation of neuroepithelial cells into medulla neuroblasts, suggesting that additional factors downstream of Notch signaling may be involved in the suppression of medulla neuroblast formation (Wang, 2011).

    The phenotypes of Notch pathway mutants are reminiscent of those of JAK/STAT mutants. For example, inactivation of either pathway led to early depletion of the OPC neuroepithelium; either pathway inhibits neuroblast formation, and ectopic activation of either pathway promotes the growth of the OPC neuroepithelium. The remarkable phenotypic similarities in Notch and JAK signaling mutant brains suggest that these pathways may act in a linear relationship such that activation of one pathway is relayed to the second, perhaps by inducing the expression of a ligand. Alternatively, these pathways may act in parallel and converge onto some key downstream effectors or target genes. Further experiments will be needed to test whether Notch interacts with JAK/STAT and if it does, to find out where the interaction occurs during the development of the optic lobe (Wang, 2011).

    The roles of Notch signaling in mammalian brain development have been studied intensely. Many Notch pathway components have been examined in knockout mice, which showed defects in brain development. Mice deficient for Notch1 or Cbf all display precocious neurogenesis during early stages of nervous system development. This has led to the view that the role of Notch signaling in the mouse brain is to maintain the progenitor state and inhibit neurogenesis. However, it is not clear from these studies whether the premature neurogenesis in Notch signaling mutant mice was caused by premature differentiation of neuroepithelial stem cells into neurons or by premature differentiation of neuroepithelial stem cells into progenitor cells, which then generated neurons. In fact, it has been proposed that Notch activation can promote the differentiation of neuroepithelial stem cells into radial glial cells, the progenitor cells that generate the majority of neurons in the cerebral cortex. This is based on the observation that ectopic Notch activation using activated forms of Notch1 and Notch3 (NICD) caused an increase in radial glial cells as compared to control. The radial glial cells resemble medulla neuroblasts in the Drosophila optic lobe in that they are both derived from neuroepithelial stem cells and undergo asymmetric division to self-renew and generate neurons, although morphologically radial glial cells are still polarized while medulla neuroblasts have lost epithelial characters and are rounded in shape. Based on the current results, it is suggested that Notch signaling maintains the pool of neuroepithelial stem cells and promotes their expansion in both Drosophila and mammals and that the precocious neurogenesis in Notch signaling mutant brains arise due to premature differentiation of the neuroepithelial stem cells into the progenitor cells (Wang, 2011).

    However, ectopic Notch activation may indeed promote progenitor cell proliferation in the brain. Ectopic neuroblasts were observed in the medulla cortex when NACT was ectopically expressed by the neuroblast/GMC driver insc-Gal4, by ubiquitous expression using hs-Gal4, or when numb15 mosaic clones were induced at later larval stages when neuroblasts normally begin to form. Since the results have shown that the Notch pathway is not essential for medulla neuroblast formation or self-renewal, the ectopic neuroblasts are a novel phenotype solely induced by ectopic Notch signaling activity. This is consistent with Notch activation promoting ectopic neuroblast formation in the central brain and VNC without being required for neuroblast self-renewal in these regions of the CNS; and Notch has been shown to be an oncogene in mammals. Since the sizes of the ectopic neuroblasts were in the range of GMC or neurons, they may resemble the transit-amplifying (TA) neuroblasts that are found in the dorsal-medial region of the central brain. The origin of these ectopic neuroblasts in the medulla cortex is not clear, but it is unlikely that they are derived from differentiated medulla neurons as ectopic expression of NACT using elav-Gal4, which is active in medulla neurons, did not result in ectopic neuroblasts and by the fact that ectopic neuroblasts can be induced in numb15 mosaic clones, which could only arise from mitotically active cells that include neuroepithelial cells, medulla neuroblasts, and ganglion mother cells (GMCs), but not neurons. The ectopic neuroblasts could be generated by a transformation of GMCs into a neuroblast identity as suggested for ectopic neuroblasts in brat mutant central brains. Ectopic Notch signaling activity may even directly promote the expansion of neuroblasts after they have differentiated from the neuroepithelial cells in the OPC. In either case, ectopic Notch signaling activity may block the normal path of neuronal differentiation and lock the cells in a proliferative state. This is indeed what was observed in numb15 mosaic clones in which numerous ectopic neuroblasts were induced in the medulla cortex without generating medulla neurons. Perhaps ectopic Notch signaling activity may also promote the proliferation of neural progenitors in vertebrates, such as the radial glial cells in the mouse brain (Wang, 2011).

    Coordinated sequential action of EGFR and Notch signaling pathways regulates proneural wave progression in the Drosophila optic lobe.

    During neurogenesis in the medulla of the Drosophila optic lobe, neuroepithelial cells are programmed to differentiate into neuroblasts at the medial edge of the developing optic lobe. The wave of differentiation progresses synchronously in a row of cells from medial to the lateral regions of the optic lobe, sweeping across the entire neuroepithelial sheet; it is preceded by the transient expression of the proneural gene lethal of scute [l(1)sc] and is thus called the proneural wave. This study found that the epidermal growth factor receptor (EGFR) signaling pathway promotes proneural wave progression. EGFR signaling is activated in neuroepithelial cells and induces l(1)sc expression. EGFR activation is regulated by transient expression of Rhomboid (Rho), which is required for the maturation of the EGF ligand Spitz. Rho expression is also regulated by the EGFR signal. The transient and spatially restricted expression of Rho generates sequential activation of EGFR signaling and assures the directional progression of the differentiation wave. This study also provides new insights into the role of Notch signaling. Expression of the Notch ligand Delta is induced by EGFR, and Notch signaling prolongs the proneural state. Notch signaling activity is downregulated by its own feedback mechanism that permits cells at proneural states to subsequently develop into neuroblasts. Thus, coordinated sequential action of the EGFR and Notch signaling pathways causes the proneural wave to progress and induce neuroblast formation in a precisely ordered manner (Yasugi, 2010).

    Loss of EGFR function in progenitor cells caused failure of L(1)sc expression and differentiation into neuroblasts (see A model of progression of the proneural wave). In addition, elevated EGFR signaling resulted in faster proneural wave progression and induced earlier neuroblast differentiation. The activation of the EGFR signal is regulated by a transient expression of Rho, which cleaves membrane-associated Spi to generate secreted active Spi. This study also demonstrated that Rho expression itself depends on EGFR function, and thus the sequential induction of the EGFR signal progresses the proneural wave. Clones of cells mutant for pnt were not recovered unless Minute was employed, suggesting that the EGFR pathway is required for the proliferation of neuroepithelial cells. However, the progression of the proneural wave is not regulated by the proliferation rate per se (Yasugi, 2010).

    The function of the Notch signaling pathway in neurogenesis is known as the lateral inhibition. A revision of this notion has recently been proposed for mouse neurogenesis, in which levels of the Notch signal oscillate in neural progenitor cells during early stages of embryogenesis, and thus no cell maintains a constant level of the signal. The oscillation depends mainly on a short lifetime and negative-feedback regulation of the Notch effecter protein Hes1, a homolog of Drosophila E(spl). This prevents precocious neuronal fate determination. The biggest difficulty in analysis of Notch signaling is the random distribution of different stages of cells in the developing ventricular zone, which is thus called a salt-and-pepper pattern. In medulla neurogenesis, however, cell differentiation is well organized spatiotemporally and the developmental process of medulla neurons can be viewed as a medial-lateral array of progressively aged cells across the optic lobe. Such features allowed the functions of Notch to be precisely analyzed. Cells are classified into four types according to their developmental stages: neuroepithelial cells expressing PatJ, neuronal progenitor I expressing a low level of Dpn, neuronal progenitor II expressing L(1)sc and neuroblasts expressing high levels of Dpn. The Notch signal is activated in neuronal progenitor I and II. The EGFR signal turns on in the neuronal progenitor II stage and progresses the stage by activating L(1)sc expression. Cells become neuroblasts when the Notch and EGFR signals are shut off. Cells stay as neuronal progenitor I when Notch signal alone is activated, whereas cells stay as neuronal progenitor II when the Notch signal is activated in conjunction with the EGFR signal. Although the Notch signal is once activated, it must be turned off to let cells differentiate into neuroblasts. In neuronal progenitor II, E(spl)-C expression is induced by Notch signaling, and the increased E(spl)-C next downregulates Dl expression and subsequent activation of the Notch signal (Yasugi, 2010).

    What does Notch do in medulla neurogenesis? It is infered that the Notch signal sustains cell fates, whereas the EGFR signal progresses the transitions of cell fate. This was well documented when a constitutively active form of each signal component was induced. EGFR, or its downstream Ras, induces expression of L(1)sc but does not fix its state, even though the constitutively active form is employed. At the same time, a constitutively active Notch sustains cell fates in a cell-autonomous manner. Constitutively active N receptors, by contrast, autonomously determine cell fates depending on the context: cells become neuronal progenitor I in the absence of EGFR and neuronal progenitor II in the presence of EGFR. The precocious neurogenesis caused by the impairment of Notch signaling suggests that Notch keeps cells in the progenitor state for a certain length of time in order to allow neuroepithelial cells to grow into a sufficient population. In the prospective spinal cord of chick embryo (Hammerle, 2007), the development from neural stem cells to neurons progresses rostrocaudally, during which the transition from proliferating progenitors to neurogenic progenitors is regulated by Notch signaling (Yasugi, 2010).

    Although Notch plays a pivotal role in determining cell fate between neural and non-neural cells, the function may be context dependent and can be classified into three categories. (1) Classical lateral inhibition is seen in CNS formation in embryogenesis and SOP formation in Drosophila. Cells that once expressed a higher level of the Notch ligand maintain their cell states and become neuroblasts. (2) Oscillatory activations are found in early development of the mouse brain (Shimojo, 2008). Progenitor cells are not destined to either cell types. (3) An association with the proneural wave found in Drosophila medulla neurogenesis as is described in this study. The Notch signal is transiently activated only once and then shuts off in a synchronized manner. The notable difference in the outcome is the ratio of neural to non-neural cells; a small number of cells from the entire population become neuroblasts or neural stem cells in the former cases (1 and 2), whereas most of the cells become neuroblasts in the latter case (3). The differences between (1) and (2) can be ascribed at least in part to the duration of development. Hes1 expression has been shown to oscillate within a period of 2 hours in the mouse, whereas in Drosophila embryogenesis, selection of neuroblasts from neuroectodermal cells takes place within a few hours. Thus, even if Drosophila E(spl) has a half-life time equivalent to Hes1, the selection process during embryogenesis probably finishes within a cycle of the oscillation. The process of medulla neuroblast formation continues for more than 1 day, but Notch signaling is activated for a much shorter period in any given cell. This raises the possibility that E(spl)/Hes1 may have a similarly short half-life but outcome would depend on the developmental context (Yasugi, 2010).

    The functions of EGFR and Notch described in this study resemble their roles in SOP formation of adult chordotonal organ development; the EGFR signal provides an inductive cue, whereas the Notch signal prevents premature SOP formation. In addition, restricted expression of rho and activation of the EGFR signal assure reiterative SOP commitment. Several neuroblasts are also sequentially differentiated from epidermal cells in adult chordotonal organs (Yasugi, 2010).

    Unpaired, a ligand of the JAK/STAT pathway is expressed in lateral neuroepithelial cells and shapes an activity gradient that is higher in lateral and lower in the medial neuroepithelium. The JAK/STAT signal acts as a negative regulator of the progression of the proneural wave (Yasugi, 2008). This report has shown that activation of both EGFR and Notch signaling pathways depends on the activity of the JAK/STAT signal. The JAK/STAT signal probably acts upstream of EGFR and Notch signals in a non-autonomous fashion. These three signals coordinate and precisely regulate the formation of neuroblasts (Yasugi, 2010).

    Conserved miR-8/miR-200 defines a glial niche that controls neuroepithelial expansion and neuroblast transition

    Neuroepithelial cell proliferation must be carefully balanced with the transition to neuroblast (neural stem cell) to control neurogenesis. This study shows that loss of the Drosophila microRNA mir-8 (the homolog of vertebrate miR-200 family) results in both excess proliferation and ectopic neuroblast transition. Unexpectedly, mir-8 is expressed in a subpopulation of optic-lobe-associated cortex glia that extend processes that ensheath the neuroepithelium, suggesting that glia cells communicate with the neuroepithelium. Evidence is provided that miR-8-positive glia express Spitz, a transforming growth factor α (TGF-α)-like ligand that triggers epidermal growth factor receptor (EGFR) activation to promote neuroepithelial proliferation and neuroblast formation. Further, these experiments suggest that miR-8 ensures both a correct glial architecture and the spatiotemporal control of Spitz protein synthesis via direct binding to Spitz 3' UTR. Together, these results establish glial-derived cues as key regulatory elements in the control of neuroepithelial cell proliferation and the neuroblast transition (Morante, 2013).

    This analysis reveals that the production by an optic-lobe-associated cortex glia of the EGFR ligand Spitz is critical for coordination of neuroepithelial proliferation and the spatiotemporal emergence of neuroblasts. External signaling from the surrounding microenvironment is a common mechanism for the regulation of stem cell number and behavior in mature tissues, but the need of a niche microenvironment during early neurogenesis was unknown (Morante, 2013).

    Flies deficient for the microRNA mir-8 exhibit brain degeneration and behavioral defects. Strikingly, the microRNA is expressed in cortex glial cells lying underneath the blood-brain-barrier (subperineurial) glial layer. These glia are of large size and produce long protrusions that ensheath the developing optic lobe neuroepithelium and can be distinguished by their selective expression of the EGFR ligand spitz as well as its modulators aos and rho (Morante, 2013).

    Genetic manipulation of mir-8, spi, or aos, in this glial cell population unveiled cell nonautonomous influences of these glia on the development of the underlying neuroepithelium. Similar glial signaling to neuroblasts in the larval Drosophila brain has been demonstrated for a class of neuroblasts that remain quiescent until nutrient-responsive satellite and cortical glia reactivate their proliferation. This study identifies another population of glia cells that sustain growth of neuroepithelial cells and the neuroepithelial-neuroblast transition via a mir-8-Spitz axis (Morante, 2013).

    Glial-mediated regulation of the neuroepithelium is reminiscent to the roles of mammalian astrocytes that are known to potently stimulate neurogenesis in cell culture and a component of endogenous neural stem cell niche in adult mammalian neurogenesis. Additionally, EGFR is also implicated in glial cell proliferation in Drosophila and human glioma often exhibits elevated EGFR signaling (Morante, 2013).

    The notion that mir-8-Spitz-positive cortex glia constitute an anatomically and functionally distinct population of surface-associated glia cells is strongly supported by the finding that RNAi knockdown of spi in subperineurial glia using moody- Gal4 has no effect on neuroepithelial development. Spitz protein is converted to its active form by the Rhomboid protease, which is also expressed by miR-8 glia. The extracellular factor Aos limits Spitz spreading and signaling level that may influence the effects of Spitz-EGFR in the responding neuroepithelium (e.g., sustaining proliferation and preventing premature or ectopic neuroblast formation). The posttranscriptional silencing of spi mRNA by the miR-8 binding to a sequence in its 3' UTR provides another layer of regulation to fine-tune the timing, localization and/or amount of Spitz protein translation. Moreover, the distinctive architecture of miR-8-Spitz-positive cortex glia appears to be regulated by endoreplication regulators dup/ Cdt1 and the microRNA miR-8. Importantly, expression of a spitz transgene lacking its 3' UTR (and hence unable of regulating by miR-8) fully rescued the undergrowth defect caused by mir-8 overexpression in the glia cells. Therefore, it is suggested that cortex glia employ a coordinated strategy that is mediated by miR-8 to ensure that: (1) the glia establish a correct architecture to provide a continuous layer of cortex glia cells that extend long processes to the neuroepithelium; and (2) correct local (or temporal) control of Spitz protein synthesis. Given the expression of rho and aos is directly induced by EGFR signaling in other context, a feed-back signaling via EGFR may also occur in miR-8-Spitz positive glial cells, thereby contributing to the fine-tuning of Spitz protein activation and secretion (Morante, 2013).

    A niche typically refers to a confined anatomical location where adult stem cells reside and provides the signals required to sustain stem cell function and number. Niches are usually composed of supporting cells that make physical contact with the stem cells and act locally. The optic-lobe-associated miR-8-Spitz-positive cortex glia appear to represent a niche that contributes signals for the growth and morphogenesis of the neuroepithelium and that constitutes a functionally distinct population of that of the bloodbrain- barrier glial cells (Morante, 2013).

    Extrinsic signaling in the coordination of neuroepithelial proliferation in the developing mammalian forebrain of Foxc1 mutant mice has also been suggested. In these mice, the meninges are reduced or absent, resulting in an expansion of the neuroepithelium due to the predominance of symmetric divisions. The meninges are a source of the retinoic acid required for the transition of neuroepithelial cells into radial glia and neurons (Siegenthaler, 2009). Furthermore, meningeal cells secrete and organize the pial basement membrane, a thin sheet of extracellular matrix that covers the brain and that is enriched in a variety of growth factors. Rupture of the basement membrane in the developing brain causes type II lissencephaly, generating ectopic precursor clusters and cortical heterotopias due to impaired attachment of the radial glia to the basement membrane, resulting in a general laminar disorganization. These defects are reminiscent to those of disrupted surface glia cells described in this study (Morante, 2013).

    In summary these findings suggest that neuroepithelial proliferation and the onset of neuroblasts in the developing optic lobe neuroepithelium are largely influenced by extrinsic cues via a miR-8-dependent mechanism in the overlying glia. The reprogramming of neuroepithelial cells into neural stem cells (neuroblasts) is associated with dramatic morphological and molecular changes, including the loss of epithelial determinants DE-Cadherin, Crumbs and PatJ, and enhanced expression of the Snail-family zinc-finger transcriptional repressor Worniu. These changes are strikingly reminiscent of the events that drive the epithelial-to-mesenchymal transition (EMT), which confers a stem-like character in mammalian epithelial cells and in cancer cells and which are regulated by the miR-200 family. Indeed, downregulation of human mir-200 genes in epithelial normal and cancer cells promotes EMT and the acquisition of 'stemness' effects that are presumed to be cell-autonomous. These findings demonstrate that miR-8 can exert its effects non-cell- autonomously, opening the possibility that microRNA of the miR-200 family may play similar roles in stem cell fate niches and/or microenvironmental regulation of metastasis (Morante, 2013).

    miR-7 buffers differentiation in the developing Drosophila visual system

    The 40,000 neurons of the medulla, the largest visual processing center of the Drosophila brain, derive from a sheet of neuroepithelial cells. During larval development, a wave of differentiation sweeps across the neuroepithelium, converting neuroepithelial cells into neuroblasts that sequentially express transcription factors specifying different neuronal cell fates. The switch from neuroepithelial cells to neuroblasts is controlled by a complex gene regulatory network and is marked by the expression of the proneural gene l'sc. This study discovered that microRNA miR-7 is expressed at the transition between neuroepithelial cells and neuroblasts. miR-7 promotes neuroepithelial cell-to-neuroblast transition by targeting downstream Notch effectors to limit Notch signaling. miR-7 acts as a buffer to ensure that a precise and stereotypical pattern of transition is maintained, even under conditions of environmental stress, echoing the role that miR-7 plays in the eye imaginal disc. This common mechanism reflects the importance of robust visual system development (Caygill, 2017).

    Drosophila vision requires the accurate specification of over 80 different types of optic lobe neurons and the establishment of precise visual circuits between the neurons of the optic lobe and the photoreceptors of the eye. The medulla is the largest visual ganglion of the brain. Medulla neurons play roles in motion detection, through input from the R1-R6 photoreceptors via the lamina, and in the perception of color, via direct input from the R7 and R8 photoreceptors. The 40,000 medulla neurons originate from a pseudostratified neuroepithelium. During early development, symmetric division expands the stem cell pool. As development progresses, the medial edge of the neuroepithelium is progressively converted into asymmetrically dividing neuroblasts. Medulla neuroblasts sequentially express a series of transcription factors that specify the differentiation of the medulla neurons (Caygill, 2017).

    The transition from neuroepithelial cells into neuroblasts occurs in a highly ordered, sequential manner in response to expression of the proneural gene, lethal of scute (l'sc). Expression of L'sc marks a two- to three-cell-wide boundary between the neuroepithelial cells and the neuroblasts, the so-called transition zone, which moves medially across the neuroepithelium, forming a proneural wave. Within the transition zone, L'sc transiently suppresses Notch activity, triggering the switch from the symmetric, proliferative division of neuroepithelial cells to the asymmetric, differentiative division of neuroblasts. Progress of the wave is regulated by the orchestrated action of the Notch, epidermal growth factor receptor (EGFR), Fat-Hippo, and JAK/STAT signaling pathways (Caygill, 2017).

    This study presents evidence here that the transition from neuroepithelial cells to neuroblasts in the developing optic lobe is buffered by the microRNA miR-7. miR-7 is expressed at the transition zone in response to epidermal growth factor (EGF) signaling and is sufficient to promote transition. miR-7 acts via repression of downstream Notch effectors to limit Notch signaling and promote timely transition. In the absence of miR-7, proneural wave progression is disrupted. This disruption becomes more severe under conditions of temperature stress, suggesting that the role of miR-7 is to act as a buffer to ensure the timely and precise transition from neuroepithelial cells to neuroblasts in the developing optic lobe (Caygill, 2017).

    This study has shown that miR-7 targets members of the E(spl) family of bHLH transcription factors to define the boundaries of the transition zone, buffering the transition from neuroepithelial cell to neuroblast in the developing optic lobe. Downregulation of Notch signaling is essential for the neuroepithelial-to-neuroblast transition. L'sc provides one mechanism for Notch downregulation. miR-7, expressed in a pattern similar to that of l'sc at the transition zone, provides another, emphasizing the importance of Notch regulation at the transition zone (Caygill, 2017).

    The transition zone of the proneural wave mediates the specification of the neuroblasts of the medulla, the largest ganglion of the adult Drosophila visual system. The medulla receives information directly from the R7 and R8 photoreceptors of the ommatidia. Similar to the specification of neuroblasts by the proneural wave in the optic lobe, photoreceptor differentiation is triggered by the movement of the morphogenetic furrow across the epithelium of the eye imaginal disc. As the furrow passes, expression of the proneural gene atonal (ato) is induced in a stripe that is later refined to the R8 cells by Notch-mediated lateral inhibition (Caygill, 2017).

    Similar to the observations in the optic lobe, miR-7 has been shown to play a role in photoreceptor differentiation. Misexpression of miR-7 results in an increase in Ato expression and R8 cell specification, while a loss of miR-7 results in a decrease in Ato expression under conditions of temperature stress. These results show that miR-7 acts to buffer the development of both the medulla and the eye, two tissues that will directly communicate in the adult brain (Caygill, 2017).

    miR-7 has been shown to target anterior open (aop; also known as yan) in the eye imaginal disc. Within the neuroepithelium, aop activity helps to repress the neuroepithelial-to-neuroblast transition. This raises the possibility that miR-7 targeting of aop could also contribute to its function in buffering the neuroepithelial-to-neuroblast transition and that aop could represent a common target during the progression of the proneural wave and the morphogenetic furrow (Caygill, 2017).

    In the adult brain, each ommatidium maps to a columnar unit within the lamina and medulla, providing a retinotopic map of the visual field. Signaling from innervating photoreceptors induces the differentiation of lamina neurons. This direct communication provides a strict control of the mapping of photoreceptor and lamina neuron numbers. In contrast, while final numbers of ommatidia and medulla neurons show some co-ordination based on nutrient availability, there is no evidence for direct communication between the eye disc and the developing medulla. The presence of miR-7 in both the eye imaginal disc and the optic lobe represents an independent but conserved buffer that operates to coordinate appropriate developmental progression in each system, in spite of external environmental fluctuations. The presence of this common buffer provides robustness within each system that may contribute to ensuring the eventual connectivity required for retinotopic mapping of the visual system (Caygill, 2017).

    Notch regulates the switch from symmetric to asymmetric neural stem cell division in the Drosophila optic lobe

    The proper balance between symmetric and asymmetric stem cell division is crucial both to maintain a population of stem cells and to prevent tumorous overgrowth. Neural stem cells in the Drosophila optic lobe originate within a polarised neuroepithelium, where they divide symmetrically. Neuroepithelial cells are transformed into asymmetrically dividing neuroblasts in a precisely regulated fashion. This cell fate transition is highly reminiscent of the switch from neuroepithelial cells to radial glial cells in the developing mammalian cerebral cortex. To identify the molecules that mediate the transition, neuroepithelial cells were microdissected, and their transcriptional profile was compared with similarly obtained optic lobe neuroblasts. Genes encoding members of the Notch pathway were found expressed in neuroepithelial cells. Notch mutant clones are extruded from the neuroepithelium and undergo premature neurogenesis. A wave of proneural gene expression is thought to regulate the timing of the transition from neuroepithelium to neuroblast. The proneural wave transiently suppresses Notch activity in neuroepithelial cells, and inhibition of Notch triggers the switch from symmetric, proliferative division, to asymmetric, differentiative division (Egger, 2010).

    In the developing mammalian cortex, neural stem cells initially divide symmetrically to produce two neural stem cells, thereby increasing the neural precursor pool. The radial glial cells subsequently divide asymmetrically to produce a neural stem cell and either a basal progenitor cell or an immature neuron. Most basal progenitor cells divide once more to generate two postmitotic neuron. The Notch signalling pathway is thought to play a role in maintaining the undifferentiated state of neuroepithelial cells, radial glia and basal progenitors, but the downstream signalling cascades activated in these cells might be differentially regulated. Neurogenesis is initiated by proneural genes, such as Mash1 and Neurogenin2 (Ngn2) (Egger, 2010).

    This study shows that this sequence of neurogenic events is remarkably similar to that seen in the development of the optic lobe in Drosophila. Notch is activated in the neuroepithelial cells, which remain undifferentiated. The proneural gene l'sc is expressed within the transition zone, and levels of Delta are increased, while Notch activity is decreased. Thus neuroepithelial cells ultimately give rise to a variety of differentiated neurons, but only after they have passed through the transition zone (Egger, 2010).

    Low levels of Delta expression were found thoughout the optic lobe neuroepithelium, with increased expression in the transition zone. Several Brd genes were found within the neuroepithelium. Negative regulation of Delta activity by the Brd proteins would be expected to further reduce the level of Delta signalling. This situation might be analogous to the oscillations in Delta and Ngn2 levels observed in vertebrates, and it will be interesting to assess whether the expression of Delta, HLHm5 or proneural genes also oscillate in flies. Strikingly, when Delta activity is inhibited throughout the epithelium, the premature transformation of the entire neuroepithelium into neuroblasts is observed. This suggests that neuroepithelial cells might both send and receive the Notch signal (Egger, 2010).

    Higher levels of Delta were observed in the L'sc positive transition zone. High levels of Delta or Serrate can inhibit Notch signalling through cis-inhibition, suggesting one possible mechanism for the downregulation of Notch signalling at the transition zone. Interestingly, very recent results suggest that cis-inhibition can create sharp boundaries and this could be the role of the high levels of Delta that were observe in the transition zone (Egger, 2010).

    Epithelial integrity might be important to maintain proliferative cell division. This study shows that Notch mutant clones are extruded from the neuroepithelium. Furthermore, expression in the optic lobe neuroepithelial cells was found of a number of genes involved in cell adhesion. Notch could regulate cell adhesion molecules at the transcriptional level, or might itself form a complex with adhesion molecules. In either case, Notch loss of function would disrupt cell adhesion and lead to the extrusion of epithelial cells. Subtypes of cadherins, such as DE-Cad, Cad99C, Fat, which were found preferentially expressed in the neuroepithelium, might be activated by Notch to maintain the neuroepithelium, and repressed by L'sc to promote neurogenesis. Notch mutant clones also upregulate expression of the neuroblast transcription factor Dpn (but not of L'sc), and divide asymmetrically only once they have delaminated from the epithelium. In contrast to Notch mutant clones, L'sc misexpression clones upregulate Dpn and switch to asymmetric division whilst still embedded within the neuroepithelium. L'sc acts, at least in part, through repression of Notch signalling, but might also induce neuroblast-specific genes directly (Egger, 2010).

    JAK/STAT signalling negatively regulates the progression of the proneural wave and neurogenesis in the optic lobe. Interestingly, the ability of Notch to maintain radial glial cell fate appears to be largely dependent on functional JAK/STAT signalling. It remains to be seen whether the Notch pathway interacts with JAK/STAT in the Drosophila optic lobe (Egger, 2010).

    This study has shown that the development of the Drosophila optic lobe parallels that of the vertebrate cerebral cortex, suggesting that the pathways regulating the transition from symmetric to asymmetric division might be conserved from flies to mammals. Identifying the effector genes that are regulated by Notch and L'sc, and the links between JAK/STAT and Notch signalling, will yield further insights into the molecular mechanisms that maintain an expanding neural stem cell pool and regulate the timely transition to differentiation (Egger, 2010).

    Changes in Notch signaling coordinates maintenance and differentiation of the Drosophila larval optic lobe neuroepithelia

    A dynamic balance between stem cell maintenance and differentiation paces generation of post-mitotic progeny during normal development and maintenance of homeostasis. Recent studies show that Notch plays a key role in regulating the identity of neuroepithelial stem cells, which generate terminally differentiated neurons that populate the adult optic lobe via the intermediate progenitor cell type called neuroblast. Thus, understanding how Notch controls neuroepithelial cell maintenance and neuroblast formation will provide critical insight into the intricate regulation of stem cell function during tissue morphogenesis. This study shows that a low level of Notch signaling functions to maintain the neuroepithelial cell identity by suppressing the expression of pointedP1 gene through the transcriptional repressor Anterior open. Increased Notch signaling, which coincides with transient cell cycle arrest but precedes the expression of PointedP1 in cells near the medial edge of neuroepithelia, defines transitioning neuroepithelial cells that are in the process of acquiring the neuroblast identity. Transient up-regulation of Notch signaling in transitioning neuroepithelial cells decreases their sensitivity to PointedP1 and prevents them from becoming converted into neuroblasts prematurely. Down-regulation of Notch signaling combined with a high level of PointedP1 trigger a synchronous conversion from transitioning neuroepithelial cells to immature neuroblasts at the medial edge of neuroepithelia. Thus, changes in Notch signaling orchestrate a dynamic balance between maintenance and conversion of neuroepithelial cells during optic lobe neurogenesis (Weng, 2012).

    A deregulated conversion of neuroepithelial cells into neuroblasts perturbs formation of the neuronal network and will almost certainly lead to visual impairment of the adult fly. Thus, a dynamic balance between neuroepithelial cell maintenance and differentiation plays a pivotal role during morphogenesis of the optic lobe. This study provides evidence that changes in Notch signaling regulate the dynamic balance between maintenance of neuroepithelial cells and formation of neuroblasts. A low level of Notch signaling maintains the neuroepithelial cell identity by triggering Aop-dependent repression of the pntP1 gene. Transient up-regulation of Notch signaling in transitioning neuroepithelial cells raises their threshold of response to PntP1 preventing them from precociously converting into immature neuroblasts. Finally, abrupt down-regulation of Notch signaling together with a high level of PntP1 trigger the conversion from transitioning neuroepithelial cells into immature neuroblasts at the medial edge of neuroepithelia. Thus, interplay between changes in Notch signaling and transient up-regulation of pntP1 orchestrates synchronous and progressive formation of neuroblasts in a medial-to-lateral orientation across the entire neuroepithelial swath (Weng, 2012).

    Lack of Notch reporter transgene expression throughout neuroepithelia located laterally from transitioning neuroepithelial cells has been perplexing in light of recent studies reporting that Notch signaling is necessary for maintenance of their identity. One possibility might be that these Notch reporter transgenes including E(spl)mγ-GFP might not contain all necessary regulatory response elements to respond to Notch signaling in most neuroepithelial cells. Alternatively, the level of Notch signaling might simply be too low to activate the expression of the Notch reporter transgene. The second hypothesis is favored for the following reasons. Since over-expression of Notchintra is sufficient to trigger robust cell autonomous expression of E(spl)mγ-GFP in neuroepithelia located laterally from transitioning neuroepithelial cells, this transgene does contain all necessary regulatory elements to respond to a high level of Notch signaling. Furthermore, the Notch ligand Delta is expressed in a low level throughout neuroepithelia located laterally from transitioning neuroepithelial cells and Delta likely functions to trans-activate Notch signaling in these cells. In the context of trans-activation of Notch signaling by Delta, the level of the ligand correlates with the level of signaling output. Taken together, it is concluded that maintenance of the neuroepithelial cell identity requires a low level of Notch signaling (Weng, 2012).

    It is proposed that Notch maintains the identity of neuroepithelial cells by activating Aop-dependent repression of the pntP1 gene. The Suppressor of Hairless protein, which is necessary for activating transcription of Notch targets genes, directly binds to the promoter of the aop gene. Furthermore, removing the Notch or aop function triggered premature conversion of neuroepithelia into neuroblasts whereas over-expressing Notch or aop prevented conversion of neuroepithelial cells into neuroblasts. Most importantly, over-expression of aop suppressed premature differentiation of Notch mutant neuroepithelial cells. Finally, heterozygosity of the pntP1 gene completely suppressed premature conversion of neuroepithelial cells into neuroblasts in a hypomorphic aop mutant genetic background. These data lead to the conclusion that Notch signaling maintains the identity of neuroepithelial cells by activating an Aop-dependent repression of pntP1. In the future, analyses of Notch and pntP1 double mutants will be necessary to confirm this regulatory mechanism (Weng, 2012).

    Down-regulation of Notch signaling is necessary for formation of neuroblasts, so transient up-regulation of Notch signaling in transitioning neuroepithelial cells appears rather counterproductive. One possibility might be that up-regulation of Notch signaling paces the conversion from transitioning neuroepithelial cells into neuroblasts by increasing their threshold of response to PntP1. Consistent with this hypothesis, constitutively activated Notch signaling prevented transitioning neuroepithelial cells from becoming converted into neuroblasts despite expressing PntP1. This hypothesis was further supported by co-expression of pntP1 overcoming the blockade by constitutively activated Notch signaling and restoring conversion of transitioning neuroepithelial cells into neuroblasts. Thus, it is proposed that up-regulation of Notch signaling in transitioning neuroepithelial cells raises their threshold of response to PntP1 and functions to prevent them from becoming converted into immature neuroblasts precociously. Such an elaborated mechanism only permits transitioning neuroepithelial cells expressing the highest level of PntP1 to convert into immature neuroblasts. This mechanism is consistent with a recent study reporting that the EGF ligand is processed and secreted by cells near the medial edge of the optic lobe neuroepithelia. As a result of simple diffusion, transitioning neuroepithelial cells at the medial edge of neuroepithelia will be exposed to the highest level of the EGF ligand and will express the highest level of PntP1. As such, EGF signaling likely creates a vector field establishing the directionality of conversion from neuroepithelial cells into neuroblasts whereas Notch signaling refines the functional output of EGF signaling by raising the threshold response to PntP1 (Weng, 2012).

    Many important questions arise from this highly plausible mechanism by which the interplay between Notch and EGF signaling paces synchronous conversion of neuroepithelial cells into neuroblasts one row at a time. This model will predict that immature neuroblasts immediately adjacent to transitioning neuroepithelial cells should secrete the processed EGF ligand. However, the antibody specific for the Rhomboid (Rho) protease required for proteolytic activation of the EGF protein is currently unavailable and a genomic fragment encompassing the rho-1 locus tagged with YFP did not show detectable expression in the larval optic lobe. Alternatively, a recent study shows that pntP1 is a direct target of Notch in vivo. Thus, up-regulation of Notch signaling might directly activate transcription of the pntP1 gene in transitioning neuroepithelial cells. Since Notch signaling becomes abruptly down-regulated at the medial edge of neuroepithelia, it is highly possible that the threshold of response to PntP1 also becomes lowered in the same cells. Thus, the pre-existing level of PntP1 protein will likely be sufficient to trigger the conversion from transitioning neuroepithelial cells into immature neuroblasts. More experiments including identification of the cell type from which the processed EGF ligand is released and a direct test to confirm the role of EGF signaling during conversion of neuroepithelia into neuroblasts will be key to distinguish these two possible mechanisms (Weng, 2012).

    The fat-hippo signaling mechanism controls tissue growth by regulating proliferation and cell death and promotes timely differentiation of optic lobe neuroepithelial cells). While inactivation of fat-hippo signaling delays conversion of neuroepithelia into neuroblasts, removal of the downstream effecter yorkie only accelerates the conversion near the medial edge of the optic lobe neuroepithelia. Thus, fat-hippo signaling likely functions as a gatekeeper to prevent over-growth of optic lobe neuroepithelia by triggering transient cell cycle arrest. Intriguingly, transient cell cycle arrest precedes increased Notch signaling in transitioning neuroepithelial cells. Detailed studies in the future will be necessary to determine whether activation of the fat-hippo signaling might contribute to increased Notch signaling in transitioning neuroepithelial cells (Weng, 2012).

    Serrate-Notch-Canoe complex mediates glial-neuroepithelial cell interactions essential during Drosophila optic lobe development

    It is firmly established that neuron-glia interactions are fundamental across species for the correct establishment of a functional brain. This study found that the glia of the Drosophila larval brain display an essential non-autonomous role during the development of the optic lobe. The optic lobe develops from neuroepithelial cells that proliferate by dividing symmetrically until they switch to asymmetric/differentiative divisions generating neuroblasts. The proneural gene lethal of scute (l'sc) is transiently activated by the Epidermal Growth Factor Receptor (EGFR)/Ras signal transduction pathway at the leading edge of a proneural wave that sweeps from medial to lateral neuroepithelium promoting this switch. This process is tightly regulated by the tissue-autonomous function within the neuroepithelium of multiple signaling pathways, including EGFR/Ras and Notch. This study shows that the Notch ligand Serrate (Ser) is expressed in the glia and it forms a complex in vivo with Notch and Canoe, which colocalize at the adherens junctions of neuroepithelial cells. This complex is crucial for glial-neuroepithelial cell interactions during optic lobe development. Ser is tissue-autonomously required in the glia where it activates Notch to regulate its proliferation, and non-autonomously in the neuroepithelium where Ser induces Notch signaling to avoid the premature activation of the EGFR/Ras pathway and hence of L'sc. Interestingly, different Notch activity reporters showed very different expression patterns in the glia and in the neuroepithelium, suggesting the existence of tissue-specific factors that promote the expression of particular Notch target genes or/and a reporter response dependent on different thresholds of Notch signaling (Perez-Gomez, 2013).

    Cno and its vertebrate homologues AF-6/Afadin localize at epithelial AJs where they regulate the linkage of AJs to the actin cytoskeleton by binding both actin and nectin family proteins. This study found that Cno colocalizes with Notch at the AJs of NE cells in the optic lobe proliferation centers. Notch also colocalizes with its ligand Ser, which was detected at the glia, highly accumulated at the interface between NE cells and the surrounding glia. Co-immunoprecipitation experiments indicate the formation of a Ser-Notch-Cno complex in vivo, and the mutant analysis shows the functional relevance of such a complex at the glia neuroepithelium interface. The data presented in this study support the hypothesis that Cno may be stabilizing Notch at the AJs of NE cells, favoring the binding of Ser present in the adjacent glial cells. Indeed, in cno lof both Notch and Ser distribution is affected; this alteration is accompanied by an abnormally advanced proneural wave, a reminiscent phenotype to that shown by Notch lof optic lobes and also a similar phenotype found in this work in Ser lof. The activation of Notch pathway is essential to maintain the integrity of the neuroepithelium and to allow the correct progression of the proneural wave. The results show that glial Ser is responsible of such activation, promoting the expression of the m7-nuclacZ reporter in NE cells. In fact, the reduction of glial Ser either by knocking down epithelial cno or by expressing DNSer in the glia led to a decrease in the expression of the m7-nuclacZ reporter in NE cells and to an ectopic activation of the Ras/PntP1 pathway and of L'sc. It is proposed that this may be ultimately the cause of the proneural wave advance observed in those genotypes. Thus, the activation of Notch in the neuroepithelium by glial Ser, in nomal conditions, would be essential to avoid a premature activation of the EGFR/Ras/PntP1 pathway and hence of L'sc. Indeed, Notch has been shown to downregulate different EGFR/Ras signaling pathway components such as Rhomboid (Rho), required for the processing of the EGFR ligand Spitz, in other developmental contexts in which both pathways are actively cross-talking. Therefore, Notch activity in NE cells could be contributing to inhibit Rho, restricting its presence to the transition zone where Rho is very locally expressed (Perez-Gomez, 2013).

    It was observed that in a WT condition Ser is present in all surface glia (perineurial and subperineurial), as shown by the expression of CD8::GFP (SerGal4>>UAS-CD8::GFP), and Notch, as tested by different reporters, is active in this tissue and highly reduced in Ser lof in the glia. This makes sense with the existence of a Ser-Notch mediated intercellular communication between the glial cells that comprise both the perineurial and subperineurial glia. Intriguingly, the knockin down and overexpression of cno in NE cells also had a clear effect on Notch activity in the glia, a reduction and an increase, respectively. This is more challenging to explain. As the cno lof in the NE led to a high reduction of both neuroepithelial Notch and glial Ser, the easiest explanation is that an 'excess' of unbound glial Ser is degraded and this impinges on the general thresholds of glial Ser, therefore causing a general reduction in the Notch activity in this tissue. This is an interesting field to explore in detail and is left open for future investigation (Perez-Gomez, 2013).

    The activity of Notch in the neuroepithelium and in medulla NBs seems controversial. For example, Notch has been shown to be active in the neuroepithelium at low/null levels or in a 'salt and pepper' patter. A weak/null activity of Notch has also been reported in NBs as well as a high activation. One possibility to conciliate all these results and apparently contradictory data is that different Notch target genes used as Notch activity reporters are, in fact, differentially activated in particular regions or tissues. The results support this proposal. Four different Notch reporters were used in this study. Whereas m7-nuclacZ was expressed throughout the neuroepithelium, Gbe+Su(H)lacZ was restricted to the transition zone, although both were expressed in medulla NBs along with mβ-CD2. In addition, mβ-CD2 was strongly activated in the glia, whereas the Gbe+Su(H)lacZ and the mδ-lacZ reporters were expressed at much lower levels at this location. Differential activation of Notch targets genes has been previously reported and tissue-specific factors could contribute to this differential expression. This is an intriguing scenario to analyze in the future. The in depth analysis of other Notch reporter genes in the developing optic lobe can contribute to further clarify this issue (Perez-Gomez, 2013).

    At third larval instar during optic lobe development, Dl is highly restricted to 2-3 cells at the transition zone in the neuroepithelium, where Dl activates Notch. This work has found that the other ligand of Notch, Ser, is expressed in the surrounding glia at this larval stage and it is strongly accumulated at the interface with NE cells. Ser activates Notch in the neuroepithelium and this, in turn, would contribute to restrict the activation of the Ras-PntP1 pathway and L'sc to the transition zone. Intriguingly, it was observed that Ser preferentially activates the Notch target gene m7-nuclacZ in the neuroepithelium whereas Dl activates other Notch target genes, including Gbe+Su(H)lacZ, in the transition zone. For example, the overexpression of Dl in NE cells caused an ectopic activation throughout the neuroepithelium of Gbe+Su(H)lacZ, along with dpn that also behaves as a Notch target in other systems, and a repression of m7-nuclacZ . In addition, the lof of Ser in the glia caused a striking decrease in the expression of m7-nuclacZ in the neuroepithelium. One possibility to explain these observations is that the pool of Notch associated to the AJs and activated by glial Ser is subject of particular posttranslational modifications or/and is associated with other AJs proteins (including Cno) that somehow make Notch more receptive to Ser and able to activate specific target genes (i.e., m7). In this regard, it is interesting to note that Dl ectopically expressed in the glia (i.e., repoGal4>>UAS-Dl) was not detected at the interface with NE cells, where glial Ser is highly present in contact with Notch, but Dl was restricted to the outermost surface glia (perineurial glia). This result strongly indicates that Dl cannot bind or has very low affinity for this pool of Notch at the AJs, hence being actively degraded in the subperineurial glia. This low affinity of Dl by Notch at this location further suggests that this pool of Notch at the AJs must be endowed with particular characteristics that ultimately could alter the activity properties of Su(H), explaining in turn the distinct expression pattern of Notch targets genes. Another possibility, which is not necessarily exclusive, to explain the differential activation of the Notch reporters is that they respond to different Notch thresholds. For example, m7-nuclacZ would require very low levels of Notch activation whereas Gbe+Su(H)lacZ would require high amounts of Notch signaling in NE cells. All these questions remain open for further investigation (Perez-Gomez, 2013).

    Fat/Hippo pathway regulates the progress of neural differentiation signaling in the Drosophila optic lobe

    A large number of neural and glial cell cell types differentiate from neuronal precursor cells during nervous system development. Two types of Drosophila optic lobe neurons, lamina and medulla neurons, are derived from the neuroepithelial (NE) cells of the outer optic anlagen. During larval development, epidermal growth factor receptor (EGFR)/Ras signaling sweeps the NE field from the medial edge and drives medulla neuroblast (NB) formation. This signal drives the transient expression of a proneural gene, lethal of scute, and its signal array is referred to as the 'proneural wave', since it is the marker of the EGFR/Ras signaling front. This study shows that the atypical cadherin Fat and the downstream Hippo pathways regulate the transduction of EGFR/Ras signaling along the NE field and, thus, ensure the progress of NB differentiation. Fat/Hippo pathway mutation also disrupts the pattern formation of the medulla structure, which is associated with the regulation of neurogenesis. A candidate for the Fat ligand, Dachsous is expressed in the posterior optic lobe, and its mutation was observed to cause a similar phenotype as fat mutation, although in a regionally restricted manner. It was also shown that Dachsous functions as the ligand in this pathway and genetically interacts with Fat in the optic lobe. These findings provide new insights into the function of the Fat/Hippo pathway, which regulates the ordered progression of neurogenesis in the complex nervous system (Kawamori, 2011).

    The Fat/Hippo pathway has been known as a tumor suppressor pathway. This study and in the report of Reddy (2010), it was shown that the loss of Fat/Hippo signaling causes a delay of NB differentiation in the optic lobe. In contrast, dachs;ft double mutation, which is expected to stabilize the Fat/Hippo pathway, causes an advance of NB differentiation. This led to the question of how the Fat/Hippo pathway controls NB differentiation (Kawamori, 2011).

    It has been reported that EGFR/Ras signaling is necessary and sufficient for NB induction, and its transduction is the driving force of the progress of the proneural wave. EGFR/Ras signaling sweeps the NE field through the gradual activation of Ras and its downstream EGF secretion by Rho. It was reasoned that this signal transduction is the target of the Fat/Hippo pathway in the control of NB differentiation. Indeed, ectopic expression of the EGFR/Ras signaling components RasV12 and rho was sufficient to induce NBs in the ft mutant background (Kawamori, 2011).

    Which step of this cycling process does Fat/Hippo pathway mutation affect? Based on ectopic expression experiments, the Fat/Hippo pathway lies upstream of Ras and Rho, and it is expected to control the process from EGF transmission to Ras activation. The phenotypic difference produced by RasV12 and rho overexpression should be noted. When rho was expressed in the ft mutant background, several NE clones with abnormal morphology remained. This phenotype was not observed when RasV12 was expressed. In this model, RasV12 drives NB differentiation in RasV12-expressing cells in a cell-autonomous manner. In contrast, Rho activates Ras signaling in neighboring cells through the secretion of an EGF ligand. Based on these phenotypic differences, the Fat/Hippo pathway is expected to control the cell-to-cell EGF transmission, including its secretion, distribution or reception at the cell surface. This hypothesis is supported by the fact that several signaling components of the EGFR/Ras pathway, including Rho and EGFR, are localized to the apical side of epithelial tissues, and it is thought that this signal is transmitted along the apical side in epithelial tissues. It has also been reported that Fat/Hippo pathway mutations enhance the expression level of several apically localized molecules, such as aPKC, PatJ, Crumbs and E-cadherin. Thus, Fat/Hippo signaling targets could include unknown apical components that are involved in EGF transmission and this could account for the incomplete NB induction by rho overexpression. rho-expressing cells secret the EGF ligand, which diffuses in the NE surface, but Fat/Hippo pathway mutation would prevent its cell-to-cell transmission and subsequent EGFR/Ras pathway activation in the receiving cells. In this hypothesis, EGF transmission would be disturbed in the NE mutant for the Fat/Hippo pathway, causing the delay of proneural wave progress (Kawamori, 2011).

    As an alternative hypothesis, the Fat/Hippo pathway could regulate signal transduction from the EGFR to Ras activation. If this is the case, the Fat/Hippo pathway regulates the intracellular signal transduction of the EGFR pathway. Many of the known targets of the Fat/Hippo pathway are components of growth regulatory, cell survival and cell adhesion molecules. There could be unknown targets that modulate other signaling pathways, including the EGFR pathway, and the NB differentiation defect would thus be caused by a failure in the activation of differentiation signals in the absence of Fat/Hippo signaling (Kawamori, 2011).

    This study shows that the Fat/Hippo pathway mutation also affected the morphological character of the NE. Fat/Hippo pathway mutant clones were induced, and they often included NE tissue with a folded morphology and disrupted the medulla structure. The results showed that the Fat/Hippo pathway functions in the regulation of NB differentiation and in NE morphology are distinct, but the two functions could affect each other. The morphological defect of the NE could affect EGFR/Ras signal transduction. The possibility is discussed that the EGF ligand could be distributed along the apical membrane of the NE. The invagination of the apical membrane of the folded NE into the inner region could prevent EGF ligand signaling. There were clones with a normal NE morphology in which NB differentiation was delayed and, thus, morphological defects are not determinate, but they could promote the delay of NB induction (Kawamori, 2011).

    How is the activity of the Fat/Hippo pathway regulated throughout the development of the optic lobe? Ft is a member of the cadherin family, and an extracellular molecule is expected to regulate its activity. Ds is a candidate for the Ft ligand that regulates planar cell polarity and Fat/Hippo signaling activity in other epithelial tissues. The expression of ds with a posterior-specific pattern in the developing optic lobe (Reddy, 2010) was confirmed. In the rescue experiments for the ds mutation, the expression of either ds lacking its intracellular domain (dsΔICD) or ft lacking its extracellular domain (ftΔECD) was sufficient to compensate for ds function, suggesting that Ds functions as a ligand and that Ft lies downstream of Ds in this context (Kawamori, 2011).

    The phenotypes of ds and ft mutants were compared to assess whether the mutation of ds by itself accounts for the phenotype of the ft mutants. In contrast to the ft mutants that exhibited altered NB differentiation in the entire outer optic anlagen, the ds mutant phenotype was regionally specific; NB differentiation was severely delayed in the posterior region, and the development of the anterior region was not significantly affected. These differences suggest that there might be some regulatory mechanisms that control Ft activity independently of Ds in the anterior region of the optic lobe (Kawamori, 2011).

    The Fat/Hippo pathway is known as a tumor suppressor pathway, and many studies related to this pathway have focused on tissue growth or cell survival. This study has reported a new function of the Fat/Hippo pathway in the regulation of neural differentiation. The Fat/Hippo pathway regulates the progress of neural differentiation signaling, and the EGFR/Ras pathway is a candidate target of this pathway. The data suggest that the Fat/Hippo pathway includes unknown targets involved in EGFR/Ras signal transduction. Further studies are required to identify the targets of the Fat/Hippo pathway and determine the interplay between Fat/Hippo and EGFR/Ras pathways, specifically in NB differentiation (Kawamori, 2011).

    The tumour suppressor L(3)mbt inhibits neuroepithelial proliferation and acts on insulator elements

    In Drosophila, defects in asymmetric cell division often result in the formation of stem cell derived tumors. This study shows that very similar terminal brain tumor phenotypes arise through a fundamentally different mechanism. Brain tumors in l(3)mbt mutants originate from overproliferation of neuroepithelial cells in the optic lobes caused by de-repression of target genes in the Salvador-Warts-Hippo (SWH) pathway. ChIP-seq was used to identify L(3)mbt binding sites, and it was shown that L(3)mbt binds to chromatin insulator elements. Mutating l(3)mbt or inhibiting expression of the insulator protein gene mod(mdg4) results in upregulation of SWH-pathway reporters. As l(3)mbt tumors are rescued by mutations in bantam or yorkie or by overexpression of expanded the deregulation of SWH pathway target genes is an essential step in brain tumor formation. Therefore, very different primary defects result in the formation of brain tumors, which behave quite similarly in their advanced stages (Richter, 2011).

    Drosophila nervous system recapitulates many steps in mammalian neurogenesis. Neurons in the adult fly brain arise from stem cells called neuroblasts which undergo repeated rounds of asymmetric cell division during larval stages. After division, one daughter cell remains a neuroblast while the other is called the ganglion mother cell (GMC) and divides just once more into two differentiating neurons. Most larval neuroblasts are inherited from the embryo but the so-called optic lobe neuroblasts (NB) located laterally on each brain lobe pass through a neuroepithelial (NE) stage and are therefore a particularly suitable model for mammalian neurogenesis. During early larval stages, the NE cells of the optic lobes (OL) proliferate and separate into the inner (IOA) and outer (OOA) optic anlagen. During late larval stages, NE cells switch to a neurogenic mode. On the medial side, they generate optic lobe neuroblasts (OL NBs), which generate the neurons of the medulla, the second optic ganglion. OL neurogenesis is controlled by a wave of lethal of scute (l(1)sc) expression passing through the neuroepithelium from medial to lateral. The activity of the Jak/STAT pathway inhibits neural wave progression and thereby controls neuroblast number. Differentiation of neuroepithelial cells also involves the Notch, Epidermal Growth Factor (EGF) and Salvador-Warts-Hippo (SWH) pathways (Richter, 2011).

    Characterization of Drosophila genes identified in brain tumor suppressor screens has demonstrated that defects in neuroblast asymmetric cell division result in the formation of stem cell derived tumors that metastasize and become aneuploid upon transplantation. These screens also identified lethal (3) malignant brain tumor (l(3)mbt), a conserved transcriptional regulator that is also required for germ-cell formation in Drosophila. L(3)mbt binds to the cell cycle regulators E2F and Rb but the relevance of these interactions is unclear. This study shows that in Drosophila, L(3)mbt regulates target genes of the Salvador-Warts-Hippo (SWH) pathway that are important in proliferation and organ size control. The SWH-pathway is regulated by the membrane proteins Expanded (Ex) and Fat, which activate a protein complex containing the kinases Hippo and Warts to phosphorylate the transcriptional co-activator Yorkie. Yorkie acts together with the transcription factors Scalloped and Homothorax to activate proliferative genes like Cyclin E and the microRNA bantam (ban) and Drosophila inhibitor of apoptosis 1 (diap1: thread). Upon phosphorylation, Yorkie is retained in the cytoplasm and its target genes are not activated. In Drosophila the main role of the SWH-pathway is to limit proliferation in imaginal discs and its absence leads to tumorous overgrowth. In vertebrates, many homologs of key pathway members are tumor suppressors indicating that this function is conserved (Richter, 2011).

    L(3)mbt contains three MBT domains which bind mono- or dimethylated histone tails. Biochemical experiments in vertebrates have suggested a role in chromatin compaction but whether this role is conserved is not known. Results published while this paper was under review have shown that germline genes are upregulated in l(3)mbt mutant brains and are necessary for tumor formation. The current data indicate that L(3)mbt is bound to insulator sequences, which affect promoter-enhancer interactions and influence transcription. In Drosophila, the proteins CTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4) and GAF are found at insulator sequences but how these factors act is largely unknown (Richter, 2011).

    The data presented in this study show that tumor formation in l(3)mbt mutants is initiated by the uncontrolled overproliferation of neuroepithelial cells in the optic lobes due to the upregulation of proliferation control genes normally repressed by the SWH-pathway. L(3)mbt is located at DNA sequences bound by chromatin insulators and we propose that the function of L(3)mbt as a chromatin insulator is essential for repressing SWH target genes and preventing brain tumor formation (Richter, 2011).

    brat, lgl and dlg were previously identified as Drosophila brain tumor suppressors. In all cases, defects in asymmetric cell division cause a huge expansion of the neuroblast pool. In l(3)mbt mutants, however, the neuroblast pool is expanded because an upregulation of SWH target genes results in a massive expansion of neuroepithelial tissue. Why those neuroblasts proliferate indefinitely upon transplantation is currently not understood for any of those mutants (Richter, 2011).

    While the SWH-pathway is essential for tumorigenesis in l(3)mbt mutants, its overactivation can not recapitulate the neuroblast tumor phenotype seen in l(3)mbt mutants (this study and Reddy, 2010). Similar to the multifactorial origin of mammalian tumors, therefore, the combined deregulation of several signaling pathways could be required. The Notch pathway could be involved as it regulates the formation of OL neuroblasts from neuroepithelia and Notch pathway gene insulator sequences are bound by L(3)mbt. Increased activity of the Jak/STAT pathway, a major regulator of OL development, was also observed. Finally, the deregulation of germline genes in l(3)mbt mutants that has been described while this manuscript was under review (Janic, 2010) could provide another exciting explanation (Richter, 2011).

    The results indicate that L(3)mbt acts on insulator elements, which isolate promoters from the activity of nearby enhancers acting on other genes. the analysis showed that L(3)mbt binding sites overlap with CP190, CTCF and BEAF-32, placing the protein into what has been called the class I of chromatin insulators (Negre, 2010; Richter, 2011 and references therein).

    The identification of a DNA consensus motif for a histone binding protein like L(3)mbt is highly unexpected as insulators are typically nucleosome free. Currently, the activity of these important transcriptional regulators could be explained in several ways. Either, they form physical barriers blocking the interaction between enhancers and promoters. Alternatively, they mimic promoters and compete with endogenous promoters for enhancer interaction. Finally, they could interact with each other or nuclear structures to form loop domains that regulate transcriptional activity. The data suggests another model in which insulators interact with histones on nearby nucleosomes and influence the structure of higher order chromatin. Importantly, in the regions flanking CTCF binding sites nucleosomes are enriched for histones that are mono- and di-methylated on H3K4 or mono-methylated on H3K9 or H4K20, the variants to which MBT domains can bind in vitro. As the human L(3)mbt homolog L3MBTL1 was shown to compact nucleosome arrays in vitro , a model becomes feasible in which simultaneous binding to insulators and the surrounding nucleosomes reduces flexibility and thereby restricts the ability of nearby enhancers to interact with promoters on the other side of the insulator. However, the data could equally well be worked into the other prevalent models for insulator activity. Since L(3)mbt is currently the only chromatin insulator besides CTCF that is conserved in vertebrates, analysis of its homologs will certainly allow to distinguish between those possibilities (Richter, 2011).

    OL development resembles vertebrate neurogenesis. Both processes consist of an initial epithelial expansion phase followed by neurogenesis through a series of asymmetric divisions. Together with previous findings, these data demonstrate that l(3)mbt and the SWH-pathway are crucial regulators of the initial neuroepithelial proliferation phase. Interestingly, the SWH-pathway has been implicated in regulating neural progenitors in the chicken embryo and it will be exciting to test the role of mammalian L(3)mbt in this process. It is remarkable that YAP is upregulated and L3MBTL3 is deleted in a subset of human medulloblastomas. Medulloblastoma is the leading cause of childhood cancer death and investigating the role of the SWH-pathway might contribute to the progress in fighting this disastrous disease (Richter, 2011).

    Temporal patterning of Drosophila medulla neuroblasts controls neural fates

    In the Drosophila optic lobes, the medulla processes visual information coming from inner photoreceptors R7 and R8 and from lamina neurons. It contains approximately 40,000 neurons belonging to more than 70 different types. This study describes how precise temporal patterning of neural progenitors generates these different neural types. Five transcription factors - Homothorax, Eyeless, Sloppy paired, Dichaete and Tailless - are sequentially expressed in a temporal cascade in each of the medulla neuroblasts as they age. Loss of Eyeless, Sloppy paired or Dichaete blocks further progression of the temporal sequence. Evidence is provided that this temporal sequence in neuroblasts, together with Notch-dependent binary fate choice, controls the diversification of the neuronal progeny. Although a temporal sequence of transcription factors had been identified in Drosophila embryonic neuroblasts, this work illustrates the generality of this strategy, with different sequences of transcription factors being used in different contexts (Li, 2013).

    In the developing medulla, the wave of conversion of neuroepithelium into neuroblasts makes it possible to visualize neuroblasts at different temporal stages in one snapshot, with newly generated neuroblasts on the lateral edge and the oldest neuroblasts on the medial edge of the expanding crescent shaped neuroblast region. An antibody screen was conducted for transcription factors expressed in the developing medulla and five transcription factors, Hth, Ey, Slp1, D and Tll, were identified that are expressed in five consecutive stripes in neuroblasts of increasing ages, with Hth expressed in newly differentiated neuroblasts, and Tll in the oldest neuroblasts. This suggests that these transcription factors are sequentially expressed in medulla neuroblasts as they age. Neighbouring transcription factor stripes show partial overlap in neuroblasts with the exception of the D and Tll stripes, which abut each other. Previous studies have reported that Hth and Ey< were expressed in medulla neuroblasts, but they had not been implicated in controlling neuroblast temporal identities. Hth and Tll also show expression in the neuroepithelium (Li, 2013).

    To address whether each neuroblast sequentially expresses the five transcription factors, their expression was examined in the neuroblast progeny. Hth, Ey and Slp1 are expressed in three different layers of neurons that correlate with birth order, that is, Hth in the first-born neurons of each lineage in the deepest layers; Ey or Slp1 in correspondingly more superficial layers, closer to the neuroblasts. This suggests that they are born sequentially in each lineage. D is expressed in two distinct populations of neurons. The more superficial population inherit D from D+ neuroblasts. D+ neurons in deeper layers (corresponding to the Hth and Ey layers) turn on D expression independently and will be discussed later. Single neuroblast clones were generated, and the expression of the transcription factors was examined in the neuroblast and its progeny. Single neuroblast clones in which the neuroblast is at the Ey+ stage include Ey+ GMCs/neurons as well as Hth+ neurons. This indicates that Ey+ neuroblasts have transited through the Hth+ stage and generated Hth+ neurons. Clones in which the neuroblast is at the D+ stage contain Slp1+ GMCs and Ey+ neurons, suggesting that D+ neuroblasts have already transited through the Slp+ and Ey+ stages. This supports the model that each medulla neuroblast sequentially expresses Hth, Ey, Slp1 and D as it ages, and sequentially produces neurons that inherit and maintain expression of the transcription factor (Li, 2013).

    slp1 and slp2 are two homologous genes arranged in tandem and function redundantly in embryonic and eye development. Slp2 is expressed in the same set of medulla neuroblasts as Slp1. Slp1 and Slp2 are referred to collectively as Slp (Li, 2013).

    Tll is expressed in the oldest medulla neuroblasts. The oldest Tll+ neuroblasts show nuclear localization of Prospero (Pros), suggesting that they undergo Pros-dependent cell-cycle exit at the end of their life, as in larval nerve cord and central brain neuroblasts. Tll+ neuroblasts and their progeny express glial cells missing (gcm), and the progeny gradually turn off Tll and turn on Repo, a glial-specific marker. These cells migrate towards deeper neuronal layers and take their final position as glial cells around the medulla neuropil. Thus, Tll+ neuroblasts correspond to previously identified glioblasts between the optic lobe and central brain that express gcm and generate medulla neuropil glia. Clones in which the neuroblast is at the Tll+ stage contain Hth+ neurons and Ey+ neurons, among others, confirming that Tll+ neuroblasts represent the final temporal stage of medulla neuroblasts rather than a separate population of glioblasts. Therefore, these data clearly show that medulla neuroblasts sequentially express five transcription factors as they age. The four earlier temporal stages generate neurons that inherit and maintain the temporal transcription factor present at their birth, although a subset of neurons born during the Ey, Slp or D neuroblast stages lose expression of the neuroblast transcription factor. At the final temporal stage, neuroblasts switch to glioblasts and then exit the cell cycle (Li, 2013).

    Whether cross-regulation among transcription factors of the neuroblast temporal sequence contributes to the transition from one transcription factor to the next was examined. Loss of hth or its cofactor, extradenticle (exd), does not affect the expression of Ey and subsequent progression of the neuroblast temporal sequence (Li, 2013).

    ey-null mutant clones were generated using a bacterial artificial chromosome (BAC) rescue construct recombined on a chromosome containing a Flip recombinase target (FRT) site in an eyJ5.71 null background. eyJ5.71 homozygous mutant larvae were also tested. In both cases, Slp expression is lost in neuroblasts, along with neuronal progeny produced by Slp+ neuroblasts, marked by the transcription factor Twin of eyeless (Toy, see below). However, neuroblast division is not affected, and Hth remains expressed in only the youngest neuroblasts and first-born neurons. Targeted ey RNA interference (RNAi) using a Vsx-Gal4 driver that is expressed in the central region of the neuroepithelium and neuroblasts gives the same phenotype. This suggests that Ey is required to turn on the next transcription factor, Slp, but is not required to repress Hth (Li, 2013).

    In clones of a deficiency mutation, slpS37A, that deletes both slp1 and slp2, neuroblasts normally transit from Hth+ to Ey+, but older neuroblasts maintain the expression of Ey and do not progress to express D or Tll, suggesting that Slp is required to repress ey and activate D (Li, 2013).

    Similarly, in D mutant clones, neuroblasts are also blocked at the Slp+ stage, and do not turn on Tll, indicating that D is required to repress slp and activate tll. Finally, in tll mutant clones, D expression is not expanded into oldest neuroblasts, suggesting that tll is not required for neuroblasts to turn off D. Thus, in the medulla neuroblast temporal sequence, ey, slp and D are each required for turning on the next transcription factor. slp and D are also required for turning off the preceding transcription factor (Li, 2013).

    Gain-of-function phenotypes of each gene were studied. However, misexpression of Hth, Ey, Slp1 or Slp2, or D in all neuroblasts or in large neuroblast clones is not sufficient to activate the next transcription factor or repress the previous transcription factor in neuroblasts. Only misexpressing tll in all neuroblasts is sufficient to repress D expression (Li, 2013).

    In summary, cross-regulation among transcription factors is required for at least some of the transitions. No cross-regulation was observed between hth and ey. Because ey is already expressed at low levels in the neuroepithelium and in Hth+ neuroblasts, an as yet unidentified factor might gradually upregulate ey and repress hth to achieve the first transition. As tll is sufficient but not required to repress D expression, additional factors must act redundantly with Tll to repress D (Li, 2013).

    The temporal sequence of neuroblasts described above could specify at least four neuron types plus glia (in fact more than ten neuron types plus glia considering that neuroblasts divide several times at each stage with overlaps between neighbouring temporal transcription factors). As this is not sufficient to generate the 70 medulla neuron types, it was asked whether another process increases diversity in the progeny neurons born from a neuroblast at a specific temporal stage. Apterous (Ap) is known to mark about half of the 70 medulla neuron types. In the larval medulla, Ap is expressed in a salt-and-pepper manner in subsets of neurons born from all temporal stages. In the progeny from Hth+ neuroblasts, all neurons seem to maintain Hth, with a subset also expressing Ap. However, only half of the neurons born from neuroblasts at other transcription factor stages maintain expression of the neuroblast transcription factor. For instance, in the progeny of Ey+ neuroblasts, Ey+ neurons are intermingled with about an equal number of Ey neurons that instead express Ap. Neuroblast clones contain intermingled Ey+ and Ap+ neurons. This is also true for the progeny of Slp+ neuroblasts: Slp1+ neurons are intermingled with Slp1 Ap+ neurons. In the progeny of D+ neuroblasts, D and Ap are co-expressed in the same neurons, and they are intermingled with neurons that express neither D nor Ap. Neurons in deeper neuronal layers (corresponding to the Ey+ and Hth+ neuron layers) also express D independently, and these neurons are Ap. The expression of Ap is stable from larval to adult stages (Li, 2013).

    The intermingling of Ap+ and Ap neurons raised the possibility that asymmetric division of GMCs gives rise to one Ap+ and one Ap neuron. Two-cell clones were generated to visualize the two daughters of a GMC. In every case, one neuron is Ap+ and the other is Ap-, suggesting that asymmetric division of GMCs diversifies medulla neuron fates by controlling Ap expression (Li, 2013).

    Asymmetric division of GMCs in Drosophila involves Notch (N)-dependent binary fate choice. In the developing medulla, the N pathway is involved in the transition from neuroepithelium to neuroblast, and loss of Su(H), the transcriptional effector of N signalling, leads to faster progression of neurogenesis and neuroblast formation. However, Su(H) mutant neuroblasts still follow the same transcription factor sequence and generate GMCs and neuronal progeny, allowing analysis of the effect of loss of N function on GMC progeny diversification. Notably, neurons completely lose Ap expression in Su(H) mutant clones. All mutant neurons born during the Hth+ stage still express Hth, but not Ap, suggesting that the NON daughters of Hth+ GMCs are the neurons expressing both Ap and Hth. In contrast to wild-type clones, all Su(H) mutant neurons born during the Ey+ neuroblast stage express Ey and none express Ap. Similarly, all mutant neurons born during the Slp+ neuroblast stage express Slp1 but lose Ap. These data suggest that, for Ey+ or Slp+ GMCs, the NOFF daughter maintains the neuroblast transcription factor expression, whereas the NON daughter loses this expression but expresses Ap. In the wild-type progeny born during the D+ neuroblast stage, Ap+ neurons co-express D. Both D and Ap are lost in Su(H) mutant clones in the D+ neuroblast progeny, confirming that D is transmitted to the Ap+ NON daughter of D+ GMCs. By contrast, the D+ Ap neurons in the deeper layers (corresponding to the NOFF progeny born during the Ey+ and Hth+ neuroblast stages, see above) are expanded in Su(H) mutant clones at the expense of Ap+ neurons. Therefore, the deeper layer of D expression is turned on independently in the NOFF daughters of Hth+ and Ey+ GMCs (Li, 2013).

    Finally, in wild type, a considerable amount of apoptotic cells were observed dispersed among neurons, suggesting that one daughter of certain GMCs undergoes apoptosis in some of the lineages. Together these data suggest that Notch-dependent asymmetric division of GMCs further diversifies neuronal identities generated by the temporal sequence of transcription factors (Li, 2013).

    How does the neuroblast transcription factor temporal sequence, together with the Notch-dependent binary fate choice, control neuronal identities in the medulla? Transcription factor markers specifically expressed in subsets of medulla neurons, but not in neuroblasts, were examined including Brain-specific homeobox (Bsh) and Drifter (Dfr), as well as other transcription factors identified in the antibody screen, for example, Lim3 and Toy. Bsh is required and sufficient for the Mi1 cell fate, and Dfr is required for the morphogenesis of nine types of medulla neurons, including Mi10, Tm3, TmY3, Tm27 and Tm27Y (Hasegawa, 2011). Investigation were carried out to identify at which neuroblast temporal stage these neurons were born by examining co-expression with the inherited neuroblast transcription factors. Then whether the neuroblast transcription factors regulate expression of these markers and neuron fates was investigated. The results for each neuroblast stage are described below (Li, 2013).

    Bsh is expressed in a subset of Hth+ neurons, suggesting that Bsh is in the NON daughter of Hth+ GMCs. Indeed, Bsh expression is lost in both Su(H) and hth mutant clones. Thus, both Notch activity and Hth are required for specifying the Mi1 fate, consistent with the previous report that Hth is required for the Mi1 fate. Ectopic expression of Hth in older neuroblasts is also sufficient to generate ectopic Bsh+ neurons, although the phenotype becomes less pronounced in later parts of the lineage. These data suggest that Hth is necessary and sufficient to specify early born neurons, but the competence to do so in response to sustained expression of Hth decreases over time. This is similar to embryonic CNS neuroblasts, where ectopic Hb is only able to specify early born neurons during a specific time window (Li, 2013).

    Lim3 is expressed in all Ap progeny of both Hth+ and Ey+ neuroblasts. Toy and Dfr are expressed in subsets of neurons born from Ey+ neuroblasts, as indicated by their expression in the Ey+ neuron progeny layer. The most superficial row of Ey+ Ap neurons express Toy (and Lim3), suggesting that they are the NOFF progeny of the last-born Ey+ GMCs. Dfr is co-expressed with Ap in two or three rows of neurons that are intermingled with Ey+ neurons, suggesting that they are the NON progeny from Ey+ GMCs. In addition to these Ap+ Dfr+ neurons, Dfr is also expressed in some later-born neurons that are Ap but express another transcription factor: Dachshund (Dac), in specific sub-regions of the medulla crescent (Li, 2013).

    Whether Ey in neuroblasts regulates Dfr expression in neurons was tested. As expected, Dfr-expressing neurons are lost in ey-null mutant clones, suggesting that they require Ey activity in neuroblasts, even though Ey is not maintained in Ap+ Dfr+ neurons. Furthermore, in slp mutant clones in which neuroblasts remain blocked in the Ey+ state, the Ap+ Dfr+ neuron population is expanded into later-born neurons, suggesting that the transition from Ey+ to Slp+ in neuroblasts is required for shutting off the production of Ap+ Dfr+ neurons. In addition, Ap+ Dfr+ neurons are lost in Su(H) mutant clones. Thus, Ey expression in neuroblasts and the Notch pathway together control the generation of Ap+ Dfr+ neurons (Li, 2013).

    In addition to its expression with Ey in the NOFF progeny of the last-born Ey+ GMCs, Toy is also expressed in Ap+ (NON) neurons in more superficial layers generated by Slp+ and D+ neuroblasts. Consistently, in Su(H) mutant clones, an expansion of Toy+ Ey+ neurons is seen in the Ey progeny layer, followed by loss of Toy in the Slp and D progeny layer (Li, 2013).

    Tests were performed to see whether Slp is required for the neuroblasts to switch from generating Toy+ Ap neurons, progeny of Ey+ neuroblasts, to generating Toy+ Ap+ neurons. Indeed, in slp mutant clones, the Toy+ Ap+ neurons largely disappear, whereas Toy+ Ap neurons expand (Li, 2013).

    WAp and Toy expression was examined in specific adult neurons. OrtC1-gal4 primarily labels Tm20 and Tm5 plus a few TmY10 neurons, and these neurons express both Ap and Toy. To examine whether Slp is required for the specification of these neuron types, wild-type or slp mutant clones were generated using the mosaic analysis with a repressible cell marker (MARCM) technique by heat-shocking for 1 h at early larval stage, and the number of OrtC1-gal4-marked neurons in the adult medulla was examined. In wild-type clones, OrtC1-gal4 marks ~100 neurons per medulla. By contrast, very few neurons are marked by OrtC1-gal4 in slp mutant clones. Slp is unlikely to directly regulate the Ort promoter because Slp expression is not maintained in Ap+ Toy+ neurons. Furthermore, the expression level of OrtC1-gal4 in lamina L3 neurons is not affected by slp mutation. These data suggest that loss of Slp expression in neuroblasts strongly affects the generation of Tm20 and Tm5 neurons (Li, 2013).

    In summary, these data show that the sequential expression of transcription factors in medulla neuroblasts controls the birth-order-dependent expression of different neuronal transcription factor markers, and thus the sequential generation of different neuron types (Li, 2013).

    Although a temporal transcription factor sequence that patterns Drosophila nerve cord neuroblasts was reported more than a decade ago, it was not clear whether the same or a similar transcription factor sequence patterns neural progenitors in other contexts. The current identification of a novel temporal transcription factor sequence patterning the Drosophila medulla suggests that temporal patterning of neural progenitors is a common theme for generating neuronal diversity, and that different transcription factor sequences might be recruited in different contexts (Li, 2013).

    There are both similarities and differences between the two neuroblast temporal sequences. In the Hb-Kr-Pdm-Cas-Grh sequence, ectopically expressing one gene is sufficient to activate the next gene, and repress the previous gene, but these cross-regulations are not necessary for the transitions, with the exception of Castor. In the Hth-Ey-Slp-D-Tll sequence, removal of Ey, Slp or D does disrupt cross-regulations necessary for temporal transitions (except the Hth-Ey transition). However, in most cases these cross-regulations are not sufficient to ensure temporal transitions, suggesting that additional timing mechanisms or factors are required (Li, 2013).

    For simplicity, the medulla neuroblasts are represented as transiting through five transcription factor stages, whereas in fact the number of stages is clearly larger than five. First, neuroblasts divide more than once while expressing a given temporal transcription factor, and each GMC can have different sub-temporal identities. Furthermore, there is considerable overlap between subsequent temporal neuroblast transcription factors: neuroblasts expressing two transcription factors are likely to generate different neuron types from neuroblasts expressing either one alone (Li, 2013).

    Although the complete lineage of medulla neuroblasts is still being investigated, this study shows how a novel temporal sequence of transcription factors is required to generate sequentially the diverse neurons that compose the medulla. The requirement for transcription factor sequences in the medulla and in embryonic neuroblasts suggests that this is a general mechanism for the generation of neuronal diversity. Interestingly, the mammalian orthologue of Slp1, FOXG1, acts in cortical progenitors to suppress early born cortical cell fates. Thus, transcription-factor-dependent temporal patterning of neural progenitors might be a common theme in both vertebrate and invertebrate systems (Li, 2013).

    A unique class of neural progenitors in the Drosophila optic lobe generates both migrating neurons and glia

    How neuronal and glial fates are specified from neural precursor cells is an important question for developmental neurobiologists. This study addresses this question in the Drosophila optic lobe, composed of the lamina, medulla, and lobula complex. It was shown that two gliogenic regions posterior to the prospective lamina also produce lamina wide-field (Lawf) neurons, which share common progenitors with lamina glia. These progenitors express neither canonical neuroblast nor lamina precursor cell markers. They bifurcate into two sub-lineages in response to Notch signaling, generating lamina glia or Lawf neurons, respectively. The newly born glia and Lawfs then migrate tangentially over substantial distances to reach their target tissue. Thus, Lawf neurogenesis, which includes a common origin with glia, as well as neuronal migration, resembles several aspects of vertebrate neurogenesis (Chen, 2016).

    Function of Nerfin-1 in preventing medulla neurons dedifferentiation requires its inhibition of Notch activity

    Drosophila larval central nervous system comprises the central brain, ventral nerve cord and optic lobe. In these regions, neuroblasts divide asymmetrically to self-renew and generate differentiated neurons or glia. To date, mechanisms of preventing neuron dedifferentiation are still unclear, especially in the optic lobe. This study shows that the zinc finger transcription factor Nerfin-1 is expressed in early stage of medulla neurons and essential for maintaining their differentiation. Loss of Nerfin-1 activates Notch signaling, which promotes neuron-to-NB reversion. Repressing Notch signaling largely rescues dedifferentiation in nerfin-1 mutant clones. Thus, it is concluded that Nerfin-1 represses Notch activity in medulla neurons and prevents them from dedifferentiation (Xu, 2017).

    Stem cells generate progeny that undergo terminal differentiation. In Drosophila CNS, the balance of self-renewal and differentiation of neural stem and progenitor cells is a central issue during development. On the other hand, the maintenance of differentiated status of post-mitotic neurons is also crucial for tissue function and homeostasis. It is obvious that mechanisms must exist to prevent the cells from dedifferentiation. Although proteins that function to keep differentiation have been well studied in other cell types, few have been implicated in post-mitotic neuronal maintenance. In the central brain, loss of Midlife crisis (Mdlc), a CCCH zinc-finger protein, results in a decrease in Pros, thus derepressing NB genes in neurons. However, it is insufficient to make neurons revert to proliferating NBs. Furthermore, as Pros is not expressed in medulla neurons, it is unclear whether Mdlc has the same function in the optic lobe. On the other hand, absence of Lola leads to neuron-to-NB reversion and tumorigenesis , but it is crucial for neuronal maintenance only in the optic lobe. Recently, a paper reported that Nerfin-1 loss induces neuron dedifferentiation in both central brain and VNC (Froldi, 2015). This paper demonstrates a conserved function for Nerfin-1 in medulla neurons in the optic lobe. These findings indicate that Nerfin-1 is expressed mainly in early-stage medulla neurons and functions to maintain their differentiated state (Xu, 2017).

    Interestingly, it was noticed that ectopic NB induced by Nerfin-1 depletion in the optic lobe appeared much earlier than that in the central brain. Considering that Lola loss causes dedifferentiation just in the optic lobe, it is speculated that the differentiated state of medulla neurons is less stable, possibly owing to absence of Pros. Furthermore, different from the mechanism in the central brain, the function of Nerfin-1 in the optic lobe requires the silencing of Notch signaling. Neither Myc knockdown nor Tor-DN misexpression inhibits dedifferentiation caused by Nerfin-1 loss in the medulla neurons. Thus, these findings identify a distinct regulatory mechanism in medulla neurons and validate different regulatory modes between the optic lobe and the rest of the CNS (Xu, 2017).

    On the other hand, cell cycle genes play important roles in cell differentiation. Among them, Cyclin E (CycE) is reported to be regulated directly by Lola-N and is involved in the neuron dedifferentiation caused by loss of Mdlc. Thus, this study also examined whether CycE is regulated directly by Nerfin-1 and controls cell differentiation independently of Notch and neuroblast genes. Interestingly, CycE expression levels were upregulated dramatically in nerfin-1159 clones, but such upregulation was mostly blocked by Notch repression. These results suggest that CycE is not a direct target of Nerfin-1 for maintaining medulla neuron differentiation. CycE acts downstream of Notch signaling or it is subsequently upregulated after cell type change (Xu, 2017).

    As Notch signaling is hyper-activated in nerfin-1 mutant clones, it was of interest to discover how it is regulated. One possibility is that Notch signaling becomes constitutively activated without the inhibition by Nerfin-1. To investigate this, Delta was knocked down upon Nerfin-1 loss and it was found that dedifferentiation was suppressed. These results indicate that Notch signaling is not constitutively activated and that it needs a ligand. Furthermore, Notch signal is both produced and received by medulla neurons. At the same time, the results show that Nerfin-1 loss induces dramatic upregulation of the expression level of Notch receptor. Thus, it is hypothesized that Nerfin-1 suppresses the expression of the Notch receptor in normal medulla neurons and inhibits Notch pathway activity. When Nerfin-1 is absent, expression levels of the Notch receptor increase strikingly. The receptors then bind to Delta from the adjacent cells and activate Notch signaling in its own. However, it is still unclear whether Notch receptor is a direct target of Nerfin-1. Therefore, subsequent studies on Nerfin-1 may help to clarify the underlying mechanisms and provide better understanding about neuronal maintenance (Xu, 2017).

    A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system

    Brain areas each generate specific neuron subtypes during development. However, underlying regional variations in neurogenesis strategies and regulatory mechanisms remain poorly understood. In Drosophila, neurons in four optic lobe ganglia originate from two neuroepithelia, the outer (OPC) and inner (IPC) proliferation centers. Using genetic manipulations, this study found that one IPC neuroepithelial domain progressively transformed into migratory progenitors that matured into neural stem cells (neuroblasts) in a second domain. Progenitors emerged by an epithelial-mesenchymal transition-like mechanism that required the Snail-family member Escargot and, in subdomains, Decapentaplegic signaling. The proneural factors Lethal of scute and Asense differentially controlled progenitor supply and maturation into neuroblasts. These switched expression from Asense to a third proneural protein, Atonal. Dichaete and Tailless mediated this transition, which was essential for generating two neuron populations at defined positions. It is proposed that this neurogenesis mode is central for setting up a new proliferative zone to facilitate spatio-temporal matching of neurogenesis and connectivity across ganglia. (Apitz, 2014).

    Recent studies have distinguished three neurogenesis modes in the Drosophila CNS. First, type I neuroblasts arise from neuroepithelia and generate GMCs, which produce neuronal and glial progeny. Second, Dpn+ type II neuroblasts in the dorsomedial central brain go through a transit-amplifying Dpn+, Ase+ population, called intermediate neural precursors, which generate GMCs and postmitotic offspring. Third, lateral OPC neuroepithelial cells bypass the neuroblast stage and generate lamina precursor cells (LPCs) that divide once to produce lamina neurons. The current results provide evidence for a fourth strategy: p-IPC neuroepithelial cells give rise to progenitors that migrate to a second neurogenic domain, where they mature into type I neuroblasts. These progenitors are distinct, as they originate from the neuroepithelium, do not express markers for neuroblasts, intermediate neural precursors, GMCs or postmitotic neurons, and acquire NSC properties after completing their migration (Apitz, 2014).

    Migratory progenitors arise from the p-IPC by a mechanism that shares cellular and molecular characteristics with EMT. On the basis of data on gastrulation and neural crest formation, EMT is commonly associated with cells adopting a mesenchymal state, enabling them to leave their epithelial tissue and migrate through the extracellular matrix to new locations. A recent study also reported an EMT-like process in the mammalian neocortex, whereby newborn neurons and intermediate progenitors delaminate from the ventricular neuroepithelium and radially migrate to the pial surface. This study observed that neuroepithelial cells at the p-IPC margins and migratory progenitors upregulated the Snail homolog Esg, whereas E-cad levels were decreased. Moreover, esg knockdown caused the formation of ectopic E-cad-expressing clusters adjacent to the p-IPC. Although this is a previously uncharacterized role of Drosophila esg, these findings are consistent with the requirement of two Snail transcription factors, Scratch1 and 2, and downregulation of E-cad in cortical EMT migration (Apitz, 2014).

    Although TGFβ signaling is well known to induce EMT, it was unclear whether it could have such a role in the brain. Two lines of evidence are consistent with a requirement of the Drosophila family member Dpp. First, it is expressed and downstream signaling is activated in dorsal and ventral p-IPC subdomains and emerging cell streams. Second, tkv mutant cells form small neuroepithelial clusters in p-IPC vicinity. Similar to the neural crest, where distinct molecular cascades control delamination in the head and trunk, region-specific regulators may also be required in p-IPC subdomains. Because neuroblasts derived from Dpp-dependent cell streams map to defined areas in the d-IPC, this pathway could potentially couple EMT and neuron subtype specification (Apitz, 2014).

    Cell migration is an essential feature of vertebrate brain development. Commonly, postmitotic immature neurons migrate from their proliferation zones to distant regions, where they further differentiate and integrate into local circuits. Examples include the radial migration of projection neurons and tangential migration of interneurons in the embryonic cortex, as well as migration of interneuron precursors in the rostral migratory stream to the olfactory bulb in adults. In contrast, IPC progenitors develop into NSCs (neuroblasts) after they migrated. A recent study found that NSCs relocating from the embryonic ventral hippocampus to the dentate gyrus act as source for adult NSCs in the subgranular zone. In addition, cerebellar granule cell precursors migrate from the rhombic lip to the external granule layer, where they proliferate during early postnatal development. The migration of neural cell types that become proliferative in a new niche could therefore constitute a more general strategy. IPC progenitors form streams of elongated, closely associated cells. Despite their different developmental state, their organization is notably similar to the neuronal chain network in the lateral walls of the subventricular zone and the rostral migratory stream in mammals, or of migratory trunk neural crest cells in chick. Further studies will need to identify the determinants directing migratory progenitors into the d-IPC (Apitz, 2014).

    Several constraints could shape a neurogenesis mode that requires migratory progenitors in the larval optic lobe. The OPC is located superficially and the IPC is positioned centrally. If medulla and lobula neurons arose by neuroepithelial duplications, these new populations would need to be integrated into an ancestral visual circuit consisting of lamina and lobula plate neurons. Cellular migration may therefore be a derived feature and serve as an essential spatial adjustment of the IPC to the newly added medulla. In principle, the migratory population could consist of immature neurons. However, migratory progenitors help to establish a new superficial proliferative niche, and to align OPC and d-IPC neuroblast positions. This in turn enables the OPC and IPC to use spatially matching birth order-driven neurogenesis patterns for establishing functionally coherent connections across ganglia (Apitz, 2014).

    IPC progenitors were primed to mature into neuroblasts, but were prevented to do so in cell streams. Consistently, progenitors showed weak cytoplasmic Mira expression and prematurely differentiated into neuroblasts following loss of Pcl. Although Dichaete has been shown to repress ase to maintain embryonic neuroectodermal cells in an undifferentiated state, this study did not identify such a role in the IPC. Future studies are therefore required to distinguish whether this block in neuroblast maturation is released in the d-IPC by cell-intrinsic mechanisms or locally acting signals (Apitz, 2014).

    The p-IPC and d-IPC consecutively expressed three proneural factors. esg-positive p-IPC neuroepithelial cells transiently expressed L'sc as they converted into progenitors. Following arrival in the d-IPC, progenitors matured into neuroblasts, which switched bHLH protein expression from Ase to Ato. This correlated with a change in cell division orientations from toward the lamina to the optic lobe surface and the generation of two lineages, distal cells and lobula plate neurons. The progression of neuroblasts through two stages is supported by the observations that progenitors solely entered the lower d-IPC, all neuroblasts were labeled with Ase in this area, and idpp reporter gene expression in a progenitor subset persisted in both lower and upper d-IPC neuroblasts and their progeny (Apitz, 2014).

    Late l'sc knockdown reduced the number of d-IPC neuroblasts and both neuron classes, whereas p-IPC formation and EMT of progenitors appeared to be unaffected. This supports the idea that l'sc promotes neuroblast formation by controlling the rate of conversion and the progenitor supply. In contrast, ase loss severely decreased the amount of lower d-IPC neuroblasts and distal cells. This revealed a central role in the maturation of progenitors into neuroblasts, endowing them with the potential to proliferate and generate a specific lineage. Although these functions are the opposite of those observed in the OPC, they align with the role of a murine Ase homolog, Achaete-scute homolog 1 (Ascl1), in the embryonic telencephalon. Ase- neuroblasts with type I proliferation patterns have not previously been described. Further underscoring the context-dependent activities of proneural bHLH factors, ato does not have the equivalent role of ase in conferring neurogenic properties to upper d-IPC neuroblasts, but acts upstream of differentiation programs controlling the projections of lobula plate neurons (Apitz, 2014).

    Although Ase and Ato each regulated distinct aspects of d-IPC development, they were not required for either the transition or the extent of their expression domains. These functions were fulfilled by Dichaete and tll, whose cross-regulatory interactions were essential for the transition from Ase+ to Ato+, Dac+ expression. To link birth order and fate, temporal identity transcription factors are sequentially expressed by neuroblasts and inherited by GMCs and their progeny born during a given developmental window. Acting as the final two members of the OPC-specific series of temporal identity factors, Dichaete is required for Tll expression, whereas tll is sufficient, but not required, to inhibit Dichaete Although OPC and d-IPC neuroblasts shared the sequential expression of Dichaete and Tll, key differences include the fact that d-IPC progeny did not maintain Dichaete, that Tll was transiently expressed in newborn progeny of the upper d-IPC and was not maintained in older lineages, that Dichaete in the lower d-IPC was not required in its own expression domain for neurogenesis, and that Dichaete was required to activate tll, and tll to repress Dichaete and ase, as well as to independently upregulate Ato and Dac. Although the mechanisms that trigger the timing of the switch require further analysis, these observations support the notion that, in the d-IPC, Dichaete and tll do not function as temporal identity factors, but as switching factors between two sequential neuroblast stages. The vertebrate homologs of Dichaete and tll, Sox2 and Tlx, are essential for adult NSC maintenance and Sox2 positively regulates Tlx expression, suiggesting that core regulatory interactions between Dichaete and tll family members may be conserved (Apitz, 2014).

    These studies uncovered molecular signatures for generating a migratory neural population by EMT and subsequent NSC development that are in part shared between the fly optic lobe and vertebrate cortical neurogenesis. The unexpected parallels suggest that ancestral gene regulatory cassettes imparting specific cellular properties may have been re-employed during vertebrate brain development. Analysis of p-IPC and d-IPC neurogenesis in the Drosophila optic lobe therefore opens new possibilities for systematically identifying genes regulating EMT, cell migration and sequential NSC specification (Apitz, 2014).

    Brain-specific-homeobox is required for the specification of neuronal types in the Drosophila optic lobe

    The Drosophila optic lobe comprises a wide variety of neurons forming laminar and columnar structures similar to the mammalian brain. The Drosophila optic lobe may provide an excellent model to investigate various processes of brain development. However, it is poorly understood how neuronal specification is regulated in the optic lobe to form a complicated structure. This study shows that the Brain-specific homeobox (Bsh) protein, which is expressed in the lamina and medulla ganglia, is involved in specifying neuronal identity. Bsh is expressed in L4 and L5 lamina neurons and in Mi1 medulla neurons. Analyses of loss-of-function and gain-of-function clones suggest that Bsh is required and largely sufficient for Mi1 specification in the medulla and L4 specification in the lamina. Additionally, Bsh is at least required for L5 specification. In the absence of Bsh, L5 is transformed into glial cells (Hasegawa, 2013).

    The establishment of precise neuronal circuits is essential for correct brain function. Complex neuronal circuits contain various types of neurons that are connected intricately with one another. Processes that result in the formation of the correct circuits include the specification of neuronal types, the extension of axons to the appropriate places, and the formation of synapses with their correct partners. The specification of neuronal types is an important process in the making of complete neuronal circuits (Hasegawa, 2013).

    The Drosophila visual system may serve as a powerful model for neuronal circuit formation because it has only a limited number of neurons but forms sufficiently complex neuronal circuits that can be analyzed comprehensively. In addition, neurogenetic tools that are available in Drosophila allow artificial manipulation of neuronal activity in temporal ly and spatially restricted manner. However, the full picture of development of the Drosophila optic lobe awaits further investigation (Hasegawa, 2013).

    The Drosophila retina is composed of 750-800 ommatidia that contain eight types of photoreceptor neurons, denoted as R1-R8. The visual information received in the retina is transmitted to the optic lobe, which is composed of four ganglia; the lamina, medulla, lobula and lobula plate. The complex neuronal circuits in the visual center process various types of visual information, such as motion, color and shape. This paper focuses on the development of the lamina and medulla (Hasegawa, 2013).

    The development of the lamina has been studied in some detail. During the third instar larva, photoreceptor neurons extend retinal axons (R axons) to the optic lobe and deliver the inductive signal Hedgehog (Hh) to the lamina precursor cells (LPCs). LPCs divide and become lamina neurons and activate the expression of Dachshund (Dac) and EGF receptor. Spitz, an EGF ligand, is delivered by R axons, received by EGFR and promotes further differentiation of lamina neurons, including the expression of Elav and the formation of lamina columns. Finally, the five types of lamina neurons, L1-L5, become closely associated with R1-R8 axons, forming a lamina cartridge. Although the differentiation of lamina neurons is understood to some extent, how the distinction among L1-L5 neurons is regulated remains unclear (Hasegawa, 2013).

    The second visual ganglion, the medulla, contains 40,000 neurons forming tangentially oriented stratifications, which are defined as 10 layers. Medulla neurons are classified by their pattern of arborizations. Some neurons arborize only in the medulla (medulla intrinsic neurons, Mi-neurons), and some send projections back to the lamina (lamina wide-field neurons, Lawf-neurons). Other neurons arborize in both the medulla and the lobula (transmedullary neurons, Tm-neurons) and in the lobula complex (transmedullary Y neurons, TmY neurons). The medulla is the largest part of the optic lobe and is thought to process both color and motion. Although the medulla is considered to play an important role in visual processing, the developmental mechanisms of the medulla remain elusive. During the third instar larva, neuroblasts (NBs) located in the outer proliferation center divide to make ganglion mother cells (GMCs), which divide to produce differentiated neurons. Expression of specific transcription factors in a subset of medulla neurons was examined (Hasegawa, 2011; Morante, 2008). It has been reported previously that neurons produced from NBs express different types of transcription factors according to their birth order to form a concentric expression pattern (Hasegawa, 2011). However, how the differentiation of the medulla neurons is controlled is still unclear (Hasegawa, 2013).

    The Bsx family transcription factors are widely conserved homeodomain proteins that are involved in various neuronal processes. For example, mouse Bsx regulates hyperphagia, locomotory behavior, growth, and nursing. Xenopus Bsx links daily cell cycle rhythms to pineal photoreceptors. The Drosophila Bsx protein, Brain-specific homeobox (Bsh), is expressed in the embryonic brain , and in the lamina and medulla neurons of larvae and adults. However, the molecular function of Drosophila bsh has not been studied (Hasegawa, 2013).

    A previous paper showed that Bsh is expressed in the medulla and that Bsh-positive neurons differentiate into a single type of medulla intrinsic neuron, Mi1. Moreover, Hth, which is expressed in Mi1 together with Bsh, is essential for Mi1 neuron identity (Hasegawa, 2011). This study shows that Bsh is also required for Mi1 neuron specification in the medulla. bsh mutant neurons were transformed to Tm-type neurons, and overexpression of Bsh induced Mi1-like neurons. Moreover, Bsh expression was required for L4 neuronal specification in the L4 neurons of the lamina, and overexpression of Bsh in the lamina induced L4-like neurons. Therefore, Bsh may have roles in neuronal type specification in both the lamina and the medulla. Relatively weak Bsh expression found in L5 lamina neurons may be required for neuronal differentiation of L5. In the absence of bsh, L5 cells were transformed into glial cells (Hasegawa, 2013).

    The previous study showed that Hth and Bsh are expressed in a concentric manner in the larval medulla. Bsh is expressed in the outer half of the Hth domain, and these Bsh/Hth double-positive neurons differentiate into a single neuronal type, Mi1 (Hasegawa, 2011). bsh is predicted to encode two isoforms of homeodomain proteins, long Bsh-PB and short Bsh-PA lacking the N-terminal domain of Bsh-PB The homeodomain is located in the C-terminal region that is shared by both isoforms. No conserved motifs are found in the N-terminal region, which is deleted in Bsh-PA (Hasegawa, 2013).

    This study has shown that Bsh is expressed in Mi1 neurons, which differentiates into Tm-type neurons in bsh mutant clones. A similar neuronal transformation from local interneuron to projection neuron is observed in hth mutant clones (Hasegawa, 2011). The number of Hth positive cells was decreased in bsh homozygous animals. However, Hth expression was not affected in bsh mutant clones at larval and adult stages, suggesting that the neuronal type change observed in bsh mutants may not have been caused by reduced expression of Hth. The previous paper showed that residual Bsh is still observed in transformed hth mutant neurons. Moreover, expressing UAS-bshPB could not rescue the defect of the hth mutation, suggesting that Bsh alone cannot induce Mi1 neuron identity and both Hth and Bsh are required for Mi1 neuron identity. By contrast, Bsh expression under the control of drf-Gal4 induced Mi1-like neurons. These results seem inconsistent, however, Mi1-like neurons induced by ectopic Bsh expression were still abnormal compared to endogenous Mi1. Expressing Bsh together with Hth induced Mi1-like neurons that were more similar to endogenous Mi1, implying that both Hth and Bsh are required to generate a complete Mi1 neuron (Hasegawa, 2013).

    Because Bsh is a homeodomain protein, the neuronal transformation observed in bsh mutants may be a homeotic transformation. Similar neuronal transformation is observed in mouse Hox gene mutants. However, the transformations observed in bsh mutant clones are striking compared to those observed in mouse Hox mutants. It is not yet known what happens downstream of Bsh and what kind of genes are expressed in transformed Tm-type neurons. It may be that Bsh represses the expression of unknown transcription factors that specify Tm-type neurons. Identification of such transcription factors will lead to insights into the mechanism of neuronal transformation found in bsh mutant (Hasegawa, 2013).

    Bsh is expressed in L4 and L5 neurons. The results suggest that bsh mutation transforms L4 neurons into L3-like neurons, and that ectopic Bsh expression induces L4-like neurons, suggesting that Bsh is necessary and sufficient for L4 neuron specification. Although induction and development of lamina neurons are understood to some extent, almost nothing is known about neuronal type specification in the lamina. It is possible to speculate that Ap acts downstream of Bsh. However, Ap alone could not induce L4-like neurons (data not shown), suggesting that Ap is not sufficient for L4 neuron formation. Ap may cooperate with Bsh and/or other factors to specify L4 neuron identity (Hasegawa, 2013).

    In bsh mutant lamina neurons, transformation into L3-like neurons was observed most often. It is not known whether this transformation is specifically oriented to L3. Other mutations that transform, for example, L1 neuron into other neurons may reveal whether L3 is the ground state of lamina neurons. At third instar, L1-L5 all express Dac, but during metamorphosis Dac expression disappears in L2 and is reduced in L5. It will be interesting to determine the mechanism that regulates the changes in Dac expression. The combination of Dac, Bsh, and Ap expression can specify L2, L4 and L5 but cannot distinguish between L1 and L3. Therefore, there must be other transcription factors that distinguish between L1 and L3 (Hasegawa, 2013).

    Using L5-Gal4, it was unexpectedly found that L5 neurons are transformed to glial cells in bsh mutant clones. The transformed glial cells are found along the endogenous glial cell layers that are situated adjacent to the L5 cells in adult and pupal lamina. They are located in the lamina cortex and ensheath lamina neuron cell bodies, and are closely positioned to L5, suggesting that they are most likely proximal satellite glial cells. Transformation of L5 to glia may suggest that their developmental mechanisms are coupled to each other. It will be interesting to see if there is developmental relationship between L5 lamina neurons and proximal satellite glial cells in the developing optic lobe (Hasegawa, 2013).

    Bsx regulates hyperphagia and locomotory behavior by regulating the expression of Npy and Agrp in the arcuate nucleus of the mouse hypothalamus. Bsx expression is repressed by repressor element silencing transcription factor (REST) in non-neuronal cells. Xenopus Bsx links daily cell cycle rhythms with pineal photoreceptors, but its downstream targets are not known. Xbsx expression peaks during the night and represses proliferation. Xbsx knockdown prevents the cell cycle exit of photoreceptor precursors that eventually undergo apoptosis. Xbsx overexpression increases the cell cycle exit of photoreceptor precursors and promotes their differentiation. Therefore, Bsx family proteins may have a general role in cell fate determination. If Bsh target genes are studied in Drosophila, insights into the molecular function of Bsh family proteins may be obtained in the future (Hasegawa, 2013).

    These results show that Bsh is expressed in the Mi1 medulla neuron and essential for Mi1 neuron identity. Overexpression of Bsh induces Mi1-like neurons that show some of the features of the Mi1 neuron. In the lamina, Bsh is expressed in the L4 and L5 neurons and is required for L4 and L5 neuron specification. Overexpression of Bsh can induce L4-like neurons. Bsh may have roles in specifying neuronal identity in both the lamina and the medulla (Hasegawa, 2013).

    A temporal mechanism that produces neuronal diversity in the Drosophila visual center

    The brain consists of various types of neurons that are generated from neural stem cells; however, the mechanisms underlying neuronal diversity remain uncertain. A recent study demonstrated that the medulla, the largest component of the Drosophila optic lobe, is a suitable model system for brain development because it shares structural features with the mammalian brain and consists of a moderate number and various types of neurons. The concentric zones in the medulla primordium that are characterized by the expression of four transcription factors, including Homothorax (Hth), Brain-specific homeobox (Bsh), Runt (Run) and Drifter (Drf/Vvl), correspond to types of medulla neurons. This study examined the mechanisms that temporally determine the neuronal types in the medulla primordium. For this purpose, transcription factors were sought that are transiently expressed in a subset of medulla neuroblasts (NBs, neuronal stem cell-like neural precursor cells) and identified five candidates [Hth, Klumpfuss (Klu), Eyeless (Ey), Sloppy paired (Slp) and Dichaete (D)]. The results of genetic experiments at least explain the temporal transition of the transcription factor expression in NBs in the order of Ey, Slp and D. The results also suggest that expression of Hth, Klu and Ey in NBs trigger the production of Hth/Bsh-, Run- and Drf-positive neurons, respectively. These results suggest that medulla neuron types are specified in a birth order-dependent manner by the action of temporal transcription factors that are sequential ly expressed in NBs (Suzuki, 2013).

    In the embryonic central nervous system, the heterochronic transcription factors suchas Hb, Kr, Pdm, Cas and Grh are expressed in NBs to regulate the temporal specification of neuronal identity. They regulate each other to achieve sequential changes in their expression in NBs without cell-extrinsic factors. However, expression of the embryonic heterochronic genes was not detected in the medulla NBs.Instead this study found that Hth, Klu, Ey, Slp and D are transiently and sequentially expressed in medulla NBs. The expression of Hth and Klu was observed in lateral NBs, while that of Ey/Slp and D was observed in intermediate and medial NBs, respectively. These observations suggest that the expression of heterochronic transcription factors changes sequentially as each NB ages, as observed in the development of the embryonic central nervous system (Suzuki, 2013).

    This study demonstrates that at least three of the temporal factors Ey, Slp and D regulate each other to form a genetic cascade that ensures the transition from Ey expression to D expression in the medulla NBs. Ey expression in NBs activates Slp, while Slp inactivates Ey expression. Similarly, Slp expression in NBs activates D expression, while D inactivates Slp expression. In fact, the expression of Slp is not strong in newer NBs in which Ey is strongly expressed, but is up regulated in older NBs in which Ey is weakly expressed in the wildtype medulla. A similar relationship is found between Slp and D, supporting the idea that Ey, Slp and D regulate each other's expression to control the transition from Ey-expression to D-expression. In the embryonic central nervous system, similar interaction is mainly observed between adjacent genes of the cascade hb-Kr-pdm-cas-grh, and this concept may also be applied to the medulla primordium. The expression pattern and function of Ey, Slp and D suggest that they are adjacent to each other in the cascade of transcription factor expression in medulla NBs (Suzuki, 2013).

    However, no such relationship was found between Hth, Klu and the other temporal factors.The sequential expression of Hth and Klu could be regulated by an unidentified mechanism that is totally different from the genetic cascade that controls the transition through Ey-Slp-D. Or, there might be unidentified temporal factors that are expressed in lateral NBs which act upstream of Hth and Klu to regulate their expression. It is necessary to identify additional transcription factors that are transiently expressed in medulla NBs (Suzuki, 2013).

    The expression of concentric transcription factors in the medulla neurons correlates with the temporal sequence of neuron production from the medulla NBs (Hasegawa, 2011). In the larval medulla primordium, the neurons are located in the order of Hth/Bsh-, Run- and Drf-positive cells from inside to outside, and these domains are adjacent to each other (Hasegawa, 2011). Given that NBs generate neurons toward the center of the developing medulla, Hth/Bsh-positive neurons are produced at first, and then Run-positive and Drf-positive neurons. Thus Hth/Bsh, Run and Drf were used as markers to examine roles of Hth, Klu, Ey, Slp and D expressed in NBs in specifying types of medulla neurons. The continuous expression of Hth and Ey from NBs to neurons and the results of clonal analyses that visualize the progeny of NBs expressing each one of the temporal transcription factors suggest that the temporal windows of NBs expressing Hth, Klu and Ey approximately correspond to the production of Hth/Bsh-, Run- and Drf- positive neurons, respectively. Indeed, the results of the genetic study suggest that Hth and Ey are necessary and sufficient to induce the production of Hth/Bsh- and Drf-positive neurons,respectively (Hasegawa, 2011, 2013). Ectopic Klu expression at least induces the produc tion of Run-positive neurons (Suzuki, 2013).

    Slp and D expression in NBs may correspond to the temporal windows that produce medulla neurons in the outer domains of the concentric zones, which are most likely produced after the production of Drf-positive neurons. The results at least suggest that Slp is necessary and sufficient and D is sufficient to repress the production of Drf-positive neurons. Identification of additional markers that are expressed in the outer concentric zones compared to the Drf-positive domain would be needed to elucidate the roles of Slp and D in specification of medulla neuron types (Suzuki, 2013).

    D mutant clones did not produce any significant phenotype except for derepression of Slp expression in NBs. Drf expression in neurons was not affected either. Since D is a Sox family transcription factor, SoxN, another Sox family transcription factor, is a potential candidate molecule that acts together with D in the medulla NBs. However, its expression was found in neuroepithelia cells and lateral NBs that overlap with Hth-positive cells but not with D-positive cells. All the potential heterochronic transcription factors examined in this study are expressed in three to five cell rows of NBs. Nevertheless, one NB has been observed to produce one Bsh- positive and one Run-positive neuron (Hasegawa, 2011). Therefore, the expression pattern of the heterochronic transcription factors is not sufficient to explain the stable production of one Bsh-positive and one Run-positive neuron from a single NB.The combinatorial action of multiple temporal factors expressed in NBs may play important roles in the specification of Bsh- and Run- positive neurons (Suzuki, 2013).

    Another possible mechanism that guarantees the production of a limited number of the same neuronal type from multiple rows of NBs expressing a temporal transcription factor could be a mutual repression between concentric transcription factors expressed in medulla neurons. For example, Hth/Bsh, Run and Drf may repress each other to restrict the number of neurons that express either of these transcription factors. However, expression of Run and Drf was not essentially affected in hth mutant clones and in clones expressing Hth (Hasegawa, 2011). Similarly, expression of Hth and Drf was not essentially affected in clones expressing run RNAi under the control of AyGal4, in which Run expression is eliminated. Hth and Run expression was not affected in drf mutant clones (Hasegawa, 2011). These results suggest that Hth/Bsh, Run and Drf do not essentially regulate each other during the formation of concentric zones in the medulla (Suzuki, 2013).

    During embryonic development, the heterochronic genes that are expressed in NBs (hb-Kr-pdm-cas-grh) are maintained and act in GMCs to specify neuronal type. Similarly, Hth and Ey are continuously expressed from NBs to neurons, suggesting that their expression may also be inherited through GMCs (Hasegawa, 2011). However, this type of regulatory mechanism may be somewhat modified in the case of Klu, Slp and D (Suzuki, 2013).

    Klu is expressed in NBs and GMCs, but not in neurons. Slp and D are predominantly detected in NBs and neurons visualized by Dpn and Elav, respectively. Occasionally, however, expression of D was found in putative GMCs, which are situated between NBs and neurons. Additionally, both D-positive and D-negative cells were found among Miranda-positive GMCs. Slp expression was not found in Miranda-positive GMCs. Finally, D is expressed in medulla neurons forming a concentric zone in addition to its expression in medial NBs. However, D expression was abolished in slp mutant NBs but remained in the mutant neurons, suggesting that D expression in medulla neurons is not inherited from the NBs. These results suggest that Slp and D expression are not maintained from NBs to neurons and that not all the temporal transcription factors expressed in NBs are inherited through GMCs. However, it is possible to speculate that Klu, Slp and D regulate expression of unidentified transcription factors in NBs that are inherited from NBs to neurons through GMCs (Suzuki, 2013).

    Integration of temporal and spatial patterning generates neural diversity

    In the Drosophila optic lobes, 800 retinotopically organized columns in the medulla act as functional units for processing visual information. The medulla contains over 80 types of neuron, which belong to two classes: uni-columnar neurons have a stoichiometry of one per column, while multi-columnar neurons contact multiple columns. This study shows that combinatorial inputs from temporal and spatial axes generate this neuronal diversity: all neuroblasts switch fates over time to produce different neurons; the neuroepithelium that generates neuroblasts is also subdivided into six compartments by the expression of specific factors (see The OPC neuroepithelium is patterned along its dorsal-ventral axis). Uni-columnar neurons are produced in all spatial compartments independently of spatial input; they innervate the neuropil where they are generated. Multi-columnar neurons are generated in smaller numbers in restricted compartments and require spatial input; the majority of their cell bodies subsequently move to cover the entire medulla. The selective integration of spatial inputs by a fixed temporal neuroblast cascade thus acts as a powerful mechanism for generating neural diversity, regulating stoichiometry and the formation of retinotopy (Erclik, 2017).

    The optic lobes, composed of the lamina, medulla and the lobula complex, are the visual processing centres of the Drosophila brain. The lamina and medulla receive input from photoreceptors in the compound eye, process information and relay it to the lobula complex and central brain. The medulla, composed of ~40,000 cells, is the largest compartment in the optic lobe and is responsible for processing both motion and colour information. It receives direct synaptic input from the two colour-detecting photoreceptors, R7 and R8. It also receives input from five types of lamina neuron that are contacted directly or indirectly by the outer photoreceptors involved in motion detection (Erclik, 2017).

    Associated with each of the ~800 sets of R7/R8 and lamina neuron projections are 800 medulla columns defined as fixed cassettes of cells that process information from one point in space. Columns represent the functional units in the medulla and propagate the retinotopic map established in the compound eye. Each column is contributed to by more than 80 neuronal types, which can be categorized into two broad classes. Uni-columnar neurons have arborizations principally limited to one medulla column and there are thus 800 cells of each uni-columnar type. Multi-columnar neurons possess wider arborizations, spreading over multiple columns. They compare information covering larger receptor fields. Although they are fewer in number, their arborizations cover the entire visual field (Erclik, 2017).

    The medulla develops from a crescent-shaped neuroepithelium, the outer proliferation centre (OPC). During the third larval instar, the OPC neuroepithelium is converted into lamina on its lateral side and into medulla neuroblasts on its medial side. A wave of neurogenesis moves through the neuroepithelial cells, transforming them into neuroblasts; the youngest neuroblasts are closest to the neuroepithelium while the oldest are adjacent to the central brain. Neuroblasts divide asymmetrically multiple times to regenerate themselves and produce a ganglion mother cell that divides once more to generate medulla neurons. Recent studies have shown that six transcription factors are expressed sequentially in neuroblasts as they age: neuroblasts first express Homothorax (Hth), then Klumpfuss (Klu), Eyeless (Ey), Sloppy-paired 1 (Slp1), Dichaete (D) and Tailless (Tll). This temporal series is reminiscent of the Hb --> Kr --> Pdm --> Cas --> Grh series observed in Drosophila ventral nerve cord neuroblasts that generates neuronal diversity in the embryo. Indeed, distinct neurons are generated by medulla neuroblasts in each temporal window. Further neuronal diversification occurs through Notch-based asymmetric division of ganglion mother cells. In total, over 20 neuronal types can theoretically be generated using combinations of temporal factors and Notch patterning mechanisms. However, little is known about how the OPC specifies the additional ~60 neuronal cell types that constitute the medulla (Erclik, 2017).

    To understand the logic underlying medulla development, late larval brains were stained with 215 antibodies generated against transcription factors and 35 genes were identifiied that are expressed in subsets of medulla progenitors and neurons. The OPC neuroepithelial crescent can be subdivided along the dorsal-ventral axis by the mutually exclusive expression of three homeodomain-containing transcription factors: Vsx1 is expressed in the central OPC (cOPC), Rx in the dorsal and ventral posterior arms of the crescent (pOPC), and Optix in the two intervening 'main arms' (mOPC). These three proteins are regionally expressed as early as the embryonic optic anlage and together mark the entire OPC neuroepithelium with sharp, non-overlapping boundaries. Indeed, these three regions grow as classic compartments: lineage trace experiments show that cells permanently marked in the early larva in one OPC region do not intermingle at later stages with cells from adjacent compartments. Of note, Vsx1 is expressed in cOPC progenitor cells and is maintained in a subset of their neuronal progeny whereas Optix and Rx are not expressed in post-mitotic medulla neurons. The OPC can be further subdivided into dorsal (D) and ventral (V) halves: a lineage trace with hedgehog-Gal4 (hh-Gal4) marks only the ventral half of the OPC, bisecting the cOPC compartment. As hedgehog is not expressed in the larval OPC, this dorsal-ventral boundary is set up in the embryo. Thus, six compartments (ventral cOPC, mOPC and pOPC and their dorsal counterparts) exist in the OPC. The pOPC compartment can be further subdivided by the expression of the wingless and dpp signalling genes. Cells in the wingless domain behave in a very distinct manner from the rest of the OPC, and have been described elsewhere (Erclik, 2017).

    The Hth --> Klu --> Ey --> Slp1 --> D --> Tll temporal progression is not affected by the compartmentalization of the OPC epithelium; the same neuroblast progression throughout the entire OPC. Thus, in the developing medulla, neuroblasts expressing the same temporal factors are generated by developmentally distinct epithelial compartments (Erclik, 2017).

    To test whether the intersection of the dorsal-ventral and temporal neuroblast axes leads to the production of distinct neural cell types, focus was placed on the progeny of Hth neuroblasts, which maintain Hth expression. In the late third instar, Hth neurons are found in a crescent that mirrors the OPC (see Distinct neuronal cell types are generated along the dorsal-ventral axis of the OPC). The NotchON (NON) progeny of Hth+ ganglion mother cells express Bsh and Ap, and they are distributed throughout the entire medulla crescent. In contrast, the NotchOFF (NOFF) progeny, which are BshHth+ neurons, express different combinations of transcription factors, and can be subdivided into three domains along the dorsal-ventral axis: (1) in the cOPC, NOFFHth+ neurons express Vsx1, Seven-Up (Svp) and Lim3; (2) in the pOPC, these neurons also express Svp and Lim3, but not Vsx1; (3) in the ventral pOPC exclusively, these neurons additionally express Teashirt (Tsh). NOFFHth+ cells are not observed in the mOPC. Rather, Cleaved-Caspase-3+ cells are intermingled with Bsh+ neurons. When cell death is prevented, Bsh+Hth+ cells become intermingled with neurons that express the NOFF marker Lim3, confirming that the NOFFHth+ progeny undergo apoptosis in the mOPC (Erclik, 2017).

    It was therefore possible to distinguish three regional populations of Hth neurons (plus one that is eliminated by apoptosis) and a fourth population that is generated throughout the OPC. The neuronal identity of each of these populations was identified, as follows. (1) Bsh is a specific marker of Mi1 uni-columnar interneurons that are generated in all regions of the OPC. (2) To determine the identity of Hth+NOFF cOPC-derived neurons, Hth+ single cell flip-out clones were generated (using hth-Gal4) in the adult medulla. The only Hth+ neurons that are also Vsx1+Svp+ are Pm3 multi-columnar local neurons. (3) For Hth+NOFF pOPC-derived neurons, 27b-Gal4 was used; it drives expression in larval pOPC Hth+NOFF neurons and is maintained to adulthood. Flip-out clones with 27b-Gal4 mark Pm1 and Pm2 neurons, as well as Hth- Tm1 uni-columnar neurons that come from a different temporal window. Both Pm1 and Pm2 neurons (but not Tm1) express Hth and Svp. Pm1 neurons also express Tsh, which only labels larval ventral pOPC neurons (Erclik, 2017).

    Thus, in addition to uni-columnar Mi1 neurons generated throughout the OPC, Hth neuroblasts generate three region-specific neuronal types: multi-columnar Pm3 neurons in the cOPC; multi-columnar Pm1 neurons in the ventral pOPC; and multi-columnar Pm2 neurons in the dorsal pOPC (Erclik, 2017).

    To determine the contribution of the temporal and spatial factors to the generation of the different neuronal fates, the factors were mutated them and whether neuronal identity was lost was examined. To test the temporal axis, hth was mutated. As previously reported, Bsh expression is lost in hth mutant clones. Loss of hth in clones also leads to the loss of the Pm3 marker Svp without affecting expression of Vsx1, indicating that Vsx1 is not sufficient to activate Svp and can only do so in the context of an Hth+ neuroblast. Hth is also required for the specification of Pm1 and Pm2 in the pOPC as Svp and Tsh expression is lost in hth mutant larval clones. Ectopic expression of Hth in older neuroblasts is not able to expand Pm1, 2 or 3 fates (on the basis of the expression of Svp) into later born neurons, although it is able to expand Bsh expression. Thus, temporal input is necessary for the specification of all Hth+ neuronal fates but only sufficient for the generation of Mi1 neurons (see Temporal and spatial inputs are required for neuronal specification in the medulla. ) (Erclik, 2017).

    Next, whether regional inputs are necessary and/or sufficient to specify neuronal fates in the progeny of Hth+ neuroblasts was determined. In Vsx1 RNA interference (RNAi) clones, Svp expression is lost in the cOPC but Bsh is unaffected. Additionally, Hth+Lim3+ cells are absent, suggesting that NOFF cells undergo apoptosis in these clones. Conversely, ectopic expression of Vsx1 leads to the expression of Svp in mOPC Hth+ neurons but does not affect Bsh expression. Therefore, Vsx1 is both necessary and sufficient for the specification of Pm3 fates in the larva. However, unlike the temporal factor Hth, Vsx1 does not affect the generation of Mi1 neurons (Erclik, 2017).

    In Rx whole mutant larvae and in mutant clones, Svp+Lim3+Hth+ larval neurons (that is, Pm1 and Pm2 neurons) in the pOPC are lost. Additionally, the Pm1 marker Tsh is lost in ventral pOPC Hth+ cells. Consistent with the Vsx1 mutant data, larval Bsh expression is not affected by the loss of Rx. In adults, the Pm1/Pm2 markers (Svp, Tsh and 27b-Gal4) are lost in the medulla (Erclik, 2017).

    Ectopic expression of Rx leads to the activation of Svp in mOPC Hth+ neurons, but does not affect the expression of Bsh. It also leads to the activation of Tsh, but only in the ventral half of mOPC Hth+ neurons, suggesting that a ventral factor acts together with Rx to specify ventral fates. Taken together, the above data show that Rx is both necessary and sufficient for the specification of Pm1/2 neurons but (like Vsx1) does not affect the generation of Mi1 neurons (Erclik, 2017).

    Finally, the role of the mOPC marker Optix in neuronal specification was examined. In Optix mutant clones, Svp is ectopically expressed in the mOPC, but Bsh expression is not affected. Of note, these ectopic Svp+ neurons fail to express the region-specific Pm markers Vsx1 or Tsh (in ventral clones), which suggests that they assume a generic Pm fate. Conversely, ectopic expression of Optix leads to the loss of Svp expressing neurons in both the cOPC and pOPC but does not affect Bsh. These NOFF neurons die by apoptosis as no Lim3+ neurons are found intermingled with Bsh+Hth+NON neurons. When apoptosis is prevented in mOPC-derived neurons, Svp is not derepressed in the persisting Hth+NOFF neurons, which suggests that Optix both represses Svp expression and promotes cell death in Hth+NOFF neurons (Erclik, 2017).

    The above data demonstrate that input from both the temporal and regional axes is required to specify neuronal fates. The temporal factor Hth is required for both Mi1 and Pm1/2/3 specification. The spatial genes are not required for the specification of NON Mi1 neurons, consistent with the observation that Mi1 is generated in all OPC compartments. The spatial genes, however, are both necessary and sufficient for the activation (Vsx1 and Rx) or repression (Optix) of the NOFF Pm1/2/3 neurons. Thus, Hth+ neuroblasts generate two types of progeny: NOFF neurons that are sensitive to spatial input (Pm1/2/3) and NON neurons that are refractory to spatial input (Mi1). Vsx1 expression in the cOPC is only maintained in Hth+NOFF neurons, suggesting that spatial information may be 'erased' in Mi1, thus allowing the same neural type to be produced throughout the OPC (Erclik, 2017).

    Do spatial genes regulate each other in the neuroepithelium? In Vsx1 mutant clones, Optix (but not Rx) is derepressed in the cOPC epithelium. Conversely, ectopic Vsx1 is sufficient to repress Optix in the mOPC and Rx in the pOPC. Similarly, Optix, but not Vsx1, is derepressed in Rx mutant clones in the pOPC epithelium and ectopic Rx is sufficient to repress Optix in the mOPC (but not Vsx1 in the cOPC). In Optix mutant clones, neither Vsx1 nor Rx are derepressed in the mOPC epithelium, but ectopic Optix is sufficient to repress both Vsx1 in the cOPC and Rx in the pOPC. The observation that Optix is not necessary to suppress Vsx1 or Rx in the mOPC neuroepithelium is surprising because Svp is activated in a subset of Hth+ neurons in the mOPC in Optix mutant clones. Nevertheless, when cell death in the mOPC is abolished, the ectopic undead NOFF neurons express Lim3 but not Svp, which confirms that Optix represses Svp expression in mOPC neurons. Taken together, these results support a model in which Optix is sufficient to repress Vsx1 and Rx, to promote the death of Hth+NOFF neurons and to repress Pm1/2/3 fates (see Spatial genes cross-regulate each other in the OPC neuroepithelium). Vsx1 and Rx act to promote Pm3 (Vsx1) or Pm1/2 (Rx) fates but can only do so in the absence of Optix (Erclik, 2017).

    These results suggest that multi-columnar neurons are generated at specific locations in the medulla crescent. However, since these neurons are required to process visual information from the entire retina in the adult medulla, how does the doral-ventral position of neuronal birth in the larval crescent correlate with their final position in the adult? Lineage-tracing experiments were performed with Vsx1-Gal4 to permanently mark neurons generated in the cOPC and with Optix-Gal4 for mOPC neurons, and the position of the cell bodies of these neurons was analyzed. In larvae, neurons from the cOPC or from the mOPC remain located in the same dorsal-ventral position where they were born. However, in adults, both populations have moved to populate the entire medulla cortex along the dorsal-ventral axis (see Neuronal movement during medulla development is restricted to multi-columnar cell types). The kinetics of cell movement during development was analyzed by following cOPC neurons. Neurons born in the cOPC remain tightly clustered until 20 h after puparium formation (P20), after which point the cell bodies spread throughout the medulla cortex. By P30 the neurons are distributed over the entire dorsal-ventral axis of the medulla cortex. In the adult, most neurons derived from the cOPC neuroepithelium are located throughout the cortex although there is an enrichment of neurons in the central region of the cortex (Erclik, 2017).

    To determine whether these observed movements involve the entire neuron or just the cell body, the initial targeting of cOPC or mOPC-derived neurons in larvae was examined before the onset of cell movement. In larvae, both populations send processes that target the entire dorsal-ventral axis of the medulla neuropi. Therefore, medulla neurons first send projections to reach their target columns throughout the entire medulla. Later, remodelling of the medulla results in extensive movement of cell bodies along the dorsal-ventral axis, leading to their even distribution in the cortex (Erclik, 2017).

    What is the underlying logic behind why some neurons move while others do not? Markers were studied for the Mi1 (Bsh), Pm2 (Hth+Svp+), Pm1 (Hth+Svp+Tsh+), and Pm3 (Vsx1+Svp+Hth+) populations of neurons through pupal stages and up to the adult. Mi1 neurons are generated evenly throughout the larval OPC and remain regularly distributed across the dorsal-ventral axis at all stages. The lineage-tracing experiment was repeated with Vsx1-Gal4 to follow Mi1 neurons produced by the cOPC. These Mi1 neurons remain exclusively in the centre of the adult medulla cortex, demonstrating that they do not move. In contrast, Pm3 neurons remain tightly clustered in the central region until P20, at which point they move to occupy the entire cortex (Erclik, 2017).

    However, not all multi-columnar neurons have cell bodies that move to occupy the entire medulla cortex. Unlike Mi1 and Pm3, adult Pm1 and Pm2 cell bodies are not located in the adult medulla cortex but instead in the medulla rim, at the edges of the cortex. Pm1 and Pm2 markers remain clustered at the ventral (Pm1) or dorsal (Pm2) posterior edges of the medulla cortex throughout all pupal stages. In the adult, both populations occupy the medulla rim from where they send long horizontal projections that reach the entire dorsal-ventral axis of the medulla neuropil. The pOPC may be a specialized region where many of the medulla rim cell types are generated. Even though most of cOPC-derived neurons move during development, a cOPC-derived multi-columnar neuron (TmY14) was identified that sends processes targeting the entire dorsal-ventral length of the medulla neuropil but whose cell bodies remain in the central medulla cortex in the adult (Erclik, 2017).

    Thus, the four populations of Hth neurons follow different kinetics: Mi1 neurons are born throughout the OPC and do not move; Pm3 neurons are born centrally and then move to distribute throughout the entire cortex; and Pm1/Pm2 neurons are born at the ventral or dorsal posterior edges of the OPC and occupy the medulla rim in adults (Erclik, 2017).

    It is noted that uni-columnar Mi1 neurons, whose cell bodies do not move, reside in the distal cortex whereas multi-columnar Pm3 neurons, which move, reside in the proximal cortex. The hypothesis was thus tested that neurons whose cell bodies are located distally in the medulla cortex represent uni-columnar neurons generated homogeneously throughout the OPC that do not move. In contrast, proximal neurons, which are fewer in number and are generated in specific subregions of the medulla OPC, would be multi-columnar and move to their final position (Erclik, 2017).

    It was first confirmed that neurons that move have their cell bodies predominantly in the proximal medulla cortex. The cell body position of neurons born ventrally that have moved dorsally was analyzed using the hh-Gal4 lineage trace: in the adult, the cell bodies found dorsally are mostly in the proximal medulla cortex, whereas the cell bodies in the ventral region are evenly distributed throughout the distal-proximal axis of the ventral cortex. They probably represent both distal uni-columnar neurons that did not move as well as proximal multi-columnar neurons that remained in the ventral region (Erclik, 2017).

    Next the pattern of movement of Tm2 uni-columnar neurons from the ventral and dorsal halves of the OPC was analyzed using the hh-based lineage-trace. The cell bodies of Tm2 neurons are located throughout the dorsal-ventral axis in the adult medulla cortex but are co-labelled with the hh lineage marker only in the ventral half. Thus, like Mi1, Tm2 uni-columnar neurons do not move. Furthermore, uni-columnar Tm1 neurons, labelled by 27b-Gal4, are born throughout the dorsal-ventral axis of the OPC crescent with distal cell bodies in the adult, suggesting that they also remain where they were born (Erclik, 2017).

    Conversely, it was asked whether neurons that are specified in only one region, such as the Vsx+ neurons of the cOPC, are multi-columnar in morphology. By sparsely labelling cOPC-derived neurons using the Vsx1-Gal4 driver, 13 distinct cell types were characterized that retain Vsx1 expression in the adult medulla. Strikingly, all are multi-columnar in morphology, further supporting the model that it is the multi-columnar neurons that move during pupal development (Erclik, 2017).

    Finally, MARCM clones were generated in the OPC neuroepithelium and visualized using cell-type-specific Gal4 drivers in the adult medulla. Two classes of adult clone distribution were observed: clones in which neurons are tightly clustered, and clones in which neurons are dispersed. Consistent with the model, the clustered clones are those labelled with uni-columnar neuronal drivers, whereas the dispersed clones are those labelled with a multi-columnar driver (Erclik, 2017).

    Taken together, these data demonstrate that neurons that do not move are uni-columnar (with cell bodies in the distal cortex), whereas most multi-columnar neurons (with cell bodies in the proximal cortex) move (Erclik, 2017).

    This study shows that combinatorial inputs from the temporal and spatial axes act together to promote neural diversity in the medulla. Previous work has shown that a temporal series of transcription factors expressed in medulla neuroblasts allows for a diversification of the cell types generated by the neuroblasts as they age. This study now shows that input from the dorsal-ventral axis leads to further diversification of the neurons made by neuroblasts; at a given temporal stage, neuroblasts produce the same uni-columnar neuronal type globally as well as smaller numbers of multi-columnar cell types regionally. This situation is reminiscent of the mode of neurogenesis in the Drosophila ventral nerve cord, in which each neuroblast also expresses a (different) temporal series of transcription factors that specifies multiple neuronal types in the lineage. Spatial cues from segment polarity, dorsal-ventral and Hox genes then intersect to impart unique identities to each of the lineages. However, neuroblasts from the different segments give rise to distinct lineages to accommodate the specific function of each segment. In contrast, in the medulla, the entire OPC contributes to framing the repeating units that form the retinotopic map. It is therefore likely that each neuroblast produces a common set of neurons that connect to each pair of incoming R7 and R8 cells, or L1-L5 lamina neurons. This serves to produce 800 medulla columns with a 1:1 stoichiometry of medulla neurons to photoreceptors. The medulla neurons that are produced by neuroblasts throughout the dorsal-ventral axis of the OPC are thus uni-columnar The production of the same neuronal type along the entire OPC could be achieved by selectively 'erasing' spatial information in uni-columnar neurons, as observed in Mi1 neurons (Erclik, 2017).

    Regional differences in the OPC confer further spatial identities to neuroblasts with the same temporal identity, and lead to specific differences in the lineages produced in the compartments along the dorsal-ventral axis of the medulla. These differences produce smaller numbers of multi-columnar neurons whose stoichiometry is much lower than 1:1. The majority of these neurons move during development to be uniformly distributed in the adult medulla cortex. This combination of regional and global neuronal specification in the medulla presents a powerful mechanism to produce the proper diversity and stoichiometry of neuronal types and generate the retinotopic map (Erclik, 2017).

    Wnt signaling specifies anteroposterior progenitor zone identity in the Drosophilavisual center

    During brain development, various types of neuronal populations are produced from different progenitor pools to produce neuronal diversity that is sufficient to establish functional neuronal circuits. However, the molecular mechanisms that specify the identity of each progenitor pool remain obscure. This study shows that Wnt signaling is essential for the specification of the identity of posterior progenitor pools in the Drosophila visual center. In the medulla, the largest component of the visual center, different types of neurons are produced from two progenitor pools: the outer proliferation center (OPC) and glial precursor cells (GPCs; also known as tips of the OPC). It was found that OPC-type neurons are produced from the GPCs at the expense of GPC-type neurons when Wnt signaling is suppressed in the GPCs. In contrast, GPC-type neurons are ectopically induced when Wnt signaling is ectopically activated in the OPC. These results suggest that Wnt signaling is necessary and sufficient for the specification of the progenitor pool identity. It was also found that Homothorax (Hth), which is temporally expressed in the OPC, is ectopically induced in the GPCs by suppression of Wnt signaling and that ectopic induction of Hth phenocopies the suppression of Wnt signaling in the GPCs. Thus, Wnt signaling is involved in regionalization of the fly visual center through the specification of the progenitor pool located posterior to the medulla by suppressing Hth expression (Suzuki, 2016).

    Ecdysone-dependent and ecdysone-independent programmed cell death in the developing optic lobe of Drosophila

    The adult optic lobe of Drosophila develops from the primordium during metamorphosis from mid-3rd larval stage to adult. Many cells die during development of the optic lobe with a peak of the number of dying cells at 24 h after puparium formation (h APF). Dying cells were observed in spatio-temporal specific clusters. This study analyzed the function of a component of the insect steroid hormone receptor, EcR, in this cell death. Expression patterns of two EcR isoforms, EcR-A and EcR-B1, were examined in the optic lobe. Expression of each isoform altered during development in isoform-specific manner. EcR-B1 was not expressed in optic lobe neurons from 0 to 6h APF, but was expressed between 9 and 48 h APF and then disappeared by 60 h APF. In each cortex, its expression was stronger in older glia-ensheathed neurons than in younger ones. EcR-B1 was also expressed in some types of glia. EcR-A was expressed in optic lobe neurons and many types of glia from 0 to 60 h APF in a different pattern from EcR-B1. Then, EcR function were genetically analyzed in the optic lobe cell death. At 0 h APF, the optic lobe cell death was independent of any EcR isoforms. In contrast, EcR-B1 was required for most optic lobe cell death after 24 h APF. It was suggested that cell death cell-autonomously required EcR-B1 expressed after puparium formation. betaFTZ-F1 was also involved in cell death in many dying-cell clusters, but not in some of them at 24 h APF. Altogether, the optic lobe cell death occurred in ecdysone-independent manner at prepupal stage and ecdysone-dependent manner after 24 h APF. The acquisition of ecdysone-dependence was not directly correlated with the initiation or increase of EcR-B1 expression (Hara, 2013).

    This study analyzed the requirement of ecdysone in the optic lobe cell death. The role of ecdysone in cell death during metamorphosis has been examined in the salivary gland, larval midgut, and two types of neurons in the VNC, vCrz neurons and RP2 neurons. In the salivary gland, ecdysone triggers cell death in vitro, and the cell death required some components of ecdysone cascade, BR-C, E93, E74 and βFTZ-F1. In the midgut, cell death was induced by injection of ecdysone, and required BR-C and E93. For the cell death in vCrz neurons and RP2 neurons, ecdysone requirement was shown using EcR mutants. Among these tissues, a requirement for EcR isoforms was addressed only in the vCrz neurons and RP2 neurons. In the vCrz neurons, the cell death occurred in EcR-A or EcR-B1 mutants, but not in EcR-B1 and EcR-B2 mutant, indicating that EcR-B2 is required for this cell death. In RP2 neurons, cell death did not require EcR-A, but EcR-B1, EcR-B2 or both. Here, it was shown that the optic lobe cell death included ecdysone-independent and dependent tissues. The ecdysone-dependent cell death required EcR-B1 (Hara, 2013).

    The number of dying cells in the optic lobe of EcR-B1 mutant animals at 24 and 36 h APF was much smaller than that in wild-type animals, but the number in EcR-A mutant animals was not. This finding showed that cell death in the optic lobe at these stages required EcR-B1, but not EcR-A (Hara, 2013).

    Dying cells were examined in the following structures; LAD, lamina anterior dying cells; LPD, lamina posterior dying cells; LUD, lamina underlying dying cells; MALD, medulla anterolateral dying cells; MAMD, medulla anteromedial dying cells; MCD, medulla cortex dying cells; MCLD, medulla cortex lateral dying cells; MCMD, medulla cortex medial dying cells; MLBD, medulla-lamina boundary dying cells; MPLD, medulla posterolateral dying cells; PMCD, posterior medulla cortex dying cells; PMD, posteromedial dying cells; T/C, dying cells in the T/C region; LopD, dying cells in the lobula plate cortex; MMC, abnormal dying cells in the medial side of the medulla cortex; MLopD, abnormal dying cells in the medial side of the lobula plate cortex. The dependence on EcR-B1 was common among all dying cells in all clusters, except the MCMD. At 24 h APF, dying cells in the LAD, LPD, LUD, MCLD and T/C region were absent in optic lobes of most EcR-B1 mutants. In these mutants at 36 h APF, dying cells in the LUD, MLBD, MPLD and the lobula plate cortex were not evident. Similarly, at 48 h APF, MPLDs were absent from the EcR-B1 mutants. These results indicated that cell death in most clusters required EcR-B1 at stages after 24 h APF. It could not be determined whether death of MCMD was dependent on EcR-B1 because it was not clear whether the abnormal dying cells included MCMD in the medial side of the medulla cortex in EcR-B1 mutants (Hara, 2013).

    In EcR-B1 mutant, a significant number of dying cells was constantly observed after 24 h APF. However, this fact does not mean that the loss of EcR-B1 delayed the timing of cell death. From 24 to 72 h APF, dying cells were mostly located in the medial side of the medulla cortex in EcR-B1 mutants and they did not include those in the clusters which would have been normally observed from 24 to 48 h APF, i.e., the LAD, LPD, LUD, MCLD, MPLD, MLBD, and dying cells in the T/C region and the lobula plate cortex. This fact strongly suggests that the optic lobe cell death was not delayed but suppressed by EcR-B1 mutation (Hara, 2013).

    In some EcR-B1 mutants, enormous dying cells were observed at 72 h APF at positions where cell death would have normally occurred: the LAD, MCLD, MPLD, MLBD and T/C region. This suggests that delayed cell death can be induced at normal position without EcR-B1 at 72 h APF in these samples. It is known that experimental suppression of cell death can lead a delayed cell death by another complementally cell death mechanism. Therefore, it is possible that a complementary mechanism was induced in these samples (Hara, 2013).

    It has been shown that a cell death initiator caspase, Dronc, had a EcRE in its promoter and EcR-B1 could induce Dronc expression. Indeed, the optic lobe cell death was suppressed in Dronc mutant in a preliminary experiment. Altogether, it is most likely that EcR-B1 directly controls cell death and consistently induces the death at right time in the optic lobe (Hara, 2013).

    In this study, requirement of EcR-B2 function was not suggested. When the functions of all EcR isoforms were inhibited after puparium formation, the number of dying cells was larger than that in EcR-B1 mutant at 24 h APF (638.6 versus 363.8). If EcR-B2 was required for the optic lobe cell death, the number would have been less than that in the mutant. However, a function of EcR-B2 in the ecdysone-dependent cell death can still not be entirely excluded since there was a possibility that RNAi was insufficient to entirely inhibit EcR function in this experimental condition (Hara, 2013).

    The number of dying cells in the optic lobes of EcR-A and EcR-B1 mutants was the same as that in wild-type animals at 0 h APF. When distribution of dying cells was examined, all clusters that were present in wild-type animals (specifically the LAD, MALD, MCD, MAMD, PMCD and dying cells in the T/C region) were also observed in the mutants. These results strongly indicated the cell death at 0 h APF was independent of both EcR-A and EcR-B1. EcR-B2 was also not required for the cell death because concurrent knockdown of all EcR isoforms via expression of hs-EcRi-11 resulted in no reduction in the number of dying cells at 0 h APF (Hara, 2013).

    The above argument is relevant only for the zygotic not with maternal EcR. The contribution of maternal EcR should be tested. The result of the heat shock-inducible EcR RNAi denied the contribution of maternal EcR mRNA of all EcR isoforms. As with maternal proteins, there was no detectable EcR-B1 in any cluster or region at 0 h APF. In contrast, EcR-A was weakly expressed in all cluster regions, and these is no information about EcR-B2. Therefore, possible roles of maternal EcR-A and EcR-B2 protein cannot be excluded. However, there are no published reports of a requirement for maternal EcR proteins during metamorphosis. Taken together, these findings indicated that cell death in the optic lobe at 0 h APF is independent of any EcR. Furthermore, it seems likely this cell death is also independent of ecdysone because the number of dying cells gradually increased from 0 to 6 h APF rather than decreased, but the ecdysone titer rapidly drops and is very low during this period (Hara, 2013).

    The period around 12 h APF may be a transient period when the ecdysone-dependence of cell death changes. In many EcR-B1 mutant optic lobes, the number of dying cells was the same as that in the wild type. In contrast, the number was reduced in a few mutant optic lobes and dying cells were absent in many of the clusters. These findings indicated that most of the cell deaths in many optic lobes was independent of EcR-B1, but some had become EcR-B1-dependent (Hara, 2013).

    There is no previous report on ecdysone-independent cell death during metamorphosis. The ecdysone-independent cell death was limited to the early phase of metamorphosis in the optic lobe. However, this timing does not necessarily indicate that all cell death is independent of ecdysone because the larval midgut and vCrz neurons die ecdysone-dependently during this period. Therefore, ecdysone independence is a unique feature of the cell death that occurs in the optic lobe. There has been no report that the cell death that occurs during embryogenesis and larval development depends on ecdysone. Hence, it is proposed that the same mechanisms that mediate cell death during embryogenesis or larval development work for cell death during the early phase of optic lobe development (Hara, 2013).

    Based on findings from many previous studies, every cell death that occurred during metamorphosis was part of the degeneration of a larval tissue and was dependent on ecdysone. These finding are understandable because ecdysone orchestrates the entire developmental process of metamorphosis. However, cell death within the optic lobe was independent of ecdysone during an early phase and then this cell death became ecdysone dependent later. This unique feature of the optic lobe cell death may be due to the fact that cell death in the optic lobe takes place during metamorphosis and is simultaneously involved in the organogenesis. So two cell death mechanisms, i.e., an organogenesis-accompanied (ecdysone-independent) mechanism and a metamorphosis-accompanied (ecdysone-dependent) mechanism may have evolved to cooperate during the optic lobe development (Hara, 2013).

    The expression pattern of EcR-A and EcR-B1 was examined in this study. Expression of each isoform altered during development in isoform specific manner. However, there was no direct relationship between EcR-B1 expression and the emergence of the cell death. At 0 h APF, when cells die independent of ecdysone, EcR-B1 was not expressed in any region with clusters of dying cells. In contrast, EcR-B1 was expressed in all regions with clusters of dying cells at 12 h APF, although cell death, at this stage, was, for the most part, ecdysone independent in all clusters, except PMD. Thus, there was a temporal gap between EcR-B1 expression and ecdysone-dependent cell death. This indicates that the expression of EcR-B1 was not a direct cause that shifted cell death from an ecdysone-independent to an ecdysone-dependent one. EcR-B1 expression would be one of the requisites to make cells competent to undergo ecdysone-dependent cell death at a later time point and another mechanism following EcR-B1 expression would be required for the shift (Hara, 2013).

    Although cell death in the optic lobe after 24 h APF required EcR-B1, the level of EcR-B1 expression varied among cluster regions during this period. For example, at 24 h APF, EcR-B1 was expressed weakly in the anterior region of the lamina cortex where LAD was located. On the lateral side of the medulla cortex where MCLD were present, EcR-B1 was expressed moderately. EcR-B1 was strongly expressed in the T/C region where many dying cells were present. The expression levels also varied among cluster regions at 36 and 48 h APF. All these findings indicate that the death decision, even for the ecdysone-dependent cell death, was not simply related to high EcR-B1 levels. This decision would be made within specific context of each cluster (Hara, 2013).

    EcR-B1 expression was correlated with glial ensheathment in the lamina cortex, medulla cortex and T/C region. In these regions, newly-born neurons derived from the OOA or IOA compose pre-ensheathed domains. As development proceeds, they become to be surrounded by glial membrane and compose ensheathed domains of mature neurons. In particular, the ensheathed domain in lamina cortex corresponds to a region with columnar structures. In the lamina cortex, EcR-B1 was weakly expressed in the pre-ensheathed domain, while strongly expressed in the ensheathed domain. In the medulla cortex and T/C region, it was not expressed in the pre-ensheathed domains, but expressed in the ensheathed domains. These facts suggest a possibility that the glial ensheathment promotes or initiates EcR-B1 expression in the process of neuronal differentiation in these regions. This possibility is supported by the fact that EcR-B1 expression became stronger after the ensheathment as development proceeded (Hara, 2013).

    With regard to cell death, the LAD, MCLD and MALD were always located near the border of the pre-ensheathed and ensheathed domains. Therefore, cell death may be linked to the entry of glial membrane in these clusters. Since this positional relationship was observed from 0 to 24 h APF, the glial ensheathement and ecdysone signaling via EcR-B1 may cooperate to induce cell death in the clusters after 12 h APF, when cell death become dependent on EcR-B1 (Hara, 2013).

    Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper

    Temporal patterning of neural progenitors is one of the core mechanisms generating neuronal diversity in the central nervous system. This study shows that, in the tips of the outer proliferation center (tOPC) of the developing Drosophila optic lobes, a unique temporal series of transcription factors not only governs the sequential production of distinct neuronal subtypes but also controls the mode of progenitor division, as well as the selective apoptosis of NotchOFF or NotchON neurons during binary cell fate decisions. Within a single lineage, intermediate precursors initially do not divide and generate only one neuron; subsequently, precursors divide, but their NotchON progeny systematically die through Reaper activity, whereas later, their NotchOFF progeny die through Hid activity. These mechanisms dictate how the tOPC produces neurons for three different optic ganglia. It is concluded that temporal patterning generates neuronal diversity by specifying both the identity and survival/death of each unique neuronal subtype (Bertet, 2014).

    Although apoptosis is a common feature of neurogenesis in both vertebrates and Drosophila, the mechanisms controlling this process are still poorly understood. For instance, several studies in Drosophila have shown that, depending on the context, Notch can either induce neurons to die or allow them to survive during binary cell fate decisions. This is the case in the antennal lobes where Notch induces apoptosis in the antero-dorsal projecting neurons lineage (adpn), whereas it promotes survival in the ventral projecting neurons lineage (vPN). In both of these cases, the entire lineage makes the same decision whether the NotchON or NotchOFF cells survive or die. This suggests that, in this system, Notch integrates spatial signals to specify neuronal survival or apoptosis (Bertet, 2014).

    This study shows that, during tOPC neurogenesis, neuronal survival is determined by the interplay between Notch and temporal patterning of progenitors. Indeed, within the same lineage, Notch signaling leads to two different fates: it first induces neurons to die, whereas later, it allows them to survive. This switch is due to the sequential expression of three highly conserved transcription factors-Dll/Dlx, Ey/Pax-6, and Slp/Fkh-in neural progenitors. These three factors have distinct functions, with Dll promoting survival of NotchOFF neurons, Ey inducing apoptosis of NotchOFF neurons, and Slp promoting survival of NotchON neurons. These data suggest that Ey induces death of NotchOFF neurons by activating the proapoptotic factor hid. Thus, Dll probably antagonizes Ey activity by preventing Ey from activating hid. The data also suggest that Notch signaling induces neuronal death by activating the proapoptotic gene rpr. Thus, Slp might promote survival of NotchON neurons by directly repressing rpr expression or by preventing Notch from activating it. In both cases, the interplay between Notch and Slp modifies the default fate of NotchON neurons, allowing them to survive. Further investigations will test these hypotheses and determine how Dll, Ey, Slp, and Notch differentially activate/repress hid and rpr (Bertet, 2014).

    Although the tOPC and the main OPC have related temporal sequences, their neurogenesis is very different. This difference is in part due to the fact that newly specified tOPC neuroblasts express Dll, which controls neuronal survival, instead of Hth. Why do tOPC neuroblasts express Dll? The tOPC, which is defined by Wg expression in the neuroepithelium, is flanked by a region expressing Dpp. Previous studies have shown that high levels of Wg and Dpp activate Dll expression in the distal cells of the Drosophila leg disc. Wg and Dpp could therefore also activate Dll in the neuroepithelium and at the beginning of the temporal series in tOPC progenitors. Another difference between the main OPC and tOPC neurogenesis is that Ey and Slp have completely different functions in these regions. Indeed, unlike in the main OPC, Ey and Slp control the survival of tOPC neurons. This suggests that autonomous and/or nonautonomous signals interact with these temporal factors and modify their function in the tOPC (Bertet, 2014).

    Finally, tOPC neuroblasts produce neurons for three different neuropils of the adult visual system, the medulla, the lobula, and the lobula plate. This ability could be due to the particular location of this region in the larval optic lobes. Indeed, the tOPC is very close to the two larval structures giving rise to the lobula and lobula plate neuropils-Dll-expressing neuroblasts are located next to the lobula plug, whereas D-expressing neuroblasts are close to the IPC. Interestingly, Dll and D neuroblasts specifically produce lobula plate neurons. This raises the possibility that these neuroblasts and/or the neurons produced by these neuroblasts receive signals from the lobula plug and the IPC, which instruct them to specifically produce lobula plate neurons. These nonautonomous signals could also modify the function of Ey and Slp in the tOPC (Bertet, 2014).

    In summary, this study demonstrates that temporal patterning of progenitors, a well-conserved mechanism from Drosophila to vertebrates, generates neural cell diversity by controlling multiple aspects of neurogenesis, including neuronal identity, Notch-mediated cell survival decisions, and the mode of intermediate precursor division. In the tOPC temporal series, some factors control two of these aspects (Ey), whereas others have a specialized function (Dll, Slp, and D). This suggests that temporal patterning does not consist of a unique series of transcription factors controlling all aspects of neurogenesis but instead consists of multiple superimposed series, each with distinct functions (Bertet, 2014).

    Signals transmitted along retinal axons in Drosophila: Hedgehog signal reception and the cell circuitry of lamina cartridge assembly

    The arrival of retinal axons in the Drosophila brain triggers the assembly of glial and neuronal precursors into a neurocrystalline array of lamina synaptic cartridges. Retinal axons arriving from the eye imaginal disc trigger the assembly of neuronal and glial precursors into precartridge ensembles in the crescent-shaped lamina target field. In the eye disc, photoreceptor cells assemble into ommatidial clusters behind the morphogenetic furrow (mf) as it moves to the anterior. The ommatidial clusters project their axon fascicles into the crescent-shaped lamina. Neuronal precursor cells of the lamina (LPCs) are incorporated into the axon target field at its anterior margin, which is demarcated by a morphological depression known as the lamina furrow. Glia precursor cells (GPCs) are generated in two domains that lie at the dorsal and ventral anterior margins of the prospective lamina. These glial precursors migrate into the lamina along an axis perpendicular to that of LPC entry. Postmitotic LPCs within the lamina axon target field express the nuclear protein Dac, as revealed by anti-Dac antibody staining. Like the eye, lamina differentiation occurs in a temporal progression on the anterioposterior axis. Axon fascicles from new ommatidial R-cell clusters arrive at the anterior margin of the lamina (adjacent to the lamina furrow) and associate with neuronal and glia precursors in a vertical lamina column assembly. At the anterior of the lamina, at the trough of the lamina furrow, LPCs await a retinal axon-mediated signal in G1-phase and enter their terminal S-phase at the posterior margin of the furrow. Postmitotic (Dac-positive) LPCs assemble into columns at the posterior margin of the furrow. In older columns at the posterior of the lamina, a subset of postmitotic LPCs express definitive neuronal markers as they become specified as the lamina neurons L1-L5. Lamina neurons L1-L4 form a stack in a superficial layer, while L5 neurons reside in a medial layer near the R1-R6 axon termini. These neurons arise at cell-type specific positions along the column's vertical axis. Lamina glial cells take up cell-type positions in the precartridge assemblies. Epithelial (E-glia) and marginal (Ma-glia) glia are located above and below the R1-R6 termini, respectively. Satellite glia are interspersed among the neurons of the L1-L4 layer. The Ma-glia and E-glia layers, both located ventral to the neuronal precursor column, sandwich the R1-R6 axon termini. The medulla neuropil serves as the target for R7/8 axons and is separated from the lamina by the medulla glia, situated just below the Ma-glia (Huang, 1998 and references).

    Hedgehog is transmitted along retinal axons to serve as the inductive signal in the brain for differentiation of lamina neurons. The target of HH is wingless, which in turn targets decapentaplegic and Distal-less. The lamina is a ganglion layer of the visual center of the brain that processes information received from R1-R6 photoreceptor neurons located in the ommatidia of compound eyes. The lamina develops in a precise order, directly coupled to the arrival of retinal axons from the eye. Ectopic hh expression in the brains of eyeless flies induces lamina differentiation, but is not sufficient to induce Elav, a late marker. This suggests that HH alone is not sufficient for the later events of lamina development that include the specification of lamina neurons (Huang, 1998).

    Hedgehog, a secreted protein, is an inductive signal delivered by retinal axons for the initial steps of lamina differentiation. In the development of many tissues, Hedgehog acts in a signal relay cascade via the induction of secondary secreted factors. Lamina neuronal precursors respond directly to Hedgehog signal reception by entering S-phase, a step that is controlled by the Hedgehog-dependent transcriptional regulator Cubitus interruptus. The terminal differentiation of neuronal precursors and the migration and differentiation of glia appear to be controlled by other retinal axon-mediated signals. Thus retinal axons impose a program of developmental events on their postsynaptic field utilizing distinct signals for different precursor populations (Huang, 1998).

    A number of markers distinguish glial and neuronal precursor cells from the corresponding mature cell types. The expression of optomotor-blind (omb) labels both glial precursors in the dorsal and ventral anlagen and mature glia that have migrated into the lamina target field. The glia cell marker Repo and the enhancer-trap lacZ insertion 3-109 are expressed by glia once they have entered the lamina target field. Cubitus interruptus (Ci), a transcriptional mediator of Hh signaling is expressed by LPCs anterior of the lamina furrow and by the postmitotic neuronal precursors within the lamina. The nuclear protein Dachshund is expressed only by neuronal precursors that have begun terminal differentiation and lie posterior to the lamina furrow. Thus, Omb and Ci label the glial and neuronal precursors, respectively, while the mature cells, following their interaction with retinal axons, additionally express Repo and Dac. In the lamina target field of eyeless mutants (mutants that project no neurons toward the optic disc), such as eyes absent (eya) or sine oculis (so), Dac expression is not detected and Repo expression is greatly diminshed (Huang, 1998). The migration and early differentiation of lamina glia are independent of Hh. Enhanced transcription of the putative Hh receptor, patched (ptc) is a universal characteristic of Hh signal reception. All classes of glia in the lamina region upregulate ptc expression in an hh-dependent fashion. These cells are thus Hh-responsive. All three classes of lamina glia, as well as medulla glia, that express a ptc-lacZ reporter construct are in close proximity to Hh-bearing retinal axons. Glia cell ptc reporter gene expression is not observed in hh- animals. This raises the question of whether Hh signal reception is responsible for the migration and/or subsequent maturation of glia cells. To determine whether the migration of glial precursors into the lamina target field is Hh-dependent, the distribution of Omb-positive cells was examined in hh- animals. In the wild type, a trail of Omb-positive cells delineates a path of glia migration from the dorsal and vental anlagen. Is glia precursor migration Hh-dependent? This was investigated by examining the distribution of Omb-positive cells in hh1 mutant animals. hh1 is a regulatory mutation that specifically affects hh expression in the visual system. In hh1 animals, approximately 12 columns of ommatidia initiate differentiation in the eye imaginal disc before the anterior progression of the morphogenetic furrow ceases. hh1 retinal axons lack Hh immunoreactivity by the time they reach the lamina target field and thus the Hh-dependent steps of LPC maturation fail to occur in hh1 animals. Omb staining reveals a relatively normal number of glia precursors in the lamina target field of hh1 animals, despite the absence of Dac induction. The Omb-positive cells are distributed uniformly along the dorsoventral axis among the retinal axon fascicles, but appear more closely spaced than in the wild type. A likely explanation for this spacing defect is the absence of the neuronal precursors that would constitute the majority of lamina cells at this point in development. To determine whether the glial precursors that enter the lamina target field in hh- animals express a retinal innervation-dependent marker, their expression of Repo was examined. In hh1 animals, the Omb-positive cells within the lamina also express Repo. Moreover, the Repo-positive cells occupy proper layers above and below the R1-R6 axon termini expected for satellite, marginal and epithelial glia, though the lack of markers specific for these three glia types precludes an unambiguous determination of glial cell type. The presence of marginal and epithelial glia is consistent with the observation that R1-R6 growth cones terminate in their proper positions between these layers in hh- animals. The ectopic expression of Hh in the brains of `eyeless' animals is sufficient to induce the initial steps of LPC maturation in the absence of retinal axons. However, neither Hh nor the Hh-mediated events of LPC maturation are sufficient for glia cell migration and maturation (Huang, 1998).

    The activities of a number of Hh signal transduction pathway components are now well characterized. Mutations at these loci have been shown to either mimic or block Hh signal reception in a cell-autonomous fashion. Examining the cellular requirements for these genes in mosaic animals should help illuminate the cellular circuitry that mediates the Hh-dependent events of lamina development. The seven-pass transmembrane protein encoded by smoothened (smo) acts as a positive effector of Hh signal reception, downstream of the Hh receptor Patched. If Hh exerts its effects directly on LPCs, it would be expected that loss of smo function should block the entry of G1-phase LPCs into S-phase and/or prevent the expression of Hh-dependent markers of lamina differentiation such as Dac. Inducing smo mutant clones reveals that with respect to lamina differentiation, smo acts cell autonomously. smo clones that extended to the posterior of the lamina are rare. It is possible that LPCs that cannot respond to Hh are not readily incorporated into the lamina and displaced by smo+ LPCs. LPCs that are unable to respond to Hh might be eliminated by cell death (Huang, 1998).

    A hallmark of Hh signal reception in many Drosophila tissues is an increase in immunoreactivity to the C-terminal portion of the protein Ci, a transcriptional mediator of Hh signaling. This enhanced Ci immunoreactivity is due to inhibition of Ci proteolytic processing, a cellular response to Hh signal reception. LPCs posterior to the lamina furrow display the enhanced Ci immunoreactivity that would be predicted for Hh signal reception by LPCs. In animals in which hh- retinal axons innervate the lamina target field, cells posterior to the lamina furrow display a level of Ci immunoreactivity equivalent to the basal level detected anterior to the furrow, indicating that the increased Ci observed in the wild type is Hh-dependent. In smo mosaic animals, smo cells either anterior or posterior to the lamina furrow display a basal level of Ci immunoreactivity, while smo + cells immediately adjacent to the portion of smo clones within the lamina display the high Hh-dependent level. The initial response of LPCs to the arrival of Hh-bearing retinal axons would appear to be entry into S-phase at the lamina furrow. To determine whether cell cycle progression is directly dependent on Hh signal reception, the incorporation of bromodeoxyuridine (BrdU) into S-phase cells was examined in smo mosaic animals. In the wild type, LPCs that have entered their terminal S-phase form a discrete and continuous band at the posterior margin of the lamina furrow. In animals lacking photoreceptor innervation (due to defective hh expression in the eye disc) or animals in which photoreceptor axons lacking functional Hh enter the lamina target field, only a low background of scattered S-phase cells are detected. It is unclear whether the products of these scattered divisions are incorporated into the lamina (i.e., that these cells are indeed LPCs). In smo 3 mosaic animals, mutant clones that include the posterior margin of the lamina furrow lack S-phase LPCs. In contrast, the scattered S-phase cells anterior to the lamina furrow, and the distribution of S-phase cells in other proliferation centers, such as the OPC, are unaffected by the loss of smo function. At the lamina furrow, smo+ cells bordering smo clones are often found in S-phase. Thus, in sum, smo+ behaves as a cell-autonomous requirement for LPCs to initiate the Hh-dependent steps of lamina differentiation (Huang, 1998).

    The Hh receptor Ptc, a multiple-pass membrane protein, and the cAMP-dependent protein kinase (PKA) normally maintain the Hh signal transduction pathway in a repressed state. Loss-of-function mutations in either of these genes mimic Hh signal reception and result in the cell autonomous activation of Hh target genes in many tissues. LPCs harboring mutations for either pka or ptc undergo differentiation cell-autonomously and independently of retinal innervation. Mutant cells anterior to the furrow do not differentiate precociously. This observation is consistent with the consequences of ectopic Hh expression in an the lamina in mutants lacking retinal innervation of the lamina. Hh expression in regions anterior to the lamina furrow does not induce precocious lamina differentiation, as though competence to respond to Hh is acquired by G1-phase LPCs at the anterior margin of the lamina furrow. Within the lamina target field, wild-type cells neighboring the pka or ptc mutant cells are never observed to express Dac. Thus activation of the Hh pathway by loss-of-function in either gene results in a strictly autonomous induction of LPC maturation. These results permit the conclusion that the terminal cell division and differentiation of LPCs both require the direct reception of the Hh signal (Huang, 1998).

    In a number of instances, pattern formation mediated by Hh is accompanied by cell division. The well-defined pattern of Hh-induced cell division in the lamina provides an opportunity to determine the point at which the Hh signal reception engages the cell cycle machinery. LPC cell cycle progression and cell fate determination are jointly controlled by the transcriptional regulator Cubitus interruptus. Biochemical and epistasis experiments have placed the zinc finger molecule Ci downstream of all other hh signaling pathway components. Ci has been shown to bind directly to the regulatory sequences of Hh-responsive genes. Should all Hh-mediated events of LPC maturation be found to depend on Ci function, it could be concluded that, at least with regard to cell proliferation and the expression of differentiation markers, there is no branchpoint within the signaling pathway. To examine the requirement for Ci, two recombinant constructs were used that result in either dominant Ci gain-of-function or loss-of-function phenotypes. Overexpression of the wild-type Ci gene results in a gain-of-function phenotype that mimics activation of the Hh signaling pathway. Expression of an amino terminal fragment of Ci (hereafter referred to as DN-Ci) results in a dominant loss-of-function phenotype, as the normal in vivo function of this portion of the molecule appears to be transcriptional repression of Hh target genes. With either construct, genetically engineered ectopic expression in the lamina region results in the expected phenotype with respect to the lamina differentiation marker Dac. Dac expression in cells posterior of the lamina furrow is strongly reduced or undetectable in cells expressing DN-Ci. Conversely, the ectopic expression of wild-type Ci results in the induction of Dac-positive cells in the lamina target field of animals lacking innervation from the developing eye. The effects observed with either construct are strictly cell autonomous. Thus the results with ectopic Ci and DN-Ci expression are consistent with the expectation that Ci modulates Hh signaling activity directly in LPCs (Huang, 1998).

    To determine whether Hh signaling acts via Ci to regulate the G1- to S-phase transition of LPCs at the lamina furrow, the incorporation of BrdU into S-phase cells was examined in animals harboring clones expressing either of the two constructs described above. Cells expressing DN-Ci at the posterior margin of the lamina furrow fail to enter S-phase. Where clones of DN-Ci-expressing cells traversed the lamina furrow, S-phase LPCs are absent, while S-phase LPCs are observed immediately outside of the clone. Moreover, the effect on cell division is limited to the LPCs at the lamina furrow. No defects are observed in other proliferation zones such as the OPC or IPC, the other major proliferation centers of the optic lobe, when they contain DN-Ci-expressing cells. Conversely, the induction of lamina differentiation by ectopic Ci expression in flies lacking retinal input into the lamina is accompanied by the entry of LPCs into S-phase at the lamina furrow. At the point when lamina differentiation is induced in the absence of retinal axons by ectopic Hh expression, ectopic Ci expression triggers a posterior-to-anterior pattern of differentiation such that S-phase LPCs are found at the anterior margin. In sum, these observations indicate that the induction of cell division by Hh occurs via the transcriptional regulation of Hh target genes (Huang, 1998).

    Neuropil pattern formation and regulation of cell adhesion molecules in Drosophila optic lobe development depend on Synaptobrevin

    To investigate a possible involvement of synaptic machinery in Drosophila visual system development, the effects of a loss of function of neuronal synaptobrevin (n-syb), a protein required for synaptic vesicle release, were studied. Expression of tetanus toxin light chain (which cleaves neuronal synaptobrevin) and genetic mosaics were used to analyze neuropil pattern formation and levels of selected neural adhesion molecules in the optic lobe. Targeted tetanus toxin light chain (TeTxLC) expression in the developing optic lobe results in disturbances of the columnar organization of visual neuropils and of photoreceptor terminal morphology. IrreC-rst immunoreactivity in neuropils is increased after widespread expression of toxin. In photoreceptors, targeted toxin expression results in increased Fasciclin II and chaoptin but not IrreC-rst immunoreactivity. Axonal pathfinding and programmed cell death are not affected. In genetic mosaics, patches of photoreceptors that lack neuronal synaptobrevin exhibit the same phenotypes observed after photoreceptor-specific toxin expression. These results demonstrate the requirement of neuronal synaptobrevin for regulation of cell adhesion molecules and development of the fine structure of the optic lobe. A possible causal link to fine-tuning processes that may include synaptic plasticity in the development of the Drosophila CNS is discussed (Hiesinger, 1999).

    The finding of an onset of n-syb expression in the first half of pupation poses the question of whether synapses actually start to function so early during optic lobe development. Neuronal activity plays a major role during vertebrate visual system development. A critical period of 1 d after eclosion has been demonstrated for experience-dependent developmental plasticity in the Drosophila. It has not yet been shown whether synaptic plasticity in the Drosophila CNS extends to pupation or whether neurotransmitters are released before any form of neuronal activity. Assuming the involvement of such processes, the following time scale would be expected: first, expression and localization of proteins of the vesicle release machinery; second, release of neurotransmitter independent or dependent on spontaneous activity; and third, release of neurotransmitter dependent on evoked activity. Given the early immunoreactivity of specific synaptic vesicle cycle proteins such as n-syb and synaptotagmin before P + 25% (25% through the pupal period), the synaptic vesicle cycle appears to be available for more than half of pupal development before first evoked photoreceptor responses occur at P + 82%. Morphological analysis has revealed a brief interval of intense synapse formation in the lamina of Musca starting ~P + 62% and peaking at P + 74%. Although this time window does not necessarily correspond to the first occurrence of synapses in the optic lobe of Drosophila, and the heterogeneity of optic lobe neurons should also be taken into consideration, it may indicate that n-syb is expressed long before synapses are morphologically recognizable (Hiesinger, 1999 and references).

    Apparently, not all processes between target selection and the establishment of functional connectivity are yet known. The demonstration of the dependence of neuropil patterning on NO release shows a process of terminal development in a similar time window as the neuropil patterning defects observed in the current study. With regard to the current study, one possibility would be the involvement of n-syb in the release of neurotransmitters or other factors before or during synapse development. histamine is synthesized in photoreceptors extending from cultured imaginal disks. Histamine or other substances released by growth cones after arrival in their target layers might exert functions necessary for the establishment of a regular terminal pattern (Hiesinger, 1999).

    Lack of functional n-syb has no obvious influence on target selection and the development of largely overlapping terminals of R7 and R8 cells. In contrast, further development of terminal fine structure between P + 25 and P + 50% is significantly disturbed, indicating its involvement in a fine-tuning process. This early onset of n-syb function shows that either n-syb is involved in nonsynaptic processes taking place soon after target recognition, or synapses form earlier in the Drosophila optic lobe than is generally believed. Because this time window occurs significantly before the observed upregulation of Fasciclin II in active toxin-expressing photoreceptors, the morphological changes do not depend on this CAM (Hiesinger, 1999).

    Cell adhesion molecules play multiple roles during optic lobe development. Most fruitfully investigated are functions during axon guidance and target recognition and synaptic plasticity. The finding of increased Fasciclin II immunoreactivity under conditions of blocked neurotransmitter release corresponds to previous studies that have shown the opposite effect with an opposite approach: apCAM is downregulated after application of serotonin, and synaptic Fasciclin II is reduced in mutants with abnormally high neuronal activity. Although it has been demonstrated that apCAM is downregulated via endocytosis, the mechanism of activity-dependent Fasciclin II downregulation at the Drosophila neuromuscular junction remains unknown. Possible downregulation mechanisms to be considered include endocytosis, extracellular cleavage, and reduced transcription or translation in combination with a continuous turnover of the protein. The upregulation of two different types of CAMs (Fasciclin II and chaoptin) in the same cell type under conditions of blocked neurotransmitter release poses the question of the specificity of the mechanism. In the absence of functional n-syb, increased numbers of docked synaptic vesicles accumulate presynaptically. Assuming that this would result in a significant sequestration of membrane material and that the synaptic vesicle cycle is continuously replenished from cell surfaces carrying adhesion molecules, deactivation of n-syb could result in decreased intake of CAMs and thus increased CAM immunoreactivity. However, current understanding of the recycling mechanism in the synaptic vesicle cycle and different localization of CAM isoforms does not support this hypothesis. Alternatively, specifically CAMs on active terminals and fibers could be downregulated to serve as markers for the competence of the synapses for sprouting (Hiesinger, 1999).

    In wild-type third instar larvae, Fasciclin II is found on R7 and R8 retinal axons. During parts of pupation, Fasciclin II is detectable at low levels on photoreceptor cell bodies. It is possible that Fasciclin II is never completely downregulated from R7 and R8 terminals but is mostly below threshold for visualization with confocal microscopy. Upregulation of Fasciclin II levels in photoreceptors lacking functional n-syb after P + 75% may thus be attributable to an accumulation of the protein, when its downregulation would normally occur via an n-syb-dependent mechanism as part of a continuous protein turnover. The finding that IrreC-rst immunoreactivity remains unaltered in photoreceptors without functional n-syb but is increased in proximal neuropils after widespread TeTxLC expression can be interpreted in two different ways: either IrreC-rst protein is not present on photoreceptor terminals at the addressed time of pupation, or the n-syb-dependent CAM downregulation mechanism has a different molecular specificity in photoreceptors than in other optic lobe cells. During axonal pathfinding IrreC-rst is expressed on photoreceptors. In pupal stages IrreC-rst is localized to rhabdomeres but not to axons and cell bodies of photoreceptors during the second half of pupation. Because rhabdomeres are unique to this cell type and seem to be a preferred localization for IrreC-rst in photoreceptors, a cell-specific distribution that excludes terminals appears more likely than a specific CAM regulation mechanism for photoreceptors. Taken together, the results presented here clearly show a requirement of n-syb for optic lobe development. Either n-syb has a previously unknown activity-independent function, or synaptic transmission is involved in optic lobe development, or both (Hiesinger, 1999 and references).

    Ig superfamily ligand and receptor pairs expressed in synaptic partners in Drosophila

    Information processing relies on precise patterns of synapses between neurons. The cellular recognition mechanisms regulating this specificity are poorly understood. In the medulla of the Drosophila visual system, different neurons form synaptic connections in different layers. This study identifies candidate cell recognition molecules underlying this specificity. Using RNA sequencing (RNA-seq), it was shown that neurons with different synaptic specificities express unique combinations of mRNAs encoding hundreds of cell surface and secreted proteins. Using RNA-seq and protein tagging, it was demonstrated that 21 paralogs of the Dpr family, a subclass of immunoglobulin (Ig)-domain containing proteins, are expressed in unique combinations in homologous neurons with different layer-specific synaptic connections. Dpr interacting proteins (DIPs), comprising nine paralogs of another subclass of Ig-containing proteins, are expressed in a complementary layer-specific fashion in a subset of synaptic partners. The study proposes that pairs of Dpr/DIP paralogs contribute to layer-specific patterns of synaptic connectivity (Tan, 2015).

    Control of synaptic connectivity by a network of Drosophila IgSF cell surface proteins

    This study defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. These findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity (Carrillo, 2015).

    Dpr-DIP matching expression in Drosophila synaptic pair

    Neurons form precise patterns of connections. The cellular recognition mechanisms regulating the selection of synaptic partners are poorly understood. As final mediators of cell-cell interactions, cell surface and secreted molecules (CSMs) are expected to play important roles in this process. To gain insight into how neurons discriminate synaptic partners, the transcriptomes were profiled of 7 closely related neurons forming distinct synaptic connections in discrete layers in the medulla neuropil of the fly visual system. The sequencing data revealed that each one of these neurons expresses a unique combination of hundreds of CSMs at the onset of synapse formation. 21 Ig domain paralogs of the defective proboscis extension response (see Drosophila Piecing Together the Extracellular Puzzle) family were shown to be expressed in a unique cell-type-specific fashion, consistent with the distinct connectivity pattern of each neuron profiled. Expression analysis of their cognate binding partners, the 9 members of the Dpr interacting protein (DIP) family, revealed complementary layer-specific expression in the medulla, suggestive of interactions between neurons expressing Dpr and those expressing DIP in the same layer. Through coexpression analysis and correlation to connectome data, neurons expressing DIP were identified as a subset of the synaptic partners of the neurons expressing Dpr. It is proposed that Dpr-DIP interactions regulate patterns of connectivity between the neurons expressing them (Marta, 2016).

    An axon scaffold induced by retinal axons directs glia to destinations in the Drosophila optic lobe

    In the developing Drosophila visual system, glia migrate into stereotyped positions within the photoreceptor axon target fields and provide positional information for photoreceptor axon guidance. Conversely, glial migration depends on photoreceptor axons, as glia precursors stall in their progenitor zones when retinal innervation is eliminated. These results support the view that this requirement for retinal innervation reflects a role of photoreceptor axons in the establishment of an axonal scaffold that guides glial cell migration. Optic lobe cortical axons extend from dorsal and ventral positions toward incoming photoreceptor axons and establish at least four separate pathways that direct glia to proper destinations in the optic lobe neuropiles. Photoreceptor axons induce the outgrowth of these scaffold axons. Most glia do not migrate when the scaffold axons are missing. Moreover, glia follow the aberrant pathways of scaffold axons that project aberrantly, as occurs in the mutant dachsous. The local absence of glia is accompanied by extensive apoptosis of optic lobe cortical neurons. These observations reveal a mechanism for coordinating photoreceptor axon arrival in the brain with the distribution of glia to multiple target destinations, where they are required for axon guidance and neuronal survival (Dearborn, 2004).

    Attempts were made to determine the location of progenitors that give rise to the distinct types of migratory glia and the neurons that form their migratory pathways. The Wingless expressing cells of the dorsal and ventral domains are located in areas of complex gene expression controlled by Wingless (Wg) signaling activity. Adjacent to the Wg domains are non-overlapping cell populations that express the TGF-ß family member Decapentaplegic (Dpp). Both the Wg- and Dpp-positive cell populations express the transcription factor Optomotor Blind (Omb). Dachsous (Ds), a Cadherin family member, is expressed in a graded fashion with respect to the Wg domains. These three genes, though expressed in different patterns, are under the control of Wg activity (Dearborn, 2004 and references therein).

    It is concluded that Drosophila optic lobe glia use axon fascicles as migratory guides and that the extension of these axon fascicles is induced by the ingrowth of photoreceptor axons from the developing retina. The migratory scaffold axons emerge from optic lobe regions that are in close proximity to sites where glial cells originate; both arise in the dorsal and ventral domains where cells express the morphogen Wingless. When the scaffold axons were eliminated by the autonomous expression of an activated Ras transgene, glia failed to migrate and stalled at the borders of their progenitor sites. Extensive cortical cell apoptosis ensued. When the scaffold axons projected aberrantly (in animals mutant for the cadherin Dachsous), glia followed the aberrant routes to incorrect destinations. The longstanding observation that glial migration does not occur in eyeless mutant Drosophila might thus be explained by an indirect mechanism in which innervation controls the establishment of an axon scaffold necessary to direct glial migration (Dearborn, 2004).

    DPP signaling controls development of the lamina glia required for retinal axon targeting in the visual system of Drosophila: A requirement for medea for expression of gcm

    The Drosophila visual system consists of the compound eyes and the optic ganglia in the brain. Among the eight photoreceptor (R) neurons, axons from the R1-R6 neurons stop between two layers of glial cells in the lamina, the most superficial ganglion in the optic lobe. Although it has been suggested that the lamina glia serve as intermediate targets of R axons, little is known about the mechanisms by which these cells develop. DPP signaling has been shown to play a key role in this process. dpp is expressed at the margin of the lamina target region, where glial precursors reside. The generation of clones mutant for Medea, the DPP signal transducer, or inhibition of DPP signaling in this region results in defects in R neuron projection patterns and in the lamina morphology; these defects are caused by defects in the differentiation of the lamina glial cells. glial cells missing is expressed shortly after glia precursors start to differentiate and migrate. Its expression depends on DPP; gcm is reduced or absent in dpp mutants or Medea clones, and ectopic activation of DPP signaling induces ectopic expression of gcm and Repo. In addition, R axon projections and lamina glia development are impaired by the expression of a dominant-negative form of gcm, suggesting that gcm indeed controls the differentiation of lamina glial cells. These results suggest that DPP signaling mediates the maturation of the lamina glia required for the correct R axon projection pattern by controlling the expression of gcm (Yoshida, 2005).

    dpp is expressed in the dorsal and ventral margins of the posterior region of the optic lobe, adjacent to the cells expressing wg, which induces dpp expression. Glial cells in the lamina target region arise from these regions and migrate into the lamina target region as they contact R axons. Axons from R1-R6 neurons stop between two rows of glial cell layers, the epithelial and marginal layers, and form the lamina plexus. The third row of glial cells, the medulla glia, is located just beneath the marginal glia. The homeodomain protein Repo is expressed in these glial cells (Yoshida, 2005).

    The expression pattern of dpp-lacZ, an enhancer-trap allele of dpp, was compared with the expression pattern of Repo. At a stage prior to glia differentiation and migration, expression of the dpp reporter is detected in the dorsal and ventral margins of the lamina target region. dpp continues to be expressed at the margins of the lamina target region throughout the third larval instar (Yoshida, 2005).

    wg at the posterior-most domain induces the expression of dpp and omb. Some wg-expressing cells extend projections towards the lamina target region. These cells extend scaffold axons along which the lamina glia migrate. Thus, it was possible that the wg signal is involved in the migration and/or differentiation of lamina glia. However, partial elimination of Wg activity with a wgts allele does not cause a specific defect in glia migration. Therefore, wg may play a role in organizing domains in the visual cortex by activating/repressing various genes, rather than contributing to the generation of specific cell types (Yoshida, 2005).

    Medea is required for lamina glia development. Medea encodes a co-SMAD and mediates a range of DPP/BMP/TGFß signaling events. In addition to dpp, four related genes -- glass bottom boat (gbb), screw, activin and activin2 -- have been identified in Drosophila. GBB signals through TKV/Saxophone (SAX) and Wishful Thinking (WIT) type I and type II receptors, respectively. Activin uses Baboon as a type I receptor, and Punt and WIT as type II receptors. Brains mutant for gbb and wit were examined, but no defects in lamina glia development were observed. It is concluded that it is highly likely that dpp is the ligand responsible for lamina glia development. However, the possibility that one or more of the DPP-related ligands acts redundantly in this process cannot be excluded (Yoshida, 2005).

    In the embryo, gcm initiates the specification of glial cells from neural cells of various lineages. gcm expression is strictly controlled to ensure the correct separation of glial versus neuronal cell fate. Analysis of the cis-regulatory elements of gcm suggests that gcm expression depends on multiple regulatory elements to allow the control of lineage-specific transcription and autoregulation. The analysis carried out in this study suggests that a different situation exists in the optic lobe; gcm is expressed in the glia and the lamina neuronal cells, and is required for the differentiation of these cell types. In addition, differentiation is controlled differently in the lamina and in the glia. In the lamina, gcm expression seems to be controlled by hh, and in the glia, by dpp. These results suggest that gcm is controlled and functioning in a different manner in the optic lobe. Uncovering the mechanisms of the control and function of gcm would probably prove an intriguing focus for future research (Yoshida, 2005).

    DPP and its vertebrate homolog BMP play crucial roles in many aspects of development by controlling patterning, cell growth and differentiation. This analysis reveals a role for DPP signaling in lamina glia differentiation in the Drosophila visual system. DPP has also been reported to function in several aspects of visual center development; for instance, DPP signaling has been shown to be involved in the proliferation and migration of the subretinal glia in eye disc development, which plays an important role in the R axon navigation. In addition, defects have been reported in the medulla neuropile in dpp mutant animals, suggesting a role for dpp in neuronal fate specification. Furthermore, tkv is expressed in lamina precursor cells just ahead of the lamina furrow, where these cells meet R axons and start to differentiate. Although this possibility is one of the things that prompted an examination of the role of DPP signaling in lamina development, no defects were uncovered when Mad or Medea clones were generated in the OPC or the lamina. Moreover, dpp appears to be expressed in the inner proliferation center (IPC), which will form the lobula, in addition to its expression in the dorsal and ventral marginal domains. Thus, dpp may be required for some aspects of lobula development. Unfortunately, this cannot be easily addressed at this moment because of a lack of appropriate markers. Further study of the requirements for dpp in the lamina, the medulla, the lobula and other cell types could lead to a more comprehensive understanding of how DPP signaling controls differentiation and other events during development of the visual system (Yoshida, 2005).

    Robo-3-mediated repulsive interactions guide R8 axons during Drosophila visual system development

    The formation of neuronal connections requires the precise guidance of developing axons toward their targets. In the Drosophila visual system, photoreceptor neurons (R cells) project from the eye into the brain. These cells are grouped into some 750 clusters comprised of eight photoreceptors or R cells each. R cells fall into three classes: R1 to R6, R7, and R8. Posterior R8 cells are the first to project axons into the brain. Using a microarray-based molecular screen as a starting point, this study identified the early and transient expression of Robo3 in R8 growth cones. Loss of Robo3 demonstrated a specific axon guidance choice point at an early stage of optic lobe innervation. In the absence of Robo3, posterior R8 growth cones inappropriately extend across Slit-expressing glial cells joining axon fascicles of the C+T lobula neurons, instead of remaining alongside the glial process as they extend into the lamina. This early repulsive function of Robo3 plays a crucial role in segregating axons and thereby contributes to the orderly assembly of columnar units comprising the fly visual system (Pappu, 2011).

    The microarray data, coupled with antibody staining and the identification of a robo3-Gal4 enhancer trap, identified Robo3 as an R8-specific guidance receptor. Robo3 expression is transient in the R8 growth cone and prolonging Robo3 expression in R8 axons results in defects in R8 targeting. The microarray analysis suggests that restricted Robo3 expression during early stages of R8 differentiation occurs downstream of the transcription factors Sens and Run. However, the expression of both Sens and Run persists beyond the expression of Robo3. For example, during mid- to late-pupal development, Sens regulates both targeting of R8 axons to their final target layer in the medulla and the expression of R8-specific opsins. Therefore, other mechanisms must exist to control the expression of Robo3 in R8 (Pappu, 2011).

    The importance of the tightly regulated expression of Robo receptors is emerging as a central theme in axon guidance. Indeed, previous studies have revealed a set of discrete posttranslational mechanisms controlling Robo functions both in vertebrates and invertebrates. For example, commissural axons in the fly embryo express Robo1 transcripts before crossing the midline, but Robo1 protein in these axons is sequestered into endosomes by the action of Commissureless protein, thereby preventing precocious repulsion from the midline and, thus, allowing these axons to cross. Subsequent up-regulation of Robo1 prevents them from recrossing. In vertebrates, alternative splicing of a divergent Robo receptor Rig1/Robo3, perhaps coupled with translational regulation, governs the switch from midline attraction to repulsion (Pappu, 2011).

    The regulated expression of Robo3 in the R8 photoreceptors is similar to its expression in the chordotonal neurons in the embryonic peripheral nervous system. Sens is activated downstream of Ato in both chordotonal and in R8 neurons, suggesting that a conserved transcriptional program regulates Robo3 expression in these neurons. In a broader sense, these findings raise the possibility that conserved regulatory cassettes exist, which link specific transcriptional hierarchies controlling neuronal differentiation with specific constellations of downstream guidance receptors controlling wiring specificity (Pappu, 2011).

    Posterior-most R8 neurons face at least three different guidance choices as they extend from the eye disk into the developing optic lobe. These early choices have a profound effect on later aspects of visual system assembly. First, R8 axons must navigate to the posterior of the eye disk and enter the optic stalk. This process is facilitated, in part, by retinal basal glial cells at the posterior edge of the eye disk. If glial cells are displaced anteriorly, R-cell axon fascicles project away from the optic stalk rather than toward it. Although it seems likely that this directional choice relies upon R8, it is not known whether posterior growth requires R8-specific functions or whether all retinal neurons are endowed with this function (Pappu, 2011).

    Second, R8 axons from each ommatidium must possess molecular mechanisms to retain their individuality. As the R8 axons extend down the optic stalk they form a tight fascicle. Fasciculation is transient, however, because R8s defasciculate as they exit the optic stalk. R8 defasciculation relies on two cell surface receptors, Flamingo (Fmi) and Golden Goal (Gogo) that are expressed in the R8 growth cones as they exit the optic stalk. Fmi and Gogo mediate repulsive interactions between R8 axons, and thus play a key role ensuring that columns remain as separate modules. These repulsive interactions between R8 axons of adjacent columns also explain why axons from later-born (anterior) R8 neurons are not affected in robo33 mutant optic lobes; only the posterior R8 axons traverse through the optic lobe with access to the glial cell boundaries that separate them from the C+T lobular neuron axons (Pappu, 2011).

    Third, in this article it was demonstrated that posterior R8 axons rely upon Robo3 to prevent inappropriate fasciculation with C+T lobula neurons. This process requires early, transient, and specific expression of Robo3 in R8 growth cones and is likely to require the reciprocal expression of Slit in the glial cells that these posterior R8 axons encounter when they enter the optic lobe. Thus, the posterior R8 axons are unique because they navigate a choice point that is not encountered by later arriving, more anterior R8 axons. The robo33 mutant phenotype described in this study is reminiscent of the loss of another Ig receptor encoded by the irregular chiasm/roughest (irre-C) locus. Whether IrreC acts in the same molecular pathway as Robo3 and, indeed, whether it acts in photoreceptor growth cones or lamina neurons is not known (Pappu, 2011).

    In summary, Fmi, Gogo, and Robo3 play crucial roles in R8s in regulating fascicle organization, which provides the structural basis for columnar organization of the visual system. Although Fmi and Gogo mediate interactions between axons of the same class of cells (R8s), Robo3 prevents axons from one class of neurons (R8s) from inappropriately associating with a different class of axons (C+T lobular neurons) projecting into the same neuropil along a different pathway. Given the cellular complexity of columns (e.g., medulla columns comprise more than 50 axons from many different neuronal subclasses) and the stereotyped organization of axons and synaptic connections within them, it is speculated that many additional cell-surface proteins must act in a coordinated fashion in space and time to promote the orderly assembly of columnar units (Pappu, 2011).

    Does Robo3 function in the R8 rely on Slit? This is indeed the most parsimonious model for Robo3 function in R8. Slit expression is detected around glial cells separating the posterior R8 growth cones from C+T lobula neurons. Although R8 projection defects seen in slit mutants are similar to those seen in robo3 mutants, they are more severe. In contrast to robo3, slit mutant optic lobes are extremely disorganized, arguing that Slit has a broader role in neuropil organization. Indeed, mutants deficient in all three Robo proteins exhibit cell migration defects, which largely phenocopy the loss of Slit. It has been proposed that Slit provides a repellent function in the optic lobe preventing cell migration between cell populations in the lamina and lobula. However, the residual Robo3 function in the robo31 hypomorphic allele available for study at that time masked the robo3 phenotypes uncovered in this study. Thus, although slit mutants uncover a broader role for Slit-Robo signaling in many aspects of optic lobe development and patterning, the unique robo3 mutants described in this study uncover a specific role for the Robo3 receptor in R8 axon guidance (Pappu, 2011).

    The repulsive role of Robo3 in R8 neurons in response to locally secreted Slit is proposed in this study to be analogous to the role of Robo1 in the guidance of ipsilateral longitudinal pioneer axons in the ventral nerve cord. Robo1 is expressed on the growth cones of ipsilateral pioneer axons and prevents these axons from crossing the midline in response to Slit secreted from midline glia. In contrast, Robo3 is not expressed in the growth cone of commissural and longitudinal pioneer axons and is dispensable for the midline crossing during the development of the embryonic ventral nerve cord. Thus, the function of Robo3 in posterior R8s is analogous to the function of Robo1 in embryonic ipsilateral pioneers. However, analyses of knock-in mutants indicate that Robo1 and Robo3 must be sensitive to cell-type-specific regulatory functions. Robo1 has a unique role in midline repulsion of ipsilateral pioneers as it cannot be functionally replaced by Robo2 or Robo3. In contrast, as reported in this study, either Robo1 or Robo2 can functionally replace Robo3 in the R8s. Thus, repulsive signaling downstream of ipsilateral pioneers in the embryo is dependent on unique structural features of the Robo1 protein, but repulsion of posterior R8 axons does not depend on unique structural features of Robo3. Instead it is the unique and context-specific expression of Robo3 that allows it to determine R8 axon guidance and in a broader context function in the orderly assembly of a subset of columnar elements in the visual circuit (Pappu, 2011).

    The highly ordered assembly of retinal axons and their synaptic partners is regulated by Hedgehog/Single-minded in the Drosophila visual system

    During development of the Drosophila visual center, photoreceptor cells extend their axons (R axons) to the lamina ganglion layer, and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal Hedgehog (Hh). This inductive mechanism helps to establish an orderly arrangement of connections between the R axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding R axons. The bHLH-PAS transcription factor Single-minded (Sim) is induced by Hh in the lamina neurons and is required for the association of lamina neurons with R axons. In sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with R axons. As a result, lamina neurons are set aside from R axons. The data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks (Umetsu, 2006).

    Most axons in the brain establish topographic maps in which the arrangement of synaptic connections maintains the relationships between neighboring cell bodies. A notable model of topographic map formation is the visual system, where the relay of visual information from the retina to the visual center must be arranged in a spatially ordered manner through the topographic connections of retinal axons with their midbrain target, which is the optic tectum (OT) in lower vertebrates and the superior colliculus (SC) in mammals. This topographic map is termed a retinotopic map. Many studies have shown that Ephrin protein family members, acting through their Eph receptors, play pivotal roles in the establishment of the retinotopic map. In the mouse and the chick, for example, the retinal ganglion cells (RGCs) extend their axons to the OT/SC, and the low-to-high anteroposterior gradient of ephrin A in the target limits the posterior extension of growth cones at various positions, dependent on the EphA level of each RGC (Umetsu, 2006).

    The Drosophila visual system has also provided insight into topographic mapping. In Drosophila, the projections of photoreceptor neurons (R cells) themselves induce development of the corresponding postsynaptic neurons. The Drosophila visual system consists of the compound eyes and the three optic ganglia: the lamina, the medulla and the lobula complex. Each of the approximately 750 ommatidial units comprising the compound eye contain six outer photoreceptors (R1-R6) and two inner photoreceptors (R7, R8). R1-R6 cells send their axons to the first optic ganglion, the lamina, whereas R7 and R8 cells send axons through the lamina to the second ganglion, the medulla. R1-R6 cells in each ommatidium make stereotypic connections with particular lamina neurons. Synaptic units in the lamina are referred to as lamina cartridges. During the initial step of the assembly of a lamina cartridge, an arriving photoreceptor axon (R axon) fascicle forms a pre-cartridge ensemble, the 'lamina column', with a set of five lamina neurons. Formation of the ensemble results in a one-to-one correspondence of ommatidia to column units, and is fundamental to the subsequent establishment of intricate synaptic connections. Development of the lamina is tightly regulated by the projection of R axons. Failure in eye formation results in concurrent loss of the lamina, as in a normal brain, lamina neurogenesis is directly coupled to the arrival of R axons. Both R cell differentiation and ommatidial assembly progress in a posterior-to-anterior direction across the eye disc. Differentiated R cells begin to send their axons to the brain in the same sequential order, reflecting their position in the retina along the anteroposterior and the dorsoventral axes. Wnt signaling plays a role in regulating projections along the dorsoventral axis (Umetsu, 2006).

    As the axons from each new row of ommatidial R cell clusters arrive in the lamina, a corresponding group of lamina precursor cells (LPCs) undergo a final division and initiate differentiation into lamina neurons. In the first step of their neurogenesis, direct contact with R axons triggers the transition of G1-phase LPCs into S phase. Both the G1-S transition and the initial specification into a lamina neuron are induced by Hedgehog (Hh), which is delivered by arriving R axons, and the next step in lamina differentiation is induced by the Spitz signaling molecule, which is also delivered by R axons. Hh expressed in R cells functions as a signal for photoreceptor development as well: secreted Hh induces anterior precursor cells to enter the pathway of R cell specification (Umetsu, 2006).

    Thus, the retinotopic map along the anteroposterior axis of the lamina seems to be established autonomously and in a posterior-to-anterior order, as newly specified R cells send their axons to the lamina layer and make lamina columns. Each ommatidial unit sends a set of R axons as a single bundle to the lamina along the pre-existing fascicle that has been just projected. Then, the axon bundles are enveloped by the processes of newly induced lamina neurons. This step is key to forming the one-to-one associations between R axon bundles and their corresponding lamina neurons. This study shows that the activity of Single-minded (Sim) is required for developing lamina neurons to establish an association with the corresponding R axons and, hence, to form the lamina column. sim encodes a basic-helix-loop-helix-PAS (bHLH-PAS) transcription factor and is induced by Hh provided by the R axons. In sim mutant brains, the developing lamina neurons fail to associate with R axon bundles, resulting in a failure to establish connections between R axons and lamina neurons. It is inferred that sim programs developing lamina neurons to express a molecule(s) that is required for the association with R axons (Umetsu, 2006).

    Retinotopic mapping in Drosophila provides unique insights into neuronal network formation not only because of its tight coupling to the control of development, but also because of the interactions between axons and neuronal cell bodies. The interactions observed stand in sharp contrast to what has been found for other models of axon guidance, where the growth cones of axons respond to a variety of attractive or repulsive guidance cues to navigate to their synaptic target cells. The cues include the netrins, Slits, semaphorins and ephrins, and the restricted expression pattern of these cues and the reactivity of growth cones play pivotal roles in the establishment of the proper synaptic connections. In this context, postsynaptic cells are seen as mere providers of guidance/adhesion molecules, passively awaiting the arrival of a growth cone. In other words, it is conceivable that presynaptic growth cones seek their targets dynamically, whereas postsynaptic cells remain static. Unlike the roles of presynaptic axons, the cellular behaviors of postsynaptic cells in the establishment of synaptic targeting are poorly understood. This study proposes another possible model for the establishment of topographic neuronal connections in which postsynaptic cells dynamically interact with presynaptic axons (Umetsu, 2006).

    Thus, Sim, a target of Hh, is required for at least the first step of lamina column formation; namely, the incorporation of developing lamina neurons into the area where R axons project and lamina columns mature, an area referred to as the assembling domain. This model for Sim is based on four observations. First, sim2/simry75 brains have a reduced number of lamina neurons in the assembling domain, leaving an abnormally large number of premature lamina neurons behind in the pre-assembling domain. Second, in clonal analysis, sim2 clones fail to be recovered in the assembling domain (similar to smo1 clones). Third, lamina neuron-specific inhibition of Sim function causes R axon bundles to be tightly packed and lamina neurons to be excluded from R axon bundles. And fourth, overexpression of sim in lamina neurons causes precocious incorporation of lamina neurons into the assembling domain (Umetsu, 2006).

    In case of overexpression, neither expansion of the assembling domain nor increase in the number of lamina neurons relative to the number of R axon bundles was observed, even though lamina neurons prematurely incorporated into the assembling domain. This is probably because a reduced number of lamina neurons were generated. In fact, loss of E2F expression was observed at the lamina furrow in NP6099-GAL4 UAS-sim brains. The onset of incorporating lamina neurons into the assembling domain might be linked to an inhibition of cell proliferation. However, this is thought to be unlikely for two reasons: (1) lamina neurons did not show any extra E2F signal in the sim mutant brain in spite of an increase in unincorporated lamina neurons; and (2) lamina neurons ectopically expressing a cell cycle-braking factor, the Drosophila p21/p27 homolog dacapo (dap) cause the precocious incorporation of lamina neurons. Thus, a direct link between cell cycle regulation and the incorporation of lamina neurons is less plausible (Umetsu, 2006).

    An alternative model, the 'time lag' model, is proposed. There appears to be a lag between the onset of sim expression and the onset of incorporation of lamina neurons. Differentiating lamina neurons are held temporarily in the pre-assembling domain and then the proper amount of lamina neurons are coordinately integrated into columns as more R axons are projected. Thus, it is speculated that a certain degree of accumulation of the Sim/dARNT heterodimer in nuclei is needed to exert cellular function. Consistent with this idea, graded accumulation of Sim is observed, with lower Sim levels in anterior (younger) lamina neuron nuclei and higher levels in posterior (older) lamina neuron nuclei. Overexpression of Sim in lamina neurons would thus cause higher levels of accumulation of the protein in young lamina neurons and facilitate their incorporation into the assembling domain. Interestingly, overexpression of the wild-type dARNT did not have any detectable effects, suggesting that Sim accumulation is a limiting factor for cell incorporation (Umetsu, 2006).

    The mechanism of neuronal maturation and that of assembly of lamina neurons are independent, although both are under the control of Hh signaling. Disruption of sim did not affect the differentiation and proliferation of lamina neurons. Correspondingly, neither the incorporation of lamina neurons into the lamina column nor the expression of sim were affected by dac mutation. The cellular function required for assembling the column or the function of Sim at the cellular level is still not known. Electron microscopic observations by have revealed an intriguing behavior of lamina neurons at the early pupal stage; large processes extending from lamina neurons engulf R1 and R6 axons of newly incoming R axon bundles. This may be the key step in lamina column formation and interaction between the R axons and lamina neurons. Sim may regulate genes required for process formation, interaction with R axons and/or events that follow shortly after, since lamina neurons seem to fail to make interactions with R axons from the beginning in the sim mutant background. Sim is expressed in the midline cells of the CNS throughout neurogenesis in the Drosophila embryo and is required for the proper differentiation of the midline cells into mature neurons and glial cells. Midline precursor cells undergo synchronized cell division and then transform into the bottle-shaped cells, in which the nuclei migrate internally and leave a cytoplasmic projection joined to the surface of the embryo. The sim mutant midline cells fail to delaminate from the epidermal cell layer. Cells do not make the normal bottle-like shape and, instead, they appear rounded. In addition, overexpression of sim can induce other cell types to exhibit midline morphology. sim may thus regulate the transcription of a set of genes required for morphological changes, which in turn are required for interaction between cells, both in the lamina and during embryonic CNS development (Umetsu, 2006).

    Although sim expression is regulated by Hh signaling, this does not answer the question of whether sim function is sufficient to confer on cells the ability to be incorporated into the assembling domain. Whether smo mutant clones can be recovered in the assembling domain was examined by forcing sim expression in smo clones using the MARCM technique. However, smo mutant clones expressing sim were not recovered in the assembling domain. This suggests that additional factors are involved in lamina neuron assembly. Hh may also contribute to specification of the difference in affinity between lamina neurons and R axons and/or between anterior and posterior lamina neurons. In Drosophila wing discs, the Hh signal differentiates the affinity of anterior compartment cells from that of the posterior compartment cells, thereby maintaining the compartment border (Umetsu, 2006).

    An active role is proposed for postsynaptic cells in making a topographic map of the Drosophila visual system. Targeted expression of the dominant-negative form of the Sim partner in the lamina neurons clearly showed a role for postsynaptic cells in assembling lamina columns. This presumably affects an early step of assembly. It is not known if Sim function is also required for later steps in more mature lamina neurons. The forced expression of the dominant-negative Sim partner in the posterior lamina neurons had no effect, although it may simply be that the level of expression of the dominant-negative form of dARNT was not sufficient to have an observable effect on Sim function. In the lamina column, the R axon bundle associates with a precisely arranged row of five lamina neurons. No mechanisms for the development and formation of this stereotypic structure have been revealed. Another signal might be provided from the R axons with lamina neurons, and/or intrinsic structures of the R axons might play a role in this architecture. An intriguing property of postsynaptic muscle cells for axonal targeting has been observed: the muscle cells bear numerous postsynaptic filopodia ('myopodia') during motoneuron targeting. They showed that postsynaptic cells actively contribute to synaptic matchmaking by direct, long-distance communication. Together with what has been learned about myopodia in neuromuscular synapse formation, the curent findings reveal an active role for postsynaptic cells for the establishment of precise neural networking (Umetsu, 2006).

    Sim belongs to the family of bHLH-PAS transcription factors, whose members function in many developmental and physiological processes, including neurogenesis, tissue development, toxin metabolism, circadian rhythms, response to hypoxia, and hormone receptor function. bHLH-PAS proteins usually function as dimeric DNA-binding protein complexes. The most common functional unit is a heterodimer. These heterodimers consist of one partner that is broadly expressed, and another whose expression is regulated spatially, temporally or by the presence of inducers. Sim and the bHLH-PAS protein dARNT heterodimerize to bind to their responsive element, the CME (CNS midline enhancer element), to activate target gene transcription. In this complex, dARNT is the general dimerization partner and Sim is the tissue-specific partner (Umetsu, 2006).

    The Drosophila Sim and mammalian Sim1 and Sim2 proteins are highly conserved in their amino-terminal halves, which contain a bHLH and a PAS domain. Murine Sim1 and Sim2 are also expressed in both proliferative and postmitotic zones of the central nervous system at different stages of neural development. These zones of expression include the longitudinal basal plate of the diencephalon (Sim1 and Sim2), the mesencephalon (Sim1), the zona limitans intrathalamica (Sim1 and Sim2) and the portion of the spinal cord that flanks the floor plate (Sim1). Sim2 maps to the region responsible for Down Syndrome (DS) on Chromosome 21. Interestingly, Sim2 is also expressed in non-neuronal tissues, including branchial arches and the developing limb, which are primordia of tissues and organs where DS abnormalities are frequently observed (Umetsu, 2006).

    Given the important roles of sim in Drosophila development and the expression of Sim2 in cell types that are affected in DS individuals, it was proposed that Sim2 may play a causative role in DS. However, because of a lack of direct evidence and the existence of other candidate genes, this remains speculative. Cells expressing sim during Drosophila development and Sim2-positive cells affected in DS seem to be able to migrate. The conserved role of Sim may enable cells to migrate and/or interact with surrounding cells in the various tissues, including the central nervous system. It will thus be intriguing to search for extra cellular targets of Sim regulation with the hope of elucidating mechanisms that underlie the behavior of Sim-expressing cells (Umetsu, 2006).

    bantam is required for optic lobe development and glial cell proliferation

    microRNAs (miRNAs) are small, conserved, non-coding RNAs that contribute to the control of many different cellular processes, including cell fate specification and growth control. Drosophila bantam, a conserved miRNA, is involved in several functions, such as stimulating proliferation and inhibiting apoptosis in the wing disc. This study reports the detailed expression pattern of bantam in the developing optic lobe, and demonstrates a new, essential role in promoting proliferation of mitotic cells in the optic lobe, including stem cells and differentiated glial cells. Changes in bantam levels autonomously affect glial cell number and distribution, and non-autonomously affect photoreceptor neuron axon projection patterns. Furthermore, bantam promotes the proliferation of mitotically active glial cells and affects their distribution, largely through down regulation of the T-box transcription factor, optomotor-blind (omb, Flybase, bifid). Expression of omb can rescue the bantam phenotype, and restore the normal glial cell number and proper glial cell positioning in most Drosophila brains. These results suggest that bantam is critical for maintaining the stem cell pools in the outer proliferation center and glial precursor cell regions of the optic lobe, and that its expression in glial cells is crucial for their proliferation and distribution (Li, 2012).

    These results provide evidence that bantam is important for stem cell maintenance in the optic lobe. First, bantam shows high expression in the OPC and GPC areas in the optic lobe, where stem cells are located. Second, bantam is critical for cell proliferation in the OPC and GPC areas. banΔ1/banΔ1 null mutants have smaller brains with a dramatic decrease in the proliferation in the OPC and GPC. On the other hand, bantam over expression causes brain size to increase, along with increased proliferation in the OPC and GPC. During development, it is very important to maintain a constant stem cell population while differentiated cells are produced. In Drosophila, the central nervous system is derived from neural stem cells called neuroblasts. The optic lobe neuroepithelia are important as they maintain the pool of optic lobe neuroblasts with symmetric division. Misregulation of the self-renewing capacity of the neuroblasts is related to brain tumors; however, the mechanism underlying the precise regulation of proliferation and differentiation of the neuroepithelia and neuroblasts is not well known. miRNAs are known to be crucial for stem cell maintenance in other tissues. When the miRNA processing machinery is affected by loss of Dicer-1 (Dcr-1), which is essential for generating mature miRNAs from their corresponding precursors, stem cells cannot be maintained and are lost rapidly in the Drosophila ovary. These dcr-1 mutant stem cells are delayed in G1 to S transition. bantam was reported to be important for germline stem cell (GSC) maintenance in adult Drosophila, but the detailed underlying mechanism remains to be determined. It will be interesting to learn how bantam affects the cell cycle machinery of stem cells in the OPC and GPC regions. bantam has been known to promote cell proliferation in other tissues as well. The ability of bantam to promote cell proliferation in various tissues suggests that bantam might target molecules that directly, but negatively, affect cell-cycle machinery. Recently, a report showed that bantam targets Mei-P26, which has ubiquitin ligase activity, causing the oncogene c-Myc to degrade in the wing imaginal disc. c-Myc can respond to different growth factors to promote cell proliferation through positive regulation of the transcription factor E2F, which is a common G1-S master regulator, and is involved in regulating the expression of a number of genes required for G1-S progress. Future experiments studying whether bantam employs this same mechanism in regulating the cell cycle of stem cells in the optic lobe will be informative (Li, 2012).

    It was also found that bantam is required for glial cell growth in the optic lobe. Glial cell numbers in the optic lobe were greatly increased, in a cell-autonomous manner, by an over expression of bantam. Conversely, a loss of bantam led to a dramatic decrease in glial cells in the optic lobe. During normal development, development of glial cells in the optic lobe is controlled by both extrinsic and intrinsic mechanisms. Glial cell numbers increase rapidly during the third instar larval stage due to the mitosis of differentiated glia, and, more significantly, the proliferation of precursor cells. bantam was found to increase proliferation of both glia precursor cells. This work also provides evidence that bantam's function on glial cell numbers is dependent on its negative regulation of omb in a small subgroup of differentiated glial cells, as evidenced by the ability of omb to rescue bantam's effect on glial cell numbers and distribution. Omb is a T-box transcription factor, highly conserved in all metazoans. The T-box family appears to play critical roles in development, including specification of the mesoderm and morphogenesis in the heart and limbs. In the Drosophila optic lobe, omb is expressed in a subgroup of glial cells that are required for their proper positioning and morphology. However, the downstream targets of omb responsible for these functions are not clear. Future experiments to determine if the same mechanism is employed in the brain need to be performed (Li, 2012).

    It is thought that bantam does not affect glial cell differentiation because the loss of bantam in null mutants still maintains Repo-positive differentiated glial cells. Transcriptional regulators, such as Glial cells missing (Gcm) and its closely related homolog Gcm2, have been well-studied for their roles in glial cell differentiation in the embryonic and postembryonic nervous system of Drosophila. Gcm/Gcm2 are considered to be at the top of the hierarchy for initiating the differentiation of all glial cells. Their downstream targets for maintaining terminal glial cell differentiation include repo, pointed and tramtrack. With antibody staining for Repo, no obvious defects were seen in larvae caused by bantam, further supporting the idea that bantam increases glial cell numbers independent of Gcm-Repo (Li, 2012).

    Besides promoting glial cell numbers, bantam also affects the mobility of glial cells, as an increase was observed in glial cells located under the lamina furrow, the migrating path for glial cells. When bantam was over-expressed, the three-layer organization of glial cells was disturbed. R-cell axon-derived signals were reported to be required for glial cell proliferation and migration in the lamina. However, the results demonstrated that glial cell defects by bantam are cell-autonomous, as neuronal over expression of bantam did not show any affect on glial cells. So far, nonstop, which encodes an ubiquitin-specific protease, was the only gene reported to be required in laminal glial cells for migration. Future experiments to determine bantam's target genes responsible for glial cell migration will be of interest (Li, 2012).

    Recognition of pre- and postsynaptic neurons via nephrin/NEPH1 homologs is a basis for the formation of the Drosophila retinotopic map

    Topographic maps, which maintain the spatial order of neurons in the order of their axonal connections, are found in many parts of the nervous system. This study focused on the communication between retinal axons and their postsynaptic partners, lamina neurons, in the first ganglion of the Drosophila visual system, as a model for the formation of topographic maps. Post-mitotic lamina precursor cells differentiate upon receiving Hedgehog signals delivered through newly arriving retinal axons and, before maturing to extend neurites, extend short processes toward retinal axons to create the lamina column. The lamina column provides the cellular basis for establishing stereotypic synapses between retinal axons and lamina neurons. This study identified two cell-adhesion molecules: Hibris, which is expressed in post-mitotic lamina precursor cells; and Roughest, which is expressed on retinal axons. Both proteins belong to the nephrin/NEPH1 family. Evidence is provided that recognition between post-mitotic lamina precursor cells and retinal axons is mediated by interactions between Hibris and Roughest. These findings revealed mechanisms by which axons of presynaptic neurons deliver signals to induce the development of postsynaptic partners at the target area. Postsynaptic partners then recognize the presynaptic axons to make ensembles, thus establishing a topographic map along the anterior/posterior axis (Sugie, 2010).

    This study shows that cell recognition between pre- and postsynaptic neurons via the Hbs-Rst interaction is required for the establishment of precise retinotopic mapping. During the development of the Drosophila visual center, presynaptic photoreceptors extend their axons to the lamina layer. Postsynaptic lamina precursor cells (pLPCs) start to differentiate in response to Hh delivered through newly arriving R axons. They then express Hbs, which interacts with Rst on R axons (see Model for the specific interaction between R axons and pLPCs mediated by an interaction between Hbs and Rst). This Hbs-Rst interaction is required for lamina column assembly, which underlies the topographic connections of the synapses along the anteroposterior axis (Sugie, 2010).

    The process of lamina column assembly is unique in that presynaptic neurons regulate the development of postsynaptic partners in the target area, and the somata of postsynaptic neurons recognize the presynaptic axons at the developing stage well before neurite formation. This mechanism appears to be an efficient and accurate way to make a topographic map along the anterior/posterior axis. In addition, unlike the well-known axon guidance process, in which growth cones search for their targets, postsynaptic cells actively contribute to the pre- and postsynaptic interactions via direct communication. The changes in the Hbs localization that are associated with rst mutation were not only observed in pLPCs adjacent to R axons, but also in pLPCs far from R axons. This finding could be ascribed to the fact that pLPCs that are distant from R axons can contact R axons through their protrusions. Hbs might be preferentially localized at the protrusions of pLPCs that interact with R axons. The behavior of pLPCs is analogous to that of developing muscle cells, which extend filopodia to the axonal targeting of innervating motoneurons (Sugie, 2010).

    Tests were performed to see whether the cell-adhesion mechanism mediated by Hbs and Rst was sufficient to rescue the sim phenotypes. Induction of exogenous hbs in pLPCs did not rescue sim loss-of-function mutants. Consistent with this finding, overexpression of sim using the NP6099-Gal4 driver caused the premature incorporation of pLPCs into the assembling domain, but overexpression of hbs did not. These results suggest that other molecules under the control of sim must be required for lamina column assembly (Sugie, 2010).

    hbs expressed in photoreceptor cells does not play an essential role in lamina column assembly. The reason that Hbs originating in R axons does not interfere with the Hbs-Rst association remains unknown. The intracellular interaction of the two proteins might be blocked in R axons as a result of alternative subcellular localization and/or steric hindrance, or additional intermediates might be required for Hbs function in pLPCs, but not in R axons (Sugie, 2010).

    Nephrin and NEPH1 homolog proteins tend to be located on opposing cell membranes so that they are brought into close apposition. This arrangement underlies the amazingly similar patterns of immunoreactivity in the eye disc, wing disc and somatic muscle as well as in the pupal optic lobe. These proteins are located in opposing cell membranes in the lamina. Consistent with previous studies, Hbs and Sns proteins were expressed in pLPCs, whereas Rst and Kirre were expressed in R axons; however, Hbs was also expressed in R axons. Recent studies have demonstrated that proteins of the nephrin and NEPH subfamilies are also expressed in neighboring cell types in vertebrate nervous systems. These observations reveal the conservation of nephrin/NEPH1 expression patterns across tissues and species (Sugie, 2010).

    Previous work has identified SYG-1, a homolog of Rst, Kirre and NEPH1, as well as SYG-2, a homolog of Hbs, Sns and nephrin, which are necessary for synaptic specificity in Caenorhabditis elegans. The first Ig domain of SYG-1 and the first five Ig domains of SYG-2 are necessary and sufficient for binding and synapse formation in vivo. Similarly, it was found that the extracellular domain of Hbs and the first Ig domain of Rst are important for the association of pLPCs with R axons. These observations show remarkable functional conservation of the restricted domains of Drosophila and C. elegans nephrin/NEPH1 homologs (Sugie, 2010).

    Further study of the preferential cell adhesion between nephrin/NEPH1 homolog proteins may reveal a common mechanism underlying the interaction between pre- and postsynaptic neurons in both Drosophila and vertebrate brains (Sugie, 2010).

    Localized netrins act as positional cues to control layer-specific targeting of photoreceptor axons in Drosophila

    A shared feature of many neural circuits is their organization into synaptic layers. However, the mechanisms that direct neurites to distinct layers remain poorly understood. This study identified a central role for Netrins and their receptor Frazzled in mediating layer-specific axon targeting in the Drosophila visual system. Frazzled is expressed and cell autonomously required in R8 photoreceptors for directing their axons to the medulla-neuropil layer M3. Netrin-B is specifically localized in this layer owing to axonal release by lamina neurons L3 and capture by target neuron-associated Frazzled. Ligand expression in L3 is sufficient to rescue R8 axon-targeting defects of Netrin mutants. R8 axons target normally despite replacement of diffusible Netrin-B by membrane-tethered ligands. Finally, Netrin localization is instructive because expression in ectopic layers can retarget R8 axons. It is proposed that provision of localized chemoattractants by intermediate target neurons represents a highly precise strategy to direct axons to a positionally defined layer (Timofeev, 2012).

    Recent studies identified at least four molecular mechanisms that control layer-specific targeting in the nervous system by cell-cell interactions independently of neural activity. First, combinatorial expression of homophilic cell surface molecules promotes the recognition and stabilization of contacts between matching branches of pre- and postsynaptic neuron subsets. For instance, four members of the immunoglobulin superfamily of cell adhesion molecules, Sidekick 1 and 2 and Dscam and DscamL, are expressed and required in subsets of bipolar, amacrine, and retinal ganglion cells for targeting to different inner plexiform sublayers (IPLs) in the chick retina. In Drosophila, the leucine-rich repeat protein Caps may play an analogous role, as it is specifically expressed in R8 cells and target layers M1-M4 and, thus, could promote homophilic interactions to stabilize connections within correct columns and layers. Second, concise temporal transcriptional control is used to regulate the levels of ubiquitous cell surface molecules and, thus, adhesiveness of afferent and target neurons to balance branch growth and targeting. This mechanism is supported by findings in the fly visual system where the transcription factor Sequoia controls R8 and R7 axon targeting by the temporal regulation of N-Cadherin (CadN) expression levels. Third, repellent guidance cues are utilized to exclude projections from some layers, as has been shown for membrane-bound Semaphorin family members and Plexin receptors in the IPL of the mouse retina. Fourth, recent studies also implicated the graded expression of extracellular matrix-bound guidance cues such as Slit in the organization of layered connections in the zebrafish tectum. The current findings for the essential role of Netrins and Fra in visual circuit assembly provide evidence for a different strategy: a localized chemoattractant guidance cue is used to single out one layer, thus providing precise positional information required for layer-specific axon targeting of cell types expressing the receptor. Unlike in the ventral nerve cord, where the Netrin/Fra guidance system controls growth across the midline, in the visual system, it mediates target recognition by promoting axon growth into but not past the Netrin-positive layer (Timofeev, 2012).

    Rescue experiments support the model that Netrins are primarily provided by the axon terminals of lamina neurons L3 in the M3 layer. During early pupal stages, Fra-positive R8 axons pause in their temporary layer at the distal medulla neuropil border. From midpupal development onward, upon release from this block, Fra-positive R8 axons are guided to the Netrin-expressing M3 layer (Timofeev, 2012).

    Axons can use intermediate target cells either along their trajectory to guide them toward their target areas or within the target area to bring putative synaptic partners into close vicinity. Although R8 axons and lamina neurons L3 terminate closely adjacent to each other in the same layer, they have been described to not form synaptic connections with each other but to share common postsynaptic partners such as the transmedullary neuron Tm9. Thus, the results suggest that layer-specific targeting of R8 axons relies on the organizing role of lamina neurons L3 as intermediate targets in the M3 layer rather than direct interactions with postsynaptic partners. Consistent with this notion, axons of lamina neurons L3 timely extend between the temporary layers of R8 and R7 axons from early pupal stages onward, and targeting of their axons is independently controlled by other cell surface molecules such as CadN. Further studies will need to identify potential Fra-positive synaptic partners in the medulla and test whether this guidance receptor equally controls targeting of their dendritic branches, thus bringing pre- and postsynaptic neurites into the same layer. Additional mechanisms likely mediate cell-cell recognition and synaptic specificity, as electron microscopic analysis showed that presynaptic sites in R8 axons are not restricted to the M3 layer but distributed along the axon (Timofeev, 2012).

    Netrins are diffusible guidance cues acting both at long range in a gradient and at short range when immobilized. Consistent with studies in the Drosophila embryo, it was observed in this study that NetB in the visual system acts at short range, as R8 axon targeting is normal when solely membrane-tethered NetB is available at near-endogenous levels. Secreted Netrins are converted into a short-range signal because they are locally released by lamina neurons L3 and prevented to diffuse away through a Fra-mediated capturing mechanism. Filopodial extensions could enable R8 growth cones to bridge the distance to NetB-expressing lamina neuron L3 axon terminals (Timofeev, 2012).

    Although in principle Netrins could be secreted by both dendritic and axonal arbors of complex neurons, the results support the notion that axon terminals are the primary release sites to achieve layer-specific expression. This may be mediated by a cargo transport machinery along polarized microtubules similar to that used by synaptic proteins or neurotransmitters. Consistently, recent findings in C. elegans identified proteins involved in motor cargo assembly and axonal transport as essential for Netrin localization and secretion. Intermediate target neurons may thus constitute an important strategy to draw afferent axons into a layer, if guidance cues are preferentially released by axon terminals and not by dendritic branches of synaptic partner neurons. Netrin-releasing lamina neurons L3 form dendritic spines in the lamina and axon terminals in the medulla. Similarly, Netrin-positive transmedullary neuron subtypes such as Tm3 and Tm20 form dendritic branches in the medulla and extend axons into the lobula. Thus, a mechanism, whereby neurons in one brain area organize the connectivity in the next, may be used at least twice in the visual system (Timofeev, 2012).

    Knockdown of fra in the target area strongly reduced NetB in the M3 layer, supporting the notion that a receptor-mediated capturing mechanism controls layer-specific Netrin accumulation. Despite the use of multiple genetic approaches, no R8 axon-targeting errors were observed when manipulating Fra levels exclusively in target . This could be attributed to the technical limitation that knockdown is incomplete owing to the activity of the ey enhancer in around 50% of medulla neurons. However, as lamina neurons L3 continue to locally release Netrins, remaining ligands may likely be sufficient to guide fully responsive R8 axons to their target layer (Timofeev, 2012).

    Unlike in the fly embryonic CNS, where Netrins are captured by Fra and presented to growth cones expressing a Netrin receptor other than Fra, or in C. elegans, where Unc-6 is captured at the dendrite tips of nociceptive neurons by Unc-40 to interact with Unc-5 (Smith, 2012), genetic analyses indicate that fra is required in R8 axons. Hence, Netrins captured by Fra-positive target neurons may either be presented to Fra-expressing R8 axons in a dynamic fashion, or R cell- and target neuron-derived Fra interact with Netrins in a ternary complex in trans. This is conceivable since (1) the vertebrate counterpart Netrin-1 shows a high binding affinity for DCC; (2) DCC can bind Netrins with multiple domains (DCC, fourth and fifth fibronectin type III domains; Netrins, Laminin N-terminal (LamNT) and three Laminin-type epidermal growth factor [EGF]-like domains); and (3) at least in cis, Netrins can bind and aggregate multiple DCC ectodomain molecules. Ligand capture and presentation by receptors have also been reported for F-spondin and lipoprotein receptor-related protein (LRP) at the vertebrate floor plate. Netrins have previously been shown to promote exocytosis and recruitment of their receptor to distinct subcellular locations on cell surfaces. Moreover, in the visual system, Netrins may increasingly draw neurites of Fra-positive target neurons into layer M3, which in turn could promote further ligand accumulation. Thus, additional feedback loops may contribute to the specific enrichment of both Netrins and Fra in the M3 layer (Timofeev, 2012).

    R8 axon targeting involves multiple successive steps: (1) the selection of the retinotopically correct column; (2) pausing in the temporary layer; (3) timely release from the temporary layer and extension of a filopodium; (4) bypassing of incorrect neuropil layers; (5) correct identification and targeting to the M3 layer; (6) stabilization of connections in the correct layer and column and transformation of growth cones into mature terminals; and (7) formation of the correct repertoire of synaptic contacts. Strong early defects would likely impact on subsequent steps (Timofeev, 2012).

    Within this sequence of events, interactions of Golden goal (Gogo) and Flamingo (Fmi) in cis within R8 axons and in trans with Fmi-positive neuronal processes in the emerging M1, M2, and lower M3 layers have been shown to contribute to the timely release of R8 growth cones from their temporary layer and, consequently, enable correct targeting to the M3 layer (steps 3 and 6). Caps may specifically promote cell-cell recognition and stabilize interactions between R8 axons and target neuron branches within their correct column and target layer (step 6). However, an alteration of adhesiveness may not be sufficient to promote the extension of filopodia toward the correct layer, and additional attractive guidance forces are required. The Netrin/Fra guidance system is well suited to play such a role by providing the necessary positive forces directing filopodia toward deeper layers and by promoting recognition of a single layer at a given position (steps 4 and 5). This notion is supported by observations that loss of Fra or Netrins causes many R8 axons to stall at the distal medulla neuropil border and to terminate at interim positions in layers M1/M2. Furthermore, ectopic expression of membrane-tethered NetB is sufficient to retarget a significant proportion of R8 axons. Unlike Caps and Gogo/Fmi, whose ectopic expression can promote targeting of some R7 axons to the M3 layer, Fra was not sufficient to redirect R7 axons from the M6 to the M3 layer. A likely explanation is that the effects of R7-specific guidance determinants cannot be overwritten, or essential cooperating receptors or downstream components of Fra present in R8 are missing in R7 cells. Furthermore, overexpression of Fra causes many R8 axons to stall at the medulla neuropil border, suggesting that tight temporal regulation of receptor levels in R8 axons is essential for the integration of an additional potential repellent input (Timofeev, 2012).

    Together, these findings in the Drosophila visual system suggest that the dynamic coordinated actions of chemotropic guidance cues and cell adhesion molecules contribute to layer-specific targeting of specific cell types. A similar molecular mechanism relying on Netrins or other localized attractive guidance cues and their receptors may be more widely used for the assembly of laminated circuits (Timofeev, 2012).

    Sequential axon-derived signals couple target survival and layer specificity in the Drosophila visual system

    Neural circuit formation relies on interactions between axons and cells within the target field. While it is well established that target-derived signals act on axons to regulate circuit assembly, the extent to which axon-derived signals control circuit formation is not known. In the Drosophila visual system, anterograde signals numerically match R1-R6 photoreceptors with their targets by controlling target proliferation and neuronal differentiation. This study demonstrates that additional axon-derived signals selectively couple target survival with layer specificity. Jelly belly (Jeb) produced by R1-R6 axons was shown to interact with its receptor, Anaplastic lymphoma kinase (Alk), on budding dendrites to control survival of L3 neurons, one of three postsynaptic targets. L3 axons then produce Netrin, which regulates the layer-specific targeting of another neuron within the same circuit. It is proposed that a cascade of axon-derived signals, regulating diverse cellular processes, provides a strategy for coordinating circuit assembly across different regions of the nervous system (Pecot, 2014).

    This study demonstrates that Jeb/Alk signaling regulates the survival of L3 neurons, one of several postsynaptic targets of R1-R6 neurons. Jeb is expressed in R1-R6 growth cones and acts at short range, prior to synapse formation, through the Alk receptor tyrosine kinase localized on budding L3 dendrites within the lamina neuropil. Jeb/Alk signaling is highly selective, as the survival of other R1-R6 postsynaptic targets (i.e., L1 and L2) is not affected when signaling is disrupted. This study also showed that, at a later stage of development, L3 growth cones produce Netrin within the medulla, which is required for the targeting of R8 growth cones to the M3 layer. It is speculated that a cascade of growth-cone-derived signals acting across different brain regions provides a general strategy for the assembly of neural circuits (Pecot, 2014).

    In many regions of the developing nervous system, neurons are produced in excess, and significant cell death occurs after axons innervate their targets. In vertebrates, it is well established that target-derived neurotrophins, such as nerve growth factor, regulate neuronal numbers. These factors are produced by target neurons in limiting amounts and locally promote survival in a retrograde manner through receptors localized on axon terminals, providing a mechanism for matching the number of axons to targets. In recent years, diverse classes of molecules have been shown to control neuronal survival during development. Anterograde sources of trophic factors may also regulate survival, as denervation has been shown to induce excessive target neuron cell death. Indeed, several signals, including BDNF, are transported, in some contexts, in an anterograde manner within axons. In addition, the overexpression of BDNF in afferents can rescue cell death within the target field, and the disruption of BDNF through function blocking antibodies has been reported to decrease the number of target neurons within the rat superior colliculus. As BDNF may be produced by both axons and cells within the superior colliculus, it remains unclear whether endogenous axon-derived BDNF, and thus anterograde signaling, is required to regulate neuron survival (Pecot, 2014).

    Although a role for target-derived retrograde trophic factors in vertebrate neural development was established many decades ago, trophic factors have only recently been shown to regulate neuronal development in Drosophila. Three Drosophila proteins, Neurotrophin 1, Neurotrophin 2, and Spatzle, are distantly related to vertebrate neurotrophins, and it has been shown that, like their vertebrate counterparts, they function as target-derived retrograde survival signals. Unlike their vertebrate homologs, however, which act through receptor tyrosine kinases, fly neurotrophins promote cell survival through Toll-like receptors (Pecot, 2014).

    Although Jeb bears no significant homology to fly or vertebrate neurotrophins, Jeb acts through a receptor tyrosine kinase, Alk, which is distantly related to vertebrate neurotrophin receptors or Trks. Alk was originally identified as part of a fusion protein associated with large cell anaplastic lymphoma. Its role in mammals remains poorly understood. Drosophila Alk was initially found to regulate visceral mesoderm development through interaction with Jeb, and subsequently, Jeb/Alk signaling has been shown to regulate diverse cellular processes. Recent studies in vertebrates and Drosophila demonstrated that disrupting Alk function causes a decrease in the number of neurons. While in the vertebrate studies Alk's mechanism of action was not established, in Drosophila, Alk was shown to antagonize pathways that restrict neurogenesis under conditions of nutrient deprivation. Whether Jeb and Alk regulate neuronal survival in contexts outside of L3 development is not known, although Alk is widely expressed in the developing visual system, and Jeb is expressed by several populations of neurons, in addition to photoreceptors (Pecot, 2014).

    The cellular specificity of the Jeb/Alk requirement is particularly surprising. Indeed, at all R1-R6 synapses containing L3 postsynaptic elements, L1 and L2 neurons each contribute a single postsynaptic element juxtaposing the same presynaptic site on R cell axons. In the absence of Jeb/Alk signaling, however, only L3 neurons die. The mechanisms that underlie this selectivity are not known. Alk is broadly expressed in the lamina, suggesting specificity may be controlled at the level of downstream signaling or that other trophic signals act redundantly with Jeb to control L1 and L2 survival. Collectively, the findings reported in this study demonstrate that anterograde Jeb/Alk signaling acts selectively to control L3 survival, providing direct evidence that anterograde signaling regulates target neuron survival in vivo (Pecot, 2014).

    Several lines of evidence indicate that signaling between Jeb, expressed by R1-R6 growth cones, and Alk, localized to budding L3 dendrites, controls L3 survival between 20-40 hr APF. First, Alk mutant L3 neurons, or wild-type L3 neurons innervated by jeb mutant R1-R6 axons, die between 20-40 hr APF. Second, R cell populations containing only R1-R6 neurons are sufficient for L3 survival. Third, Alk and Jeb are expressed in a complementary fashion at the appropriate time on budding L3 dendrites and R1-R6 growth cones, respectively. And finally, L3 degeneration begins within budding L3 dendrites juxtaposed to R1-R6 growth cones. The temporal requirement for Alk/Jeb signaling corresponds to a critical and fascinating phase of lamina circuit assembly (Pecot, 2014).

    R1-R6 growth cones form connections with lamina neurons in three discrete steps. First, R1-R6 growth cones from the same ommatidium associate with a single cartridge of differentiating lamina neurons. Second, through a highly stereotyped reassortment process occurring between 24-38 hr APF, these six growth cones diverge from one another and project locally to six different developing cartridges. As a consequence of this rearrangement, the R1-R6 cells that 'see' the same point in space form connections with L1, L2, and L3 neurons within the same cartridge. And third, R1-R6 then commence synapse formation at 45 hr APF, and this process continues until eclosion (~96 hr). Thus, L3 death in jeb and Alk mutants occurs prior to synapse formation, during the process of R1-R6 growth cone rearrangement. The suppression of L3 death by expression of the caspase inhibitor p35 argues that during normal development Jeb/Alk signaling acts to inhibit caspase activity. Which caspases contribute to L3 death, and whether caspases antagonize other cellular processes necessary for wiring, is not known. Regardless of how Jeb/Alk signaling functions at the molecular level, it acts to ensure that visual input from R1-R6 neurons is transmitted to the L3 pathway (Pecot, 2014).

    These findings and the work of others suggest a logic underlying neural circuit assembly within the Drosophila visual system. The retina, lamina, and medulla are distinct yet interconnected regions comprising columnar modules (i.e., ommatidia, cartridges, and columns, respectively) that are matched topographically between each region. Within each module, intrinsic mechanisms and intercellular interactions control cell fate determination. For instance, R8 neurons provide a discrete locally acting signal to induce R7 development in the developing retina, while in the medulla, Notch/Delta interactions between daughter cells generated from the same ganglion mother cell promote acquisition of distinct cell fates. Superimposed upon these interactions are axon-derived signals that coordinate development between matched modules from different regions. Together, these mechanisms organize the assembly of columnar units in multiple regions (i.e., super columns), each processing visual information captured from a discrete region of the visual field. Indeed, the modular assembly of these super columns spanning different regions of the visual system reflects the function of these circuits in the parallel processing of visual information (Pecot, 2014).

    R cell growth cones produce signals that regulate diverse cellular processes in the developing lamina. Hedgehog drives lamina neuronal precursors through their final division; cell adhesion proteins promote the association of columns of lamina neurons with R cell axon fascicles; EGF induces lamina neuron differentiation; a yet-to-be-identified signal regulates the development of lamina glia; and Jeb selectively regulates L3 survival. Thus, axon-derived signals act at multiple levels and in a cell-type-specific manner to regulate target development (Pecot, 2014).

    Axon-derived signals also coordinate circuit assembly across topographically matched modules. Within medulla columns, L3 growth cones produce Netrin in the M3 layer, which controls the targeting of R8 growth cones to M3. Importantly, Netrin production by L3 occurs after Jeb, released from R1-R6 cells in topographically corresponding lamina cartridges, promotes L3 survival. Thus, Netrin indirectly relies upon prior Jeb signaling. As the L3 and R8 axon terminals within each medulla column transmit information captured from the same point in space to the same layer (M3) and share several postsynaptic targets, the developmental mechanisms giving rise to this circuit may reflect functional relationships between these neurons. Thus, signals produced by axons coordinate assembly of circuits between different brain regions (Pecot, 2014).

    It is envisioned that intercellular signaling cascades, analogous to what are described in this study, organize other circuit modules in the fly visual system [e.g., ON (L1) and OFF (L2) circuits] comprising different cell types. As many regions of the vertebrate nervous system, including the neocortex, spinal cord, and retina, are also arranged in a hierarchically repetitive fashion, this raises the intriguing possibility that similar strategies may coordinate the development of these structures (Pecot, 2014).

    Analyzing dendritic morphology in columns and layers

    In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (1) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (2) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (3) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants (Ting, 2017).

    Birth order dependent growth cone segregation determines synaptic layer identity in the visual system

    The precise recognition of appropriate synaptic partner neurons is a critical step during neural circuit assembly. However, little is known about the developmental context in which recognition specificity is important to establish synaptic contacts. This study shows that in the Drosophila visual system, sequential segregation of photoreceptor afferents, reflecting their birth order, lead to differential positioning of their growth cones in the early target region. By combining loss- and gain-of-function analyses it was demonstrated that relative differences in the expression of the transcription factor Sequoia regulate R cell growth cone segregation. This initial growth cone positioning is consolidated via cell-adhesion molecule Capricious in R8 axons. Further, the initial growth cone positioning was shown to determine synaptic layer selection through proximity-based axon-target interactions. Taken together, this study demonstrates that birth order dependent pre-patterning of afferent growth cones is an essential pre-requisite for the identification of synaptic partner neurons during visual map formation in Drosophila (Kulkarni, 2016).

    This study demonstrates that the early, birth order dependent, segregation of R cell growth cones determines later synaptic layer identity in the Drosophila visual system. Small inter-ommatidial differences in Sequoia levels organize R cell growth cones within a layer whereas large intra-ommatidial differences segregate growth cones between layers. Changes in the positioning of growth cones directly correlate with changes in synaptic layer selection without affecting the expression of known cell-type specific targeting molecules (see Role of early growth cone patterning in synaptic layer selection). These results highlight the importance of initial afferent growth cone positioning for visual map formation prior to synaptic partner recognition (Kulkarni, 2016).

    An early shift in the R8 growth cones to R7 position, induced by a short pulse of Sequoia expression, allows them to recognize the R7 target cell Dm8 as synaptic partner later during the development. Similarly, if R7 growth cones fail to segregate from R8 growth cones, they terminate together with R8 axons in the M3 synaptic layer independent of their intrinsic differentiation and targeting program. This extension of Frazzled-negative R7 axons towards layer M3 could be explained by the default setting of R7 axons to tightly fasciculate and follow the R8 pioneer axons towards the target region (Kulkarni, 2016).

    R cell growth cone segregation can be controlled by axon-target interactions or axon-axon interactions or both. Although there is no experimental demonstration for direct R7-R8 afferent interactions, the following set of data indicate that such interactions occur during development. Support for direct interaction between R7-R8 afferents comes from Maurel-Zaffran (2001), where the expression of LAR as membrane tethered ligand in R8 cells alone (using an R8-specific Gal4 driver line) could induce a response from R7 axons, indicating a direct signalling between R8-R7 axons. Further, induced Capricious expression in R8 and R7 cells, in Capricious null background (therefore resulting in a target region without any Capricious expression) is sufficient to mis-target R7 axons to layer M3, again indicating a direct R7-R8 afferent interaction (Berger-Müller, 2013). The current study shows that final target layer of sequoia mutant R7 axons depends on the targeting of R8 axons, further suggesting that mis-targeting of sequoia mutant R7 axons to ectopic synaptic layer is the consequence of segregation defect rather than a change in target layer recognition (Kulkarni, 2016).

    Interactions among afferent axons have been implicated in the assembly of visual and olfactory circuits in vertebrates as well as invertebrates. It has recently been shown that Eph-Ephrin signalling mediates local sorting of RGC axons in mammalian visual system. Notch signalling was demonstrated to play a role in spacing of DCN cluster neuron axons via neighbour axon interactions. But, whether these afferent interactions influence synaptic partner recognition is not known (Kulkarni, 2016).

    This study shows that in the Drosophila visual system, relative levels of Sequoia determine the segregation of afferent R7/R8 growth cones within or between layers. By creating Seqhigh-Seqlow R cell combinations using Sequoia gain-of-function R7 mosaics it was observed that difference in Sequoia levels among neighbouring cells could induce growth cone segregation. The endogenous differences in Sequoia levels most likely arise as a result of the temporal sequence of R cell specification, suggesting a self-patterning mechanism in early visual circuit assembly. How the relative differences in Sequoia levels in the nuclei of R cells translate into growth cone segregation remains elusive. This study has tested candidate signalling pathways including Semaphorin/Plexin, TGF-beta ligand Activin and its receptor Baboon and Notch but did not find evidence for a critical role in initial growth cone segregation. This suggests a so far unknown molecular mechanism in which the growth state of an axon is directly coupled to differential growth cone adhesion. As it was possible to demonstrate a cell-autonomous function of Sequoia in R8 for columnar segregation as well as in R7 for layer segregation, a mechanistic model related to the concept of cell competition is envisioned, in which strong cell-cell interactions induce cell-autonomous responses (Kulkarni, 2016).

    The initial segregation of afferent growth cones into distinct positions is then consolidated by expression of Capricious in R8 axons in the same posterior-to-anterior pattern in which they arrive in the medulla. It is speculated that Capricious mediated growth cone consolidation serves two purposes: 1. It removes the temporal difference in the arrival of R8 axons and 2. It maintains R8 axons in the position where they are responsive to subsequent NetrinB signal provided by L3 neurites. This is supported by two different sets of results: First, as was shown in this study, the displacement of R8 growth cones to deeper medulla position leads to their mis-targeting to layer M6 in spite of normal Frazzled expression in these R8 cells. Second, the ectopic expression of Frazzled in R7 cells cannot re-direct them to M3 layer in response to localized NetrinB signal present in the superficial position but R8 axons can be re-directed to a different layer (M1/M2) by ectopic expression of localized NetrinB in a position deeper to the superficial R8 medulla position. Taken together, these observations suggest that M3 layer targeting via L3-mediated NetrinB signalling requires R8 axons to be positioned superficially in the medulla further underscoring the importance of R8 growth cone consolidation in this position (Kulkarni, 2016).

    Previous studies have identified several molecules necessary for M6 targeting of R7 axons, including Liprin-alpha/beta/gamma, PTP69D and D-Lar. The loss of these molecules specifically affects the stabilization of R7 growth cones during the second step of targeting. Additionally, these molecules along with N-Cadherin have been shown to be critical for establishment of synaptic contacts between pre and post-synaptic neurons. This study confirmed previous observations, that R7 growth cones are in close proximity with their primary post-synaptic target neurons, Dm8, at the end of growth cone segregation. This raises the possibility that targeting of R7 axons to M6 layer, later in the development, could be the direct result of R7->Dm8 contacts mediated by N-Cadherin. Recently it was shown that N-Cadherin function is necessary for stabilizing R7 growth cones in the deeper medulla position but not for targeting and subsequent extension of R7 axons to M6 layer seems to be a result of passive dislocation. Additional support for the role of N-Cadherin in the formation and maintenance of R7-->Dm8 contacts, following their initial segregation from R8 growth cones, comes from the observation that early expression of Sequoia in CadN mutant R7 cells under weak elav-Gal4 driver can rescue the mis-targeting of R7 growth cones in the superficial medulla position along with R8 growth cones at 24 hr APF, but fails to rescue the later mis-targeting to layer M3 eventually resulting in a mis-targeting phenotype identical to CadN mutant R7 axons. Interestingly, the R8 growth cones initially mis-positioned in the deeper medulla eventually mis-target to layer M6 and form synaptic contacts with Dm8. In addition, these R8 cells, with axons mis-targeted to layer M6, do not show changes in any of their known cell-type specific molecules including early specifier of cell identity (Senseless), guidance receptors (Frazzled, Capricious) and sensory receptors (Rh6). In addition, no expression of R7 specific molecules (Prospero, R3, Rh4) can be detected. Thus, the R8 cells interact with Dm8 neurons most likely via ubiquitously expressed molecules such as N-Cadherin expressed in both, R7 as well as R8, cells. This is supported by the observation that N-Cadherin is required for stabilization of R8 axons at the layer M6 (Kulkarni, 2016).

    It was observed that R8 axons form functional synapses with Dm8, a known R7 target neuron, in the layer M6. This raises the fundamental question of how synaptic layer selection influences synaptic partner recognition. The cellular complexity of potential post-synaptic target layer encountered by ingrowing R cell axons has not been fully determined, leaving room for selective recognition for synaptogenesis within a layer. In fact, it has been shown that within M6, R7 axons form synapses with Dm8 but not with Tm5c which also arborize the M6 layer. In addition, this study has identified various medulla columnar neurons within M6 that are not contacted by R7 axons. Similarly in layer M3 R8 and L3 select distinct post-synaptic partners (Kulkarni, 2016).

    The types of neurons present in the medulla at the time of R8 and R7 axon innervation have not been fully identified. Based on published data, the medulla neurons are generated in temporal fashion and therefore they likely innervate the medulla at different time points. Experiments presented in this study support a developmental scenario in which the medulla context for arriving R cell axons reduces the complexity of synaptic partner selection. For example R8 and L3 have different arrival times at M3, thereby would encounter a different local environment of potential post-synaptic partners competent for synaptogenesis. It is plausible that some form of temporal co-ordination of afferent axons and their post-synaptic partner cell neurites would actually simplify the synaptic partner matching. The concept of temporal identity would argue that R7 and R8 axons arriving at the same medulla position approximately the same time, as shown in the Sequoia gain-of-function background, will pick the same synaptic partners exemplified by Dm8. Support for such proximity-based axon-target interaction for synaptogenesis comes from earlier analysis of ectopic axons in Drosophila as well as Zebrafish (Kulkarni, 2016).

    From an evolutionary perspective, such proximity-induced synapse formation has several advantages over mechanisms that require regulation and expression of distinct sets of cell recognition molecules. Considering R7 as the most recently added cell to the precursor ommatidium: During development, R7 is recruited using mechanisms similar to R8 and therefore possesses default R8 specification program. However, this default R8 program is suppressed to facilitate R7 specification. Thus, a temporally separated, novel R7 cell is generated with basic neuronal differentiation similar to that of an R8 cell. Interestingly the temporal difference in the R8/R7 differentiation is then translated into Sequoia mediated layer segregation of their growth cones, with Sequoia expression being part of common differentiation program. Thus, the evolutionary recent R7 cell seems to recognize its synaptic targets via pan neuronal molecules like N-Cadherin as part of the default neuronal differentiation program, instead of the invention of an additional recognition code (Kulkarni, 2016).

    Visual circuit assembly requires fine tuning of the novel Ig transmembrane protein Borderless

    Establishment of synaptic connections in the neuropils of the developing nervous system requires the coordination of specific neurite-neurite interactions (i.e., axon-axon, dendrite-dendrite and axon-dendrite interactions). The molecular mechanisms underlying coordination of neurite-neurite interactions for circuit assembly are incompletely understood. This study identified a novel Ig superfamily transmembrane protein that was named Borderless (Bdl), as a novel regulator of neurite-neurite interactions in Drosophila. Bdl induces homotypic cell-cell adhesion in vitro and mediates neurite-neurite interactions in the developing visual system. Bdl interacts physically and genetically with the Ig transmembrane protein Turtle, a key regulator of axonal tiling. These results also show that the receptor tyrosine phosphatase leukocyte common antigen-related protein (LAR) negatively regulates Bdl to control synaptic-layer selection. It is proposed that precise regulation of Bdl action coordinates neurite-neurite interactions for circuit formation in Drosophila (Cameron, 2013).

    The presence of numerous axons and dendrites in the neuropils of the developing CNS makes it a daunting task for establishing specific synaptic connections. Studies over the last two decades have identified a number of cell-surface recognition molecules that mediate specific neurite-neurite interactions for circuit assembly. That many cell-surface recognition molecules are present broadly in developing neuropils throughout embryonic development, however, raises the question how the action of cell-surface recognition molecules is modulated temporally to ensure accuracy in circuit formation (Cameron, 2013).

    The assembly of visual circuits in Drosophila is an attractive model for understanding the general mechanisms underlying spatiotemporal control of neurite-neurite interactions. The Drosophila adult visual system is comprised of the compound eye and the optic lobe. The compound eye consists of ∼800 ommatidia, each containing six outer photoreceptor neurons (R1-R6) for processing motion and two inner photoreceptor neurons (R7 and R8) for processing color. R1-R6 axons form synaptic connections in the superficial lamina layer, and R7 and R8 axons project through the lamina into the deeper medulla layer, where they are organized into ∼800 regularly spaced columns. Each R7 and R8 axon from the same ommatidium terminate in a topographic manner in two synaptic layers within the same column. The R8 axon terminates within the M3 layer, and the R7 axon terminates in the deeper M6 layer (Cameron, 2013).

    Visual circuit assembly in Drosophila involves complex neurite-neurite interactions. Specific recognition between R-cell axons and their target layers in the optic lobe have been shown to be required for synaptic-layer selection. Visual circuit assembly also requires the interactions among R-cell axons. Selection of postsynaptic targets by R1-R6 axons in the lamina requires specific axon-axon interactions. The assembly of medulla columns requires modulation of both heterotypic and homotypic axon-axon adhesion. For instance, receptor tyrosine phosphatases LAR and protein tyrosine phosphatase 69D (PTP69D) are reported to be involved in negatively regulating the adhesion between R7 and R8 axons for facilitating R7 synaptic-layer selection. And Ig-superfamily transmembrane proteins Dscam2 and Turtle (Tutl) prevent homotypic axon-axon terminal adhesion for tiling L1 and R7 axons, respectively. The exact mechanisms by which those cell-surface recognition molecules negatively regulate axon-axon adhesion, however, remain unknown (Cameron, 2013).

    The role of a novel Ig-superfamily transmembrane protein Borderless (Bdl) in Drosophila was investigated in this study. Bdl is expressed in the developing visual system, and functions as a cell-surface recognition molecule to mediate neurite-neurite interactions. The receptor tyrosine phosphatase LAR and the Ig-superfamily transmembrane protein Tutl are key regulators of Bdl-mediated axon-axon interactions in controlling synaptic-layer selection and axonal tiling, respectively. The results shed new light on spatiotemporal control of cell-surface recognition molecules for coordinating circuit assembly (Cameron, 2013).

    Tiling and self-avoidance, two cellular mechanisms discovered in the early 1980s, are important for patterning neuronal circuitry. Previous studies have identified several cell-surface recognition molecules, such as Dscam, Tutl, Protocadherins, MEGF10, and MEGF11, that mediate homotypic neurite-neurite interactions in tiling and self-avoidance. These cell-surface recognition molecules may act by mediating homotypic repulsion or de-adhesion between adjacent same-type neurites. For instance, molecular and genetic analyses of fly Dscam1 support a role for Dscam1 in mediating homotypic repulsion in dendritic self-avoidance, whereas mammalian Dscams appear to mediate de-adhesion by interfering with some unknown cell-type-specific cell adhesion molecules. The exact mechanisms by which these cell-surface recognition molecules mediate homotypic repulsion or de-adhesion, however, remains elusive (Cameron, 2013).

    Several lines of evidence implicate Bdl as a target of Tutl in regulating R7 axonal tiling. First, overexpression of Bdl induced an R7 tiling phenotype similar to that in tutl mutants. Second, Tutl associates with Bdl in cultured cells. And third, loss of bdl rescued the tiling phenotype in tutl mutants. It is proposed that Tutl-mediated surface recognition counteracts the affinity between adjacent R7 axonal terminals by interacting with Bdl. The association of Tutl with Bdl may downregulate the level and/or adhesive activity of Bdl, thus allowing the separation of adjacent R7 axonal terminals. Since co-overexpression of Tutl and Bdl did not affect Bdl-mediated cell-cell aggregation in culture nor the Bdl-overexpression-induced tiling phenotype in flies, it is speculated that the regulation of Bdl by Tutl requires the involvement of additional regulatory molecules. Future studies are needed to determine the exact mechanism by which Tutl downregulates the function of Bdl. It will also be of interest to determine whether other cell-surface recognition molecules implicated in tiling and self-avoidance (e.g., Dscam and Protocadherins), function similarly to modulate certain cell adhesion molecules (Cameron, 2013).

    The receptor tyrosine phosphatase LAR and its mammalian homologs have been shown to play important roles in axon guidance, neuronal target selection, and presynaptic development. In the developing Drosophila visual system, LAR is required for target selection of R1-R6 axons in the lamina, and synaptic-layer selection of R7 axons in the medulla. The action of LAR in R7 synaptic-layer selection reportedly involves both stabilization of axon-target interactions and down-regulation of adhesion between R7 and R8 axons. LAR-mediated axon-target interactions may involve the binding between LAR on R7 axons and an unknown ligand in the target layer, which in turn modulates the interaction between LAR and its cytoplasmic domain-binding partner Liprin to stabilize axon-target interactions. It is also reported that LAR negatively regulates an unknown cell adhesion molecule to decrease adhesion between R7 and R8 axons for facilitating synaptic-layer selection of R7 axons (Cameron, 2013).

    The current results suggest strongly that LAR downregulates adhesion between R7 and R8 axons by negatively regulating Bdl. That LAR inhibited Bdl-mediated cell-cell adhesion without affecting the level of Bdl suggests that LAR inhibits adhesive activity of Bdl. Although the role of LAR in mediating axon-target interactions requires its binding to Liprin via the cytoplasmic domain, negative regulation of Bdl by LAR appears to involve a Liprin-independent mechanism. This is supported by in vitro analysis showing that a LAR mutant lacking the cytoplasmic domain also inhibited Bdl-mediated adhesion. Consistently, a previous study showed that R8-specific expression of a truncated LAR mutant lacking the cytoplasmic domain in LAR mutants could partially rescue the R7 mistargeting phenotype. LAR may directly modulate Bdl to downregulate R7-R8 adhesion, or act indirectly by interacting with other proteins. Future studies are needed to distinguish between these possibilities (Cameron, 2013).

    Although negative regulation of Bdl-mediated axon-axon interactions is necessary for R7 synaptic-layer selection and tiling, it remains unclear how the presence of Bdl contributes to the formation of the R-cell axonal projection pattern in the fly visual system. Cell adhesion molecules, such as NCAM/FasII and L1-CAM/Neuroglian, have been shown to mediate selective fasciculation in axonal pathfinding. Similarly, Bdl-mediated axon-axon interactions may facilitate the projections of R7 and/or R1-R6 axons along the pioneer R8 axon. That the R-cell projection pattern remained normal in bdl mutants may be due to the presence of redundant genes. Functional redundancy among different cell adhesion molecules seems to be common in the developing nervous system, which may account for no or subtle phenotypes in mutants defective in a number of cell adhesion molecules (Cameron, 2013).

    In conclusion, this study study identifies Bdl as a novel and important regulator of neurite-neurite interactions in the developing visual system. Tuning of Bdl-mediated axon-axon interactions in axonal tiling and synaptic-layer selection presents an excellent example for modulating the action of cell adhesion molecules in ensuring accuracy in circuit assembly. It is highly likely that similar mechanisms are employed for circuit assembly in mammalian nervous systems (Cameron, 2013).

    Multiple interactions control synaptic layer specificity in the Drosophila visual system

    How neurons form synapses within specific layers remains poorly understood. In the Drosophila medulla, neurons target to discrete layers in a precise fashion. This study demonstrates that the targeting of L3 neurons to a specific layer occurs in two steps. Initially, L3 growth cones project to a common domain in the outer medulla, overlapping with the growth cones of other neurons destined for a different layer through the redundant functions of N-Cadherin (CadN) and Semaphorin-1a (Sema-1a). CadN mediates adhesion within the domain and Sema-1a mediates repulsion through Plexin A (PlexA) expressed in an adjacent region. Subsequently, L3 growth cones segregate from the domain into their target layer in part through Sema-1a/PlexA-dependent remodeling. Together, these results and recent studies argue that the early medulla is organized into common domains, comprising processes bound for different layers, and that discrete layers later emerge through successive interactions between processes within domains and developing layers (Pecot, 2013).

    Although the growth cones of L1, L3, and L5 neurons target to different layers, they initially overlap within a common domain in the outer medulla. Based on biochemical interactions and the mistargeting phenotypes and protein expression patterns described in this paper, it is envisioned that CadN-dependent adhesive interactions restrict processes to the outer medulla and that PlexA-expressing tangential neurons prevent Sema-1a expressing growth cones from projecting into the inner medulla. L2 and L4 growth cones also appear to initially target to a common domain within the distal outer medulla, but do not require Sema-1a and CadN for this targeting step and thus utilize an alternative mechanism. Interestingly, the morphology of L2 and L4 neurons does rely on Sema-1a and CadN function, indicating that within lamina neurons, these molecules regulate different aspects of targeting. This is supported by the expression of Sema-1a and CadN in all lamina neuron subclasses during development (Pecot, 2013).

    In mice separate channels encoding light increments (ON) and decrements (OFF) are spawned in the outer retina and relayed to different sublaminas of the inner plexiform layer (IPL). The current findings are reminiscent of recent studies in the mouse IPL (Matsuoka, 2011) in which Kolodkin and colleagues demonstrated that the processes of different subclasses of PlexA4-expressing amacrine cells are segregated to different OFF layers and that this requires both PlexA4 and Sema6A. Although these proteins act in a more traditional fashion as a receptor and ligand, respectively, they are expressed in a complementary fashion early in development when the developing neuropil is very thin, with PlexA expressed in the nascent OFF layer and Sema6A in the developing ON layers. This raises the intriguing possibility that, as in the medulla, different cells initially target to common domains, from which they then segregate into discrete layers. As Cadherin proteins are differentially expressed in a layered fashion in the developing IPL and defects in targeting are incomplete in both Sema6A and PlexA4 mutants (Matsuoka, 2011), it is possible that, as in the medulla, Semaphorin/Plexin repulsion acts in parallel with cadherin-based adhesion to control layer-specific patterning within the developing IPL (Pecot, 2013).

    Taken together, these studies suggest that the restriction of processes to a common domain prior to their segregation into distinct layers may be a developmental strategy used in both the medulla and the vertebrate IPL. This step-wise process may represent a more general strategy for reducing the molecular diversity required to establish synaptic connections by limiting the potential synaptic partners that growth cones and nascent dendritic arbors encounter within the developing neuropil (Pecot, 2013).

    After targeting to a common domain within the outer medulla, L3 growth cones undergo stereotyped changes in shape and position that lead to segregation into the M3 layer. Initially, L3 growth cones are spear-like, spanning much of the depth of the incipient outer medulla. They then expand and elaborate a myriad of filopodia before resolving into flattened synaptic terminals within the M3 layer. This transformation is marked by two prominent steps: extension of processes from one side of the lateral region of the growth cone into the incipient M3 layer and retraction of the leading edge of the growth cone from the incipient M5 layer (part of the domain shared by L1 and L5 growth cones) (Pecot, 2013).

    It has been suggested that CadN may regulate the extension within M3, as this step is partially perturbed in CadN mutant growth cones. However, as CadN mutations affect the initial position of L3 growth cones within the outer medulla, the extension defect within the M3 layer may be indirect. By contrast, in sema-1a mutant growth cones, initial targeting is indistinguishable from wild-type, so defects in retraction away from the incipient M5 layer are likely to reflect a direct role for Sema-1a in this later step in growth cone reorganization. PlexA RNAi phenocopies a sema-1a null mutation and, thus, PlexA is also required for retraction and is likely to function on medulla tangential fibers, where it is most strongly expressed. In support of this, the tip of the L3 growth cone that retracts is in close proximity to these PlexA-expressing fibers (Pecot, 2013).

    The function of Sema-1a/PlexA signaling in sculpting L3 growth cones appears to be distinct mechanistically from the earlier role it plays in confining the growth cones to a common domain. During initial targeting, PlexA acts as a barrier to L3 growth cones and prevents them from projecting beyond the outer medulla. Thus, at this early step, Sema-1a/PlexA interaction provides a stop signal for the leading edge of L3 (uncovered in double mutants with CadN). In the second step, however, Sema-1a/PlexA signaling promotes retraction into the M3 layer. How these diverse outputs of Sema-1a/PlexA signaling arise is unclear. Sema-1a may be coupled to different downstream effectors at each step, modified by association with other receptor subunits, or may be modulated by other extracellular signaling pathways (Pecot, 2013).

    CadN may also play a role in the retraction of L3 growth cones away from the domain shared with L1 and L5 growth cones. In early pupal stages, disrupting CadN function, while leaving growth cone morphology largely spear-like, causes L3 axons to project deeper within the medulla. Under these conditions, Sema-1a function is sufficient to prevent the growth cones from extending beyond the outer medulla. Subsequently, CadN mutant L3 growth cones fail to move away from the outer medulla's proximal edge into the developing M3 layer and thus remain within the most proximal layer, M6. This suggests that CadN, while acting in parallel with Sema-1a to restrict L3 growth cones to the outer medulla initially, may also be required at later stages for movement of the L3 leading edge into the M3 layer. As CadN has been shown previously to regulate neurite outgrowth over cultured astrocytes, it may be required for L3 growth cones to move along adjacent processes. However, the initial projection of L3 axons into the medulla is not affected by CadN mutations, indicating that other components control this process. It also remains possible that the defect in growth cone retraction results indirectly from CadN's earlier role in targeting; this earlier role may account for the defects in growth cone extension within M3 (Pecot, 2013).

    Disrupting CadN function in different neurons affects targeting in unique ways. For example, L5 axons lacking CadN target to the proper layer, but extend inappropriately within the layer into neighboring columns (Nern, 2008). In addition, CadN mutant R7 growth cones display abnormal morphology and, in contrast to mutant L3 growth cones, initially target correctly, but retract to a more superficial medulla region. Collectively, these findings demonstrate that CadN regulates divergent features of growth cone targeting in different contexts. This likely reflects molecular diversity between different growth cones and illustrates the importance of understanding how molecules act in combination to generate target specificity (Pecot, 2013).

    These studies add to previous findings suggesting that column assembly relies on a precisely orchestrated sequence of interactions between different neuronal cell types (Nern, 2008; Timofeev, 2012). This study shows that, as L1, L3, and L5 growth cones expressing Sema-1a enter the medulla, they meet the processes of newly arriving tangential fibers expressing PlexA, which acting in parallel with CadN, prevents extension of these growth cones into the inner medulla. This timing may permit other Sema-1a-expressing growth cones to extend into the inner medulla at earlier stages; these growth cones may then use Sema-1a/PlexA signaling for patterning connections in the inner medulla or deeper neuropils of the lobula complex. Subsequent sculpting of the L3 growth cone, mediated by Sema-1a/PlexA and perhaps CadN, leads to its reorganization into an expanded terminal within M3. As L3 growth cones become restricted to the M3 layer, Netrin, secreted from L3 growth cones, becomes concentrated within the M3 layer, and this, in turn, attracts R8 growth cones to the M3 layer, as recently described by Salecker and colleagues (Timofeev, 2012; Pecot, 2013 and references therein).

    Given the extraordinary cellular complexity of the medulla neuropil, with over 100 different neurons forming connections in different medulla layers, and the few mechanistic clues to layer specific targeting that have emerged so far, a complex interplay between different sets of neurons is envisioned to be required to assemble the medulla circuit. The availability of specific markers for many of these neurons, techniques to follow the expression of even widely expressed proteins at the single cell level as is described in this study, and the ability to genetically manipulate single cells during development provide a robust system for uncovering the molecular logic regulating the layered assembly of axon terminals, dendritic arbors, and synaptic connectivity (Pecot, 2013).

    Identifying functional connections of the inner photoreceptors in Drosophila using Tango-Trace

    In Drosophila, the four inner photoreceptor neurons exhibit overlapping but distinct spectral sensitivities and mediate behaviors that reflect spectral preference. A genetic strategy, Tango-Trace, permits the identification of the connections of the four chromatic photoreceptors. Each of the four stochastically distributed chromatic photoreceptor subtypes make distinct connections in the medulla with four different TmY cells. Moreover, each class of TmY cells forms a retinotopic map in both the medulla and the lobula complex, generating four overlapping topographic maps that could carry different color information. Thus, the four inner photoreceptors transmit spectral information through distinct channels that may converge in both the medulla and lobula complex. These projections could provide an anatomic basis for color vision and may relay information about color to motion sensitive areas. Moreover, the Tango-Trace strategy may be applied more generally to identify neural circuits in the fly brain (Jagadish, 2014).

    Visual stimuli are detected by photoreceptors in the retina and transmitted to the brain to generate an internal representation of the visual world. The brain must then translate this representation of stimulus features into visually guided behaviors. In Drosophila, the retina resembles a crystalline lattice comprised of 750 precisely ordered units, the ommatidia. Each ommatidium contains eight photoreceptor neurons (R1-R8). The outer photoreceptor neurons, R1-R6, express the Rh1 opsin and are thought to receive achromatic visual stimuli that ultimately inform the fly about the form, position, and movement of objects in the visual world. There are two types of ommatidia that differ in the opsins expressed by the inner photoreceptor, R7 and R8. In Pale (p) ommatidia, R7 expresses the near UV-sensitive opsin Rh3 and the R8 cell expresses the blue-sensitive opsin Rh5. In Yellow (y) ommatidia, R7 cells contain the far UV-sensitive opsin Rh4, whereas the R8 cell expresses the green-sensitive opsin Rh6. The existence of the four types of inner photoreceptor neurons, each with overlapping but distinct spectral sensitivities, has implicated these neurons in the recognition of chromatic visual information (Jagadish, 2014).

    Drosophila exhibit phototactic behaviors, strongly preferring UV to green light, a preference that is not observed in flies in which neurotransmitter release is blocked in R7 cells. These observations suggest that the chromatic inner photoreceptors elicit behaviors that reflect spectral preference, but the response to light of distinct wavelengths does not constitute color vision. Color vision requires the ability to distinguish light of distinct spectral composition independent of intensity. The principle of univariance argues that a single photoreceptor cannot distinguish different wavelengths from different intensities of light. Color vision therefore requires a neural system capable of comparing the inputs from photoreceptor neurons in the retina that exhibit different spectral sensitivities. In mammals, this comparison is apparent early in the visual pathway, with a subset of retinal ganglion cells exhibiting color opponency, a feature that reflects opposing neural responses to input from different types of photoreceptor cells. In flies, connections between photoreceptors do not facilitate a comparison of inputs. Color vision would therefore require a comparison of different photoreceptor inputs in downstream visual processing centers. However, the connections of the four inner photoreceptors are largely unidentified and it is unknown whether p and y ommatidia project to identical or distinct downstream circuits (Jagadish, 2014).

    The achromatic R1-R6 neurons project axons to cartridges within the lamina, an optic lobe structure immediately below the retina. The R7 and R8 axons course through the lamina and synapse on second-order neurons within a column in the medulla. The R7 and R8 cells from a single ommatidium project axons to the same column and the topographic organization of the columns maintains retinotopic order. Anatomic studies continue to reveal a vast complexity of richly arborizing intrinsic neurons and projection neurons in the medulla that have seriously hindered the identification of synaptic partners of R7 and R8 neurons. EM studies suggest connections between R7, a medullary projection neuron Tm5 and a medullary intrinsic neuron Dm8. Moreover, genetic studies have shown that Dm8 is necessary for UV spectral preference. Tm neurons in the medulla project their axons to only one of the two neuropils of the lobula complex. TmY cells in the medulla project a branched axon to both the lobula and the lobula plate. The lobula plate contains neurons that respond strongly to motion and loom sensitive neurons have been identified in the lobula plate, but the function of the remaining neural structures within the lobula complex remains obscure (Jagadish, 2014).

    In other insects such as bees that exhibit clear behavioral evidence for color vision, color opponent neurons have been identified with electrophysiologic recordings in the inner layers of both the medulla and lobula as well as by imaging studies in the anterior optic tubercle. Color opponent neurons provide an anatomic substrate for a comparison of the different photoreceptors. Efforts to identify the chromatic neural circuits that could underlie color vision in Drosophila have failed to identify color opponent neurons in the optic lobe. Moreover, behavioral studies have not provided convincing evidence for color vision in the fly (Jagadish, 2014).

    A genetic strategy, Tango-Trace, has been developed that permits tracing of functional synaptic connections of the R7 and R8 photoreceptor neurons in the optic lobe of Drosophila. These studies reveal that each of the four stochastically distributed chromatic photoreceptor subtypes makes a different functional synaptic connection with four different TmY cells in the medulla. The four distinct TmY cells all project to the innermost layer of the lobula and extend axons more diffusely to multiple layers within the lobula plate. The observation that the four inner photoreceptors transmit spectral information through distinct channels that can connect to one another in both the medulla and the lobula complex may provide the anatomic substrate for color vision. Moreover, the Tango-Trace strategy used to trace connections in the dense medullary neuropil may be applied more generally to identify neural circuits in the fly brain (Jagadish, 2014).

    The stochastically distributed inner photoreceptor subtypes each contact a unique postsynaptic TmY cell. The TmY cells, although anatomically distinct in the proximal medulla, share several anatomic features in the lobula and lobula plate. In the lobula plate, each TmY projects to all four layers, whereas in the lobula, arborizations are restricted to the innermost layer. Moreover, each of the four different TmY cells maintains a retinotopic map from the medulla to the lobula complex. Registered images of isosurfaces of different TmYs show that contiguous maps are likely to overlap. Thus, four overlapping topographic maps transmit different color information from individual inner photoreceptor subtypes in the retina to the lobula complex. These observations demonstrate that the four inner photoreceptor subtypes process chromatic visual information in separate and parallel pathways. A given photoreceptor, however, cannot distinguish wavelength differences from intensity differences and color vision therefore requires that signals from the different dedicated TmYs are compared by convergent processing downstream (Jagadish, 2014).

    In the trichromatic mammalian retina, each of the different cones synapse on a different bipolar cell, a feature most clearly illustrated by the S-type cones that synapse on postsynaptic S-ON bipolar cells. Thus, independent parallel information channels continue from the cone to bipolar cell and L versus M opponency, as well as L-M versus S opponency is observed in the retinal ganglion cells (Nassi, 2009). This functional organization in the mammalian retina resembles the parallel channels between the inner photoreceptors of the fly and the four TmY cells. Opponency in Drosophila would therefore be apparent in downstream targets including the proximal medulla or lobula complex. In the honeybee, several types of color opponent neurons have been detected by electrophysiologic recordings in the medulla and lobula, two sites of axon arborization we observe for the four TmY cells. Whatever the site of integration of the four distinct photoreceptor channels, the identification of independent output neurons for the four photoreceptor types suggests an anatomic substrate for the integration necessary for color analysis (Jagadish, 2014).

    Individual inner photoreceptors express only one of the four rhodopsin subtypes, dictated by the stochastic expression of specific transcription factors. Each photoreceptor then projects retinotopically to a single column in the medulla. The stochastically distributed photoreceptor subtypes then contact a unique postsynaptic TmY target with precision. How is this map established? In one model, all columns might contain dendrites from all four potential TmY targets, but an identity code of synaptic specificity cues between photoreceptors and TmY cells assures that only the correct synapses form. Alternatively, each photoreceptor subtype may induce the differentiation of its cognate TmY target, thereby matching pre- and postsynaptic partners. Finally, the specificity of connections may be initiated by columnar guidance cues and TmY receptors (Jagadish, 2014).

    The four TmY cells postsynaptic to each of the chromatic photoreceptor types arborize across all layers of the lobula plate and this may afford a new site of integration of inner and outer photoreceptor signals. Inner photoreceptors provide inputs to motion-sensitive pathways by forming direct, electrical connections with the axon terminals of outer photoreceptors, enhancing the robustness of motion vision. These connections arise at an early stage in visual processing that necessarily precedes comparisons between inner photoreceptor signals and therefore cannot provide color information to motion detecting circuits. Rather, these early connections can broaden the spectral tuning of these pathways. The lobula plate outputs of the TmY cells identified in this study may act redundantly with these peripheral interactions, providing another neural mechanism to enhance motion detection. However, as the arbors of these TmY cells span small numbers of neighboring columns in the proximal medulla, these cells might also represent a site of integration between different spectral inputs. Thus, the projections of these cells to the lobula plate could relay spatially restricted information about color to motion sensitive areas. Although turning responses evoked by optic flow are color insensitive, lobula plate neurons in fruit flies and larger Diptera can be tuned to motion in different directions, complex patterns of optic flow, as well as looming objects. Moreover, distinct motion sensitive circuits guide different behavioral responses. Thus, the pathways defined by these TmY cells might adjust the sensitivity of lobula plate neurons to motion in a color-dependent fashion, providing a mechanism for tuning particular motion-sensitive behaviors to specific spectral environments (Jagadish, 2014).

    Tango-Trace was developed to identify postsynaptic targets of the chromatic photoreceptor neurons. The modular design of Tango-Trace allows a more general application to identify postsynaptic partners of virtually any genetically defined subpopulation of presynaptic cells. A variant Tango mapping approach has been developed to identify sites of dopamine-mediated neuromodulation in the brain. The only requirement for Tango tracing is the knowledge of the neurotransmitter released by the presynaptic neuron allowing the identification and genetic modification of its cognate GPCR essential for Tango-Trace. Tango-Trace affords several advantages as a general approach to trace neural pathways in the fly brain. First, the identification of postsynaptic targets relies on exogenous genes introduced into the fly to create a novel signaling pathway independent of the activation of potential confounding endogenous cell-signaling events. Moreover, this approach transforms rapidly adapting signaling events into a more stable and amplifiable cellular response. In addition, the transcriptional readout of the Tango system not only permits the expression of reporters that allow the visualization of neurons, but also manipulatable effectors including ion channels as well as Ca2+ indicators in restricted populations of postsynaptic neurons. Tango-Trace has particular advantages in the fly brain where tangled neuropils, without apparent structure, predominate and the anatomic proximity of processes cannot reliably predict functional connections. GRASP, which depends upon synaptic level proximity, affords an alternative tracing approach but does not directly report a functional synapse, an important feature inherent in Tango-Trace (Jagadish, 2014).

    One concern remaining with the Tango approach surrounds the question of sensitivity. This study detected four TmY cells and two interneurons, Dm8 and Mia, postsynaptic to the inner photoreceptors. The complete repertoire of neurons in the medulla, postsynaptic to the inner photoreceptors is not known and therefore the effectiveness of labeling for all postsynaptic targets cannot be determined. Over 70 neuronal cell types have been identified in the medulla and the identification of direct targets of R7 and R8 by electron microscopic reconstruction has preliminarily described Dm8, Tm5, Tm9 as well as L1, L2, L3, Dm2, Mi4, Mi9, Mi15, and Tm20 as postsynaptic targets. At present, the EM reconstruction of medulla columns is incomplete because current efforts have focused on the circuits involved in motion detection downstream of outer photoreceptors. Thus, resolution of apparent discrepancies among the various EM reconstruction efforts, and between the EM data and Tango trace, may emerge upon a complete EM reconstruction of a medulla column. The inability to detect targets other than the four TmYs in Tango- Trace may reflect inefficiencies or bias in the identification of postsynaptic targets. This notwithstanding, the identification of the four distinct TmYs and the independent confirmation of their connectivity by functional imaging argues strongly for their participation in specific parallel pathways postsynaptic to the chromatic photoreceptors (Jagadish, 2014).

    Mapping chromatic pathways in the Drosophila visual system

    In Drosophila, color vision and wavelength-selective behaviors are mediated by the compound eye's narrow-spectrum photoreceptors, R7 and R8, and their downstream neurons, Tm5a/b/c and Tm20, in the second optic neuropil, or medulla. These chromatic Tm neurons project axons to a deeper optic neuropil, the lobula, which in insects has been implicated in processing and relaying color information to the central brain. The synaptic targets of the chromatic Tm neurons in the lobula are not known, however. Using a modified GRASP (GFP reconstitution across synaptic partners) method to probe connections between the chromatic Tm neurons and 28 known and novel types of lobula neurons, the visual projection neurons LT11 and LC14, and the lobula intrinsic neurons Li3 and Li4, were identified anatomically as synaptic targets of the chromatic Tm neurons. Single-cell GRASP analyses revealed that Li4 receives synaptic contacts from over 90% of all four types of chromatic Tm neurons while LT11 is postsynaptic to the chromatic Tm neurons with only modest selectivity and at a lower frequency and density. To visualize synaptic contacts at the ultrastructural level, a 'two-tag' double labeling method was developed and applied to label LT11's dendrites and the mitochondria in Tm5c's presynaptic terminals. Serial electron microscopic reconstruction confirmed that LT11 receives direct contacts from Tm5c. This method would be generally applicable to map the connections of large complex neurons in Drosophila and other animals (Lin, 2015).

    The developmental rules of neural superposition in Drosophila

    Complicated neuronal circuits can be genetically encoded, but the underlying developmental algorithms remain largely unknown. This study describes a developmental algorithm for the specification of synaptic partner cells through axonal sorting in theDrosophila visual map. This approach combines intravital imaging of growth cone dynamics in developing brains of intact pupae and data-driven computational modeling. These analyses suggest that three simple rules are sufficient to generate the seemingly complex neural superposition wiring of the fly visual map without an elaborate molecular matchmaking code. This computational model explains robust and precise wiring in a crowded brain region despite extensive growth cone overlaps and provides a framework for matching molecular mechanisms with the rules they execute. Finally, ordered geometric axon terminal arrangements that are not required for neural superposition are a side product of the developmental algorithm, thus elucidating neural circuit connectivity that remained unexplained based on adult structure and function alone (Langen, 2015).

    A central question in neuroscience is how neural circuits self-organize into functional structures during development. The wiring of compound eyes to the brain of flies provides a fascinating model system for studying this question. In particular, the neural superposition eye, such as found in advanced flies, is characterized by a complicated wiring diagram: each point in visual space is captured by multiple photoreceptors from different ommatidia that converge upon the same synaptic unit (cartridge) in the brain; different photoreceptors within the same ommatidium view different points in visual space and project to neighboring cartridges. The correct pooling of axon terminals viewing the same point in space into a single cartridge increases sensitivity without loss of spatial resolution compared with simpler, ancestral eye types. The developmental process underlying neural superposition is remarkable, because each individual axon, among thousands of neighboring axons in the brain, must be sorted together with those few axons that receive input from the same point in visual space (Langen, 2015).

    A classic model of neural superposition is found in the Drosophila compound eye, which contains ~800 ommatidia. Each ommatidium projects a bundle of eight photoreceptor (retinula or R-cell) axons into the brain. Six of these photoreceptors, R1-R6 (the focus of the current study) form the primary visual map in the lamina (first optic neuropil) of the fly brain. The R1-R6 axons from one bundle that receive input from six different points in visual space are denoted A-F (Langen, 2015).

    After neural superposition is established, the R-cells have a precise organization of the six subtypes around the circumference of cartridges, that is, R1 neighbors R2, which neighbors R3, etc., referred to as 'rotational stereotypy'. The precision of rotational stereotypy is noteworthy, as the six axon terminals in a cartridge carry the same input information and synapse with the same postsynaptic target cells. Hence, rotational stereotypy is not a functional requirement for neural superposition and increases the demands placed on the sorting problem from 800 cartridges to 4,800 (800 × 6 R1-R6) precise terminal positions. The role, development, and evolutionary origin of this wiring precision are unknown (Langen, 2015).

    The neural superposition wiring diagram has a 'canonical' pattern of six R-cell axon terminals per cartridge. An equator from anterior to posterior divides the compound eye, as well as the wiring pattern in the lamina, into dorsal and ventral halves. The wiring patterns in each half of the lamina are opposite to one another with respect to the equator axis. As a consequence, six rows of "non-canonical" cartridges exist at the equator that contain stereotypic compositions of seven or eight R1-R6 cell axon terminals. The three different types of equator cartridges also exhibit rotational stereotypy, each with a distinct pattern. As in the case of canonical cartridges, the function of the rotational stereotypic arrangement of photoreceptor terminals within the equator cartridges is unknown. It is unclear which common developmental rules or mechanisms might robustly encode the canonical cartridges, as well as the three types of equator cartridges (Langen, 2015).

    The Drosophila visual system is an example of a genetically encoded neural circuit in which a developmental sorting step precedes and ensures synaptic specificity between input neurons and their targets. Many aspects of the developmental sorting step have been characterized in detail, including the formation of an initial grid by lamina cells. Previous studies have suggested the possibility of simple developmental rules underlying this sorting process. Furthermore, work in recent years has revealed molecular mechanistic insight into how differential adhesion of guidance receptors may play a key role in growth cone sorting. However, no rule set or algorithm has been formulated that is sufficient to generate precise neural superposition in canonical cartridges and equator cartridges. Two key challenges have been (1) the inability to monitor the dynamic sorting process live in developing flies and (2) lack of quantitative, data-driven models to conceptualize or test understanding of this apparently complicated process (Langen, 2015).

    This study reports live imaging of R1-R6 growth cone dynamics in intact developing pupa and the derivation of a model that summarizes the conceptual understanding of the development of neural superposition. It is proposed that three simple rules are sufficient to provide a solution to the neural superposition sorting problem. Systematic tests of these rules in a computational model reveal that the same rule set leads to precise superposition and the three types of cartridges observed at the equator (Langen, 2015).

    This study describes a developmental algorithm for the axonal sorting of ~4,800 presynaptic cells in the primary visual map of Drosophila. The work suggests that the neural superposition wiring diagram found in adult fly brains can be established through simple, local pattern formation principles without the need for an elaborate molecular matchmaking code. Codification of the developmental algorithm reveals quantitative constraints and provides a conceptual framework for molecular mechanisms that execute these rules (Langen, 2015).

    The current findings, together with previous studies, support the following developmental algorithm.

    Rule 1: The Scaffolding Rule: Incoming rows of axon bundles from individual ommatidia are organized in a repeating pattern of evenly spaced semi-circles. This pattern and spacing of original axon arrival points provide a scaffold that remains stable during the entire process of growth cone sorting and is required for neural superposition. The future target areas are encircled by the anchored heels and thus already defined prior to growth cone movements. How the precision of the scaffold pattern develops is unknown. The scaffold is likely to instruct the extension angle through non-autonomous R-cell interactions within a bundle. Such intra-bundle interactions have been proposed to play a more prominent role than do interactions across bundles (Schwabe, 2013). To which extent the geometric arrangement of heels observed in the scaffold is influenced by their axonal arrangements within the bundles or by other cells within the target area is unclear (Langen, 2015).

    Rule 2: The Extension Rule: All R1-R6 growth cones extend synchronously with speeds and angles specific to their R-cell subtype during the 5-10 hr of extension. The extension is unaffected by highly varying environments at the equator and thus is unlikely to depend on R-cell front interactions. However, precise extension dynamics may require permissive R-cell heel interactions and recognition of other cells that are equally distributed across the equator as instructive guides. It is unlikely that R-cell growth cones simply extend toward attractive cues at the target regions because the growth cones can overlap with several target regions throughout their sorting (including the target regions closest to the heels). Based on these observations, it is considered that the extension process of the bipolar R-cell growth cones differs from the classic view of growth cone movements toward attractive targets (Langen, 2015).

    Rule 3: The Stop Rule: The target regions defined by the scaffold (and the L-cells therein) provide possible, but poor, targets for R-cell fronts to stop extending, because those R-cell fronts overlap with multiple targets simultaneously and throughout their extension. In addition, all R-cell fronts increasingly overlap with other R-cell fronts throughout their extension. The computational model reveals that stop rules based on R-cell front overlap function even without any target-derived cues and are more robust than a 'target only' model under the same conditions. A target model using coincidence detection of overlap with other R fronts, as well as target L-cells, performs best. R-cell front interaction is predicted to be part of the stop rule because of reduced robustness at the equator and because of the rotational stereotypy of R-cell terminal positions within cartridges. These two observations also argue against 'no interaction' stop rules. However, the results do not rule out the existence of a synchronously applied stop signal that could act as part of a combinatorial stop rule. The precise nature and molecular correlate of the stop rule remain unknown (Langen, 2015).

    Previous work has revealed important insights into further constraints of these rules. Most importantly, the 180°rotation of a single bundle results in 180° rotated extension angles. This finding is consistent with the model. In addition, differential subtype dependencies have been unraveled, where R1, R2, R5 and R6 targeting depend on R3 and R4, but not the other way round. Whether this dependency arises from the scaffolding, extension, or stop rule remains to be determined. It is not yet known whether reconciliation of the current model with these observations arises from constraining existing rules or requires new ones (Langen, 2015).

    After growth cone sorting is complete, a process of centripetal growth commences synchronously from all R-cell fronts and these then generate R-cell terminal columns orthogonal to the lamina plexus. This columnar extension preserves and freezes the relative positions of R-cell fronts in the lamina plexus; the resulting columns of R-cell terminals then define the adult lamina (Langen, 2015).

    Complicated wiring diagrams can originate through the iterative execution of simple rules. Early brain development is associated with genetically encoded pattern formation rules, while later phases of synapse specification often depend on neuronal activity. It is unclear which level of synaptic partner specification can be achieved through simple, genetically encoded developmental rules. This study has focused on the identification of such rules and their quantitative constraints using the genetically hard-wired Drosophila visual map as a model (Langen, 2015).

    Much previous elegant work has focused on searching for molecular codes underlying synaptic partner specification. Such codes may be characterized by either many molecular cues (e.g., olfactory systems) or fewer molecular cues that are dynamically localized (e.g., the fly's visual system). The current work on identifying an underlying developmental algorithm provides a framework for matching these molecular mechanisms with the rules they execute. For example, recent studies on guidance receptors of the Cadherin family have provided strong evidence for a role of differential adhesion in R-cell growth cone sorting (Schwabe, 2013; Schwabe, 2014). Specifically, R-cell growth cones interact through differential adhesion of the protocadherin Flamingo, both within the same bundle. The current data are consistent with the idea that Flamingo-dependent differential adhesion between R-cell heels prior to extension determines the extension angle (thus exercising a role in the scaffolding rule). In contrast, interactions between moving R-cell fronts are unlikely to instruct extension itself (no role in the extension rule). However, studies on the guidance receptor N-Cadherin suggest a role for the interaction of R-cell growth cones with L-cells in the target cartridge. These findings are consistent with a role of N-Cadherin-mediated interactions between R-cell fronts and L-cells as part of the stop rule and thereby indicate that L-cell interactions contribute to the stop rule. These interpretations of roles of Flamingo in the R-cell heel (as part of the scaffolding rule) and N-Cadherin in the R-cell front (as part of the stop rule) are further supported by their subcellular localization within the growth cone (Schwabe, 2013; Langen, 2015 and references therein).

    Finally, the model supports R-cell front interactions as part of the stop rule. It is unclear to which degree this interaction is based on differential adhesion. How molecular signal integration is implemented to utilize the substantial increasing overlap of R-cell fronts as a stop signal remains to be discovered (Langen, 2015).

    Both the equator and the rotational stereotypy of R-cell terminals have received little recent attention in the study of growth cone sorting and neural superposition, perhaps because they appear to be complications of an already complicated wiring problem. In particular, the findings of four types of rotational stereotypy within cartridges across the entire lamina have not been addressed in the literature since their discovery more than 40 years ago. The stereotypic arrangement of R1-R6 terminals in cartridges that encode precise neural superposition increases the apparent number of target slots 6-fold; yet, this arrangement is not required for neural superposition, given that all six carry the same information and synapse with the same output neurons. This study shows that evolutionary selection of the developmental algorithm that ensures precise axon sorting required for neural superposition wiring is sufficient to establish rotational stereotypy. While it is possible that rotational stereotypy may serve a function independent of neural superposition, selection for such a putative unknown function is not required to explain its occurrence. Hence, the fly's visual map provides an example for a neuronal circuit whose connectivity map can only be understood through its developmental context. Knowledge of a circuit's developmental algorithm may more generally help to explain aspects of neuronal circuits that cannot be derived from the study of the adult wiring diagrams alone (Lengen, 2015).

    Cholinergic circuits integrate neighboring visual signals in a Drosophila motion detection pathway

    Detecting motion is a feature of all advanced visual systems, nowhere more so than in flying animals, like insects. In flies, an influential autocorrelation model for motion detection, the elementary motion detector circuit (EMD), compares visual signals from neighboring photoreceptors to derive information on motion direction and velocity. This information is fed by two types of interneuron, L1 and L2, in the first optic neuropile, or lamina, to downstream local motion detectors in columns of the second neuropile, the medulla. Despite receiving carefully matched photoreceptor inputs, L1 and L2 drive distinct, separable pathways responding preferentially to moving 'on' and 'off' edges, respectively. Serial electron microscopy (EM) identifies two types of transmedulla (Tm) target neurons, Tm1 and Tm2, that receive apparently matched synaptic inputs from L2. Tm2 neurons also receive inputs from two retinotopically posterior neighboring columns via L4, a third type of lamina neuron. Light microscopy reveals that the connections in these L2/L4/Tm2 circuits are highly determinate. Single-cell transcript profiling suggests that nicotinic acetylcholine receptors mediate transmission within the L2/L4/Tm2 circuits, whereas L1 is apparently glutamatergic. It is proposed that Tm2 integrates sign-conserving inputs from neighboring columns to mediate the detection of front-to-back motion generated during forward motion (Takemura, 2011).

    Given that both L2 and L4 express Choline acetyltransferase (Cha) and are thus genotypically qualified to synthesize acetylcholine and provide cholinergic input to Tm2, the expression of acetylcholine receptors in Tm2 was profiled. This proved more complex than for L2 and L4. In addition to Dα7 and Dβ1 nAcR shared with L2 and L4, Tm2 also expressed Dα1/2 and Dβ2 nAcR. The exclusive expression of nicotinic rather than muscarinic receptors (nAcR not mAcR) in Tm2 suggests that both L2 and L4 provide fast excitatory inputs to Tm2. It was also found that Tm2 expressed Cha but not VGlut, indicating that, like L2 and L4, Tm2 is also genotypically cholinergic. In summary, these data predict that both synaptic connections in the L2/L4/Tm2 network are mediated by excitatory acetylcholine systems, and therefore sign-conserving (Takemura, 2011).

    While either the L1 or L2 channel alone can mediate rudimentary motion detection, each also responds differentially in walking flies, and in head-yaw assays the L2 pathway is preferentially tuned to front-to-back motion. Although the connections between L4 and L2 along the anteroposterior direction might account for this front-to-back preference, these connections are reciprocal so that information also flows from posterior to anterior, while L2's activity fails to reveal asymmetrical responses. Between L2's two targets, only Tm2 receives two additional L4 inputs from neighboring posterior columns; Tm1 does not. These L2/L4/Tm2 connections are highly determinate, underscoring a critical role in connecting neighboring L2 channels along the AP direction, in what is arguably the most important motion direction for flies since it occurs during forward flight. Interestingly, other flies have a Tm neuron closely resembling Drosophila's Tm2 morphologically, for example Tm1 in the calliphorid Phaenicia. This is proposed to receive L2 inputs, suggesting that an L2/L4/Tm2 network might be conserved in higher Diptera (Takemura, 2011).

    Tm2 could conceivably serve as half of the EMD's multiplier stage, comparing the temporally delayed input from collateral L4s with the cognate signal from L2. However, electrophysiological investigations on calliphorid 'Tm1' neurons, which resemble morphologically Drosophila's Tm2, have yet to provide strong evidence for this role. An alternative interpretation is that the L2/L4/Tm2 network serves instead as a prefilter in the preprocessing stage while Tm2's output feeds into the multiplier stage. The topology and sign-conserving nature of L4/Tm2 connections suggest the spatial summation of neighboring visual signals, which could increase light sensitivity at the expense of spatial acuity. It has been suggested that under low luminance conditions, neighboring visual signals are pooled prior to their interaction at the multiplier stage, while at higher luminance levels nearest-neighbor interactions dominate motion detection. Alternatively, the L4/Tm2 connections could convert visual signals sampled from the hexagonal ommatidial array into an orthogonal coordinate upon which motion signals can be derived. Differentiating between these possibilities must await future investigations that combine genetic and electrophysiological approaches (Takemura, 2011).

    The temporal tuning of the Drosophila motion detectors is determined by the dynamics of their input elements

    Detecting the direction of motion contained in the visual scene is crucial for many behaviors. However, because single photoreceptors only signal local luminance changes, motion detection requires a comparison of signals from neighboring photoreceptors across time in downstream neuronal circuits. For signals to coincide on readout neurons that thus become motion and direction selective, different input lines need to be delayed with respect to each other. Classical models of motion detection rely on non-linear interactions between two inputs after different temporal filtering. However, recent studies have suggested the requirement for at least three, not only two, input signals. This study comprehensively characterized the spatiotemporal response properties of all columnar input elements to the elementary motion detectors in the fruit fly, T4 and T5 cells, via two-photon calcium imaging. Between these input neurons, large differences were found in temporal dynamics. Based on this, computer simulations show that only a small subset of possible arrangements of these input elements maps onto a recently proposed algorithmic three-input model in a way that generates a highly direction-selective motion detector, suggesting plausible network architectures. Moreover, modulating the motion detection system by octopamine-receptor activation, the temporal tuning of T4 and T5 cells was found to be shifted toward higher frequencies, and this shift can be fully explained by the concomitant speeding of the input elements (Arenz, 2017).

    The emergence of directional selectivity in the visual motion pathway of Drosophila

    The perception of visual motion is critical for animal navigation, and flies are a prominent model system for exploring this neural computation. In Drosophila, the T4 cells of the medulla are directionally selective and necessary for ON motion behavioral responses. To examine the emergence of directional selectivity, genetic driver lines were developed for the neuron types with the most synapses onto T4 cells. Using calcium imaging, it was found that these neuron types are not directionally selective and that selectivity arises in the T4 dendrites. By silencing each input neuron type, which neurons are necessary for T4 directional selectivity and ON motion behavioral responses were identified. Tthe sign of the connections between these neurons and T4 cells were determined using neuronal photoactivation. These results indicate a computational architecture for motion detection that is a hybrid of classic theoretical models (Strother, 2017).

    Transgenic line for the identification of cholinergic release sites in Drosophila melanogaster

    The identification of neurotransmitter type used by a neuron is important for the functional dissection of neuronal circuits. In the model organism Drosophila melanogaster, several methods for discerning the neurotransmitter systems are available. This study expanded the toolbox for the identification of cholinergic neurons by generating a new line FRT-STOP-FRT-VAChT::HA that is a conditional tagged knock-in of the VAChT gene in its endogenous locus. Importantly, in comparison to already available tools for the detection of cholinergic neurons, the FRT-STOP-FRT-VAChT::HA allele also allows for identification of the subcellular localization of the cholinergic presynaptic release sites in a cell-specific manner. The newly generated FRT-STOP-FRT-VAChT::HA line was used to characterize the Mi1 and Tm3 neurons in the fly visual system and found that VAChT is present in the axons of the both cell types, suggesting that Mi1 and Tm3 neurons provide cholinergic input to the elementary motion detectors, the T4 neurons (Pankova,, 2017).

    Orientation selectivity sharpens motion detection in Drosophila

    Detecting the orientation and movement of edges in a scene is critical to visually guided behaviors of many animals. What are the circuit algorithms that allow the brain to extract such behaviorally vital visual cues? Using in vivo two-photon calcium imaging in Drosophila, this study describes direction selective signals in the dendrites of T4 and T5 neurons, detectors of local motion. This circuit performs selective amplification of local light inputs, an observation that constrains motion detection models and confirms a core prediction of the Hassenstein-Reichardt correlator (HRC). These neurons are also orientation selective, responding strongly to static features that are orthogonal to their preferred axis of motion, a tuning property not predicted by the HRC. This coincident extraction of orientation and direction sharpens directional tuning through surround inhibition and reveals a striking parallel between visual processing in flies and vertebrate cortex, suggesting a universal strategy for motion processing (Fisher, 2015).

    Cellular evidence for efference copy in Drosophila visuomotor processing

    Each time a locomoting fly turns, the visual image sweeps over the retina and generates a motion stimulus. Classic behavioral experiments have suggested that flies use active neural-circuit mechanisms to suppress the perception of self-generated visual motion during intended turns. Direct electrophysiological evidence, however, has been lacking. This study found that visual neurons in Drosophila receive motor-related inputs during rapid flight turns. These inputs arrive with a sign and latency appropriate for suppressing each targeted cell's visual response to the turn. Precise measurements of behavioral and neuronal response latencies support the idea that motor-related inputs to optic flow-processing cells represent internal predictions of the expected visual drive induced by voluntary turns. Motor-related inputs to small object-selective visual neurons could reflect either proprioceptive feedback from the turn or internally generated signals. These results in Drosophila echo the suppression of visual perception during rapid eye movements in primates, demonstrating common functional principles of sensorimotor processing across phyla (Kim, 2015).

    Humans scanning a visual scene show periods of stable gaze punctuated by rapid eye movements called saccades. During saccades, the visual image translates briskly over the retina and the nervous system employs mechanisms to suppress the perception of such self-induced motion stimuli so as to help perceive the outside environment as stationary. Primate saccades represent just one example in which sensorimotor processing must distinguish between self-generated sensory stimulation, also known as reafference, from externally generated stimulation, or exafference. In a few model systems, there is now even a developed understanding of how neuronal circuits distinguish exafference from reafference. For example, male crickets have an identified interneuron that activates during chirps and inhibits auditory neurons to prevent them from responding to the chirp, whereas weakly electric fish use circuitry in a cerebellum-like structure to subtract the predicted sensory input, resulting from the animal's own electric organ discharge from the incoming electrical sensory stream. One common scenario in which the suppression of reafference is essential, but remains poorly understood, is in the inhibition of stability reflexes during voluntary locomotor turns. Consider a flying fly. Analogously with human eye movements, the fly punctuates periods of stable flight with rapid turns called body saccades. Between saccades, the fly employs an optokinetic reflex, also known as the optomotor response, to help maintain stable flight. In this reflex, wide-field visual motion, say, to the right, is interpreted as being caused by an erroneous turn of the fly to the left (perhaps caused by a gust of wind or noise in the flight motor) and a corrective rightward turn is elicited. Although this reflex is important for stability, if it were always active, it would act against any intended change to the locomotor trajectory. This begs the question of how then do flies ever turn (Kim, 2015).

    Guided by simple behavioral experiments, von Holst and Mittelstaedt famously postulated that with each motor command to initiate a voluntary locomotor turn, also known as an efference, flies send a copy of the command, an efference copy, to their visual system. This efference-copy signal was postulated to have the correct sign and magnitude for silencing the reafferent visual input caused by voluntary turns, thereby preventing the optomotor response from kicking in. Subsequent behavioral experiments have continued to argue that locomoting insects send efference copies to their visual system or make use of internal models in their visuomotor processing; however, electrophysiological evidence has been scarce (Kim, 2015).

    This study found that Drosophila visual neurons received motor-related inputs during voluntary body saccades. These inputs were similar in magnitude, but opposite in sign, to the expected reafferent visual input caused by saccades. Responses of wide field-sensitive neurons in the visual lobe and a new class of small object-selective cells in the central brain were strongly suppressed during intended flight turns. Other visual cells, whose receptive-field properties were such that they should not respond to wide-field motion during saccades, were only mildly affected. These findings demonstrate cell type-tailored signals that are appropriate for silencing reafferent visual responses during voluntary locomotor turns in flies, as predicted by von Holst and Mittelstaedt 65 years ago (Kim, 2015).

    Drosophila visual neurons receive both visually driven and motor-related inputs with voluntary saccades. Motor-related inputs were tailored in sign and latency to effectively suppress neuronal responses to the reafferent visual motion resulting from saccades. Motor-related modulations are observed in cells that arborize in two late stages of visual processing, the lobula plate and the optic glomeruli, and these modulations are sufficiently strong to cancel, rather than just weakly modulate, visual signaling (Kim, 2015).

    Von Holst and Mittelstaedt were careful to distinguish silencing of expected visual motion, the computation they believed to take place in fly brains, from complete blindness during voluntary turns. At the cellular level, some visual neurons, such as VS1-2 cells, did not receive prominent motor-related inputs (SRPs or saccade related potentials) during voluntary saccades, and other cells, such as horizontal system north (HSN) cells, were modified in a direction-selective manner that should have allowed them to still respond to unexpected (exafferent) visual motion. Thus, although flies suppress visual input during rapid flight turns, it is unlikely that they are completely blind supporting von Holst and Mittelstaedt's model. In many scenarios, it might make sense for motion-sensitive neurons to actually sense reafference; however, because HSN cells likely contribute to optomotor stability, eliminating reafference in their output signal during voluntary turns is sensible. It has been argued, based on behavioral experiments, that flying flies completely ignore certain visual motion during saccades or selectively ignore visual motion depending on whether this motion is in the expected or unexpected direction. The current data provide a plausible cellular explanation for these behavioral results (Kim, 2015).

    Although the fly visual system, overall, shows cell type-specific and direction-selective silencing, it is not yet clear whether von Holst and Mittelstaedt's computation is instantiated in its full form in the Vm of single cells. To do so, each visual neuron would receive a saccade-associated input that is not just of the correct sign, but also of the correct time-varying magnitude to exactly cancel the expected visual drive associated with each voluntary turn. Activating such a negative-image input would require that fly brains instantiate a forward model to predict the visual drive that each neuron will experience from a given saccade. In realizing such a model, flies should scale their silencing signals by an internal estimate of the velocity time course of the upcoming saccade (using a so-called inverse model), as well as by how strongly the current visual environment (for example, a forest or a fog) is expected to drive each visual neuron during each saccade. If flies make use of internal models of this sort, it will be important to determine how they are implemented in the nervous system and how widespread their influence is on sensory processing and behavior (Kim, 2015).

    At face value, the fact that SRPs grew in magnitude when flies generated saccades in the context of a preferred steady-state stimulus compared with a uniformly lit screen and the fact that this growth of SRP magnitude was quantitatively matched to the level of ongoing visual drive supports the forward model idea. Note, however, that if saccade-related inputs activate a consistent membrane conductance, independent of visual context, for saccades of a certain direction and size, then saccade-related potentials will naturally grow in size as the cell's Vm moves further away from the reversal potential associated with that conductance. Given that ongoing visual stimulation causes cells to depolarize or hyperpolarize from the resting Vm, SRPs may grow in magnitude as a result of this reason alone. Future work will be needed to differentiate this simple biophysical explanation for why SRPs grow in magnitude during ongoing visual drive, which may represent a rudimentary implementation of a forward model, from a more sophisticated process in which the strength of motor-related conductances in visual neurons are actively scaled on the basis of the structure of the visual environment. Notably, HS cells appear to show similar visual responses to moving natural scenes over a wide range of contrast levels and arrangements of local features40. This fact may allow an efference copy system to get away with injecting silencing signals that have a consistent time-varying profile for saccades of a given direction and magnitude, independent of the structure of the visual environment. Flies may also continuously calibrate the strength of their efference copy signal based on the difference between predicted and experienced sensory feedback (Kim, 2015).

    Regardless of whether motor-related inputs are scaled by the structure of the visual environment, an important associated question is whether, in a fixed visual environment, the motor-related inputs are scaled by the magnitude and duration of each saccade. Because torque is not measured in current platform, even if L-R WBA acts as a decent proxy, and because tethered-flight saccades are known to have altered dynamics relative to free-flight saccades, it is difficult to provide a definitive answer to this question with current methods (Kim, 2015).

    Classically, only neurons that contribute to optomotor stability, such as HSN cells, should be silenced during voluntary turns. This study found that small object-selective optic-glomeruli interneurons (OGINs) were also silenced. Why might this be? Object-selective OGINs may contribute to behaviors such as small object avoidance during flight or tracking of conspecifics during Drosophila courtship. Distinguishing reafference from exafference would seem to be critical for such object-orienting behaviors to prevent behavioral responses to object motion on the retina caused by the fly's own movements. When a locomoting fly turns in a cluttered visual environment, the image of the entire cluttered panorama, not just that of the small object, translates globally on the retina, and such a global stimulus, simulated by the grating in receptive field mapping experiments, will not excite these OGINs. Thus, the native stimulus selectivity of these cells already helps to distinguish exafferent from reafferent object motion. However, if a single object were situated on a sparse background, such as a spider against a homogenous blue sky, OGINs may very well respond to the reafferent motion of such an object during a locomotor turn and the motor-related silencing mechanism that is described would abrogate this deleterious sensory response (Kim, 2015).

    Mechanistically, SRPs in OGINs are consistent with an inhibitory input that arrives on each saccade, either directly to these cells or to upstream neurons, to reduce feedforward excitatory drive. Because OGINs are spiking cells (although their spikes are often very small when measured at the soma), the role of SRPs in these neurons may be to simply eliminate spike output during saccades rather than to activate a precisely time-varying negative-image input. Indeed, SRPs in OGINs lasted ~400-500 ms, which is longer than the time course of the expected reafference during a typical saccade. If OGIN SRPs are the result of mechanosensory feedback associated with saccades, this feedback signal would be expected to be prolonged in tethered flight, as tethered-flight saccades lasted longer (~300-500 ms) than free-flight saccades (~50-150 ms). By contrast, HSN cells are non-spiking neurons that signal both with hyperpolarizations and depolarizations of Vm. As such, HSN cells received both depolarizing and hyperpolarizing SRPs, whose duration (~150-200 ms) was more closely matched to the expected reafference from a typical saccade. SRPs in HSN cells may serve a function closer to that of a negative image of the expected visual drive (Kim, 2015).

    The average SRP in HSN cells begins ~30 ms before the wings initiate a saccade, arguing for an internal (rather than sensory feedback) origin for these signals. However, if the sole function of motor-related inputs to HSN cells were to silence visual reafference, one might expect the efference-copy signal to kick in only after the body starts turning, once reafferent visual input is arriving at the cell. One intriguing possibility is that the early component of the motor-related input to HSN cells might help to actually drive the voluntary turn by injecting a small pre-charging signal into the optomotor reflex system, hijacking its natural coupling to the neck and flight motor systems. The sign of this early component is consistent with this possibility, although a rigorous test will require a specific manipulation of the SRPs to the optomotor system, which likely includes many more cells than just HS cells. Flies stabilize flight not just with vision, but also with mechanosensory inputs from their modified hindwings, called halteres, and a similar pre-charging idea was postulated to occur in the haltere stability system during turns, a hypothesis that should now be revisited (Kim, 2015).

    Dynamic modulations of visual signaling have been studied primarily during saccadic eye movements in primates. The current results open the door to studying cellular mechanisms for similar processes in Drosophila. For vision research, flies have already offered key insights into state-dependent sensory processing and the circuit basis for direction selectivity. Drosophila may now help in understanding how brains build forward models and how they use these models to modify sensory processing. The tiny fly brain may not perform these tasks in exactly the same manner as the primate brain. However, the advanced genetic and physiological tools in Drosophila should allow for a detailed cellular- and circuit-level description of how the fly brain models and predicts the outside world. This description could yield insight on how similar predictive processes are implemented in all brains (Kim, 2015).

    Cross-modal influence of mechanosensory input on gaze responses to visual motion in Drosophila

    Animals typically combine inertial and visual information to stabilize their gaze against confounding self-generated visual motion, and to maintain a level gaze when the body is perturbed by external forces. In vertebrates, an inner ear vestibular system provides information about body rotations and accelerations, but gaze stabilization is less understood in insects, which lack a vestibular organ. In flies, the halteres, reduced hindwings imbued with hundreds of mechanosensory cells, sense inertial forces and provide input to neck motoneurons that control gaze. These neck motoneurons also receive input from the visual system. Head movement responses to visual motion and physical rotations of the body have been measured independently, but how inertial information might influence gaze responses to visual motion has not been fully explored. In this study, the head movement responses to visual motion were measured in intact and haltere-ablated tethered flies to explore the haltere's role in modulating visually-guided head movements in the absence of rotation. It is noted that visually-guided head movements occur only during flight. Although halteres are not necessary for head movements, the amplitude of the response is smaller in haltereless flies at higher speeds of visual motion. This modulation occurred in the absence of rotational body movements, demonstrating that the inertial forces associated with straight tethered flight are important for gaze-control behavior. The cross-modal influence of halteres on the fly's responses to fast visual motion indicates that the haltere's role in gaze stabilization extends beyond its canonical function as a sensor of angular rotations of the thorax (Mureli, 2017).

    Optogenetic control of fly optomotor responses.

    When confronted with a large-field stimulus rotating around the vertical body axis, flies display a following behavior called 'optomotor response.' As neural control elements, the large tangential horizontal system (HS) cells of the lobula plate have been prime candidates for long. This study applied optogenetic stimulation of HS cells to evaluate their behavioral role in Drosophila. To minimize interference of the optical activation of channelrhodopsin-2 with the visual perception of the flies, a bistable variant was used called ChR2-C128S. By applying pulses of blue and yellow light, it was first demonstrated electrophysiologically that lobula plate tangential cells can be activated and deactivated repeatedly with no evident change in depolarization strength over trials. It was next shown that selective optogenetic activation of HS cells elicits robust yaw head movements and yaw turning responses in fixed and tethered flying flies, respectively (Haikala, 2013).

    To safely navigate through space particularly fast-moving animals face the challenging need of constantly integrating changing information about the environment and self-motion. Flies are capable of impressively robust, precise, and fast flight maneuvers that largely depend on visual and mechanosensory information (Haikala, 2013).

    Depending on the species, a bilateral set of 20-60 large neurons, termed lobula plate tangential cells (LPTCs), located in the posterior part of the optic lobes, are considered critical for vision-based estimation of self-motion. LPTCs are individually identifiable and have large receptive fields, sometimes covering more than one hemisphere of visual space. Their most distinguishing characteristic is their directional tuning to visual wide-field motion, termed optic flow (Haikala, 2013).

    Among the LPTCs, three horizontal system (HS) cells sensitive to horizontal wide-field motion, such as occurring during rotation about the vertical body axis, have long been prime candidates to control compensatory yaw head movements and body turns of the fly. This suggestive notion rests on the following observations: (1) Mutant fruit flies in which LPTCs are missing or defective show a strong reduction in their optomotor response. (2) Cutting HS cell axons in flies or laser ablation of HS/vertical system precursor cells in larvae significantly affects the optomotor response of adult flies. (3) Extracellular electrical stimulation of the lobula plate region where HS cells are located elicits yaw turning responses. While all of these findings support the idea that HS cells control optomotor responses, it has not been conclusively shown that activation of HS cells is sufficient to evoke yaw optomotor behavior (Haikala, 2013).

    This study analyzed the role of HS cells for head movements and flight-turning responses by stimulating them optogenetically in Drosophila. When a fly is visually stimulated by a pattern rotating around the vertical axis, its HS cells depolarize on the side where motion progresses from front to back, and the fly displays syndirectional head movements and flight turns. If HS cells control both these optomotor responses, unilateral optogenetic depolarization of HS cells alone should mimic these behaviors. To avoid behavioral artifacts due to activation of photoreceptors during optical stimulation of HS cells, this study generated flies expressing the bistable channelrhodopsin-2 variant ChR2-C128S, a depolarizing cation channel that can be switched between an open and closed configuration with brief light pulses of different wavelengths. Expressing ChR2-C128S in HS cells via a selective driver line enabled control of the cells in a similar way as visual motion does. When HS cells were activated in one brain hemisphere, flies turned their heads toward the stimulated side. Likewise, tethered flying flies displayed unilateral wing beat changes indicative of flight-turning responses in the same direction. The results strongly suggest that HS cell activity alone is sufficient to elicit yaw head movements and flight-turning responses in Drosophila (Haikala, 2013).

    In recent years, optogenetic methods have been successfully applied in a number of studies, involving different species such as Caenorhabditis elegans, zebrafish, and mice. Nonetheless, its use in Drosophila has been rather limited, in particular for analyzing visual circuits, perhaps in part because light delivery to activate light-sensitive ion channels inevitably leads to direct stimulation of the photoreceptors and thus interferes with the visual processing to be studied. To avoid this problem, optogenetic stimulation has been performed in blind flies. This strategy, however, raises potential concerns as to whether the circuits are compromised by visual deprivation. Moreover, it precludes the simultaneous visual probing of the system. In this respect, the development of switchable, bistable channelrhodopsin-2 variants offers a powerful solution. As this study has shown, one of these variants (ChR2-C128S) allows for prolonged and repeated excitation of large motion-sensitive neurons in the Drosophila visual system by delivery of pulses of light with the appropriate wavelength (Haikala, 2013).

    While previous work in Drosophila and Calliphora has already put forward the idea that HS cells control yaw optomotor behavior, well in agreement with their visual response properties, only one study actually tested their requirement directly by lesioning HS cell axons unilaterally and recording the yaw torque response to visual motion. The resulting behavioral phenotype nicely supports the assumed role of HS cells. Nonetheless, it was acknowledged that axons of other lobula plate output neurons could have also been severed. The current complementary experiments indicate that unilateral optogenetic stimulation of the three HS cells is sufficient to evoke both yaw head movements as well as flight-turning responses. While light pulses were visually perceived by flies and led to transient physiological and behavioral artifacts, consistent long-lasting responses were only observed in animals expressing functional ChR2-C128S, in agreement with the bistable nature of this channel. The direction of the behavioral responses toward the stimulated side can be readily interpreted such that the flies attempt to counteract perceived unintended yaw body rotations. The weaker response levels in visually intact flies compared to actual visually evoked behavior and robust optogenetic responses in blind flies could be explained by unspecific prolonged perturbances of visual circuits by blue light pulses that potentially attenuate optogenetic effects in visually intact flies. Alternatively, stationary visual signals from the inactive arena that provide stable reference points might antagonize the effect of HS cell activation. It should also be noted that visual stimuli were presented to both eyes, leading to hyperpolarization of HS cells in one hemisphere and depolarization of HS cells in the other hemisphere. In contrast, optogenetic stimulation of HS cells involved the cells within only one hemisphere. The information provided by unilateral HS cell activation is partly ambiguous and might signal yaw rotation, but also sideway or forward translation. In fact, HS cells have been shown to encode translating optic flow provided by both eyes during intersaccadic flight intervals, which may serve to extract depth information. Furthermore, HS cells might be functionally specialized, and processing the outputs in various combinations could be used to recover different aspects of self-motion-induced optic flow. For instance, HSN and HSE with dorsal and equatorial receptive fields, respectively, receive contralateral input tuned to back-to-front motion, rendering them sensitive to sideward translation in addition to yaw rotation, particularly so when activity is bilaterally subtracted. In contrast, HSS lacks contralateral input, and summating HSS activity from both hemispheres in higher processing centers might be used to recover parameters associated with forward translation. Although it is not possible to resolve such functional specializations, the fact that unilateral stimulation of all three HS cells produces clear turning responses toward the stimulated side supports the notion that the HSE and HSN cells, and potentially the HSS cell, are important for controlling compensatory yaw rotations of head and body. Nonetheless, it is well known that HS cells are part of a larger network of tangential cells in the lobula plate. There is no doubt that under natural conditions, more dynamic and finer-grained activity patterns in HS and perhaps other neurons than was possible to induce optogenetically are required to reduce ambiguities related to horizontal optic flow and to guide flies safely through their environment. It is also interesting to note that neither cessation of moving patterns nor ChR2-C128S channel closure by longer-wavelength light, both leading to repolarization of HS cells, immediately reestablishes baseline head angle and wing beat amplitudes. This might indicate that these behavioral parameters require active stimulation along the opposite direction and the concomitant depolarization of the contralateral HS cells to return to baseline (Haikala, 2013).

    The results contribute to an emerging picture of how visually guided behavioral patterns are generated in flies. Certain visual cues contained in the raw image sequence such as direction of motion are extracted by intricate parallel circuits of the optic lobes and integrated and conveyed toward appropriate motor circuits via a cohort of wide-field projection neurons, each of which is endowed with a unique visual tuning profile. As an analogous example to the HS system, FOMA-1 cells of the lobula complex are tuned to looming stimuli and consequently elicit escape responses when activated (de Vries, 2012). In blow flies, part of the pathway downstream of the HS cells has been identified for the neck motor system via a motor neuron of the ventral cervical nerve, constituting a straight-forward link between unilateral HS cell activation and neck muscle contraction. In contrast, much less is known about the circuits underlying turning behavior during flight. Unilateral change of wing beat amplitude is controlled by small steering muscles, some of which have been shown to respond to horizontal wide-field motion in a direction-selective way. Such muscles are therefore probable downstream targets of the HS system, but the neural elements that convey the visual information to them remain to be functionally characterized (Haikala, 2013).

    The fact that HS cell responses are remarkably robust while optomotor behavior can be quite variable as explicitly shown, for instance, for Calliphora illustrates that visual reflexes are not unimodal all-or-nothing events, but rather are subject to multimodal modification as well as to central gating. This might reflect the ethological need to conditionally modify or disable optomotor compensation by conflicting visual cues, other sensory modalities, or during certain internally generated flight maneuvers like saccades. Identification of further constituents of the optomotor pathways would provide opportunities to elucidate the neural basis for such multimodal stimulus integration, modulation, and gating of the optomotor response. To this end, flies expressing bistable channelrhodopsin-2 in combination with specific driver lines can help to establish the participation of candidate neurons. Using channelrhodopsin-2 variants with longer time constants than ChR2-C128S, the consequence of activating certain neurons could perhaps even be tested in freely behaving, unrestrained animals (Haikala, 2013).

    A directional tuning map of Drosophila elementary motion detectors

    The extraction of directional motion information from changing retinal images is one of the earliest and most important processing steps in any visual system. In the fly optic lobe, two parallel processing streams have been anatomically described, leading from two first-order interneurons, L1 and L2, via T4 and T5 cells onto large, wide-field motion-sensitive interneurons of the lobula plate. Therefore, T4 and T5 cells are thought to have a pivotal role in motion processing; however, owing to their small size, it is difficult to obtain electrical recordings of T4 and T5 cells, leaving their visual response properties largely unknown. This problem was circumvented by means of optical recording from these cells in Drosophila, using the genetically encoded calcium indicator GCaMP5. This study finds that specific subpopulations of T4 and T5 cells are directionally tuned to one of the four cardinal directions; that is, front-to-back, back-to-front, upwards and downwards. Depending on their preferred direction, T4 and T5 cells terminate in specific sublayers of the lobula plate. T4 and T5 functionally segregate with respect to contrast polarity: whereas T4 cells selectively respond to moving brightness increments (ON edges), T5 cells only respond to moving brightness decrements (OFF edges). When the output from T4 or T5 cells is blocked, the responses of postsynaptic lobula plate neurons to moving ON (T4 block) or OFF edges (T5 block) are selectively compromised. The same effects are seen in turning responses of tethered walking flies. Thus, starting with L1 and L2, the visual input is split into separate ON and OFF pathways, and motion along all four cardinal directions is computed separately within each pathway. The output of these eight different motion detectors is then sorted such that ON (T4) and OFF (T5) motion detectors with the same directional tuning converge in the same layer of the lobula plate, jointly providing the input to downstream circuits and motion-driven behaviours (Maisak, 2013).

    Most of the neurons in the fly brain are dedicated to image processing. The respective part of the head ganglion, called the optic lobe, consists of several layers of neuropile called lamina, medulla, lobula and lobula plate, all built from repetitive columns arranged in a retinotopic way. Each column houses a set of identified neurons that, on the basis of Golgi staining, have been described anatomically, first by Santiago Ramon y Cajal (see Cajal, 1915), in great detail. Owing to their small size, however, most of these columnar neurons have never been recorded from electrophysiologically. Therefore, their specific functional role in visual processing is still largely unknown. This fact is contrasted by rather detailed functional models about visual processing inferred from behavioural studies and recordings from the large, electrophysiologically accessible output neurons of the fly lobula plate (tangential cells). As the most prominent example of such models, the Reichardt detector derives directional motion information from primary sensory signals by multiplying the output from adjacent photoreceptors after asymmetric temporal filtering. This model makes a number of rather counter-intuitive predictions all of which have been confirmed experimentally. Yet, the neurons corresponding to most of the circuit elements of the Reichardt detector have not been identified so far. This study focused on a set of neurons called T4 and T5 cells which, on the basis of circumstantial evidence, have long been speculated to be involved in motion detection. However, it is unclear to what extent T4 and T5 cells are directionally selective or whether direction selectivity is computed or enhanced within the dendrites of the tangential cells. Another important question concerns the functional separation between T4 and T5 cells; that is, whether they carry equivalent signals, maybe one being excitatory and the other inhibitory on the tangential cells, or whether they segregate into directional- and non-directional pathways or into separate ON- and OFF-motion channels (Maisak, 2013).

    To answer these questions, Gal4-driver lines specific for T4 and T5 cells were combined with GCaMP5 (Akerboom, 2012) and the visual response properties were optically recorded using two-photon fluorescence microscopy. In a first series of experiments, a driver line labelling both T4 and T5 cells was used. A confocal image revealed clear labelling in the medulla (T4 cell dendrites), in the lobula (T5 cell dendrites), as well as in four distinct layers of the lobula plate, representing the terminal arborizations of the four subpopulations of both T4 and T5 cells. These four layers of the lobula plate can also be seen in the two-photon microscope when the calcium indicator GCaMP5 is expressed. After stimulation of the fly with grating motion along four cardinal directions (front-to-back, back-to-front, upwards and downwards), activity is confined to mostly one of the four layers, depending on the direction in which the grating is moving. The outcome of all four stimulus conditions can be combined into a single image by assigning a particular colour to each pixel depending on the stimulus direction to which it responded most strongly. From these experiments it is clear that the four subpopulations of T4 and T5 cells produce selective calcium signals depending on the stimulus direction, in agreement with previous deoxyglucose labelling. Sudden changes of the overall luminance evokes no responses in any of the layers. However, gratings flickering in counter-phase lead to layer-specific responses, depending on the orientation of the grating (Maisak, 2013).

    The retinotopic arrangement of this input to the lobula plate is demonstrated by experiments where a dark edge was moved within a small area of the visual field only. Depending on the position of this area, activity of T4 and T5 cells is confined to different positions within the lobula plate. Consequently, when moving a bright vertical edge horizontally from back to front, activity of T4 and T5 cells is elicited sequentially in layer 2 of the lobula plate. These two experiments also demonstrate that T4 and T5 cells indeed signal motion locally. Next, the question of where direction selectivity of T4 and T5 cells arises was investigated; that is, whether it is already present in the dendrite, or whether it is generated by synaptic interactions within the lobula plate. This question is hard to answer, as the dendrites of both T4 and T5 cells form a dense mesh within the proximal layer of the medulla (T4) and the lobula (T5), respectively. However, signals within the inner chiasm where individual processes of T4 and T5 cells can be resolved in some preparations show a clear selectivity for motion in one over the other directions. Such signals are as directionally selective as the ones measured within the lobula plate, demonstrating that the signals delivered from the dendrites of T4 and T5 cells are already directionally selective (Maisak, 2013).

    To assess the particular contribution of T4 and T5 cells to the signals observed in the above experiments, driver lines specific for T4 and T5 cells, respectively, were used. Applying the same stimulus protocol and data evaluation as described above, identical results were obtained as before for both the T4- as well as the T5-specific driver line. It is concluded that T4 and T5 cells each provide directionally selective signals to the lobula plate. Thus, both T4 and T5 cells can be grouped, according to their preferred direction, into four subclasses covering all four cardinal directions, reminiscent of ON–OFF ganglion cells of the rabbit retina (Maisak, 2013).

    Next whether T4 cells respond differently to T5 cells was addressed. To answer this question, moving edges instead of gratings were used with either positive (ON edge, brightness increment) or negative (OFF edge, brightness decrement) contrast polarity as visual stimuli. It was found that T4 cells strongly responded to moving ON edges, but showed little or no response to moving OFF edges. This is true for T4 cells terminating in each of the four layers. The opposite was found for T5 cells. T5 cells selectively responded to moving OFF edges and mostly failed to respond to moving ON edges. Again, this was found for T5 cells in each of the four layers. Next, whether there are any other differences in the response properties between T4 and T5 cells was addressed by testing the velocity tuning of both cell populations by means of stimulating flies with grating motion along the horizontal axis from the front to the back at various velocities covering two orders of magnitude. T4 cells revealed a maximum response at a stimulus velocity of 30° s-1, corresponding to a temporal frequency of 1 Hz. T5 cell responses showed a similar dependency on stimulus velocity, again with a peak at a temporal frequency of 1 Hz. Thus, there is no obvious difference in the velocity tuning between T4 and T5 cells. As another possibility, T4 cells might functionally differ from T5 cells with respect to their directional tuning width. To test this, flies were stimulated with gratings moving into 12 different directions, and the relative change of fluorescence was evaluated in all four layers of the lobula plate. Using the T4-specific driver line, an approximate half width of 60-90° of the tuning curve was found, with the peak responses in each layer shifted by 90°. No decrease of calcium was detectable for grating motion opposite to the preferred direction of the respective layer. When the experiments were repeated using the T5-specific driver line, a similar dependence of the relative change of fluorescence was found on the stimulus direction. It is concluded that T4 cells have the same velocity and orientation tuning as T5 cells. The only functional difference that was detected remains their selectivity for contrast polarity (Maisak, 2013).

    The finding about the different preference of T4 and T5 cells for the polarity of a moving contrast makes the strong prediction that selective blockade of T4 or T5 cells should selectively compromise the responses of downstream lobula plate tangential cells to either ON or OFF edges. To test this prediction, the output of either T4 or T5 cells was blocked via expression of the light chain of tetanus toxin, and the responses of tangential cells via somatic whole-cell patch was recorded to moving ON and OFF edges. In response to moving ON edges, strong and reliable directional responses were observed in all control flies. However, T4-block flies showed a strongly reduced response to ON edges, whereas the responses of T5-block flies were at the level of control flies. When moving OFF edges were used, control flies again responded with a large amplitude. However, the responses of T4-block flies were at the level of control flies, whereas the responses of T5-block flies were strongly reduced. These findings are reminiscent of the phenotypes obtained from blocking lamina cells L1 and L2 (Joesch, 2010) and demonstrate that T4 and T5 cells are indeed the motion-coding intermediaries for these contrast polarities on their way to the tangential cells of the lobula plate. Whether the residual responses to ON edges in T4-block flies and to OFF edges in T5-block flies are due to an incomplete signal separation between the two pathways or due to an incomplete genetic block in both fly lines is currently unclear (Maisak, 2013).

    To address the question of whether T4 and T5 cells are the only motion detectors of the fly visual system, or whether they represent one cell class, in parallel to other motion-sensitive elements, tethered flies walking on an air-suspended sphere were used, and and they were stimulated by ON and OFF edges moving in opposite directions. As in the previous experiments, T4 and T5 cells were blocked specifically by selective expression of the light chain of tetanus toxin. During balanced motion, control flies did not show significant turning responses to either side. T4-block flies, however, strongly followed the direction of the moving OFF edges, whereas T5-block flies followed the direction of the moving ON edges. In summary, the selective preference of T4-block flies for OFF edges and of T5-block flies for ON edges not only corroborates the findings about the selective preference of T4 and T5 cells for different contrast polarities, but also demonstrates that the signals of T4 and T5 cells are indeed the major, if not exclusive, inputs to downstream circuits and motion-driven behaviours (Maisak, 2013).

    Almost a hundred years after T4 and T5 cells have been anatomically described, this study reports their functional properties in a systematic way. Using calcium as a proxy for membrane voltage, this study found that both T4 and T5 cells respond to visual motion in a directionally selective manner and provide these signals to each of the four layers of the lobula plate, depending on their preferred direction. Both cell types show identical velocity and orientation tuning which matches the one of the tangential cells. The strong direction selectivity of both T4 and T5 cells is unexpected, as previous studies had concluded that the high degree of direction selectivity of tangential cells is due to a push–pull configuration of weakly directional input with opposite preferred direction. Furthermore, as the preferred direction of T4 and T5 cells matches the preferred direction of the tangential cells branching within corresponding layers, it is currently unclear which neurons are responsible for the null-direction response of the tangential cells. As for the functional separation between T4 and T5 cells, this study found that T4 cells selectively respond to brightness increments, whereas T5 cells exclusively respond to moving brightness decrements. Interestingly, parallel ON and OFF motion pathways had been previously postulated on the basis of selective silencing of lamina neurons L1 and L2 (Joesch, 2010). Studies using apparent motion stimuli to probe the underlying computational structure arrived at controversial conclusions: whereas some studies concluded that there was a separate handling of ON and OFF events by motion detectors, others did not favour such a strict separation. The present study directly demonstrates the existence of separate ON and OFF motion detectors, as represented by T4 and T5 cells, respectively. Furthermore, the results anatomically confine the essential processing steps of elementary motion detection -- that is, asymmetric temporal filtering and nonlinear interaction -- to the neuropile between the axon terminals of lamina neurons L1 and L2 (Joesch, 2013) and the dendrites of directionally selective T4 and T5 cells. The dendrites of T4 and T5 cells might well be the place where signals from neighbouring columns interact in a nonlinear way, similar to the dendrites of starburst amacrine cells of the vertebrate retina (Maisak, 2013).

    RNA-seq transcriptome analysis of direction-selective T4/T5 neurons in Drosophila

    Neuronal computation underlying detection of visual motion has been studied for more than a half-century. In Drosophila, direction-selective T4/T5 neurons show supralinear signal amplification in response to stimuli moving in their preferred direction, in agreement with the prediction made by the Hassenstein-Reichardt detector. Nevertheless, the molecular mechanism explaining how the Hassenstein-Reichardt model is implemented in T4/T5 cells has not been identified yet. The present study utilized cell type-specific transcriptome profiling with RNA-seq to obtain a complete gene expression profile of T4/T5 neurons. The expression was analyzed of genes that affect neuronal computational properties and can underlie the molecular implementation of the core features of the Hassenstein-Reichardt model to the dendrites of T4/T5 neurons. Furthermore, the acquired RNA-seq data was used to examine the neurotransmitter system used by T4/T5 neurons. Surprisingly, co-expression of the cholinergic markers and the vesicular GABA transporter was observed in T4/T5 neurons. Previously undetected expression of vesicular GABA transporter was documented in T4/T5 cells using VGAT-LexA knock-in line. The provided gene expression dataset can serve as a useful source for studying the properties of direction-selective T4/T5 neurons on the molecular level (Pankova, 1016).

    Complementary mechanisms create direction selectivity in the fly

    How neurons become sensitive to the direction of visual motion represents a classic example of neural computation. Two alternative mechanisms have been discussed in the literature so far: preferred direction enhancement, by which responses are amplified when stimuli move along the preferred direction of the cell, and null direction suppression, where one signal inhibits the response to the subsequent one when stimuli move along the opposite, i.e. null direction. Along the processing chain in the Drosophila optic lobe, directional responses first appear in T4 and T5 cells. Visually stimulating sequences of individual columns in the optic lobe with a telescope while recording from single T4 neurons, this study found both mechanisms at work implemented in different sub-regions of the receptive field. This finding explains the high degree of directional selectivity found already in the fly's primary motion-sensing neurons and marks an important step in understanding of elementary motion detection (Haag, 2016).

    Candidate neural substrates for off-edge motion detection in Drosophila

    In the visual motion pathways contained within the fly's optic lobe, two cell types-T4 and T5-are the first known relay neurons to signal small-field direction-selective motion responses (see Graphical abstract). These cells then feed into large tangential cells that signal wide-field motion. Recent studies have identified two types of columnar neurons in the second neuropil, or medulla, that relay input to T4 from L1, the ON-channel neuron in the first neuropil, or lamina, thus providing a candidate substrate for the elementary motion detector (EMD). Interneurons relaying the OFF channel from L1's partner, L2, to T5 are so far not known, however. This study report that multiple types of transmedulla (Tm) neurons provide unexpectedly complex inputs to T5 at their terminals in the third neuropil, or lobula. From the L2 pathway, single-column input comes from Tm1 and Tm2 and multiple-column input from Tm4 cells. Additional input to T5 comes from Tm9, the medulla target of a third lamina interneuron, L3, providing a candidate substrate for L3's combinatorial action with L2. Most numerous, Tm2 and Tm9's input synapses are spatially segregated on T5's dendritic arbor, providing candidate anatomical substrates for the two arms of a T5 EMD circuit; Tm1 and Tm2 provide a second. Transcript profiling indicates that T5 expresses both nicotinic and muscarinic cholinoceptors, qualifying T5 to receive cholinergic inputs from Tm9 and Tm2, which both express choline acetyltransferase (ChAT). It is hypothesized that T5 computes small-field motion signals by integrating multiple cholinergic Tm inputs using nicotinic and muscarinic cholinoceptors (Shinomiya, 2014).

    The L2 pathway has been identified as the substrate for detecting moving dark edges. L2's partner cell, L4, and its common medulla target, Tm2, are both essential components of this dark-edge pathway prior to the computation of directionality, which occurs first in the dendrites of T5. Tm2 and L4 cells both respond with a nondirectional increase in activity to moving dark (OFF) edges, and silencing of both either singly or in combination also abolishes the response to moving dark edges in downstream LPTCs. The anatomical receptive fields described in this report for the input terminals to T5 cell dendrites are compatible with Tm2's being wired as one of the two arms of an EMD circui and with Tm1, Tm9, or both as the other. The existence of two EMD circuits would require that the inputs from Tm1/Tm2 and Tm9/Tm2 have aligned vector angles, since each T5 receives both circuits but must respond to dark-edge motion in only one of the four cardinal directions. This alignment is clear for three of the T5 cells plotted in detail, but less so for T5-08. Clearly, additional detailed plots of T5 anatomical receptive fields are needed (Shinomiya, 2014).

    Even though this scheme is still highly speculative, nonlinear interaction between two input arms of the EMD circuits is the computational basis of local motion-detection models, while specific computational models favor different types of interactions: either multiplication or facilitation for the Hassenstein-Reichardt model or inhibition for the Barlow-Levick model (see Motion sensing in vision) (Shinomiya, 2014).

    A parallel may be seen in the vertebrate retina. There, turtle B10 bipolar cells use ionotropic and metabotropic glutamate receptors to signal, respectively, L and M cone inputs, a dual deployment that has been suggested to form the basis for B10 red-ON, blue/green-OFF color opponency color opponency. The expression of both nicotinic and muscarinic cholinoceptors in T5 may provide a similar means to integrate multiple cholinergic Tm inputs and so compute small-field motion signals. Vertebrate muscarinic receptors are coupled to G proteins and various downstream signaling pathways to regulate a broad spectrum of cellular functions, including neuronal excitability. In Drosophila, agonist activation of mAchR-A acts via the IP3 pathway to increase calcium release from internal stores, which elsewhere is reported in turn to activate high-conductance calcium-dependent potassium (BK) channels (the Slowpoke channel), leading to membrane hyperpolarization. It was therefore interesting that, using single-cell RT-PCR, slowpoke transcripts were detected in both T4 and T5, consistent with a previous immunohistochemical study that Slowpoke is expressed in the optic lobe, including the lobula. It is proposed that these two postsynaptic events, mAchR-A-mediated increased intracellular calcium and mAchR activation of Slowpoke channels, occur at postsynaptic sites activated by different inputs distributed over the T5 dendritic arbor. Given the relative temporal inflexibility of an excitatory nicotinic cholinoceptor synaptic response and the fact that their anatomy qualifies Tm2/Tm9 and Tm2/Tm1 input pairs as two independent pairs of EMD input arms to T5 that share Tm2, it seems most reasonable that T5 uses the nicotinic receptor inputs for fast excitation from Tm2 (as the instantaneous signal) and compares this with a slow inhibitory input from either Tm9 or Tm1 (as the delayed signal). This arrangement could allow T5 to inherit motion information with two temporal characteristics provided that Tm1 and Tm9 have their own time delay. The coupling of the inhibitory inputs to secondary messenger system also provides a potential mechanism to adapt the temporal delay filter. In addition, the activation of Drosophila mAchR-A in cultured cells also induces a secondary calcium influx, potentially originating from an extracellular calcium pool, while the activation of vertebrate muscarinic receptors has been shown to inhibit potassium channels, including those of the Kv7 (KCNQ/M) type, and to lower the excitability threshold. It is therefore possible that an interaction between the nicotinic and muscarinic inputs provides some form of multiplication, as suggested in the Hassenstein-Reichardt model. Further definition of the roles of the Tm1, Tm2, and Tm9 input cells as input arms to different EMD circuits and determination of the synaptic mechanisms for detecting moving dark edges must obviously await additional genetic silencing experiments and electrophysiological recordings from all these cells, which this study now clearly identifies as synaptic inputs to T5 in Drosophila (Shinomiya, 2014).

    Functional specialization of neural input elements to the Drosophila ON motion detector

    The neural apparatus for detecting the direction of visual movement consists of two spatially separated input lines that are asymmetrically filtered in time and then interact in a nonlinear way. However, the cellular implementation of this computation remains elusive. Recent connectomic data of the Drosophila optic lobe has suggested a neural circuit for the detection of moving bright edges (ON motion) with medulla cells Mi1 and Tm3 providing spatially offset input to direction-selective T4 cells, thereby forming the two input lines of a motion detector. Electrophysiological characterization of Mi1 and Tm3 revealed different temporal filtering properties and proposed them to correspond to the delayed and direct input, respectively. This hypothesis was tested by silencing either Mi1 or Tm3 cells and using electrophysiological recordings and behavioral responses of flies as a readout. It was shown that Mi1 is a necessary element of the ON pathway under all stimulus conditions. In contrast, Tm3 is specifically required only for the detection of fast ON motion in the preferred direction. This study thereby provides first functional evidence that Mi1 and Tm3 are key elements of the ON pathway and uncover an unexpected functional specialization of these two cell types. The results thus require an elaboration of the currently prevailing model for ON motion detection and highlight the importance of functional studies for neural circuit breaking (Ammer, 2015).

    A common evolutionary origin for the ON- and OFF-edge motion detection pathways of the Drosophila visual system

    Two candidate pathways for ON- and OFF-edge motion detection in the visual system act via circuits that use respectively either T4 or T5, two cell types of the fourth neuropil, or lobula plate (LOP), that exhibit narrow-field direction-selective responses and provide input to wide-field tangential neurons. T4 or T5 both have four subtypes that terminate one each in the four strata of the LOP. Representatives are reported in a wide range of Diptera, and both cell types exhibit various similarities in: (1) the morphology of their dendritic arbors; (2) their four morphological and functional subtypes; (3) their cholinergic profile in Drosophila; (4) their input from the pathways of L3 cells in the first neuropil, or lamina (LA), and by one of a pair of LA cells, L1 (to the T4 pathway) and L2 (to the T5 pathway); and (5) their innervation by a single, wide-field contralateral tangential neuron from the central brain. Progenitors of both also express the gene atonal early in their proliferation from the inner anlage of the developing optic lobe, being alone among many other cell type progeny to do so. Yet T4 receives input in the second neuropil, or medulla (ME), and T5 in the third neuropil or lobula (LO). This study suggests that these two cell types were originally one, that their ancestral cell population duplicated and split to innervate separate ME and LO neuropils, and that a fiber crossing-the internal chiasma-arose between the two neuropils (Shinomiya, 2015).

    Neural mechanisms for Drosophila contrast vision

    Spatial contrast, the difference in adjacent luminance values, provides information about objects, textures, and motion and supports diverse visual behaviors. Contrast computation is therefore an essential element of visual processing. The underlying mechanisms, however, are poorly understood. In human psychophysics, contrast illusions are means to explore such computations, but humans offer limited experimental access. Via behavioral experiments in Drosophila, this study shows that flies are also susceptible to contrast illusions. Using genetic silencing techniques, electrophysiology, and modeling, the mechanisms and neuronal correlates underlying the behavior were dissected. Results indicate that spatial contrast computation involves lateral inhibition within the same pathway that computes motion of luminance increments (ON pathway). Yet motion-blind flies, in which downstream motion-sensitive neurons needed for optomotor behavior were silenced, exhibit fully intact contrast responses. In conclusion, spatial contrast and motion cues are first computed by overlapping neuronal circuits which subsequently feed into parallel visual processing streams (Bahl, 2015).

    Direction selectivity in Drosophila emerges from preferred-direction enhancement and null-direction suppression

    Across animal phyla, motion vision relies on neurons that respond preferentially to stimuli moving in one, preferred direction over the opposite, null direction. In the elementary motion detector of Drosophila, direction selectivity emerges in two neuron types, T4 and T5, but the computational algorithm underlying this selectivity remains unknown. This study found that the receptive fields of both T4 and T5 exhibit spatiotemporally offset light-preferring and dark-preferring subfields, each obliquely oriented in spacetime. In a linear-nonlinear modeling framework, the spatiotemporal organization of the T5 receptive field predicts the activity of T5 in response to motion stimuli. These findings demonstrate that direction selectivity emerges from the enhancement of responses to motion in the preferred direction, as well as the suppression of responses to motion in the null direction. Thus, remarkably, T5 incorporates the essential algorithmic strategies used by the Hassenstein-Reichardt correlator and the Barlow-Levick detector. The model developed in this paper for T5 also provides an algorithmic explanation for the selectivity of T5 for moving dark edges: the model captures all two- and three-point spacetime correlations relevant to motion in this stimulus class. More broadly, the findings reveal the contribution of input pathway visual processing, specifically center-surround, temporally biphasic receptive fields, to the generation of direction selectivity in T5. As the spatiotemporal receptive field of T5 in Drosophila is common to the simple cell in vertebrate visual cortex, this stimulus-response model of T5 will inform efforts in an experimentally tractable context to identify more detailed, mechanistic models of a prevalent computation (Leong, 2016).

    In vivo imaging reveals composite coding for diagonal motion in the Drosophila visual system

    Understanding information coding is important for resolving the functions of visual neural circuits. The motion vision system is a classic model for studying information coding as it contains a concise and complete information-processing circuit. In Drosophila, the axon terminals of motion-detection neurons (T4 and T5) project to the lobula plate, which comprises four regions that respond to the four cardinal directions of motion. The lobula plate thus represents a topographic map on a transverse plane. This enables study of the coding of diagonal motion by investigating its response pattern. By using in vivo two-photon calcium imaging, the axon terminals of T4 and T5 cells in the lobula plate were found to be activated during diagonal motion. Further experiments showed that the response to diagonal motion is distributed over the following two regions compared to the cardinal directions of motion-a diagonal motion selective response region and a non-selective response region-which overlap with the response regions of the two vector-correlated cardinal directions of motion. Interestingly, the sizes of the non-selective response regions are linearly correlated with the angle of the diagonal motion. These results revealed that the Drosophila visual system employs a composite coding for diagonal motion that includes both independent coding and vector decomposition coding (Yue, 2016).

    Direct measurement of correlation responses in Drosophila elementary motion detectors reveals fast timescale tuning

    Animals estimate visual motion by integrating light intensity information over time and space. The integration requires nonlinear processing, which makes motion estimation circuitry sensitive to specific spatiotemporal correlations that signify visual motion. Classical models of motion estimation weight these correlations to produce direction-selective signals. However, the correlational algorithms they describe have not been directly measured in elementary motion-detecting neurons (EMDs). This study employed stimuli to directly measure responses to pairwise correlations in Drosophila's EMD neurons, T4 and T5. Activity in these neurons was required for behavioral responses to pairwise correlations and was predictive of those responses. The pattern of neural responses in the EMDs was inconsistent with one classical model of motion detection, and the timescale and selectivity of correlation responses constrained the temporal filtering properties in potential models. These results reveal how neural responses to pairwise correlations drive visual behavior in this canonical motion-detecting circuit (Salazar-Gatzimas, 2016).

    Multiple redundant medulla projection neurons mediate color vision in Drosophila

    The receptor mechanism for color vision has been extensively studied. In contrast, the circuit(s) that transform(s) photoreceptor signals into color percepts to guide behavior remain(s) poorly characterized. Using intersectional genetics to inactivate identified subsets of neurons in the optic lobe, this study has uncovered the first-order interneurons that are functionally required for hue discrimination in Drosophila. A novel aversive operant conditioning assay was developed for intensity-independent color discrimination (true color vision) in Drosophila. Single flying flies are magnetically tethered in an arena surrounded by blue and green LEDs (light-emitting diodes). The flies' optomotor response is used to determine the blue-green isoluminant intensity. Flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies are successfully trained in this paradigm when conditioned to avoid either blue or green. Functional color entrainment requires the function of the narrow-spectrum photoreceptors R8 and/or R7, and is within a limited range, intensity independent, suggesting that it is mediated by a color vision system. The medulla projection neurons, Tm5a/b/c and Tm20, receive direct inputs from R7 or R8 photoreceptors and indirect input from the broad-spectrum photoreceptors R1-R6 via the lamina neuron L3. Genetically inactivating these four classes of medulla projection neurons abolished color learning. However, inactivation of subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. It is hypothesized that flies represent color along multiple axes at the first synapse in the fly visual system. The apparent redundancy in learned color discrimination sharply contrasts with innate ultraviolet (UV) spectral preference, which is dominated by a single pathway from the amacrine neuron Dm8 to the Tm5c projection neurons (Melnattur, 2014).

    Processing properties of ON and OFF pathways for Drosophila motion detection

    The algorithms and neural circuits that process spatio-temporal changes in luminance to extract visual motion cues have been the focus of intense research. An influential model, the Hassenstein-Reichardt correlator, relies on differential temporal filtering of two spatially separated input channels, delaying one input signal with respect to the other. Motion in a particular direction causes these delayed and non-delayed luminance signals to arrive simultaneously at a subsequent processing step in the brain; these signals are then nonlinearly amplified to produce a direction-selective response. Recent work in Drosophila has identified two parallel pathways that selectively respond to either moving light or dark edges. Each of these pathways requires two critical processing steps to be applied to incoming signals: differential delay between the spatial input channels, and distinct processing of brightness increment and decrement signals. This study demonstrates, using in vivo patch-clamp recordings, that four medulla neurons implement these two processing steps. The neurons Mi1 and Tm3 respond selectively to brightness increments, with the response of Mi1 delayed relative to Tm3. Conversely, Tm1 and Tm2 respond selectively to brightness decrements, with the response of Tm1 delayed compared with Tm2. Remarkably, constraining Hassenstein-Reichardt correlator models using these measurements produces outputs consistent with previously measured properties of motion detectors, including temporal frequency tuning and specificity for light versus dark edges. It is proposed that Mi1 and Tm3 perform critical processing of the delayed and non-delayed input channels of the correlator responsible for the detection of light edges, while Tm1 and Tm2 play analogous roles in the detection of moving dark edges. The data show that specific medulla neurons possess response properties that allow them to implement the algorithmic steps that precede the correlative operation in the Hassenstein-Reichardt correlator, revealing elements of the long-sought neural substrates of motion detection in the fly (Behnia, 2014).

    Behavioural and electrophysiological studies in flies have demonstrated that visual motion responses display the fundamental signatures predicted by the Hassenstein-Reichardt correlator (HRC). In Drosophila, photoreceptors R1-R6 are required for motion detection, and synapse onto three lamina monopolar cells L1, L2 and L3, which provide inputs to distinct motion pathways (Behnia, 2014).

    Since L1 and L2 relay information about both contrast increments and decrements to motion pathways, this study focused on medulla neurons that link L1 and L2 to T4 and T5 to identify potential sites of half-wave rectification and delay. Similarly, Tm1 and Tm2 are the main synaptic targets of L2. Furthermore, Mi1 and Tm3 together constitute over 80% of the presynaptic inputs to T4. L1 feeds into pathways involved in detecting moving light edges while L2, with contributions from L1 and L3, is involved in detecting moving dark edges. Offset of light differed in magnitude: in Mi1, the offset hyperpolarization amplitude was only 11% of the onset depolarization, while in Tm3 this fraction was 36.6%. Deeper in the optic lobe, two direction-selective neuronal types, T4 and T5, are also differentially tuned: T4 cells respond to moving light edges while T5 cells respond to moving dark edges. Both T4 and T5 are required for downstream, direction-selective responses of the visual system output cells called lobula plate tangential cells. According to the HRC model, these light and dark edge direction-selective pathways each require two processing steps: differential temporal delay and nonlinear amplification. Moreover, these two pathways must process changes in contrast differently to respond differentially to light and dark edges. One such asymmetric mechanism is 'half-wave rectification', where inputs of one polarity are amplified and inputs of the opposite polarity are suppressed (Behnia, 2014).

    Since L1 and L2 relay information about both contrast increments and decrements (they hyperpolarize in response to light increments and depolarize in response to decrements) and act as inputs to motion pathways, focus was placed on medulla neurons that link L1 and L2 to T4 and T5 to identify potential sites of half-wave rectification and delay. Electron microscopic reconstruction of the medulla has identified columnar neurons types Tm3 and Mi1 as receiving the large majority of synapses from L1. Similarly, Tm1 and Tm2 are the main synaptic targets of L2. Furthermore, Mi1 and Tm3 together constitute over 80% of the presynaptic inputs to T4 and both Tm1 and Tm2 provide input to T5. Based on their innervation patterns, Mi1 and Tm3 have been proposed to be core components of the motion detector for light edges involving T4. Similarly, Tm1 and Tm2 are likely candidates for analogous roles in a motion detector for dark edges (Behnia, 2014).

    Since changes in luminance are central to motion detection, the responses of Mi1, Tm3, Tm1 and Tm2 to step changes in light intensity were examined by performing whole-cell current-clamp recordings on awake immobilized fruit flies. Both Mi1 and Tm3 responded with a strong, transient depolarization at the onset of a 1 s light step, and then transiently hyperpolarized to below pre-stimulus levels at light offset. The responses to onset and offset of light differed in magnitude: in Mi1, the offset hyperpolarization amplitude was only 11% of the onset depolarization, while in Tm3 this fraction was 36.6%. A brief flash of light elicited a sharper depolarization in both cells, with the offset hyperpolarization terminating the depolarization phase of the response. The responses observed in Tm1 and Tm2 were similar to each other, yet were strikingly different from those in Mi1 and Tm3. Tm1 and Tm2 hyperpolarized at light onset, and depolarized strongly at light offset. The hyperpolarization of Tm1 evoked by stimulus onset was 26.1% as large as the depolarization evoked at offset; for Tm2, this number was 17.7%. Finally, rapid sequential presentations of light caused repolarization of these cells while their membrane potential was still peaking or decaying from a previous flash. Thus, Mi1 and Tm3, the postsynaptic targets of L1, respond mostly to brightness increments. Conversely, Tm1 and Tm2, the postsynaptic targets of L2, respond most strongly to brightness decrements, consistent with calcium imaging studies of Tm2. All four cells showed asymmetries in their responses to brightness changes, consistent with a role in conferring edge selectivity to each pathway. In addition, whether these medulla neurons could relay long-term information about contrast to downstream circuitry was examined by characterizing responses evoked by 5 s brightness increments or decrements presented on an intermediate grey background. All four neurons displayed a sustained response for both brightness increments and decrements, consistent with observations that motion responses can be evoked even when a sequential change in luminance at two points in space occurs with a delay period of up to 10 s in experiments using apparent motion stimuli (Behnia, 2014).

    In HRC models, the individual inputs to the cells that perform the nonlinear amplification step are not themselves direction selective. Therefore the responses were tested of Mi1, Tm3, Tm1 and Tm2 to motion stimuli, using light and dark bars moving in different directions, under conditions that evoke strong responses from lobula plate tangential cells. All four neurons responded to moving bars with a sharp depolarization but the amplitude of these responses was independent of the direction of motion. Thus, Mi1, Tm3, Tm1 and Tm2 are not direction selective under these conditions, consistent with these cells acting upstream of the nonlinear correlation stage of motion detection, as recently reported for Tm2 (Behnia, 2014).

    Next whether Mi1 and Tm3, or Tm1 and Tm2, have different response latencies that would allow them to differentially delay responses to contrast changes was examined. To quantitatively capture the responses of these neurons to dynamic stimuli spanning a wide range of contrast values and time-scales, an approximately Gaussian-distributed random flicker stimulus with a 50% contrast and an exponential correlation time of 10 ms was used. Standard procedures were used to extract the linear filter that best described the temporal properties of the response. The responses of Mi1 and Tm3 to the noise stimuli were very similar, with temporal filters that comprised a large positive lobe reflecting a sign-conserving relationship between the contrast input and the neural response. Mean Tm1 and Tm2 responses to these noise stimuli were also similar to one another, with temporal filters that included a large negative lobe, reflecting a sign-inversion between the contrast input and the neural response. For Mi1 the average peak response time was 71 ms after a contrast change while it was 53 ms for Tm3. Thus, a difference of 18 ms existed between the peak times of the filters for Mi1 and Tm3. Similarly, the average peak time was 56 ms for Tm1 and 43 ms for Tm2. The difference in latency between the two cells was 13 ms:. Thus, in both cases, there was a small but significant temporal offset, with Mi1 exhibiting a delayed response compared with Tm3, and Tm1 being delayed relative to Tm2. Notably, these peak delay differences are not much smaller than delays inferred from some lobula plate tangential cells recordings and behavioural experiments (Behnia, 2014).

    It was next asked whether neuronal responses to this stochastic stimulus were linear or whether different gains were applied to brightness increments and decrements. The noise stimulus was convolved with the corresponding filters for each neuron type to obtain the predicted linear response of each neuron. The linear predictions were then compared with the actual response to define the instantaneous nonlinearity for each neuron. Consistent with the light step results, the nonlinearities extracted for Mi1 and Tm3 revealed that these cells respond more strongly to brightness increments than to decrements. Similarly, both Tm1 and Tm2 neurons were less hyperpolarized in response to brightness increments and more depolarized in response to brightness decrements than predicted by the linear model. The noise stimuli evoked smaller response asymmetries than those observed with brightness steps, possibly because these stimuli use smaller changes in intensity than the step stimuli. Such differences in gain for brightness increments and decrements reflect partial half-wave rectification, a central feature of models that selectively respond to one contrast polarity (Behnia, 2014).

    Can the dynamics of the linear filters and the extent of the nonlinearities that were measured account for well-characterized properties of motion detecting pathways? One hallmark of the HRC is that it displays a peak response to a defined temporal frequency, creating a temporal frequency optimum. Because of its structure, the output of an HRC is not proportional to the speed of motion, but rather increases to a maximum value, before decaying at faster speeds. The shape of this tuning curve depends on the temporal properties of its two input channels. Two separate model correlators were constructed, one that used Mi1 and Tm3 filters and nonlinearities as the two channels preceding multiplication and subtraction, and a second one that used Tm1 and Tm2 filters and nonlinearities. Whether these model motion detectors produced temporal frequency tuning curves similar to those previously measured in flies was measured. When these models were presented with moving sine waves of 20% contrast at various contrast frequencies, a peak response was observed at approximately 1 Hz for both the Mi1/Tm3 and the Tm1/Tm2 models. This computed temporal frequency optimum compares favourably with the optima measured in blowflies and Drosophila (Behnia, 2014).

    Another measured feature of these two motion pathways is their selectivity for edges of particular contrast polarity. The model correlators were presented with light and dark edges of 100% contrast, moving across a grey background. The Tm1/Tm2 model was highly selective for dark edges over a range of speeds. The Mi1/Tm3 model was only mildly selective for light edges, owing to the more linear responses measured in Mi1 and Tm3 compared with Tm1 and Tm2. These modelling results are consistent with experimentally measured high selectivity of the dark edge motion pathway, and a more modest selectivity of the light edge motion pathway (Behnia, 2014).

    Taken together, these data are consistent with a model in which Mi1 and Tm3 represent central components of the input channels of a correlator detecting moving light edges, while Tm1 and Tm2 represent analogous components for a correlator that is tuned to detect moving dark edges. The asymmetric responses of these four neurons to brightness increments and decrements corroborates previous studies that argued for separate processing of ON and OFF inputs by distinct channels to explain the segregation between light and dark edge processing. This situation is similar to separate processing of ON and OFF signals by bipolar cells in the vertebrate retina (Behnia, 2014).

    The relative delays measured between the peak responses in these cells is roughly ten times smaller than previously calculated for idealized motion detector models that fit a host of experimental data. In classic HRC models, input to one channel is not filtered, while input to the second channel is low-pass filtered with a time constant of τ. In these models, the maximum response occurs at a temporal frequency of 1/2πτ, so that the delay for a 1 Hz optimum is τ ~ 150 m. However, since both of the measured filters act as band-pass filters, they suppress high-frequency inputs, while still producing delay differences between the channels. Thus, when input channels contain both these measured filters, a peak timing difference of ~15 ms can result in a temporal frequency optimum of 1 Hz. Furthermore, two considerations might lengthen the actual relative delays between pathways. First, somatic recordings were performed that may only approximate the true axonal response of the neurons. Second, the synapses between Mi1/Tm3 and T4, and those between Tm1/Tm2 and T5 could impose additional delays to either input channel before a correlation operation (Behnia, 2014).

    Anatomical reconstruction of the Drosophila medulla connected the predicted spatial receptive fields of Mi1 and Tm3 cells to the dendritic arbors of individual T4 cells with known directional preferences. According to predictions derived from that analysis, if Mi1 signals are delayed relative to those of Tm3, as the recordings indicate, the observed direction selectivity in T4 could be accomplished by combining Mi1 and Tm3 inputs with opposite signs onto T4 (one inhibitory and the other excitatory). Such an arrangement could be similar to the motion detection model proposed to explain direction selective responses in the vertebrate retina (Behnia, 2014).

    Given the cellular and synaptic complexity of medulla circuitry, as well as the wealth of distinct behaviours that are guided by visual motion, additional cell types are likely to play computational roles in Drosophila elementary motion detectors. Nonetheless, the data show that Mi1, Tm3, Tm1 and Tm2 possess response properties that are consistent with implementing the algorithmic steps that precede the correlation operations in the motion detecting pathways in Drosophila (Behnia, 2014).

    Neural circuit to integrate opposing motions in the visual field

    When navigating in their environment, animals use visual motion cues as feedback signals that are elicited by their own motion. Such signals are provided by wide-field neurons sampling motion directions at multiple image points as the animal maneuvers. Each one of these neurons responds selectively to a specific optic flow-field representing the spatial distribution of motion vectors on the retina. This study describes the discovery of a group of local, inhibitory interneurons in the fruit fly Drosophila key for filtering these cues. Using anatomy, molecular characterization, activity manipulation, and physiological recordings, this study demonstrates that these interneurons convey direction-selective inhibition to wide-field neurons with opposite preferred direction and provides evidence for how their connectivity enables the computation required for integrating opposing motions. The results indicate that, rather than sharpening directional selectivity per se, these circuit elements reduce noise by eliminating non-specific responses to complex visual information (Mauss, 2015).

    Diverse sensory experiences can result in largely overlapping patterns of activation within sensory circuits yet require fundamentally different behavioral responses. An underlying key operation is the extraction of features relevant for specific behaviors by hierarchical layers of neuronal networks with increasing selectivity. A well-studied example of such feature extraction is the computation of the optic flow associated with self-motion -- that is, the feedback motion cues created by an animal progressing through its environment. Across many animals studied, motion-sensitive neurons covering large receptive fields (those that receive input from cues spanning the visual field) tend to be motion opponent, i.e., are excited by motion along one and inhibited along the opposite direction. However, the functional significance of motion opponency is unclear and has to date not been experimentally challenged. This problem was addressed in Drosophila, which has emerged as a powerful model system to study the mechanisms underlying motion vision (Mauss, 2015).

    The Drosophila optic lobe consists of four neuropiles called lamina, medulla, lobula, and lobula plate. Each of these neuropiles is built from about 750 repetitive columns arranged in a retinotopic way. Monopolar L1 and L2 cells, among others, receive photoreceptor input in the lamina and feed into two motion pathways. Within each pathway, the direction of motion is computed separately, with the L1-pathway selectively processing motion of brightness increments (ON) and the L2-pathway motion of brightness decrements (OFF). The outputs of the ON and OFF pathways are represented by arrays of small-field T4 and T5 cells, respectively. Each T4 and T5 cell is tuned to one of four cardinal directions and terminates in one of the four layers of the lobula plate such that opposite directions are represented in adjacent layers (layer 1: front to back; layer 2: back to front; layer 3: upward; layer 4: downward). These directions match the preferred directions of wide-field motion-sensitive tangential cells that extend their dendrites in the respective layers: horizontal system cells with dendrites in layer 1 depolarize during front-to-back motion and hyperpolarize during back-to-front motion, Hx cells in layer 2 exhibit the opposite tuning, and vertical system (VS) cells with dendrites mostly in layer 4 depolarize primarily during downward and hyperpolarize during upward motion. With T4/T5 cells blocked, tangential cells lose all of their motion sensitivity, and flies become completely motion blind. Combining optogenetic stimulation of T4/T5 cells with various pharmacological antagonists, the connections between T4/T5 and tangential cells have recently been characterized as monosynaptic, excitatory, and cholinergic. T4/T5 cells thus account for the depolarization of the tangential cells during preferred direction motion. What remains unclear is the mechanism and functional role of subtracting information about motion in the opposite or null direction (Mauss, 2015).

    This study characterize a hitherto unknown class of vertical system lobula plate intrinsic (LPi) neurons and demonstrates how they contribute to motion opponency. First, anatomical and molecular characterization, as well as combined optogenetic stimulation and electrophysiological recordings, reveal that LPi neurons are bi-stratified and inhibit tangential cells in single lobula plate layers via glutamatergic synapses. Second, two-photon calcium imaging demonstrated that LPi neurons are activated in response to motion directions similar to their presumed T4/T5 inputs and opposite to their postsynaptic targets. Third, genetically silencing LPi cell output selectively abolishes null direction inhibitory potential changes in tangential cells. It is therefore concluded that LPi neurons hyperpolarize tangential cells during null direction motion through sign-inverting layer interactions, thus forming the cellular basis of motion opponency in the fly. As a final point, the identification of LPi neurons enabled the long-sought functional relevance of motion opponency to be experimentally addressed. As blocking the activity of LPi neurons renders their postsynaptic wide-field motion-sensitive neurons responsive to a variety of moving patterns, these experiments suggest that motion opponency is essential for flow-field selectivity, thereby improving the ability to reliably estimate self-motion trajectories based on complex visual information (Mauss, 2015).

    Motion detection is a fundamental function of all higher visual systems. It is a paradigmatic model for sensory feature extraction since motion information is not explicitly encoded in the single receptor response but has to be computed by downstream neural circuits. Motion detection can be described as a two- stage process: In the first stage, direction-selective signals are generated by correlating the output from neighboring photoreceptors after asymmetric temporal filtering. Neural substrates corresponding to these correlators are, for instance, the T4/T5 cells of the fly optic lobes and the dendrites of starburst amacrine cells in the mammalian retina. In the second stage, signals from oppositely tuned correlators are subtracted from each other, giving rise to a fully opponent output. This processing step is implemented in the fly optic lobe on the dendrites of the lobula plate tangential cells, which receive two kinds of inputs: (1) a direct excitatory input from T4/T5 cells terminating within the same lobula plate layer, giving rise to depolarization during preferred direction motion; and (2) as shown in this study, an indirect inhibitory input via bi-stratified LPi neurons from T4/T5 cells terminating in the adjacent layer, causing hyperpolarization during null direction motion (Mauss, 2015).

    GABAergic inhibition has been shown to shape response properties of interneurons in early visual processing by mediating lateral antagonistic effects in Drosophila. Work in the Calliphora visual system has ascribed a more specialized role for GABAergic transmission in mediating null direction inhibition, based on experiments using picrotoxinin as a GABA receptor antagonist. Unexpectedly, in the same context, glutamate has been identified as the underlying neurotransmitter in Drosophila. This discrepancy is perhaps due to neglecting the action of the pharmacologic compound as a rather unspecific chloride channel blocker in earlier work. It should also be noted that, in Calliphora, picrotoxinin application was shown to have two effects on tangential cell motion processing: preferred direction depolarization was enlarged, and null direction hyperpolarization was replaced by noticeable depolarization. This was interpreted as evidence for weak directional tuning of the inputs, i.e., the later identified T4/T5 cells. A similar result was observed in Drosophila. The LPi3-4 block in Drosophila, however, did not produce a prominent null direction depolarization, and preferred direction excitation was indistinguishable from the control condition. Since a recent study demonstrated narrow directional tuning of the T4/T5 cells, rendering postsynaptic directional response sharpening unnecessary, it is suggested that picrotoxinin off-target effects on glutamate or GABA receptors in the upstream circuit are responsible for this inconsistency, and genetic LPi block represents a more suitable approach to eliminate null direction inhibition (Mauss, 2015).

    This analysis focused on the LPi3-4 neurons and their postsynaptic partners in layer 4, the VS cells, because of their experimental accessibility. However, the current findings can be most likely extended to the other layers. Tangential cells with dendrites in layer 3 have been identified in other fly species. Such so-called V2 cells are motion opponent with preference to upward flow, in agreement with their presumed inputs from excitatory layer 3 T4/T5 cells. Since the data indicate that the LPi4-3 neurons convey glutamatergic signals selective for downward motion to lobula plate layer 3, it seems plausible that a motion-opponent wiring complementary to the LPi3-4/VS cell connectivity exists as well. The preference of LPi4-3 cells to ON over OFF edges is unexpected because in contrast to tangential cells, LPi4-3 neurons appear to be able to differentiate between T4/ON and T5/OFF input. Whether this finding hints toward an ON-selective null direction inhibition in layer 3 postsynaptic cells, perhaps dictated by certain natural stimulus statistics, or whether it reflects merely a bias of the driver line for an ON-selective LPi4-3 subgroup remains to be investigated. Some presynaptic swellings of the complementary LPi3-4 cells also exhibited polarity preference, but at present, it is unclear whether this indicates a similar T4/ T5 selectivity on a cell-by-cell basis or stochastic sampling of inputs. The functional architecture of lobula plate layers 1 and 2 strongly resembles the one of layers 3 and 4 with a 90 directional tuning shift: motion-opponent HS cells with a preference for front-to-back motion ramify their dendrites exclusively in layer 1, while motion-opponent Hx cells that prefer back-to-front motion confine their dendrites to layer 2. Therefore the existence of at least two complementary horizontal LPi cell types is anticipated in those layers too. It thus seems that global motion information is processed initially in two segregated horizontal and vertical subsystems with little direct interaction. Rather than representing the cardinal directions in a clock- or counter-clockwise manner, the four lobula plate layers are arranged such that opposite directions are represented side by side. This functional organization might serve to facilitate efficient nearest-neighbor interactions of motion-opponent signals (Mauss, 2015).

    Similar to the fly lobula plate, the dorsal lateral geniculate nucleus (dLGN) in mammals relays direction-selective signals from the retina to higher brain centers (Cruz-Martin, 2014). Some fundamental parallels in the organization of the two brain regions seem to exist. Their input channels, T4/T5 neurons in flies and ON/OFF direction-selective ganglion cells in mammals, predominantly encode the four cardinal directions of motion up, down, left, and right. The anatomical separation of the vertical and horizontal subsystems in flies seems to be mirrored, at least to a degree, in the dLGN, where opposing horizontal direction information resides in the superficial region of mouse dLGN, segregated from vertical motion. Moreover, a feed-forward inhibitory principle to generate motion opponency that this study describes in the fly might also prevail in the dLGN, where directionally selective output neurons were suggested to integrate opposing signals from retinal ganglion cells, possibly directly and indirectly via local inhibitory neurons. However, many mammalian dLGN neurons are also orientation selective, potentially obtaining this property by integrating opponent excitatory direction-selective input (Mauss, 2015).

    Associated with their proposed role as matched filters for sensing the optic flow generated by an animal's self-motion, in contrast to dLGN neurons, lobula plate tangential cells have large receptive fields, in some cases covering more than 100 degrees of visual space. Independent movement, e.g., originating from conspecifics or foliage, thus poses a challenge to the system by providing excitatory drive to tangential cells not associated with self-motion. The current experiments with intact and silenced LPi neurons support the idea that such inputs are attenuated by antagonistic signals from oppositely moving objects elsewhere in the visual scene. Perhaps more importantly, different flight maneuvers generate ambiguous optic flow patterns in sub-parts of the receptive field. For instance, both lift and forward translation cause downward optic flow in the ventral visual field, while only the latter produces upward flow dorsally. Taking into account excitation only, a reliable distinction between those patterns, especially under varying stimulus intensities, i.e., contrasts as experienced in natural scenes, seems inconceivable. This study has demonstrated that LPi cells strongly reduce such ambiguities, most likely by cancelling the excitation caused in one part of the dendrite by inhibition in another part. Motion opponency is thus reminiscent of other neural opponent mechanisms. In the classical example of color opponency, neural comparison discriminates sensory signals that are ambiguous at the level of photoreceptors in terms of wavelength and stimulus intensity. Notably, while color vision requires at least two separate measurements at any point in space, motion opponency disambiguates different optic flowfields derived from the same photoreceptor responses. Given that wide-field motion-sensitive neurons in various other systems are also motion opponent, it is suggested that such a mechanism might be universally required to increase sensitivity and selectivity for optic flow-fields associated with selfmotion. Similar neural comparators might be widely used for the extraction of equally complex sensory features (Mauss, 2015).

    Nonlinear circuits for naturalistic visual motion estimation
    Many animals use visual signals to estimate motion. Canonical models suppose that animals estimate motion by cross-correlating pairs of spatiotemporally separated visual signals, but recent experiments indicate that humans and flies perceive motion from higher-order correlations that signify motion in natural environments. This study shows how biologically plausible processing motifs in neural circuits could be tuned to extract this information. Known aspects of Drosophila's visual circuitry can embody this tuning and predict fly behavior. Segregating motion signals into ON/OFF channels can enhance estimation accuracy by accounting for natural light/dark asymmetries. Furthermore, a diversity of inputs to motion detecting neurons can provide access to more complex higher-order correlations. Collectively, these results illustrate how non-canonical computations improve motion estimation with naturalistic inputs. This argues that the complexity of the fly's motion computations, implemented in its elaborate circuits, represents a valuable feature of its visual motion estimator (Fitzgerald, 2015).

    The metabolism of histamine in the Drosophila optic lobe involves an ommatidial pathway: β-alanine recycles through the retina

    Flies recycle the photoreceptor neurotransmitter histamine by conjugating it to β-alanine to form β-alanyl-histamine (carcinine). The conjugation is regulated by Ebony, while Tan hydrolyses carcinine, releasing histamine and β-alanine. In Drosophila, β-alanine synthesis occurs either from uracil or from the decarboxylation of aspartate but detailed roles for the enzymes responsible remain unclear. Immunohistochemically detected β-alanine is present throughout the fly's entire brain, and is enhanced in the retina especially in the pseudocone, pigment and photoreceptor cells of the ommatidia. HPLC determinations reveal 10.7 ng of β-alanine in the wild-type head, roughly five times more than histamine. When wild-type flies drink uracil their head β-alanine increases more than after drinking l-aspartic acid, indicating the effectiveness of the uracil pathway. Mutants of black, which lack aspartate decarboxylase, cannot synthesize β-alanine from l-aspartate but can still synthesize it efficiently from uracil. The findings of this study demonstrate a novel function for pigment cells, which not only screen ommatidia from stray light but also store and transport β-alanine and carcinine. This role is consistent with a β-alanine-dependent histamine recycling pathway occurring not only in the photoreceptor terminals in the lamina neuropile, where carcinine occurs in marginal glia, but vertically via a long pathway that involves the retina. The lamina's marginal glia are also a hub involved in the storage and/or disposal of carcinine and β-alanine (Borycz, 2012).

    The histamine recycling pathway in photoreceptors faces two major physiological demands. First, histamine is released at high rates, which if unopposed would deplete the eye within seconds. Second, the demands on histamine recycling vary greatly from moment to moment, at least within the time frame of 100 ms, depending on the light stimulus conditions that result from the fly's own activity and changes in its ambient light conditions. To maintain a constant supply of histamine may therefore require not only a fast recycling pathway via carcinine but also storage sites for the neurotransmitter, as well as β-alanine and their conjugate carcinine. These storage sites seem to be the marginal and fenestrated glia and, in the retina, the pigment cells. The fenestrated glia have already been recognized as such a site, and three interrelated candidate functions, recycling, spillover and reserve, have been identified. It is imagined that a rapid reuptake pathway is processed via epithelial glia and their capitate projections in the lamina. The additional storage sites in the ommatidium and cartridge are possibly responsible for the slower supply of histamine to photoreceptors for re-release, the supply of β-alanine for synthesis of carcinine in the epithelial and proximal satellite glia, or the return of carcinine to the photoreceptor for the liberation of both (Borycz, 2012).

    Synaptic circuits and their variations within different columns in the visual system of Drosophila

    This study reconstructed the synaptic circuits of seven columns in the second neuropil or medulla behind the fly's compound eye. These neurons embody some of the most stereotyped circuits in one of the most miniaturized of animal brains. The reconstructions allow study of variations between circuits in the medulla's neighboring columns. This variation in the number of synapses and the types of their synaptic partners has previously been little addressed because methods that visualize multiple circuits have not resolved detailed connections, and existing connectomic studies, which can see such connections, have not so far examined multiple reconstructions of the same circuit. This study addresses the omission by comparing the circuits common to all seven columns to assess variation in their connection strengths and the resultant rates of several different and distinct types of connection error. Error rates reveal that, overall, :lt;1% of contacts are not part of a consensus circuit, and those contacts that supplement (E+) or are missing from it (E-) were classified. Autapses, in which the same cell is both presynaptic and postsynaptic at the same synapse, are occasionally seen; two cells in particular, Dm9 and Mi1, form >/=20-fold more autapses than do other neurons. These results delimit the accuracy of developmental events that establish and normally maintain synaptic circuits with such precision, and thereby address the operation of such circuits. They also establish a precedent for error rates that will be required in the new science of connectomics (Takemura, 2015).

    Comprehensive characterization of the major presynaptic elements to the Drosophila OFF motion detector

    Estimating motion is a fundamental task for the visual system of sighted animals. In the Drosophila optic lobe, direction-selective T4 and T5 cells respond to moving brightness increments (ON) and decrements (OFF), respectively. Current algorithmic models of the circuit are based on the interaction of two differentially filtered signals. However, electron microscopy studies have shown that T5 cells receive their major input from four classes of neurons: Tm1, Tm2, Tm4, and Tm9. Using two-photon calcium imaging, this study demonstrates that T5 is the first direction-selective stage within the OFF pathway. The four cells provide an array of spatiotemporal filters to T5. Silencing their synaptic output in various combinations, it was found that all input elements are involved in OFF motion detection to varying degrees. This comprehensive survey challenges the simplified view of how neural systems compute the direction of motion and suggests that an intricate interplay of many signals results in direction selectivity (Serbe, 2016).

    Direct neural pathways convey distinct visual information to mushroom bodies

    Previous studies have identified that visual and olfactory associative memories of Drosophila share the mushroom body (MB) circuit. Despite well-characterized odor representations in the Drosophila MB, the MB circuit for visual information is totally unknown. This study shows that a small subset of MB Kenyon cells (KCs) selectively responds to visual but not olfactory stimulation. The dendrites of these atypical KCs form a ventral accessory calyx (vAC), distinct from the main calyx that receives olfactory input. Two types of visual projection neurons (VPNs) directly connecting the optic lobes and the vAC were identified. Strikingly, these VPNs are differentially required for visual memories of color and brightness. The segregation of visual and olfactory domains in the MB allows independent processing of distinct sensory memories and may be a conserved form of sensory representations among insects (Vogt, 2016).

    Automatic segmentation of Drosophila neural compartments using GAL4 expression data reveals novel visual pathways
    Identifying distinct anatomical structures within the brain and developing genetic tools to target them are fundamental steps for understanding brain function. This study hypothesized that enhancer expression patterns can be used to automatically identify functional units such as neuropils and fiber tracts. Two recent, genome-scale Drosophila GAL4 libraries and associated confocal image datasets were used to segment large brain regions into smaller subvolumes. Obtained results (available at Straw Lab BrainCode) support this hypothesis because regions with well-known anatomy, namely the antennal lobes and central complex, were automatically segmented into familiar compartments. The basis for the structural assignment is clustering of voxels based on patterns of enhancer expression. These initial clusters are agglomerated to make hierarchical predictions of structure. The algorithm was applied to central brain regions receiving input from the optic lobes. Based on the automated segmentation and manual validation, promising driver lines for 11 previously identified and 14 novel types of visual projection neurons and their associated optic glomeruli could be identified and are available for sharing. The same strategy can be used in other brain regions and likely other species, including vertebrates (Panser, 2016).

    Subcellular imaging of voltage and calcium signals reveals neural processing in vivo

    A mechanistic understanding of neural computation requires determining how information is processed as it passes through neurons and across synapses. However, it has been challenging to measure membrane potential changes in axons and dendrites in vivo. This study used in vivo, two-photon imaging of novel genetically encoded voltage indicators, as well as calcium imaging, to measure sensory stimulus-evoked signals in the Drosophila visual system with subcellular resolution. Across synapses, major transformations were found in the kinetics, amplitude, and sign of voltage responses to light. Distinct relationships were described between voltage and calcium signals in different neuronal compartments, a substrate for local computation. Finally, it was demonstrated that ON and OFF selectivity, a key feature of visual processing across species, emerges through the transformation of membrane potential into intracellular calcium concentration. By imaging voltage and calcium signals to map information flow with subcellular resolution, this study illuminates where and how critical computations arise (Yang, 2016).

    Visual projection neurons in the Drosophila lobula link feature detection to distinct behavioral programs

    Visual projection neurons (VPNs) provide an anatomical connection between early visual processing and higher brain regions. This study characterized lobula columnar (LC) cells, a class of Drosophila VPNs that project to distinct central brain structures called optic glomeruli. This study anatomically describes 22 different LC types and show that, for several types, optogenetic activation in freely moving flies evokes specific behaviors. The activation phenotypes of two LC types closely resemble natural avoidance behaviors triggered by a visual loom. In vivo two-photon calcium imaging reveals that these LC types respond to looming stimuli, while another type does not, but instead responds to the motion of a small object. Activation of LC neurons on only one side of the brain can result in attractive or aversive turning behaviors depending on the cell type. These results indicate that LC neurons convey information on the presence and location of visual features relevant for specific behaviors (Wu, 2016).

    This report presents anatomical and functional studies of lobula columnar (LC) cells, prominent visual projection neurons from the lobula to target regions in the central brain called optic glomeruli. Comprehensive anatomical analyses of the dendritic arbors and central brain projections of LC neurons support the notion that these cells encode diverse visual stimuli, distinct for each LC cell type, and convey this information to cell-type specific downstream circuits. Precise genetic tools that target individual LC cell types allowed exploration of the behavioral consequences of optogenetic activation of these cell types. Activating cells of single LC neuron types was often sufficient to evoke a range of coordinated behaviors in freely behaving flies. Using two-photon calcium imaging from head-fixed flies, two LC cell types with activation phenotypes similar to avoidance responses, were shown to selectively encode visual looming, a stimulus that also evokes similar avoidance behaviors, while a third cell type responded strongly to a small moving object. These results suggest that LC cell types encode visual features that are relevant for specific behaviors. Activation of LC cells in only one brain hemisphere can result in either an attractive or repulsive directional turning response, depending on cell type. Thus which LC neuron channel is activated determines the valence of the behavior, whereas comparison across the brain by two such channels of the same type provides information about the location of relevant visual features (Wu, 2016).

    Anatomical properties of LC neurons have been previously described both in Drosophila and other Diptera. This work extends these studies by providing a comprehensive description of LC neurons in Drosophila, including the identification of several previously unreported cell types. Further, these anatomical analyses with were combined the generation of highly specific genetic markers (split-GAL4 lines) for each cell type. Each of the 22 LC types described has morphologically distinct dendritic arbors in the lobula with stereotyped arbor stratification, size and shape. As observed in the medulla, where synapse-level connectomics data are available for many cell types, different layer patterns and arbor shapes are likely to reflect differences in synaptic connectivity and neuronal computation. Arbors of LC neurons are found in all lobula strata, though with large differences between layers. Only LC4 (and perhaps LPLC1 and LPLC2) cells are potentially postsynaptic to neurons in the most distal lobula layer, Lo1, while other strata such as Lo4 and Lo5B include processes of more than half of the LC types. The presence of at least some LC dendrites in each lobula layer implies that all of the about 50 different interneuron types that convey visual information from the medulla, and to a lesser extent from the lobula plate, to the lobula, are potentially presynaptic to some LC cells, although a far smaller number is likely presynaptic to any single LC cell type. The predicted differences in the synaptic inputs to different LC cell types also suggest that they will differ in their responses to visual stimuli. Thus, individual LC neuron types are expected to encode specific visual stimuli, while the population of all LC cell types together should signal a wide range of behaviorally relevant visual features (Wu, 2016).

    The visual responses of several LC cell types measured using two-photon calcium imaging support the expectation that different types selectively respond to different visual features. The three LC neuron types examined preferentially responded to distinct stimuli, with either a dark looming stimulus (LC6 and LC16) or a small moving object (LC11) evoking the strongest measured responses. LC6 and LC16 showed stronger responses to a dark expanding disc than to related stimuli such as an expanding bright disk or a darkening stimulus that lacks the expanding motion. The reduction in the LC6 and LC16 responses when the edge motion is removed from the stimulus is precisely what is expected of loom-sensitive neurons and is reminiscent of behavioral studies in houseflies showing that darkening contrast combined with edge motion is the most effective stimulus for triggering takeoffs. Consistent with their similar responses in the imaging experiments, LC6 and LC16 have very similar lobula layer patterns while LC11 has a different arbor stratification indicating that LC11 receives inputs from a different set of medulla cell types than LC6 and LC16 (Wu, 2016).

    It is likely that the selectivity for visual stimuli observed in LC neuron responses is both a property of the stimulus selectivity of their inputs-some selectivity was seen while imaging in the dendrites of a few LC cell types-and specific computations implemented by individual LC neuron types. In addition, cells post-synaptic to the LC cells may integrate the responses of several individual LC neurons of the same type to provide more robust detection of specific visual features. For example, while LC6 and LC16 cells as populations are strongly excited by dark looming stimuli, it is currently unknown whether individual LC6 and LC16 neurons, which have dendritic extents well below the maximum size of the looming stimuli, and also well below the size known to elicit maximal behavioral responses, show the same response properties. The anatomical data and genetic reagents provide a starting point for the additional functional and ultra-structural studies that will be required to elucidate the circuit mechanisms that produce the response properties of these and other LC cell types (Wu, 2016).

    The suggestion that LC cells are feature-responsive neurons has been partly based on the apparent dramatic reduction in retinotopy between LC neuron dendrites, which have a retinotopic arrangement in the lobula, and their axons, which appear to discard this spatial information as they converge onto optic glomeruli, the cell-type specific target regions in the central brain. This study extended previous analyses of LC neuron arbor convergence by directly visualizing multiple single LC cells in a glomerulus in the same fly. These experiments revealed no detectable retinotopy of LC cell processes in most glomeruli even at this cellular level of resolution. It is possible that the responses of individual LC cells carry information about retinotopic position; given the comparatively small size of LC dendrites (the lateral spread of even the largest LC cells covers less than 20% of visual columns) and the retinotopic distribution of these dendrites in the lobula it would be surprising if they did not. Such retinotopic responses could for example be relevant for those LC cell types that appear to have presynaptic sites in the lobula and are thus likely to provide input to retinotopically organized circuits. However, with the caveat that synapse-level connectivity was not examined, for most LCs the available anatomical information appears to support the view that much retinotopic information is discarded at the glomerulus level. Consistent with this anatomical observation, the calcium imaging experiments from single LC cell types revealed visual responses to localized stimuli that could be measured throughout a cross-section of the glomerulus without clear retinotopic arrangement of the responding axons. Because of the columnar nature and apparently restricted visual field of the dendrites of LC neurons, the features computed by individual LC neurons are likely to be well defined in subregions of the eye, with perhaps downstream circuits required to integrate these locally-extracted features, as discussed above for looming. There is currently little insight into how these computations are initiated in the optic glomeruli and this remains an exciting area for future investigation (Wu, 2016).

    Unlike the other LC neurons, it was found that LC10, and to a lesser extent LC9, cells retain some retinotopic information in the arrangement of their axon terminals indicating that the loss of retinotopy is not a necessary consequence of axonal convergence onto a glomerular target region. More specifically, it was observed that the order of LC10 axonal terminals in the anterior optic tubercle (AOTu) along the DV axis matches the sequence of AP positions of the corresponding dendrites in the lobula. This organization could facilitate synaptic interactions of LC10 cells corresponding to different azimuthal positions in the visual field with distinct target cells. Consistent with a possible general role of the AOTu in the processing or the relaying of retinotopic information, retinotopic responses have recently been observed in the dendrites of central complex neurons that, mainly based on work in other insects, are thought to be synaptic targets of output neurons of the lateral zone of the AOTu (Wu, 2016).

    It was found that, independent of the presence or absence of retinotopy at the glomerulus level, positional information can be extracted from the differential activity of LC cells between the two optic lobes. It was directly demonstrated this capability by genetically restricting optogenetic LC neuron activation to only one optic lobe. This unilateral activation evoked directional turning responses relative to the activated brain side. Thus, LC neuron signaling appears to convey information on both different visual features and their location. This may further extend the similarities to the antennal lobes where differences in odorant receptor neuron activity between the left and right antennal lobes may contribute to odorant tracking (Wu, 2016).

    Activation of different types of LC neurons can induce distinct behaviors including jumping, reaching, wing extension, forward walking, backward walking and turning. While specific activation phenotypes have been reported for a variety of cell types and behaviors, many of these studies have focused on command-like neurons thought to orchestrate specific motor programs. By contrast, the activation phenotypes reported in this study result from the optogenetic stimulation of different types of related visual projection neurons. A plausible interpretation of these results is that activation of LC neurons can mimic the presence of the visual features that these neurons normally respond to and thus elicits behavioral responses associated with these fictive stimuli. This possibility is supported by several lines of evidence from studies of LC6 and LC16. First, optogenetic depolarization of each of these cell types evokes a specific behavioral response-backward walking for LC16 and jumping for LC6-that resembles a similar natural avoidance or escape behavior. Second, backward walking and jumping can both also be elicited by presentation of a predator-mimicking visual loom and, third, in calcium imaging experiments both LC16 and LC6 showed a preferential response to a similar looming stimulus compared to a number of related stimuli. Although this study did not explore LC10 response properties, it is noted that LC10-activation phenotypes also show similarities to natural behaviors: movements resembling the directed foreleg extension displayed during activation-evoked reaching occur, for example, during gap-climbing behavior and in aggressive fly-fly interactions (Wu, 2016).

    Overall, the LC neuron activation phenotypes that were observed suggest that the encoding of visual information at the level of LC neurons is sufficiently specialized to contribute to distinct behavioral responses in a cell-type dependent fashion. However, patterns of LC neuron activation that produce more refined fictive stimuli than were employed in the current work will be required to fully explore the LC neuron behavioral repertoire. Likewise, more comprehensive physiological studies of the response properties of the LC cell types will be needed (Wu, 2016).

    How does LC cell activation evoke specific behavioral responses? In the simplest scenario, LC neuron depolarization could directly activate a single postsynaptic premotor descending interneuron that would then in turn trigger the observed behavior. This appears plausible in some cases: for example, activation of LC4 neurons (called ColA cells in larger flies) might evoke a jumping response via activation of the Giant Fiber (GF) cells, a pair of large descending neurons known to be postsynaptic to ColA and LC4 and which have a known role in escape behavior. For other LC cell types, there is currently no evidence suggesting a direct connection to descending neurons. For example, candidate descending neurons for the LC16 backward walking response, the moon-walker descending interneurons, do not have dendrites in or near the LC16 glomerulus. Responses to diverse visual stimuli, some of which may derive from LC neuron activity, have also been observed in higher order brain centers without direct connections to LC neurons such as the central complex (Wu, 2016).

    The activation experiments also provide several indications that the signaling downstream of LC neurons is likely to be more complex; for example, activation of a single LC cell type can give rise to multiple behaviors such as reaching, wing extension and turning for LC10, or backward walking and turning for LC16. Changes of the spatial pattern of LC neuron activation, as in the stochastic labeling experiments of this study, can further modify activation phenotypes. For example, unilateral LC16 activation primarily evokes turning away from the location of LC16 activation, not backward walking, suggesting that the relative differences in LC16 activity between the two eyes can guide the direction of motor output through downstream signaling. Furthermore, several different LC neuron types may contribute to the same or similar behaviors, as suggested by the jumping phenotypes of LC4, LC6, LC15, LPLC1 and LPLC2. Presumably, visual signals and other information downstream of LC neurons are integrated to select appropriate behavioral actions. Such additional processing is also suggested by the cases of neurons with overlapping response properties but distinct activation phenotypes such as LC6 and LC16. It is also noted that some responses to LC neuron activation appear to be context dependent; for example, reduced forward walking was observed for several LC cell types on the platform of the single-fly assay that is much smaller than the arena used in the arena assay (Wu, 2016).

    In addition, this study examined onoy the behavior of standing or walking flies and LC neuron signaling might have different consequences depending on the behavioral state. For example, looming stimuli can also elicit avoidance responses in flying flies, but these responses differ from the takeoff or retreat behaviors of walking animals. Therefore, while LC cell activity appears to convey visual information that is specialized for sets of related behavioral responses, LC neurons do not appear to instruct a single behavioral output (Wu, 2016).

    The most common activation phenotypes observed in the screen were apparent avoidance responses. Furthermore, in addition to the LC cells examined in this study, other VPNs may also contribute to avoidance behaviors. This predominance of avoidance phenotypes is perhaps not unexpected. Since escape responses have to be fast and reliably executed under many different conditions, neurons that signal features that can evoke escape may be particularly likely to show phenotypes in an activation screen. Given the importance of predator avoidance for fly survival, it appears plausible that a considerable fraction of visual output neurons might be utilized for the detection of visual threats ranging from looming to small objects. Furthermore, it is likely that CsChrimson-mediated depolarization of an entire population of LC neurons is more similar to the pattern of neuronal activity induced by an imminent collision, and thus responses of many individual loom-sensitive neurons, so it is not surprising that the activation screen revealed at least two looming-sensitive neuron types (Wu, 2016).

    The escape-inducing neurons that were identifed in this study could provide inputs to different escape response pathways, such as long- and short-mode escape, or act as multiple inputs to the same downstream circuits. Interestingly, neurons with avoidance-like activation phenotypes project to two separate groups of adjacent glomeruli, one in the dorsal Posterior Ventrolateral Protocerebrum (PVLP; LC6, LC16 and also LC15) and one more ventral and medial (LC4, LPLC1 and LPLC2), the latter two with dendrites in the lobula and lobula plate. This spatial organization may facilitate synaptic interactions of functionally related LC neuron types with common downstream pathways for a specific behavior. The second group is close to dendritic branches of the GF, large descending neurons required for short-mode responses in Drosophila and a postsynaptic partner of LC4/ColA and possibly also the two LPLC cell types. LC6 terminals do not overlap with GF dendrites and LC6 cells may play a role in the GF-independent escape pathways that have been proposed in both Drosophila and housefly. Parallel neuronal pathways involved in escape behaviors have been identified or postulated in both vertebrates and invertebrates, but a contribution of several identified visual projection neurons to such pathways, as suggested by the activation screen, has not been previously reported. Different visual output neurons with distinct tuning of their response properties to looming parameters such as speed, size, luminance change or edge detection might have evolved to ensure robust responses to avoid predators or collisions. It is, however, currently not known whether LPLC1, LPLC2, LC4 and LC15 are indeed sensitive to looming stimuli and if so, whether their response details differ from LC16, LC6 and each other. Nevertheless, the identification of these neurons opens the possibility to examine the potential contribution of several visual pathways to avoidance behaviors (Wu, 2016).

    LC neurons are a subset of the about a hundred VPN cell types that relay the output of optic lobe circuits to targets in the central brain. These data strongly support existing proposals for LC cell types as feature-detecting neurons, which have been mainly based on the distinct anatomical properties of LC cells. While these anatomical features distinguish LC neurons from many other VPNs, an association of VPN pathways with specific behaviors is not unique to LC cell types. The notion that individual neuronal pathways are tuned for specific behavioral requirements is a prominent theme in invertebrate neuroethology, with these neurons described as 'matched filters' for behaviorally relevant features of the external world. A number of previously studied VPN pathways, outside of the LC subgroup, have been described as encoding specific behaviorally related visual stimuli. In particular, very similar to the current results for LC6 and LC16, a group of tangential cells of the lobula and lobula plate (Foma-1 neurons) were found to respond to looming visual stimuli and, upon optogenetic activation, trigger escape responses. And perhaps most famously, the long-studied lobula plate tangential cells (LPTCs), such as the HS and VS cells, integrate local motion signals so as to preferentially respond to global optic flow patterns that are remarkably similar to visual motion encountered during specific behavioral movements. These findings are consistent with the idea that, at the outputs of the fly visual system, VPN pathways are found whose encoding properties are already well matched to particular fly behaviors or groups of behaviors. Matching the response properties of these deep sensory circuits to behavioral needs may be a general evolutionary solution to the challenge of dealing with the complexity of the visual world with limited resources (Wu, 2016).

    LC neurons have long been recognized as a potential entry point for the circuit-level study of visual responses outside of the canonical motion detection pathways. This study has provided a comprehensive anatomical description of LC cell types and genetic reagents to facilitate such further investigations. It was also shown that activation of several LC cell types results in avoidance behaviors and that some of these same LC types respond to stimuli that can elicit such behaviors. Other LC neurons appear to mediate attractive behavioral responses. This work provides a starting point for exploring the circuit mechanisms both upstream and downstream of LC neurons (Wu, 2016).

    Object-detecting neurons in Drosophila

    Many animals rely on vision to detect objects such as conspecifics, predators, and prey. Hypercomplex cells found in feline cortex and small target motion detectors found in dragonfly and hoverfly optic lobes demonstrate robust tuning for small objects, with weak or no response to larger objects or movement of the visual panorama. However, the relationship among anatomical, molecular, and functional properties of object detection circuitry is not understood. This study characterized a specialized object detector in Drosophila, the lobula columnar neuron LC11. By imaging calcium dynamics with two-photon excitation microscopy, it was shown that LC11 responds to the omni-directional movement of a small object darker than the background, with little or no responses to static flicker, vertically elongated bars, or panoramic gratings. LC11 dendrites innervate multiple layers of the lobula, and each dendrite spans enough columns to sample 75 degrees of visual space, yet the area that evokes calcium responses is only 20 degrees wide and shows robust responses to a 2.2 degrees object spanning less than half of one facet of the compound eye. The dendrites of neighboring LC11s encode object motion retinotopically, but the axon terminals fuse into a glomerular structure in the central brain where retinotopy is lost. Blocking inhibitory ionic currents abolishes small object sensitivity and facilitates responses to elongated bars and gratings. These results reveal high-acuity object motion detection in the Drosophila optic lobe (Keles, 2017).

    The comprehensive connectome of a neural substrate for 'ON' motion detection in Drosophila

    Analysing computations in neural circuits often uses simplified models because the actual neuronal implementation is not known. For example, a problem in vision, how the eye detects image motion, has long been analysed using Hassenstein-Reichardt (HR) detector or Barlow-Levick (BL) models. These both simulate motion detection well, but the exact neuronal circuits undertaking these tasks remain elusive. This study reconstructed a comprehensive connectome of the circuits of Drosophila's motion-sensing T4 cells using a novel EM technique. Complex T4 inputs were uncovered, and putative excitatory inputs cluster were revealed at T4's dendrite shafts, while inhibitory inputs localize to the bases. Consistent with a previous study, this. study revealed that Mi1 and Tm3 cells provide most synaptic contacts onto T4. It was not possible, however, to reproduce the spatial offset between these cells reported previously. This comprehensive connectome reveals complex circuits that include candidate anatomical substrates for both HR and BL types of motion detectors (Takemura, 2017).

    Development of the anterior visual input pathway to the Drosophila central complex

    The anterior visual pathway (AVP) conducts visual information from the medulla of the optic lobe via the anterior optic tubercle (AOTU) and bulb (BU) to the ellipsoid body (EB) of the central complex. This paper analyzes the formation of the AVP from early larval to adult stages. The immature fiber tracts of the AVP, formed by secondary neurons of lineages DALcl1/2 and DALv2, assemble into structurally distinct primordia of the AOTU, BU, and EB within the late larval brain. During the early pupal period (P6-P48) these primordia grow in size and differentiate into the definitive subcompartments of the AOTU, BU, and EB. The primordium of the EB has a complex composition. DALv2 neurons form the anterior EB primordium, which starts out as a bilateral structure, then crosses the midline between P6 and P12, and subsequently bends to adopt the ring shape of the mature EB. Columnar neurons of the central complex, generated by the type II lineages DM1-4, form the posterior EB primordium. Starting out as an integral part of the fan-shaped body (FB) primordium, the posterior EB primordium moves forward and merges with the anterior EB primordium. This paper documents the extension of neuropil glia around the nascent EB and BU and analyzes the relationship of primary and secondary neurons of the AVP lineages (Lovick, 2017).


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    Genes involved in organ development

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