The Interactive Fly

Genes involved in tissue and organ development

The Eye-Antenna Disc and Eye Morphogenesis

  • Genes expressed in eye morphogenesis
  • Genes expressed in antennal morphogenesis
  • Bolwig's Organ, the Hofbauer-Buchner Eyelet and Ocelli
  • Structure of the eye
  • Development of the eye

    Eye-antenna disc
  • Specification of the eye versus the antenna
  • 5D imaging via light sheet microscopy reveals cell dynamics during the eye-antenna disc primordium formation in Drosophila
  • Transformation of eye to antenna by misexpression of a single gene
  • Notch-dependent epithelial fold determines boundary formation between developmental fields in the Drosophila antenna
  • Identification of genomic enhancers through spatial integration of single-cell transcriptomics and epigenomics
  • Single cell RNA sequencing of the adult Drosophila eye reveals distinct clusters and novel marker genes for all major cell types
  • Allocation of distinct organ fates from a precursor field requires a shift in expression and function of gene regulatory networks
  • Patterning mechanisms diversify neuroepithelial domains in the Drosophila optic placode
  • Avalanches during epithelial tissue growth; Uniform Growth and a Drosophila eye disc model

    Eye development
  • A quantitative analysis of growth control in the Drosophila eye disc
  • Atonal and EGFR signalling orchestrate rok- and Drak-dependent adherens junction remodelling during ommatidia morphogenesis
  • Mapping gene regulatory networks in Drosophila eye development by large-scale transcriptome perturbations and motif inference Comparative transcriptome analyses of the Drosophila pupal eye
  • Stochastic spineless expression creates the retinal mosaic for colour vision
  • Binary cell fate decisions and fate transformation in the Drosophila larval eye
  • The spatiotemporal order of signaling events unveils the logic of development signaling
  • New regulators of Drosophila eye development identified from temporal transcriptome changes
  • dachshund potentiates Hedgehog signaling during Drosophila retinogenesis
  • Wingless mediated apoptosis: How cone cells direct the death of peripheral ommatidia in the developing Drosophila eye
  • Drosophila DDX3/Belle exerts its function outside of the Wnt/Wingless signaling pathway
  • Polycomb group genes are required to maintain a binary fate choice in the Drosophila eye
  • Single-base pair differences in a shared motif determine differential Rhodopsin expression
  • Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila
  • An eye on trafficking genes: Identification of four eye color mutations in Drosophila
  • A model of the spatio-temporal dynamics of Drosophila eye disc development
  • Increased avidity for Dpp/BMP2 maintains the proliferation of progenitors-like cells in the Drosophila eye
  • The Drosophila functional Smad suppressing element fuss, a homologue of the human Skor genes, retains pro-oncogenic properties of the Ski/Sno family
  • The cuticular nature of corneal lenses in Drosophila melanogaster
  • Developmentally regulated autophagy is required for eye formation in Drosophila
  • A population of G2-arrested cells are selected as sensory organ precursors for the interommatidial bristles of the Drosophila eye
  • mir-279/996 cluster represses receptor tyrosine kinase signaling to determine cell fates in the Drosophila eye
  • The phosphoinositide phosphatase Sac1 regulates cell shape and microtubule stability in the developing Drosophila eye
  • Cellular homeostasis in the Drosophila retina requires the lipid phosphatase Sac1
  • ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet-mediated degeneration
  • Role of the smallish gene during Drosophila eye development
  • Novel approach to quantitative spatial gene expression uncovers genetic stochasticity in the developing Drosophila eye
  • Activating and repressing gene expression between chromosomes during stochastic fate specification
  • STRIPAK-PP2A regulates Hippo-Yorkie signaling to suppress retinal fate in the Drosophila eye disc peripodial epithelium
  • Tissue-specific Expression of Ribosomal Protein Paralogue eRpL22-like in Drosophila melanogaster Eye Development
  • Ultrastructural evidence for the origin of the subretinal pigment shield in the compound eye of Drosophila melanogaster
  • Early lineage segregation of the retinal basal glia in the Drosophila eye disc
  • Quantitative Relationships Between Growth, Differentiation, and Shape That Control Drosophila Eye Development and Its Variation
  • Geometry of gene regulatory dynamics
  • Histone H3.3 K27M and K36M mutations de-repress transposable elements through perturbation of antagonistic chromatin marks
  • The timing of cell fate decisions is crucial for initiating pattern formation in the Drosophila eye
  • The Blimp-1 transcription factor acts in non-neuronal cells to regulate terminal differentiation of the Drosophila eye
  • Blimp-1/PRDM1 and Hr3/RORbeta specify the blue-sensitive photoreceptor subtype in Drosophila by repressing the hippo pathway
  • Motif 1 Binding Protein suppresses wingless to promote eye fate in Drosophila
  • Tiling mechanisms of the Drosophila compound eye through geometrical tessellation
  • Probing the conserved roles of cut in the development and function of optically different insect compound eyes
  • The Notch pathway regulates the Second Mitotic Wave cell cycle independently of bHLH proteins

    Photoreceptor development
  • Role of Tau, a microtubule associated protein, in Drosophila photoreceptor morphogenesis
  • The actomyosin machinery is required for Drosophila retinal lumen formation
  • Does Unc-GFP uncover ciliary structures in the rhabdomeric eye of Drosophila?
  • Ire1 supports normal ER differentiation in developing Drosophila photoreceptors
  • Past1 modulates Drosophila eye development
  • Apical accumulation of the Sevenless receptor tyrosine kinase during Drosophila eye development is promoted by the small GTPase Rap1
  • Integrins are required for synchronous ommatidial rotation in the Drosophila eye linking planar cell polarity signalling to the extracellular matrix
  • Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila
  • Nonsynaptic Transmission Mediates Light Context-Dependent Odor Responses in Drosophila melanogaster
  • Generation and characterization of inner photoreceptor-specific enhancer-trap lines using a novel piggyBac-Gal4 element in Drosophila
  • Identification of Genes Involved in the Differentiation of R7y and R7p Photoreceptor Cells in Drosophila
  • Hydroxylated sphingolipid biosynthesis regulates photoreceptor apical domain morphogenesis
  • The role of Ire1 in Drosophila eye pigmentation revealed by an RNase dead allele
  • A combinatorial cis-regulatory logic restricts color-sensing Rhodopsins to specific photoreceptor subsets in Drosophila
  • Calcium waves facilitate and coordinate the contraction of endfeet actin stress fibers in Drosophila interommatidial cells
  • Emergence of a geometric pattern of cell fates from tissue-scale mechanics in the Drosophila eye
  • The exit of axons and glial membrane from the developing Drosophila retina requires integrins
  • Brain connectivity inversely scales with developmental temperature in Drosophila<
  • Tissue fluidity mediated by adherens junction dynamics promotes planar cell polarity-driven ommatidial rotation
  • Prickle isoform participation in distinct polarization events in the Drosophila eye

    Damage Response and Regeneration
  • Fate mapping during regeneration: cells that undergo compensatory proliferation in damaged Drosophila eye imaginal discs differentiate into multiple retinal accessory cell types
  • Dpp/TGFβ-superfamily play a dual conserved role in mediating the damage response in the retina
  • The Clock:Cycle complex is a major transcriptional regulator of Drosophila photoreceptors that protects the eye from retinal degeneration and oxidative stress
  • Chemical interference with DSIF complex formation lowers synthesis of mutant huntingtin gene products and curtails mutant phenotypes

    Aging
  • An increase in mitochondrial TOM activates apoptosis to drive retinal neurodegeneration
  • Dietary restriction and the transcription factor Clock delay eye aging to extend lifespan in Drosophila Melanogaster

    Vision
  • Contribution of photoreceptor subtypes to spectral wavelength preference in Drosophila
  • Color discrimination with broadband photoreceptors
  • Genetic dissection reveals two separate retinal substrates for polarization vision in Drosophila
  • Identifying functional connections of the inner photoreceptors in Drosophila using Tango-Trace
  • Loss of Na/K-ATPase in Drosophila photoreceptors leads to blindness and age-dependent neurodegeneration
  • Histamine Recycling Is Mediated by CarT, a Carcinine Transporter in Drosophila Photoreceptors
  • Anatomical reconstruction and functional imaging reveal an ordered array of skylight polarization detectors in Drosophila
  • Fly photoreceptors encode phase congruency
  • Small fruit flies sacrifice temporal acuity to maintain contrast sensitivity
  • Phototactic T-maze Behavioral Assay for Comparing the Functionality of Color-sensitive Photoreceptor Subtypes in the Drosophila Visual System
  • Interaction of "chromatic" and "achromatic" circuits in Drosophila color opponent processing
  • High-speed imaging of light-induced photoreceptor microsaccades in compound eyes
  • Binocular mirror-symmetric microsaccadic sampling enables Drosophila hyperacute 3D vision
  • Muscles that move the retina augment compound eye vision in Drosophila


    Structure of the eye

    The adult eye consists of an array of approximately 800 hexagonal ommatidia, or facets. There is also an array of sensory hairs projecting from the surface.

    Each ommatidium contains eight photoreceptor cells, each associated with a rhabomere. A rhabomere is a rod-like element containing photoreceptor elements (see The Drosophila Adult Ommatidium: Illustration and explanation with Quicktime animation). Photoreceptor cells one through six of each ommatidium are placed radially around cells 7 and 8, forming an irregular trapezoid. Rhadomeres of cells seven and eight occupy a central position, cell seven above cell eight. Each ommatidium is surrounded by two primary pigment cells and these are surrounded by six secondary pigment cells, shared by adjacent ommatidia. Thus each ommatidium contains a total of 22 cells, making the total number of cells in each eye over 16,000 (Ready, 1976). From each mature cluster a bundle of eight axons runs posteriorly into a pre-optic stalk. For information on inductive relationships between the projecting axons and the innervated medula of the brain optic lobe see the brain site.

    In the differentiation of the eye, R8 is the first cell population to have its fate determined. Subsequently and successively comes the differentiation of three cell pairs: R2/5, R3/4 and R1/6. Following this is the differentiation of R7 and the surrounding cone cells.

    Development of the eye

    The eye is derived from the eye-antennal disc. The disc itself arises from approximately 20 cells of the optic primordium in the embryonic blastoderm. The disc is formed by invagination at stage 12, to produce a flattened sac of epithelium. By the third instar larva the disc contains about 2000 cells. During the middle of the third instar phase, a dorsal ventral furrow forms, advancing from posterior to anterior. The furrow, whose progression requires hedgehog function, is the site where commitment to photoreceptor fate is initiated. hh activates the expression of decapentaplegic and the proneural gene atonal in the furrow. ato expression is refined to the future R8 photoreceptor and is required for the development of this cell. The area anterior to the furrow is rich in synchronously dividing cells, but lacks a pattern. The furrow itself is caused by a shortening of cells at its center. The area posterior to the furrow shows preclusters of cells, each with a recognizable core of five cells, corresponding to cells 2, 3, 4, 5 and 8 of the photoreceptor. These photoreceptors and accessory cells are recruited to each ommatidial cluster (composed of developing photoreceptors) by waves of expression of the ligand spitz (spi) for the EGF receptor. A diagram is presented describing the progression of the morphogenetic furrow across the eye disc.

    In addition to the dorso-ventral furrow described above, there is an axis of dorso-ventral symmetry. Associated with the advancing boundary of cluster formation, the area ahead of this boundary is indented along the anterior posterior axis. This groove can be seen in the early third instar disc. It is this anterior-posterior groove that structures the DV symmetry of the eye (Ready, 1976). A diagram is presented describing the specification of the eye disc primordium and the establishment of dorsal/ventral asymmetry.

    Cells in the Drosophila eye are determined by inductive signalling. A model of eye development has been built that explains how simple intercellular signals could specify the diverse cell types that constitute the ommatidium. This model arises from the observation that the Drosophila homologue of the EGF receptor (EGF-R) is used reiteratively to trigger the differentiation of each of the cell types -- successive rounds of EGF-R activation recruit first the photoreceptors, then cone and finally pigment cells. It seems that a cell's identity is not determined by the specific signal that induces it, but is instead a function of the state of the cell that receives the signal. EGF-R signalling is activated by the ligand, Spitz, and inhibited by the secreted protein, Argos. Spitz is initially produced by the central cells in the ommatidium and diffuses over a small distance. Argos has a longer range, allowing it to block more distal cells from being activated by low levels of Spitz; This interplay between a short-range activator and a long-range inhibitor is termed 'remote inhibition'. Since inductive signalling is common in many organisms and its components have been conserved, it is possible that the logic of signalling may also be conserved (Freeman, 1997).

    Specification of the eye versus the antenna

    The Drosophila compound eye is specified by the concerted action of seven nuclear factors: Twin of eyeless (Toy), Eyeless (Ey), Eyes absent (Eya), Sine oculis (So), Dachshund (Dac), Eye gone (Eyg), and Optix (Opt). These factors have been called 'master control' proteins because loss-of-function mutants lack eyes and ectopic expression can direct ectopic eye development. However, inactivation of these genes does not cause the presumptive eye to change identity. Surprisingly, several of these eye specification genes are not coexpressed in the same embryonic cells -- or even in the presumptive eye. Surprisingly, the EGF Receptor and Notch signaling pathways have homeotic functions that are genetically upstream of the eye specification genes; specification occurs much later than previously thought -- not during embryonic development but in the second larval stage (Kumar, 2001).

    The Egfr and Notch pathways function in the specification or determination of the eye. An ey-GAL4 driver was used to express target proteins; this element drives expression first in the eye and antenna anlagen in the embryo (by stage 11) and then in regions ahead of the furrow in just the eye imaginal disc. Egfr function was removed in this domain by expressing a dominant negative form of the receptor. Both the eye and antenna were deleted from eclosed adults indicating that both structures require Egfr signaling for their specification, determination, or survival. Under these conditions, the larval discs do not form, making analysis of later developmental phenotypes impossible. The same phenotype was obtained with dominant negative Ras indicating that this activity is Ras dependent. Wild-type and activated forms of several components of the Ras pathway were expressed in the eye anlagen using the same driver and it was found that hyperactivation of many elements leads to the homeotic transformation of the eye into a morphologically complete antenna. Homeotic transformation of the eye to antenna can also be induced by the Egfr ligand Spitz but not by two other known activators. The membrane bound version of Spitz does not induce homeotic transformations, suggesting a requirement for paracrine signaling. Wild-type and constitutively active forms of the Egfr and two other Drosophila RTKs (Breathless [Btl] and Heartless [Htl]) were expressed but only Egfr is able to induce the transformation. Expression of the constitutively active version of Egfr gives a significantly stronger phenotype than the wild-type version of Egfr, suggesting that the level of Egfr signaling is important for maintaining the balance between eye and antennal identities. The downstream elements of the pathway that can induce this transformation include Ras, Raf, and PntP1, while neither MEK, MAPK, nor PntP2 induced this effect in this assay. Aop, Tramtrack (Ttk), and BarH1/H2, each of which mediates negative feedback inhibition of Egfr signaling, delete the eye. The failure of Mek, Mapk, and PntP2 to induce this transformation reflects the existence of actual branch points in the pathway. However, it is also possible that the quantitative levels of expression of these three elements are not limiting for this signal at this time and place; indeed, their phosphorylation states may be more relevant (Kumar, 2001).

    Notch and Egfr have been shown to often antagonize each other during cell fate decisions in the fly eye. Notch function was removed with a dominant negative form and results similar to the effects of Egfr signal hyperactivation were obtained. Consistent with this, when an activated form of Notch was expressed, the size of the eye was reduced and there were severe dysmorphies. Expression of dominant negative transgenes of the ligands Delta (Dl) or Serrate (Ser) also results in the eye to antenna transformation. Elevated expression levels of both Su(H) and many of the proteins of the E(spl) complex (m4, m7, m8, m8DN, malpha, mß, mgamma, and mdelta) were also expressed but no effect on either eye or antenna disc development was observed. However, homeotic eye to antenna transformations occurred when Mastermind (Mam) was expressed using a dominant negative construct. Mam is a member of the neurogenic gene group that encodes a nuclear protein of unknown function. These results suggest a Su(H) and E(spl)C independent pathway for eye and antenna disc development that involves Mam (Kumar, 2001).

    The Wg and Hh pathways have been shown to regulate the size and shape of the eye. Overexpression of a transgene containing the full-length wild-type Cubitus interruptus (Ci) protein, a downstream component of Hh signaling, can mediate eye to antenna homeotic transformations. It is uncertain if these data show a positive or negative Hedgehog signal; Ci may be cleaved into the repressor form, thereby mimicking the effects on eye development seen in loss of Hedgehog signaling. Overexpression of Wg results in the near complete deletion of the eye. Downstream of Wg lie two transcription factors: Sloppy Paired 1 and 2 (Slp1, Slp2). The effect of Wg on eye specification appears to be mediated through Slp2 but not Slp1. Taken together, these observations suggest that along with Egfr and Notch, both Wg and Hh signaling function during eye and antenna disc specification (Kumar, 2001).

    Do Egfr and Notch Act upstream of the eye specification genes? A molecular epistasy study was undertaken, examining the expression of some of the eye and antennal specification genes in the transforming conditions during the third larval stage (before cell types differentiate). In eye specification gene mutants (such as ey), ommatidial development is blocked, but the eye disc remains in a reduced form. Conditions that produce eye to antenna transformations, whether through hyperactivation of Egfr or downregulation of Notch signaling, show a complete replacement of the eye disc with an antenna disc. Distal-less (Dll) and Spalt-Major are normally expressed within subdomains of the antenna disc and are required for antenna development. Dll and SalM are expressed in the correct locations in the transformed antenna disc suggesting that both endogenous and transformed antenna are also both morphologically and molecularly equivalent (Kumar, 2001).

    The transcription of five of the seven known eye specification genes (toy, ey, eya, so, and eyg) was examined. In transforming conditions, transcription levels of all five of the seven genes are below the levels of detection. This is consistent with both Egfr and Notch signaling acting genetically upstream to both the eye and antennal specification genes. The downregulation of ey suggests that the ey-GAL4 driver may also be downregulated via an autoregulatory mechanism. That the transformation occurs despite this may reflect a phenocritical period for the eye-antenna transformation; once the transformation has occurred the system is refractory to the loss of Egfr signaling (Kumar, 2001).

    When and where are the eye and antenna specified? The seven known eye specification genes are thought to act in a genetic and biochemical complex; by pairwise tests, their products have been shown to either directly regulate each other's transcription or to interact at the protein level, or both. From the few published reports of the early expression patterns of eye specification genes and from fate mapping experiments, it has been suggested that eye versus antennal fate specification occurs during the latter stages of embryogenesis. These concepts lead to a straightforward hypothesis: at some point in the developing embryo, the seven eye specification genes' products are coexpressed in the presumptive eye and act to specify its fate. A similar event (with the action of different genes) also specifies the antenna (Kumar, 2001).

    If this hypothesis is true, then three predictions should hold: (1) At some time during embryonic development, there should be two domains of expression of the eye specification genes that correspond to the future eyes, and anterior to these, should be two domains of antenna specification gene expression marking and acting to direct antenna fate. These gene products should be specific to the future structures they mark, and should not be found elsewhere. (2) The eye specification genes should be coexpressed in the same cells. This is known to be true of toy, ey, and eyg. (3) The phenocritical period for the eye to antenna transforming function of Egfr and Notch pathway signaling should be coincident with, or earlier than, the time at which the eye and antenna specification genes are first specifically coexpressed. All three of these predictions were tested (Kumar, 2001).

    To test the first prediction above, embryos were collected (at 1 hr intervals from 1 to 16 hr after egg deposition, AED) and analyzed for expression of the canonical eye specification gene ey (Pax6) and the antenna specification protein Dll. Dll is first detected at 7 hr in the leg imaginal disc primordia and in several segments in the embryonic head. ey transcription in the eye imaginal disc is first detectable at 11 hr while Dll is seen in an adjacent region as well as other sites. In latter stages of embryogenesis, the eye imaginal disc invaginates and assumes a more dorsal-medial position within the embryonic head, just above the developing embryonic brain. Regions of ey expression are observed that correspond to this. Furthermore, this ey expression corresponds to domains of Escargot expression (Esg), a general imaginal disc marker. However, Dll expression is more anterior and it is not clear if these sites correspond to the presumptive antennae. It thus appears that ey is expressed in both the presumptive eye and antenna by 13 hr and remains there through the last embryonic time point observed, and that Dll is not expressed in the future antenna at any embryonic time. It is also quite clear that ey is expressed in many sites in the embryo that will never form eye (such as the segmental grooves). In short, the position of the presumptive eye or antenna during embryonic development cannot be distinguish based on the specific expression of their respective 'master control' genes—neither ey nor Dll expression are sufficient to specify the eye or the antenna; therefore, prediction 1 (above) does not hold true (Kumar, 2001).

    Are the eye specification genes coexpressed during embryonic development? The expression pattern of Eya and Dac proteins and so transcription were examined at 1 hr time points (from 1 to 16 hr AED) and it was found that none of these three eye specification genes is coexpressed with ey within the presumptive eye. The fact that these genes are not expressed within the same cells during embryonic development precludes any possibility that their products act in a multiprotein complex critical for eye specification in the embryo and, thus, prediction 2 (above) does not hold true either. However, eye specification might occur later in development (Kumar, 2001).

    The eye specification genes are first coexpressed in the second larval stage. In second stage larva, the eye specification gene products are completely segregated into the eye portion of eye-antennal disc, but the antennal marker Dll is evenly expressed in both the eye and antennal segments. Interestingly, the expression patterns of the eye specification genes are still not completely overlapping. For instance, toy appears to be expressed throughout the entire eye field while both eya and dac are expressed just in the posterior portions of the eye disc. In the third larval stage, the eye specification genes remain within the eye portion and Dll is now segregated to just the antennal segments (Kumar, 2001).

    The phenocritical period for eye to antenna transformation is in the second larval stage. Use was made of the cold sensitivity of the GAL4 protein to determine the phenocritical period. GAL4 is a yeast protein and is fully functional at 25°C but is less active at 18°C. Flies of the ey-GAL4/UAS-SerDN genotype were raised at 18°C, shifted to 25°C for a consecutive series of 24 hr periods, and then returned to 18°C until late third instar imaginal discs could be examined. The use of the dominant negative Ser construct in this experiment effectively eliminates Notch pathway function in the developing eye. Indistinguishable transformations were observed in other experiments with constructs that either hyperactivate Egfr pathway signaling or inactivate Notch. The developing eye-antennal complex is completely normal if kept continuously at 18°C (negative control) while constant exposure to 25°C temperatures resulted in the eye to antenna transformation (positive control). These controls confirm that the cold sensitivity of GAL4 protein activity is sufficient to control the transformation (Kumar, 2001).

    Temperature shifts during the embryonic and first larval stages failed to induce any effects. The eye-antennal discs are completely normal. This is consistent with the expression data suggesting that the eye is not specified during embryogenesis. A temperature shift during the first half of the second larval stage results in a reduced eye field, but no transformation to antenna. Interestingly, this phenotype is similar to that seen in ey mutant homozygotes. The eye to antenna transformation is fully induced in all cases when the temperature shift occurs during the latter half of the second larval stage. The transformed antenna expresses Dll in an identical pattern as seen in the endogenous antenna. Subsequent temperature shifts during the earliest phase of the third larval stage do not result in a complete transformation. Interestingly, loss of Notch during the next day of the third larval stage results in defects in the regulation of the morphogenetic furrow. These results clearly indicate that the phenocritical period is chiefly within the latter half of the second larval stage. This phenocritical period does not predate the expression of any of the eye specification genes, but it is coincident with their first coexpression and, thus, prediction 3 (above) holds true (Kumar, 2001).

    Is the presumptive eye actually transformed into a second antenna under these conditions, or does the eye degenerate and get replaced by regrowth from elsewhere? The former interpretation (transformation) is favored because in hundreds of transformed L2 disc complexes dissected, a degenerating eye disc, or a small (presumably regrowing) antennal disc was never observed. In all cases, the transformed antenna is equal in size to the normal one. Indeed, both are somewhat larger than normal. This might suggest that a fixed number of cells that normally distribute preferentially to the eye are now equally allocated to both antennae (Kumar, 2001).

    The notch pathway signals differentially in the eye and antenna primordia in the second larval stage. Loss of Notch activity during the second larval stage results in the transformation of the eye into an antenna. Thus, it is predicted that Notch signaling should be elevated in the presumptive eye versus the antenna at the critical time. Cells that are actively receiving a Notch signal upregulate Notch protein expression. Thus elevated Notch antigen expression can be used as a reporter of elevated Notch signaling. Notch and ey expression were examined in imaginal discs from first, second, and third stage larvae. Both Notch and ey are expressed throughout the entire eye-antennal disc anlagen during the first larval stage. By the second larval stage, Notch is differentially upregulated within the presumptive eye. Interestingly, Notch appears especially active along the eye margins and midline, where it is thought to regulate retinal polarity. In contrast, ey appears to be exclusively within the eye field. In the third larval stage, Notch expression is upregulated in the morphogenetic furrow, where it acts to control ommatidial spacing while ey remains upregulated ahead of the furrow (Kumar, 2001).

    Therefore, Egfr signaling promotes an antennal fate while Notch signaling promotes an eye fate. This role for Notch is consistent with the observation that removal of Notch signaling can partially inhibit compound eye development. Furthermore, several of the eye and antennal specification genes (ey, toy, eya, so, eyg, salM, and Dll) are downstream of the Egfr and Notch inputs. Wg and Hh pathway signaling affect this specification. The eye specification genes form a regulatory network and the direct control of any one of these genes may affect the others. Thus, which (if any) of the known eye specification genes is a direct target of Notch or Egfr signals may require direct biochemical assays (Kumar, 2001).

    While activating Egfr or blocking Notch signals transforms the eye cleanly into an antenna, the reciprocal transformation is not complete, suggesting that there may be additional positive regulators of eye fate. The reciprocal transformation experiment could not be conducted (i.e., antenna to eye switch via hyperactivation of Notch or downregulation of Egfr signaling solely within the antennal anlagen). Unlike the ey-GAL4 driver, there is not an equivalent known driver that is expressed solely with the antennal anlagen. All known antennal-determining genes are also expressed in other imaginal discs. For instance, the Dll-GAL4 driver is expressed in several places within the embryonic head and leg imaginal disc. Expression of Egfr or Notch constructs with this driver results only in embryonic lethality. It may be that the antenna can be changed to an eye via alterations of Egfr or Notch signaling provided that the appropriate tools for their missexpression are available (Kumar, 2001).

    Why do homozygous mutants for eye specification genes not transform the eye into an antenna? While it may be that some alleles are not nulls (e.g., ey1), a more interesting possibility is that there may be functional redundancy in some cases -- particularly that of ey and toy. Thus, only when both genetic functions are eliminated will a true null condition exist. Just such a situation confused the phenotypic analysis of two other twin homeodomain proteins, engrailed and invected. Unfortunately, mutations of the toy gene do not yet exist (Kumar, 2001).

    How do the eye specification genes function? Published genetic epistasy and biochemical interaction data suggest that the seven known eye specification genes' products interact at the transcriptional and protein levels to direct cells toward eye fate. This requires that they are expressed in the same cells. Furthermore, it has been suggested that many, if not all, of these genes are 'master regulators' of eye fate -- that is, they are both necessary and sufficient for eye specification. Many very compelling experiments have been described showing the induction of ectopic eyes through the ectopic expression of these genes alone or in synergistic combinations. It is suggested that these genes come under separate regulation by different patterning signals in early development and that there are overlapping domains. Only when all of the domains coincide (during the second larval stage) do eye specification genes specify the eye. This seems to be the simplest explanation since the eye specification genes form a very tight genetic, biochemical, and transcriptional regulatory network suggesting that they are together required for eye specification. It may be that the final coexpression of the eye specification genes' products (and the exclusion of the antennal specification factors) is the last step required to allow the morphogenetic furrow to initiate in response to the next local expression of hh and for the final specification of retinal cell types and pattern (Kumar, 2001).

    5D imaging via light sheet microscopy reveals cell dynamics during the eye-antenna disc primordium formation in Drosophila

    Although the eye-antenna disc primordium primordium (EADP) is thought to be derived from the visual primordium, which is defined by so expression, ey and so expressions do not overlap. Consistently, loss of so does not affect ey and toy expressions and the formation of the EADP. Other genes in the retinal determination network, namely, dachshund (dac) and eyes absent (eya), are not expressed in the EADP. These observations strongly suggest that the EADP formation is independent of the retinal determination network genes so, dac and eya. Its origin and the course of formation need further clarification. 5D images of engrailed (en) and eye gone (eyg) gene expressions during the course of the (EADP) formation of Drosophila embryos from embryonic stages 13 through 16 were recorded via light sheet microscopy and analyzed to reveal the cell dynamics involved in the development of the EADP. Detailed analysis of the time-lapsed images revealed the process of EADP formation and its invagination trajectory, which involved an inversion of the EADP anterior-posterior axis relative to the body. Furthermore, analysis of the en-expression pattern in the EADP provided strong evidence that the EADP is derived from one of the en-expressing head segments (Huang, 2017).

    Transformation of eye to antenna by misexpression of a single gene

    In Drosophila, the eye and antenna originate from a single epithelium termed the eye-antennal imaginal disc. Illumination of the mechanisms that subdivide this epithelium into eye and antenna would enhance understanding of the mechanisms that restrict stem cell fate. This study shows that Dorsal interacting protein 3 (Dip3), a transcription factor required for eye development, alters fate determination when misexpressed in the early eye-antennal disc, and this observation has been taken advantage of to gain new insight into the mechanisms controlling the eye-antennal switch. Dip3 misexpression yields extra antennae by two distinct mechanisms: the splitting of the antennal field into multiple antennal domains (antennal duplication), and the transformation of the eye disc to an antennal fate. Antennal duplication requires Dip3-induced under proliferation of the eye disc and concurrent over proliferation of the antennal disc. While previous studies have shown that overgrowth of the antennal disc can lead to antennal duplication, these results show that overgrowth is not sufficient for antennal duplication, which may require additional signals perhaps from the eye disc. Eye-to-antennal transformation appears to result from the combination of antennal selector gene activation, eye determination gene repression, and cell cycle perturbation in the eye disc. Both antennal duplication and eye-to-antennal transformation are suppressed by the expression of genes that drive the cell cycle providing support for tight coupling of cell fate determination and cell cycle control. The finding that this transformation occurs only in the eye disc, and not in other imaginal discs, suggests a close developmental and therefore evolutionary relationship between eyes and antennae (Duong, 2008).

    Dip3 is able to bind DNA in a sequence specific manner and activate transcription directly. Dip3 possesses an N-terminal MADF domain and a C-terminal BESS domain, an architecture that is conserved in at least 14 Drosophila proteins, including Adf-1 and Stonewall. The MADF domain directs sequence specific DNA binding to a site consisting of multiple trinucleotide repeats, while the BESS domain directs a variety of protein-protein interactions, including interactions with itself, with Dorsal, and with a TBP-associated factor (Bhaskar, 2002).

    Antagonism between the N and EGFR signaling pathways influences developmental fate in the eye-antennal disc leading to a loss of eye tissue and the appearance of extra antennae. Although this phenotype was originally suspected to represent eye-to-antennal transformation, subsequent analysis suggests that it most likely represents antennal duplication. Specifically, the absence of the N signal leads to a failure in eye disc proliferation resulting in compensatory over-proliferation of the antennal disc and its subdivision into multiple antennae. Consistent with the idea that the extra antennae result from under-proliferation of the eye field, it was found that the phenotype was largely suppressed by over-expression of CycE to drive the cell cycle (Duong, 2008).

    In this study, it was found that inhibition of eye disc growth leads to antennal duplication. But in addition, it was shown that the same treatment that leads to antennal duplication can also direct the transformation of eyes to antennae. These two phenotypes are anatomically distinct. This anatomical distinction is evident in adults: antennae resulting from antennal duplication are found anterior to the antennal foramen, while the antennae resulting from eye-to-antenna transformation are found posterior to the antennal foramen. It is also apparent in larval eye-antennal imaginal discs: antennal duplication discs exhibit multiple circular dac expression domains within a single sac of epithelium (the antennal disc), while eye-to-antennal transformation discs exhibit two or more circular dac expression domains spread over both the eye and antennal discs. The two types of discs display distinct molecular signatures as well: the antennal duplication discs exhibit duplicated Dll expression domains, while the eye discs undergoing transformation to antennae lack Dll expression (Duong, 2008).

    Perhaps the most persuasive evidence that Dip3 can direct eye-to-antennal transformation is provided by the observation of eyes that are only partially transformed to antennae since is very difficult to reconcile these partial transformations with the idea of antennal duplication. In some cases, proximal antennal segments tipped with eye tissue are observed. In accord with this phenotype, some third instar larval eye discs display a central domain of Elav-positive differentiating photoreceptors surrounded by a circular dac domain (Duong, 2008).

    These arguments support the idea that antennal duplication and eye-to-antennal transformation are mechanistically distinct phenomena, and the remainder of the discussion assumes this to be the case. However, the possibility that these two phenotypes are two manifestations of a single mechanism cannot be excluded. For example, the discs exhibiting duplicated Dll domains may represent complete transformations, while the discs lacking duplicated Dll domains, but containing Elav may represent partial transformations (Duong, 2008).

    The data show that discs undergoing antennal duplication as a result of Dip3 expression are comprised of a severely diminished eye region and an enlarged antennal region. As shown by BrdU labeling experiments, these antennal duplication discs most likely result from suppression by Dip3 of cell proliferation in the eye field leading to overproliferation of the antennal disc. This conclusion is supported by the ability of factors that drive cell proliferation (e.g., Cyclin E) to alleviate the Dip3 misexpression defect (Duong, 2008).

    Many experimental manipulations that reduce the size of the eye disc (e.g., surgical excision, induction of cell death, or suppression of cell proliferation) lead to enlargement and duplication of the antennal primordium. How might reduction of the eye field lead to antennal field over-growth? One possibility is that the eye field produces a growth inhibitory signal. Alternatively, the eye field and the antennal field may compete with each other for limited nutrients or growth factors. In support of this latter possibility, recent studies of the role of dMyc in wing development have demonstrated growth competition between groups of imaginal disc cells (Duong, 2008).

    While the results imply that antennal disc overgrowth is required for antennal duplication, overgrowth is thought not to be sufficient for duplication. This conclusion derives from experiments in which an antennal disc specific driver is used to direct over-expression of CycE or Nact. This resulted in antennal overgrowth without concurrent reduction in the eye disc. In this case, antennal duplication was not observed. Thus, in addition to antennal overgrowth, antennal duplication also appears to require reduction or elimination of the eye disc. Regulatory signals from the eye disc may act to prevent antennal duplication (Duong, 2008).

    The eye and antenna discs differ in several respects: (1) Specific expression of the organ-specification genes. The eye disc expresses the retinal determination gene network (RDGN) genes, including eyeless (ey), twin of eyeless (toy), eyes absent (eya), sine oculis (so), and dachshund (dac), while the antennal disc expresses Dll and hth. hth is also expressed in the eye disc but in a distinct pattern from that seen in the antennal disc. In the second instar eye disc, hth is expressed throughout the eye disc, and collaborates with ey and teashirt (tsh) to promote cell proliferation. The hth expression domain later retracts to only the anterior-most region of the eye disc. This pattern is different from the circular expression pattern observed in the antennal disc. (2) In the antennal disc, dpp is expressed in a dorsal anterior wedge and wg is expressed in a ventral anterior wedge. The intersection of Dpp and Wg signaling is required to specify the proximodistal axis in the leg and antenna. In the early eye disc, Wg and Dpp signaling may overlap. But as the disc grows in size, the wg and dpp expression domain are separated, so that there is probably no intersection between high levels of Wg and Dpp signaling. (3) Whereas the partial overlap of Dll and hth expression domains in the antennal disc is important for proximodistal axis specification, there is no Dll expression in the eye disc. Dll expression in the center of the antennal and leg discs is induced by the combination of high levels of Dpp and Wg signaling. Because there is no overlap of Dpp and Wg signaling in the eye disc, Dll is not induced (Duong, 2008).

    Therefore, efficient transformation of the eye disc into an antennal disc requires at least three things: (1) repression of the eye fate pathway; (2) activation the antennal fate pathway; and (3) the intersection of Dpp and Wg signaling, mimicking the situation in the antenna and leg disc that induces proximodistal axis formation. Any one of these three conditions by itself is not sufficient. (1) Loss of the RDGN genes leads only to the loss of the eye. However, if apoptosis is blocked, or cell proliferation is induced, in the ey2 mutant (ey>p35 or ey>Nact in ey2), then Dll can be induced in the eye disc and extra antenna are formed. The induction of Dll is not ubiquitous in the eye disc, suggesting that the loss of ey does not autonomously lead to the expression of Dll and the transformation to the antennal fate. (2) Simply expressing the antennal determining genes Dll or hth in the eye disc does not change the eye fate into antennal fate. It was found that uniform expression of Dll in the eye disc (ey>Dll) resulted in mild eye reduction, whereas ey>hth completely abolished eye development. E132>Dll caused the formation of small antenna in the eye in about 46% of flies, whereas ptc>Dll and C68a>Dll induced extra antenna but not within the eye field. Therefore, although Dll and hth are important determinants for antennal identity, it is their specific spatial expression patterns that determine antennal development. (3) Creating the intersection of Wg and Dpp signaling does not change the eye into antenna. Such manipulation in the leg disc turned on vg and transdetermined the leg disc into wing disc. Therefore, the specific genes induced by Dpp and Wg signaling may depend on disc-specific factors. In the eye disc, turning on Wg signaling in the dpp expressing morphogenetic furrow only blocked furrow progression (Duong, 2008).

    In this study, it was found that the ectopic expression of a single gene, Dip3, can cause eye-to-antenna transformation. Dip3 apparently satisfied all three requirements. (1) Overexpression of Dip3 repressed (non-cell-autonomously) ey and dac. The repression of ey may be due to the induction of ct. The ability of Dip3 to simultaneously repress multiple retinal determination genes is completely consistent with the many known cross-regulatory interactions between these genes. (2) ey>Dip3 turned on ct and hth. (3) By blocking cell proliferation, ey>dip3 reduced the eye field size and allowed the intersection of Dpp and Wg signaling. Furthermore, ey>Dip3 induced en, which probably created an ectopic A/P border and induced ectopic dpp/wg expression (Duong, 2008).

    Interference with cell cycle progression appears to be a common link between the two phenotypes described in this study. In the case of antennal duplication, interference with eye disc growth leads to antennal disc overgrowth, which is a prerequisite for duplication. In the case of eye-to-antenna transformation, eye disc undergrowth allows the required intersection between Dpp and Wg signaling (Duong, 2008).

    The observation that Dip3 misexpression can transform the eye field, but not other tissues, to an antennal fate suggests a close evolutionary relationship between the eye and the antenna. Previous studies have emphasized the homology between antennae and legs. The findings presented here that misexpression of a single transcription factor, namely Dip3, can transform eyes to antennae provides support for the notion that the eye and antenna may also, in some sense, be homologous to one another. Previous evidence in support of this idea comes from the observation that similar spatial arrangements of Wg and Dpp signaling along with a temporal cue provided by the ecdysone signal are required for the formation of the eye and the mechanosensory auditory organ. Small mechanosensory sensilla, such as Johnston's organ and the chordotonal organs (stretch receptors) are thought to represent the earliest evolving sense organs. Perhaps the eye resulted from a duplication and specialization of such a sensillum (Duong, 2008).

    Notch-dependent epithelial fold determines boundary formation between developmental fields in the Drosophila antenna

    Compartment boundary formation plays an important role in development by separating adjacent developmental fields. Drosophila imaginal discs have proven valuable for studying the mechanisms of boundary formation. This study examined the boundary separating the proximal A1 segment and the distal segments, defined respectively by Lim1 and Dll expression in the eye-antenna disc. Sharp segregation of the Lim1 and Dll expression domains precedes activation of Notch at the Dll/Lim1 interface. By repressing bantam miRNA and elevating the actin regulator Enabled, Notch signaling then induces actomyosin-dependent apical constriction and epithelial fold. Disruption of Notch signaling or the actomyosin network reduces apical constriction and epithelial fold, so that Dll and Lim1 cells become intermingled. These results demonstrate a new mechanism of boundary formation by actomyosin-dependent tissue folding, which provides a physical barrier to prevent mixing of cells from adjacent developmental fields (Ku, 2017).

    This study attempted to unravel the molecular and cellular mechanisms of boundary formation in the Drosophila head. Focus was placed on the antennal A1 fold that separates the A1 and A2-Ar segments. The results showed that the expression of the selector genes Lim1 and Dll, which are expressed in A1 and A2-Ar, respectively, was sharply segregated. This step was followed by differential expression of Dl, Ser and Fng, as well as activation of N signaling at the interface between A1 and A2. N signaling then induced apical constriction and epithelial fold, possibly through repression of bantam to allow levels of the bantam target Ena to become elevated, with this latter inducing the actomyosin network. The actomyosin-dependent epithelial fold then provided a mechanical force to prevent cell mixing. When N signaling or actomyosin was disrupted, or when bantam was overexpressed, the epithelial fold was disrupted and Dll and Lim1 cells become mixed. Thus this study describes a clear temporal and causal sequence of events leading from selector gene expression to the establishment of a lineage-restricting boundary (Ku, 2017).

    Sharp segregation of Dll/Lim1 expressions began before formation of the A1 fold, suggesting that fold formation is not the driving force for segregation of Dll/Lim1 expression. Instead, the fold functions to safeguard the segregated lineages from mixing. Whether Dll/Lim1 segregated expression is due to direct or indirect antagonism between the two proteins is not known (Ku, 2017).

    Actomyosin-dependent apical constriction is an important mechanism for tissue morphogenesis in diverse developmental processes, e.g., gastrulation in vertebrates, neural closure and Drosophila gastrulation, as well as dorsal closure and formation of the ventral furrow and segmental groove in embryos. This study describes a new function of actomyosin, i.e., the formation of lineage-restricting boundaries via apical constriction during development (Ku, 2017).

    This actomyosin-dependent epithelial fold provides a mechanism distinctly different from other known types of boundary formation. The cells at the A1 fold still undergo mitosis, suggesting that mitotic quiescence is not involved. Perhaps epithelial fold as a lineage barrier is needed in situations in which mitotic quiescence does not happen. Mechanically and physically, epithelial folds could serve as stronger barriers than intercellular cables when mitotic activity is not suppressed. The drastic and sustained morphological changes, including reduced apical area and cell volume, may be accompanied by increased cortical tension of cells along the A1 fold, with such high interfacial tension then preventing cell intermingling and ensuring Dll and Lim1 cell segregation. Although similar to actomyosin boundaries, the epithelial fold in the A1 boundary is distinctly different from the supracellular actomyosin cable structure in fly parasegmental borders, the wing D/V border, and the interrhombomeric boundaries of vertebrates. The adherens junction protein Echinoid, which is known to promote the formation of supracellular actomyosin cables, is not involved in A1 fold formation. Although actomyosin is enriched in a ring of cells in the A1 fold, it does not exert a centripetal force to close the ring, unlike the circumferential cable described in dorsal closure and wound healing (see review. In the A1 fold, the constricting cells become smaller in both their apical and basolateral domains, thus differing from ventral furrow cells where cell volume remains constant (Ku, 2017).

    A tissue fold probably provides a strong physical or mechanical barrier to prevent cell mixing. In addition, whereas in a flat tissue where the boundary involves only one to two rows of cells, the tissue fold involves more cells engaging in cell-cell communication. The close apposition of cells within the fold may allow efficient signaling within a small volume. This may be an evolutionarily conserved mechanism for boundary formation that corresponds to stable morphological constrictions such as the joints in the antennae and leg segments (Ku, 2017).

    Although N signaling has been reported to be involved in many developmental processes, a role in inducing actomyosin-dependent apical constriction and epithelial fold is a novel described function for N. For the A1 boundary, N activity is possibly mediated through repression of bantam and consequent upregulation of Ena. In the wing D/V boundary, N signaling is also mediated through bantam and Ena, but the outcome is formation of actomyosin cables, i.e., without apical constriction and epithelial fold [19]. Thus, the N/bantam/Ena pathway for tissue morphological changes is apparently context-dependent (Ku, 2017).

    Tissue constriction also occurs later in joint formation of the legs and antennae. N activation also occurs in the joints of the leg disc and is required for joint formation. This role is conserved from holometabolous insects like the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum to the hemimetabolous cricket Gryllus bimaculatus. It is possible that for segmented structures that telescope out in the P/D axis, like the antennae, legs, proboscis and genitalia, N signaling is used to demarcate the boundaries between segments, which are characterized by tissue constriction. N-dependent epithelial fold morphogenesis has also been reported in mice cilia body development without affecting cell fate, suggesting that such N-dependent regulation in morphogenesis is evolutionarily-conserved (Ku, 2017).

    It is proposed that N signaling is important in all boundaries that involve stable tissue morphogenesis. For those boundaries corresponding to stable morphological constrictions, e.g., the joints in insect appendages, N acts via actomyosin-mediated epithelial fold. The wing D/V boundary represents a different type of stable tissue morphogenesis. It becomes bent into the wing margin and involves N signaling via actomyosin cables, rather than apical constriction. In contrast, actomyosin-dependent apical constrictions do not involved N signaling and are involved in transient tissue morphogenesis, such as gastrulation in vertebrates, neural closure, Drosophila gastrulation, dorsal closure, as well as formation of the ventral furrow, eye disc morphogenetic furrow, and segmental groove in embryos (Ku, 2017).

    N signaling is also involved in the boundary between new bud and the parent body of Hydra, where it is required for sharpening of the gene expression boundary and tissue constriction at the base of the bud [78]. Whether the role of N in these tissue constrictions is due to actomyosin-dependent apical constriction and epithelial fold is not known (Ku, 2017).

    Boundaries may be established early in development. As the tissue grows in size through cell divisions and growth, boundary maintenance become essential. This study found that N activity is maintained by actomyosin, suggesting feedback regulation to stably maintain the boundary. Mechanical tension generated by actomyosin networks has been suggested to enhance actomyosin assembly in a feedback manner. Interestingly, the N-mediated wing A/P and D/V boundaries, which form actomyosin cables rather than tissue folds, did not exhibit such positive feedback regulation. Instead, the stability of the Drosophila wing D/V boundary is maintained by a complex gene regulatory network involving N, Wg, N ligands and Cut. Perhaps this is necessary for a boundary not involving tissue morphogenesis (Ku, 2017).

    The segmented appendages of arthropods (antennae, legs, mouth parts) are homologous structures of common evolutionary origin. It has been proposed that the generalized arthropod appendage is composed of a proximal segment called the coxopodite and a distal segment called the telopodite, either of which can further develop into more segments. The coxopodite is believed to be an extension of the body wall, whereas the telopodite represents the true limb, and thus represents an evolutionary addition. Dll mutants lack all distal segments except for the coxa in legs and the A1 segment in antennae. Lineage tracing studies have shown that Dll-expressing cells contributed to all parts of the legs except the coxa. These results indicate that the leg coxa and antenna A1 segment correspond to the Dll-independent coxopodite, and that Dll is the selector gene for the telopodite. Therefore, the antennal A1 fold is the boundary between the coxopodite and telopodite. It is postulated that the same N-mediated epithelial fold mechanism also operates in the coxopodite/telopodite boundary of legs and other appendages (Ku, 2017).

    Identification of genomic enhancers through spatial integration of single-cell transcriptomics and epigenomics

    Single-cell technologies allow measuring chromatin accessibility and gene expression in each cell, but jointly utilizing both layers to map bona fide gene regulatory networks and enhancers remains challenging. This study generated independent single-cell RNA-seq and single-cell ATAC-seq atlases of the Drosophila eye-antennal disc and spatially integrate the data into a virtual latent space that mimics the organization of the 2D tissue using ScoMAP (Single-Cell Omics Mapping into spatial Axes using Pseudotime ordering). To validate spatially predicted enhancers, a large collection of enhancer-reporter lines and identify ~85% of enhancers in which chromatin accessibility and enhancer activity are coupled. Next, infer enhancer-to-gene relationships were inferred in the virtual space, finding that genes are mostly regulated by multiple, often redundant, enhancers. Exploiting cell type-specific enhancers, cell type-specific effects of bulk-derived chromatin accessibility QTLs were deconvoluted. Finally, Prospero was found to drive neuronal differentiation through the binding of a GGG motif. In summary, a comprehensive spatial characterization of gene regulation is provided in a 2D tissue (Bravo González-Blas, 2020).

    Cellular identity is defined by Gene Regulatory Networks (GRNs), in which transcription factors bind to enhancers and promoters to regulate target gene expression, ultimately resulting in a cell type-specific transcriptome. Single-cell technologies provide new opportunities to study the mechanisms underlying cell identity. Particularly, single-cell transcriptomics allow measuring gene expression in each cell, while single-cell epigenomics, such as single-cell ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing), serves as a read-out of chromatin accessibility. Although these technologies and computational approaches are recently evolving to include spatial information, most approaches currently target single-cell transcriptomes. It remains a challenge how to exploit single-cell epigenomic data for resolving spatiotemporal enhancer activity and GRN dynamics, both experimentally and computationally (Bravo González-Blas, 2020).

    In addition, while ATAC-seq is a powerful tool for predicting candidate enhancers, not all accessible regions correspond to functionally active enhancers. For example, accessible sites can correspond to ubiquitously accessible promoters or binding sites for insulator proteins; to repressed or inactive regions due to binding of repressive transcription factors; or to primed regions that are accessible across a tissue, but become only specifically activated in a subset of cell types. Importantly, single-cell ATAC-seq has not been fully exploited to explore these aspects yet. While most scATAC-seq studies have been carried out in mammalian systems, in which enhancer testing is not trivial, Cusanovich (2018) evaluated 31 cell type-specific enhancers predicted from scATAC-seq in the Drosophila embryo, finding that ~ 74% showed the expected activity patterns (Bravo González-Blas, 2020).

    Another current challenge in the field of single-cell regulatory genomics is how to integrate epigenomic and transcriptomic information. Although some experimental approaches have been developed for profiling both the epigenome and the transcriptome of the same cell, currently either the quality of the measurements, or the throughput, is still significantly lower compared to each independent single-cell assay. For example, sci-CAR (Single-cell Combinatorial Indexing Chromatin Accessibility and mRNA) or SNARE-seq (Single-Nucleus Chromatin Accessibility and mRNA Expression sequencing) on human cells achieved a median of 1,000-4,000 UMIs (Unique Molecular Identifiers) and 1,500-3,000 fragments per cell, while the coverage with non-integrative methods, such as 10x, is around 20,000 UMIs and 10,000 fragments per cell for scRNA-seq and scATAC-seq, respectively. Methods that achieve high sensitivity, such as scCAT-seq (single-cell Chromatin Accessibility and Transcriptome sequencing), are based on microwell plates rather than droplet microfluidics, making their throughput limited (Bravo González-Blas, 2020).

    Given the current limitations of combined omics methods, the computational integration of independent high-sensitivity assays provides a valuable alternative. For example, Seurat and Liger have been used to integrate independently sequenced single-cell transcriptomes and single-cell epigenomes. Nevertheless, these methods require the 'conversion' of the genomic region accessibility matrix into a gene-based matrix, and how to perform such a conversion is an unresolved issue. Some studies have used the accessibility around the Transcription Start Site (TSS) as proxy for gene expression (Bravo González-Blas, 2019); others aggregate the accessibility regions that are co-accessible (i.e., correlated) with the TSS of the gene in a certain space (Pliner et al., 2018). However, promoter accessibility is not always correlated with gene expression. Furthermore, enhancers can be located very far from their target genes-upstream or downstream, up to 1 Mbp in mammalian genomes, or up to 100-200 kb in Drosophila , often with intervening non-target genes in between-and relationships between enhancers and target genes are often not one-to-one (i.e., an enhancer can have multiple targets, and a gene can be regulated by more than one enhancer) (Shlyueva et al., 2014). Enhancer-promoter interactions can also be predicted using Hi-C approaches at the bulk level (Ghavi-Helm et al , 2019); however, these methods have limited sensitivity at single-cell resolution (Bravo González-Blas, 2020).

    The Drosophila third-instar larval eye-antennal disc provides an ideal biological system for the spatial modeling of gene regulation at single-cell resolution. The eye-antennal disc comprises complex, dynamic, and spatially restricted cell populations in two dimensions. The antennal disc consists of four concentric rings (A1, A2, A3, and arista), each with a different transcriptome and different combinations of master regulators. For example, both Hth and Cut regulate the outer antennal rings (A1 and A2), with additional expression of Dll in A2, while Dll, Ss, and Dan/Danr are key for the development of the inner rings (A3 and arista), among others. On the other hand, a continuous cellular differentiation process from anterior to posterior occurs in the eye disc, in which progenitor cells differentiate into neuronal (i.e., photoreceptors) and non-neuronal (i.e., cone cells, bristle, and pigment cells) cell types. This differentiation wave is driven by the morphogenetic furrow (MF). Posterior to the MF, R8 photoreceptors are specified first, and then, they sequentially recruit R2/R5, R3/R4, and R7 photoreceptors and cone cells to form hexagonally packed units called ommatidia. In summary, the heterogeneity of cell types and differentiation trajectories results in diverse-static and dynamic-GRNs, which can be modeled with a combination of experimental and computational approaches (Bravo González-Blas, 2020).

    This work first generated a scRNA-seq and a scATAC-seq atlas of the eye-antennal disc. Second, taking advantage of the fact that the disc proper is a 2D tissue, these single-cell profiles were spatially mapped on a latent space that mimics the eye-antennal disc, called the virtual eye-antennal disc. Next, by exploiting publicly available enhancer-reporter data, the relationship between enhancer accessibility and activity was assessed. Third, these virtual cells, for which both epigenomic and transcriptomic data are available, were used to derive links between enhancers and target genes using a new regression approach. Fourth, a panel of 50 bulk ATAC-seq profiles across inbred lines was used to predict cell type-specific caQTLs (chromatin accessibility QTLs). Finally, these findings were used to characterize the role of Prospero in the accessibility of photoreceptor enhancers. In summary, a comprehensive characterization is provided of gene regulation in the eye-antennal disc, using a strategy that is applicable to other tissues and organisms. These results can be explored as a resource on SCope and the UCSC Genome Browser (), and an R package, called ScoMAP (Single-Cell Omics Mapping into spatial Axes using Pseudotime ordering) is provided, to spatially integrate single-cell omics data and infer enhancer-to-gene relationships (Bravo González-Blas, 2020).

    This work presents a semi-supervised approach to map omics data into a virtual template by extracting axial information via pseudotime ordering, available as an R package called ScoMAP. The main limitations of this approach are that (1) it can be currently only applied to 1D or 2D tissues, (2) it requires a priori information about at least one landmark between the real and the virtual cells and the direction of the axis, and (3) it assumes symmetry around the axes, meaning that other gradients may be lost as cells are spread randomly in each bin. Nevertheless, the spatial gene expression atlas resulting from the mapping of scRNA-seq accurately recapitulates known gene expression patterns and allows to generate virtual gene expression profiles for any gene, at a resolution comparable with novoSpaRc (Bravo González-Blas, 2020).

    Whereas spatial inference has been reported based on scRNA-seq data, this work generates the first spatial map of a tissue from scATAC-seq data. This accessibility atlas effectively predicts enhancer-reporter activity for more than 700 enhancers from the Janelia FlyLight Project, with ~85% of enhancers showing matching accessibility and activity patterns. The remaining enhancers (~ 15%) are binding sites of the epithelial pioneer transcription factor Grainyhead, which primes these regions in all the epithelial cells without resulting in enhancer activity. Indeed, pioneer transcription factors are able to displace nucleosomes, resulting in an ATAC-seq signal; and despite that they are necessary, their binding is not sufficient for activity (Jacobs, 2018). Thus, enhancer accessibility can be achieved either by the binding of pioneer factors or through the cooperative binding of multiple TFs. These results highlight both the power of using scATAC-seq as a proxy of enhancer activity and the need for caution when dealing with pioneer factors (Bravo González-Blas, 2020).

    The virtual map also acts as a latent space in which scATAC-seq and scRNA-seq data are available for each virtual cell. While experimental approaches for the simultaneous profiling of epigenome and transcriptome are emerging, these do not achieve the same throughout and sensitivity compared with the independent assays yet. Computationally, Granja (2019) has taken a similar approach, in which cells are mapped into the same latent space and for each single-cell transcriptome, the aggregate scATAC-seq profile of the closest neighbors is assigned. The resulting integrated profiles allow inferring relationships between enhancers and target genes. While Pliner (2018) has tackled this problem uniquely using scATAC-seq data, Granja (2019) used Pearson correlation between the chromatin accessibility and gene expression. This work extends this approach by also using random forest models to assess non-linear relationships. Of note, these approaches are not robust to pioneer sites, whose accessibility and activity are unpaired. For example, in the current approach a validated intronic enhancer of Atonal and Grainyhead in sca is missed, as the enhancer is ubiquitously accessible while only functional in the morphogenetic furrow, where the gene is expressed. Nevertheless, for the remaining 85% of the enhancers in which accessibility and activity are coupled, in this system, this study has been able to reconstruct novel and validated enhancer-to-target gene links (Bravo González-Blas, 2020).

    The predicted links between enhancers and target genes support that (1) the probability of an enhancer regulating a gene decreases exponentially with the distance and the number of non-intervening genes in between, as also reported by others, and (2) genes are regulated by several-and in some cases, redundant-enhancers, with a median of 22 enhancers linked to each gene. Indeed, Cannavo (2016) reported in the Drosophila embryo that ~64% of the mesodermal loci have redundant (or shadow) enhancers, of which ~ 60% contain more than one pair of shadow enhancers. In agreement, this study finds that ~80% of the genes are regulated by shadow enhancers (6,937 out of 8,307 genes), out of which ~72% are regulated by at least three shadow enhancers (4,900 out of 6,937 genes). Transcription factors are more tightly regulated, being linked with a higher number of enhancers (with an average of 13 positive links per gene) and having almost twice the number of redundant enhancers compared with non-TFs genes. As abnormalities in the expression of transcription factor genes can have more severe phenotypes compared with final effector genes, having more-and redundant-enhancers may provide evolutionary robustness. In addition, the majority of shadow enhancers are partially redundant, meaning that they can be uniquely essential on other developmental stages or tissues, or under adverse environmental conditions (Bravo González-Blas, 2020).

    Of note, almost ~50% of the inferred links are negatively correlated with their target genes. While polycomb-mediated repression has been shown to reduce region accessibility, other studies suggest that, although repressed enhancers are less accessible than active enhancers, they still show accessibility compared with the non-regulatory genome (Bozek, 2019). Such effect can be observed in the embryonic eve stripe 2 enhancer, which is active (and more accessible) in the second embryonic stripe, while repressed (and less accessible) in the rest. Meanwhile, in the eye-antennal disc, where it is not active nor repressed, there is no accessibility. Thus, accessible regions do not only correspond to primed or active enhancers, promoters, and insulators, but also to repressed enhancers (Bravo González-Blas, 2020).

    Several works have focused on the inference of GRNs from single-cell data, mostly exploiting scRNA-seq to infer co-expression patterns between TFs and potential target genes. In an attempt to reduce the number of false-positive targets due to activating cascade effects, SCENIC, which additionally evaluates the enrichment of binding sites for the TF around the TSS of the putative target gene, was introduced. On the other hand, other studies have exploited single-cell ATAC-seq to find target enhancers with binding sites for specific TFs. For example, chromVAR aggregates regulatory regions based on motif enrichment and then evaluates these modules on single-cell ATAC-seq data, while cisTopic (Bravo Gonzalez-Blas, 2019) performs motif enrichment on sets of co-accessible enhancers inferred from scATAC-seq profiles (i.e., topics) to find common master regulators. However, none of these approaches incorporates knowledge about the TF nor target gene expression. This study has aimed to integrate all these layers-transcription factor binding sites, chromatin, and gene expression-to infer GRNs, by deriving co-expression modules between genes and transcription factors (from the scRNA-seq data) and pruning them based on the enrichment of the TF motif in the enhancers that regulate these genes (based on the enhancer-to-target gene links derived from the integration of scATAC and scRNA-seq data). Such networks de facto have enhancers, rather than genes, as nodes (i.e., TF-Enhancer-Gene networks) (Bravo González-Blas, 2020).

    As bulk profiles may mask true biological signal (due to the proportions of the different cell types), single-cell data have been used to deconvolute cell type-specific signals from bulk RNA-seq data, permitting to exploit large cohorts with bulk omics data, complemented with only one single-cell reference atlas. This study has investigated the impact of genomic variation on cell type-specific enhancers. For example, the relevance was revealed of Atonal binding sites for opening Johnston's organ precursor-specific regions and the GGG motif, previously unlinked to any transcription factor, for opening photoreceptor regions. Interestingly, Atonal has been shown to be a key transcription factor for the specification of sensory neuronsand bHLH proteins have been proposed to act as pioneer transcription factors in certain contexts, such as the mammalian family member Ascl1 (Bravo González-Blas, 2020).

    The importance of the GGG motif in neuronal enhancers was evident in most of the current analyses; however, its interpretation was a challenge because the binding TFs were unknown. While yeast one-hybrid (Y1H) experiments have been previously used to reverse-engineer which transcription factors can bind a motif of interest, lowly expressed TFs may be underrepresented in the cDNA library and interactions that occur in vivo may be missed (such as those dependent of post-transcriptional modifications). This study has used a novel in vivo approach, in which the changes that overexpression of potential TF candidates causes in chromatin accessibility at the bulk ATAC-seq level were evaluated. Although this strategy allows to characterize the effects of TF overexpression directly on the tissue of interest, it also has limitations, such as the limited throughput of in vivo genetic screens (one TF per experiment, compared to dozens of TFs that can be tested by Y1H or Perturb-ATAC in vitro). This requires making a stringent selection of potential candidates that can be further bounded by the existence of compatible tools, such as UAS-TF lines. In addition, the changes in chromatin may not be direct, but these effects can be partially ruled out using external data available, such as ChIP-seq (Bravo González-Blas, 2020).

    This study found that the neuronal precursor transcription factor Prospero acts as the strongest binder of the GGG motif, followed by Nerfin-1 and l(3)neo38. In fact, overexpression of each of them, but especially Prospero, results in the opening of GGG regions; and all three transcription factors, especially Pros and Nerfin-1, can bind to the GGG motif. Based on the expression of these transcription factors, it is hypothesized that Nerfin-1-and l(3)neo38-are the early binders of the GGG motif, while Pros can bind to these regions in the late-born photoreceptors, where it is expressed. In fact, Pros and Nerfin-1 have been reported to share direct targets during CNS differentiation and have been found to be key regulators during the photoreceptor and retinal differentiation in other organisms, such as zebrafish, chicken, and mammals (Bravo González-Blas, 2020).

    In summary, this study provides a comprehensive and user-friendly single-cell resource of the Drosophila's eye-antennal disc. It is envisioned that these computational strategies and enhancer resources will be of value not only to the Drosophila community, but also to the field of single-cell regulatory genomics in general (Bravo González-Blas, 2020).

    Single cell RNA sequencing of the adult Drosophila eye reveals distinct clusters and novel marker genes for all major cell types

    The adult Drosophila eye is a powerful model system for phototransduction and neurodegeneration research. However, single cell resolution transcriptomic data are lacking for this tissue. This study presents single cell RNA-seq data on 1-day male and female, 3-day and 7-day old male adult eyes, covering early to mature adult eyes. All major cell types, including photoreceptors, cone and pigment cells in the adult eye were captured and identified. The data sets identified novel cell type specific marker genes, some of which were validated in vivo. R7 and R8 photoreceptors form clusters that reflect their specific Rhodopsin expression and the specific Rhodopsin expression by each R7 and R8 cluster is the major determinant to their clustering. The transcriptomic data presented in this report will facilitate a deeper mechanistic understanding of the adult fly eye as a model system (Yeung, 2022).

    This report presentS single-cell resolution transcriptomic data of Drosophila adult eyes from 1-day, 3-day, and 7-day-old Canton S flies. scRNA-seq from the adult eye poses several technical challenges. First, the adult retina is firmly attached to the hydrophobic lens and cuticle. Physical removal of the lens without damaging the underlying retinal cells is extremely difficult. When left intact, the lens and cuticle is a solid barrier to chemicals and enzymes used for dissociation. Second, adult eye cells are elongated and adhere to each other in vivo and this makes dissociating the adult eye into viable single cells much more challenging than many other tissues. Two of the data sets are from aged animals and aging is known to correlate with a decrease in the total number of transcripts per cell. In fact, a general decrease was observed in the number of genes detected and number of reads from our 1-day-old to 7-day-old data sets. The lower number of transcripts per cell poses a difficulty in single-cell RNA-sequencing where transcripts in each cell may not be captured, converted to cDNA and sequenced. This may lead to a loss of detected transcripts or loss of detected cells due to low transcript numbers. Despite these challenges, the quality control metrics the data sets indicate that the samples are of high quality (>80% viability and >600 median genes per cell) and the data sets captured over 6000 cells each. The scRNA-seq data sets show that all major cell types in the eye are captured and different cell types form clear and distinct clusters. The clustering observed in the data sets accurately reflects the known cell types in the eye. For example, it was observed that R7 and R8 photoreceptors are well separated and the pale and yellow R7 and R8 subtypes form their own distinct clusters. Such a high-resolution representation of the different cell types in the Drosophila adult eye has not been reported (Yeung, 2022).

    The data sets were compared in two ways. First, Monocle 3 was used to find any pseudotime trajectories between the three male time points. The results revealed that the transcriptomes of 1-day to 7-day-old adult eyes do not change substantially, and no clear trajectories could be plotted. Second, the 1-day-old adult male and female eye data sets were compared. The quality control metrics are similar between the two data sets. Harmony integration reveals that the male and female data sets are highly similar and only two sex-specific genes were discovered in the data sets (Yeung, 2022).

    Although this study has captured and sequenced all major cell types in the eye, including all photoreceptor subtypes, all pigment cell types and cone cells, it was observed that cone cells and primary pigment cells are underrepresented in the data sets. Cone cells and primary pigment cells are positioned most apically and are located next to the cuticle/lens of the eye. Since the dissociation protocol uses proteinase on adult eyes with the lenses intact, the cone cells and primary pigment cells would be the cell types that are least exposed to the proteinase. This may account, at least in part, for why fewer cone and primary pigment cells dissociated into solution for sequencing were observed when compared to the other cell types. Attempts were made to increase dissociation incubation time or adding mechanical dissociation steps to the protocol but both modifications result in a dramatic decrease in cell viability, leading to poor quality scRNA-seq results. It was reasoned that high viability for most cells is of greater value than producing a dataset with good representation of all cell types but with severely compromised viability and quality. Despite primary pigment and cone cells being underrepresented, they still form their own clusters and can be identified in the Seurat UMAP clustering. These were validated as primary pigment cells using the novel primary pigment cell and cone cell marker genes, wrapper and CG5597, respectively (Yeung, 2022).

    The majority of the ommatidia in the eye can be classified as either pale or yellow based on Rhodopsin expression in R7 and R8 cells (Rh3-expressing R7s and Rh5-expressing R8s are pale; Rh4-expressing R7s and Rh6-expressing R8s are yellow). The ratio of pale to yellow ommatidia in an adult eye is about 30:70. However, the ratios of captured pale:yellow R7s and R8s are not in the expected ratios (10:90 to 13:87 for R8s and about 50:50 for R7 in the data sets). While Rh6-expressing R8s are well represented in the data sets, the number of Rh5-expressing R8s are low and are consistently underrepresented. The number of pale or yellow R7s do not appear to be obviously underrepresented like pale R8s. Immunostaining of Canton-S adult eyes show the expected ratios of pale to yellow R7s and R8s; thus the discrepancy between the expected ratios and the realized results are not due to the input tissue. It is not likely that R7s and R8s changed their Rhodopsin expression to a different Rhodopsin during sample preparation since the sample was treated with Actinomycin D, which inhibits all transcription. Therefore, it is hypothesized that Rh5-expressing R8s may be preferentially lost during sample preparation or there may be a bias against Rh5 expressing R8s in the data acquisition pipeline. It is possible that adult Rh5 expressing R8 are especially sensitive to the dissociation conditions for scRNA-seq and thus are lost during sample preparation and filtered out. For R7s, it is hypothesized that there may be a bias for capturing pale R7s. However, the possibility that there may be a bias against yellow R7s in the pipeline must be considered (Yeung, 2022).

    Although a single nuclear RNA-seq (snRNA-seq) data set of whole adult Drosophila heads, which includes the eyes, was previously reported, our data presented in this study are single-cell RNA-seq data (scRNA-seq). The transcriptome data from snRNA-seq and scRNA-seq differ. First, because snRNA-seq are nuclear in origin, the extracted RNA molecules are biased toward unspliced versions. In contrast, scRNA-seq should not have a bias toward spliced or unspliced RNA. Bias for unspliced forms may cause complications in RNA velocity analyses which elucidate the dynamics of changes in transcriptome using spliced and unspliced transcripts. The established scRNA-seq protocol can also be adapted for a different, more sensitive scRNA-seq technique such as SMART-seq to detect any cell type-specific splicing. Second, snRNA-seq data capture fewer RNA molecules per cell compared to scRNA-seq because each nucleus is smaller than a whole cell. Thus for aging tissues, where the total number of transcripts decrease, scRNA-seq offer an advantage over snRNA-seq. Third, studies comparing snRNA-seq and scRNA-seq in human liver and human brain tissues show that certain cell type-specific transcripts are only present in scRNA-seq but not in snRNA-seq and vice versa. The cell-type marker gene lists were compared with the lists from published snRNA-seq. Although there is overlap between the photoreceptor marker gene lists from the two data sets, CG2082 and santa-maria, an R8 marker and secondary and tertiary pigment cell marker genes, respectively, identified and in vivo validated in this work were not identified as marker genes for R8 and pigment cells in the published snRNA-seq. Differences in marker gene lists between the two data sets may be expected since different methods were employed (single nucleus vs single cell). In summary, these overlaps and differences suggest that thw scRNA-seq and the published snRNA-seq are complementary (Yeung, 2022).

    Seurat analyses and UMAP clustering of the data sets identified all photoreceptor subtypes and a substantial number of pigment cells in all time points. Each identified cell type cluster expresses known marker genes for the corresponding adult cell type. Interestingly, most previously characterized cell-type specific marker genes from larval eye discs are no longer expressed in the same specific cell types in the adult eye. The identities of clusters were validated using T2A-Gal4 drivers of marker genes and immunostaining with antibodies recognizing known markers of specific cell types. Differential gene expression and FeaturePlot analyses of the data sets produce many potential novel markers for each identified cell type. GO-Term enrichment analyses on the marker lists of well-represented cell types show photoreceptors and pigment cells have enriched GO-terms relating to phototransduction and pigment production, respectively. To further test the validity of the cluster annotation and the specificity of cell type-specific markers, several candidates were validated in vivo, including CG2082 for adult R8s, igl for both R7 and R8, Zasp66 for adult photoreceptors, CG5597 for cone cells, wrapper for primary pigment cells and santa-maria for secondary and tertiary pigment cells. In vivo validations of marker genes in this report all show specific expression patterns that closely match the expected expression pattern predicted from Seurat FeaturePlots. It is likely that most of the other potential marker genes identified in this study will also follow the expected expression pattern. Interestingly, the in vivo validated marker genes in this study are either uncharacterized (e.g., CG2082, CG5597) or their functions in the corresponding cell types are unclear (e.g., wrapper in primary pigment cells). Except for santa-maria, eye phenotypes have not been previously reported for the validated marker genes. Further studies of these marker genes may lead to new insights in the maintenance or function of the adult eye (Yeung, 2022).

    The scRNA-seq data sets have identified numerous novel marker genes for each major cell type in the adult eye. Prior to this study, cell type-specific markers for the adult eye were very limited. The novel marker genes identified here address the lack of cell type-specific markers for this stage. The cell type-specific marker genes also provide novel targets for constructing new reagents and genetic tools (e.g., for driving transgenes). Many marker genes are conserved in humans and the conserved human homologs have associated disease phenotypes. These marker genes are likely to benefit research that uses the adult eye as a model system. The genes discovered by the data sets may play key roles in the function or maintenance of the adult eye and thus may play key roles in phototransduction and neurodegeneration (Yeung, 2022).

    Interestingly, it was noticed that the T2A-Gal4 driven reporters often show a more specific expression pattern than suggested by the FeaturePlots. FeaturePlots show relative expression of selected genes but not the absolute expression. Thus, for genes that are expressed weakly, any fluctuation in expression levels may be exaggerated. SoupX was used to remove ambient RNA from the data sets but it may not remove all ambient RNA (e.g., ninaE). Thus the FeaturePlots may show cells with unexpected expression of cell type markers simply due to the presence of ambient RNA sequenced in that cell. Despite these caveats, the FeaturePlots still show a clear enrichment of cell type markers in the expected cell clusters and in vivo staining results show T2A-Gal4 driven reporters are expressed only in the expected cell type as suggested by the FeaturePlots. For example, CG2082 > reporter is expressed in R8 only, which matches the CG2082 expression enrichment in R8s as shown in the FeaturePlots. Although some CG2082 positive cells are seen in R1-6 in the FeaturePlots, no reporter expression in R1-6 was observed in the adult eye. This suggests that CG2082 is not strongly expressed or has no expression in R1-6 cells but CG2082 is strongly expressed in R8 cells. Taken together, the FeaturePlots are accurate predictors of the in vivo expression patterns of marker genes in cell type clusters where the expression is enriched (Yeung, 2022).

    The scRNA-seq results show that ninaE expression is not restricted to R1-6 cells as expected but it is also expressed to a lower degree in all other clusters. In contrast, the other Rhodopsins (Rh3-6) do not show nearly as much expression in unexpected cell types. There are at least two hypotheses that may explain this observation. First, all eye cells may express ninaE transcripts but NinaE protein is translated only in R1-6. Second, ninaE transcripts are expressed only by R1-6 and the ninaE transcripts detected in non-R1-6 cells represent ambient RNA. Of the two hypotheses, the second is favored for two reasons. First, R1-6 account for six different cells per ommatidium. This outnumbers all other cell types per ommatidium. Second, NinaE proteins are one of the most highly abundant proteins in the adult eye. This was expected as NinaE proteins are localized to the entire R1-6 rhabdomeres and R1-6 rhabdomeres are large and span nearly the entire depth of the retina. Therefore, ninaE transcripts are expected to be highly abundant in R1-6. Indeed, all of the datasets show that ninaE is the most abundant transcript. Since ninaE is the most highly expressed gene in the eye, it is predicted to contribute to ambient RNA more than any other gene (Yeung, 2022).

    In the adult eye, R1-6 photoreceptors are needed for motion detection while pale and yellow R7s and R8s are needed for color detection. Pale and yellow R7 and R8s are used to detect different colors and UV light (Rh3: ~345 nm UV light, Rh4: ~375 nm UV light, Rh5: green, Rh6: blue). In all time points, Seurat clustering segregated R1-6 as one cluster that is distinct from R7s and R8s, which form their own distinct clusters. R7s and R8s are further separated into clusters that represent their pale/yellow subtypes. It is thought that specific wavelengths of light activate the expressed Rhodopsins in each photoreceptor, which then activates a downstream phototransduction cascade that is common to all photoreceptors. One implication of these observations is that all photoreceptor subtypes may be transcriptionally similar and which Rhodopsin is expressed may be the main difference between subtypes. Consistent with this model, Seurat clustering strongly reflects the type of opsin expressed by each photoreceptor subtype at all time points (Yeung, 2022).

    To test this model,the effects of R7 and R8 specific Rhodopsins, Rh3-6, were removed in the clustering process. Removal of Rh3 and Rh4 causes the R7 clusters to collapse and Rh3 and Rh4 R7s are intermixed with one another. Similarly, when Rh5 and Rh6 are removed, all R8s cells are intermixed. When Rh3 to Rh6 are removed, R7 and R8 clusters collapse into one loose cluster. These results suggest that the major drivers of R7 and R8 clustering are the R7 and R8 specific Rhodopsins and apart from Rhodopsin expression, R7 and R8 cells are very similar transcriptionally. This implies that other than Rh3-6, there should be very few markers that distinguishes R7 and R8. Indeed, differential gene and FeaturePlot analyses of R7 and R8 marker genes identified very few R7 or R8 specific marker genes; instead, the majority of novel markers for R7 and R8 identified in this work are expressed in both subtypes. Interestingly, it was noticed that some but not all R7 and R8 cells are mixed with R1-6 clusters after Rh3-6 are removed. This also suggests that Rh3-6 are determinants in separating R7 and R8 from R1-6. But the lack of a complete intermixing of R7, R8, and R1-6 when Rh3-6 are removed suggests other genes also contribute to photoreceptor clustering (Yeung, 2022).

    Although R1-6 cells form a distinct cluster and Seurat can call marker genes for the R1-6 clusters for all time points, FeaturePlot analyses showed these markers are not specific to R1-6; instead, most are specific to all photoreceptors. It was observed that the R1-6 clusters have ninaE as their top marker while Rh3-6 were not called as markers in the R1-6 clusters in all time points. Therefore, it is predicted that if ninaE was not a major ambient RNA, it would be a definitive R1-6 marker gene in the FeaturePlots. However, ninaE is not a major contributor to R1-6 clustering as the removal of ninaE does not change the clustering of R1-6 clusters. Other contributors (e.g., R8-specific and R7/8-specific markers) may also contribute to defining the R1-6, R7, and R8 photoreceptor clusters. In the case of R7 and R8, our Rh removal results suggest that R7 and R8 are transcriptionally similar to one another and this may account for the lack of R7-specific markers. It is hypothesized that R1-6 cell clusters are also somewhat transcriptionally similar to R7 and R8. Thus, R1-6 specific marker genes may be rare and may not be detected in the data sets, similar to the lack of R7-specific markers (Yeung, 2022).

    In summary, this work presents transcriptomic data of Drosophila adult eyes prepared from whole cells at a single-cell resolution. The data sets show that there are very few sex-specific differences between male and female adult eyes. Analyses of our data sets show that Rhodopsin expression is a major contributor to the transcriptomic differences between photoreceptor subtypes. Finally, we identified numerous marker genes with differential expression analyses for all the major cell types in the adult fly eye and novel markers were verified for each major cell type in the adult eye in vivo. The function of many of the cell type-specific marker genes are unknown but they may perform critical roles in the maintenance and function of the adult eye. In addition, many of the marker genes are conserved and have been associated with human diseases. These marker genes will also serve as valuable tools for generating cell type-specific reagents for field (Yeung, 2022).

    Avalanches during epithelial tissue growth; Uniform Growth and a Drosophila eye disc model

    Epithelial tissues constitute an exotic type of active matter with non-linear properties reminiscent of amorphous materials. In the context of a proliferating epithelium, modeled by the quasistatic vertex model, this study identified novel discrete tissue scale rearrangements, i.e. cellular rearrangement avalanches, which are a form of collective cell movement. During the avalanches, the vast majority of cells retain their neighbors, and the resulting cellular trajectories are radial in the periphery, a vortex in the core. After the onset of these avalanches, the epithelial area grows discontinuously. The avalanches are found to be stochastic, and their strength is correlated with the density of cells in the tissue. Overall, avalanches redistribute accumulated local spatial pressure along the tissue. Furthermore, the distribution of avalanche magnitudes is found to obey a power law, with an exponent consistent with sheer induced avalanches in amorphous materials. To understand the role of avalanches in organ development, epithelial growth of the Drosophila eye disc during the third instar was simulated using a computational model, which includes both chemical and mechanistic signaling. During the third instar, the morphogenetic furrow (MF), a ~10 cell wide wave of apical area constriction propagates through the epithelium. These simulations are used to understand the details of the growth process, the effect of the MF on the growth dynamics on the tissue scale, and to make predictions for experimental observations. The avalanches are found to depend on the strength of the apical constriction of cells in the MF, with a stronger apical constriction leading to less frequent and more pronounced avalanches. The results herein highlight the dependence of simulated tissue growth dynamics on relaxation timescales, and serve as a guide for in vitro experiments (Courcoubetis, 2022).

    Allocation of distinct organ fates from a precursor field requires a shift in expression and function of gene regulatory networks

    A common occurrence in metazoan development is the rise of multiple tissues/organs from a single uniform precursor field. One example is the anterior forebrain of vertebrates, which produces the eyes, hypothalamus, diencephalon, and telencephalon. Another instance is the Drosophila wing disc, which generates the adult wing blade, the hinge, and the thorax. Gene regulatory networks (GRNs) that are comprised of signaling pathways and batteries of transcription factors parcel the undifferentiated field into discrete territories. This simple model is challenged by two observations. First, many GRN members that are thought to control the fate of one organ are actually expressed throughout the entire precursor field at earlier points in development. Second, each GRN can simultaneously promote one of the possible fates choices while repressing the other alternatives. It is therefore unclear how GRNs function to allocate tissue fates if their members are uniformly expressed and competing with each other within the same populations of cells. This paradigm was addressed by studying fate specification in the Drosophila eye-antennal disc. The disc, which begins its development as a homogeneous precursor field, produces a number of adult structures including the compound eyes, the ocelli, the antennae, the maxillary palps, and the surrounding head epidermis. Several selector genes that control the fates of the eye and antenna, respectively, are first expressed throughout the entire eye-antennal disc. This study shows that during early stages, these genes are tasked with promoting the growth of the entire field. Upon segregation to distinct territories within the disc, each GRN continues to promote growth while taking on the additional roles of promoting distinct primary fates and repressing alternate fates. The timing of both expression pattern restriction and expansion of functional duties is an elemental requirement for allocating fates within a single field (Palliyil, 2018).

    Patterning mechanisms diversify neuroepithelial domains in the Drosophila optic placode

    The central nervous system develops from monolayered neuroepithelial sheets. In a first step patterning mechanisms subdivide the seemingly uniform epithelia into domains allowing an increase of neuronal diversity in a tightly controlled spatial and temporal manner. In Drosophila, neuroepithelial patterning of the embryonic optic placode gives rise to the larval eye primordium, consisting of two photoreceptor (PR) precursor types (primary and secondary), as well as the optic lobe primordium, which during larval and pupal stages develops into the prominent optic ganglia. This study characterize a genetic network that regulates the balance between larval eye and optic lobe precursors, as well as between primary and secondary PR precursors. In a first step the proneural factor Atonal (Ato) specifies larval eye precursors, while the orphan nuclear receptor Tailless (Tll) is crucial for the specification of optic lobe precursors. The Hedgehog and Notch signaling pathways act upstream of Ato and Tll to coordinate neural precursor specification in a timely manner. The correct spatial placement of the boundary between Ato and Tll in turn is required to control the precise number of primary and secondary PR precursors. In a second step, Notch signaling also controls a binary cell fate decision, thus, acts at the top of a cascade of transcription factor interactions to define photoreceptor subtype identity. This model serves as an example of how combinatorial action of cell extrinsic and cell intrinsic factors control neural tissue patterning (Mishra, 2018).

    In the fruit fly Drosophila melanogaster, all parts of the visual system develop from an optic placode, which forms in the dorsolateral region of the embryonic head ectoderm. During embryogenesis, neuroepithelial cells of the optic placode are patterned to form two subdomains. The ventroposterior domain gives rise to the primordium of the larval eye and consists of two photoreceptor (PR) precursor types (primary and secondary precursors), whereas the dorsal domain harbors neuroepithelial precursors that generate the optic lobe of the adult visual system. The basic helix-loop-helix transcription factor Atonal (Ato) promotes PR precursor cell fate in the larval eye primordium. The orphan nuclear receptor Tailless (Tll) is confined to the optic lobe primordium and maintains non-PR cell fate. Hedgehog (Hh) and Notch (N) signaling are critical during the early phase of optic lobe patterning. The secreted Hh protein is required for the specification of various neuronal and non-neuronal cell types, while Notch acts as neurogenic factor preventing ectodermal cells from becoming neuronal precursors by a process termed lateral inhibition. In the optic placode Ato expression is promoted by Hh and the retinal determination genes sine oculis (so) and eyes absent (eya). Notch delimits the number of PR precursors and maintains a pool of non-PR precursors. Ato is initially expressed in all PR precursors in the placode and its expression gets progressively restricted to primary precursors. In a second step, primary precursors recruit secondary precursors via EGFR signaling: primary precursors express the EGFR ligand Spitz, which is required in secondary precursors to promote their survival. After this initial specification of primary and secondary PR precursors, the transcription factors Senseless (Sens), Spalt (Sal), Seven-up (Svp) and Orthodenticle (Otd) coordinate PR subtype specification. Sens and Spalt are expressed in primary PR precursors, while Svp contributes to the differentiation of secondary PR precursors. By the end of embryogenesis, primary PR precursors have fully differentiated into blue-tuned Rhodopsin5 PRs (Rh5), while secondary PR precursors have differentiated into green-tuned Rhodopsin6 PRs (Rh6). While the functional genetic interactions of transcription factors controlling PR subtype specification has been thoroughly studied, it remains unknown how the placode is initially patterned by the interplay of Hh and Notch signaling pathways. Similarly, the mechanisms of how ato and tll-expressing domains are set up to ensure the correct number of primary and secondary PR precursors as well as non-PR precursors of the optic lobe primordium remain unknown (Mishra, 2018).

    This study describes the genetic mechanism of neuroepithelial patterning and acquisition of PR versus non-PR cell fate in the embryonic optic placode and provide the link to subsequent PR subtype identity specification. The non-overlapping expression patterns of ato and tll in the optic placode specifically mark domains giving rise to the larval eye precursors (marked by Ato) and the optic lobe primordium (marked by Tll). ato expression in the larval eye primordium is temporally dynamic and can be subdivided into an early ato expression domain, including all presumptive PR precursors and a late ato domain, restricted to presumptive primary PR precursors. The ato expression domain directly forms a boundary adjacent to tll expressing precursors of the optic lobe primordium. tll is both necessary and sufficient to delimit primary PR precursors by regulating ato expression. Hh signaling regulates the cell number in the optic placode and controls PR subtype specification in an ato- and sens-dependent manner. Finally, this study also shows that Notch has two temporally distinct roles in larval eye development. Initially, Notch represses ato expression by promoting tll expression and later, Notch controls the binary cell fate decision of primary versus secondary PR precursors by repressing sens expression. In summary, this study has identified a network of genetic interactions between cell-intrinsic and cell-extrinsic developmental cues patterning neuroepithelial cells of the optic placode and ensuring the timely specification of neuronal subtypes during development (Mishra, 2018).

    Neurogenic placodes are transient structures that are formed by epithelial thickenings of the embryonic ectoderm and give rise to most neurons and other components of the sensory nervous system. In vertebrates, cranial placodes form essential components of the sensory organs and generate neuronal diversity in the peripheral nervous system. How neuronal diversity is generated varies from system to system, and different gene regulatory networks have been proposed for each particular type of neuron. Interestingly, some transcription factors, like Atonal, play an evolutionary conserved role during neurogenesis both in Drosophila and in vertebrates (Mishra, 2018).

    Neuroepithelial patterning of the Drosophila optic placode exhibits unique segregation of larval eye and optic lobe precursors during embryogenesis. This study has identified genetic mechanisms that control early and late steps in specifying PR versus non-PR cell fate that ensure the expression of precursor cell fate determinants. During germband extension at stage 10, transcriptional regulators (so, eya, ato and tll) show complex and partially overlapping expression patterns in the optic placode. Their interactions with the Notch and Hh signaling pathways define distinct PR and non-PR domains of the larval eye and optic lobe primordium. Intriguingly, the results show a spatial organization of distinct precursor domains, supporting a new model of how the subdivision of precursor domains emerges. In agreement with previous studies initially the entire posterior ventral tip expresses Ato, defining the population of cells that give rise to PR precursors, while neuroepithelial precursors for the presumptive optic lobe are defined by Tll-expression in the anterior domain of the optic placode. Subsequently, Ato expression ceases in the ventral most cells and thus gets restricted to about four primary PR precursors that are located directly adjacent to the Tll expression domain. Hence, a few cell rows are between the primary PR precursors and the ventral most edge of the optic placode. This is in agreement with a recent observation on the transcriptional regulation of ato during larval eye formation. Thus, primary PR precursors are directly adjacent to the Tll-expressing cells while the Ato and Tll negative domain of secondary PR precursors is located at the posterior ventral most tip of the optic placode. Setting the Tll-Ato boundary is critical to define the number of putative secondary PR precursors, which can be recruited into the larval eye, probably via EGFR signaling. A model is proposed during which coordinated action of Hh, Notch and Tll restricts the initially broad expression of Ato to primary PR precursors (see Ato to primary PR precursors). Lack of Tll results in a de-repression of Ato and results in an increased number of primary PR precursors, which in turn recruit secondary PR precursors. Interestingly, while tll mutants show an increase in both primary and secondary PR precursors, the ratio between both subtypes is maintained. This notion further displays similarities of ommatidal formation in the adult eye-antennal imaginal disc, where Ato expressing R8-precursors recruit R1-R6. In the eye-antennal disc, specification of R8-precursors determines the total number of ommatida and therefore also the total number of PRs, the ratio of R8 to outer PRs however always remains the same. Thus, the initial specification of primary PR precursors defines the total number of PRs in the larval eye similarly to R8 PRs, and the ratio of founder versus recruited cells remains constant. Interestingly, the maintenance of primary versus secondary PR precursor ratio is also maintained in ptc mutants further supporting this model (Mishra, 2018).

    During photoreceptor development in the eye-antennal imaginal disc hh is expressed in the posterior margin and is required for the initiation and progression of the morphogenetic furrow as well as the regulation of ato expression. During embryogenesis the loss of hh results in a complete loss of the larval eye, while increasing Hh signaling (by means of mutating ptc) generates supernumerary PRs in the larval eye. During early stages, an increase of Ato expression was found in ptc mutants suggesting that similarly to the eye-antennal disc Hh positively regulates ato expression. The observed increase of Ato-expressing cells is not due to a reduction of Tll but is likely due to increased cell proliferation in ptc mutants. Hh also controls proliferation during the formation of the Drosophila compound eye (Mishra, 2018).

    During embryonic nervous system development Notch dependent lateral inhibition selects individual neuroectodermal cells to become neuroblasts. Notch represses neuroblast cell fate and promotes ectodermal cell fate. During compound eye development, Notch regulates Ato expression and acts through lateral inhibition to select Ato expressing R8 PR precursors. Similarly, during Drosophila larval eye development, Notch is required for regulating PR cell number by maintaining epithelial cell fate of the optic lobe primordium. Inhibiting Notch signaling leads to a complete transformation of the optic placode to PRs of the larval eye. In the absence of Notch signaling, Ato expression is expanded in the optic placode and as a result the total number of PRs is increased. Despite the increase of the overall PR-number the number of secondary PR precursors is significantly decreased or lost in the absence of Notch activity. In the compound eye Notch promotes R7 cell fate by repressing the R8-specific transcription factor Sens. It was also proposed that genetic interaction between Notch and Sens is required for sensory organ precursor (SOP) selection in the proneural field in a spatio-temporal manner. This study found that during PR subtype specification Notch represses Sens expression, thereby controlling the binary cell fate decision of primary versus secondary PR precursors. Therefore, in the absence of Notch signaling, Sens expression represses the secondary PR precursor fate. As a result, all PR precursors are transformed and acquire primary PR precursor identity. In conclusion, this study observed that Notch is essential for two aspects during optic placode patterning. First, Notch activity is critical for balancing neuroepithelial versus PR cell fate mediated through Tll-regulated Ato expression. Second, Notch regulates the binary cell fate decision of primary versus secondary PR precursor cell fate through the regulation of Sens expression (Mishra, 2018).

    A quantitative analysis of growth control in the Drosophila eye disc

    The size and shape of organs is species-specific, and even in species in which organ size is strongly influenced by environmental cues, such as nutrition or temperature, it follows defined rules. Therefore, mechanisms must exist to ensure a tight control of organ size within a given species, while being flexible enough to allow for the evolution of different organ sizes in different species. This study combined computational modelling and quantitative measurements to analyse growth control in the Drosophila eye disc. It was found that the area growth rate declines inversely proportional to the increasing total eye disc area. Two growth laws were identified and found to be consistent with the growth data that would explain the extraordinary robustness and evolutionary plasticity of the growth process and thus of the final adult eye size. The study discusses how each of these laws constrains the set of candidate biological mechanisms for growth control in the Drosophila eye disc (Vollmer, 2016).

    Atonal and EGFR signalling orchestrate rok- and Drak-dependent adherens junction remodelling during ommatidia morphogenesis

    Morphogenesis of epithelial tissues relies on the interplay between cell division, differentiation and regulated changes in cell shape, intercalation and sorting. These processes are often studied individually in relatively simple epithelia that lack the complexity found during organogenesis when these processes might all coexist simultaneously. To address this issue, this study makes use of the developing fly retinal neuroepithelium. Retinal morphogenesis relies on a coordinated sequence of interdependent morphogenetic events that includes apical cell constriction, localized alignment of groups of cells and ommatidia morphogenesis coupled to neurogenesis. Live imaging was used to document the sequence of adherens junction (AJ) remodelling events required to generate the fly ommatidium. In this context, it was demonstrated that the kinases Rok and Drak function redundantly during Myosin II-dependent cell constriction, subsequent multicellular alignment and AJ remodelling. In addition, it was shown that early multicellular patterning characterized by cell alignment is promoted by the conserved transcription factor Atonal (Ato). Further ommatidium patterning requires the epidermal growth factor receptor (EGFR) signalling pathway, which transcriptionally governs Rho-kinase (rok) and Death-associated protein kinase related (Drak)-dependent AJ remodelling while also promoting neurogenesis. In conclusion, this work reveals an important role for Drak in regulating AJ remodelling during retinal morphogenesis. It also sheds new light on the interplay between Ato, EGFR-dependent transcription and AJ remodelling in a system in which neurogenesis is coupled with cell shape changes and regulated steps of cell intercalation (Robertson, 2013).

    In Drosophila, Rok seems to be the main kinase responsible for phosphorylating the Myosin regulatory light chain (Sqh) during epithelial patterning and apical cell constriction. This is the case for the activation of MyoII during intercalation as germband extension proceeds, but also during various instances of compartment boundary formation and cell sorting situations in the embryo and in the wing imaginal disc. The current work reveals that in the constricting cells of the MF, Rok functions redundantly with Drak, a kinase recently shown to phosphorylate Sqh both in vitro and in vivo (Neubueser, 2010). It is noteworthy that previous work has shown that RhoGEF2 is not required for cell constriction in the MF, suggesting that perhaps another guanine exchange factor (GEF) might function redundantly with RhoGEF2 to promote cell constriction. These data on Drak reinforce the idea that redundancies exist in this context. Because the RhoA (Rho1 -- FlyBase) loss of function abolishes this cell response entirely, it would be expected that Drak function is regulated by RhoA. In addition, the current data indicate that Drak acts redundantly with Rok during MyoII-dependent multicellular alignment and AJ remodelling during ommatidia patterning. It will be interesting to test whether Drak functions in other instances of epithelial cell constriction or MyoII-dependent steps of AJ remodelling in other developmental contexts in Drosophila (Robertson, 2013).

    This study demonstrates a two-tiered mechanism regulating the planar polarization of MyoII and Baz. In the constricting cells in the posterior compartment, MyoII and Baz are segregated from one another and this is exacerbated by the wave of cell constriction in the MF. Upon Ato-dependent transcription in the MF cells, this segregated pattern of expression is harnessed and these factors become planar polarized at the posterior margin of the MF. This is independent of the core planar polarity pathway including the Fz receptor and is accompanied by a striking step of multicellular alignment. Previous work has demonstrated that Ato upregulates E-Cad transcription at the posterior boundary of the MF. In addition, apical constriction leads to an increase in E-Cad density at the ZA. The current data are therefore consistent with both hh-dependent constriction and ato-dependent transcriptional upregulation of E-Cad promoting differential adhesion, thus leading to a situation in which the ato+ cells maximize AJ contacts between themselves and minimize contact with the flanking cells that express much less E-Cad at their ZA. This typically leads to a preferential accumulation of cortical MyoII at the corresponding interface. Such actomyosin cables are correlated with increased interfacial tension, and it is proposed that this is in turn responsible for promoting cell alignment. Unfortunately, the very small diameter of these constricted cells precludes direct measurements of the AJ-associated tension using laser ablation experiments (Robertson, 2013).

    Supra-cellular cables of MyoII have been previously associated with cell alignment in various epithelia and have also been observed at the boundary of sorted clones, whereby cells align at a MyoII-enriched interface. Interestingly, this study found that the actomyosin cable defining the posterior boundary of the MF is also preferentially enriched for Rok, a component of the T1, MyoII-positive AJ in the ventral epidermis (Simoes Sde, 2010). This indicates an important commonality between actomyosin cable formation during cell sorting and the process of cell intercalation. However, unlike during intercalation, this study found that in the developing retina baz is largely dispensable for directing the pattern of E-Cad and actomyosin planar polarization. Further work will therefore be required to understand better the relationship between Baz and E-Cad at the ZA during ommatidia morphogenesis. It is speculated that the creation of a high E-Cad versus low E-Cad boundary in the wake of the MF might be sufficient to promote Rok and MyoII enrichment at the posterior AJs. This posterior Rok and MyoII enrichment might perhaps prevent E-Cad accumulation by promoting E-Cad endocytosis, as has been recently shown in the fly embryo (Robertson, 2013).

    This study has used live imaging to define a conserved step of ommatidia patterning that consists of the coalescence of the ommatidial cells' AJs into a central vertex to form a 6-cell rosette. The corresponding steps of AJ remodelling require Rok, Drak, Baz and MyoII, a situation compatible with mechanisms previously identified during cell intercalation in the developing fly embryo. The steps of AJ remodelling required to transform lines of cells into 5-cell pre-clusters are transcriptionally regulated downstream of EGFR in a ligand-dependent manner. Interestingly, in the eye EGFR signalling is activated in the cells that form lines and type1-arcs in the wake of the MF and, thus, are undergoing AJ remodelling. Previous work examining tracheal morphogenesis in the fly has demonstrated that interfaces between cells with low levels versus high levels of EGFR signalling correlate with MyoII-dependent AJ remodelling in the tracheal placode. This situation resembles that which is described in this study in the wake of the MF. In the eye, however, it was found that EGFR signalling is not required to initiate cell alignment. Nevertheless, taken together with work in the tracheal placode and previous studies related to multicellular patterning in the developing eye, this work indicates a conserved function for the EGFR signalling pathway in promoting MyoII-dependent AJ remodelling. This leaves open several interesting questions; for example, it is not presently clear how EGFR signalling can promote discrete AJ suppression and elongation. It is, however, tempting to speculate that previously described links between EGFR signalling and the expression of E-Cad or Rho1 might play a role during this process (Robertson, 2013).

    Mapping gene regulatory networks in Drosophila eye development by large-scale transcriptome perturbations and motif inference

    Genome control is operated by transcription factors (TFs) controlling their target genes by binding to promoters and enhancers. Conceptually, the interactions between TFs, their binding sites, and their functional targets are represented by gene regulatory networks (GRNs). Deciphering in vivo GRNs underlying organ development in an unbiased genome-wide setting involves identifying both functional TF-gene interactions and physical TF-DNA interactions. To reverse engineer the GRNs of eye development in Drosophila, this study performed RNA-seq across 72 genetic perturbations and sorted cell types and inferred a coexpression network. Next, direct TF-DNA interactions were derived using computational motif inference, ultimately connecting 241 TFs to 5,632 direct target genes through 24,926 enhancers. Using this network, network motifs, cis-regulatory codes, and regulators of eye development were found. The predicted target regions of Grainyhead were validated by ChIP-seq and this factor was identified as a general cofactor in the eye network, being bound to thousands of nucleosome-free regions (Potier, 2014).

    The development of the Drosophila eye is a classical model system to study neuronal differentiation and patterning. The TFs that represent the core of the retinal determination network are Eyeless (Ey), Twin of Eyeless (Toy), Dachsund (Dac), Sine Oculis (So), and Eyes Absent (Eya). Although many regulatory interactions are known between these TFs, as they intensively cross-regulate each other, knowledge about interactions with downstream target genes and of other TFs involved in the eye-antennal gene regulatory network (GRN) is sparse. This study aimed at combining classical reverse genetics-starting from a mutant allele and analyze its (molecular) phenotype-with genomics. Doing so, attempts were made to unveil genetic regulatory interactions in an unbiased way, and many regulators of the eye and antennal developmental programs were identified; most of these did not require or use any mutation or direct perturbation (Potier, 2014).

    The mapping approach began by systematically perturbing the developmental system. Attempts were made to include multiple perturbations into one data matrix to obtain a broad spectrum of expression profile changes. These perturbations included TF mutants, TF overexpression, TF knockdown, and cell sorting (Potier, 2014).

    Eye-antennal discs were dissected at the stage where in the WT discs about half of the eye disc contains pluripotent cells that are dividing asynchronously, while the other half contains differentiating PR neurons, in consecutive stages of differentiation. Simultaneously, the antennal disc contains neuronal precursors that are undergoing specification. The expression changes induced by the perturbations often result from a shift in proportion of cell types. This is trivial for the cell-sorting experiments; for example, the GMR>GFP-positive cells show, as expected, a very strong enrichment of genes related to PR differentiation. TF mutants and TF perturbations can also result in cell type shifts; for example, overexpression of Atonal yields more R8 PRs, and the glass mutant results in fewer differentiated PRs. Other TF perturbations cause changes in gene expression downstream of the TF without changing the cell type composition, such as Retained, which disturbs axonal projection. The key technique that was applied, however, was not to compare each TF perturbation with WT discs to identify differentially expressed genes. Rather, linear and nonlinear correlations of gene expression profiles were used across the entire vector of 72 gene expression measurements. This TF-gene coexpression network contains both direct and indirect edges, and although this network is informative, a second layer of predicted TF-DNA interactions was added, thus making this a direct GRN. To increase the sensitivity, a very large collection was used of TF motifs, also including position weight matrices derived for yeast and vertebrate TFs and including computationally derived motifs (e.g., highly conserved words). Using motif-motif similarity measures and TF-TF orthology relationships, each motif was linked to a candidate binding factor. This yielded a large network with 335 TFs and their predicted direct targets. The only functional network of comparable size and comparable directedness to this in vivo network is the TH17 GRN that was derived in vitro in a recent study (Yosef, 2013). That study used a microarray time course of naive CD4+T cells differentiating into TH17. From these gene expression data, they derived TF-gene interactions by clustering and filtered those with TF-DNA interactions obtained by ChIP-seq data, TF perturbations, and cis-regulatory sequence analysis (Potier, 2014).

    The predicted direct and functional eye-antennal GRN includes many previously reported interactions, such as known target genes for Eyeless and Sine Oculis. Target genes in the network were also found for late factors (e.g., Glass, Onecut) and very late factors (e.g., Pph13). The fact that information was captured at different time points during development is because several cell populations were sorted that are loosely correlated with the temporal axis of development, consisting of undifferentiated pluripotent cells anterior to the furrow, all PR cells undergoing differentiation posterior to the MF, R8 PR cells, and late populations of chp-positive cells. However, the temporal information encoded in the network is limited to these broad domains, and a more detailed reconstruction of the time axis would require higher resolution cell sorting or microdissection experiments. Although the perturbed TFs were mainly chosen for their development of the retina, master regulators of antennal development, such as aristaless were also identified (Potier, 2014).

    Interestingly, general factors like Grainyhead were found that were ubiquitously expressed. Grh was found as one of the TFs with the largest number of target enhancers and its binding correlates with open chromatin. Previous studies have shown that Grainyhead may interact with Polycomb and Trithorax proteins to regulate (both activate and repress) target gene expression. It is speculated that this observed correlation can be explained by the fact that Grh is present ubiquitously in the eye disc, thus yielding many sequence fragments from bound and nucleosome-free enhancers by FAIRE-seq (Potier, 2014).

    It is well known that network motifs such as FFLs play an important role in biological networks. One such network motif was examined in more detail, namely the TF pair Glass-Lozenge, and their common targets. These TFs constitute a double-feedback loop (Glass regulates Lozenge, Lozenge regulates Glass, and they together regulate 36 targets). For this network motif, it was found that Glass and Lozenge motifs co-occur at the same enhancer, where they furthermore overlap; this may indicate competition for binding between Glass and Lozenge. Given that Lozenge, an important regulator of cone cell differentiation, could be a repressor and Glass, an important regulator of PR differentiation, could be an activator, such a competition at the CRM level could indeed be a plausible mechanism for their regulatory action (Potier, 2014).

    Another interesting feature that can be derived from a GRN is the proportion of autoregulatory TFs (108 autoregulatory TFs in the eye network) and the proportion of activating versus repressive TFs. A recent large-scale study in yeast found a small majority of yeast TFs to have a repressive role. In that study, each individual TF was perturbed, thereby providing information on positive versus negative edges from the TF to its direct predicted targets, whereby TF-DNA information was used from ChIP-chip data. Since the eye GRN was started from a coexpression TF-gene network, the correlations between TFs and their candidate targets were revisited, and 151 TFs were found that have their motif enriched in the positively correlated target genes, but not in the negatively correlated targets, and 127 TFs showing the opposite; 62 TFs show enrichment in both. This finding agrees, to some extent, with the results in yeast concerning the high amounts of gene-specific repressors. On the other hand, the eye network suggests relative more TFs with a dual activator/repressor function, while the yeast study found only a few such cases (Potier, 2014).

    In conclusion, starting from an expression matrix derived from large-scale perturbations and combining TF-gene coexpression with TF-DNA interactions based on motif inference enabled drawing an extensive eye-antennal GRN. All predicted regulatory interactions, target genes, and candidate regulatory regions are stored in a Neo4J database and can be queried from a laboratory website. The database can also be accessed directly from Cytoscape using the CyNeo4j plugin or can be queried programmatically using the Neo4j query language Cypher. Although many known regulators and cis-regulatory elements were uncovered and several other ones were revealed, a large part of the predicted network, including how the dynamics of the developmental program are encoded in the cis-regulatory regions and in the topology of the network, remains to be explored (Potier, 2014).

    Comparative transcriptome analyses of the Drosophila pupal eye

    Tissue function is dependent on correct cellular organization and behavior. As a result, the identification and study of genes that contribute to tissue morphogenesis is of paramount importance to the fields of cell and developmental biology. The Drosophila melanogaster pupal eye that has a highly stereotyped arrangement of cells. In addition, the pupal eye is postmitotic that allows for the study of tissue morphogenesis independent from any effects of proliferation. While the changes in cell morphology and organization that occur throughout pupal eye development are well documented, less is known about the corresponding transcriptional changes that choreograph these processes. To identify these transcriptional changes, wild-type Canton S pupal eyes were dissected, and RNA-sequencing was performed. This analyses identified differential expression of many loci that are documented regulators of pupal eye morphogenesis and contribute to multiple biological processes including signaling, axon projection, adhesion, and cell survival. Differential expression of genes not previously implicated in pupal eye morphogenesis were identified such as components of the Toll pathway, several non-classical cadherins, and components of the muscle sarcomere, which could suggest these loci function as novel patterning factors (DeAngelis, 2021)

    The actomyosin machinery is required for Drosophila retinal lumen formation

    Multicellular tubes consist of polarized cells wrapped around a central lumen and are essential structures underlying many developmental and physiological functions. In Drosophila compound eyes, each ommatidium forms a luminal matrix, the inter-rhabdomeral space, to shape and separate the key phototransduction organelles, the rhabdomeres, for proper visual perception. In an enhancer screen to define mechanisms of retina lumen formation, Actin5C was identifed as a key molecule. The results demonstrate that the disruption of lumen formation upon the reduction of Actin5C is not linked to any discernible defect in microvillus formation, the rhabdomere terminal web (RTW), or the overall morphogenesis and basal extension of the rhabdomere. Second, the failure of proper lumen formation is not the result of previously identified processes of retinal lumen formation: Prominin localization, expansion of the apical membrane, or secretion of the luminal matrix. Rather, the phenotype observed with Actin5C is phenocopied upon the decrease of the individual components of non-muscle myosin II (MyoII) and its upstream activators. In photoreceptor cells MyoII localizes to the base of the rhabdomeres, overlapping with the actin filaments of the RTW. Consistent with the well-established roll of actomyosin-mediated cellular contraction, reduction of MyoII results in reduced distance between apical membranes as measured by a decrease in lumen diameter (see Model for Drosophila retinal lumen formation). Together, these results indicate the actomyosin machinery coordinates with the localization of apical membrane components and the secretion of an extracellular matrix to overcome apical membrane adhesion to initiate and expand the retinal lumen (Nie, 2014; Pubmed

    Does Unc-GFP uncover ciliary structures in the rhabdomeric eye of Drosophila?

    The Uncoordinated (Unc) gene product, a potential ortholog of orofaciodigital syndrome 1 (Ofd1), is involved in the assembly of the ciliary axoneme in Drosophila and it is, therefore, constrained to cell types that have ciliary structures, namely type 1 sensory neurons and male germ cells. This study shows that evenly spaced Unc-GFP spots are present in the eye imaginal discs of third instar larvae. These spots are restricted to the R8 photoreceptor cell of each ommatidium in association with mother centrioles. This finding is unexpected since the Drosophila eye is of rhabdomeric type and should lack ciliary structures (Gottardo, 2016).

    Ire1 supports normal ER differentiation in developing Drosophila photoreceptors

    The endoplasmic reticulum (ER) serves virtually all aspects of cell physiology and, by pathways incompletely understood, is dynamically remodeled to meet changing cell needs. Inositol-requiring enzyme 1 (Ire1), a conserved core of the Unfolded Protein Response (UPR), participates in ER remodeling and is particularly required during the differentiation of cells devoted to intense secretory activity, "professional" secretory cells. This study characterize Ire1's role in ER differentiation in developing Drosophila compound eye photoreceptors (R cells). As part of normal development, R cells take a turn as professional secretory cells with a massive secretory effort that builds the photosensitive membrane organelle, the rhabdomere. Rough ER sheets proliferate as rhabdomere biogenesis culminates and Ire1 is required for normal ER differentiation. Ire1 is active early in R cell development and is required in anticipation of peak biosynthesis. Without Ire1, rough ER sheets are strongly reduced and the extensive cortical ER network at the rhabdomere base, the subrhabdomere cisterna (SRC), fails. Instead, ER proliferates in persistent, ribosome-poor tubular tangles. A phase of Ire1 activity early in R cell development thus shapes dynamic ER (Xu, 2016).

    From its initial specification in the morphogenetic furrow, to its adult service as a photosensor, a developing Drosophila R cell plays many roles, calling upon a sequence of conserved, core cell functions that target the developmental task at hand. In the last quarter of pupal life that task is the enormous expansion of the photosensory membrane via secretory delivery of membrane rich in rhodopsin and allied elements of phototransduction. This study shows a corresponding expansion of stacked rER meets this secretory challenge; at this developmental stage, R cells are typical of cells devoted to intensive secretion, 'professional' secretory cells, generally. As seen with other professional secretory cells, this study found that R cells require Ire1 for normal ER expansion and differentiation. When normal cells proliferate rER stacks, Ire1 mutant ER proliferates in a dense, chaotic tangle of reticulon-rich tubules. The mechanisms by which Ire1 supports normal R cell ER differentiation remain unknown and in view of its multiple and far-reaching effectors are likely to be multiple. Shown in this study recapitulated in R cells, a common theme in Ire1 regulation of ER differentiation is a requirement in anticipation of secretory activity, suggesting Ire1 builds in secretory capacity as part of normal organelle programming. It is unlikely Ire1 acts alone and temporal overlap of Ire1 activity, shown in this study, and also by (Coelho, 2013), with that of PERK, a second conserved UPR pathway suggests they may cooperate in ER programming. The profuse tangle of reticulon-rich tubules that arises during peak secretory effort, resembles the pathology seen in Ire1 mutant yeast upon ER stress: in response to unfolded protein stress, normal yeast proliferate ER sheets but, although normal in the absence of stress, stressed Ire1 mutant yeast expand ER in dense, reticulon-rich tangles. Together, results suggest the possibility that Ire1 contributes essential shaping activity to a program of ER expansion and, in its absence, an unbalanced drive expands dysmorphic ER (Xu, 2016).

    Shown in this study, and previously (Coelho, 2013), Ire1 loss compromises Rh1 production and secretory delivery needed to build the rhabdomere. Prior work has shown that Ire1's contribution to these tasks is Xbp1-independent, which is in accord with the current study, and now has been extended to ER shaping; R cell ER is normal in severe Xbp1 hypomorphs. Previously, it has been shown that Ire1 contributes to Rh1 delivery via degradation of fatty acid transporter, fatp, mRNA: Ire1 loss elevates fatp mRNA and its reduction using RNAi rescues Rh1 delivery (Coelho, 2013). As elevated levels of phosphatidic acid (PA) have been shown to disrupt R cell apical membrane transport, it is proposed that increased fatp elevates PA and thus degrades Rh1 delivery (Coelho, 2013). Abnormal, expanded rER is seen in R cells with increased PA, but appears distinct from the tangled tubular ER seen in this study. Tubulated ER seen in this study also differs from the dilated ER lumens commonly noted when misfolded secretory protein products over-accumulate, e.g., in Akita mice that accumulate misfolded proinsulin, and distinct from the strongly amplified, well-formed rER seen in ninaA mutants caused by lumenal Rh1 accumulation. Failure to assemble expanded rER sheets with accumulation of irregular, ribosome-poor ER tubules in Ire1 mutant R cells is reasonably the ultrastructural substrate for the severe reduction of Rh1 levels and growth deficit generally; without a definitive measure of reduced Rh1 synthesis, it is possible that reduced Rh1 levels are attributable to enhanced ER-Associated Degradation (ERAD) in an out of control ER. Loss of a normal SRC in Ire1 mutants plausibly contributes to the failure to deliver secretory traffic to the growing rhabdomere. Collapse of normal ER morphology is thus catastrophic for multiple cell activities (Xu, 2016).

    The mechanism by which lower temperature rescues ER differentiation in Ire1 mutant cells remains to be determined, but may be connected to numerous observations showing basal, Ire1-independent, ER capacity supports many aspects of normal cell development and physiology. Indeed, despite profound ER disruption during peak rhabdomere growth, mutant R cells are viable and execute a wide range of normal cell physiologies, including normal cell polarity, cell fate specification and the complex choreography that assembles ommatidia. Similarly, Ire1 null nematodes are viable and morphologically normal, but die when challenged with mutations in other UPR branches or tunicamycin. Mouse hepatocytes lacking Ire1 α show reduced rER content but are otherwise phenotypically normal; however upon ER stress they fail to maintain lipid homeostasis. The gut of mice lacking Ire1beta is phenotypically normal, but sensitized to experimental colitis. In the absence of stress, ER morphology is normal in yeast ire1 mutants. It is speculated that when pupal development is slowed more than, twofold, 9-10 days at 19°C, versus 4 days at 25°C, basal ER capacity meets secretory demand (Xu, 2016).

    The distribution of peripheral ER between sheet and tubule domains has been likened to a "tug-of-war", with activity promoting ER sheets poised against tubule promoting activity, particularly that of membrane curvature-inducing reticulons. The near disappearance of rER sheets and concomitant emergence of dense tubular tangles in Ire1 mutants suggests a runaway win for tubule promotion, potentially due to a failure of sheet-forming activities, an abnormal regulation of tubule formation, or a combination of both. Ribosomes promote ER sheets and it is possible the loss ribosomes in mutant R cells destabilizes rER. Alternately, abnormal, dense Rtnl1 accumulations seen in mutant R cells may reflect a cannibalization of ER sheets by misregulated Rtnl1 with a consequent reduction in Rh1 synthesis; misregulated Rtnl1 could also account for the loss of the SRC cortical ER network. The dynamic, stereotyped differentiation of ER morphology underlying Drosophila R cell differentiation presents a genetically accessible system to investigate how developmental programs shape ER (Xu, 2016).

    Past1 modulates Drosophila eye development

    Endocytosis is a multi-step process involving a large number of proteins, both general factors, such as clathrin and adaptor protein complexes, and unique proteins, which modulate specialized endocytic processes, like the EHD proteins. EHDs are a family of Eps15 Homology Domain containing proteins that consists of four mammalian homologs, one C. elegans, one Drosophila melanogaster and two plants orthologs. These membrane-associated proteins are involved in different steps of endocytic trafficking pathways. The Drosophila EHD ortholog, PAST1, has been shown to associate predominantly with the plasma membrane. Mutations in Past1 result in defects in endocytosis, male sterility, temperature sensitivity and premature death of the flies. Also, Past1 genetically interacts with Notch. The present study investigated the role of PAST1 in the developing fly eye. In mutant flies lacking PAST1, abnormal differentiation of photoreceptors R1, R6 and R7 was evident, with partial penetrance. Likewise, five cone cells were present instead of four. Expression of transgenic PAST1 resulted in a dominant negative effect, with a phenotype similar to that of the deletion mutant, and appearance of additional inter-ommatidial pigment cells. These results strongly suggest a role for PAST1 in differentiation of photoreceptors R1/R6/R7 and cone cells of the fly ommatidia (Dorot, 2017).

    In the Past1 null mutant flies no external eye abnormalities were observed, but the development of photoreceptors R1, R6 and R7, as well as of cone cells, was abnormal. In flies overexpressing PAST1 in the eye external abnormalities were noted, as well as an abnormal number of photoreceptors, cone cells and IPCs. Defects in ommatidium development accompanied by no external abnormalities in the eye have been previously reported for other mutations. For example, the external eye of mutants in Muscleblind (Mbl), a regulator of alternative splicing, appear to be normal, yet tangential sections revealed that they harbored ommatidia defects. Another example involves Crumbs (Crb), a transmembrane protein which is essential for biogenesis of adherens junctions and for establishing apical-basal polarity in Drosophila epithelia by downregulating endocytosis of Notch and Delta during eye development. External morphology of mutant Crb (crbS87-2) was reported to be normal. However, internal structure of adult CrbS87-2 mutant ommatidia was found to be defective with shortened and bulkier rhabdomeres, often in contact with each other rather than being distinct as in wild type eyes (Dorot, 2017).

    Interestingly in Past1 null flies, the ommatidia always contained at least one R7 photoreceptor, with some having two R7 photoreceptors with a concomitant loss of either R1 or R6 or both. These results imply that differentiation to R1 or R6 was abrogated and instead, at least one R7 photoreceptor was developed. No change in Boss staining was detected in R7 cells of eye discs from Past1 null mutants or from transgenic GFP-PAST1 flies, while in Past1 null mutants the ommatidia always contained at least four cone cells and some ommatidia had five cone cells (Dorot, 2017).

    Previously work has shown that Past1 genetically interacts with Notch in the wing. The results of the present study indicated that Past1 plays a role during eye development, which is known to be regulated by Notch. Interestingly, endocytosis of Notch seemed abnormal in early-mid pupal mutant eyes. Thus, higher level of Notch staining was detected at the vicinity of the plasma membrane of mutant pigment cells, indicating abrogated endocytosis. This result implies abnormal internalization of Notch in the absence of PAST1 and hints to the possibility that PAST1 modulates internalization of Notch during eye development. (Dorot, 2017).

    The results well fit the model suggested by Tomlinson (2011) for the development of photoreceptors R1/R6/R7 and cone cells. According to this model Notch overexpression in either R1 or R6 photoreceptors, leads to development of one of these cells into an extra R7 photoreceptor. Additionally, overexpression of Notch in R1 or R6 combined with an absence of Sev leads to the appearance of an extra cone cell. It is proposed that lack of PAST1 elevates Notch activation in R1 and R6 photoreceptors, which subsequently develop into R7 or into cone cells. It is hypothesized that PAST1 negatively regulates Notch signaling in R1 and R6. Since penetrance of Past1 mutation in the eye is low, PAST1 is, most probably, not a major regulator of Notch signaling, but fine-tunes it. Further experiments are needed to establish the role of PAST1 in endocytosis of Notch (Dorot, 2017).

    Eyes of flies overexpressing PAST1 were rough with regions of fused ommatidia and exhibited extra IPCs. Mutations that abrogate the normal cell death process of IPCs have been shown to lead to rough eyes. More so, overexpression of Notch (NFL or NICD) in the pupal retina led to appearance of extra IPCs. Therefore, it is assumed that the rough eyes phenotype in the PAST1 transgenic flies is due to abnormal PCD of IPCs (Dorot, 2017).

    This study examined the ability of Garland cells overexpressing PAST1 to internalize fluorescently labeled Texas-Red avidin, as was examined for Past1 mutants. Garland cells overexpressing Past1 displayed attenuated endocytosis. Overexpression of PAST1 had a dominant negative effect, showing approximately 50-60% attenuation in endocytosis. A similar dominant negative effect has also been previously shown in HeLa cells overexpressing the mammalian EHD2, leading to attenuation of plasma membrane internalization (Dorot, 2017).

    In conclusion, the results presented in this work highlight the importance of PAST1 for development of R1,R6 and R7 photoreceptors of the fly ommatidia (Dorot, 2017).

    Cell surface molecule, Klingon, mediates the refinement of synaptic specificity in the Drosophila visual system

    In the Drosophila brain, neurons form genetically specified synaptic connections with defined neuronal targets. It is proposed that each central nervous system neuron expresses specific cell surface proteins, which act as identification tags. Through an RNAi screen of cell surface molecules in the Drosophila visual system, this study found that the cell adhesion molecule Klingon (Klg) plays an important role in repressing the ectopic formation of extended axons, preventing the formation of excessive synapses. Cell-specific manipulation of klg showed that Klg is required in both photoreceptors and the glia, suggesting that the balanced homophilic interaction between photoreceptor axons and the glia is required for normal synapse formation. Previous studies suggested that Klg binds to cDIP and genetic analyses indicate that cDIP is required in glia for ectopic synaptic repression. These data suggest that Klg play a critical role together with cDIP in refining synaptic specificity and preventing unnecessary connections in the brain (Shimozono, 2019).

    Apical accumulation of the Sevenless receptor tyrosine kinase during Drosophila eye development is promoted by the small GTPase Rap1

    The Ras/MAPK-signaling pathway plays pivotal roles during development of metazoans by controlling cell proliferation and cell differentiation elicited, in several instances, by receptor tyrosine kinases (RTKs). While the internal mechanism of RTK-driven Ras/MAPK signaling is well understood, far less is known regarding its interplay with other co-required signaling events involved in developmental decisions. In a genetic screen designed to identify new regulators of RTK/Ras/MAPK signaling during Drosophila eye development, the small GTPase Rap1, PDZ-GEF, and Canoe as components contributing to Ras/MAPK-mediated R7 cell differentiation. Rap1 signaling has recently been found to participate in assembling cadherin-based adherens junctions in various fly epithelial tissues. This study shows that Rap1 activity is required for the integrity of the apical domains of developing photoreceptor cells and that reduced Rap1 signaling hampers the apical accumulation of the Sevenless RTK in presumptive R7 cells. It thus appears that, in addition to its role in cell-cell adhesion, Rap1 signaling controls the partitioning of the epithelial cell membrane, which in turn influences signaling events that rely on apico-basal cell polarity (Baril, 2014).

    This report describes a genetic screen in Drosophila for dominant modifiers of a CNK-dependent rough eye phenotype. Two of those modifiers, Rap1 and PDZ-GEF, were characterized to further shed light on the mechanism by which Rap1-mediated events influence photoreceptor cell development (Baril, 2014).

    Given the role Connector eNhancer of KSR (CNK) plays in RTK-elicited Ras/MAPK signaling, mutations in loci encoding general components of this pathway in flies were recovered in a CNK C-terminal-dependent (CCT) screen. They correspond to Star [S; trafficking factor for the EGFR ligand Spitz, Egfr, daughter of sevenless (dos; Gab2 homolog), Son of sevenless (Sos; RasGEF), Ras85D, rl/mapk, ksr, and pointed (pnt; ETS domain transcription factor mediating MAPK activity). Mutations in two genes previously identified in RTK-dependent screens, but of unclear function, were also isolated. These are kismet [kis; chromatin remodeler] and multiple ankyrin repeats single KH domain [mask; putative RNA-binding protein (Baril, 2014).

    Mutant alleles not identified in classical RTK/MAPK-dependent genetic screens, but for which a functional link to RTK signaling in flies or in other organisms had been established were also recovered. These include Btk family kinase at 29A [Btk29A; the single representative of Tec family kinases]; Delta (Dl); and three Rap1 pathway loci, PDZ-GEF, Rap1, and canoe (cno). Finally, mutations were isolated in four additional loci not previously reported to influence RTK/MAPK signaling: Pre-mRNA-processing factor 19 (Prp19), BREFeldin A sensitivity 1 (Bre1), NEM sensitive factor 2 (Nsf2), and second mitotic wave missing (swm) (Baril, 2014).

    The Prp19 locus encodes a core spliceosome component. A specific role for these proteins has been unvailed in the Ras/MAPK pathway. Indeed, several splicing factors, including Prp19 and Prp8, were identified in a genome-wide RNAi screen in S2 cells for modulators of Ras-induced MAPK activation. Incidentally, a single mutant allele of Prp8 was also recovered in the CCT screen. Characterization of their implication in the pathway revealed that they specifically regulate MAPK protein levels by controlling the alternative splicing of selected introns of the mapk pre-mRNAs. It is thus likely that Prp19 alleles were recovered in the CCT screen because of their impact on endogenous MAPK levels during eye development (Baril, 2014).

    The association that the last three genes (Bre1, Nsf2, and swm) might have with respect to CCT activity is less clear. Bre1 encodes a RING finger-containing E3 ligase mediating histone H2B monoubiquitination. This modification contributes to specific histone epigenetic changes such as histone H3K4 and H3K79 methylation that correlate with transcriptional activation. A role for Bre1 in Notch- and Wingless-dependent gene expression has been reported, but whether it acts similarly downstream of RTK signaling is not known. Given the concerted, yet distinct role Notch and EGFR signaling play in morphogenetic furrow progression and thereby in eye development, it could well be that, as for Dl, Bre1 was recovered primarily for its function in Notch signaling (Baril, 2014).

    Nsf2 encodes an AAA ATPase involved in vesicular trafficking and synaptic vesicle release. Previous genetic studies have also associated this gene with Notch and Wingless signaling, and thus this could be the basis for the isolation of Nsf2 alleles. Alternatively, Nsf2 activity could be required in trafficking events directly involved in RTK signaling (Baril, 2014).

    The swm gene [aka Su(Rux)2B encodes a novel protein that comprises a CCCH zinc finger and a RNA recognition motif. SWM localizes to the nucleus and was found to play multiple roles during Drosophila development, although its precise molecular function is not known. During eye development, Swm regulates the proliferation of undifferentiated cells by controlling their G1/S transition. In particular, third instar eye discs deprived of SWM activity are reduced in size and, as epitomized by the gene name, they lack the second mitotic wave, which corresponds to a row of cells located at a few cells distance posterior to the morphogenetic furrow that undergo a unique and synchronous round of cell division. This event increases the pool of uncommitted cells used for completing ommatidial assembly. Both Notch and EGFR signaling are essential for cell cycle progression of the uncommitted cells in the second mitotic wave, but act at distinct steps. Whether the swm alleles were recovered because of their impact on the second mitotic wave or for another role of SWM in differentiating cells remains to be investigated (Baril, 2014).

    The ability of the CCT screen to identify mutations in three loci linked to Rap1 signaling strongly suggests a functional relationship between CNK and Rap1 activity. Yet, no evidence was found for physical association between CNK and Rap1 or PDZ-GEF, and thus the molecular underpinning of this relationship is currently not known. One possibility for their genetic interactions could be through their separate roles in RTK-mediated events. Rap1 signaling promotes adherens junction formation in differentiating photoreceptor cells, which contributes to their clustering. This phenomenon, in turn, is thought to enable the cells to respond to extracellular cues promoting differentiation. By lowering Rap1 activity, cohesive contacts between differentiating cells would be suboptimal and thereby would impede ommatidial assembly to some degree. In this scenario, the impact of CCT expression on photoreceptor cell differentiation would be exacerbated by heterozygous mutations in Rap1-signaling components as these would reduce the sensitivity of developing cells to differentiation cues (Baril, 2014).

    Interestingly, it has been noted that loss of Rap1 activity does not prevent EGFR-induced MAPK activation per se, and thus Rap1 does not appear to work like Ras as a direct RAF activator. However, the data for this conclusion were based on small Rap1 mutant clones that were close or within the morphogenetic furrow. This work was extended by producing larger clones depleted in Rap1 or PDZ-GEF activity. These clones covered the zone where R7 cell commitment normally occurs. Markedly, it was found that reduced Rap1 signaling in this area considerably decreased MAPK activity as well as global pTyr levels and thereby mimicked the loss of RTK activity. Consistent with this, a strong impairment in R7 cell fate specification was observed (Baril, 2014).

    In agreement with thes findings, Mavromatakis (2012) recently showed by genetic means that R7 cell fate specification had an absolute requirement in Rap1 activity. According to their model, R7 cell precursors sense higher Notch signaling owing to their position in the developing ommatidium, which at this stage is antagonistic to Ras/MAPK-mediated neuronal differentiation. To counteract Notch signaling, Mavromatakis (2012) proposed that presumptive R7 cells turn on two RTKs (EGFR and SEV) to produce higher MAPK activity. Intriguingly, their work suggested that Rap1 was required downstream of SEV, although they could not distinguish whether Rap1 acted through the canonical MAPK pathway or parallel to it. This was investigated and it was found Rap1 does not seem to work directly through the MAPK pathway since ectopic expression of Rap1V12 during eye development or in cultured S2 cells did not promote MAPK phosphorylation. Moreover, depletion of Rap1 or PDZ-GEF by RNAi in S2 cells had no consequence on MAPK activation induced by SEV or EGFR. Although the precise mechanism by which Rap1 influences signaling downstream of SEV remains to be delineated, the combined data suggest that Rap1 works at two distinct levels in SEV-mediated signaling, that is, upstream of SEV by modulating the apical localization of SEV and downstream of SEV by a mechanism that has yet to be characterized (Baril, 2014).

    Adherens junctions form a belt-like microdomain that encircles epithelial cells apically and that play a major role in cell-cell adhesion, motility, and polarity. One of the core structural components of adherens junctions is Ecad, which is a transmembrane glycoprotein that forms Ca2+-dependent homophilic interactions between adjacent cells. The intracellular portion of Ecad is complexed to the catenins that, in turn, mediate linkage to the actomyosin cytoskeleton. Studies conducted over the past 10 years in both vertebrate and invertebrate organisms demonstrate the critical role that Rap1 signaling plays in modulating the connections of adherens junctions to the actomyosin network, which then influence cell-cell adhesion, cell shape, and cell migration (Baril, 2014).

    Although the current data are consistent with this view, they also hint at a new role for Rap1 signaling that is to control apical domain formation in developing photoreceptor cells. Given that adherens junctions may act as physical barriers between apical and basolateral membrane compartments, the influence of Rap1 on adherens junction dynamics could represent the mechanism by which Rap1 exerts its effect on the apical domain compartment. A more exciting alternative would be that Rap1 activity directly controls the formation of the apical domain. Work conducted in fly embryos by Choi (2013) recently provided evidence supporting this model. Indeed, not only did that study find that Rap1 activity is essential for establishing the apico-basal polarity of cellularizing embryos, but their data also suggest that it has a direct impact on the apical localization of Bazooka, a member of the Par complex, which then orchestrates apical domain assembly. Whether Rap1 signaling has a direct influence on cell polarity during eye development is still unclear. Nonetheless, further characterization of the impact that Rap1 signaling has on apical domain formation/maintenance should reveal novel aspects by which cell compartmentalization is brought about and regulated as well as how it connects to downstream signaling events in epithelial cells (Baril, 2014).

    Integrins are required for synchronous ommatidial rotation in the Drosophila eye linking planar cell polarity signalling to the extracellular matrix

    Integrins mediate the anchorage between cells and their environment, the extracellular matrix (ECM), and form transmembrane links between the ECM and the cytoskeleton, a conserved feature throughout development and morphogenesis of epithelial organs. This study demonstrates that integrins and components of the ECM are required during the planar cell polarity (PCP) signalling-regulated cell movement of ommatidial rotation in the Drosophila eye. The loss-of-function mutations of integrins or ECM components cause defects in rotation, with mutant clusters rotating asynchronously compared to wild-type clusters. Initially, mutant clusters tend to rotate faster, and at later stages they fail to be synchronous with their neighbours, leading to aberrant rotation angles and resulting in a disorganized ommatidial arrangement in adult eyes. It was further demonstrated that integrin localization changes dynamically during the rotation process. The data suggest that core Frizzled/PCP factors, acting through RhoA and Rho kinase, regulate the function/activity of integrins and that integrins thus contribute to the complex interaction network of PCP signalling, cell adhesion and cytoskeletal elements required for a precise and synchronous 90 degrees rotation movement (Thuveson, 2019).

    Nonsynaptic Transmission Mediates Light Context-Dependent Odor Responses in Drosophila melanogaster

    Recent connectome analyses of the entire synaptic circuit in the nervous system have provided tremendous insights into how neural processing occurs through the synaptic relay of neural information. Conversely, the extent to which ephaptic transmission which does not depend on the synapses contributes to the relay of neural information, especially beyond a distance between adjacent neurons and to neural processing remains unclear. This study shows that ephaptic transmission mediated by extracellular potential changes in female Drosophila melanogaster can reach >200 μm, equivalent to the depth of its brain. Furthermore, ephaptic transmission driven by retinal photoreceptor cells mediates light-evoked firing rate increases in olfactory sensory neurons. These results indicate that ephaptic transmission contributes to sensory responses that can change momentarily in a context-dependent manner (Ikeda, 2022).

    This study found that ephaptic transmission of light information from photoreceptor cells in the retina mediates the increase in firing rate in the olfactory sensory neurons (OSNs) during odor stimulations. This study has not revealed whether the ephaptic transmission directly changes the firing rate of the OSNs. Amputation of the antennal nerve abolished the firing rate increases during sustained light, suggesting that once the light information might be received by neurons in the brain, the information would be relayed by the neurons through the antennal nerve to the antenna, resulting in the firing rate increases in the OSNs (Ikeda, 2022).

    While ephaptic coupling has been reported earlier, such as between neighboring neurons within the same sensillum, or between Purkinje cells, which is at a distance of <100 μm, this study shows that ephaptic transmission reaches >200 μm in vivo, equivalent to the depth of the entire fly brain, beyond the distance between neighboring neurons. Light stimulations cause -10 mV field potential deflections in a retina. If endogenous fields in the brain are neglected, light stimulations may induce ~33.3 mV/mm electric field between the retina and center of the brain (0 mV), since the distance between them is ~300 μm. This electric field is strong enough to modulate neural activities, as even weaker electric fields (<0.5 mV/mm) changed the firing patterns of neurons in vitro (Ikeda, 2022).

    In rodents, the firing rate of cerebellar Purkinje cells either decreased or increased when a current was injected into the extracellular field around their axons, causing field potential changes of 0.2 mV. In insects, odor-evoked field potential oscillations whose amplitude is comparable with that caused by the current injection in the rodents, are induced by synchronous firing of olfactory neurons in the antennal lobe which are mediated by GABAergic neurons forming reciprocal synapses with excitatory projection neurons. Changes in the extracellular field potential are commonly observed in many nervous systems. While such extracellular field potential activities have been considered as a side effect of synchronized spiking of neurons, this study suggests that such field potential changes evoked by a sensory stimulus can control the excitability of distant neurons, in addition to adjacent neurons. As ephaptic transmission is more effective at a short distance, the ephaptic transmission from the retinae may contribute significantly to firing rate changes in downstream neurons of the photoreceptor cells in the optic lobe (Ikeda, 2022).

    This study also revealed that odor responses of OSNs were clearly modulated when light conditions changed transiently. This mechanism may help flies switch attention to newly presented sensory cues or maintain attention toward those remaining after the change. Turning the light on, for example, reduces the firing rates of the OSNs, which may enable the flies to pay more attention to visual information, whereas turning the light off increases the firing rates of the OSNs, which may help them attend to olfactory sensory cues (Ikeda, 2022).

    Recent connectome analyses have revealed the entire synaptic network in the CNS in Drosophila and provides insight into how neural information is subject to synaptic relays to determine the behavioral output. This study has shown that ephaptic relays also contribute to modulating the firing rate of distant neurons and modify the sensory responses that can change momentarily in a context-dependent manner should also be considered. To build an integrated model of the fly brain, ephaptic relay of neural information should be considered. The compound eye-antenna model would be a suitable model to determine the role of ephaptic transmission in neural processing (Ikeda, 2022).

    Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila

    A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. This study shows that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, these data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions (Koca, 2023).

    This study demonstrates that dAbl regulates cell motility during OR. Although loss of Abl function interferes with multiple aspects of photoreceptor development and morphogenesis, overexpression of dAbl in developing ommatidial clusters in eye discs affects specifically OR, suggesting that dAbl has a defined function in rotation. During OR, dAbl appears to have an inhibitory role, as ommatidial clusters with increased dAbl levels under-rotate, whereas dAbl mutant ommatidia tend to rotate faster (Koca, 2023).

    The localization pattern of dAbl posterior to the MF provides further insight about its role in OR. dAbl becomes apically localized in photoreceptors R8, R2/R5, and R4, following a steady phase of rotation, at the time when clusters slow down and refine their motility until the completion of the 90° angle. Prominent Abl localization within the apical plane of specific photoreceptors suggests that Abl is likely to have a local function in the apical junctional domain. Under-rotation features observed upon dAbl overexpression are consistent with the notion that dAbl becomes apically localized in specific R cells, toward the later stages of OR, to slow down the process. Interestingly, there is a differential localization of dAbl between R3 and R4 in the apical junctional domain. Considering the role of the R3/R4 pair and associated signaling pathways in OR, it is tempting to speculate that this differential dAbl localization is comparable to the requirement of the Nmo kinase within R3/R4, with Nmo providing a directional impulse to rotation in R433 and dAbl regulating its slowing down. The data argue that dAbl activity within R3/R4 pairs is indeed important for fine-tuning rotation. Knockdown and overexpression of dAbl in R3/R4 pairs lead to over-rotation and under-rotation, respectively, during the active rotation process in eye discs, suggesting that Abl activity negatively regulates rotation. Specifically, knockdown of Abl in R3/R4 leads to over-rotation of ommatidia, which, taken together with the WT localization of Abl being restricted to the R4 apical junctional domain, suggests that Abl is required in R4 within the apical region to slow down rotation. In the case of under-rotation caused by m Δ0.5>Abl overexpression, apical dAbl was detected in both cells of the R3/R4 pair and, importantly, temporally earlier in this background compared with WT, suggesting that early dAbl expression in both cells causes an under-rotation phenotype by interfering with rotation. Taken together, these observations are consistent with the hypothesis that the timing and specificity of apical localization of dAbl in R4 is critical for its normal function in OR (Koca, 2023).

    Notably, Abl overexpression does not appear to affect ommatidial chirality and the localization of PCP factors, as Fmi expression and localization remain intact. Furthermore, Abl overexpression causes a specific and severe under-rotation defect, unlikely resulting from deregulation of core PCP factors, which are commonly associated with random ommatidial chirality and rotation. It is most likely that Abl overexpression, under sev- or m Δ0.5-Gal4 drivers, is temporally too late to interfere with Fz/PCP signaling-mediated R3/R4 cell fate decisions, and thus specifically affects OR (Koca, 2023).

    Fz/PCP signaling appears dispensable for the R4-specific apical dAbl localization, as the pattern is maintained in core PCP mutant ommatidia. Yet dAbl does synergize with Fmi, when co-overexpressed in the R3/R4 pair, in a rotation specific manner. This OR-associated functional interaction of Abl with membrane-associated core PCP factors, along with the localization pattern of Abl in the apical domain further suggests that dAbl activity is important in R4 in the apical junctional domain. The results identify Notch and Notch signaling in R4 as critical for apical dAbl localization. Notch over-activation within the R3/R4 pair (via expression of stable isoforms of the receptor) induces apical dAbl localization in both cells of the pair. In contrast, expression in R3/R4 pairs of a version of Notch deficient in Delta binding, the key Notch ligand in the eye, and thus interference with ligand induced Notch activation, leads to a loss of apical dAbl in R4. Similarly, reduction of Notch levels in R3/R4 cells (via RNAi-mediated knockdown) also causes a marked decrease in apical dAbl levels in R4. As Notch-dependent transcription is still active in these backgrounds, the combination of these results suggests that Notch-mediated dAbl apical localization is rather direct, and not via a secondary mechanism through transcriptional regulation. This conclusion is corroborated by the co-immunoprecipitation experiments (Koca, 2023).

    Several experimental lines support the hypothesis that the Notch receptor physically recruits dAbl to the membrane. In salivary glands, Notch overexpression augments junctional dAbl localization, leaving total dAbl levels unaffected. dAbl co-immunoprecipitates with Notch in third-instar larval eye disc extracts, supporting a membrane-associated Notch-Abl interaction in vivo, independent of nuclear Notch signaling activity. The sev>Abl GOF rotation phenotype is markedly suppressed upon removal of one copy of Notch, further supporting the idea that a functional N-Abl signaling module in the apical domain of R4 regulates OR (Koca, 2023).

    dAbl localization appears to be within the apical region and not restricted to the apical membrane ring. There may be multiple reasons for this. As the Notch receptor is cleaved upon ligand binding and its intracellular domain is released to the cytoplasm, distribution of Abl molecules in the apical region may be broader than restricted to the transmembrane fraction of Notch. Abl-Notch interactions likely last after Notch cleavage, considering efficient Abl co-immunoprecipitation with the Notch ICD. Abl can also interact with actomyosin cytoskeletal elements, which are apically enriched in R cells (Koca, 2023).

    As the apical diameter of R cells in this region is less than 2 μm, the imaging resolution does not separate the membrane Abl signal from the juxta-membrane cytoplasmic signal. Notably, in Notch overexpression contexts, Abl signal is often detected as a ring at the apical membrane, likely attributable to the presence of more uncleaved membrane-associated Notch. Furthermore, it is possible to detect and quantify Abl at junctions in salivary glands, and thus document the increased levels of membrane-associated Abl upon higher Notch levels. All these data are consistent with the notion that Abl is specifically recruited to the apical junctional membrane domain by Notch (Koca, 2023).

    In Drosophila, dAbl has been suggested to act downstream of Notch during axonal pathfinding in embryos. Compelling evidence suggests that a non-canonical Notch signaling branch, which does not entail nuclear Notch activity, instructs axonal pathfinding and axon-guidance-specific genetic interactions between dAbl and Notch argue that a non-canonical Notch signaling pathway via dAbl may be at work in this context (Koca, 2023).

    The results are in accordance with these observations and provide further evidence for a non-canonical Notch-Abl signaling module during morphogenesis. Recently, a non-canonical Notch pathway has been reported in the regulation of adherens junction organization during human vascular barrier formation, with the transmembrane domain of Notch forming complexes with the tyrosine phosphatase LAR, vascular endothelial cadherin, and Rac1GEF Trio to confer barrier function in human engineered microvessels. The Notch transmembrane domain requires the cleavage of the Notch extracellular and intracellular domains in this context. The data during OR indicate that apical dAbl recruitment in R4 similarly requires Notch activation by Delta. Whether the transmembrane domain of Notch is an essential component of dAbl recruitment and/or regulation remains to be confirmed. There is a growing body of evidence that Notch uses alternative downstream signaling events to regulate cellular morphogenesis and organization, besides canonical transcriptional target gene regulation. (Koca, 2023).

    Abl appears to affect junctional N-cad and Arm levels in the R3/R4 pair. N-cad mutants show OR defects. Although the mechanism of N-cad involvement remains unclear, N-cad and/or Arm at the R3/R4 boundary could mediate the communication between these cells to determine relative force generation or other directional behavior to give the rotation direction or impulse/force. Such mechanisms have been suggested in border cell migration through E-cad (Koca, 2023).

    N-cad mutant ommatidia appear to over-rotate unlike Abl-overexpressing ommatidia (in which N-cad is downregulated at the R3/R4 border). Although this seems like a discrepancy, Abl overexpression by m Δ0.5-Gal4 (unlike N-cad mutations) is spatially and temporally restricted to R3/R4s, possibly accounting for the differences observed in these backgrounds. Furthermore, Abl likely affects OR via regulating several downstream effectors, including cytoskeletal regulators, in parallel to N-cad and thus has a more complex impact on OR than N-cad alone (Koca, 2023).

    The observation that the non-phosphorylatable isoform of Arm/β-catenin, ArmY667F, rescues the Abl GOF defects, supports the idea that Arm is a key and direct target of dAbl in the OR context. dAbl is involved in the regulation of multi-cellular reorganization in the context of Drosophila germband elongation through the phosphorylation of Arm/β-catenin on tyrosine 667 (Y667), by which it controls adherens junction turnover to promote convergent extension cell movements (Koca, 2023).

    The data argue that dAbl may similarly be involved in regulating Arm/β-catenin dynamics through the same residue during the OR process. The under-rotation phenotype associated with the dAbl GOF (sev>Abl) showed a trend toward rescue by co-(over)expression of Arm-WT and ArmY667E, which is likely due to the fact that exogenously overexpressed Arm isoforms compete with endogenous Arm for dAbl binding. Further experiments will be needed to test these hypotheses (Koca, 2023).

    The requirement of Abl in R4 for accurate rotation suggests that it acts antagonistically to Nemo which is enriched at junctions in R4 early via core PCP factors and its function is to promote rotation (Koca, 2023).

    There is a temporal sequence of apical plane enrichment of factors in R4 with Nemo first to initiate rotation, and Abl a few hours later to slow it down. It was originally proposed that OR is a two-step process, with an initial fast rotation to 45° and a subsequent slower step to achieve the full 90°. However, this idea goes back to the identification of the original allele of nemo, which is a hypomorph, and only affected the rotation process partially (Koca, 2023).

    Recent live imaging studies documenting OR dynamics have established that rotation is continuous with comparable speed throughout. Similarly, there is growing evidence that for rotation to occur correctly, adherens junctions need to be dynamically regulated at the interface between all photoreceptors and the non-rotating inter-ommatidial cells, and possibly between individual inter-ommatidial cells. It is thus very likely that Abl overexpression with m Δ0.5 and sev drivers interferes with rotation by affecting adherens junction regulation and dynamics in all or multiple R cells, like Nemo (Koca, 2023).

    Localization of Abl within the apical plane of R4, as well as R2/R5, is detected at late stages of rotation (from rows 7 and 8 onward), when rotation needs to be slowed down and stopped at 90°, indicating that Abl has a role at the late phases of the process, to terminate rotation. There are additional cues that appear to signal within ommatidia to stop rotation. For example, EGFR signaling via Argos (the original allele of argos being 'roulette/rlt') certainly feeds into slowing down rotation, as without the inhibitory EGFR ligand, argosrlt mutant clusters rotate beyond 90° (as the name 'roulette' indicates). Similarly, Scabrous (Sca), a secreted fibrinogen-like factor, has been suggested to regulate the properties of the extracellular matrix to create a barrier to rotation (Koca, 2023).

    Although the mechanism of Sca function remains unknown, a direct involvement of the ECM in rotation has been reported with a specific link of Integrin signaling and ECM in the OR process. A model is thus emerging that suggests the degree of rotation depends on an interplay between multiple signaling pathways, including Notch-Abl signaling, and their regulatory input to cell adhesion and cytoskeletal elements (Koca, 2023).

    Notch signaling in R3/R4 pairs is critical to coordinate OR via its feeding into the transcriptional regulation of argos, with Notch signaling directly promoting the transcription of argos, the inhibitory ligand to EGFR, required to fine-tune EGFR signaling activity during OR. This study shows that Notch signaling regulates OR via apical junctional recruitment of dAbl in R4, linking Notch activity to non-canonical, Abl-mediated Notch signaling and associated local cellular processes, with Abl modulating cadherin/β-catenin-based junctional complexes. Involvement of Notch signaling in cellular morphogenesis has been suggested in various contexts, including Drosophila oogenesis and neuronal pathfinding, zebrafish sensory organ development and human vascular barrier formation among others (Koca, 2023).

    Besides the reported Notch signaling-mediated transcriptional inputs into adhesion and cytoskeletal dynamics a direct link from the Notch receptor to cell adhesion has been revealed (Koca, 2023).

    This work also suggests a direct input from Notch signaling to cell adhesion dynamics. Many regulators of OR show conservation across developmental processes in vertebrates. The role of Notch signaling in OR suggests a potential involvement for Notch in PCP-mediated morphogenetic events in vertebrates, which has not been reported thus far. Similarly, Abl kinase may have a role in such processes in its interaction with PCP and Notch signaling pathways. Strikingly, the mouse abl-/- arg-/- double mutants exhibit defects in neurulation and delays in neural tube closure, a process generally requiring PCP-regulated features (Koca, 2023).

    The work described here provides insight into Notch-Abl signaling in a tissue remodeling, cell motility process. Although all data are consistent with the proposed model, this model is generated by inference from analyses of static fixed tissue samples, genetics, and biochemical studies. As it involves a cell motility process, it would be desirable to analyze the respective mutant genotypes via live imaging in vivo, including studies applying FRAP and other technologies. This would allow a more complete understanding of how Abl affects junctional dynamics during OR. Future studies will be needed to provide insight into the mechanistic details of how Notch and Abl cooperate in regulating junctional complexes and their dynamics during OR and other morphogenetic developmental and disease processes (Koca, 2023).

    Generation and characterization of inner photoreceptor-specific enhancer-trap lines using a novel piggyBac-Gal4 element in Drosophila

    The Drosophila inner photoreceptors R7 and R8 are responsible for color vision and their differentiation starts at the third instar larval stage. Only a handful of genes with R7 or R8-cell-specific expression are known. An enhancer-trap screen was performed using a novel piggyBac transposable element, pBGay, carrying a Gal4 sequence under the control of the P promoter to identify novel genes expressed specifically in R7 or R8 cells. From this screen, three lines were analyzed in detail: piggyBac(AC109) and piggyBac(AC783) are expressed in R8 cells and piggyBac(AC887) is expressed in R7 cells at the third instar larval stage and pupal stages. Molecular analysis showed that the piggyBac elements were inserted into the first intron of CG14160 and CG7985 genes and the second intron of unzipped. The expression pattern was demonstrated in the developing eye imaginal disc, pupal retina as well as the adult retina. The photoreceptor-specific expression of these genes is reported for the first time and it is proposed that these lines are useful tools for studying the development of the visual system (Terzioglu Kara, 2020).

    Identification of Genes Involved in the Differentiation of R7y and R7p Photoreceptor Cells in Drosophila

    The R7 and R8 photoreceptor cells of the Drosophila compound eye mediate color vision. Throughout the majority of the eye, these cells occur in two principal types of ommatidia. Approximately 35% of ommatidia are of the pale type and express Rh3 in R7 cells and Rh5 in R8 cells. The remaining 65% are of the yellow type and express Rh4 in R7 cells and Rh6 in R8 cells. The specification of an R8 cell in a pale or yellow ommatidium depends on the fate of the adjacent R7 cell. However, pale and yellow R7 cells are specified by a stochastic process that requires the genes spineless, tango and klumpfuss. To identify additional genes involved in this process genetic screens were performed using a collection of 480 P{EP} transposon insertion strains. Genes were identified in gain of function and loss of function screens that significantly altered the percentage of Rh3 expressing R7 cells (Rh3%) from wild-type. 36 strains resulted in altered Rh3% in the gain of function screen where the P{EP} insertion strains were crossed to a sevEP-GAL4 driver line. 53 strains resulted in altered Rh3 percent in the heterozygous loss of function screen. 4 strains showed effects that differed between the two screens, suggesting that the effect found in the gain of function screen was either larger than, or potentially masked by, the P{EP} insertion alone. Analyses of homozygotes validated many of the candidates identified. These results suggest that R7 cell fate specification is sensitive to perturbations in mRNA transcription, splicing and localization, growth inhibition, post-translational protein modification, cleavage and secretion, hedgehog signaling, ubiquitin protease activity, GTPase activation, actin and cytoskeletal regulation, and Ser/Thr kinase activity, among other diverse signaling and cell biological processes (Earl, 2020).

    Hydroxylated sphingolipid biosynthesis regulates photoreceptor apical domain morphogenesis

    Apical domains of epithelial cells often undergo dramatic changes during morphogenesis to form specialized structures, such as microvilli. This study addressed the role of lipids during morphogenesis of the rhabdomere, the microvilli-based photosensitive organelle of Drosophila photoreceptor cells. Shotgun lipidomics analysis performed on mutant alleles of the polarity regulator crumbs, exhibiting varying rhabdomeric growth defects, revealed a correlation between increased abundance of hydroxylated sphingolipids and abnormal rhabdomeric growth. This could be attributed to an up-regulation of fatty acid hydroxylase transcription. Indeed, direct genetic perturbation of the hydroxylated sphingolipid metabolism modulated rhabdomere growth in a crumbs mutant background. One of the pathways targeted by sphingolipid metabolism turned out to be the secretory route of newly synthesized Rhodopsin, a major rhabdomeric protein. In particular, altered biosynthesis of hydroxylated sphingolipids impaired apical trafficking via Rab11, and thus apical membrane growth. The intersection of lipid metabolic pathways with apical domain growth provides a new facet to understanding of apical growth during morphogenesis (Hebbar, 2020).

    Despite the importance of intact microvilli for the homeostasis of many epithelia, regulators controlling microvilli formation are only partially understood. This study used the rhabdomere of Drosophila PRCs, a highly expanded and elaborated apical membrane, as a model to dissect mechanisms controlling the formation of microvilli. It was shown that increased transcription of fatty acid 2-hydroylase (fa2h), followed by increased levels of hydroxylated sphingolipids, is associated with defective rhabdomere morphogenesis. This is, at least partially, due to impaired apical trafficking of Rh1, an important structural component of the rhabdomeral membrane. These results also contribute to understanding of the role of crb in rhabdomere morphogenesis. They imply that increased levels of hydroxylated sphingolipids particularly impact one aspect of rhabdomeral growth, namely the addition of new microvilli, resulting in the extension of the rhabdomere along the proximodistal axis. Rhabdomeral thickness, determined by the length of the microvilli, is not obviously affected by changes in these lipids (Hebbar, 2020).

    The connection between hydroxylated sphingolipid metabolism and apical domain morphogenesis is not unprecedented. Increased hydroxylation of sphingolipids was observed to be one of the major lipidomic changes during the polarization of MDCK cells, and inhibition of sphingolipid biosynthesis resulted in a reduced number of apical microvilli on their apical surface. Likewise, the Caenorhabditis elegans orthologue of fa2h, fath-1, was identified in a screen for genes affecting intestinal polarity. Until now, however, the link between hydroxylated sphingolipids and their metabolism and polarization of epithelial cells was limited to circumstantial evidence. This study now clearly demonstrate that manipulating hydroxylated sphingolipid metabolism, at least in a sensitized genetic background, is sufficient to modulate rhabdomere/apical domain extension (Hebbar, 2020).

    How do hydroxylated sphingolipids and their metabolism affect rhabdomeric/apical domain growth? Given the low abundance of hydroxylated sphingolipids in the overall fly eye lipidome, it is considered unlikely that they play any major structural role in rhabdomere morphogenesis, e.g., by promoting the stability of Rhodopsin in the rhabdomeric membrane. The conclusion is favored that afa2h and thereby hydroxylated sphingolipids play a role as regulators of apical trafficking. This conclusion is based on two findings: (1) less newly synthesized Rh1 is delivered to the rhabdomeres upon overexpression of afa2h, as revealed by blue light-induced chromophore supply (BLICS) assays, which mimic Rh1 pulse-chase experiments; and (2) a similar defect in Rh1 trafficking has been observed in crb mutant PRCs , which have increased levels of afa2h expression. In particular, an effect on post-Golgi trafficking of Rh1 via the Rab11-mediated pathway was identified. Accumulation of cytoplasmic vesicles is observed upon genetic perturbation of Rab11 and/or genes that encode for Rab11 interacting proteins. However, no obvious accumulation of similar vesicles was observed in crb mutant PRCs. Thus, the trafficking defect observed upon afa2h overexpression cannot be attributed to a general increase/decrease in Rab11 compartments, but rather alludes to a defect in sorting apical cargo. These results are consistent with studies in C. elegans, where apical compartments labeled with Rab11 and Rab7 are affected upon loss of fath-1 (Li, 2018; Hebbar, 2020 and references therein).

    Hydroxylated sphingolipids, as glycosphingolipids, are proposed to regulate the sorting of apically directed vesicles by combining different polarity cues (Zhang, 2011). In line with this, it is proposed that increased afa2h transcription (and hence an increase in hydroxylated sphingolipids) prevents apical membrane (rhabdomere) growth by inhibiting trafficking of Rh1 via the apical Rab11 compartments. By regulating the amount of Rh1 delivered to the rhabdomere, afa2h modulates the amount of rhabdomeric membrane in Drosophila PRCs, and hence has an impact on rhabdomere growth. Whether afa2h additionally promotes stability of Rhodopsin in the rhabdomeric membrane via the presence of more hydroxylated sphingolipids could not be addressed here. Interestingly, exogenously labeled hydroxylated fatty acids (the precursors of hydroxylated sphingolipids) preferentially distributed to apically localized membrane-bound compartments in the epithelial cells of the C. elegans gut (Li et al, 2018) (Hebbar, 2020).

    Unexpectedly, afa2h overexpression or knockdown in an otherwise wild-type background does not result in an obvious rhabdomeric extension phenotypes. This may be because Rh1 trafficking depends on the concerted action of Rab1, Rab6, Rab11, Rip11, and MyoV as well as the Rab-effector proteins such as Parcas (Rab11GEF) and Rab11 interacting proteins such as dRip11, and to the amount of F-actin. Although sphingolipids can induce changes in the cytoskeleton, no overt changes in the staining of F-actin were observed in the rhabdomere. Therefore, it is speculated that increased afa2h acts to fine-tune growth by slowing down delivery of Rh1 and hence addition of new rhabdomeric membrane (Hebbar, 2020).

    Yet, in a sensitized background, i.e., in cells homozygous mutant for crb, which have impaired Rh1 trafficking or upon carotenoid depletion, which also reduces Rh1 levels, afa2h overexpression now decreases Rh1 delivery via Rab11. Crb normally limits oxidative stress, and the resulting oxidative status (low oxidative stress) normally limits afa2h expression. In crb mutants, however, an altered oxidative status (increased oxidative stress) causes an up-regulation of afa2h transcription, which, in turn, results in severely reduced Rh1 delivery and improperly extended rhabdomeres. The afa2h dependence of rhabdomeric growth described in this study is only one aspect of the pleiotropic cellular response to an altered redox status of increased oxidative stress signaling due to loss of crb. Interestingly, loss of crb in epithelial cells of larval salivary glands impairs Rab6-, Rab11-, and Rab30-dependent apical trafficking and hence apical membrane homeostasis. Whether this is also caused by increased oxidative stress and/or defects in sphingolipid metabolism remains to be analyzed (Hebbar, 2020).

    In conclusion, this work elucidates an interplay between oxidative stress, lipid metabolism, and apical domain growth during PRC morphogenesis. It will be interesting to investigate whether this link between cell metabolism and apical membrane growth and morphogenesis is a more widespread phenomenon in epithelial biology (Hebbar, 2020).

    Fate mapping during regeneration: cells that undergo compensatory proliferation in damaged Drosophila eye imaginal discs differentiate into multiple retinal accessory cell types

    Regeneration of tissues that have been damaged by cell loss requires new growth, often via proliferation of precursor cells followed by differentiation to replace loss of specific cell types. When regeneration occurs after normal differentiation of the tissue is complete, developmental pathways driving differentiation must be re-activated. How proliferation and differentiation are induced and balanced during regeneration is not well understood. To investigate these processes, a paradigm was used for tissue damage and regeneration in the developing Drosophila melanogaster eye. Previous studies have demonstrated that tissue damage resulting from extensive cell death stimulates quiescent, undifferentiated cells in the developing larval eye to re-enter the cell cycle and proliferate. Whether these cells are restricted to certain fates or can contribute to all retinal cell types and thus potentially be fully regenerative is not known. This study found by fate mapping experiments that these cells are competent to differentiate into all accessory cell types in the retina but do not differentiate into photoreceptors, likely because cell cycle re-entry in response to damage occurs after photoreceptor differentiation has completed. It is concluded that the ability to re-enter the cell cycle in response to tissue damage in the developing Drosophila eye is not restricted to precursors of a specific cell type and that cell cycle re-entry following damage does not disrupt developmental programs that control differentiation (Meserve, 2018).

    Dpp/TGFβ-superfamily play a dual conserved role in mediating the damage response in the retina

    Retinal homeostasis relies on intricate coordination of cell death and survival in response to stress and damage. Signaling mechanisms that coordinate this process in the adult retina remain poorly understood. This study identified Decapentaplegic (Dpp) signaling in Drosophila and its mammalian homologue Transforming Growth Factor-beta (TGFβ) superfamily, that includes TGFβ and Bone Morphogenetic Protein (BMP) signaling arms, as central mediators of retinal neuronal death and tissue survival following acute damage. Using a Drosophila model for UV-induced retinal damage, this study showed that Dpp released from immune cells promotes tissue loss after UV-induced retinal damage. Interestingly, a dynamic response of retinal cells was found to this signal: in an early phase, Dpp-mediated stimulation of Saxophone/Smox signaling promotes apoptosis, while at a later stage, stimulation of the Thickveins/Mad axis promotes tissue repair and survival. This dual role is conserved in the mammalian retina through the TGFβ/BMP signaling, as supplementation of BMP4 or inhibition of TGFβ using small molecules promotes retinal cell survival, while inhibition of BMP negatively affects cell survival after light-induced photoreceptor damage and NMDA induced inner retinal neuronal damage. These data identify key evolutionarily conserved mechanisms by which retinal homeostasis is maintained (Kramer, 2021).

    The Clock:Cycle complex is a major transcriptional regulator of Drosophila photoreceptors that protects the eye from retinal degeneration and oxidative stress

    The aging eye experiences physiological changes that include decreased visual function and increased risk of retinal degeneration. Although there are transcriptomic signatures in the aging retina that correlate with these physiological changes, the gene regulatory mechanisms that contribute to cellular homeostasis during aging remain to be determined. This study integrated ATAC-seq and RNA-seq data to identify 57 transcription factors that showed differential activity in aging Drosophila photoreceptors. These 57 age-regulated transcription factors include two circadian regulators, Clock and Cycle, that showed sustained increased activity during aging. When the Clock:Cycle complex was disrupted by expressing a dominant negative version of Clock (ClkDN) in adult photoreceptors, changes were observed in expression of 15-20% of genes including key components of the phototransduction machinery and many eye-specific transcription factors. Using ATAC-seq, expression of ClkDN in photoreceptors was shown to lead to changes in activity of 37 transcription factors and causes a progressive decrease in global levels of chromatin accessibility in photoreceptors. Supporting a key role for Clock-dependent transcription in the eye, expression of ClkDN in photoreceptors also induced light-dependent retinal degeneration and increased oxidative stress, independent of light exposure. Together, these data suggests that the circadian regulators Clock and Cycle act as neuroprotective factors in the aging eye by directing gene regulatory networks that maintain expression of the phototransduction machinery and counteract oxidative stress (Jauregui-Lozano, 2022).

    Chemical interference with DSIF complex formation lowers synthesis of mutant huntingtin gene products and curtails mutant phenotypes
    Earlier work has shown that siRNA-mediated reduction of the SUPT4H (Drosophila Spt4) or SUPT5H (Drosophila Spt5) proteins, which interact to form the DSIF complex and facilitate transcript elongation by RNA polymerase II (RNAPII), can decrease expression of mutant gene alleles containing nucleotide repeat expansions differentially. Using luminescence and fluorescence assays, this study identified chemical compounds that interfere with the SUPT4H-SUPT5H interaction, and then their effects were investigated on synthesis of mRNA and protein encoded by mutant alleles containing repeat expansions in the huntingtin gene (HTT), which causes the inherited neurodegenerative disorder, Huntington's Disease (HD). This study reports that such chemical interference can differentially affect expression of HTT mutant alleles, and that a prototypical chemical, 6-azauridine (6-AZA), that targets the SUPT4H-SUPT5H interaction can modify the biological response to mutant HTT gene expression. Selective and dose-dependent effects of 6-AZA on expression of HTT alleles containing nucleotide repeat expansions were seen in multiple types of cells cultured in vitro, and in a Drosophila melanogaster animal model for HD. Lowering of mutant HD protein and mitigation of the Drosophila 'rough eye' phenotype associated with degeneration of photoreceptor neurons in vivo were observed. These findings indicate that chemical interference with DSIF complex formation can decrease biochemical and phenotypic effects of nucleotide repeat expansions (Deng, 2022).

    Expansion of the number of contiguous nucleotide repeats that normally exist within certain human genes is the cause of multiple human diseases. Earlier work has shown that expression of alleles containing nucleotide repeat expansions can be reduced differentially by inhibiting production of SUPT4H or SUPT5H, highly conserved cellular proteins that interact to form the transcription elongation complex, DSIF (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor). DSIF assists in the elongation of mRNA molecules by attaching to RNA polymerase II (RNAPII) via an SUPT5H binding site and forming a structural clamp that maintains RNAPII occupancy of template DNA as the polymerase proceeds along the template . A decrease in production or function of SUPT4H or SUPT5H has been found to decrease synthesis of transcripts encoded by genes containing nucleotide repeat expansions including HTT, the gene that causes Huntington's Disease, the C9orf72 locus associated with amyotrophic lateral sclerosis and frontotemporal dementia, and NOP56, the gene associated with spinocerebellar atrophy type 36 (SCA36), and it has been suggested that SUPT4H or SUPT5H may be a target for treatment of certain diseases caused by nucleotide repeat expansions. As interaction between SUPT4H and SUPT5H to form the DSIF complex is required for these proteins to form the structural clamp that maintains RNAPII on DNA template, this study sought to identify compounds that interfere with the SUPT4H-SUPT5H interaction and to elucidate their effects on mutant HTT gene products. This study describes the results of experiments aimed at: 1) identifying chemicals that can interfere with the SUPT4H/5H interaction, 2) determining whether chemical interference with the interaction recapitulates the effects of decreasing SUPT4H or SUPT5H on expression of genes containing expanded nucleotide repeats, and 3) determining whether chemical interference with the interaction has phenotypic effects (Deng, 2022).

    Decreasing the expression of the SUPT4H or SUPT5H components of the DSIF complex can lower production of mRNAs encoded by mutant gene alleles containing nucleotide repeat expansions, and also can modify phenotypes associated with repeat expansions. These findings have led to proposals that that chemical or genetic targeting of SUPT4H or SUPT5H may be useful therapeutically. The results reported in this study indicate that chemical interference with the interaction of SUPT4H and SUPT5H is achievable, that such interference -which has been confirmed by two independent reporter assays and a direct biochemical assay-can lower the abundance of mutant HTT gene products in cultured cells and an HD animal model, and that chemical targeting of DSIF complex formation can mitigate phenotypic effects of repeat expansions. However, the broad and essential biochemical functions of DSIF, raise the prospect that therapeutic targeting of DSIF may be challenging. As SUPT4H and SUPT5H can act individually, as well as in complex with each other, the effects of targeting DSIF also may differ from the effects of targeting its individual components (Deng, 2022).

    Compounds of multiple chemical classes potentially may interfere with the SUPT4H-SUPT5H interaction. Among the compounds identified by the screening assays was 6-azauridine, a previously studied nucleoside inhibitor of de novo uridine-5'-monophosphate productive pathway and consequently of nucleic acid synthesis and cell division. Addition of uridine to cell cultures reversed the effects of approximately equimolar amounts of 6-AZA on global nucleic acid synthesis without affecting mutant HTT expression, demonstrating the distinctness of these two effects of the compound (Deng, 2022).

    Loss of medium spiny neurons (MSNs) in the striatum is a characteristic feature of HD and other neurodegenerative diseases. This study used CRISPR/Cas9 gene editing methodology to shorten the number of HTT gene CAG repeats in HD patient MSNs to a nonpathological length, and found that shortening of repeats in these congenic cells was associated with diminished sensitivity to H2O2 exposure. Treatment with 6-AZA partially reversed the incremental sensitivity of cells containing expanded repeats, but did not affect H2O2 sensitivity in cells containing shorter repeats (Deng, 2022).

    Analogous partial reversal of phenotypic effects of mutant HTT expression was observed also in the adult Drosophila compound eye, which has been widely used as a model for Huntington's Disease and other human neurodegenerative disorders. No loss of Drosophila larval viability was detected at a 6-AZA concentration that rescued animals displaying the rough eye phenotype. However, the ability of uridine supplementation to reverse the global effects of 6-AZA on nucleic acid synthesis in cell culture raises the possibility that such supplementation may prove useful also in mammalian models during in vivo studies (Deng, 2022).

    Whereas the pathogenic effects of repeat expansions in HD and certain other diseases have been observed most clearly in neuronal cells, they are also evident in non-CNS tissues. In the current experiments, they were observed in MSNs, in neuronal cells, in blood cells, and in photoreceptor cells of the eye-and in replicating and nonreplicating cells. Whereas chemical interference with the SUPT4H-SUPT5H interaction has the potential for affecting multiple tissues simultaneously, differences in the length of repeats as well as tissue-specific factors unrelated to DSIF may influence the results of such interference (Deng, 2022).

    Quantitative Proteomic and Metabolomic Profiling Reveals Altered Mitochondrial Metabolism and Folate Biosynthesis Pathways in the Aging Drosophila Eye

    Aging is associated with increased risk of ocular disease, suggesting that age-associated molecular changes in the eye increase its vulnerability to damage. Although there are common pathways involved in aging at an organismal level, different tissues and cell types exhibit specific changes in gene expression with advanced age. Drosophila melanogaster is an established model system for studying aging and neurodegenerative disease that also provides a valuable model for studying age-associated ocular disease. Flies, like humans, exhibit decreased visual function and increased risk of retinal degeneration with age. This study profiled the aging proteome and metabolome of the Drosophila eye and compared these data with age-associated transcriptomic changes from both eyes and photoreceptors to identify alterations in pathways that could lead to age-related phenotypes in the eye. Of note, the proteomic and metabolomic changes observed in the aging eye are distinct from those observed in the head or whole fly, suggesting that tissue-specific changes in protein abundance and metabolism occur in the aging fly. This integration of the proteomic, metabolomic, and transcriptomic data reveals that changes in metabolism, potentially due to decreases in availability of B vitamins, together with chronic activation of the immune response, may underpin many of the events observed in the aging Drosophila eye. It is proposed that targeting these pathways in the genetically tractable Drosophila system may help to identify potential neuroprotective approaches for neurodegenerative and age-related ocular diseases. Data are available via ProteomeXchange with identifier PXD027090 (Hall, 2021).

    An increase in mitochondrial TOM activates apoptosis to drive retinal neurodegeneration

    Intronic polymorphic TOMM40 variants increasing TOMM40 mRNA expression are strongly correlated to late onset Alzheimer's Disease. The gene product, hTomm40, encoded in the APOE gene cluster, is a core component of TOM, the translocase that imports nascent proteins across the mitochondrial outer membrane. This study used Drosophila melanogaster eyes as an in vivo model to investigate the relationship between elevated Tom40 (the Drosophila homologue of hTomm40) expression and neurodegeneration. Evidence is provided that an overabundance of Tom40 in mitochondria invokes caspase-dependent cell death in a dose-dependent manner, leading to degeneration of the primarily neuronal eye tissue. Degeneration is contingent on the availability of co-assembling TOM components, indicating that an increase in assembled TOM is the factor that triggers apoptosis and degeneration in a neural setting. Eye death is not contingent on inner membrane translocase components, suggesting it is unlikely to be a direct consequence of impaired import. Another effect of heightened Tom40 expression is upregulation and co-association of a mitochondrial oxidative stress biomarker, DmHsp22, implicated in extension of lifespan, providing new insight into the balance between cell survival and death. Activation of regulated death pathways, culminating in eye degeneration, suggests a possible causal route from TOMM40 polymorphisms to neurodegenerative disease (Periasamy, 2022).

    Dietary restriction and the transcription factor Clock delay eye aging to extend lifespan in Drosophila Melanogaster

    Many vital processes in the eye are under circadian regulation, and circadian dysfunction has emerged as a potential driver of eye aging. Dietary restriction is one of the most robust lifespan-extending therapies and amplifies circadian rhythms with age. This study demonstrates that dietary restriction extends lifespan in Drosophila melanogaster by promoting circadian homeostatic processes that protect the visual system from age- and light-associated damage. Altering the positive limb core molecular clock transcription factor, CLOCK, or CLOCK-output genes, accelerates visual senescence, induces a systemic immune response, and shortens lifespan. Flies subjected to dietary restriction are protected from the lifespan-shortening effects of photoreceptor activation. Inversely, photoreceptor inactivation, achieved via mutating rhodopsin or housing flies in constant darkness, primarily extends the lifespan of flies reared on a high-nutrient diet. These findings establish the eye as a diet-sensitive modulator of lifespan and indicates that vision is an antagonistically pleiotropic process that contributes to organismal aging (Hodge, 2022).

    Progressive declines in circadian rhythms are one of the most common hallmarks of aging observed across most lifeforms. Quantifying the strength, or amplitude, of circadian rhythms is an accurate metric for predicting chronological age. Many cellular processes involved in aging (e.g., metabolism, cellular proliferation, DNA repair mechanisms, etc.) display robust cyclic activities. Both genetic and environmental disruptions to circadian rhythms are associated with accelerated aging and reduced longevity. These observations suggest that circadian rhythms may not merely be a biomarker of aging; rather, declines in circadian rhythms might play a causal role. The observation that DR and DR-memetics, such as calorie restriction and time-restricted feeding, improve biological rhythms suggests that clocks may play a fundamental role in mediating their lifespan-extending benefits (Kato, 2022).

    This study identified circadian processes that are selectively amplified by DR. The findings demonstrate that DR amplifies circadian homeostatic processes in the eye, some of which are required for DR to delay visual senescence and improve longevity in Drosophila. Disrupting CLK function within photoreceptors accelerates visual declines and shortens lifespan, while overexpressing wild-type CLK protects against age-associated declines in vision and rescues AL-dependent declines in photoreceptor function. These data also demonstrate that photoreceptor stress has deleterious effects on organismal health; overstimulation of the photoreceptors induced a systemic immune response and reduced longevity (Kato, 2022).

    Among the more interesting and unexpecting findings of this study is the observation that the Drosophila eye influences systemic immune responses, as elevated AMP expression was observed in the bodies of flies overexpressing CLK-Δ pan-neuronally and in flies with forced photoreceptor degeneration (ATPα-RNAi). It is possible that GAL4 misexpression may promote inflammatory responses in the fly bodies, although this study found a reduction in systemic inflammation in the rhodopsin-null lines suggesting that this phenomenon can originate at the photoreceptor. These systemic immune responses correlated with lifespan changes (increased body AMP expression is associated with declines in longevity and vice versa), similar to what is observed with chronic inflammation or “inflammaging” in other models. However, it cannot be concluded whether neuronal or eye-mediated increases in systemic inflammation are causal to aging in other tissues. Furthermore, the mechanisms by which the Drosophila eye, and, more specifically, the photoreceptor influence systemic immune responses are unclear. It is speculated that photoreceptor degeneration may disrupt the retinal-blood barrier such that damage signals from the eye propagate through to the hemolymph and activate AMP expression in distal tissues. Future studies are aimed at elucidating this mechanism, and its effect on longevity (Kato, 2022).

    The findings of this study establish the eye as a diet-sensitive regulator of lifespan. DR's neuroprotective role in the photoreceptors appears to be mediated via the transcription factor CLK, which promotes the rhythmic oscillation of genes involved in the suppression of phototoxic cell stress. Given that CLK transcriptionally regulates circadian and non-circadian transcripts, future investigations may determine whether the time-of-day regulation of these genes by CLK is germane to promoting eye health with age. These studies may also examine whether the DR-mediated benefits on visual senescence and photoreceptor viability are mediated solely by CLK as a transcription factor (as demonstrated here) or whether circadian clock function (rhythmic output) is required. The findings also support the notion that age-related declines in the visual system impose a high cost on an organism's physiology. Perhaps this provides an alternative hypothesis for why several cave-dwelling animals, whose visual systems have undergone regressive evolution (e.g., cave-dwelling fish and naked-mole rats), are especially long-lived. Failing to develop a visual system may act as a pro-survival mechanism allowing organisms to avoid the damage and inflammation triggered by age-related retinal degeneration. Ultimately, developing a visual system, which is critical for reproduction and survival, may be detrimental to an organism later in life. Thus, vision may be an example of an antagonistically pleiotropic mechanism that shapes lifespan (Kato, 2022).

    Role of Tau, a microtubule associated protein, in Drosophila photoreceptor morphogenesis

    Cell polarity genes have important functions in photoreceptor morphogenesis. Based on recent discovery of stabilized microtubule cytoskeleton in developing photoreceptors and its role in photoreceptor cell polarity, microtubule associated proteins might have important roles in controlling cell polarity proteins' localizations in developing photoreceptors. Tau, a microtubule associated protein, was analyzed in this study to find its potential role in photoreceptor cell polarity. Tau colocalizes with acetylated/stabilized microtubules in developing pupal photoreceptors. Although it is known that tau mutant photoreceptor has no defects in early eye differentiation and development, it shows dramatic disruptions of cell polarity proteins, adherens junctions, and the stable microtubules in developing pupal photoreceptors. This role of Tau in cell polarity proteins localization in photoreceptor cells during the photoreceptor morphogenesis was further supported by Tau's overexpression studies. Tau overexpression caused dramatic expansions of apical membrane domains where the polarity proteins localize in the developing pupal photoreceptors. It is also found that Tau's role in photoreceptor cell polarity depends on Par-1 kinase. Furthermore, a strong genetic interaction between tau and crumbs was found. Tau was found to have a crucial role in cell polarity protein localization during pupal photoreceptor morphogenesis stage, but not in early eye development including eye cell differentiation (Nam, 2016).

    Stochastic spineless expression creates the retinal mosaic for colour vision

    Drosophila colour vision is achieved by R7 and R8 photoreceptor cells present in every ommatidium. The fly retina contains two types of ommatidia, called 'pale' and 'yellow', defined by different rhodopsin pairs expressed in R7 and R8 cells. Similar to the human cone photoreceptors, these ommatidial subtypes are distributed stochastically in the retina. The choice between pale versus yellow ommatidia is made in R7 cells, which then impose their fate onto R8. The Drosophila dioxin receptor Spineless is both necessary and sufficient for the formation of the ommatidial mosaic. A short burst of spineless expression at mid-pupation in a large subset of R7 cells precedes rhodopsin expression. In spineless mutants, all R7 and most R8 cells adopt the pale fate, whereas overexpression of spineless is sufficient to induce the yellow R7 fate. Therefore, this study suggests that the entire retinal mosaic required for colour vision is defined by the stochastic expression of a single transcription factor, Spineless (Wernet, 2006).

    The ability to discriminate between colours has evolved independently in vertebrates and invertebrates. However, despite the obvious differences in eye development and design, both flies and humans have developed retinal mosaics where classes of photoreceptor cells (PRs) with different spectral sensitivity are randomly distributed. The compound eye of Drosophila consists of ~800 optical units (ommatidia), each containing eight PRs in addition to accessory cells. In each ommatidium, the six 'outer PRs' (R1-R6) function like the vertebrate rod cells, as they are required for motion detection in dim light. These cells express the broad-spectrum rhodopsin, Rh1. The 'inner PRs' (R7 and R8) may be viewed as the equivalent of the colour-sensitive vertebrate cone cells, which express a range of different rhodopsin molecules (Wernet, 2006).

    The general rule of sensory receptor exclusion also applies to Drosophila ommatidia, where only one rhodopsin gene is expressed by a given PR. The expression of inner PR rhodopsins can be used to distinguish three ommatidial subtypes. Two of the subtypes are distributed randomly throughout the retina: ~30% of ommatidia express ultraviolet-sensitive Rh3 in R7 cells and blue-sensitive Rh5 in R8 cells, and therefore are specialized in the detection of short wavelengths ('pale' ommatidia). The remaining ~70% express another ultraviolet-sensitive opsin (Rh4) in R7 and green-sensitive Rh6 in R8, making them more responsive to longer wavelengths ('yellow' ommatidia). The coupled expression of Rh3/Rh5 or Rh4/Rh6 within the same ommatidium results from communication between R7 and R8. In the dorsal rim area (DRA), a third type of ommatidia exists in which both R7 and R8 express ultraviolet-sensitive Rh3. These ommatidia are used to detect the e-vector of polarized sunlight for orientation. Spatially localized polarized light detectors and stochastically distributed colour-sensitive ommatidia therefore reflect two fundamentally different specification strategies that shape the retinal mosaic of Drosophila (Wernet, 2006).

    The current model for specifying colour-sensitive ommatidia combines stochastic and instructive steps. First, a subset of R7 (pale R7, pR7) stochastically chooses Rh3 expression over the 'R7 default', Rh4. Second, these cells then impose the p fate (Rh5) onto R8 (pale R8, pR8) of the same ommatidium (Wernet, 2006).

    This study reports the identification of spineless (ss) as a key regulatory gene for establishing the retinal mosaic. ss encodes the Drosophila homologue of the human arylhydrocarbon ('dioxin') receptor, a member of the bHLH-PAS (basic helix-loop-helix- Period-Arnt-Single-minded) family of transcription factors. At mid-pupation, ss is stochastically expressed in a majority of R7 that seem to correspond to the y subtype. ss is both necessary and sufficient to specify the yellow R7 (yR7) fate and subsequently the entire y ommatidia; pR7 cells are thus specified by default, and stochastic expression of ss represents the key regulatory event defining the retinal mosaic required for fly colour vision (Wernet, 2006).

    homothorax (hth) has been identified as the key regulatory gene necessary and sufficient for the specification of DRA ommatidia. ss and hth cause similar homeotic phenotypes: that is, complete (hth) or partial (ss, 'aristapedia') transformation of antennae into legs. Therefore, a potential role of ss in ommatidial subtype specification was tested by generating whole-mutant eyes, as well as mitotic clones, lacking ss function using the null allele ssD115.7 and the ey-FLP/FRT technique. Owing to ey-FLP expression in the antennal imaginal disc, ss mutant flies showed a strong aristapedia phenotyp, but lacked any obvious morphological eye phenotype. However, expression of rhodopsin genes was severely affected. In wild-type eyes, Rh3 is found in ~30% of R7 cells, as well as in both R7 and R8 of DRA-ommatidia, whereas the remaining ~70% of R7 contain Rh4. In ss mutant eyes, Rh4 was completely absent, whereas Rh3 was expanded into all R7 cells. The total number of ommatidia was not reduced, indicating that R7 cells were mis-specified into pR7, rather than yR7 being specifically eliminated. ss mutant mitotic clones were morphologically wild type; however, Rh3 was always present in mutant R7 cells (marked by the absence of ß-galactosidase (ß-gal) expression), whereas Rh4 was always lost (Wernet, 2006).

    To test whether the R7 ss phenotype was cell autonomous, individual mutant R7 cells were generated using the MARCM technique. All mutant R7 cells [marked by the presence of green fluorescent protein (GFP) expression] contained Rh3 and never Rh4, demonstrating that ss is required cell autonomously in R7 to induce Rh4 expression. DRA ommatidia were correctly specified in ss mutant eyes, since Rh3 was expressed normally in both DRA R7 and R8 cells. Therefore, ss is necessary for the establishment of the yR7 subtype without affecting PR fate specification (Wernet, 2006).

    The ommatidial subtypes are first specified in R7, which then instruct R8. Therefore, ss mutant eyes should exhibit a rhodopsin phenotype in R8. In wild types, ~30% of R8 cells contain Rh5, and the remaining ~70% contain Rh6. In ss mutant eyes, the large majority (up to 95%) of R8 contained Rh5, with some R8 still containing Rh6. However, most of these remaining yR8 cells were located in the dorsal third of the eye. In this part of the retina, instruction of pale R8 (pR8) by pR7 is less efficient, resulting in ommatidia with odd-coupled (Rh3/Rh6) rhodopsin expression. In ss mutants, the frequency of such ommatidia was significantly increased in the dorsal region. To test whether the R8 opsin phenotype of ss mutants resulted from the inability of some mutant R7 cells to properly instruct R8, rather than from ss being directly required in R8, sevenless; spineless (sev; ss) double-mutant eyes were generated (Wernet, 2006).

    These eyes, which lacked R7 cells, always exhibited the sev single-mutant phenotype, with virtually all R8 cells containing Rh6. This indicates that ss is required in R7 for the formation of the yR7 subtype, and consequently for the formation of yR8, without being directly required in R8 PRs (Wernet, 2006).

    Whether ss was also sufficient to induce the y ommatidial subtype was tested. Overexpression of ss in all developing PRs using a strong LGMR (long glass multiple reporter)-Gal4 driver and UAS-ss (LGMR.ss flies) resulted in a rough eye phenotype, as well as a dramatic rhodopsin phenotype: Rh4 was activated in all PRs throughout the eye (R1-R6 as well as R7 and R8), as revealed by ectopic expression of an Rh4-GFP reporter in many PRs per ommatidium compared with wild type. To avoid the strong phenotype in the eye, ss was misexpressed using the weaker, variegated GMR driver, sGMR (short GMR)-Gal431 (sGMR.ss flies). This led to strong ectopic induction of Rh4 in many PRs without severely affecting retinal morphology. This ectopic induction of Rh4 was also observed in sev mutants, and was thus independent of R7. Rh3 was still detected in some R7 in sGMR.ss flies, presumably due to the lack of variegated Gal4 expression in these cells, whereas Rh4 was expanded to some outer PRs. However, co-localization of Rh3 and Rh4 was never observed, confirming that gain of Rh4 in R7 cells always leads to the exclusion of Rh3. In contrast, gain of Rh4 in outer PRs did not lead to the exclusion of Rh1; frequent coexpression of Rh1 and Rh4 was observed (Wernet, 2006).

    Using an Rh4-lacZ reporter construct in LGMR.ss flies, it was found that ß-gal-positive PR axons projected to both lamina and medulla, confirming the expansion of Rh4 into outer PRs. However, Rh4-expressing outer PRs were not transformed into genuine R7 cells, since they maintained their lamina projections. Notably, DRA inner PRs were the only cells not expressing Rh4, suggesting that the DRA fate, specified by the gene hth, antagonizes ss function. Expression of Rh3 and Rh5 was completely lost (including in the DRA, where no rhodopsin was detected), while Rh6 expression was found in most R8 cells. This resulted in R8 coexpressing Rh4 and Rh6, demonstrating that the 'one sensory receptor per cell' rule can be broken in Drosophila PRs, as has been shown in other insects. Therefore, ectopic induction of the yR7 fate by ss specifically excludes the formation of pR7 cells. As a consequence, R8 cells expressing Rh5 are not induced, with most R8 expressing Rh6. Rh6 was never found in outer PRs, supporting the hypothesis that ss is required only in R7 for the choice between Rh3 and Rh4, and not directly in R8 for the Rh6 choice. In LGMR.ss flies, the specification of outer versus inner PRs (markers spalt and seven up) or of R7 versus R8 (prospero and senseless) was normal. Thus, ss acts by segregating ommatidial subtypes downstream of early PR specification events (Wernet, 2006).

    Colour PR cell fate determination seems to be a late event in PR differentiation. To test whether ss can transform the R7 fate at late stages of development, the PanR7-Gal4 driver (which is also expressed in DRA R8 cells) was used. Late mis-expression of ss induced the y fate (Rh4) in all R7 cells, whereas Rh3 was absent. Opsin expression in the DRA was also altered, with Homothorax-positive cells (both R7 and R8) now expressing Rh4. Hence, it is possible to reprogramme the R7 fate at later stages of differentiation, as PanR7-Gal4 becomes activated at the time of rhodopsin expression. Surprisingly, expression of R8 rhodopsins outside the DRA was not affected, since the distribution of Rh5 and Rh6 resembled the wild type. As a result, many ommatidia manifested the very unusual coupling of Rh4 in R7 and Rh5 in R8. Therefore, although ss is able to reprogramme all R7 late in development, R8 cannot revert their fate once they have been instructed to become pR8, and they maintain Rh5. Two antagonistic genes expressed in either of the two R8 subtypes have been identified that act together as a molecular consolidation system responsible for this inertia of R8. To confirm that late expression of ss exclusively in R7 is sufficient to transform R7, ss was mis-expressed in ssD115.7 mutants using PanR7-Gal4. This was sufficient to induce Rh4 and to repress Rh3. R8 were again not reprogrammed and exhibited the ss mutant phenotype, with many R8 cells expressing Rh5 (Wernet, 2006).

    All of the results presented above strongly indicated that ss must be expressed in the y subtype of R7 at some point during pupal development. Since several attempts to generate an anti-Spineless antibody had failed, in situ hybridization was used to detect ss messenger RNA in the retina at mid-pupation. At ~50% pupation, ss mRNA was detected in four neuronal cells per ommatidial cluster, one PR and three bristle cells. The PR was also labelled by anti-Prospero, confirming its identity as R7. Although the expression levels of ss in bristle cells seemed uniformly high, levels of ss expression varied considerably among R7 cells, ranging from very faint to very strong in 60%-80% of R7. A 1.6 kilobase 'eye enhancer' fragment (sseye) was also identified within the ss promoter that drives PR-specific expression. After crossing ss eye-Gal4 to UAS-ß-gal::NLS (nuclear localization sequence) reporters, PR-specific ss expression was first detected at mid-pupation-that is, approximately one day before rhodopsins are expressed, and before any visible molecular or morphological distinction between ommatidial subtypes. A single PR per ommatidium, which was identified as R7 through co-staining with Prospero, expressed ss. Thus, the ss eye enhancer recapitulates endogenous ss expression in PRs. ss expression was detected in 60%-80% of R7, correlating well with the distribution of Rh4 in adult retina. Like Rh4-expressing ommatidia, ß-gal-positive ommatidia were more abundant in the dorsal half of the eye, and no ß-gal expression was detected in the DRA (marked by Homothorax), where Rh4 is also never expressed. ss eye-Gal4 expression was detectable for only ~2 h at midpupation (Wernet, 2006).

    Although it was not possible to directly co-stain for ss and Rh4 (which starts to be expressed one day later during pupation), it seems that at mid-pupation a short pulse of ss is deployed in a large subset of R7, which will become yR7 (Wernet, 2006).

    Whether a short pulse of ectopic ss expression was able to modify the entire retinal mosaic was tested using a heat shock-Gal4 driver (hs-Gal4) to temporally control ss expression (hs.ss flies). A 30-min heat shock at ~50% pupation indeed resulted in an increase of Rh4 expression with a concomitant reduction of Rh3 in adults. The phenotype varied extensively, from only R7 cells expressing Rh4 (~25% of the flies analysed had Rh4 in most R7), to almost every PR expressing Rh4. In contrast, a 30-min pulse of ss in one-day-old adult flies had no effect. Heat shocks during larval or early pupal stages were lethal. Thus, PRs are extremely sensitive to a short pulse of ss during mid-pupation, at the time when endogenous ss is normally expressed. To further study the mechanism of the stochastic choice between p and y ommatidia, the retinal mosaic was examined in different mutant backgrounds. Flies heterozygous for ssD115.7 had fewer Rh4-expressing R7 cells. Since the ssD115.7 allele affects only the ss coding sequence, heterozygous flies have two functional promoters, only one of which produces a functional protein, suggesting that the non-productive promoter might sequester limiting factor(s) that regulate(s) the expression levels of ss. If this hypothesis is correct, addition of extra copies of the ss promoter should have a similar effect. Indeed, the addition of two functional copies of the ss eye enhancer (ss eye-Gal4) in an otherwise wild-type background also caused a significant reduction of the yR7 subtype. Therefore, the level of Spineless expression is important for the induction of the yR7 fate, which is less efficient in cells where the amount of Spineless is reduced (Wernet, 2006).

    Retinal patterning in Drosophila reveals an original mechanism for how PR mosaics can be generated: stochastic expression of a single transcription factor (Spineless) acts as a binary switch that transforms the seemingly homogeneous compound eye into a mosaic, distinguishing p and y subtypes. However, subtype specification and rhodopsin expression can be separated, since ss expression in yR7 has ceased well before the time of rhodopsin expression. Additional factors are therefore required downstream of ss to ensure expression of adult p- and y-specific markers such as rhodopsins and additional screening pigments. A revised two-step model is proposed for the stochastic specification of p and y ommatidia. First, R7 are stochastically divided into two subtypes by the induction of ss in yR7. ss-positive R7 express Rh4, whereas the remaining R7 choose the pR7 fate and express Rh3 by default. Second, only those R7 cells that did not express ss (pR7) retain the ability to induce the pR8 fate (Rh5), whereas yR8 express Rh6 by default. The 'default states' of R7 (Rh3) and R8 (Rh6) therefore belong to opposite subtypes. Expression of R8 rhodopsin genes is maintained by a bistable regulatory loop containing the genes warts and melted. Notably, the localized specification of polarization-sensitive DRA ommatidia by hth antagonizes the stochastic choice executed by ss, placing these two genes into a new regulatory relationship during retinal patterning. Therefore, the role of the transcription factor Spineless is to generate the retinal mosaic required for fly colour vision by distinguishing yR7 from pR7 cell fates, and preventing R7 from instructing the underlying R8 cells. Mosaic expression of sensory receptors has been described in detail for the olfactory system of both vertebrates and insects, and random PR mosaics have been described for humans and amphibians, as well as insects. Two transcription factors have been shown to regulate the specification of blue versus red/green cone cell fates in mammals. Upon mutation of either - the human nuclear receptor NR2E3 (also known as PNR) or the rodent thyroid hormone ß2 receptor - the number of blue cones is dramatically increased at the expense of green cones, leading to 'enhanced S-cone syndrome'. It should be noted that this retinal phenotype bears important similarity to the altered ommatidial mosaic in Drosophila ss mutants, where long wavelength-sensitive y ommatidia are lost at the expense of the short wavelength-sensitive p type (Wernet, 2006).

    The stochastic cell fate choice occurs at the level of the ss promoter: the very short pulse of ss expression at mid-pupation is not only controlled temporally, but its levels are also critical, and only ~70% of R7 receive enough Spineless to commit to the yR7 fate. Elucidating the mechanism that controls ss expression will shed some light into the fascinating process of stochastic gene expression, and the identification of its downstream targets will provide insights into consolidation and maintenance of cell fates (Wernet, 2006).

    The role of Ire1 in Drosophila eye pigmentation revealed by an RNase dead allele

    Ire1 is an endoplasmic reticulum (ER) transmembrane RNase that cleaves substrate mRNAs to help cells adapt to ER stress. Because there are cell types with physiological ER stress, loss of Ire1 results in metabolic and developmental defects in diverse organisms. In Drosophila, Ire1 mutants show developmental defects at early larval stages and in pupal eye photoreceptor differentiation. These Drosophila studies relied on a single Ire1 loss of function allele with a Piggybac insertion in the coding sequence. This study reports that an Ire1 allele with a specific impairment in the RNase domain, H890A, unmasks previously unrecognized Ire1 phenotypes in Drosophila eye pigmentation. Specifically, it was found that the adult eye pigmentation is altered, and the pigment granules are compromised in Ire1(H890A) homozygous mosaic eyes. Furthermore, the Ire1(H890A) mutant eyes had dramatically reduced Rhodopsin-1 protein levels. Drosophila eye pigment granules are most notably associated with late endosome/lysosomal defects. These results indicate that the loss of Ire1, which would impair ER homeostasis, also results in altered adult eye pigmentation (Mitra, 2021).

    A combinatorial cis-regulatory logic restricts color-sensing Rhodopsins to specific photoreceptor subsets in Drosophila

    Color vision in Drosophila melanogaster is based on the expression of five different color-sensing Rhodopsin proteins in distinct subtypes of photoreceptor neurons. Promoter regions of less than 300 base pairs are sufficient to reproduce the unique, photoreceptor subtype-specific rhodopsin expression patterns. It has been proposed that the rhodopsin promoters have a bipartite structure: the distal promoter region directs the highly restricted expression in a specific photoreceptor subtype, while the proximal core promoter region provides general activation in all photoreceptors. To distinguish between these two models, the expression patterns were analyzed of a set of hybrid promoters that combine the distal promoter region of one rhodopsin with the proximal core promoter region of another rhodopsin. It was found that the function of the proximal core promoter regions extends beyond providing general activation: these regions play a previously underappreciated role in generating the non-overlapping expression patterns of the different rhodopsins. Therefore, cis-regulatory motifs in both the distal and the proximal core promoter regions recruit transcription factors that generate the unique rhodopsin patterns in a combinatorial manner. This combinatorial regulatory logic is compared to the regulatory logic of olfactory receptor genes and potential implications for the evolution of rhodopsins is discussed (Poupault, 2021).

    Calcium waves facilitate and coordinate the contraction of endfeet actin stress fibers in Drosophila interommatidial cells

    Actomyosin contraction shapes the Drosophila eye's panoramic view. The convex curvature of the retinal epithelium, organized in ∼800 close-packed ommatidia, depends upon a fourfold condensation of the retinal floor mediated by contraction of actin stress fibers in the endfeet of interommatidial cells (IOCs). How these tensile forces are coordinated is not known. This study discovered a novel phenomenon: Ca2+ waves regularly propagate across the IOC network in pupal and adult eyes. Genetic evidence demonstrates that IOC waves are independent of phototransduction, but require inositol 1,4,5-triphosphate receptor (IP3R), suggesting these waves are mediated by Ca2+ releases from ER stores. Removal of IP3R disrupts stress fibers in IOC endfeet and increases the basal retinal surface by ∼40%, linking IOC waves to facilitating stress fiber contraction and floor morphogenesis. Further, IP3R loss disrupts the organization of a collagen IV network underneath the IOC endfeet, implicating ECM and its interaction with stress fibers in eye morphogenesis. It is proposed that coordinated cytosolic Ca2+ increases in IOC waves promote stress fiber contractions, ensuring an organized application of the planar tensile forces that condense the retinal floor (Ready, 2021).

    Emergence of a geometric pattern of cell fates from tissue-scale mechanics in the Drosophila eye

    Pattern formation of biological structures involves the arrangement of different types of cells in an ordered spatial configuration. This study investigated the mechanism of patterning the Drosophila eye epithelium into a precise triangular grid of photoreceptor clusters called ommatidia. Previous studies had led to a long-standing biochemical model whereby a reaction-diffusion process is templated by recently formed ommatidia to propagate a molecular prepattern across the eye. This study finds that the templating mechanism is instead, mechanochemical in origin; newly born columns of differentiating ommatidia serve as a template to spatially pattern flows that move epithelial cells into position to form each new column of ommatidia. Cell flow is generated by a source and sink, corresponding to narrow zones of cell dilation and contraction respectively, that straddle the growing wavefront of ommatidia. The newly formed lattice grid of ommatidia cells are immobile, deflecting and focusing the flow of other cells. Thus, the self-organization of a regular pattern of cell fates in an epithelium is mechanically driven (Gallagher, 2022).

    The exit of axons and glial membrane from the developing Drosophila retina requires integrins

    Coordinated development of neurons and glia is essential for the establishment of neuronal circuits during embryonic development. In the developing Drosophila visual system, photoreceptor (R cell) axons and wrapping glial (WG) membrane extend from the eye disc through the optic stalk into the optic lobe. Extensive studies have identified a number of genes that control the establishment of R-cell axonal projection pattern in the optic lobe. The molecular mechanisms directing the exit of R-cell axons and WG membrane from the eye disc, however, remain unknown. This study shows that integrins are required in R cells for the extension of R-cell axons and WG membrane from the eye disc into the optic stalk. Knockdown of integrins in R cells but not WG caused the stalling of both R-cell axons and WG membrane in the eye disc. Interfering with the function of Rhea (i.e. the Drosophila ortholog of vertebrate talin and a key player of integrin-mediated adhesion), caused an identical stalling phenotype. These results support a key role for integrins on R-cell axons in directing R-cell axons and WG membrane to exit the eye disc (Ren, 2022).

    Brain connectivity inversely scales with developmental temperature in Drosophila

    Variability of synapse numbers and partners despite identical genes reveals the limits of genetic determinism. This study used developmental temperature as a non-genetic perturbation to study variability of brain wiring and behavior in Drosophila. Unexpectedly, slower development at lower temperatures increases axo-dendritic branching, synapse numbers, and non-canonical synaptic partnerships of various neurons, while maintaining robust ratios of canonical synapses. Using R7 photoreceptors as a model, this study showed that changing the relative availability of synaptic partners using a DIPγ mutant that ablates R7's preferred partner leads to temperature-dependent recruitment of non-canonical partners to reach normal synapse numbers. Hence, R7 synaptic specificity is not absolute but based on the relative availability of postsynaptic partners and presynaptic control of synapse numbers. Behaviorally, movement precision is temperature robust, while movement activity is optimized for the developmentally encountered temperature. These findings suggest genetically encoded relative and scalable synapse formation to develop functional, but not identical, brains and behaviors (Kiral, 2021).

    Tissue fluidity mediated by adherens junction dynamics promotes planar cell polarity-driven ommatidial rotation

    The phenomenon of tissue fluidity-cells' ability to rearrange relative to each other in confluent tissues-has been linked to several morphogenetic processes and diseases, yet few molecular regulators of tissue fluidity are known. Ommatidial rotation (OR), directed by planar cell polarity signaling, occurs during Drosophila eye morphogenesis and shares many features with polarized cellular migration in vertebrates. This study utilized in vivo live imaging analysis tools to quantify dynamic cellular morphologies during OR, revealing that OR is driven autonomously by ommatidial cell clusters rotating in successive pulses within a permissive substrate. Through analysis of a rotation-specific nemo mutant, this study demonstrated that precise regulation of junctional E-cadherin levels is critical for modulating the mechanical properties of the tissue to allow rotation to progress. This study defines Nemo as a molecular tool to induce a transition from solid-like tissues to more viscoelastic tissues broadening molecular understanding of tissue fluidity (Founounou, 2021).

    Stratum is required for both apical and basolateral transport through stable expression of Rab10 and Rab35 in Drosophila photoreceptors

    Post-Golgi transport for specific membrane domains, also termed polarized transport, is essential for the construction and maintenance of polarized cells. Highly polarized Drosophila photoreceptors serve as a good model system for studying the mechanisms underlying polarized transport. The Mss4 Drosophila ortholog, Stratum (Strat), controls basal restriction of basement membrane proteins in follicle cells, and Rab8 acts downstream of Strat. This study investigated the function of Strat in fly photoreceptors and found that polarized transport in both the basolateral and rhabdomere membrane domains was inhibited in Strat-deficient photoreceptors. 79% and 55% reductions in Rab10 and Rab35 levels, respectively, were also observed but no reduction in Rab11 levels in whole-eye homozygous clones of Strat(null). Moreover, Rab35 was localized in the rhabdomere, and loss of Rab35 resulted in impaired Rh1 transport to the rhabdomere. These results indicate that Strat is essential for the stable expression of Rab10 and Rab35, which regulate basolateral and rhabdomere transport, respectively, in fly photoreceptors (Ochi, 2022).

    Prickle isoform participation in distinct polarization events in the Drosophila eye

    Planar cell polarity (PCP) signaling regulates several polarization events during development of ommatidia in the Drosophila eye, including directing chirality by polarizing a cell fate choice and determining the direction and extent of ommatidial rotation. The pksple isoform of the PCP protein Prickle is known to participate in the R3/R4 cell fate decision, but the control of other polarization events and the potential contributions of the three Pk isoforms have not been clarified. By characterizing expression and subcellular localization of individual isoforms together with re-analyzing isoform specific phenotypes, this study showed that the R3/R4 fate decision, its coordination with rotation direction, and completion of rotation to a final ±90° rotation angle are separable polarization decisions with distinct Pk isoform requirements and contributions. Both pksple and pkpk can enforce robust R3/R4 fate decisions, but only pksple can correctly orient them along the dorsal-ventral axis. In contrast, pksple and pkpk can fully and interchangeably sustain coordination of rotation direction and rotation to completion. It is proposed that expression dynamics and competitive interactions determine isoform participation in these processes. It is proposed that the selective requirement for pksple to orient the R3/R4 decision and their interchangeability for coordination and completion of rotation reflects their previously described differential interaction with the Fat/Dachsous system which is known to be required for orientation of R3/R4 decisions but not for coordination or completion of rotation (Cho, 2022).

    Contribution of photoreceptor subtypes to spectral wavelength preference in Drosophila

    The visual systems of most species contain photoreceptors with distinct spectral sensitivities that allow animals to distinguish lights by their spectral composition. In Drosophila, photoreceptors R1-R6 have the same spectral sensitivity throughout the eye and are responsible for motion detection. In contrast, photoreceptors R7 and R8 exhibit heterogeneity and are important for color vision (see Normalized spectral quantum sensitivity of the different rhodopsins in the photoreceptor subtypes R7p, R7y, R8p, R8y, and R1-R6). This study investigated how photoreceptor types contribute to the attractiveness of light by blocking the function of certain subsets and by measuring differential phototaxis between spectrally different lights. In a 'UV vs. blue' choice, flies with only R1-R6, as well as flies with only R7/R8 photoreceptors, preferred blue, suggesting a nonadditive interaction between the two major subsystems. Flies defective for UV-sensitive R7 function preferred blue, whereas flies defective for either type of R8 (blue- or green-sensitive) preferred UV. In a 'blue vs. green' choice, flies defective for R8 (blue) preferred green, whereas those defective for R8 (green) preferred blue. Involvement of all photoreceptors [R1-R6, R7, R8 (blue), R8 (green)] distinguishes phototaxis from motion detection that is mediated exclusively by R1-R6 (Yamaguchi, 2010).

    Phototaxis consists of at least three behavioral components: (1) detection of an object or a light source in visual space; (2) the attractiveness or aversiveness of the light stimulus at this location; and (3) a motor output (goal-directed walking and turning). Genetic dissection of the visual input to phototaxis relies on the assumption that phototaxis stays intact even if some of these inputs are deleted, i.e., that the mutants can still detect the locations of the two stimuli and move toward or away from them. Although in mutants affecting the R1-R6 subsystem, walking speed, turning, and orientation toward stationary objects are affected, these behaviors are sufficiently intact to support phototaxis. In the experiments reported in this study, light intensities were chosen such that all mutants had at least one photoreceptor type that could detect the light sources and guide walking and turning behavior. This allowed measuring of the second component, the differential attractiveness of the two stimuli, which determined the choice between the two monochromatic lights depending upon the photoreceptor types available in the respective fly strains. Flies could judge the attractiveness of a light source on the basis of its spectral composition and/or spectrally weighted brightness. In the following paragraphs the contributions of the four types of photoreceptors R1-R6, R7, R8p, and R8y to the attractiveness function are discussed (Yamaguchi, 2010).

    Blocking or removing any one of the photoreceptor types shifts the preference away from the point of neutrality in at least one of the choice tests. Silencing the R1-R6 cells leaves the flies with a modest blue preference in the UV/B choice, and replacing their broadly sensitive opsin Rh1 by the UV-sensitive opsin Rh3 shifts the preference in the B/G choice to blue. Removing or silencing R7 in the presence of R1-R6 shifts the preference in the UV/B choice to blue and in the B/G choice to green. Inactivating the green-sensitive Rh6 opsin in R8y cells shifts the preference in the B/G choice to blue and in the UV/B choice to UV. Inactivating blue-sensitive Rh5 in the R8p cells causes a green preference in the B/G choice and a UV preference in the UV/B choice (Yamaguchi, 2010).

    Each of the three photoreceptor subsystems (R1-R6, R7, R8) alone can drive phototaxis. Flies with only photoreceptors R1-R6 (sev rh52 rh61 triple mutants) show a blue preference in the UV/B choice, implying that this subsystem not only mediates optomotor responses and orientation but also can mediate the attractiveness function in phototaxis, i.e., that, with only R1-R6 photoreceptors, flies compare the quantum flux of two light sources 180° apart (Yamaguchi, 2010).

    The R8 subsystem alone can also mediate the attractiveness function in phototaxis (sev ninaE17), as had been shown before for the sev rdgB double mutant. The sev ninaE17 flies have a pronounced green preference in the B/G choice test, consistent with the absorption spectrum of Rh6 expressed in all R8 photoreceptors (Yamaguchi, 2010).

    Flies that have only R7 photoreceptors operating (ninaE17, rh52, rh61) show phototaxis. Surprisingly, they have a very strong preference for blue in both choice tests. This could be explained if R7p inhibit R7y photoreceptors. Otherwise, the flies would show a strong UV preference in the UV/B choice. As an alternative explanation, however, it is possible that phototaxis is mediated by the ocelli in flies with only R7 photoreceptors (Yamaguchi, 2010).

    As long as only one of the central photoreceptor subsystems R7 or R8 is inactivated, the results can be explained by a model in which the respective photoreceptors contribute roughly additively to the attractiveness function. In the honeybee, all three subtypes of photoreceptors (UV, blue, and green) also feed into phototaxis. Spectral mixing experiments in phototaxis are consistent with simple summation of quantal fluxes, and the action spectra of phototactic behavior also suggest pooling of all three photoreceptor types. Color information, i.e., comparison between different photoreceptors, does not appear to be used in honeybee phototaxis (Yamaguchi, 2010 and references therein).

    A simple summation model thus can no longer explain the results of all of the genetic manipulations of photoreceptor types in Drosophila presented in this study. When all four photoreceptor types are intact, UV light is more attractive than would be expected from the sum of the UV attractiveness values of the two isolated major subsystems, R1-R6 and R7/R8. In the UV/B choice test that is balanced for wild type, both flies with only the R1-R6 subsystem (sev rh52 rh61) and flies with only the R7/R8 subsystem (rh1 > shits or ninaE17) show a blue preference. It is thus necessary to postulate that an interaction between the two major retinal subsystems R1-R6 and R7/R8 enhances UV attractiveness. The interaction cannot be explained by an attenuation of the attractiveness of blue because no increased blue preference of the two mutants is observed in the B/G choice. The interaction can unambiguously be traced to the R7 cells. In fact, the strength of the contribution of R7 to the attractiveness function in the UV/B choice depends upon which of the other photoreceptor types are functional. No effect of R7 on spectral preference can be detected without a functional R1-R6 subsystem (comparing ninaE17 and sev ninaE17 in both choice tests. A moderate effect is found in the presence of R1-R6 and R8 (comparing wild type and sev; whereas the effect of R7 is large in the absence of the R8 subsystem (comparing rh52 rh61 and sev rh52 rh61 in UV/B choice (Yamaguchi, 2010).

    The most parsimonious explanation of the data is to assume that, in the presence of a functional R8 subsystem, neither the R1-R6 nor the R7 subsystems on their own have a direct input to the attractiveness function. In the absence of R7 (mutant sev), the R1-R6 subsystem seems not to matter for attractiveness although the R8 subsystem is lacking spectral sensitivity in the UV. Likewise, in the absence of a functional R1-R6 subsystem, the R7 subsystem seems not to contribute. Interestingly, as soon as R8y photoreceptors (or R8p and R8y) are inactivated, the other two subsystems exert their influence on the attractiveness function independently of each other. A full model of these interactions would require a more complete investigation (Yamaguchi, 2010).

    R1-R6 rhabdomeres degenerate in ninaE17 mutants, and this degeneration may have secondary effects on the R7/R8 cells. However, there is no significant difference between ninaE17 mutants and flies expressing rh1>shits, in which R1-R6 function is disrupted without affecting rhabdomere structures. Moreover, it was recently reported that R8y are functional in ninaE17 mutants because circadian entrainment to red light, which is still observed in ninaE17, is abolished in ninaE17 rh61 double mutants (Yamaguchi, 2010).

    The data reveal a further deviation from a simple summation of photoreceptor inputs to the attractiveness function. In the meltGOF mutant, green-sensitive R8y cells are transformed into blue-sensitive R8p cells. This should shift their preference in the UV/B choice to blue. Yet, a UV shift is observed. In contrast, in the absence of R7 cells, the transformation of R8y to R8p photoreceptors (comparison between sev and sev meltGOF) increases the blue preference in the UV/B choice as would be expected from spectral sensitivities. This might indicate that R8p cells enhance the input of the UV channel (R1-R6 × R7) to the attractiveness function. These findings, however, should be treated with caution as unknown developmental defects in the sev and meltGOF mutants might account for the phenotypes (Yamaguchi, 2010).

    Using a different phototaxis paradigm, Jacob (1977) proposed a model to explain the nonadditive effects observed in his phototaxis experiments. An inhibition of R1-R6 by R7/R8 was postulated and it was suggested that R7 cells function only when the R1-R6 system is intact. The current results ask for revision of this model. No general inhibition of the R1-R6 receptor subsystem by R7/R8 was found. The data suggest that neither the R1-R6 nor the R7 subsystems have access to the attractiveness function on their own, except in the absence of functional R8y photoreceptors. Moreover, R8p cells in the current tests had an effect only in the presence of functional R8y cells (Yamaguchi, 2010).

    Rh3-expressing R8 cells in the dorsal rim area account for about 10% of all Rh3 expressing cells. These R8 are still present in sev flies, but are switched off in panR7> shits flies. The comparison of these two lines does not reveal an effect of the Rh3-expressing R8 cells in the UV/B choice. Taking these cells into consideration therefore does not change any of the above conclusions (Yamaguchi, 2010).

    This study has shown that all four photoreceptor types [R1-R6, R7(p, y), R8p, and R8y] are involved in phototaxis, in contrast to motion detection, which relies exclusively on R1-R6 photoreceptors. The wild-type attractiveness function cannot be described as the sum of the attractiveness functions of flies lacking one or more functional photoreceptor types. A multiplicative interaction is observed between photoreceptors R1-R6 and R7. In the absence of functional R1-R6 or R7, the attractiveness function is governed by R8. Only in the absence of R8 and one of the other two subsystems does the remaining subsystem govern the attractiveness function. A recent study on the neuronal substrate of spectral preference identified postsynaptic interneurons in the medulla (Gao, 2008) that are good candidates for mediating some of these interactions. Their involvement in differential phototaxis can now be tested (Yamaguchi, 2010).

    The attractiveness function of differential phototaxis is easy to record, robust, and sensitive (for example, Rh5-expressing blue-sensitive cells account for only ~4% of all photoreceptors, yet yield a significant phenotype when they are not functional). Differential phototaxis may lend itself to mutant screens affecting other chromaticity computations in the brain, including color vision, at the circuit level (Yamaguchi, 2010).

    Color discrimination with broadband photoreceptors

    Color vision is commonly assumed to rely on photoreceptors tuned to narrow spectral ranges. In the ommatidium of Drosophila, the four types of so-called inner photoreceptors express different narrow-band opsins. In contrast, the outer photoreceptors have a broadband spectral sensitivity and were thought to exclusively mediate achromatic vision. Using computational models and behavioral experiments, this study demonstrated that the broadband outer photoreceptors contribute to color vision in Drosophila. The model of opponent processing that includes the opsin of the outer photoreceptors scored the best fit to wavelength discrimination data. To experimentally uncover the contribution of individual photoreceptor types, phototransduction of targeted photoreceptor combinations were restored in a blind mutant. Dichromatic flies with only broadband photoreceptors and one additional receptor type can discriminate different colors, indicating the existence of a specific output comparison of the outer and inner photoreceptors. Furthermore, blocking interneurons postsynaptic to the outer photoreceptors specifically impaired color but not intensity discrimination. These findings show that receptors with a complex and broad spectral sensitivity can contribute to color vision and reveal that chromatic and achromatic circuits in the fly share common photoreceptors (Schnaitmann, 2013).

    Combining modeling with genetic manipulations and behavioral experiments, this study identified the photoreceptor types for blue/green discrimination in Drosophila. Functional color discrimination with the opsin pairs Rh1-Rh4 and Rh4-Rh6 indicates that postreceptoral computations underlying color vision may occur within an optic cartridge deriving from a single ommatidium. Neuronal comparison of differential receptor outputs may be through color opponent mechanisms. TM5 cells in the medulla neuropil are a candidate for color opponent cells comparing Rh1 and Rh4 signals, since they integrate the outputs of lamina monopolar cells (especially L3) and R7. Alternatively, the postreceptoral comparisons might take place further downstream in the optic neuropils. Future physiological studies will be necessary to further elucidate this neuronal computation (Schnaitmann, 2013).

    The findings of this study redress the longstanding assumption that solely narrow-band inner photoreceptors mediate color vision. The sensitivity of Rh1 covers a wide spectral range, but it is not uniform. While this spectral sensitivity is not optimal to represent colors, it nevertheless provides information about differences in wavelength composition. This is in line with the rescued color discrimination with the dichromatic opsin pair Rh1-Rh4. Considering the sufficiency of inner photoreceptors for blue/green discrimination, the role of the outer photoreceptors may be to create an additional opponency dimension for enhanced color discrimination in specific wavelength regions (Schnaitmann, 2013).

    The outer photoreceptors have predominant functions in achromatic vision, such as motion detection. Exploitation of the outer photoreceptor pathway for multiple visual functions is advantageous for animals with limited neuronal resources. A recently discovered contribution of Drosophila R7/R8 to motion detection corroborates the current findings of a differential use strategy. Downstream mechanisms for decoding converged color and motion information await future studies (Schnaitmann, 2013).

    Genetic dissection reveals two separate retinal substrates for polarization vision in Drosophila

    Linearly polarized light originates from atmospheric scattering or surface reflections and is perceived by insects, spiders, cephalopods, crustaceans, and some vertebrates. Thus, the neural basis underlying how this fundamental quality of light is detected is of broad interest. Morphologically unique, polarization-sensitive ommatidia exist in the dorsal periphery of many insect retinas, forming the dorsal rim area (DRA). However, much less is known about the retinal substrates of behavioral responses to polarized reflections. Drosophila exhibits polarotactic behavior, spontaneously aligning with the e-vector of linearly polarized light, when stimuli are presented either dorsally or ventrally. By combining behavioral experiments with genetic dissection and ultrastructural analyses, it was shown that distinct photoreceptors mediate the two behaviors: inner photoreceptors R7+R8 of DRA ommatidia are necessary and sufficient for dorsal polarotaxis, whereas ventral responses are mediated by combinations of outer and inner photoreceptors, both of which manifest previously unknown features that render them polarization sensitive. It is concluded that Drosophila uses separate retinal pathways for the detection of linearly polarized light emanating from the sky or from shiny surfaces. This work establishes a behavioral paradigm that will enable genetic dissection of the circuits underlying polarization vision (Warnet, 2012).

    This study defines the retinal substrates for both dorsal and ventral polarization vision in Drosophila. The DRA is necessary and sufficient for dorsal polarotactic responses, a result that strengthens studies in other insects, concluding that this region mediates responses to celestial polarized light. In addition, this work defines the retinal substrate for responses to ventral polarotactic stimuli, as would occur naturally by reflections from shiny surfaces like water or leaves. This work resolves the differences between previous behavioral studies of polarotactic behavior in flies by demonstrating that flies possess separate detectors to respond to distinct wavelengths, and sources, of polarized light (Warnet, 2012).

    A ventral POL region has previously been described in the backswimmer Notonecta, which uses polarized reflections to locate water bodies. In this insect, inner photoreceptors in a small ventral region form orthogonal analyzer pairs with untwisted rhabdomeres much like a DRA. Drosophila uses a different strategy by exploiting the fact that photoreceptors with moderate or weak twist still provide enough PS to serve as polarization analyzers. In this way, other visual senses, such as the detection of motion and spectral cues, should be affected only minimally by the polarization of light Hence, unlike Notonecta with its specialized ventral retina, the generalist Drosophila incorporates ventral POL detectors while preserving other critical visual capacities (Warnet, 2012).

    An interesting feature of this design is that different classes of photoreceptors form ventral POL analyzers depending on stimulus wavelength. Two subtypes are named 'pale' (p) and 'yellow' (y) and are randomly distributed across the retina. R7 cells each express one of two UV opsins rh3 (R7p), or rh4 (R7y), whereas the underlying R8 cells express either rh5 (R8p) or rh6 (R8y). Due to this chromatic heterogeneity, inner photoreceptors are thought to mediate color vision. In the UV range, R7p cells are necessary for normal polarotactic responses; correspondingly, ventral R7 cells are described with moderate to high estimated PS. However, sufficiency experiments also revealed the involvement of outer photoreceptors in polarization vision. Whereas R1-R3 appear to be weakly polarization sensitive, R4, R5, and possibly R6 show pronounced estimated PS due to reduced rhabdomeric twist. These results are consistent with intracellular recordings in Calliphora describing two classes of R1-R6, one of which retains some PS, even in the UV (Warnet, 2012).

    Whereas R4 to R6 with their pronounced PS provide a basis for ventral polarotaxis via the outer photoreceptors, the contribution of R8 is less clear. It was found that R8 rhabdomeres twist strongly and, thus, R8 cells are expected to have low PS. In contrast, behavioral tests demonstrate that R8 can rescue polarotaxis in norpA mutants. Consistent with this, rare cases were found of very short R8 rhabdomeres exhibiting small twist ranges and correspondingly high expected PS. However, we cannot exclude the possibility that the apparent behavioral contributions of R8 could reflect low-level expression of the driver lines in R1-R6 (Warnet, 2012).

    In larger flies, R7y and R8y as well as R1-R6 contain a UV-sensitizing pigment. Because this molecule is not covalently linked to the opsin protein, its function is independent of microvillar orientation, thereby diminishing PS in the UV range. In addition, these cells contain a C40 carotinoid, which both gives them their yellow appearance and induces anomalous dichroism, which further reduces PS. This may explain why R7p, but not R7y, can mediate ventral UV polarotaxis in Drosophila. The data describe an unexpected new role for 'pale' ommatidia outside the DRA (Warnet, 2012).

    Moreover, the behavioral data confirm that in R1-R6 the UV-sensitizing pigment does not completely eliminate PS in the UV, as was previously reported. The contributions of the outer photoreceptors therefore become more pronounced when polarized green light is presented. That cellular contributions to ventral POL vision differ as a function of wavelength is particularly interesting because reflections from leaves contain much less UV (and more green light) than reflections from water. Hence, activation of distinct combinations of photoreceptors might convey specific meanings to the fly (Warnet, 2012).

    The combination of polarization-sensitive outer and inner photoreceptors represents a new analyzer design, differing from those described in the DRA and the ventral retina of Notonecta. In particular, morphological data does not reveal an orthogonal organization of ventral analyzers. However, comparison between these channels might still increase quality and robustness of the signal. Nothing is known about the subtype-specific connectivity of R7p/R8p and their postsynaptic partners, and no electrophysiological data on polarization-opponent interneurons, or 'compass neurons', exist in flies. By establishing Drosophila as a model of polarization vision, these studies will enable genetic screens using quantitative behavioral assays to allow a complete dissection of the neural circuits involved in responding to this fundamental quality of light (Warnet, 2012).

    Binary cell fate decisions and fate transformation in the Drosophila larval eye

    The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs) make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5) or the green-sensitive Rhodopsin 6 (Rh6). Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. This study shows that interplay of two transcription factors Senseless (Sens) and homeodomain transcription factor Hazy [PvuII-PstI homology 13, Pph13] control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. These findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner (Mishra, 2013).

    In the larval eye, determination of primary or secondary precursors to acquire either Rh5-PR or Rh6-PR identity depends on the transcription factors Sal, Svp and Otd. Primary as well as secondary precursors have the developmental potential to express Rh5 or Rh6. During differentiation, a pulsed expression of Sens acts as a trigger to initiate a distinct developmental program: Sens acts genetically in a feedforward loop to inhibit the Rh6-PR cell-fate determinant Svp and to promote the Rh5-PR cell-fate determinant Sal. Similarly, in the adult retina, differentiation of 'inner' PRs R7 and R8 requires sens and sal. Sal is necessary for Sens expression in R8-PRs and misexpression of Sal is sufficient to induce Sens expression in the 'outer' PRs R1-R6 (Mishra, 2013).

    Svp is exclusively expressed in R3/R4 and R1/R6 pairs of the outer PRs in early retina development. Initially, Sal is expressed in the R3/R4 PRs in order to promote Svp expression. Later, Svp represses Sal in R3/R4 PRs in order to prevent the transformation of R3/R4 into R7. Similarly in larval PRs Svp is repressing Sal in secondary precursors (Mishra, 2013).

    Intriguingly, in R8 development in the adult retina Sens also provides two temporally separable functions: First, during R8 specification, lack of Sens in precursors results in a transformation of the cell into R2/R5 fate; second, during differentiation, Sens counteracts Pros to inhibit R7 cell fate and promotes R8 cell fate. Thus, Sens is an early genetic switch in R8-PRs and larval Rh5-PRs that represses an alternate cell fate (Mishra, 2013).

    The lack of Sens results in a larval eye composed of only Rh6-PRs. Thus, the default state for both primary and secondary precursors is to differentiate into Rh6-expressing PRs. Rh6 is also the default state in adult R8 PRs: In the absence of R7 PRs (e.g. sevenless mutants) that send a signal to a subset of underlying R8 PRs, the majority of R8 PRs express Rh6. Thus, the genetic pathway initiated by the Sens pulse ensures that primary precursors choose a distinct developmental pathway by repressing the Rh6 ground state. The mechanisms that initiate and control this pulse of Sens remain to be discovered (Mishra, 2013).

    In larval PRs as well as in the formation of sensory organ precursors (SOP) in the wing, Sens functions as a binary switch between two alternative cell fates. In the larval eye, this switch occurs when Sens is expressed in one cell type and not in the other. However, during wing disc development the cell fate choice in SOP formation is controlled by the levels, and not the presence or absence of Sens expression: high levels of Sens act synergistically with proneural genes to promote a neuronal fate, while in neighboring cells, low levels of Sens repress proneural gene expression, thereby promoting a non-SOP fate. Thus, Sens uses distinct molecular mechanisms to act as a switch between Rh5 versus Rh6-PR cell fate and SOP versus non-SOP cell fate (Mishra, 2013).

    Transcription factors regulate developmental programs in a context- dependent fashion. An example is Sens, which has distinct functions in BO and eyelet development. First, during embryonic development, Sens acts as a key cell fate determinant by regulating transcription factors controlling PR-subtype specification. Second, during metamorphosis Sens inhibits ecdysone-induced apoptotic cell death. Third, in the adult eyelet Sens promotes Rh6 expression. Interestingly, the pro-survival function of Sens appears to be a conserved feature of Sens in other tissues and also in other animal species. In the salivary gland of Drosophila, Sens acts also as a survival factor of the salivary gland cells under the control of the bHLH transcription factor Sage. pag-3, a C.elegans homolog of Sens is involved in touch neuron gene expression and coordinated movement (Jia, 1996; Jia, 1997). Pag-3 was shown to act as a cell-survival factor in the ventral nerve cord and involved in the neuroblast cell fate and may affect neuronal differentiation of certain interneurons and motorneurons. In mice, Gfi1 is expressed in many neuronal precursors and differentiating neurons during embryonic development and is required for proper differentiation and maintenance of inner ear hair cells. Gfi1 mutant mice lose all cochlear hair cells through apoptosis, suggesting that its loss causes programmed cell death (Wallis, 2003). Taken together, these findings support that Sens and its orthologs function in cell fate determination and cell differentiation both in nervous system formation, but also play an essential role in the suppression of apoptosis (Mishra, 2013).

    Hazy plays distinct roles in larval PRs and during metamorphosis. First, Hazy is essential during embryogenesis for proper PR differentiation. This early function of Hazy is essential for PRs to differentiate properly during embryogenesis, to express Rhodopsins and to subsequently maintain Rhodopsin expression during larval stages. This function of Hazy is similar to its role in rhabdomere formation in adult PRs and subsequent promotion of Rh6 expression, although it is not required for Rh5 in the adult retina. It is likely that Hazy exerts this function by binding to the RCSI site of the rhodopsin promoters, as has been suggested for the adult retin. Second, during metamorphosis Hazy is required in Rh6-PRs to repress sens, thus allowing these cells to undergo apoptosis. This highlights the reuse of a small number of TFs for distinct functions in the same cell type at distinct time points of PR development. How these temporally distinct developmental programs are controlled on a molecular level remains unresolved. It seems likely that the competence of the cell to respond to a specific transcription factor changes during development (Mishra, 2013).

    rh5 and rh6 are expressed in different PRs at different developmental stages: rh5 is expressed in the larval eye and in the adult retina, whereas rh6 is expressed in the larval eye, the adult eyelet and the adult retina. However, the gene regulatory networks controlling rhodopsin expression are distinct in these organs. In the adult retina, a bi-stable feedback loop of the growth regulator melted and the tumor suppressor warts acts to specify Rh5 versus Rh6 cell fate, respectively, while in the larva, Sens, Sal, Svp and Otd control Rh5 versus Rh6 identity whereas Hazy has been shown to maintain Rhodopsin expression. A third genetic program acts downstream of EcR during metamorphosis in Rh5-PRs to switch to Rh6, which requires Sens (Mishra, 2013).

    An intriguing question is how the developmental pathways to specify Rh5- or Rh6-cell fates converge on the regulatory sequences of these two genes. It seems likely that parts of the regulatory machinery acting on the rh5 and rh6 promoters are shared between the larval eye, adult retina and eyelet, especially as short minimal promoters are functional in all three different contexts. Future experiments will show how the activity of the identified trans-acting factors is integrated on these promoters to yield context-specific outcomes (Mishra, 2013).

    The spatiotemporal order of signaling events unveils the logic of development signaling

    Animals from worms and insects to birds and mammals show distinct body plans; however, the embryonic development of diverse body plans with tissues and organs within is controlled by a surprisingly few signaling pathways. It is well recognized that combinatorial use of and dynamic interactions among signaling pathways follow specific logic to control complex and accurate developmental signaling and patterning, but it remains elusive what such logic is, or even, what it looks like. This study developed a computational model for Drosophila eye development with innovated methods to reveal how interactions among multiple pathways control the dynamically generated hexagonal array of R8 cells. Two novel findings were obtained. First, the coupling between the long-range inductive signals produced by the proneural Hedgehog signaling and the short-range restrictive signals produced by the antineural Notch and EGFR signaling is essential for generating accurately spaced R8s. Second, the spatiotemporal orders of key signaling events reveal a robust pattern of lateral inhibition conducted by Atonal-coordinated Notch and EGFR signaling to collectively determine R8 patterning. This pattern, stipulating the orders of signaling and comparable to the protocols of communication, may help decipher the well-appreciated but poorly defined logic of developmental signaling (Zhu, 2016).

    New regulators of Drosophila eye development identified from temporal transcriptome changes

    In the last larval instar, uncommitted progenitor cells in the Drosophila eye primordium start to adopt individual retinal cell fates, arrest their growth and proliferation, and initiate terminal differentiation into photoreceptor neurons and other retinal cell types. To explore the regulation of these processes, mRNA-Seq studies of the larval eye and antennal primordial were performed at multiple developmental stages. A total of 10,893 fly genes were expressed during these stages and could be adaptively clustered into gene groups, some of whose expression increases or decreases in parallel with the cessation of proliferation and onset of differentiation. Using in situ hybridization of a sample of 98 genes to verify spatial and temporal expression patterns, it is estimated that 534 genes or more are transcriptionally upregulated during retinal differentiation, and 1367 or more downregulated as progenitor cells differentiate. Each group of co-expressed genes is enriched for regulatory motifs recognized by co-expressed transcription factors, suggesting that they represent coherent transcriptional regulatory programs. Using available mutant strains, novel roles are described for the transcription factors SoxNeuro (SoxN), H6-like homeobox (Hmx), CG10253, without children (woc), Structure specific recognition protein (Ssrp), and multisex combs (mxc) (Quiquand, 2021).

    dachshund potentiates Hedgehog signaling during Drosophila retinogenesis

    Proper organ patterning depends on a tight coordination between cell proliferation and differentiation. The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signaling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. This study shows that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, dac was demonstrated to enhance Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis (Bras-Pereira, 2016).

    Wingless mediated apoptosis: How cone cells direct the death of peripheral ommatidia in the developing Drosophila eye

    Morphogen gradients play pervasive roles in development, and understanding how they are established and decoded is a major goal of contemporary developmental biology. This study examined how a Wingless (Wg) morphogen gradient patterns the peripheral specialization of the fly eye. The outermost specialization is the pigment rim; a thick band of pigment cells that circumscribes the eye and optically insulates the sides of the retina. It results from the coalescence of pigment cells that survive the death of the outermost row of developing ommatidia. This study investigated here how the Wg target genes expressed in the moribund ommatidia direct the intercellular signaling, the morphogenetic movements, and ultimately the ommatidial death. A salient feature of this process is the secondary expression of the Wg morphogen elicited in the ommatidia by the primary Wg signal. Neither the primary nor secondary sources of Wg alone are able to promote ommatidial death, but together they suffice to drive the apoptosis. This represents an unusual gradient read-out process in which a morphogen induces its own expression in its target cells to generate a concentration spike required to push the local cellular responses to the next threshold response (Kumar, 2015).

    This paper used the Drosophila eye as a model system with which to study how morphogen gradients can be converted into sharply constrained tissue patterns. The action of the Wg morphogen gradient was examined and it was asked how the highest threshold response, the death of the peripheral ommatidia, is orchestrated. Three observations argue that the secondary Wg expressed by the cone cells combines with the primary Wg from the head capsule to generate a sufficient concentration to kill the ommatidia. First, when the Wg pathway is activated in all cone cells (pros->arm* were arm* is an N-terminally truncated form of Armadillo, a constitutive, cell autonomous activator of the Wg transduction pathway) there is an extended zone of apoptosis in the region where the primary Wg source is known to be high. Second, when the secondary Wg (that secreted by the cone cells) is removed the extended band of ommatidial death is lost. Third, when a level of Wg equivalent to that normally found in the peripheral regions is supplied to pros-arm* eyes all ommatidia now die. Thus, this represents a novel gradient read-out mechanism in which the primary morphogen (Wg derived from the head capsule) elicits a secondary morphogen expression (Wg expressed by the cone cells) in the target cells. Thereafter, the two sources unite to generate the high local morphogen concentration needed to direct the appropriate cell behaviors at that position (Kumar, 2015).

    If there is a permissive zone in the periphery (∼3 ommatidial rows) in which the ommatidia will die if cone cell Wg expression occurs, then this raises the question of how the cone cells responses are normally tightly restricted to the peripheral-most row of ommatidia to ensure that only these ommatidia die. The following describes 1he mechanisms likely responsible for this restriction (Kumar, 2015).

    (1) The high threshold of the ommatidial response: It is surmised that the cone cells have a high threshold response to the morphogen, and the initial responses to the primary Wg source (diffusing from the head capsule) is restricted to the outermost ommatidia. However, it can be envisioned that the secondary Wg secreted by the outer cone cells could diffuse and elicit the same output in the next ommatidial row, and an extreme view could see a relay mechanism in which even more internal rows of ommatidia could express Wg in their cone cells (Kumar, 2015).

    (2) The role played by Notum:
    The expression of Notum is similar to Snail family transcription factors in that it is expressed in the cone cells and 2°/3° PCs of the outermost ommatidia, and since Notum functions to inhibit the free diffusion of Wg, it likely acts to prevent Wg diffusion into more interior ommatidia. Indeed previous studies have shown that in notum mutant clones the zone of death expanded out into more interior rows. Thus Notum (and other mechanisms for preventing Wg diffusion) is seen as playing a critical role in restricting the ommatidial death to the outermost row of ommatidia (Kumar, 2015).

    (3) Combining the high threshold response with the restriction of Wg diffusion: Consider the primary Wg diffusing from the head capsule. It enters the outer row of ommatidia and is of sufficient concentration to elicit the appropriate responses (the various expressions in the cone cells and 2°/3° PCs) but not at a level high enough to kill the ommatidia. The cone cells of the outermost row now begin to secrete the secondary Wg, but the concomitant expression of Notum by the cone cells and 2°/3° PCs of these ommatidia provide a barrier to the movement of both the primary and secondary sources of Wg. This restriction of Wg movement not only protects the more internal ommatidia, but ensures that the high levels of morphogen are constrained in the outermost ommatidia to provide the requisite signal for apoptosis (Kumar, 2015).

    In addition to uncovering the synergy between the Wg derived from the head capsule and the cone cells, a number of phenomena relating to the behavior of the various cell types have been detected (Kumar, 2015).

    (1) The early cone cell death: Following the collapse of the cone cells, the ommatidial apoptosis program begins with the death of cone cells themselves, followed ∼two hours later by the other ommatidial cells. This precocious cone cell death may represent a lower apoptosis threshold for these cells, but it is noted that they are sources of Wg secretion and likely experience autocrine and paracrine (between cone cells of the same ommatidium) Wg signaling as they collapse, and as such are more likely to reach the critical Wg activation level before the other cells (Kumar, 2015).

    (2) The cone cell immunity to death: In pros-arm* eyes, in which all cone cell nuclei fall to the photoreceptor layer and express Wg, there is a wide swath of extended death at the periphery in which all cells of the ommatidia die (including the cone cells). But upon prevention of the cone cell nuclear fall by the expression of esg RNAi, the cone cells survive while the photoreceptors in the extended peripheral zone still die. In these ommatidia, levels of Wg needed to drive apoptosis are achieved, but the cone cells appear invulnerable to it. Whether this invulnerability results from the absence of Snail family transcription factors needed to prime the cone cells for the death signal, or whether by remaining in the apical location they somehow avoid the full level of Wg exposure remains unclear (Kumar, 2015).

    (3) The fall of the cone cell nuclei: The maintenance of cone cell cell-bodies in the appropriate apical location is seemingly critical for the ommatidial stability and integrity, as their fall leads to the disruption of corneal lens units and delamination of photoreceptors. This fall appears to be directed by their expression of Snail family transcription factors. In pros-arm* eyes, the expression of esg.RNAi prevents the fall, and correspondingly the ectopic expression of esg in otherwise wild type cone cells engenders their nuclear fall (albeit prematurely). It was asked whether the fall of the cone cell nuclei resulted from a wholesale collapse of the apical junctions of the cone cells, but D/E-cadherin staining showed a normal apical junction pattern many hours after the nuclei had migrated basally. Thus it does not appear that the cone cells nuclei move basally because the cells lose their apical attachments, rather it is inferred that expression of the Snail family transcription factors reprograms some other behaviors of the cone cells. Such a behavior could be a switch in cell-type affinity. If cone cells normally maintain an apical location by adhesive differences with the photoreceptors, and if these adhesive differences are switched, then cone cell plasma membranes will then preferentially move to the photoreceptor layer. Since the nucleus defines the site of maximum cell body profile with corresponding maximum membrane area, then the fall of the nuclei may simply result from the cone cells acquiring an adhesive affinity with the photoreceptors. Other mechanisms can also be envisaged, in which, for example, motor machinery of the cell is used to reposition the cone cell nuclei in the more basal location (Kumar, 2015).

    An appropriate Gal4 driver line is not available to activate gene expression selectively in the 1° PCs, and the mechanism of their death remains unresolved. In GMR.wg eyes, their death was observed coincident with the photoreceptors (following the apoptosis of the cone cells) and it is surmised that it is the high level of Wg derived from head capsule and the cone cells that directs their death. However, there are a number of indications from that offer clues to a more nuanced understanding of their behavior. Initially the nuclei of the 1° PCs flank the clustered photoreceptor nuclei in their more apical region, but when the cone cell nuclei fall, those of the 1° PCs are shunted more basally. This movement deeper into the photoreceptor layer may play a role in their death. A similar argument can be made from the analysis of * eyes in which 1° PCs are lost, but when Snail family transcription factors are removed from this background, the cone cell nuclei do not fall, and the 1° PCs do not die. Hence the 1° PCs behave in a similar manner as the cone cells; if their position is maintained they do not die even though ambient Wg concentrations are sufficient for their death. This may indicate a general principle; that cells need to be in the correct topological position to experience the death signal (Kumar, 2015).

    Furthermore, in * eyes, the cone cell nuclear fall is accompanied by the loss of the 1° PCs even though the cone cells themselves do not die. The removal of Wg expression from the pros-arm* cone cells rescues the 1° PCs indicating that their loss is normally triggered by the cone cell Wg expression, and it is suspected that the low-level apoptosis seen in the main body of pros-arm* eyes may represent the death of the 1° PCs. If this is the case, then this suggests that the 1° PCs have a lower threshold Wg response for their apoptosis than the cone cells and photoreceptors (Kumar, 2015).

    The death of the photoreceptors appears to simply require the additive of effects of the two Wg sources to trigger their death. But another feature has emerged from these studies – the idea that chronic exposure to sub-lethal levels of Wg triggers photoreceptor degeneration. Consider pros-arm*/esgRNAi eyes; here the photoreceptor death occurs only at the widened zone of peripheral apoptosis, but in the main body of the adult eyes ommatidia show degenerate rhabdomere-like tissue in the apical retinas. The presence of rhabdomere-like tissue suggests the differentiation and subsequent degeneration of the photoreceptors leaving them alive but in a runtish condition. Since this phenomenon is Wg dependent (it is absent when wgRNAi is additionally included) it is inferred that the persistent Wg expression from the cone cells chronically signals to the photoreceptors. Indeed, when GMR.wg/GMR.P35 eyes (in which the apoptosis mechanism is suppressed and the photoreceptors are therefore subject to chronic Wg exposure), were examined a similar degenerate phenotype occurred. This observation suggests another function for the removal of the outer-most row of ommatidia: if they were not removed, chronic exposure to high levels of Wg emanating from the head capsule would lead them to deteriorate into a runtish condition (Kumar, 2015).

    A striking feature of the peripheral patterning mechanism is the timing aspect. The peripheral ommatidia are exposed to head capsule-derived Wg from the time of their birth. And yet they only respond to this Wg signal at defined times. The first occurs shortly after pupation when ac/da transcription is repressed and hth expression is induced. This corresponds with the surge in ecdysone expression that occurs in the animals at this time. The second response is the death of ommatidia at 42 h APF and this mechanism is closely tied with the large peak of ecdysone expression that occurs in the second day of pupation. Thus, it is speculated that Wg provides the spatial signal for peripheral patterning, but that the hormone system of the fly provides the temporal cue that determines when the spatial information can be utilized (Kumar, 2015).

    It is concluded that the periphery of the fly eye is an excellent model system with which to study how morphogen gradients are decoded into discrete tissue types, and this study has delved into the mechanism that precisely restricts the spatial positioning of one of those tissue types. An intricate mechanism has been uncovered in which initial threshold responses lead to the local boosting of the morphogen signal while at the same time upregulating mechanisms to prevent the spread of the morphogen. Evidence is also provided to support the idea that appropriate spatial, temporal and topological context is required for the peripheral ommatidia to undergo developmental apoptosis (Kumar, 2015).

    Drosophila DDX3/Belle exerts its function outside of the Wnt/Wingless signaling pathway

    The helicases human DDX3 and Drosophila Belle (Bel) are part of a well-defined subfamily of the DEAD-box helicases. Individual subfamily-members perform a myriad of functions in nuclear and cytosolic RNA metabolism. It has also been reported that DDX3X is involved in cell signaling, including IFN-alpha and IFN-beta inducing pathways upon viral infection as well as in Wnt signaling. This study used a collection of EMS-induced bel alleles recovered from a Wingless (Wg) suppressor screen to analyze the role of the Drosophila homolog of DDX3 in Wg/Wnt signaling. These EMS alleles, as well as a P-element induced null allele and RNAi-mediated knock down of bel, all suppressed the phenotype of ectopic Wg signaling in the eye. However, they did not affect the expression of known Wg target genes like senseless, Distalless or wingful/Notum. Ectopic Wg signaling in eye imaginal discs induces apoptosis by increasing grim expression. Mutations in bel revert grim expression to wild-type levels. Together, these results indicate that Bel does not function as a core component in the Drosophila Wg pathway, and that mutations affecting its helicase function suppress the effects of ectopic Wg signaling downstream of the canonical pathway (Jenny, 2016).

    Polycomb group genes are required to maintain a binary fate choice in the Drosophila eye

    Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, R7 photoreceptor neuron development has been examined in the fly eye. R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp), as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens), as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). PcG genes were removed specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. This study shows that loss of the PcG genes Sce, Scm, or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. This fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, this study shows that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. It is concluded that cells can use PcG genes specifically to maintain a subset of their binary fate choices (Finley, 2015).

    Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, R7 photoreceptor neuron development has been examined in the fly eye . R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp) , as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens) , as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). PcG genes were removed specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. This study shows that loss of the PcG genes Sce , Scm , or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. This fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, this study shows that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. It is concluded that cells can use PcG genes specifically to maintain a subset of their binary fate choices (Finley, 2015).

    The GMR-FLP/MARCM system allowed allowed the removal of Sce and Scm function specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the limited number of binary fate choices that R1/R6/R7 precursors face. In order to adopt the R7 fate, these precursors must choose to: (1) become neurons in response to high RTK activity-they would otherwise become non-neuronal cells; (2) fail to express Svp in response to N activity-they would otherwise become R1/R6s; and (3) fail to express Sens in response to N activity-they would otherwise become R8s. Loss of Sce or Scm from R7s specifically was found to compromises maintenance of the last of these choices. By contrast, no evidence was found that PcG genes maintain either of the other two choices. Sce mutant R7s were examined throughout larval and pupal development and none were found none misexpressed Svp, nor were Sce or Scm mutant R7s that displayed other R1/R6 characteristics found, such as large rhabdomeres positioned at the periphery of the ommatidium or expression of the R1-R6-specific rhodopsin Rh1. While loss of the Abelson kinase was recently shown to cause R neurons to lose expression of the neuronal marker Elav and switch to a non-neuronal pigment cell fate, this study found that Sce and Scm mutant R1/R6s and R7s maintain expression of Elav and the photoreceptor-specific protein Chaoptin, indicating that their commitment to a neuronal fate is also independent of PcG gene function. It is concluded that R7s use Sce and Scm to maintain repression of one but not all alternative binary fate choices (Finley, 2015).

    The Sens-encoding region is bound by Pc in Drosophila embryos and by Sce in Drosophila larvae , suggesting that Sens is directly regulated by these proteins in at least some cell types. However, because of the technical difficulty in isolating sufficient quantities of chromatin specifically from R7 cells, it was not possible to determine whether PcG proteins bind the Sens locus in R7s. It remains possible, therefore, that Sce, Scm, and Pc maintain Sens repression indirectly in R7s-however, the evidence suggests that they do so independently of their canonical role in repressing Hox genes (Finley, 2015).

    Considerable differences were observed in the strengths of the R7 defects caused by loss of Sce, Scm, Pc, or Psc. One possibility is that these proteins do not contribute equally to PRC1's gene-silencing ability. Indeed, the fly genome contains a second Psc-related gene that plays a redundant role with Psc in some cells, possibly accounting for the lack of defect in Psc mutant R7s. Alternatively, the different wild-type PcG proteins may perdure to different degrees within the mutant R7 clones (the cells that divide to generate the mutant R1/R6/R7 precursors contain a wild-type copy of the mutant gene). Attempts were made, but it was not possible to measure the time course of Sce and Scm protein levels in Sce and Scm mutant R7s, respectively, to test their perdurance directly. However, this thought that perdurance is likely, as this study found that Gal80 perdures until early pupal development within GMR-FLP/MARCM-induced R7 clones (Finley, 2015).

    Sce and Scm were found to be required to maintain Sens repression in R7s generated either in the presence or absence of N activity. What might be regulating the deployment of Sce and Scm in these cells? One possibility is that Sce and Scm repress Sens in R1/R6/R7 precursors by default, since these cells never normally express Sens. However, it was found that neither Sce nor Scm is required to maintain the repression of Sens that is established by Svp. Alternatively, Sce and Scm may be deployed to repress Sens as part of a cell's initial commitment to the R7 fate. As mentioned above, wild-type Sce or Scm protein is likely to perdure in newly created homozygous Sce or Scm mutant R7s, respectively, leaving open the possibility that these genes are required not only for the maintenance but also for the establishment of the R7 fate. Previous work showed that the NF-YC subunit of the heterotrimeric transcription factor nuclear factor Y (NF-Y) is also required to maintain Sens repression in R7s. Like the PcG proteins, NF-YC is broadly expressed in all photoreceptor neurons and is not sufficient to cause R7s to adopt R8 fates, indicating that NF-YC is not responsible for the specific role of PcG proteins in R7s. However, the resemblance between the R7 defects caused by loss of Sce, Scm, and NF-YC suggests that NF-Y may participate in PRC1 function. In support of this possibility, loss of the NF-YA subunit from Caenorhabditis elegans also causes defects similar to those caused by loss of the PcG gene sop-2, including derepression of the Hox gene egl-5 (Finley, 2015).

    PcG proteins have been shown to silence many regulators of development in addition to their canonical Hox targets, suggesting that PcG proteins are likely to play broad roles in maintaining cell fate commitments. However, whether PcG proteins are used to maintain specific binary fate choices as cells differentiate is unclear. In fact, the opposite is true during stem cell differentiation, when the repression of terminal differentiation genes by PcG proteins must instead be relieved. This paper has have identified a role for PRC1-associated PcG proteins in maintaining a specific binary fate choice made during adoption of the R7 fate-a choice that does not involve Hox gene regulation or misregulation. The same PRC1-associated proteins are not required to maintain two other binary fate choices that R7s must make. It is concluded that PcG genes are indeed used to maintain some though not all binary fate choices (Finley, 2015).

    Single-base pair differences in a shared motif determine differential Rhodopsin expression

    The final identity and functional properties of a neuron are specified by terminal differentiation genes, which are controlled by specific motifs in compact regulatory regions. To determine how these sequences integrate inputs from transcription factors that specify cell types, this study compared the regulatory mechanism of Drosophila Rhodopsin genes that are expressed in subsets of photoreceptors to that of phototransduction genes that are expressed broadly, in all photoreceptors. Both sets of genes share an 11-base pair (bp) activator motif. Broadly expressed genes contain a palindromic version that mediates expression in all photoreceptors. In contrast, each Rhodopsin exhibits characteristic single-bp substitutions that break the symmetry of the palindrome and generate activator or repressor motifs critical for restricting expression to photoreceptor subsets. Sensory neuron subtypes can therefore evolve through single-bp changes in short regulatory motifs, allowing the discrimination of a wide spectrum of stimuli (Rister, 2015).

    In the visual system, different photoreceptor neurons express specific light-sensing pigments; however, common downstream factors amplify and convert the response to the visual stimulus into a neuronal signal. For instance, each unit eye (ommatidium) of the Drosophila retina contains eight photoreceptors (R1 to R8) that express different light-sensing Rhodopsins (Rhs) that are restricted to specific photoreceptor subsets. Outer photoreceptors R1 to R6 express Rh1. Inner photoreceptors R7 and R8 express either Rh3 in pR7s coupled with Rh5 in pR8s, or Rh4 in yR7s with Rh6 in yR8s. R1 to R8 all share broadly expressed phototransduction factors that amplify and convert the response to the visual stimulus into a neuronal signal (Rister, 2015).

    This study examined the cis-regulatory mechanisms that distinguish restricted from broad expression patterns for Rhodopsins and downstream phototransduction factors, respectively. All Rhs share the conserved Rhodopsin Core Sequence I (RCSI), which resembles the palindromic P3 motif (TAATYNRATTA), an optimal binding site for paired-class homeodomain proteins. Almost all known broadly expressed phototransduction genes contain a P3 motif in their proximal promoter. The presence of a conserved P3/RCSI motif within 100 base pairs (bps) of the Rh transcription start site (TSS) is significantly associated with enrichment in adult eyes. P3/RCSI is required for activation in photoreceptors because its mutation caused either a loss or a strong reduction in expression of 16 broad or restricted reporters, with the exception of Arr1. Moreover, expression of 10 out of 15 reporters was lost in mutants for the photoreceptor-specific transcription factor Pph13, a paired-class homeodomain protein that binds P3 and the Rh6 RCSI in vitro (Rister, 2015).

    Because each Rh promoter has a highly conserved RCSI variant, the sufficiency of P3 and RCSI were tested to determine the significance of the specific differences between perfectly palindromic (P3) and imperfect motifs (RCSI). Four copies of the P3 motif (including four neighboring bps for spacing; the contribution of these additional bps was only tested for Rh4) from the broadly expressed ninaC, rdgA, or trpl drove broad expression in all photoreceptors, consistent with previous results. In sharp contrast, multimerized RCSI motifs drove expression in subsets of photoreceptors. The RCSI of Rh3 and Rh6 contains a K50 motif, a binding site for K50 homeodomain proteins such as the Dve repressor or the Otd activator. Expression of [Rh3 RCSI]4 and [Rh6 RCSI]4 was biased to inner photoreceptors: [Rh3 RCSI]4 mediated restricted expression in R8 and R7, with a strong bias toward the pR7 subset, where Rh3 is normally expressed. This pattern is complementary to the expression of Dve, which is indeed responsible for the restricted expression as [Rh3 RCSI]4 drove a broad, P3-like pattern in dve mutants. [Rh6 RCSI]4 drove restricted expression in R8s and R7s; expression in R1 to R6 was very weak in comparison to P3 motifs, which was due to dve-dependent repression (Rister, 2015).

    [Rh1 RCSI]4 drove variable expression in R1 to R6, where Rh1 is expressed. This outer photoreceptor-specific pattern is complementary to the inner photoreceptor expression of [Rh3 RCSI]4. Rh4 has the same RCSI as Rh1. However, adding the synergistic 3' RCSII motif led to expression in yR7s, where Rh4 is expressed. Although [Rh5 RCSI]4 was not sufficient for reporter expression, adding three K50 motifs to a single Rh5 RCSI ([K50]3 + [Rh5 RCSI]1) led to expression in R8 and pR7 photoreceptors (Rister, 2015).

    In summary, the RCSI motifs of specific Rhs differ from palindromic P3 motifs in broadly expressed genes: They drive expression that is biased toward the endogenous Rh expression patterns. It is shown below that full subtype specificity and activation often requires the repetition of motifs that are present in the RCSI (Rister, 2015).

    As specific RCSI motifs directed restricted expression in different photoreceptor subsets, albeit with incomplete subtype specificity and with some variability in expression levels, it was asked whether the single-bp differences are required for subtype specificity in a wild-type promoter context and which other motifs are required for full restriction. The K50 (Otd/Dve) motifs (TAATCC) were mutated to Q50 (Pph13) motifs (TAATTG/A) to disrupt repression while preserving RCSI-mediated activation. Mutating the Rh3 RCSI resulted in an expansion to yR7s, where Dve is present at low levels. Mutating the Rh6 RCSI caused derepression in R1 to R6 and the ocelli. Rh3 and Rh6 have K50/Dve repressor motifs repeated upstream, and mutation of individual motifs also caused derepression in yR7s and R1 to R6 and the ocelli, respectively. Taken together, single-bp changes create K50 motifs in the Rh3 and Rh6 RCSI, which are required for subtype-specific expression together with their upstream repeats (Rister, 2015).

    The importance of the disrupted P3 palindrome-i.e., the imperfect 3' homeodomain binding motif in the RCSIs of Rh1 toRh5 was examined. Creating a palindromic motif in theRh3 RCSI (TAATCCAATTC->TAATCCAATTA) caused derepression in yR7s that depended on Pph13. Therefore, derepression appears to be due to increased activation through the newly created Q50/Pph13 site. The same ATTC→ATTA mutation in the Rh5 RCSI led to partial derepression in yR8s. This single-bp change created abinding site for the activator Otd (AGATTA), and indeed derepression in yR8s was lost in otd mutants, as was activation in pR8s (Rister, 2015).

    The 3' ATTC motif in the RCSI of Rh3 and Rh5 is repeated upstream. Mutating the upstream repeat without creating a Q50/Pph13 site (ATTC->CAAA) also caused derepression in yR7s (Rh3) or yR8s (Rh5). Mutating both ATTCs of Rh5 enhanced derepression into almost all yR8s. Therefore, this study has identified repressor motifs in the RCSIs of four Rhs (K50/Dve motifs in Rh3/Rh6 and ATTC motifs in Rh3/Rh5). These motifs are repeated upstream within less than 100 bps and are required for full subtype specificity (Rister, 2015).

    A single-bp ATTT->ATTA mutation in the Rh4 RCSI caused derepression in R1 to R6, pR7s, and the ocelli. The correct pattern was restored by crossing the mutant Rh4 reporter in a Pph13 mutant background, indicating that the A -> T change prevents Pph13 from overcoming repression in the 'wrong' photoreceptor subsets, as was the case for Rh3 and Rh6. The same mutation in the Rh1 RCSI caused no detectable derepression. Replacing two bps in the RCSI of the ocelli-specific Rh2 to obtain a Q50/Pph13 site led to derepression in R1 to R6 photoreceptors that depended on Pph13 (Rister, 2015).

    In vivo data revealed that a cell-fate decision requires single-bp differences in RCSI motifs. They complement previous findings in cell culture that subtle sequence differences in a glucocorticoid receptor or nuclear factor κB (NF-κB) binding site can specify the mode of transcriptional regulatio and that small differences in binding-site sequences can lead to distinct Hox specificities in vivo and in vitro. (1) Single bps in RCSI prevent binding of dimers of broadly expressed activators such as Pph13, tipping the balance of activator/repressor binding. This weakened activation allows repressors to prevent activation in other photoreceptor subtypes. (2) They generate specific combinations of overlapping activator and repressor motifs, often repeated upstream to provide robust expression and full subtype specificity. Creating overlap of activator and repressor motifs is an efficient way of blocking a key activator site in the 'wrong' cell types that express a repressor, especially because the RCSI motifs are very close to the transcription start site and repression there could block other activators. The precise tuning of RCSI motifs within their respective promoter context leads to incompatibility in other Rh promoters, as revealed by RCSI swap experiments: Replacing a given RCSI with another one resulted in two main outcomes: loss of expression or derepression in specific subsets of photoreceptors (Rister, 2015).

    The RCSI/P3 motif resembles 'terminal selector' motifs that allow the coordinated expression of effector genes that define a particular neuron type. Yet, RCSI motifs exhibit additional layers of regulation that are integrated in a single regulatory element, as their sequence is modified for subtype specificity. Mutating a cis-regulatory motif in many cases appears to be the shortest evolutionary path toward a novel phenotype. Although it was found that it is possible in some cases to eliminate ectopic expression by removing the broadly expressed activator Pph13, this simultaneously causes a loss of expression of several broad phototransduction genes, defects in photoreceptor morphology, and a severe loss of light sensitivity (Rister, 2015).

    It is proposed that the modification of a P3-type motif into different RCSI-type motifs allowed partitioning Rh expression to different subtypes of photoreceptors. This opened the possibility to discriminate wavelengths and likely conveyed a selective advantage. In this model, P3 motifs represent a positive regulatory element shared by ancestral genes that were expressed in all photoreceptors. This regulation is conserved, as the promoter of the long-wavelength Rh, as well as Gβ76C that are both expressed in all photoreceptors in the beetle Tribolium, contain a palindromic P3-type motif and depend on Pph13 (Rister, 2015).

    Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila

    Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, this study examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096) genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, this study proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, alpha5, beta5 and beta6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In all, these findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development (Velentzas, 2013).

    A population of G2-arrested cells are selected as sensory organ precursors for the interommatidial bristles of the Drosophila eye

    Cell cycle progression and differentiation are highly coordinated during the development of multicellular organisms. The mechanisms by which these processes are coordinated and how their coordination contributes to normal development are not fully understood. This study determined the developmental fate of a population of precursor cells in the developing Drosophila melanogaster retina that arrest in G2 phase of the cell cycle and investigated whether cell cycle phase-specific arrest influences the fate of these cells. Retinal precursor cells that arrest in G2 during larval development were shown to be selected as sensory organ precursors (SOPs) during pupal development and undergo two cell divisions to generate the four-cell interommatidial mechanosensory bristles. While G2 arrest is not required for bristle development, preventing G2 arrest results in incorrect bristle positioning in the adult eye. It is concluded that G2-arrested cells provide a positional cue during development to ensure proper spacing of bristles in the eye. The results suggest that the control of cell cycle progression refines cell fate decisions and that the relationship between these two processes is not necessarily deterministic (Meserve, 2017).

    High throughput measurement of locomotor sensitization to volatilized cocaine in Drosophila melanogaster

    This study developed a new behavioral test, FlyBong, which combines delivery of volatilized cocaine (vCOC) to individually housed flies with objective quantification of their locomotor activity. There are two main advantages of FlyBong: it is high-throughput and it allows for comparisons of locomotor activity of individual flies before and after single or multiple exposures. At the population level, exposure to vCOC leads to transient and concentration-dependent increase in locomotor activity, representing sensitivity to an acute dose. A second exposure leads to further increase in locomotion, representing locomotor sensitization. FlyBong was validated by showing that locomotor sensitization at either the population or individual level is absent in the mutants for circadian genes period (per), Clock (Clk), and cycle (cyc). The locomotor sensitization that is present in timeless (tim) and pigment dispersing factor (pdf) mutant flies is in large part not cocaine specific, but derived from increased sensitivity to warm air. Circadian genes are not only integral part of the neural mechanism that is required for development of locomotor sensitization, but in addition, they modulate the intensity of locomotor sensitization as a function of the time of day. Motor-activating effects of cocaine are sexually dimorphic and require a functional dopaminergic transporter. FlyBong is a new and improved method for inducing and measuring locomotor sensitization to cocaine in individual Drosophila. Because of its high-throughput nature, FlyBong can be used in genetic screens or in selection experiments aimed at the unbiased identification of functional genes involved in acute or chronic effects of volatilized psychoactive substances (Filosevic, 2018).

    Role of the smallish gene during Drosophila eye development

    Smallish (Smash; CG43427), the Drosophila homologue of human LIM domain only 7 (LMO7), is a key regulator of Drosophila embryogenesis associated with planer cell polarity and actomyosin contractility at the zonula adherence. Although smash mRNA is expressed in several tissues during Drosophila development, only Smash function at the adherence junction in the embryonic epithelial cells has been reported. This study demonstrated that the knockdown of smash in eye imaginal discs induced morphological aberrations in adult compound eyes that were associated with increased apoptosis. Furthermore, immunohistochemical analyses revealed that Smash localized to the nucleus in several tissues, including eye imaginal discs. The knockdown of smash in eye imaginal discs down-regulated the expression of the otefin and bocksbeutel genes as well as the Drosophila homologue of the emerin gene, which is a target of LMO7. Collectively, these results indicate that Smash functions in proper Drosophila eye development mediated by the regulation of ote and bocks gene expression (Tanaka, 2018).

    Novel approach to quantitative spatial gene expression uncovers genetic stochasticity in the developing Drosophila eye

    Robustness in development allows for the accumulation of genetically based variation in expression. However, this variation is usually examined in response to large perturbations, and examination of this variation has been limited to being spatial, or quantitative, but because of technical restrictions not both. This study bridged these gaps by investigating replicated quantitative spatial gene expression using rigorous statistical models, in different genotypes, sexes, and species (Drosophila melanogaster and D. simulans). Using this type of quantitative approach with molecular developmental data allows for comparison among conditions, such as different genetic backgrounds. This approach was applied to the morphogenetic furrow, a wave of differentiation that patterns the developing eye disc. Within the morphogenetic furrow, focus was placed on four genes, hairy, atonal, hedgehog, and Delta. Hybridization chain reaction quantitatively measures spatial gene expression, co-staining for all four genes simultaneously. Considerable variation was found in the spatial expression pattern of these genes in the eye between species, genotypes, and sexes. It was also found that there has been evolution of the regulatory relationship between these genes and that their spatial interrelationships have evolved between species. This variation has no phenotypic effect, and could be buffered by network thresholds or compensation from other genes. Both of these mechanisms could potentially be contributing to long term developmental systems drift (Ali, 2019).

    Activating and repressing gene expression between chromosomes during stochastic fate specification

    DNA elements act across long genomic distances to regulate gene expression. During transvection in Drosophila, DNA elements on one allele of a gene act between chromosomes to regulate expression of the other allele. Little is known about the biological roles and developmental regulation of transvection. The stochastic expression of spineless (ss) in photoreceptors in the fly eye was investigated to understand transvection. It was determine a biological role for transvection in regulating expression of naturally occurring ss alleles. DNA elements were identified required for activating and repressing transvection. Different enhancers participate in transvection at different times during development to promote gene expression and specify cell fates. Bringing a silencer element on a heterologous chromosome into proximity with the ss locus "reconstitutes" the gene, leading to repression. These studies show that transvection regulates gene expression via distinct DNA elements at specific timepoints in development, with implications for genome organization and architecture (Urban, 2023).

    STRIPAK-PP2A regulates Hippo-Yorkie signaling to suppress retinal fate in the Drosophila eye disc peripodial epithelium

    The specification of organs, tissues and cell types results from cell fate restrictions enacted by nuclear transcription factors under the control of conserved signaling pathways. The progenitor epithelium of the Drosophila compound eye, the eye imaginal disc, is a premier model for the study of such processes. Early in development, apposing cells of the eye disc are established as either retinal progenitors or support cells of the peripodial epithelium (PE), in a process whose genetic and mechanistic determinants are poorly understood. This study identified Protein Phosphatase 2A (PP2A), and specifically a STRIPAK-PP2A complex that includes the scaffolding and substrate-specificity components Cka, Strip and SLMAP, as a critical player in the retina-PE fate choice. These factors suppress ectopic retina formation in the presumptive PE and do so via the Hippo signaling axis. STRIPAK-PP2A negatively regulates Hpo kinase, and consequently its substrate Wts, to release the transcriptional co-activator Yki into the nucleus. Thus, a modular higher-order PP2A complex refines the activity of this general phosphatase to act in a precise specification of cell fate (Neal, 2020).

    Tissue-specific Expression of Ribosomal Protein Paralogue eRpL22-like in Drosophila melanogaster Eye Development

    Differences in core or tissue-specific ribosomal protein (Rp) composition within ribosomes contribute to ribosome heterogeneity and functional variability. Yet, the degree to which ribosome heterogeneity modulates development is unknown. The Drosophila melanogaster eRpL22 family contains structurally diverse paralogues, eRpL22 and eRpL22-like. Unlike ubiquitously expressed eRpL22, eRpL22-like expression is tissue-specific, notably within the male germline and the eye. This study investigated expression within the developing eye to uncover tissue/cell types where specific paralogue roles might be defined. Immunohistochemistry analysis confirms ubiquitous eRpL22 expression throughout eye development. In larvae, eRpL22-like is ubiquitously expressed, but highly enriched in the peripodial epithelium (PE). In early pupae, eRpL22-like is broadly distributed in multiple cell types, but later, is primarily enriched in interommatidial hair cells (IoHC). Adult patterns include the ring of accessory cells around ommatidia. Adult retinae IoHC patterning phenotypes (shown by scanning electron microscopy) may be linked to RNAi-mediated eRpL22-like depletion within larval PE. Immunoblots and polysome profile analyses show multiple variants of eRpL22-like across development, with the variant at the expected molecular mass co-sedimenting with active ribosomes. These data reveal differential patterns of eRpL22-like expression relative to eRpL22 and suggest a specific role for eRpL22-like in developmental patterning of the eye (Gershman, 2020).

    Ultrastructural evidence for the origin of the subretinal pigment shield in the compound eye of Drosophila melanogaster

    Little morphological information is available about subretinal pigment shields in insect compound eyes, especially ultrastructural information. The latter is however needed in order to detect possible smallest projections that emanate from pigment-granule-bearing cells and pass through the basal matrix (BM), but that are not visible in light micrographs. Previous work on the subretinal pigment shield in Drosophila melanogaster suggests that the pigment cell population located below the BM is closely associated with secondary and tertiary pigment cells. Whether these cells stay in connection throughout life with the subretinal regions via thin projections that pass through the fenestrae of the BM, or whether the subretinal parts later become separated during eye development remained so far unknown. This investigation of the periphery of the BM by three-dimensional reconstruction based on serial-sectioning transmission electron microscopy has revealed that the secondary and tertiary pigment cells possess thin projections that pass through the fenestrae of the BM and thus connect the cellular regions above and below the BM in the adult compound eye. The subretinal pigment shield of D. melanogaster is therefore of retinal origin and is not composed of additional subretinal pigment cells. The maintained bond allows the active displacement of pigment granules below the BM during the process of dark and light adaptation of the compound eye (Mohr, 2020).

    Early lineage segregation of the retinal basal glia in the Drosophila eye disc

    The retinal basal glia (RBG) is a group of glia that migrates from the optic stalk into the third instar larval eye disc while the photoreceptor cells (PR) are differentiating. The RBGs are grouped into three major classes based on molecular and morphological characteristics: surface glia (SG), wrapping glia (WG) and carpet glia (CG). The SGs migrate and divide. The WGs are postmitotic and wraps PR axons. The CGs have giant nucleus and extensive membrane extension that each covers half of the eye disc. This study used lineage tracing methods to determine the lineage relationships among these glia subtypes and the temporal profile of the lineage decisions for RBG development. The CG lineage was found to segregate from the other RBG very early in the embryonic stage. It has been proposed that the SGs migrate under the CG membrane, which prevented SGs from contacting with the PR axons lying above the CG membrane. Upon passing the front of the CG membrane, which is slightly behind the morphogenetic furrow that marks the front of PR differentiation, the migrating SG contact the nascent PR axon, which in turn release FGF to induce SGs' differentiation into WG. Interestingly, it was found that SGs are equally distributed apical and basal to the CG membrane, so that the apical SGs are not prevented from contacting PR axons by CG membrane. Clonal analysis reveals that the apical and basal RBG are derived from distinct lineages determined before they enter the eye disc. Moreover, the basal SG lack the competence to respond to FGFR signaling, preventing its differentiation into WG. Thes findings suggest that this novel glia-to-glia differentiation is both dependent on early lineage decision and on a yet unidentified regulatory mechanism, which can provide spatiotemporal coordination of WG differentiation with the progressive differentiation of photoreceptor neurons (Tsao, 2020). r

    Quantitative Relationships Between Growth, Differentiation, and Shape That Control Drosophila Eye Development and Its Variation

    The size of organs is critical for their function and often a defining trait of a species. Still, how organs reach a species-specific size or how this size varies during evolution are problems not yet solved. This study investigated the conditions that ensure growth termination, variation of final size and the stability of the process for developmental systems that grow and differentiate simultaneously. Specifically, a theoretical model is presented for the development of the Drosophila eye, a system where a wave of differentiation sweeps across a growing primordium. This model, which describes the system in a simplified form, predicts universal relationships linking final eye size and developmental time to a single parameter which integrates genetically-controlled variables, the rates of cell proliferation and differentiation, with geometrical factors. The predictions of the theoretical model showed good agreement with previously published experimental results. A new computational model was developed that recapitulates the process more realistically and concordance was found between this model and theory as well, but only when the primordium is circular. However, when the primordium is elliptical both models show discrepancies. This difference is explained by the mechanical interactions between cells, an aspect that is not included in the theoretical model. Globally, this work defines the quantitative relationships between rates of growth and differentiation and organ primordium size that ensure growth termination (and, thereby, specify final eye size) and determine the duration of the process; identifies geometrical dependencies of both size and developmental time; and uncovers potential instabilities of the system which might constraint developmental strategies to evolve eyes of different size (Lobo-Cabrera, 2021).

    Geometry of gene regulatory dynamics

    Embryonic development leads to the reproducible and ordered appearance of complexity from egg to adult. The successive differentiation of different cell types that elaborate this complexity results from the activity of gene networks and was likened by Waddington to a flow through a landscape in which valleys represent alternative fates. Geometric methods allow the formal representation of such landscapes and codify the types of behaviors that result from systems of differential equations. Results from Smale and coworkers imply that systems encompassing gene network models can be represented as potential gradients with a Riemann metric, justifying the Waddington metaphor. This study extends the representation to include parameter dependence and enumerate all three-way cellular decisions realizable by tuning at most two parameters, which can be generalized to include spatial coordinates in a tissue. All diagrams of cell states vs. model parameters are thereby enumerated. The study unifies a number of standard models for spatial pattern formation by expressing them in potential form (i.e., as topographic elevation). Turing systems appear nonpotential, yet in suitable variables the dynamics are low dimensional and potential. A time-independent embedding recovers the original variables. Lateral inhibition is described by a saddle point with many unstable directions. A model for the patterning of the Drosophila eye appears as relaxation in a bistable potential. Geometric reasoning provides intuitive dynamic models for development that are well adapted to fit time-lapse data (Rand, 2021).

    Histone H3.3 K27M and K36M mutations de-repress transposable elements through perturbation of antagonistic chromatin marks
    Histone H3.3 lysine-to-methionine substitutions K27M and K36M impair the deposition of opposing chromatin marks, H3K27me3/me2 and H3K36me3/me2. This study shows that these mutations induce hypotrophic and disorganized eyes in Drosophila eye primordia. Restriction of H3K27me3 spread in H3.3K27M and its redistribution in H3.3K36M result in transcriptional deregulation of PRC2-targeted eye development and of piRNA biogenesis genes, including krimp. Notably, both mutants promote redistribution of H3K36me2 away from repetitive regions into active genes, which associate with retrotransposon derepression in eye discs. Aberrant expression of krimp represses LINE retrotransposons but does not contribute to the eye phenotype. Depletion of H3K36me2 methyltransferase ash1 in H3.3K27M, and of PRC2 component E(z) in H3.3K36M, restores the expression of eye developmental genes and normal eye growth, showing that redistribution of antagonistic marks contributes to K-to-M pathogenesis. These results implicate a novel function for H3K36me2 and showcase convergent downstream effects of oncohistones that target opposing epigenetic marks (Chaouch, 2021).

    The timing of cell fate decisions is crucial for initiating pattern formation in the Drosophila eye
    The eye-antennal disc of Drosophila is composed of three cell layers: a columnar epithelium called the disc proper (DP); an overlying sheet of squamous cells called the peripodial epithelium (PE); and a strip of cuboidal cells that joins the other two cellular sheets to each other and comprises the outer margin (M) of the disc. The M cells play an important role in patterning the eye because it is here that the Hedgehog (Hh), Decapentaplegic (Dpp) and JAK/STAT pathways function to initiate pattern formation. Dpp signaling is lost from the margin of eyes absent (eya) mutant discs and, as a result, the initiation of retinal patterning is blocked. Based on these observations, Eya has been proposed to control the initiation of the morphogenetic furrow via regulation of Dpp signaling within the M. This study shows that the failure in pattern formation surprisingly results from M cells prematurely adopting a head epidermis fate. This switch in fate normally takes place during pupal development after the eye has been patterned. These results suggest that the timing of cell fate decisions is essential for correct eye development (Weasner, 2022).

    The Blimp-1 transcription factor acts in non-neuronal cells to regulate terminal differentiation of the Drosophila eye

    The formation of a functional organ such as the eye requires specification of the correct cell types and their terminal differentiation into cells with the appropriate morphologies and functions. This study shows that the zinc-finger transcription factor Blimp-1 acts in secondary and tertiary pigment cells in the Drosophila retina to promote the formation of a bi-convex corneal lens with normal refractive power, and in cone cells to enable complete extension of the photoreceptor rhabdomeres. Blimp-1 expression depends on the hormone ecdysone, and loss of ecdysone signaling causes similar differentiation defects. Timely termination of Blimp-1 expression is also important, as its overexpression in the eye has deleterious effects. Transcriptomic analysis revealed that Blimp-1 regulates the expression of many structural and secreted proteins in the retina. Blimp-1 may function in part by repressing another transcription factor; Slow border cells is highly upregulated in the absence of Blimp-1, and its overexpression reproduces many of the effects of removing Blimp-1. This work provides insight into the transcriptional networks and cellular interactions that produce the structures necessary for visual function (Wang, 2022).

    The Blimp-1 transcription factor has been shown to play important regulatory roles in the development of many cell types and organs in both flies and mammals. The only reported function for Blimp-1 in the mouse retina is to stabilize the photoreceptor cell fate; however, this study found that Drosophila Blimp-1 acts at a later stage of retinal development to promote the terminal differentiation of several cell types. It has no visible function in photoreceptors, but acts in the cone cells to enable normal photoreceptor rhabdomere extension and in the higher order pigment cells to promote the formation of a biconvex corneal lens. Blimp-1 controls the terminal differentiation of these cells by regulating a battery of genes that encode likely structural components of the eye as well as Slbo and other transcription factors. Upregulation of Blimp-1 expression in the retina requires ecdysone signaling, illustrating a role for steroid hormones in regulating sensory organ morphogenesis (Wang, 2022).

    Although humans and Drosophila have shared common ancestors only in the very distant past and their lenses have distinct molecular compositions, both species evolved bi-convex lens shapes that enable them to focus light effectively onto the retina. The fly corneal lens cannot change its curvature through muscle contractions as the human lens does; to compensate for its fixed parameters, the fly retina is able to detect light throughout its depth. However, the thickness of the retina is insufficient to compensate for the massive reduction in the refractive power of the corneal lens resulting from the plano-convex shape caused by Blimp-1 loss-of-function, especially as loss of Blimp-1 also reduces retinal thickness. Loss of curvature on the external surface of Blimp-1 mutant corneal lenses is more detrimental to their refractive power than flattening of the internal surface facing the pseudocone would be, as the refractive indices of the corneal lens and pseudocone are similar to each other, but quite different from that of air (Wang, 2022).

    Although major corneal lens proteins such as Crystallin and Retinin are produced by the cone and primary pigment cells, Blimp-1 acts in the more peripheral secondary and tertiary pigment cells to generate the external curvature of the corneal lens. Blimp-1 has been shown to regulate chitin deposition during embryonic cuticle formation and tracheal maturation, and chitin is a major component of the corneal lens that may determine its shape. Blimp-1 also controls the expression of numerous cuticle proteins that could be secreted from higher-order pigment cells to give the periphery of the corneal lens a composition distinct from the center. Besides regulating the expression of direct structural components of the corneal lens, Blimp-1 could contribute to the bi-convex shape by regulating the forces that pigment cells exert on the periphery of the corneal lens through its effects on the expression of cell surface and cytoskeletal proteins (Wang, 2022).

    In addition to its effect on corneal lens shape, Blimp-1 loss-of-function causes a defect in photoreceptor morphology. Instead of extending throughout the depth of the retina, rhabdomeres are absent from the proximal retina, where the photoreceptors form stalk-like structures lacking rhabdomere markers. Photoreceptor nuclei are also displaced to the proximal ends of the rhabdomeres. During development, photoreceptors interact with cone cells as they undergo a 90 ° rotation of their apical membrane surface. Microvilli projecting from this apical membrane subsequently form rhabdomeres that extend proximally down the retina, growing to ~90 &mi;m in length. As cone cells are known to structurally and physiologically support photoreceptors during development, loss of Blimp-1 in these cells may affect their ability to contribute to photoreceptor development. Genes encoding cell surface, cytoskeletal and secreted proteins that this study found to be regulated by Blimp-1, including trol, could promote rhabdomere extension or attachment to an ECM produced by the cone cell feet (Wang, 2022).

    Loss of Blimp-1 leads to a dramatic upregulation of the transcription factor Slbo in non-neuronal retinal cells. Overexpressing slbo in the eye results in a plano-convex lens and shortened rhabdomeres, similar to what is observed in Blimp-1 mutants. slbo overexpression could thus contribute to the Blimp-1 loss-of-function phenotypes. However, removing slbo is not sufficient to restore normal retinal development in the absence of Blimp-1, indicating that at least one crucial target gene requires direct input from Blimp-1 in addition to the absence of Slbo. Two distinct mechanisms, one regulated by Slbo and one by loss of Blimp-1 independently of Slbo, can thus lead to this constellation of phenotypes (Wang, 2022).

    Most previous studies of slbo have focused on its requirement for normal border cell migration in the ovary and have shown that its overexpression is detrimental to most other tissues. As the eye is quite distinct from the ovary in its structure and morphogenesis, it is surprising that only a single transcription factor prevents slbo from being expressed there. It is more common to find a factor that is poised for expression when a single repressor is removed in tissues that share a common origin or developmental pathway. For example, knot is required for wing patterning but is repressed by Ultrabithorax in the haltere disc, which is a structure homologous to the wing disc. It is not known what activators drive slbo expression in the absence of repression by Blimp-1. In the ovary, slbo is activated by JAK/STAT signaling, which acts early in eye development to promote growth of the larval eye disc, but is not known to function in the pupal retina. The mouse slbo homologue Cebpa is activated by Blimp-1 in uterine tissues, suggesting possible conservation of the regulation of C/EBP genes by Blimp-1, but not of the direction of this effect (Wang, 2022).

    Given the presence of Blimp-1 at the periphery of the developing mouse cornea, these results raise the intriguing possibility that its role in regulating the curvature of refractive surfaces is conserved. Further investigation of this hypothesis may provide insight into the genesis of the refractive disorders that afflict a growing number of people (Wang, 2022).

    Blimp-1/PRDM1 and Hr3/RORbeta specify the blue-sensitive photoreceptor subtype in Drosophila by repressing the hippo pathway

    Bunker, J., Bashir, M., Bailey, S., Boodram, P., Perry, A., Delaney, R., Tsachaki, M., Sprecher, S. G., Nelson, E., Call, G. B. and Rister, J. (2023). Blimp-1/PRDM1 and Hr3/RORbeta specify the blue-sensitive photoreceptor subtype in Drosophila by repressing the hippo pathway. Front Cell Dev Biol 11: 1058961. PubMed ID: 36960411

    During terminal differentiation of the mammalian retina, transcription factors control binary cell fate decisions that generate functionally distinct subtypes of photoreceptor neurons. For instance, Otx2 and RORbeta activate the expression of the transcriptional repressor Blimp-1/PRDM1 that represses bipolar interneuron fate and promotes rod photoreceptor fate. Moreover, Otx2 and Crx promote expression of the nuclear receptor Nrl that promotes rod photoreceptor fate and represses cone photoreceptor fate. Mutations in these four transcription factors cause severe eye diseases such as retinitis pigmentosa. This study shows that a post-mitotic binary fate decision in Drosophila color photoreceptor subtype specification requires ecdysone signaling and involves orthologs of these transcription factors: Drosophila Blimp-1/PRDM1 and Hr3/RORbeta promote blue-sensitive (Rh5) photoreceptor fate and repress green-sensitive (Rh6) photoreceptor fate through the transcriptional repression of warts/LATS, the nexus of the phylogenetically conserved Hippo tumor suppressor pathway. Moreover, this study identified a novel interaction between Blimp-1 and warts, whereby Blimp-1 represses a warts intronic enhancer in blue-sensitive photoreceptors and thereby gives rise to specific expression of warts in green-sensitive photoreceptors. Together, these results reveal that conserved transcriptional regulators play key roles in terminal cell fate decisions in both the Drosophila and the mammalian retina, and the mechanistic insights further deepen understanding of how Hippo pathway signaling is repurposed to control photoreceptor fates for Drosophila color vision (Bunker, 2023).

    Motif 1 Binding Protein suppresses wingless to promote eye fate in Drosophila

    The phenomenon of RNA polymerase II (Pol II) pausing at transcription start site (TSS) is one of the key rate-limiting steps in regulating genome-wide gene expression. In Drosophila embryo, Pol II pausing is known to regulate the developmental control genes expression, however, the functional implication of Pol II pausing during later developmental time windows remains largely unknown. A highly conserved zinc finger transcription factor, Motif 1 Binding Protein (M1BP), is known to orchestrate promoter-proximal pausing. This study found a new role of M1BP in regulating Drosophila eye development. Downregulation of M1BP function suppresses eye fate resulting in a reduced eye or a "no-eye" phenotype. The eye suppression function of M1BP has no domain constraint in the developing eye. Downregulation of M1BP results in more than two-fold induction of wingless (wg) gene expression along with robust induction of Homothorax (Hth), a negative regulator of eye fate. The loss-of-eye phenotype of M1BP downregulation is dependent on Wg upregulation as downregulation of both M1BP and wg, by using wgRNAi, shows a significant rescue of a reduced eye or a "no-eye" phenotype, which is accompanied by normalizing of wg and hth expression levels in the eye imaginal disc. Ectopic induction of Wg is known to trigger developmental cell death. Upregulation of wg as a result of downregulation of M1BP also induces apoptotic cell death, which can be significantly restored by blocking caspase-mediated cell death. These data strongly imply that transcriptional regulation of wg by Pol II pausing factor M1BP may be one of the important regulatory mechanism(s) during Drosophila eye development (Raj, 2020).

    Pol II pausing near the transcription start site has been identified as a key step in optimizing transcription of many genes in metazoans. It has been proposed that pausing allows the coupling of transcription and RNA processing. Pausing can contribute to dynamic regulation of gene expression in response to developmental and environmental signals, and can function to repress transcription. The genome-wide studies have revealed that  ~10-40% of all genes in mammalian embryonic stem cells and Drosophila have paused promoters. In Drosophila, while the phenomenon of promoter proximal pausing has been well studied in regulation of genes encoding the heat shock proteins (Hsp) and different components involved in immune response pathways, it is also proposed to play important role in regulating the gene expression during early developmental events such as patterning, sex determination etc. So far, the sequence-specific transcription factors such as GAGA factor and M1BP, and other regulators HEXIM, LARP7 (La Ribonucleoprotein 7, Transcriptional Regulator) have been implicated in dictating Pol II pausing in Drosophila. However, the biological relevance of transcriptional pausing and the exact mechanism by which the regulatory factors may contribute in pausing of Pol II is not fully understood (Raj, 2020).

    M1BP regulates retinal determination and MF progression in developing eye This study tested fthe role of transcription pausing factor, M1BP during Drosophila eye development. Downregulation of M1BP levels in the developing eye was found to result in strong suppression of eye fate, however, gain-of-function of M1BP did not affect the eye fate suggesting that optimum levels of M1BP are required for Drosophila eye development. Furthermore, no domain constraint in eye suppression function was seen when M1BP levels were downregulated. In addition, when M1BP levels were downregulated (ey > M1BPRNAi) the expression of retinal determination and differentiation genes were strongly downregulated. Interestingly, it was found that protein encoded by RD genes were downregulated in ey > M1BPRNAi background. Therefore, M1BP may not be affecting RD gene expression directly (Raj, 2020).

    During eye development, a wave of differentiation, emanates from the posterior margin of the developing eye imaginal disc, which sweeps anteriorly across the retinal primordium. The crest of this wave is referred to as the MF, which results in retinal differentiation behind it. The two signals dpp and hh plays an important role in initiation and progression of MF. Downregulation of M1BP affects retinal differentiation as well as progression of MF. It suggests that M1BP role is to promote retinal differentiation as well as MF progression. Also, M1BP downregulates the level of negative regulator(s) of the eye fate. This study screened for the genes, which may serve as target for M1BP mediated transcriptional pausing mechanism in Drosophila eye imaginal disc (Raj, 2020).

    The protein encoded by Drosophila wg gene, a member of Wg/WNT signaling pathway, act short range inducer, which organizes the pattern of cells at a distance in the embryo. Since M1BP downregulation resulted in blocking retinal differentiation and MF progression, targets were sought of M1BP transcriptional pausing function using the candidate gene approach. It was found that wg-lacZ reporter, which serves as a transcriptional read out for Wg, exhibits robust induction in eye imaginal discs where M1BP levels were downregulated. This observation was further validated by qPCR approach which showed that there is a 2.2-fold increase in wg gene expression. Furthermore, in high throughput microarray screen carried out in S2R + cells, wg was also identified as a target whose expression is downregulated by M1BP using M1BP RNAi. According to microarray analysis, wg shows a 5.5-fold change when cells are treated with M1BPRNAi (Raj, 2020).

    To validate the results from qPCR approach as well induction of wg-lacZ reporter expression in ey > M1BPRNAi eye imaginal disc, this study also employed bioinformatics analysis to determine if there are M1BP binding sites in the wingless (wg) gene. The M1BP binding sequence (YGGTCACACTR) has been reported earlier. This sequence was used for MEME analysis to screen for M1BP binding sites were found in the wg gene and regulatory region (Raj, 2020).

    Wg, a ligand for evolutionarily conserved Wg/WNT signaling pathway, is known to act as a negative regulator of eye development. During Drosophila eye development, Wg activity promotes head specific fate by negatively regulating MF progression in the differentiating eye imaginal disc. Wg regulates expression of downstream gene hth, which encodes a MEIS class of transcription factor, and act as a negative regulator of eye development. This study found that in ey > M1BPRNAi background, robust induction of wg transcription also accompanies ectopic induction of hth along with the suppression of the eye fate. Further, downregulation of wg levels, using wgRNAi, in ey > M1BPRNAi background rescued the eye suppression phenotype. This data clearly suggested that M1BP downregulates levels of wg, which in turn regulate expression of hth in the developing Drosophila eye (Raj, 2020).

    Higher levels of Wg are known to trigger developmental cell death in the developing eye field. Interestingly, in ey > M1BPRNAi eye discs, the eye field was significantly reduced. Since, majority of the cell death is triggered by the activation of caspase-dependent cell death, blocking caspase-dependent cell death by ectopic expression of anti-apoptotic P35 transgene in ey > M1BPRNAi background showed rescue of eye suppression phenotype. However, these P35 mediated rescues of ey > M1BPRNAi were not as significant as seen with wgRNAi. This suggests that Wg might be regulating eye fate through hth induction and eye field size by triggering caspase mediated cell death. In order to rule out that these in ey > M1BPRNAi phenotypes are not affected by reduced cell proliferation rates, levels were tested of pH3 in these developing eye fields. It was found that cell proliferation rates were not affected by this transcriptional pausing mechanism in the developing eye (Raj, 2020).

    These results imply that the transcription pausing function of M1BP in regulating Wg signaling may play a critical role in Drosophila eye development. However, other factors and signaling pathways involved in regulating the M1BP function at the mechanistic level is yet to be determined. In order to further understand whether M1BP mediated transcriptional regulation is also implicated during development of other imaginal discs in Drosophila, the downregulation of M1BP function was studied in bi-Gal4 domains of wing imaginal disc. Whether this role of M1BP in regulating wg gene expression is exclusive to developing eye disc or it extends to other larval imaginal disc was tested. A bi-GAL4 driver which drives the expression of a transgene in wing imaginal disc was used. Downregulation of M1BP in bi-Gal4 expression domains of wing (bi > M1BPRNAi) exhibits ectopic upregulation wg expression in the pouch region of the wing imaginal disc. Furthermore, M1BP expression levels are downregulated in the wing pouch region, which corresponds to the bi-Gal4 expression domain. These results suggested that the transcription pausing function of M1BP may have similar target in the eye and wing imaginal disc. Recently, HEXIM, another transcriptional regulator associated with pol II pausing, has been reported to affect wing development in Drosophila by regulating Hh signaling. In Drosophila wing imaginal disc, HEXIM knockdown causes developmental defects by inducing ectopic expression of hh and its transcriptional effector cubitus interuptus (ci), which triggers apoptosis. This suggests that the regulatory factors involved in Pol II pausing are important in maintaining the expression levels of different signaling pathways during development in Drosophila (Raj, 2020).

    A number of highly conserved transcriptional pausing and elongation factors such as Spt5 precisely regulate transcription during Drosophila embryogenesis. The Spt5W049 missense mutation causes defects in the anterior-posterior patterning and segmental patterning during embryogenesis. Interestingly, the mutant allele of Spt5 (foggym806) in Zebrafish also causes multiple developmental defects such as discrete problems with pigmentation, tail outgrowth, ear formation and cardiac differentiation. These studies suggest that the regulatory mechanism in Pol II pausing during fly development are also conserved in higher organisms. The Drosophila compound eye shares similarities with the vertebrate eye at the level of genetic machinery as well as the processes of differentiation. Therefore, the information generated in Drosophila can be extrapolated to higher organisms. Since Wnt signaling is known to induce programmed cell death in patterning the vasculature of the vertebrate eye, it will be important to study what molecules other than M1BP can prevent Wg signaling from inducing cell death during early eye development (Raj, 2020).

    Tiling mechanisms of the Drosophila compound eye through geometrical tessellation

    Tiling patterns are observed in many biological structures. The compound eye is an interesting example of tiling and is often constructed by hexagonal arrays of ommatidia, the optical unit of the compound eye. Hexagonal tiling may be common due to mechanical restrictions such as structural robustness, minimal boundary length, and space-filling efficiency. However, some insects exhibit tetragonal facets. Some aquatic crustaceans, such as shrimp and lobsters, have evolved with tetragonal facets. Mantis shrimp is an insightful example as its compound eye has a tetragonal midband region sandwiched between hexagonal hemispheres. This casts doubt on the naive explanation that hexagonal tiles recur in nature because of their mechanical stability. Similarly, tetragonal tiling patterns are also observed in some Drosophila small-eye mutants, whereas the wild-type eyes are hexagonal, suggesting that the ommatidial tiling is not simply explained by such mechanical restrictions. If so, how are the hexagonal and tetragonal patterns controlled during development? This study demonstrates that geometrical tessellation determines the ommatidial tiling patterns. In small-eye mutants, the hexagonal pattern is transformed into a tetragonal pattern as the relative positions of neighboring ommatidia are stretched along the dorsal-ventral axis. It is proposed that the regular distribution of ommatidia and their uniform growth collectively play an essential role in the establishment of tetragonal and hexagonal tiling patterns in compound eyes (Hayashi, 2022).

    Probing the conserved roles of cut in the development and function of optically different insect compound eyes

    Astonishing functional diversity exists among arthropod eyes, yet eye development relies on deeply conserved genes. This phenomenon is best understood for early events, whereas fewer investigations have focused on the influence of later transcriptional regulators on diverse eye organizations and the contribution of critical support cells, such as Semper cells (SCs). As SCs in Drosophila melanogaster secrete the lens and function as glia, they are critical components of ommatidia. In this study RNAi-based knockdowns were performed of the transcription factor cut (CUX in vertebrates), a marker of SCs, the function of which has remained untested in these cell types. To probe for the conserved roles of cut, two optically different compound eyes were tested: the apposition optics of D. melanogaster and the superposition optics of the diving beetle Thermonectus marmoratus. In both cases, multiple aspects of ocular formation were disrupted, including lens facet organization and optics as well as photoreceptor morphogenesis. Together, these findings support the possibility of a generalized role for SCs in arthropod ommatidial form and function and introduces Cut as a central player in mediating this role (Rathore, 2023).

    The Notch pathway regulates the Second Mitotic Wave cell cycle independently of bHLH proteins

    Notch regulates both neurogenesis and cell cycle activity to coordinate precursor cell generation in the differentiating Drosophila eye. Mosaic analysis with mitotic clones mutant for Notch components was used to identify the pathway of Notch signaling that regulates the cell cycle in the Second Mitotic Wave. Although S phase entry depends on Notch signaling and on the transcription factor Su(H), the transcriptional co-activator Mam and the bHLH repressor genes of the E(spl)-Complex were not essential, although these are Su(H) coactivators and targets during the regulation of neurogenesis. The Second Mitotic Wave showed little dependence on ubiquitin ligases neuralized or mindbomb, and although the ligand Delta is required non-autonomously, partial cell cycle activity occurred in the absence of known Notch ligands. This study found that myc was not essential for the Second Mitotic Wave. The Second Mitotic Wave did not require the HLH protein Extra macrochaetae, and the bHLH protein Daughterless was required only cell-nonautonomously. Similar cell cycle phenotypes for Daughterless and Atonal were consistent with requirement for neuronal differentiation to stimulate Delta expression, affecting Notch activity in the Second Mitotic Wave indirectly. Therefore Notch signaling acts to regulate the Second Mitotic Wave without activating bHLH gene targets (Bhattacharya, 2017).

    Differences were observed between how Notch signaling regulates the SMW compared to how Notch regulates photoreceptor differentiation (see Model of the cell cycle and cell fate specification in the morphogenetic furrow). The Notch pathway suppresses the specification of photoreceptor cells in a manner similar to regulation of neurogenesis by Notch in many other tissues. That is, activation of the transmembrane ligand Delta by the ubiquitin ligase Neuralized leads allows Delta to activate Notch, leading to release of the Notch intracellular domain, which then acts in a nuclear complex with Su(H) and Mastermind to induce the transcription of the E(spl)-C family of transcriptional repressors and prevent neural fate specification and differentiation. By contrast to the pathway regulating neural differentiation, cell cycle entry in the SMW occurred in the absence of the E(spl)-C. This suggested that a distinct transcriptional target of Su(H) is involved, but unusually, this Su(H) function did not seem to require the co-activator Mam. Although most studies find that Mam is required for transcriptional activation by Su(H), mutations in mam often have weaker phenotypes than other neurogenic genes. It is possible that Mam protein might exhibit exceptionally strong perdurance, but there is also in vitro evidence for mam-independent transcriptional activation by Su(H) (Bhattacharya, 2017).

    SMW entry also occurred in the absence of neur. In the case of neurogenesis, cells lacking neur show a weaker neurogenic phenotype than cells lacking other components of the pathway, both in the embryonic CNS and in the eye, which is thought to reflect the activities of two other ubiquitin ligases, Mindbomb and Mindbomb2. No effect was found on the SMW of mutated mib1, but neither mib2 nor cells lacking two or more of these possibly redundant ubiquitin ligases were examined (Bhattacharya, 2017).

    Eoles of ligands were examined in more detail. It has been reported that SMW entry depended on Delta, which is also the ligand necessary for regulation of neurogenesis in the eye as in other parts of the nervous system, but although their figures show an obvious reduction in cell cycle entry in the clones of cells lacking Dl, some cell cycle entry still seems to occur. Those experiments were repeated using cells lacking both Dl and the other known ligand Ser, with similar results, ie, cell cycle entry was obviously disrupted in clones lacking both Notch ligands, nevertheless some cells still entered the cell cycle. A potential complicating factor is that reducing Notch function typically results in excessive neurogenesis, raising the question of whether some Dl Ser mutant cells were prevented from entering the cell cycle by differentiation as photoreceptors. The excess neurogenesis of such clones was partially suppressed by expression of Argos or Ras-DN. Although there is clearly a considerable disruption of the SMW in the absence of N ligands, there is also clear evidence of cell cycle entry, occurring at approximately the same time as the normal SMW (Bhattacharya, 2017).

    There are at least three possible explanations for these findings. First, it possible that another ligand besides Dl or Ser activates N in the SMW. Secondly, it has been reported that Dl can signal across several cell diameters using filopodia. During neurogenesis, rescue of Dl mutant clones appears to extend beyond cells immediately adjacent to wild type cells. Signaling across many cell diameters would be required to account for the cell cycle progression seen in Dl Ser mutant clones. Thirdly, Notch can be activated independently of ligands if delivered to certain cellular compartments. Recently it has been suggested that such ligand-independent activation is suppressed by cis-inactivation, in which ligands inhibit N signaling in the same cells, so that ligand-independent activation can be revealed in cells lacking Dl and Ser. Cis-inactivation is potentially an important contributor to the patterning of Notch-mediated lateral inhibition during neurogenesis, but direct evidence of such a role has not yet been obtained. If ligand-independent signaling is occurring in Dl Ser clones, some other pathway must be active near the SMW to explain why ligand-independent signaling cell cycle entry was limited to cells at the same stage posterior to the furrow as those that undergo the normal, ligand-dependent SMW (Bhattacharya, 2017).

    The cell cycle targets of Notch are of some interest. Many Drosophila cell cycles depend on regulated transcription of Cyclin E. Cyclin E is clearly required for the SMW suggested that Cyclin E might not be limiting for SMW cell cycle entry, however, because Cyclin E was seen to accumulate more in the differentiating photoreceptor precursors that do not enter the SMW. Alternatively, the lack of Cyclin E accumulation in SMW cells might reflect Cyclin E protein instability in S phase, with accumulation only in differentiating cells that can't enter the cell cycle. A Cyclin E transcriptional reporter appears to accumulate in the same cells as the Cyclin E protein, however, which is not indicative of post-translational regulation (Bhattacharya, 2017).

    It has been suggested that Cyclin A was part of the Notch dependent machinery promoting cell cycle entry in the SMW, although it was concluded that it could not be the only important Notch target. What was observed, however, was dependence of Cyclin A protein levels on Notch signaling that exactly paralleled the dependence of S-phase entry on Notch signaling in various genotypes. Like Cyclin B, Cyclin A protein is degraded by APC/Cyclosome activity during G1, so that Cyclin A protein cannot accumulate until the G1/S transition. Therefore these results are also expected if Cyclin A simply accumulates whenever the G1/S transition has occurred, and is not a direct target of Notch signaling (Bhattacharya, 2017).

    In mammalian cells, c-Myc appears to be an important Notch target. Indeed, human Notch was first identified as a proto-oncogene that causes leukemia through c-Myc. c-Myc is an activator of the nucleolar protein fibrillarin, which transiently increases during the SMW. Although Dmyc is important for growth in Drosophila, so that clones of myc null mutant cells in the eye disc are small, they nevertheless enter the SMW at the normal time, indicating that myc is unlikely to be the critical Notch target in the SMW (Bhattacharya, 2017).

    Emc is a transcriptional target of N signaling in the eye as in other tissues. Even though Emc protein levels seem to be less affected by N signaling than is emc RNA, it has been suggested that N regulates the eye cell cycle by upregulating emc, thereby inhibiting Da, which is proposed to inhibit cell cycle entry. Contrary to this model, previous studies have reported that Da is in fact required for the SMW. We investigated this potential role for emc directly, finding no evidence that emc was required for the SMW. These studies confirmed previous findings that da is required for S phase entry into the SMW, although not apparently for cell cycle activity anterior to the morphogenetic furrow (Bhattacharya, 2017).

    The cell-autonomy of da requirement in the cell cycle had not been determined previously. da mutant cells sometimes enter the SMW at the posterior boundaries of da clones, where they were adjacent to wild type cells. This suggested that da was required for the production of a short-range non-autonomous signal that is required for SMW entry, and could be provided to da mutant cells that were near to wild type cells (Bhattacharya, 2017).

    It is instructive to compare the effect of da on the cell cycle to that of ato. Ato is the heterodimer partner of Da that is required for R8 specification. Da is also required independently of ato to specify R2,R3,R4 and R5. Interestingly, this requirement in R2-5 seems to be incomplete, since some da mutant R2-5 cells do differentiate at reduced frequencies. Because of this requirement for da outside R8, R1-7 photoreceptor cells can differentiate near borders of ato mutant clones, but fewer photoreceptors are expected near the borders of da mutant clones, and mostly of the R1, R6 and R7 cell types. Proneural genes often promote Notch ligand expression, and accordingly ato is required for normal Dl expression during these stages of eye development. Reduced Dl expression is a plausible explanation for the absence of the SMW within ato clones, and the more extensive requirement for da in photoreceptor differentiation and Dl expression may explain why non-autonomous rescue of the SMW is more pronounced near the borders of ato clones than near the borders of da clones (Bhattacharya, 2017).

    A model is present for SMW. Unpatterned proliferation of the eye imaginal disc is terminated ahead of the morphogenetic furrow by Hh and Dpp signaling. Hh and Dpp affect many aspects of eye development including expression of master regulators of retinal determination such as Eyeless, Teashirt, Dachshund, and Sine Oculis, as well as Homothorax. Cells posterior to the furrow can still respond to growth, because extra cell cycles are driven in unspecified cells by overexpression of CycD/cdk4, Inr, or myc, as well as by Cyclin E. It is possible that Notch regulates cellular growth in the SMW, since there appears to be a small but discernible increase in nucleolar size at this stage, and progenitor cells are not noticeably smaller after dividing, indicating that growth accompanies the SMW cell division. The SMW, however, does not depend on myc. It appears to depend on a transcriptional target of Su(H) outside of the E(spl)-C of bHLH genes and that can be transcribed in the absence of Mam. The possibility of a function of Su(H)/Nicd other than transcription cannot be ruled out. The SMW clearly depends on CycE, but CycE may not be the N-dependent gene required for SMW entry, because it is not obviously elevated in SMW cells. Contrary to a recent proposal, the SMW does not depend on the HLH gene emc and is not inhibited by the bHLH protein Da. Instead da is required for entry into the SMW. The positive requirement for da is cell-nonautonomous, as is the requirement for its heterodimer partner ato, and consistent with the role of ato and da in promoting Dl expression during retinal differentiation, which is likely to account for the cell cycle defect. Surprisingly, the requirement for Dl in the SMW cell cycle was not absolute, either because an unidentified ligand exists, long-range cell-nonautonomy occurs, or Dl (and Ser) may contribute to cis-inactivation of N, so that some ligand-independent Notch signaling could occur in the unphysiological circumstance that Notch ligands are completely absent (Bhattacharya, 2017).

    The mir-279/996 cluster represses receptor tyrosine kinase signaling to determine cell fates in the Drosophila eye

    Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling, mediated by the Epidermal Growth Factor receptor (EGFR) and Sevenless receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that normal Drosophila eye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of mir-279/996 in non-neural cells of the developing eye. Moreover, mir-279/996 mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, since mir-279/996 nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. mir-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion of mir-279/996 sufficiently de-represses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutants boss and sev, which otherwise completely lack this cell fate. Altogether, this study reveals a rare setting of developmental cell specification that substantially requires miRNA control (Duan, 2018).

    The phosphoinositide phosphatase Sac1 regulates cell shape and microtubule stability in the developing Drosophila eye

    Epithelial patterning in the developing Drosophila melanogaster eye requires the Neph1 homolog Roughest (Rst), an immunoglobulin family cell surface adhesion molecule expressed in interommatidial cells (IOCs). This study using a novel temperature-sensitive (ts) allele, showed that the phosphoinositide phosphatase Sac1 is also required for IOC patterning. Sac1ts mutants have rough eyes and retinal patterning defects that resemble rst mutants. Sac1ts retinas exhibit elevated levels of phosphatidylinositol 4-phosphate (PI4P), consistent with the role of Sac1 as a PI4P phosphatase. Indeed, genetic rescue and interaction experiments reveal that restriction of PI4P levels by Sac1 is crucial for normal eye development. Rst is delivered to the cell surface in Sac1ts mutants. However, Sac1ts mutant IOCs exhibit severe defects in microtubule organization, associated with accumulation of Rst and the exocyst subunit Sec8 in enlarged intracellular vesicles upon cold fixation ex vivo. Together, these data reveal a novel requirement for Sac1 in promoting microtubule stability and suggest that Rst trafficking occurs in a microtubule- and exocyst-dependent manner (Del Bel, 2018).

    Undifferentiated epithelial cells are patterned and specified during development to yield highly ordered tissues composed of multiple cell types. Patterning and differentiation within an epithelium are driven in part by cell surface adhesion molecules that promote intercellular interactions. Defects in cell-cell adhesion lead to developmental abnormalities and contribute to disease progression. For example, the mammalian Irre cell recognition module (IRM) adhesion proteins NEPH1 (KIRREL) and nephrin (NPHS1) are required in the kidney for development and maintenance of the filtration barrier or slit diaphragm. Despite their importance in animal development and physiology, little is known about how trafficking and delivery of cell surface adhesion molecules such as IRM proteins is achieved (Del Bel, 2018).

    The adult Drosophila eye contains ~750 individual units called ommatidia. Prior to pupariation, each ommatidium consists of eight photoreceptor (PR) cells and four cone cells, surrounded by undifferentiated interommatidial cells (IOCs). During the first half of pupal eye development, 0-42 h after puparium formation (APF), IOCs undergo dynamic morphogenetic changes to give rise to two primary pigment cells (1°pc), six secondary pigment cells (2°pc), three tertiary pigment cells (3°pc) and three bristles, arranged in a honeycomb lattice. These cells support and optically isolate individual ommatidia (Del Bel, 2018).

    Specification and organization of Drosophila retinal cells requires IRM protein function. 1°pc express the nephrin homologs Hibris (Hbs) and Sticks and stones (Sns), whereas IOCs express the NEPH1 homologs Roughest (Rst) and Kin of Irre (Kirre, also called Dumbfounded). Between 24 and 30 h APF, all four proteins localize to the plasma membrane (PM) of these cells at the 1°pc:IOC border, and heterophilic binding of Rst and Kirre with Hbs and Sns is needed for specification and morphogenesis of 2°pc and 3°pc (2°/3°pc). Mutations that affect cell surface accumulation of Rst result in rough eyes and reduced levels of pigmentation as a result of defects in 2°/3°pc differentiation (Del Bel, 2018).

    Phosphoinositides, or phosphatidylinositol (PI) phosphates (PIPs), regulate essential cellular processes such as membrane trafficking and actin cytoskeletal organization. In the canonical PIP pathway, PI is phosphorylated by PI 4-kinases (PI4Ks) to generate PI 4-phosphate (PI4P), which is a precursor for other PIPs, including PI 4,5-bisphosphate [PI(4,5)P2], and serves as a potent signaling molecule, for example by recruiting effectors to the Golgi body to promote membrane trafficking. Downregulation of PI4P in specific membrane compartments also drives cellular events. For example, in budding yeast (Saccharomyces cerevisiae), PI4P dephosphorylation is required for targeted delivery of cargo to the PM by the exocyst complex. Drosophila has three PI4Ks, including a single type II enzyme (PI4KII) that generates PI4P at the trans-Golgi network (TGN) and on endosomes. PI4P levels are kept in check by the phosphoinositide phosphatase Sac1, a conserved transmembrane protein present in endoplasmic reticulum and Golgi membranes (Del Bel, 2018).

    Previous work has shown that Sac1 is required for normal eye development in Drosophila (Wei, 2003b). Sac1 is essential for viability in Drosophila (Wei, 2003a,b). Flies transheterozygous for a hypomorphic allele and a null allele exhibit rough eyes with necrotic patches and drown in the food soon after eclosing (Wei, 2003b). Using the hypomorphic allele, which is shown to be temperature sensitive (ts), it was demonstrated that Sac1 plays a crucial role in patterning the retinal epithelium. IOCs of Sac1ts flies cultured at 25°C exhibit a dramatic increase in PI4P levels and decreased levels of PM PI(4,5)P2. Although Rst is present at the cell surface of these IOCs, fixation on ice ex vivo results in re-distribution of Rst to enlarged structures containing the exocyst complex subunit Sec8, suggesting a microtubule defect. Indeed, Sac1ts IOCs contain sparse, disorganized microtubules (MTs) that disappear when fixed on ice. These results thus identify a novel link between Sac1, PIP levels and microtubule stability in the developing Drosophila eye. Because Sac1 is conserved, these findings suggest a possible role for MT regulation by Sac1 in human development and disease (Del Bel, 2018).

    Conditional alleles are powerful tools for studying the functions of essential genes. Using a temperature-sensitive allele of Sac1, this study shows that proper phospholipid regulation by Sac1 is required for normal development of the Drosophila retinal epithelium (Del Bel, 2018).

    The results indicate that retinal patterning is highly sensitive to elevated levels of PI4P. Loss of Sac1 leads to increased Golgi PI4P and IOC patterning defects that are rescued by expression of catalytically active Sac1. Deletion of PI4KII suppresses Sac1ts, whereas expression of a WT PI4KII transgene exacerbates the observed defects. Thus, Sac1 and PI4KII regulate a pool of PI4P that is required for retinal patterning. Loss of PI4KII itself results in mild patterning defects (i.e. occasional extra bristles and fused ommatidia), indicating that reducing PI4P levels also has consequences for retinal development. Although these data point to a clear role for PI4KII in signaling required for 2°/3°pc adhesion and patterning, the experiments do not rule out possible contributions of other PI4Ks to this process. Moreover, it is anticipated that, in addition to the Golgi, other pools of PI4P at the PM and elsewhere may be regulated by Sac1 (Del Bel, 2018).

    Because PI4P is phosphorylated by PIP 5-kinases to produce PI(4,5)P2, Sac1ts mutants might be expected to show increased levels of PI(4,5)P2 as well as PI4P. Strikingly, however, this study found that Sac1ts mutants exhibit reduced levels of the PI(4,5)P2 marker PLCΔ-PH at the PM. Although this result is counterintuitive, similar observations have been reported in budding yeast, and the same same phenomenon in Drosophila is observed embryos homozygous for a lethal allele of Sac1. This observation is in contrast to other Drosophila tissues, where Sac1 loss was reported to have no effect on PI(4,5)P2 levels (Forrest, 2013; Yavari, 2010). It is possible that PIP 5-kinases lack access to the increased PI4P in Sac1 mutants, thereby preventing the expected increase in PI(4,5)P2, or perhaps increased PI4P triggers a feedback mechanism that activates PIP phosphatases or phospholipases, leading to PI(4,5)P2 breakdown. Thus, in addition to elevated PI4P levels, reduced levels of PI(4,5)P2 might contribute to the IOC patterning defects in Sac1ts. Indeed, in preliminary results a genetic interaction was observed between Sac1 and the PI4P 5-kinase skittles, which could stem from reduced PI(4,5)P2 or increased PI4P levels, or a combination of the two (Del Bel, 2018).

    Drosophila Sac1ts mutants exhibit rough eyes, ommatidial mispatterning and abnormal 2°/3°pc morphology, phenotypes that resemble mutants for the IRM protein Rst. Maintenance of Rst at the 1°pc:IOC border is sensitive to ice fixation in Sac1ts mutant retinas. Because MTs are labile under cold conditions, this suggested a MT defect in Sac1ts. MTs are disorganized in Sac1ts mutant IOCs fixed at RT, and even more dramatically affected in mutant IOCs fixed on ice. Moreover, Sac1ts IOCs fixed on ice display enlarged Sec8-positive vesicles containing Rst, suggesting that Rst travels to the cell surface via an exocyst- and MT-associated pathway that is sensitive to cold fixation in Sac1ts (Del Bel, 2018).

    The simplest model to explain these data is that Rst is constantly turned over at the PM and requires continuous replenishment from internal stores, as has been reported for mammalian IRM proteins. This turnover appears to occur in both WT and Sac1ts, as this study observed similar uptake of anti-Rst antibody in each. It remains possible that subtle defects in the activity or turnover of Rst (or other proteins) at the PM contribute to the observed defects in Sac1ts. Indeed, compromised expression of the differentiation marker BA12-lacZ was observed in Sac1ts IOCs. Defects in BA12-lacZ expression, as well as IOC number and specification, also occur in RstD mutants, which show a delay in Rst accumulation at the 1°pc:IOC border. Relevant to this point, it is noted that when quantification of Rst distribution after RT or cold fixation was independently validated at a later date, a slight but significant reduction was observed in Rst accumulation in Sac1ts following RT fixation. However, as this difference was not observed in earlier experiments, it is not possible to conclude that there is a reproducibly significant decrease in Rst accumulation in Sac1ts (Del Bel, 2018).

    Sac1 was originally identified as a suppressor of yeast actin mutants. Thus, much of the literature has focused on how Sac1 loss affects the actin cytoskeleton. This observed that retinal cells in Drosophila Sac1ts mutants exhibit normal actin organization during early stages of pupal eye development (24-30 h APF), yet show defects in MT stability and organization. Interestingly, both increased levels of PI4P and reduced levels of PI(4,5)P2 have previously been associated with defects in MT organization. The molecular mechanisms involved remain unknown, and further investigation will be required to determine the link between Sac1 and MT stability. However, the current observations suggest that regulation of the MT cytoskeleton by Sac1 may be more important when moving from fungal to animal models (Del Bel, 2018).

    In summary, this study has characterized a requirement for Sac1 in maintaining PI4P and PI(4,5)P2 levels and promoting normal development of the Drosophila retina. Additionally, it was demonstrated that Rst distribution in Sac1ts is sensitive to cold fixation, and that Sac1ts exhibits a microtubule defect at 24 h APF. The results indicate that Rst is delivered to the PM via exocyst- and microtubule-based trafficking (which is cold sensitive in Sac1ts) and is turned over at the PM, similar to its mammalian homologs. Further investigation will be required to unravel precisely how Sac1 regulates microtubules, and how this contributes to normal retinal development (Del Bel, 2018).

    Cellular homeostasis in the Drosophila retina requires the lipid phosphatase Sac1

    The complex functions of cellular membranes, and thus overall cell physiology, depend on the distribution of crucial lipid species. Sac1 is an essential, conserved, ER-localized phosphatase whose substrate, phosphatidylinositol 4-phosphate (PI4P), coordinates secretory trafficking and plasma membrane function. PI4P from multiple pools is delivered to Sac1 by oxysterol binding protein and related proteins in exchange for other lipids and sterols, which places Sac1 at the intersection of multiple lipid distribution pathways. However, much remains unknown about the roles of Sac1 in subcellular homeostasis and organismal development. Using a temperature-sensitive allele (Sac1ts), this study shows that Sac1 is required for structural integrity of the Drosophila retinal floor. The βps-integrin Myospheroid, which is necessary for basal cell adhesion, is mislocalized in Sac1(ts) retinas. In addition, the adhesion proteins Roughest and Kirre, which coordinate apical retinal cell patterning at an earlier stage, accumulate within Sac1(ts) retinal cells due to impaired endo-lysosomal degradation. Moreover, Sac1 is required for ER homeostasis in Drosophila retinal cells. Together, these data illustrate the importance of Sac1 in regulating multiple aspects of cellular homeostasis during tissue development (Griffiths, 2020).

    Although they comprise a minor fraction of total cellular phospholipid content, phosphoinositides, also known as phosphatidylinositol phosphates (PIPs), act as essential coordinators of membrane function and identity (Balla, 2013). PIPs are derived from the precursor phosphatidylinositol, whose inositol head group can be phosphorylated at any of three positions to yield seven unique PIP species that recruit distinct sets of effector proteins. Through the localized activity of PIP kinases and phosphatases, these species are interconverted to maintain enrichment in different membranes and to regulate numerous PIP effector-driven processes (Balla, 2013; Griffiths, 2020).

    Sac1 is a conserved phosphatase whose substrate, phosphatidylinositol 4-phosphate (PI4P), coordinates multiple stages in secretory trafficking, participates in cellular signaling pathways, and acts as the precursor for PI(4,5)P2 at the plasma membrane (PM). PI4P is produced in the PM and Golgi, respectively, by two conserved type III PI 4-kinases (PI4Ks), PI4KIIIα, and PI4KIIIβ. In addition, a type II PI4K (PI4KIIα) produces PI4P in the trans-Golgi network (TGN) and on endosomes, where it is important for endosomal trafficking (Griffiths, 2020).

    In contrast to the distribution of PI4Ks and PI4P, Sac1 localizes primarily to the ER, as well as the cis-Golgi under growth-limiting conditions. Although seemingly capable of acting in trans on PI4P in neighboring membranes in some scenarios, Sac1 appears to predominantly depend on delivery of PI4P to the ER via nonvesicular lipid transport at membrane contact sites (MCS). For instance, oxysterol-binding protein (OSBP), which localizes to ER-trans-Golgi MCS through interactions with the ER-resident vesicle-associated membrane protein-associated protein VAP as well as PI4P in the trans-Golgi, delivers PI4P from the trans-Golgi to the ER in exchange for sterols. Hydrolysis of incoming PI4P by Sac1 maintains a low concentration of PI4P in the ER that is necessary for sustained PI4P/sterol countertransport in vitro, although this relationship appears more nuanced in vivo. OSBP-related proteins (ORPs), which are encoded by 11 genes in humans and three in flies, function similarly to OSBP but differ in their localization and lipid-binding preferences. Despite its essential function, how Sac1 regulates different aspects of cellular homeostasis during animal development is not fully understood (Griffiths, 2020).

    In Drosophila, null Sac1 mutants exhibit embryonic lethality due to defects in cell shape and ectopically activated JNK signaling that prevent dorsal closure. JNK signaling defects are also observed in Sac1 clones in larval imaginal discs. Moreover, Sac1 regulates Hedgehog signaling by inhibiting recruitment and activation of Smoothened at the PM in a PI4P-dependent manner (Yavari, 2010; Jiang, 2016). Sac1 is also required for axonal pathfinding in the embryonic central nervous system, as well as for axonal transport and synaptogenesis in larval neurons (Lee, 2011; Forrest, 2013; Griffiths, 2020).

    In addition, loss of Sac1 causes severe tissue disorganization and degeneration during eye development. The Drosophila eye is composed of ~750 unit eyes called ommatidia. Presumptive ommatidia arise early in pupal development, where they initially comprise clusters of medial/basal photoreceptors and apical cone cells surrounded by a disordered pool of undifferentiated interommatidial cells (IOCs). During the first half of the ∼96-h pupal stage, two IOCs per ommatidium differentiate into primary pigment cells (1°pc), which encircle the cone cells. The remaining IOCs subsequently differentiate into a lattice of secondary and tertiary pc (2°/3°pc) and sensory bristles that separate neighboring ommatidia or are removed by apoptosis by 42 h after puparium formation (APF). Changes in IOC shape and position during this stage require the Irre cell recognition module (IRM) adhesion proteins Roughest (Rst) and Hibris (Hbs), as well as their paralogues Kirre and Sticks and stones (Sns). Rst/Kirre and Hbs/Sns are orthologues of mammalian Neph1 and nephrin, which are needed for formation of the renal slit diaphragm as well as during myoblast fusion. After IOC patterning, during late stages of pupal eye development (42-96 h APF), the retina elongates fivefold, and laminated corneal lenses with underlying gelatinous pseudocones are secreted, giving the eye its characteristic adult appearance (Griffiths, 2020).

    Previously work has examined the role of Sac1 in the developing Drosophila eye using a hypomorphic Sac1 allele that is temperature sensitive (Sac1ts). Sac1ts flies develop morphologically normal eyes when reared at 18°C, but display a rough eye phenotype caused by defective IOC sorting when reared at or above 23.5°C. This study shows that Sac1ts eyes exhibit structural defects at the retinal floor and mislocalization of the βps-integrin Myospheroid (Mys), which is required for retinal floor adhesion. This defect is not due to a loss of cell polarity, as apical adherens junctions are unaffected. However, a novel secondary defect was identified in the distribution of Rst and Kirre, which are apical transmembrane proteins. At 42 h APF, Sac1ts 2°/3°pc contain an excess of intracellular Rst and Kirre due to impaired endo-lysosomal trafficking and degradation. Sac1ts 2°/3°pc also accumulate PI4P and F-actin on enlarged, basal endosomes and exhibit ER stress. Thus, this study has identified novel roles for Sac1 in regulating cellular homeostasis during tissue morphogenesis (Griffiths, 2020).

    The Drosophila pupal eye represents a powerful system to examine protein trafficking and turnover. Patterning of retinal cells requires spatially and temporally regulated expression as well as correct subcellular distribution of cell surface proteins that mediate cell-cell contacts and determine tissue architecture. Dysregulation of these processes can produce structural defects, which frequently persist in the adult eye. This study has taken advantage of these circumstances to demonstrate the importance of Sac1 in basal delivery of the βps-integrin Mys, which is required for retinal floor integrity, as well as endo-lysosomal regulation and turnover of the apical patterning determinants Rst and Kirre. The results also highlight the importance of Drosophila Sac1 in ER homeostasis, as had been reported in yeast. This could be due to deregulation of PI4P, phosphatidylserine, and sterol levels, which would be expected to disrupt ER membrane charge and lipid order (Griffiths, 2020).

    Given the similarities between mys mutants and Sac1ts, loss of Mys at the basal grommets in Sac1ts likely causes the retinal floor defects observed in the adult eye. In addition, this phenotype resembles the basal retinal degeneration observed in an ALS-associated vap mutant, suggesting the underlying cause could be similar. However, it is unclear why basal distribution of Mys is perturbed while apical polarity is not. In the Drosophila follicular epithelium, Rab10 activity has been shown to be important for the distribution of basement membrane proteins independent of overall apical-basal polarity, in a manner dependent on PI(4,5)P2 at the apical PM. Previously study observed a decrease in apical PI(4,5)P2 abundance in Sac1ts retinas at 24 h APF (Del Bel, 2018), which it is speculated could perturb basal trafficking. Alternatively, aberrant distribution of basal F-actin in Sac1ts could inhibit localization of Mys to the grommets. Why some transmembrane proteins are sensitive to reduced Sac1 activity while others are not remains an open question. It is also unclear whether Mys mislocalization is linked to endosome dysfunction in Sac1ts (Griffiths, 2020).

    Whereas PI3P and PI(3,5)P2 are the canonical phosphoinositide regulators of endosomal progression (Wallroth, 2018), PI4P production has also emerged as an important factor in cargo delivery to lysosomes. In mammalian cells, PI4P is generated on late endosomes by type II PI4Ks (Baba, 2019). PI4KIIα is important for Golgi-to-lysosome trafficking of LIMP-2, as well as PM-to-lysosome trafficking of LAMP-1, and these proteins accumulate in enlarged endosomes when PI4KIIα levels are reduced. Furthermore, in macrophages, PI4KIIα-mediated PI4P enrichment on phagosomes occurs concurrently with Rab7 recruitment and is necessary for phagosome acidification and subsequent fusion with lysosomes (Griffiths, 2020).

    This study has shown that Sac1-dependent depletion of PI4P is also important for endosomal trafficking and degradation of transmembrane proteins from the PM. This is consistent with a recent report by Mao and colleagues (Mao, 2019), who found that in multiple larval Drosophila tissues, loss of VAP, which recruits OSBP and a subset of ORPs to MCS, increases endosomal PI4P levels and inhibits autophagic degradation. Null vap mutants exhibit decreased lysosomal acidification, as well as an increase in the abundance of lysosomes, endosomes, autolysosomes, autophagosomes, and Ref(2)P (Mao, 2019). The authors propose that increased PI4P abundance up-regulates endosome formation and progression, which causes lysosomes to become oversaturated with incoming cargo. Indeed, loss of Ubiquilin, which contributes to lysosome acidification, also delays autophagy and causes Ref(2)P buildup. Notably, this study observed increased Ref(2)P abundance in Sac1ts retinas, which suggests a similar delay in autophagy. It is a compelling notion that increased PI4P levels in Sac1ts could promote excessive fusion of endosomes with lysosomes, which would replicate the effect described by Mao (2019). However, the accumulation of Rst and Kirre in Sac1ts, which do not appear to be concentrated in lysosomes based on the lack of colocalization between Rst and Arl8, could also be caused by impaired endosomal progression or maturation, though this might stem from downstream lysosomal dysfunction. Indeed, the enlarged endosomes observed in Sac1ts lacked both Vps16a and Arl8, suggesting they were not caused by excessive fusion with lysosomes. Further analysis of PI4P in endosomal dynamics and maturation is warranted to determine the precise role of Sac1 in late stages of protein degradation (Griffiths, 2020).

    This study also found that reduced Sac1 function leads to basal accumulation of F-actin-positive enlarged endosomes. In mammalian cells, loss of both VAP isoforms has been shown to induce F-actin comet formation on endosomes via PI4P-dependent recruitment of the WASH-ARP2/3 complex. Notably, these do not resemble the more uniform F-actin coating on Sac1ts endosomes. Rather, the structures that were observed appear more reminiscent of a phenomenon termed actin-flashing, wherein phagosomes become coated in F-actin by WASP-ARP2/3 to delay fusion with lysosomes. Endosomal phenotypes similar to those in Sac1ts have also been observed when Arf6 activity is perturbed; increased Arf6 activity activates PIP5K, which has been shown to produce PI(4,5)P2 on endosomes and lead to F-actin polymerization via WASP, whereas loss of Arf6 increases endosomal PI4P levels and perturbs endosomal recycling. Intriguingly, in Caenorhabditis elegans, Sac1 inhibits Arf6 by sequestering the Arf6-GEF Bris-1. However, it is unknown whether this interaction is conserved or, more broadly, how Sac1 influences F-actin polymerization on endosomes (Griffiths, 2020).

    It is noteworthy that enlarged endosomes were restricted to basal regions in Sac1ts. Positioning of endosomes and lysosomes is mediated by bidirectional transport along microtubules, which influences their acidity and function. In mammalian cells, Rab7 recruits RILP, which activates endosomal dynein motors to promote minus end-directed transport toward perinuclear microtubule organizing centers. PI4P is also required for RILP recruitment, which implies that excess PI4P could lead to perinuclear endosome accumulation. Although the single Drosophila RILP orthologue has been shown to bind Arl8 rather than Rab7, it is possible that PI4P influences late endosome transport through analogous Rab7 effectors. Additionally, previous work has shown that Sac1ts 2°/3°pc precursors contain unstable microtubules at 24 h APF (Del Bel, 2018), which could affect microtubule-based endosome positioning later in development (although this study was unable to detect microtubule defects by immunostaining at 42 h APF). However, it is also possible that enlarged endosomes accumulate basally for other reasons or are simply excluded from narrower apical-medial regions on the basis of size. It remains to be discerned whether Rst accumulation and the appearance of enlarged endosomes, which co-occurred between 24 and 42 h APF, share a causal basis or represent distinct, parallel phenotypes of reduced Sac1 activity (Griffiths, 2020).

    Given the phenotypic similarities between Sac1ts and vap mutants (Mao, 2019), it was surprising that osbp did not affect Mys distribution or cause severe Rst accumulation. However, this is reminiscent of previous results from Drosophila neurons, where loss of Vap but not OSBP caused protein accumulation and ER stress (Moustaqim-Barrette, 2014). It is possible that, as in yeast where the presence of one out of seven OSBP homologues is sufficient for viability, OSBP functions redundantly with one or more ORPs in regulating the endosomal pathway. Indeed, CG1513, which is synthetically lethal in combination with osbp (Moustaqim-Barrette, 2014), encodes an orthologue of mammalian ORP9, which functions similarly to OSBP in sterol-PI4P exchange at ER-Golgi MCS. Alternatively, CG3860 encodes an orthologue of mammalian ORP2, which localizes to late endosomes in HeLa cells and influences sterol levels in endosomes and the PM, although countertransport of PI4P has not been shown. Mammalian ORP2 also binds ORP1L, which acts at ER-endosome MCS and promotes endosome transport, though it is unclear whether such a role is conserved in Drosophila, which lack an ORP1L orthologue. Further characterization of the Drosophila ORPs is thus needed to clarify their respective contributions to lipid homeostasis and endosomal progression (Griffiths, 2020).

    Recent years have seen a proliferation of research into Sac1's roles in lipid homeostasis and the importance of PI4P regulation, as well as the development of novel probes and methods for studying phosphoinositides in vivo. This study has provided new insights into Sac1's function in protein delivery and turnover in a developing tissue, which will serve as groundwork for further investigations into the significance of Sac1 in cell physiology, organismal development, and ultimately cellular homeostasis in human health and disease (Griffiths, 2020).

    Motif 1 Binding Protein suppresses wingless to promote eye fate in Drosophila

    The phenomenon of RNA polymerase II (Pol II) pausing at transcription start site (TSS) is one of the key rate-limiting steps in regulating genome-wide gene expression. In Drosophila embryo, Pol II pausing is known to regulate the developmental control genes expression, however, the functional implication of Pol II pausing during later developmental time windows remains largely unknown. A highly conserved zinc finger transcription factor, Motif 1 Binding Protein (M1BP), is known to orchestrate promoter-proximal pausing. This study found a new role of M1BP in regulating Drosophila eye development. Downregulation of M1BP function suppresses eye fate resulting in a reduced eye or a "no-eye" phenotype. The eye suppression function of M1BP has no domain constraint in the developing eye. Downregulation of M1BP results in more than two-fold induction of wingless (wg) gene expression along with robust induction of Homothorax (Hth), a negative regulator of eye fate. The loss-of-eye phenotype of M1BP downregulation is dependent on Wg upregulation as downregulation of both M1BP and wg, by using wgRNAi, shows a significant rescue of a reduced eye or a "no-eye" phenotype, which is accompanied by normalizing of wg and hth expression levels in the eye imaginal disc. Ectopic induction of Wg is known to trigger developmental cell death. Upregulation of wg as a result of downregulation of M1BP also induces apoptotic cell death, which can be significantly restored by blocking caspase-mediated cell death. These data strongly imply that transcriptional regulation of wg by Pol II pausing factor M1BP may be one of the important regulatory mechanism(s) during Drosophila eye development (Raj, 2020).

    Pol II pausing near the transcription start site has been identified as a key step in optimizing transcription of many genes in metazoans. It has been proposed that pausing allows the coupling of transcription and RNA processing. Pausing can contribute to dynamic regulation of gene expression in response to developmental and environmental signals, and can function to repress transcription. The genome-wide studies have revealed that  ~10-40% of all genes in mammalian embryonic stem cells and Drosophila have paused promoters. In Drosophila, while the phenomenon of promoter proximal pausing has been well studied in regulation of genes encoding the heat shock proteins (Hsp) and different components involved in immune response pathways, it is also proposed to play important role in regulating the gene expression during early developmental events such as patterning, sex determination etc. So far, the sequence-specific transcription factors such as GAGA factor and M1BP, and other regulators HEXIM, LARP7 (La Ribonucleoprotein 7, Transcriptional Regulator) have been implicated in dictating Pol II pausing in Drosophila. However, the biological relevance of transcriptional pausing and the exact mechanism by which the regulatory factors may contribute in pausing of Pol II is not fully understood (Raj, 2020).

    M1BP regulates retinal determination and MF progression in developing eye This study tested fthe role of transcription pausing factor, M1BP during Drosophila eye development. Downregulation of M1BP levels in the developing eye was found to result in strong suppression of eye fate, however, gain-of-function of M1BP did not affect the eye fate suggesting that optimum levels of M1BP are required for Drosophila eye development. Furthermore, no domain constraint in eye suppression function was seen when M1BP levels were downregulated. In addition, when M1BP levels were downregulated (ey > M1BPRNAi) the expression of retinal determination and differentiation genes were strongly downregulated. Interestingly, it was found that protein encoded by RD genes were downregulated in ey > M1BPRNAi background. Therefore, M1BP may not be affecting RD gene expression directly (Raj, 2020).

    During eye development, a wave of differentiation, emanates from the posterior margin of the developing eye imaginal disc, which sweeps anteriorly across the retinal primordium. The crest of this wave is referred to as the MF, which results in retinal differentiation behind it. The two signals dpp and hh plays an important role in initiation and progression of MF. Downregulation of M1BP affects retinal differentiation as well as progression of MF. It suggests that M1BP role is to promote retinal differentiation as well as MF progression. Also, M1BP downregulates the level of negative regulator(s) of the eye fate. This study screened for the genes, which may serve as target for M1BP mediated transcriptional pausing mechanism in Drosophila eye imaginal disc (Raj, 2020).

    The protein encoded by Drosophila wg gene, a member of Wg/WNT signaling pathway, act short range inducer, which organizes the pattern of cells at a distance in the embryo. Since M1BP downregulation resulted in blocking retinal differentiation and MF progression, targets were sought of M1BP transcriptional pausing function using the candidate gene approach. It was found that wg-lacZ reporter, which serves as a transcriptional read out for Wg, exhibits robust induction in eye imaginal discs where M1BP levels were downregulated. This observation was further validated by qPCR approach which showed that there is a 2.2-fold increase in wg gene expression. Furthermore, in high throughput microarray screen carried out in S2R + cells, wg was also identified as a target whose expression is downregulated by M1BP using M1BP RNAi. According to microarray analysis, wg shows a 5.5-fold change when cells are treated with M1BPRNAi (Raj, 2020).

    To validate the results from qPCR approach as well induction of wg-lacZ reporter expression in ey > M1BPRNAi eye imaginal disc, this study also employed bioinformatics analysis to determine if there are M1BP binding sites in the wingless (wg) gene. The M1BP binding sequence (YGGTCACACTR) has been reported earlier. This sequence was used for MEME analysis to screen for M1BP binding sites were found in the wg gene and regulatory region (Raj, 2020).

    Wg, a ligand for evolutionarily conserved Wg/WNT signaling pathway, is known to act as a negative regulator of eye development. During Drosophila eye development, Wg activity promotes head specific fate by negatively regulating MF progression in the differentiating eye imaginal disc. Wg regulates expression of downstream gene hth, which encodes a MEIS class of transcription factor, and act as a negative regulator of eye development. This study found that in ey > M1BPRNAi background, robust induction of wg transcription also accompanies ectopic induction of hth along with the suppression of the eye fate. Further, downregulation of wg levels, using wgRNAi, in ey > M1BPRNAi background rescued the eye suppression phenotype. This data clearly suggested that M1BP downregulates levels of wg, which in turn regulate expression of hth in the developing Drosophila eye (Raj, 2020).

    Higher levels of Wg are known to trigger developmental cell death in the developing eye field. Interestingly, in ey > M1BPRNAi eye discs, the eye field was significantly reduced. Since, majority of the cell death is triggered by the activation of caspase-dependent cell death, blocking caspase-dependent cell death by ectopic expression of anti-apoptotic P35 transgene in ey > M1BPRNAi background showed rescue of eye suppression phenotype. However, these P35 mediated rescues of ey > M1BPRNAi were not as significant as seen with wgRNAi. This suggests that Wg might be regulating eye fate through hth induction and eye field size by triggering caspase mediated cell death. In order to rule out that these in ey > M1BPRNAi phenotypes are not affected by reduced cell proliferation rates, levels were tested of pH3 in these developing eye fields. It was found that cell proliferation rates were not affected by this transcriptional pausing mechanism in the developing eye (Raj, 2020).

    These results imply that the transcription pausing function of M1BP in regulating Wg signaling may play a critical role in Drosophila eye development. However, other factors and signaling pathways involved in regulating the M1BP function at the mechanistic level is yet to be determined. In order to further understand whether M1BP mediated transcriptional regulation is also implicated during development of other imaginal discs in Drosophila, the downregulation of M1BP function was studied in bi-Gal4 domains of wing imaginal disc. Whether this role of M1BP in regulating wg gene expression is exclusive to developing eye disc or it extends to other larval imaginal disc was tested. A bi-GAL4 driver which drives the expression of a transgene in wing imaginal disc was used. Downregulation of M1BP in bi-Gal4 expression domains of wing (bi > M1BPRNAi) exhibits ectopic upregulation wg expression in the pouch region of the wing imaginal disc. Furthermore, M1BP expression levels are downregulated in the wing pouch region, which corresponds to the bi-Gal4 expression domain. These results suggested that the transcription pausing function of M1BP may have similar target in the eye and wing imaginal disc. Recently, HEXIM, another transcriptional regulator associated with pol II pausing, has been reported to affect wing development in Drosophila by regulating Hh signaling. In Drosophila wing imaginal disc, HEXIM knockdown causes developmental defects by inducing ectopic expression of hh and its transcriptional effector cubitus interuptus (ci), which triggers apoptosis. This suggests that the regulatory factors involved in Pol II pausing are important in maintaining the expression levels of different signaling pathways during development in Drosophila (Raj, 2020).

    A number of highly conserved transcriptional pausing and elongation factors such as Spt5 precisely regulate transcription during Drosophila embryogenesis. The Spt5W049 missense mutation causes defects in the anterior-posterior patterning and segmental patterning during embryogenesis. Interestingly, the mutant allele of Spt5 (foggym806) in Zebrafish also causes multiple developmental defects such as discrete problems with pigmentation, tail outgrowth, ear formation and cardiac differentiation. These studies suggest that the regulatory mechanism in Pol II pausing during fly development are also conserved in higher organisms. The Drosophila compound eye shares similarities with the vertebrate eye at the level of genetic machinery as well as the processes of differentiation. Therefore, the information generated in Drosophila can be extrapolated to higher organisms. Since Wnt signaling is known to induce programmed cell death in patterning the vasculature of the vertebrate eye, it will be important to study what molecules other than M1BP can prevent Wg signaling from inducing cell death during early eye development (Raj, 2020).

    Buffered EGFR signaling regulated by spitz-to-argos expression ratio is a critical factor for patterning the Drosophila eye

    The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays a critical role in regulating tissue patterning. Drosophila EGFR signaling achieves specificity through multiple ligands and feedback loops to finetune signaling outcomes spatiotemporally. The principal Drosophila EGF ligand, cleaved Spitz, and the negative feedback regulator, Argos are diffusible and can act both in a cell autonomous and non-autonomous manner. The expression dose of Spitz and Argos early in photoreceptor cell fate determination has been shown to be critical in patterning the Drosophila eye, but the exact identity of the cells expressing these genes in the larval eye disc has been elusive. Using single molecule RNA Fluorescence in situ Hybridization (smFISH), this study revealed an intriguing differential expression of spitz and argos mRNA in the Drosophila third instar eye imaginal disc indicative of directional non-autonomous EGFR signaling. By genetically tuning EGFR signaling, it was shown that rather than absolute levels of expression, the ratio of expression of spitz-to-argos to be a critical determinant of the final adult eye phenotype. Proximate effects on EGFR signaling in terms of cell cycle and differentiation markers are affected differently in the different perturbations. Proper ommatidial patterning is robust to thresholds around a tightly maintained wildtype spitz-to-argos ratio, and breaks down beyond. This provides a powerful instance of developmental buffering against gene expression fluctuations (Pasnuri, 2023).

    Developmental pathways have evolved mechanisms to monitor positional information in order to generate reproducible organismal patterns. These pathways are robust and insensitive to small changes in individual processes involved. Spatial differentiation, where a population of cells undergo deterministic molecular differentiation, brings about spatial patterns. Redundancy of mechanism and negative feedback are two ways in which reliability in pattern formation is brought about. Lateral inhibition by diffusible molecules is another mechanism that can be used to generate patterns. For systems which do not depend on developmental history, environmental makeup determines their molecular differentiation contributing towards generating a pattern (Pasnuri, 2023).

    This paper proposes a mechanism where relative expression levels of principal EGFR ligand, Spitz and negative feedback regulator Argos determines the extent of EGFR activation which is crucial for the periodic ommatidial pattern. The data suggests that it is not the absolute gene expression but the balance between gene networks on the whole which may contribute towards pattern formation. GMR-Gal4 is expressed in all cells posterior to the morphogenetic furrow and any expression cassette under the UAS is expressed strongly. Whereas, Elav-Gal4 is expressed only in neuronal cells and the expression is strongest towards the posterior end of the eye disc. While Elav-Gal4 expression occurs in differentiated neurons starting with R8 in the eye disc (for which EGFR signaling is not needed), but beyond R8 specification, Spitz and Argos levels are important for subsequent PR cell differentiation and also in the pupal stages. Although an equally drastic reduction was observed in argos expression when UAS-spitz dsRNA driven by GMR-Gal4 and Elav-Gal4, the eye discs show reduced dpERK staining in both cases. The reduction was lower when Elav-Gal4 driver was used, corresponding to the absence of phenotype in the adult eye. The eye discs expressing EGFRCA construct under GMR-Gal4 Gal80ts with spitz-to-argos ratio near 1, showed a discontinuous S-phase band after the morphogenetic furrow indicating a lower population of cells entering the second mitotic wave. Fewer cells entering the second mitotic wave leaves the tissue field with fewer uncommitted cells to make cell fate decisions. This can affect pattern formation to a great extent. This could also explain fewer number of bristle cells in the rough adult eyes as bristles cell fate is assigned from cells arising from the second mitotic wave. It should also be noted that the rough eye phenotype for EGFRDN is rather different from EGFRCA and shows a profusion of bristles. This mechanism of relative expression determining phenotype supports older work on the importance of spitz-to-argos dose as a critical determinant of eye patterning. Indeed mRNA levels may not be always predictive of protein levels, and both translational regulation and post-translational modifications may well affect biological function. However, under conditions of stress or where specific transcriptional programmes bring about developmental outcomes, transcript levels may be thought to be well-correlated to protein levels. In addition, experiments using smFISH allows clear identification of the cells that are expressing specific genes, where the diffusible protein end-products may not provide as conclusive answers. We did attempt performing antibody staining for Spitz and Argos but such relative measures cannot be used to comment on expression stoichiometry. This study shows a clear differential expression of spitz and argos mRNA in the early eye field contributing to photoreceptor fate determination and also addresses the sensitivity of the system to the heterogeneity in the expression levels of gene networks and makes developmental programs robust. It has to be noted of course that signaling via EGFR is not the only pathway determining the ommatidial pattern in the eye. For example, Notch is known to play an important role in the initiation of neural development and also in ommatidial rotation. Buffered regulation of genes in different developmental pathways that crosstalk can decrease sensitivity to variations in a gene network and can help explain other reproducible and stereotypical patterns generated throughout the development (Pasnuri, 2023).

    ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet-mediated degeneration

    Animals have evolved multiple mechanisms to protect themselves from the cumulative effects of age-related cellular damage. This study revealed an unexpected link between the TNF (tumour necrosis factor) inflammatory pathway, triggered by the metalloprotease ADAM17/TACE, and a lipid droplet (LD)-mediated mechanism of protecting retinal cells from age-related degeneration. Loss of ADAM17, TNF and the TNF receptor Grindelwald in pigmented glial cells of the Drosophila retina leads to age-related degeneration of both glia and neurons, preceded by an abnormal accumulation of glial LDs. The glial LDs initially buffer the cells against damage caused by glial and neuronally generated reactive oxygen species (ROS), but in later life the LDs dissipate, leading to the release of toxic peroxidated lipids. Finally, this study demonstrates the existence of a conserved pathway in human iPS-derived microglia-like cells, which are central players in neurodegeneration. Overall, this study has discovered a pathway mediated by TNF signalling acting not as a trigger of inflammation, but as a cytoprotective factor in the retina (Muliyil, 2020).

    This paper reports a previously unrecognised role of ADAM17 and TNF in protecting Drosophila retinal cells from age- and activity-related degeneration. Loss of ADAM17 and TNF signalling in retinal glial cells causes an abnormal accumulation of LDs in young glial cells. These LDs disperse by about 2 weeks after eclosion (middle age for flies), and their loss coincides with the onset of severe glial and neuronal cell death. By 4 weeks of age, no intact glia or neurons remain. Cell death depends on neuronal activity: retinal degeneration and, to a lesser extent, LD accumulation are rescued in flies reared fully in the dark. LD accumulation does not merely precede, but is actually responsible for subsequent degeneration, because preventing the accumulation of LDs fully rescues cell death. The data indicate that Eiger/TNF released by ADAM17 acts specifically through the Grindelwald TNF receptor. Loss of ADAM17-mediated TNF signalling also leads to elevated production of mitochondrial ROS in glial cells, causing activation of the JNK pathway and elevated lipogenic gene expression. Together, these changes trigger cell death through the production of toxic peroxidated lipids. Importantly, toxicity is also contributed to by ROS generated by normal activity of neighbouring neurons. Finally, this study shows that a similar signalling module is conserved in mammalian cells: when ADAM17 is inhibited in human iPSC-derived microglial-like cells, the same series of events is seen: LD accumulation, elevated mitochondrial ROS and high levels of toxic peroxidated lipids (Muliyil, 2020).

    It is proposed that TNF is an autocrine trophic factor that protects retinal pigmented glial cells from age-related cumulative damage caused by the ROS that are normal by-products of neuronal activity. This ADAM17/TNF protection system is located specifically in retinal glial cells, but its role is to protect both glia and neighbouring neurons. In the absence of this TNF cytoprotective pathway, severe early-onset retinal neurodegeneration is seen. The data imply that cells die by being overwhelmed by toxic peroxidated lipids when abnormal accumulations of LDs disperse. This occurs in Drosophila middle age, when LDs stop accumulating and begin to disperse, triggering the cytotoxic phase of the ADAM17-/- phenotype. It is important to emphasise that despite the ADAM17/TNF protection system being located specifically in retinal glial cells, there is neuronal involvement. Not only does TNF indirectly protect against neurodegeneration, but photoreceptor neurons are also significant sources of the ROS that generate the toxic peroxidated lipids in glia. More generally, this work provides a model for investigating more widely the functional links between ageing, cellular stress, lipid droplet accumulation and neurodegeneration. Indeed, in the light of the discovery that the pathway discovered in Drosophila is conserved in human microglia-like cells, it is significant that lipid droplets have been reported to accumulate in human microglia, cells that are increasingly prominent in the pathology of Alzheimer's disease and other neurodegenerative conditions (Muliyil, 2020).

    In a Drosophila model of neuronal mitochondrionopathies, abnormal neuronal ROS production led to elevated neuronal lipid production, followed by transfer of the lipids to the PGCs, where LDs accumulated. In that work, lipase expression in neurons suppressed LD accumulation; in contrast, suppression was only observed when lipase was expressed in PGCs, not neurons, suggesting that in the case of ADAM17 mutants, the primary source of accumulating lipids is the glial cells. Despite not being able to detect a role for neuronal lipid production in ADAM17-/- mutants, it was found that photoreceptor neurons are significant sources of the ROS that generate the toxic peroxidated lipids in glia. Despite these differences between this work and what has been previously reported, a growing body of work points to a close coupling between ROS, the JNK pathway, lipid droplets and cellular degeneration, a relationship conserved in mammals. This study did not investigate the involvement of SREBP in mediating LD accumulation caused by ADAM17 loss, but its well-established connection with stress-induced and JNK-mediated lipid synthesis suggests that it is a likely additional shared component of this conserved regulatory axis (Muliyil, 2020).

    It has become clear that LDs are much more than simply passive storage vessels for cellular lipids; they have multiple regulatory functions. Indeed, although this study highlighted a developing picture of an LD/ROS-dependent trigger of cell death, in other contexts LDs have protective functions against oxidative damage, both in flies and mammals. This may occur by providing an environment that shields fatty acids from peroxidation by ROS and/or by sequestering toxic peroxidated lipids. Although this superficially appears to contradict the theme of LD/ROS toxicity, it is important to recall that in LD-related cell death is not simultaneous with LD accumulation. In fact, degeneration temporally correlates with the dispersal of LDs in middle age, rather than their earlier accumulation. Together, the strands of evidence from several studies suggest that it is the combination of elevated ROS and the dispersal of abnormally high quantities of lipids from previously accumulated LDs that trigger death. This suggests that cells die by being overwhelmed by toxic peroxidated lipids when abnormal accumulations of LDs break down in the presence of high levels of ROS. Experiments with Brummer lipase are consistent with this idea: the Brummer lipase was expressed from early in development, thereby preventing abnormal LD accumulation, and this protected against cell death. This sequence of events implies the existence of a metabolic switch, when LDs stop accumulating and begin to disperse, triggering the toxic phase of ADAM17 loss. It will be interesting in the future and may provide insights into the normal ageing process, to understand the molecular mechanism of this age- and/or activity-dependent change (Muliyil, 2020).

    ADAM17 is one of the most important shedding enzymes in humans, responsible for the proteolytic release of a vast array of cell surface signals, receptors and other proteins. Because of its role in signalling by both TNF and ligands of the EGF receptor, it has been the focus of major pharmaceutical efforts, with a view to treating inflammatory diseases and cancer. It is therefore surprising that it has been very little studied in Drosophila. This is the first report of Drosophila ADAM17 mutants. This study also confirmed for the first time that Drosophila ADAM17 is indeed an active metalloprotease, able to shed cell surface proteins including the Drosophila TNF homologue Eiger. The only other description of Drosophila ADAM17 function is mechanistically consistent with the data, despite relating to a different physiological context. In that case, ADAM17 was shown to cause the release of soluble TNF from the fat body so that it can act as a long range adipokine (Agrawal, 2016). Other ADAM17 substrates in different developmental or physiological contexts cannot be ruled out, although the relatively subtle phenotype of null mutant flies implies that ADAM17 does not have essential functions that lead to obvious defects when mutated. Moreover, wdevelopmental defects or LD accumulation were not observed in any neuronal or non-neuronal ADAM17-/- larval tissues, suggesting that the mechanism reported in this study is both age and tissue specific (Muliyil, 2020).

    Although TNF is sometimes viewed as a specific cell death-promoting signal, and the pathways by which it activates caspase-induced apoptosis have been studied extensively in flies and mammals, the response to TNF is in fact very diverse, depending on the biological context. Indeed, its most well-studied role in mammals is as the primary inflammatory cytokine, released by macrophages and other immune cells, and triggering the release of other cytokines, acting as a chemoattractant, stimulating phagocytosis, and promoting other inflammatory responses. However, TNF has not previously been shown to have trophic activity, protecting cells in the nervous system from stress-induced damage, although a link with the Nrf2/Keap1 redox pathway in cardiomyocytes provides an interesting parallel (Muliyil, 2020).

    In conclusion, this work highlights three important biological concepts. The first is to identify a new function for the ADAM17/TNF pathway in a cytoprotective role that protects Drosophila retinal cells against age- and activity-dependent degeneration. This contrasts with its well-established roles in inflammation and cell death. Secondly, the existence is highlighted of a glia-centric cellular pathway by which the breakdown of accumulated LDs and ROS together participate in promoting stress-induced and age-related cell death. Finally, this study has shown that the core phenomenon of the ADAM17 protease, acting to regulate the homeostatic relationship between ROS and LD biosynthesis, is conserved in human microglial cells, which themselves are intimately involved in neuroprotection (Muliyil, 2020).

    Loss of Na/K-ATPase in Drosophila photoreceptors leads to blindness and age-dependent neurodegeneration

    The activity of Na+/K+-ATPase establishes transmembrane ion gradients and is essential to cell function and survival. Either dysregulation or deficiency of neuronal Na+/K+-ATPase has been implicated in the pathogenesis of many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and rapid-onset dystonia Parkinsonism. However, genetic evidence that directly links neuronal Na+/K+-ATPase deficiency to in vivo neurodegeneration has been lacking. This study used Drosophila photoreceptors to investigate the cell-autonomous effects of neuronal Na+/K+ ATPase. Loss of ATPα, an α subunit of Na+/K+-ATPase, in photoreceptors through UAS/Gal4-mediated RNAi eliminated the light-triggered depolarization of the photoreceptors, rendering the fly virtually blind in behavioral assays. Intracellular recordings indicated that ATPα knockdown photoreceptors were already depolarized in the dark, which was due to a loss of intracellular K+. Importantly, ATPα knockdown resulted in the degeneration of photoreceptors in older flies. This degeneration was independent of light and showed characteristics of apoptotic/hybrid cell death as observed via electron microscopy analysis. Loss of Nrv3, a Na+/K+-ATPase β subunit (see Nervana 1 and Nervana 2), partially reproduced the signaling and degenerative defects observed in ATPα knockdown flies. Thus, the loss of Na+/K+-ATPase not only eradicates visual function but also causes age-dependent degeneration in photoreceptors, confirming the link between neuronal Na+/K+ ATPase deficiency and in vivo neurodegeneration. This work also establishes Drosophila photoreceptors as a genetic model for studying the cell-autonomous mechanisms underlying neuronal Na+/K+ ATPase deficiency-mediated neurodegeneration (Luan, 2014).

    The Na+/K+-ATPase transports Na+ and K+ against their concentration gradients across the cell membrane to maintain a low Na+ and high K+ concentration within the cells. These ion gradients determine the resting membrane potential and form the basis of the excitability of neurons. The Na+ gradient also provides the driving force for various secondary active transporters that import glucose, amino acids, and other nutrients into the cell. Additionally, the ion concentrations maintained by Na+/K+-ATPase are important for regulating cellular volume and preventing cells such as neurons from swelling and lysing (Luan, 2014).

    Considering the importance of Na+/K+-ATPase in basic cellular functions, it is not surprising that either dysregulation or deficiency of neuronal Na+/K+-ATPase was observed in many neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD) and rapid-onset dystonia Parkinsonism (RDP). Thus, disrupting normal Na+/K+-ATPase activity in neurons has been proposed to contribute to the pathogenesis of neurodegeneration. Nevertheless, the link between the disrupting neuronal Na+/K+-ATPase activity and neuronal dysfunction/degeneration has yet to be clarified (Luan, 2014).

    Na+/K+-ATPase is composed of at least two subunits: a large catalytic α subunit and a regulatory, single-transmembrane-domain β subunit. Mammals have three α-subunit and two β-subunit genes and may express six structurally distinct Na+/K+-ATPase isoforms. In the brain, although the α3 and β2 subunits are expressed predominantly in neurons, the α2 and β1 subunits are found primarily in glia, and the α1 subunit is ubiquitously expressed. The Na+/K+-ATPase in glia is required to maintain a low K+ level in the neuronal environment and thus has a large impact on neuronal function and survival. Na+/K+-ATPase inhibitors like ouabain act on all Na+/K+-ATPase isoforms and cannot differentiate between the cell-autonomous effects of the Na+/K+-ATPase in neurons from those derived from the neighboring glia. Thus, genetic approaches are needed to modulate the Na+/K+-ATPase level in neurons to investigate the function of neuronal Na+/K+-ATPase. Genetic studies on the impact of neuronal Na+/K+-ATPase deficiency in the past decade, which were mostly based on characterization of heterozygous mutant mice of the α3 subunit, have identified defects in the function of central brain neurons but have not provided direct evidence of neurodegeneration (Luan, 2014).

    The Drosophila visual system expresses only one type of α subunit, ATPα, and three β subunits, Nrv1–3. This study used Drosophila photoreceptors as a genetic model to study the cell-autonomous functions of neuronal Na+/K+-ATPase. Although ATPα mutants in Drosophila exhibit extensive neurodegeneration, these mutants were not used because the degeneration is due to the loss of Na+/K+-ATPase not only in neurons but also in neighboring non-neuronal cells. Instead, using a UAS/Gal4-mediated RNAi approach, knocked down ATPα and Nrv1-3 were generated specifically in photoreceptors, and the impact of this knockdown was assessed on visual signaling and photoreceptor integrity in the fly (Luan, 2014).

    Na+/K+-ATPase activity establishes and maintains the characteristic transmembrane gradients of Na+ and K+, which underlie essentially all vertebrate and invertebrate cellular physiology. Although the importance of Na+/K+-ATPase to the function and survival of both neurons and non-excitable cells has been demonstrated by decades of pharmacological studies, the involvement of neuronal Na+/K+ ATPase defects in neurodegeneration has yet to be demonstrated in vivo. This cell-specific RNAi study reveals for the first time that Na+/K+-ATPase is essential for normal neuronal function of Drosophila photoreceptors. More importantly, this work provides in vivo evidence that links neuronal Na+/K+-ATPase deficiency to age-dependent neurodegeneration (Luan, 2014).

    Genetic studies on neuronal Na+/K+-ATPase function in the past decade, which were primarily based on the characterization of mice heterozygous for a mutation of the α3 subunit, have focused on central brain neurons; however, the importance of functional Na+/K+-ATPase in sensory neurons remains largely unknown. This study shows that knockdown of ATPα in Drosophila photoreceptor neurons abolishes their response to light, resulting in complete blindness in the fly. These results confirm the importance of Na+/K+-ATPase in animal sensory functions. The light response of Drosophila photoreceptors is mediated by cation influx (mostly Na+) through light-stimulated TRP channels. Photoreceptors in ATPα knockdown flies were unresponsive to light for two reasons. First, without a sufficient K+ gradient across the cell membrane, the cell has no negative resting membrane potential and, thus, no electrical driving force for cation influx through TRP channels. Second, in the absence of Na+/K+-ATPase, photoreceptors may have already accumulated a high intracellular level of Na+ in the dark, which prevents extracellular Na+ flow into the cell through TRP channels during light stimulation. However, loss of the light response may not be attributed to morphological defects in the photoreceptor. First, temporally controlled knockdown of ATPα in the adult stage excludes the involvement of obvious developmental problems. Second, the light response of photoreceptors was abolished in 1-day-old ATPα knockdown flies despite the overall normal shape of the rhabdomeres. Based on these findings, it is concluded that loss of the light response is independent of degeneration in ATPα knockdown photoreceptors (Luan, 2014).

    Drosophila photoreceptors have been used as a genetic model for retinal degeneration studies. Photoreceptor degeneration in many mutants is caused by defects in the regulation of the visual transduction cascade and is light-dependent. In ATPα knockdown photoreceptors, however, the visual cascade may not mediate or regulate degeneration because light deprivation does not change the severity of neurodegeneration in 10-day-old flies. Instead, the progressive degeneration in ATPα knockdown photoreceptors has demonstrated characteristics of apoptotic/necrosis hybrid cell death that are reminiscent of those observed in Na+/K+-ATPase-inhibited mammalian cells. When Na+/K+-ATPase is inhibited by ouabain, mammalian cells undergo hybrid cell death, which has been attributed to a loss of intracellular K+ ions. In vitro studies suggest that the depletion of intracellular K+ may induce apoptosis or act as a necessary cofactor to promote apoptosis. It is estimated that the intracellular levels of K+ in ATPα knockdown photoreceptors could be as low as 10 mM, which is comparable to the 50%-80% decrease in K+ observed in ouabain-treated mammalian cells. Thus, the low levels of intracellular K+ could have contributed to the degeneration of ATPα knockdown photoreceptors. Additionally, Ca2 + overload may also have a role in photoreceptor degeneration. The depolarization of the membrane potential in ATPα knockdown photoreceptors may activate voltage-gated Ca2 + channels and a reversed operation of the Na+-Ca2 + exchanger. Both activities will increase the intracellular Ca2 + concentration and could promote necrotic cell death. Finally, accumulation of Na+ inside ATPα knockdown photoreceptors impairs the driving force of nutrient import through the secondary membrane transporters, which may also play a role in cell degeneration. Hybrid cell death, an intermediate form of cell death falling along an apoptosis-necrosis continuum, can also be found in the neurodegeneration caused by excitotoxicity and ischemia. Therefore, further studies on the role Na+/K+-ATPase in hybrid cell death will elucidate the mechanism of the cell death bearing both apoptotic and necrotic features in different neuropathological conditions (Luan, 2014).

    Until now, most in vivo studies on Na+/K+-ATPase-related neurodegeneration have relied on either pharmacological agents (Bignami, 1966 and Lees, 1994) or Drosophila ATPα mutants. Those studies have suggested that both dysregulation and deficiency of Na+/K+-ATPase lead to extensive neurodegeneration. However, the degeneration observed in those studies could be partially derived from defects in non-neuronal tissues and cells in the brain. For example, Na+/K+-ATPase in the blood-brain-barrier participates in the maintenance of water and ion homeostasis in the central nervous system (CNS), which is critical for neuronal function and survival. In the Drosophila auditory organ (Johnston's organ), Roy found that knocking down ATPα in scolopale cells, principal support cells that enclose neuronal dendrites, results in neuronal dysfunction and complete deafness. Additionally, a defect of Na+/K+-ATPase in astrocytes could be responsible for neonatal seizures and spongiform encephalopathy. In common neurodegenerative disorders such as AD, PD and RPD, however, Na+/K+-ATPase is only reduced in specific subgroups of neurons. To better mimic the neuropathological conditions of neurodegenerative diseases to study this degeneration mechanism, it is necessary to specifically downregulate Na+/K+-ATPase in particular neurons to avoid perturbations in other cells (Luan, 2014).

    Because homozygous mutations in the mouse α3 subunit of Na+/K+-ATPase cause neonatal lethality, genetic studies on neuronal isoforms of Na+/K+-ATPase have so far been primarily based on the characterization of heterozygous α3 mutants. Those mouse studies, however, have not revealed direct evidence of neurodegeneration in the brain most likely due to the relatively moderate reduction of Na+/K+-ATPase activity in the heterozygous mutants. This in vivo model using Drosophila photoreceptors, which mimics the neuropathological conditions of those neurodegenerative disorders, could be a valuable tool for further investigating the mechanism of neuronal Na+/K+-ATPase deficiency-mediated neurodegeneration. In addition to the genetic tools for gene modulation, Drosophila utilizes simple assays such as ERG, phototaxis and optomotor responses, and the optical neutralization assay to evaluate both the function and morphology of photoreceptor neurons. The loss of Na+/K+-ATPase in photoreceptors does not change the environmental K+ levels, allowing the study the cell-autonomous effects of neuronal Na+/K+-ATPase deficiency. Taking advantage of this simple and convenient neuronal model may allow identification of the key players in Na+/K+-ATPase deficiency-mediated neurodegeneration, which will thereby guide in the design of new therapeutic strategies for neurodegenerative disorders (Luan, 2014).

    This study has provided evidence that either dysregulation or deficiency of neuronal Na+/K+-ATPase causes abnormal depolarization of neurons by disrupting the intracellular ion balance instead of extracellular ion homeostasis, which leads to neuronal dysfunction and behavioral abnormality. Furthermore, disrupted neuronal Na+/K+-ATPase activity triggers progressive neurodegeneration. Therefore, this study suggests that early intervention against dysregulation or deficiency of neuronal Na+/K+-ATPase may alleviate the progression of neurodegenerative disorders (Luan, 2014).

    Histamine Recycling Is Mediated by CarT, a Carcinine Transporter in Drosophila Photoreceptors

    Histamine is an important chemical messenger that regulates multiple physiological processes in both vertebrate and invertebrate animals. Even so, how glial cells and neurons recycle histamine remains to be elucidated. Drosophila photoreceptor neurons use histamine as a neurotransmitter, and the released histamine is recycled through neighboring glia, where it is conjugated to beta-alanine to form carcinine. However, how carcinine is then returned to the photoreceptor remains unclear. In an mRNA-seq screen for photoreceptor cell-enriched transporters, CG9317, an SLC22 transporter family protein, was identified and named CarT (Carcinine Transporter). S2 cells that express CarT are able to take up carcinine in vitro. In the compound eye, CarT is exclusively localized to photoreceptor terminals. Null mutations of cart alter the content of histamine and its metabolites. Moreover, null cart mutants are defective in photoreceptor synaptic transmission and lack phototaxis. These findings reveal that CarT is required for histamine recycling at histaminergic photoreceptors and provide evidence for a CarT-dependent neurotransmitter trafficking pathway between glial cells and photoreceptor terminals (Xu, 2015).

    Anatomical reconstruction and functional imaging reveal an ordered array of skylight polarization detectors in Drosophila

    Drosophila photoreceptors R7 and R8 in the dorsal rim area (DRA) of the compound eye are specialized to detect the electric vector (e-vector) of linearly polarized light. These photoreceptors are arranged in stacked pairs with identical fields of view and spectral sensitivities, but mutually orthogonal microvillar orientations. This anatomical arrangement in larger flies suggests that the DRA constitutes a detector for skylight polarization, in which different e-vectors maximally excite different positions in the array. To test the hypothesis, responses to polarized light of varying e-vector angles in the terminals of R7/8 cells using genetically encoded calcium indicators were measured. Data confirm a progression of preferred e-vector angles from anterior to posterior in the DRA, and a strict orthogonality between the e-vector preferences of paired R7/8 cells. Decreased activity in photoreceptors was observed in response to flashes of light polarized orthogonally to their preferred e-vector angle, suggesting reciprocal inhibition between photoreceptors in the same medullar column, which may serve to increase polarization contrast. Together, these results indicate that the polarization-vision system relies on a spatial map of preferred e-vector angles at the earliest stage of sensory processing (Weir, 2016).

    Circuit mechanisms underlying chromatic encoding in Drosophila photoreceptors

    Spectral information is commonly processed in the brain through generation of antagonistic responses to different wavelengths. In many species, these color opponent signals arise as early as photoreceptor terminals.This study measured the spectral tuning of photoreceptors in Drosophila. In addition to a previously described pathway comparing wavelengths at each point in space, A horizontal-cell-mediated pathway similar to that was found found in mammals. This pathway enables additional spectral comparisons through lateral inhibition, expanding the range of chromatic encoding in the fly. Together, these two pathways enable efficient decorrelation and dimensionality reduction of photoreceptor signals while retaining maximal chromatic information. A biologically constrained model accounts for the findings and predicts a spatio-chromatic receptive field for fly photoreceptor outputs, with a color opponent center and broadband surround. This dual mechanism combines motifs of both an insect-specific visual circuit and an evolutionarily convergent circuit architecture, endowing flies with the ability to extract chromatic information at distinct spatial resolutions (Heath, 2020).

    Fly photoreceptors encode phase congruency

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although it is now known that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. This study reverse-engineered the neural code of Drosophila photoreceptors and shows for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. To mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli (Friederich, 2016).

    Small fruit flies sacrifice temporal acuity to maintain contrast sensitivity

    Holometabolous insects, like fruit flies, grow primarily during larval development. Scarce larval feeding is common in nature and generates smaller adults. Despite the importance of vision to flies, eye size scales proportionately with body size, and smaller eyes confer poorer vision due to smaller optics. Variable larval feeding, therefore, causes within-species differences in visual processing, which have gone largely unnoticed due to ad libitum feeding in the lab that results in generally large adults. Do smaller eyes have smaller ommatidial lenses, reducing sensitivity, or broader inter-ommatidial angles, reducing acuity? And to what extent might neural processes adapt to these optical challenges with temporal and spatial summation? To understand this in the fruit fly, a distribution of body lengths (1.67-2.34mm; n=24) and eye lengths (0.33-0.44mm; n=24) was generated, resembling the distribution of wild-caught flies, by removing larvae from food during their third instar. Smaller eyes have substantially fewer and smaller ommatidia separated by slightly wider inter-ommatidial angles. This corresponds to a greater loss in contrast sensitivity than spatial acuity. Using a flight arena and psychophysics paradigm, it was found that smaller flies lose little spatial acuity, and recover contrast sensitivity by sacrificing temporal acuity at the neural level. Therefore, smaller flies sacrifice contrast sensitivity to maintain spatial acuity optically, but recover contrast sensitivity, almost completely, by sacrificing temporal acuity neurally (Currea, 2018).

    Phototactic T-maze Behavioral Assay for Comparing the Functionality of Color-sensitive Photoreceptor Subtypes in the Drosophila Visual System

    The Drosophila retina contains light-sensitive photoreceptors (R cells) with distinct spectral sensitivities that allow them to distinguish light by its spectral composition. R7 and R8 photoreceptors are important for color vision, and can be further classified into pale (p) or yellow (y) subtypes depending on the rhodopsin expressed. While both R7y and R7p are sensitive to UV light, R8y and R8p detect light in the green and blue spectrum, respectively. The ability of R7 and R8 photoreceptors to distinguish different spectral sensitivities and the natural preference for Drosophila towards light sources (phototaxis), allow for the development of a phototactic T-maze assay that compares the functionality of different R7 and R8 subtypes. A "UV vs. blue" choice can compare the functionalities of R7p and R8p photoreceptors, while a "UV vs. green" choice can compare the functionalities of R7y and R8y photoreceptors. Additionally, a "blue vs. green" choice could be used to compare R8p and R8y photoreceptors, while a "dark vs. light" choice could be used to determine overall vision functionality. The phototactic T-maze assay presented in this study is a robust, straight-forward and an inexpensive method to study genetic and developmental factors that contribute to the individual functionality of R7 and R8 photoreceptors, and is especially useful when performing large-scale genetic screens (Shaw, 2020).

    Interaction of "chromatic" and "achromatic" circuits in Drosophila color opponent processing

    Color vision is an important sensory capability of humans and many animals. It relies on color opponent processing in visual circuits that gradually compare the signals of photoreceptors with different spectral sensitivities. In Drosophila, this comparison begins already in the presynaptic terminals of UV-sensitive R7 and longer wavelength-sensitive R8 inner photoreceptors that inhibit each other in the medulla. How downstream neurons process their signals is unknown. This study reports that the second order medulla interneuron Dm8 is inhibited when flies are stimulated with UV light and strongly excited in response to a broad range of longer wavelength (VIS) stimuli. Inhibition to UV light is mediated by histaminergic input from R7 and expression of the histamine receptor ort in Dm8, as previously suggested. However, two additional excitatory inputs antagonize the R7 input. First, activation of R8 leads to excitation of Dm8 by non-canonical photoreceptor signaling and cholinergic neurotransmission in the visual circuitry. Second, activation of outer photoreceptors R1-R6 with broad spectral sensitivity causes excitation in Dm8 through the cholinergic medulla interneuron Mi1, which is known for its major contribution to the detection of spatial luminance contrast and visual motion. In summary, Dm8 mediates a second step in UV/VIS color opponent processing in Drosophila by integrating input from all types of photoreceptors. These results demonstrate novel insights into the circuit integration of R1-R6 into color opponent processing and reveal that chromatic and achromatic circuitries of the fly visual system interact more extensively than previously thought (Pagni, 2021).

    High-speed imaging of light-induced photoreceptor microsaccades in compound eyes

    ide compound eyes, photoreceptors contract to light changes, sharpening retinal images of the moving world in time. Current methods to measure these so-called photoreceptor microsaccades in living insects are spatially limited and technically challenging. this study present goniometric high-speed deep pseudopupil (GHS-DPP) microscopy to assess how the rhabdomeric insect photoreceptors and their microsaccades are organised across the compound eyes. This method enables non-invasive rhabdomere orientation mapping, whilst their microsaccades can be locally light-activated, revealing the eyes' underlying active sampling motifs. By comparing the microsaccades in wild-type Drosophila's open rhabdom eyes to spam-mutant eyes, reverted to an ancestral fused rhabdom state, and honeybee's fused rhabdom eyes, this study showed how different eye types sample light information. These results show different ways compound eyes initiate the conversion of spatial light patterns in the environment into temporal neural signals and highlight how this active sampling can evolve with insects' visual needs (Kemppainen, 2022a).

    Binocular mirror-symmetric microsaccadic sampling enables Drosophila hyperacute 3D vision

    To move efficiently, animals must continuously work out their x,y,z positions with respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes' optical image disparity is not understood because this fundamental information-limiting step has not been investigated in vivo over the eyes' whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enable Drosophila superresolution three-dimensional vision and proposes neural computations for accurately predicting these flies' depth-perception dynamics, limits, and visual behaviors (Kemppainen, 2022b).

    Fenk, L. M., Avritzer, S. C., Weisman, J. L., Nair, A., Randt, L. D., Mohren, T. L., Siwanowicz, I. and Maimon, G. (2022). Muscles that move the retina augment compound eye vision in Drosophila. Nature. PubMed ID: 36289333

    Muscles that move the retina augment compound eye vision in Drosophila

    Most animals have compound eyes, with tens to thousands of lenses attached rigidly to the exoskeleton. A natural assumption is that all of these species must resort to moving either their head or their body to actively change their visual input. However, classic anatomy has revealed that flies have muscles poised to move their retinas under the stable lenses of each compound eye. This study shows that Drosophila use their retinal muscles to smoothly track visual motion, which helps to stabilize the retinal image, and also to perform small saccades when viewing a stationary scene. When the retina moves, visual receptive fields shift accordingly, and even the smallest retinal saccades activate visual neurons. Using a head-fixed behavioural paradigm, it was found that Drosophila perform binocular, vergence movements of their retinas-which could enhance depth perception-when crossing gaps, and impairing the physiology of retinal motor neurons alters gap-crossing trajectories during free behaviour. That flies evolved an ability to actuate their retinas suggests that moving the eye independently of the head is broadly paramount for animals. The similarities of smooth and saccadic movements of the Drosophila retina and the vertebrate eye highlight a notable example of convergent evolution (Fenk, 2022).

    An eye on trafficking genes: Identification of four eye color mutations in Drosophila

    Genes that code for proteins involved in organelle biogenesis and intracellular trafficking produce products that are critical in normal cell function. Conserved orthologs of these are present in most or all eukaryotes including Drosophila melanogaster. Some of these genes were originally identified as eye color mutants with decreases in both types of pigments found in the fly eye. Using these criteria, this study molecularly mapped and evaluated the genome sequences of four eye color mutations: chocolate, maroon, mahogany, and red Malpighian tubules. Mapping was performed using deletion analysis and complementation tests. chocolate is an allele of the VhaAC39-1 gene, which is an ortholog of the Vacuolar H+ ATPase AC39 subunit 1. maroon corresponds to the Vps16A gene and its product is part of the HOPS complex, which participates in transport and in organelle fusion. red Malpighian tubule is the CG12207 gene, which encodes a protein of unknown function that includes a LysM domain. mahogany is the CG13646 gene, which is predicted to be an amino acid transporter. The strategy of identifying eye color genes based on perturbations in quantities of both types of eye color pigments has proven useful in identifying proteins involved in trafficking and biogenesis of lysosome related organelles. Mutants of these genes can provide valuable in vivo models to understand these processes (Grant, 2016).

    A model of the spatio-temporal dynamics of Drosophila eye disc development

    Patterning and growth are linked during early development and have to be tightly controlled to result in a functional tissue or organ. During the development of the Drosophila eye, this linkage is particularly clear: the growth of the eye primordium mainly results from proliferating cells ahead of the morphogenetic furrow (MF), a moving signaling wave that sweeps across the tissue from the posterior to the anterior side, that induces proliferating cells anterior to it to differentiate and become cell cycle quiescent in its wake. Therefore, final eye disc size depends on the proliferation rate of undifferentiated cells and on the speed with which the MF sweeps across the eye disc. A spatio-temporal model of the growing eye disc was developed in this study based on the regulatory interactions controlled by the signals Decapentaplegic (Dpp), Hedgehog (Hh) and the transcription factor Homothorax (Hth) and how the signaling patterns affect the movement of the MF and impact on eye disc growth was explored. Published and new quantitative data were used to parameterize the model. In particular, two crucial parameter values, the degradation rate of Hth and the diffusion coefficient of Hh, were measured. The model is able to reproduce the linear movement of the MF and the termination of growth of the primordium. It was further shown that the model can explain several mutant phenotypes, but fails to reproduce the previously observed scaling of the Dpp gradient in the anterior compartment (Fried, 2016).

    Increased avidity for Dpp/BMP2 maintains the proliferation of progenitors-like cells in the Drosophila eye

    During organ development, the progenitor state is transient, and depends on specific combinations of transcription factors and extracellular signals. Not surprisingly, abnormal maintenance of progenitor transcription factors may lead to tissue overgrowth, and the concurrence of signals from the local environment is often critical to trigger this overgrowth. Therefore, identifying specific combinations of transcription factors/signals promoting (or opposing) proliferation in progenitors is essential to understand normal development and disease. This study used the Drosophila eye as a model where the transcription factors hth and tsh are transiently expressed in eye progenitors causing the expansion of the progenitor pool. However, if their co-expression is maintained experimentally, cell proliferation continues and differentiation is halted. Hth+Tsh-induced tissue overgrowth was shown to require the BMP2 Dpp and the abnormal hyperactivation of its pathway. Rather than using autocrine Dpp expression, Hth+Tsh cells increase their avidity for Dpp, produced locally, by upregulating extracellular matrix components. During normal development, Dpp represses hth and tsh ensuring that the progenitor state is transient. However, cells in which Hth+Tsh expression is forcibly maintained use Dpp to enhance their proliferation (Neto, 2016).

    Abnormal maintenance of transcription factors that promote an undifferentiated, proliferative state is often an initiating event in tumors. However, abnormal growth is dependent on specific non-autonomous signals provided by the microenvironment. This study used an experimental system that results in continuous growth to identify these signals and the mechanism of action. In this system, the GAL4-driven maintenance during eye development of hth and tsh, two transcription factors normally transiently co-expressed in eye progenitors, cause cells to increase their avidity for Dpp. This, in turn, leads to a hyper-activation of the pathway, which is necessary to maintain the proliferative/undifferentiated phenotype. The increased avidity for Dpp was shown to be mediated, at least partly, through increased expression of the proteoglycans components encoded by dally and dlp, functionally modified by slf (Neto, 2016).

    Progenitor cells, forced to maintain Hth and Tsh (hth+tsh progenitor-like cells) trap Dpp produced at local sources, which then causes an increased in intracellular signaling. The mechanism responsible of this trapping seems to be the increase of extracellular matrix (ECM) components. First, a cell-autonomous increase was found in dally transcription and Dlp membrane levels, the two glypican moieties of heparane sulphate proteoglycans. Second, the RNAi-mediated attenuation of sfl function, a gene encoding an enzyme required for the biosynthesis of these proteoglycans, is required for the overgrowth/eye-suppression phenotype induced by hth+tsh maintenance. A third line of support comes from examination of the effects of hth+tsh or hth+tsh+slf RNAi on the pMad profiles. Considering that the Dpp production remains unaltered, hth+tsh tissue shows an increase in both pMad signal amplitude and range, which is consistent with the increase in proteoglycans simultaneously augmenting Dpp diffusion and stability. On the contrary, reducing proteoglycan biosynthesis in hth+tsh+slf RNAi cells results in the retraction of the pMad signaling range back towards control values, which again is expected if Dpp's diffusion depends on proteoglycans (Neto, 2016).

    By forcing the expression of hth and tsh in eye precursors, these cells are exposed to signaling levels higher than they would normally encounter. This is because during normal eye development Dpp, produced at the furrow, represses first hth and then, closer to the furrow, also tsh, so that the cells approaching the furrow and receiving the highest Dpp levels no longer co-express hth and tsh. The loss of hth marks the transition between proliferation/undifferentiation and cell quiescence/commitment. This transition coincides with a transient proliferative wave (the so-called 'first mitotic wave') that precedes entry into G1. This transition zone corresponds to a region where low, but not null, levels of Hth and pMad signals overlap. If the interaction between hth+tsh and the Dpp pathway described in this study were to hold also in the zone of hth/Dpp signal overlap during normal eye development (remember that hth-positive cells co-express normally tsh too), one prediction would be that the mitotic wave would be lost if either hth or dpp-signaling were removed. Indeed this has been shown to be the case: RNAi-mediated attenuation of hth or abrogation of Dpp signaling result in the loss of the first mitotic wave. However, it is not thought that the mechanisms driving Dpp-mediated proliferation of optix> hth+tsh cells are necessarily the same as those operating normally in hth+tsh-expressing progenitors during eye development, because of the following experiment. Discs were generated expressing in their dorsal domain an RNAi targeting Hth's partner, the Pbx gene extradenticle (exd). In the absence of Exd, Hth is degraded. Therefore, a depletion of Exd causes an effective loss of Hth. Knowing that in optix>hth+tsh the stability and diffusion of Dpp were increased, the prediction would be that the loss of hth (in exd-depleted cells) should cause a decrease in both the stability and diffusion of Dpp. However, when the dorsal ('exd-') with the ventral ('exd+') pMad profiles of D>exdRNAi discs was quantified, it was found that both the stability and diffusion of Dpp increased by the loss of hth. This result suggests that during normal eye development hth (perhaps together with tsh) influences Dpp signaling, but the mechanisms described in this study as triggered by forced hth+tsh expression are likely different (Neto, 2016).

    The upregulation of dally and dlp by hth+tsh is likely the consequence of the transcriptional activity of Hth+Tsh in partnership with the YAP/TAZ homologue, Yki, as previous work showed that loss of the protocadherin genes fat (ft) and dachsous (ds) , which causes the activation of Yki, results in an upregulation of dally and dlp in the wing primordium. In fact, previous studies have found, in imaginal tissues, binding of Yki and Hth to nearby sites on the dlp locus, suggesting that some of this regulation might be direct. All these data make Yki a necessary component of the molecular machinery responsible for the increased avidity of hth+tsh cells for Dpp. However, in the eye primordium, the overexpression of Yki induces a different phenotype than hth+tsh. More importantly, in the eye primordium, yki+ clones do not cause the autonomous upregulation of pMad signal that hth+tsh clones do. Therefore, a specific stoichiometry among Hth, Tsh and Yki is likely necessary to induce the Dpp signaling-dependent properties of hth+tsh cells, at least in the developing eye. Alternatively, Hth and Tsh may activate Yki-independent targets that would be required for the full expression of the phenotype. Recently, another study has found that Yki and the Dpp pathway synergize in stimulating tissue overgrowth, both in eye and wing primordia, through the physical association between Yki and Mad. The current results suggest that hth+tsh progenitor-like cells establish a positive feedback, in which the growth promoting activity of the Hth:Tsh:Yki complex would be enhanced by increasing levels of pMad activated by Dpp. This feedback would be region-specific, as it depends on sources of Dpp that are localized within the eye primordium. Further work is needed to investigate the molecular mechanisms behind this feedback. Finally, it has been shown recently that tissue growth promoted by the PI3K/PTEN and TSC/TOR nutrient-sensing pathways also requires Dally, which, in turn, increases the avidity of the growing tissue for Dpp. Therefore, increasing the avidity for Dpp by augmenting proteoglycan levels may be a common strategy of tissues to sustain their growth (Neto, 2016).

    The Drosophila functional Smad suppressing element fuss, a homologue of the human Skor genes, retains pro-oncogenic properties of the Ski/Sno family

    Over the years Ski and Sno have been found to be involved in cancer progression e.g. in oesophageal squamous cell carcinoma, melanoma, oestrogen receptor-positive breast carcinoma, colorectal carcinoma, and leukaemia. Often, their prooncogenic features have been linked to their ability of inhibiting the anti-proliferative action of TGF-β signalling. Recently, not only pro-oncogenic but also anti-oncogenic functions of Ski/Sno proteins have been revealed. Besides Ski and Sno, which are ubiquitously expressed other members of Ski/Sno proteins exist which show highly specific neuronal expression, the SKI Family Transcriptional Corepressors (Skor). Among others Skor1 and Skor2 are involved in the development of Purkinje neurons and a mutation of Skor1 has been found to be associated with restless legs syndrome. But neither Skor1 nor Skor2 have been reported to be involved in cancer progression. Using overexpression studies in the Drosophila eye imaginal disc, this study analysed if the Drosophila Skor homologue Fuss has retained the potential to inhibit differentiation and induce increased proliferation. Fuss expressed in cells posterior to the morphogenetic furrow, impairs photoreceptor axon pathfinding and inhibits differentiation of accessory cells. However, if its expression is induced prior to eye differentiation, Fuss might inhibit the differentiating function of Dpp signalling and might maintain proliferative action of Wg signalling, which is reminiscent of the Ski/Sno protein function in cancer (Rass, 2022).

    Negative regulators of the TGF-β signalling pathway are inhibitory Smads (I-Smads), Smurfs and the Ski/Sno protein family. Proteins of the latter group possess two structural domains: the Ski/Sno homology domain and the SMAD4-binding domain. With the help of these domains, Ski/Sno proteins can interact, among others, with R-Smads, N-CoR, Sin3a, SMAD4 and the histone deacetylase HDAC1 and this complex leads to transcriptional repression of target genes. By their expression domains, Ski/Sno proteins can be further subdivided into ubiquitously expressed genes (human Ski and Sno), and mainly neuronally expressed genes, the SKI Family Transcriptional Corepressors (Skor1 and Skor2). The Ski/Sno proteins fulfil a wide range of different physiological functions such as axonal morphogenesis, Purkinje cell development, myogenesis and mammary gland alveogenesis (Rass, 2022).

    However, the Ski/Sno proteins were not discovered by their physiological functions but via the transforming capability of the viral ski (v-ski) homologue found in the Sloan-Kettering virus. The first evidence that Ski/Sno proteins possess oncogenic capabilities came from overexpression experiments, where it was shown that the truncation of v-ski is not responsible for the transformation of chicken embryo fibroblasts, but that overexpression of v-ski, Ski or Sno is sufficient for this transformation. Despite this background, their role in carcinogenesis is still not fully understood, if not even contradictory at times. Ski and Sno have been found to be upregulated in different types of cancer e.g. oesophagus squamous cell carcinoma, melanoma, and colorectal cancer. Further evidence for a pro-oncogenic role was found in downregulation analyses of Sno or Ski. This downregulation resulted in decreased tumour growth in breast cancer cells and pancreatic cancer cells. But as stated before, there is some objection that Ski and Sno function purely as oncogenes. Mice, which were heterozygous mutant for Ski or Sno, showed an increased level of tumour induction after carcinogen treatment. In metastatic non-small cell lung cancer, Ski expression is significantly reduced, whereas increased expression of Ski in these cells reduced the invasiveness inhibiting epithelial-mesenchymal transition. Therefore, this could reflect that the outcome of Ski or Sno expression in cancer cells is dependent on the cell type or the actual status of the cancer cells and cancer cells often exploit Ski or Sno to inhibit the anti-proliferative effects of TGF-β signalling. Whereas Ski or Sno have been found to be involved in a lot of different cancer types, there is sparse evidence for deregulation of Skor proteins in cancer cells. Endogenously, Skor proteins have been linked to neurodevelopmental processes. After Skor1 overexpression, genes involved in axonal guidance or post-synapse assembly were differentially expressed. Skor2 is important for cerebellar Purkinje cell differentiation as in Skor2 knockout mice dendrite formation of Purkinje cells was impaired. Pathophysiologically, Skor1 has mainly been linked to restless leg syndrome and localized scleroderma (Rass, 2022).

    In Drosophila melanogaster, only one homologue of Ski and Sno, which is designated Snoo, and one homologue of Skor1 and Skor2, which is designated Fuss, exist. It has been recently shown that Fuss is interacting with SMAD4 and HDAC1. In overexpression assays, Fuss can inhibit Dpp signalling and endogenously, the Fuss/HDAC1 complex is required for bitter gustatory neuron differentiation and fuss mutant flies pause more often during walking. However, this study was interested whether the Skor/Fuss proteins retained their ability to inhibit differentiation and induce increased proliferation. For this purpose, Fuss was overexpressed in differentiating cells of the eye imaginal disc, an excellent model tissue to study regulatory gene function in the context of carcinogenesis. This overexpression impaired photoreceptor axon guidance and inhibited the differentiation of accessory cells such as cone cells and primary pigment cells, which are all transformed into a basal pigment cell type. In a second approach, fuss overexpressing clones were generated early during development in the eye imaginal discs, when cells are still proliferating. This resulted in vast outgrowths of undifferentiated tissue of the eye imaginal disc because fuss overexpression most likely inhibited Dpp-signalling, a member of the TGF-β superfamily. This work shows that Fuss retained the ability of Ski/Sno proteins to inhibit the antiproliferative effects of TGF-β signalling by analogous inhibition of Dpp-signalling, allowing proliferation to be sustained (Rass, 2022).

    The overexpression of fuss posterior to the morphogenetic furrow with the GMR-Gal4 driver line resulted in a nearly complete loss of all cell types in the adult eye. During development, photoreceptor axons were not able to target the appropriate layers of the optic lobe anymore and cone cells, primary pigment cells and bristle cells were transformed into a basal pigment cell fate. This transformation was caused by the inhibition of sv expression, which is crucial for accessory cell differentiation. Additionally, increased apoptosis during pupal development lead to the removal of photoreceptors and lastly adult eyes only consisted of cells containing pigment granules. This lack of differentiation cannot be explained by the Dpp inhibiting role Fuss exerts, when overexpressed, because inhibiting the Dpp signaling pathway via knockdown of Tkv or Med had no effect. Photoreceptor axon guidance is impaired, if Dpp signaling is disrupted in photoreceptors by the expression of the inhibitory Smad Dad. Thus, the observed photoreceptor axon guidance phenotype, when fuss is overexpressed with GMR, could indeed be a result of Dpp signaling inhibition. However, the loss of nearly all eye cell types is due to other effects (e.g. downregulation of sv and apoptosis) than Dpp signaling repression alone, because loss of Dpp signaling behind the morphogenetic furrow only results in mild patterning defects of the pupal retina. Nonetheless, the inhibition of cell differentiation has already been shown in other cancer models e.g., when two copies of the constitutive active form of the receptor tyrosine kinase dRETMEN2B are expressed with the GMR-Gal4 line, pupal retinas are devoid of any distinguishable cell types. This phenotype is indistinguishable from the phenotype of the pupal retinas generated by the overexpression of fuss via GMR-Gal4. In a screen for novel oncogenes from breast cancer patients, human transgenes have been overexpressed with the GMR-Gal4 driver line. Overexpression of human RPS12, a subunit of the small ribosomal subunit, whose expression is increased in various cancer types, leads also to a glazed eye phenotype. Therefore, different oncogenes can result in different outcomes when expressed with the GMR-Gal4 driver line and are not always leading to massive tissue overgrowth like the Yorkie overexpression. Most importantly, with this approach to overexpress fuss in cells which already were destined for acquiring a cell fate and have left the cell cycle, it was not possible to induce increased proliferation anymore, but could prevent cell differentiation (Rass, 2022).

    Consequently, a more pluripotent cell type in the eye imaginal disc was used and fuss overexpressing clones were induced prior to the formation of the morphogenetic furrow. These results allowed the assumption that in this context, fuss overexpressing clones do not react to the antiproliferative effects of the Dpp morphogen anymore. Instead, wg expression and thus, proliferation promotion might be maintained. This leads to outgrowths of clonal tissue from the eye imaginal disc of third instar larvae, which showed an increased number of mitotic events. If these flies survived to adulthood, undifferentiated, extra tissue was visible in the complex eye (Rass, 2022).

    An analogous mechanism can be observed in tumors which overexpress Ski or Sno. The TGF-β signaling pathway is also anti-proliferative, but this action is inhibited by the increased presence of Ski/Sno proteins. Therefore, the molecular mode of action is similar to the human Ski/Sno proteins. The function of Ski and Sno is highly context dependent, as they can fulfill an anti-oncogenic or pro-oncogenic role depending on the cancer type or status of the cancer. This was also observed with fuss overexpressing clones. Only when induced 48h after egg laying, was additional tissue found in late third instar larvae and only in eye imaginal discs, because there, Dpp counteracts the proliferative effects of Wg signaling. When fuss is overexpressed in the wing disc or after induction of the morphogenetic furrow differentiation is inhibited, this results in a wing with truncated veins or in a smooth eye surface. This is also underlined by RNAseq data from eye and wing imaginal discs, where fuss was overexpressed with the GMR-Gal4 and Nub-Gal4 driver line, respectively. In the eye dataset, wg expression in eye imaginal discs is not significantly different from control eye discs, whereas wg expression in fuss overexpression wing discs is significantly reduced in contrast to control wing discs (Rass, 2022).

    Thus, this study showed that the Skor protein Fuss in Drosophila melanogaster still retained the function of the Ski/Sno proteins by inhibiting differentiation but inducing hyperproliferation. But the hallmarks of real tumorigenesis are lacking, because at some point during pupal development, proliferation stops, and these cells become protruding head tissue as it could be observed in complex eyes of surviving flies. Furthermore, there was no evidence of an epithelial-mesenchymal transition because fuss overexpressing clones maintained their epithelial fate. It will be of high interest if future studies can find similar results in overexpression studies for the vertebrate Skor proteins or detect increased expression of these proteins in specific cancer types (Rass, 2022).

    The cuticular nature of corneal lenses in Drosophila melanogaster

    The dioptric visual system relies on precisely focusing lenses that project light onto a neural retina. While the proteins that constitute the lenses of many vertebrates are relatively well characterized, less is known about the proteins that constitute invertebrate lenses, especially the lens facets in insect compound eyes. To address this question, this study used mass spectrophotometry to define the major proteins that comprise the corneal lenses from the adult Drosophila melanogaster compound eye. This led to the identification of four cuticular proteins: two previously identified lens proteins, drosocrystallin and retinin, and two newly identified proteins, Cpr66D and Cpr72Ec. To determine which ommatidial cells contribute each of these proteins to the lens, in situ hybridization was conducted at 50% pupal development, a key age for lens secretion. The results confirm previous reports that drosocrystallin and retinin are expressed in the two primary corneagenous cells-cone cells and primary pigment cells. Cpr72Ec and Cpr66D, on the other hand, are more highly expressed in higher order interommatidial pigment cells. These data suggest that the complementary expression of cuticular proteins give rise to the center vs periphery of the corneal lens facet, possibly facilitating a refractive gradient that is known to reduce spherical aberration. Moreover, these studies provide a framework for future studies aimed at understanding the cuticular basis of corneal lens function in holometabolous insect eyes (Stahl, 2017).

    Developmentally regulated autophagy is required for eye formation in Drosophila

    This study demonstrates that autophagy, an evolutionarily conserved self-degradation process of eukaryotic cells, is essential for eye development in Drosophila. Autophagic structures accumulate in a specific pattern in the developing eye disc, predominantly in the morphogenetic furrow (MF) and differentiation zone. Silencing of several autophagy genes (Atg) in the eye primordium severely affects the morphology of the adult eye through triggering ectopic cell death. In Atg mutant genetic backgrounds however genetic compensatory mechanisms largely rescue autophagic activity in, and thereby normal morphogenesis of, this organ. The results also show that in the eye disc the expression of a key autophagy gene, Atg8a, is controlled in a complex manner by the anterior Hox paralog lab (labial), a master regulator of early development. Atg8a transcription is repressed in front of, while activated along, the MF by labial. The amount of autophagic structures then remains elevated behind the moving MF. These results indicate that eye development in Drosophila depends on the cell death-suppressing and differentiating effects of the autophagic process. This novel, developmentally regulated function of autophagy in the morphogenesis of the compound eye may shed light on a more fundamental role for cellular self-digestion in differentiation and organ formation than previously thought (Billes, 2018).


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    Genes involved in organ development

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