The Interactive Fly

Genes involved in tissue and organ development

Central Nervous System (CNS) - Ventral Cord (page 2 | page 1)

  • Determination of neuroblast identity in the neurectoderm
  • Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells
  • Spindle alignment is achieved without rotation after the first cell cycle in Drosophila embryonic neuroblasts
  • Patterns of embryonic neurogenesis in a primitive wingless insect, the silverfish; comparision with those in flying insects
  • Using Fas2 to chart the structure of the neuropile
  • Glia and axonogenesis
  • Embryonic origins of a motor system: Motor dendrites form a myotopic map in Drosophila
  • Developmental architecture of adult-specific lineages in the ventral CNS of Drosophila
  • Lineage and birth date specify motor neuron targeting and dendritic architecture in adult Drosophila
  • Lineage mapping identifies molecular and architectural similarities between the larval and adult Drosophila central nervous system
  • Neuron hemilineages provide the functional ground plan for the Drosophila ventral nervous system
  • Condensation of the CNS in Drosophila is inhibited by blocking hemocyte migration or neural activity
  • Polarity and intracellular compartmentalization of Drosophila neurons
  • A pair of interneurons influences the choice between feeding and locomotion in Drosophila
  • A group of segmental premotor interneurons regulates the speed of axial locomotion in Drosophila larvae
  • Even-skipped+ interneurons are core components of a sensorimotor circuit that maintains left-right symmetric muscle contraction amplitude
  • A circuit mechanism for the propagation of waves of muscle contraction in Drosophila
  • Postmating circuitry modulates salt taste processing to increase reproductive output in Drosophila
  • MicroRNA-encoded behavior in Drosophila
  • Anterior-posterior gradient in neural stem and daughter cell proliferation governed by spatial and temporal Hox control
  • Temporal cohorts of lineage-related neurons perform analogous functions in distinct sensorimotor circuits
  • Neural circuitry that evokes escape behavior upon activation of nociceptive sensory neurons in Drosophila larvae
  • Neuronal cell fate specification by the molecular convergence of different spatio-temporal cues on a common initiator terminal selector gene
  • Gap junction-mediated signaling from motor neurons regulates motor generation in the central circuits of larval Drosophila

    Glia and axonogenesis

    Separate sections of The Interactive Fly group genes according to their involvement inglia morphogenesis and axonogenesis.

    Determination of neuroblast identity in the neurectoderm

    The Drosophila central nervous system (CNS) develops from a bilateral neuroectoderm that lies on either side of a narrow strip of ventral midline cells. Single neuroectodermal cells delaminate from the surface epithelium, in a fixed pattern, and move into the interior of the embryo to form neural precursor cells called neuroblasts. The early neuroblasts form an orthogonal grid of four rows (1, 3, 5, and 7) along the anterior-posterior (AP) axis and three columns (ventral, intermediate, and dorsal) along the dorsoventral (DV) axis. Subsequently, each neuroblast expresses a characteristic combination of genes and contributes a stereotyped family of neurons and glia to the CNS. Thus the earliest steps in patterning the CNS are the formation and specification of neuroblasts.

    Neuroblast formation is regulated by two phenotypically opposite classes of genes: Proneural genes promote neuroblast formation, whereas the neurogenic genes inhibit neuroblast formation. Proneural genes encode a family of basic helix-loop-helix transcription factors that are expressed in 4-6 cell clusters at specific positions within the neuroectoderm. Embryos lacking the proneural genes achaete/scute or lethal of scute have a reduced number of neuroblasts (for review, see Skeath, 1994). Conversely, neurogenic genes are expressed uniformly in the neuroectoderm, and embryos that lack any one neurogenic gene function, such as Notch or Delta, develop an excess number of neuroblasts (for review, see Campos-Ortega, 1995).

    Neuroblast identity is determined in the neuroectoderm. Neuroblasts delaminate in five waves spanning approximately three hours. The generation of neuronal diversity begins with the specification of unique neuroblast identities along both the anterior-posterior (AP) and dorsal-ventral (DV) axes. The pair-rule genes wingless, hedgehog, gooseberry, and engrailed are expressed in stripes of neuroectoderm that subdivide the AP axis. These genes are required for establishing AP row identity within the neuroectoderm and neuroblasts (Chu-LaGraff, 1993; Zhang, 1994; Skeath, 1995; Bhat, 1996; Matsuzaki, 1996; Bhat, 1997 and McDonald, 1997). For example, gooseberry is expressed in row 5 neuroectoderm. Embryos lacking gooseberry function have a transformation of row 5 into row 3 neuroectoderm and neuroblast identity, whereas misexpression of gooseberry results in the converse row 3 to row 5 transformation (Zhang, 1994 and Skeath, 1995). Similarly, wingless encodes a protein secreted from row 5 and required for specifying the fate of the adjacent rows 4 and 6 neuroectoderm and neuroblasts (Chu-LaGraff, 1993). For information on the expression of segmentation genes and neuroblast identity genes in specific neuroblasts, see Chris Doe's Hyper-Neuroblast map.

    Three genes are expressed in restricted domains along the DV axis within the neuroectoderm: ventral nervous system defective (vnd) is an NK2 class homeobox gene expressed in the ventral column neuroectoderm (Jimenez, 1995 and Mellerick, 1995) and muscle segment homeobox (msh) is a homeobox gene expressed in the dorsal column neuroectoderm and neuroblasts (D'Alessio, 1996 and Isshiki, 1997). Mutations in vnd cause defects in neuroblast formation and lead to severe defects later in neurogenesis (White, 1983 and Skeath, 1994). Mutations in msh result in a partial transformation of dorsal neuroblasts into a more ventral or intermediate column identity, without affecting neuroblast formation (Isshiki, 1997). Signaling via the EGF receptor is required to establish ventral and/or intermediate column fates in the neuroectoderm (Rutledge, 1992; Raz, 1993; Schweitzer, 1995; Skeath, 1998; Udolph, 1998 and Yagi, 1998). A newly cloned homeobox gene, intermediate neuroblasts defective (ind) is the first gene known to be expressed specifically in the intermediate column of neuroectoderm and neuroblasts. ind function is required for the establishment of intermediate column identity in the neuroectoderm, and for the formation of intermediate column neuroblasts. There is a hierarchical cascade of transcriptional repression. Vnd represses ind expression to establish the ventral boundary of ind transcription, and ind represses msh to establish the ventral boundary of msh transcription. The homeobox genes expressed in columns within the Drosophila neuroectoderm--vnd, ind, and msh--each have gene homologs expressed in corresponding domains along the DV axis of the vertebrate neural ectoderm. On this basis it appears that fundamental molecular mechanisms of DV patterning may be similar in Drosophila and vertebrates (Weiss, 1998 and McDonald, 1998).

    Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells

    Stem cell asymmetric division requires tight control of spindle orientation. To study this key process, Drosophila larval neural stem cells (NBs) engineered to express fluorescent reporters for microtubules, pericentriolar material (PCM), and centrioles, were examined. Early in the cell cycle, the two centrosomes become unequal: one organizes an aster that stays near the apical cortex for most of the cell cycle, while the other loses PCM and microtubule-organizing activity, and moves extensively throughout the cell until shortly before mitosis when, located near the basal cortex, it recruits PCM and organizes the second mitotic aster. Upon division, the apical centrosome remains in the stem cell, while the other goes into the differentiating daughter. Apical aster maintenance requires the function of Pins. These results reveal that spindle orientation in Drosophila larval NBs is determined very early in the cell cycle, and is mediated by asymmetric centrosome function (Rebollo, 2007).

    Immediately after cytokinesis, the single dot revealed by both PCM and centriole reporters splits in two, strongly suggesting that centrosome duplication has taken place. The YFP-Asl marker, like other centriolar markers in Drosophila, does not allow for resolution of individual centrioles within a centrosome in larval NBs. Therefore, timing of centriole duplication in these cells remains uncertain. The two resulting centrosomes migrate together within the single major aster of the cell to the apical cortex. Later on, one of the centrosomes loses PCM and starts to migrate. At this early time point in the cell cycle, unequal centrosome fate is already established: one, apical, will remain in the stem cell; the other will go into the differentiating daughter. Migration of the downregulated centrosome (revealed by the centriolar marker), initially within the apical side of the cell and more basally later on, occupies most of the cell cycle and is the most variable stage, its duration being dependent on cell-cycle length. The apical centrosome organizes the only aster found in the NB for most of the cell cycle. As mitosis onset approaches, the moving downregulated centrosome becomes stabilized at the basal side and starts to accumulate PCM and organize the second aster. As a direct consequence, the spindle is assembled already in alignment with the polarity axis of the cell. In Drosophila male germline stem cells (Yamashita, 2007), one of the centrosomes is also consistently located adjacent to the hub from early interphase onward. Only this centrosome maintains a robust aster through the cell cycle. The other, associated with only a few microtubules, moves away from the hub and is inherited by the gonialblast. In these cells, the oldest centriole is always in the centrosome that is proximal to the hub and is therefore retained by the stem cell (Yamashita, 2007). It has not yet been possible to determine which of the two centrosomes contains the oldest centriole in larval neural stem cells. In pins NBs, unequal centrosome fate and function are established, but, eventually, the stable, aster-forming apical centrosome is downregulated and starts to behave like the other, migrating across the cell. Like the other too, it organizes an aster only shortly before NEB. The place of assembly of the two asters in pins mutant NBs is not fixed and consequently spindle orientation is randomized, and so is the size difference between the two daughter cells (Rebollo, 2007).

    It is still unclear how NB polarity is maintained from one cycle to the next; a distinct Baz apical crescent is only assembled at prophase. The permanent positioning of the NB centrosome in the apical side of the cell, through the cell cycle, suggests that it could be contributing to specifying the apical cortex after mitosis (Rebollo, 2007).

    In summary, four main conclusions can be derived from these observations: (1) the two centrosomes of asymmetrically dividing Drosophila larval NBs become unequal early in the cell cycle in terms of mobility, MTOC activity, and fate; (2) such elaborated unequal centrosome regulation provides a means to position the asters, thus ensuring spindle alignment along the polarity axis of the cell; (3) Pins contributes to spindle orientation in NBs by preventing the downregulation of the MTOC capability of the apical centrosome, thus maintaining the apical aster in place, and (4) spindle orientation is predetermined and can be accurately predicted as soon as the aster reaches the apical cortex during the initial stages of the cell cycle. Altogether, these observations reveal that asymmetry in Drosophila neural stem cells goes beyond the polarized localization of a number of protein complexes during mitosis and may affect entire organelles such as the centrosome, which exerts a major effect on cell architecture and function throughout the cell cycle (Rebollo, 2007).

    Spindle alignment is achieved without rotation after the first cell cycle in Drosophila embryonic neuroblasts

    Spindle alignment along the apicobasal polarity axis is mandatory for proper self-renewing asymmetric division in Drosophila neuroblasts (NBs). In embryonic NBs, spindles have been reported to assemble orthogonally to the polarity axis and later rotate to align with it. In larval NBs, spindles assemble directly aligned with the axis owing to the differential spatiotemporal control of the microtubule organising activity of their centrosomes. This study has recorded embryonic NBs that express centrosome and microtubule reporters, from delamination up to the fourth cell cycle, by two-photon confocal microscopy, and it was found that the switch between these two spindle orientation modes occurs in the second cell cycle of the NB, the first that follows delamination. Therefore, predetermined spindle orientation is not restricted to larval NBs. On the contrary, it actually applies to all but the first cell cycle of embryonic NBs (Rebollo, 2009).

    In terms of centrosome behaviour and microtubule-organising center (MTOC) activity, NBs in their first cell cycle are somewhat between the neuroectodermal cells from which they derive and older NBs. In epidermoblasts and delaminating NBs, centrosomes duplicate long before mitosis and both centrosomes have feeble MTOC activity. MTOC activity has been shown to be very weak during interphase in many Drosophila cell types (Rogers, 2008). At mitosis onset, the centrosomes of epidermoblasts and delaminating NBs start to gain MTOC activity and to migrate to opposite sides of the nucleus defining a line that is nearly orthogonal to the apicobasal axis along which the spindle assembles. However, while spindle orientation remains unchanged through mitosis in epidermoblast, it changes in the newly differentiated NB at metaphase to an apicobasal orientation. In most cases, rotation occurs in the direction that positions the slightly larger aster on the apical side. Completion of the first cytokinesis results in the basal delivery of the first GMC and leaves the NB centrosome on the apical side of the cell. This is the landmark of the switch to the predetermined alignment mode in which differential centrosome behaviour leads to spindle assembly directly along the apicobasal axis, and in which no major spindle rotation occurs. The apical centrosome contains a considerable amount of PCM, organises a large microtubule aster, and stays at the apical cortex of the cell. The other centrosome, almost totally devoid of PCM and microtubules, moves away from the apical cortex and remains motile, mostly around the basal half of the cell, until mitosis onset, when it starts to accumulate PCM and to nucleate microtubules near to the basal cortex. As a result, the spindle assembles directly along the apicobasal axis, and once more, but this time without significant spindle rotation, asymmetric division delivers a basal GMC. This process of asymmetric centrosome behaviour and aligned spindle assembly is repeated in the following cycles in the embryo and indeed in larval NBs. This mode has been observed in all the post-delamination NB cell cycles that could be unequivocally identified in the embryo by two-photon microscopy. It is therefore likely that such a mode operates in all but the first cell cycle in Drosophila NBs (Rebollo, 2009).

    When a NB delaminates from the epithelium, the apical stalk carries the Par complex from the apical cortex of the corresponding epidermoblast, which triggers the recruitment cascade that establishes apicobasal polarity during the first round of NB asymmetric cell division. However, because the Par complex and other known polarity markers fade away from the cortex after mitosis, it is unclear how cortical polarity orientation is passed on in the following cell cycles. The current results strongly suggest that the aster that stays anchored to the cortex during interphase might convey such information (Rebollo, 2009).

    Patterns of embryonic neurogenesis in a primitive wingless insect, the silverfish; comparision with those in flying insects

    Neurogenesis was examined in the central nervous system of embryos of the primitively wingless insect, the silverfish, Ctenolepisma longicaudata, using staining with toluidine blue and the incorporation of bromodeoxyuridine. The silverfish has the same number and positioning of neuroblasts as seen in more advanced insects and the relative order in which the different neuroblasts segregate from the neuroectoderm is highly conserved between Ctenolepisma and the grasshopper, Schistocerca. Of the 31 different neuroblasts found in a thoracic segment, one (NB 6-3) has a much longer proliferative period in silverfish. Of the remainder, 14 have similar proliferative phases, while 16 neuroblasts have extended their proliferative period by 10% of the duration of embryogenesis (10%E) or greater in the grasshopper, as compared with the silverfish. Both insects have similar periods of abdominal neurogenesis except that in the silverfish terminal ganglion, a prominent set of neuroblasts continues dividing until close to hatching, possibly reflecting the importance of cercal sensory input in this insect. This comparison between silverfish and grasshopper shows that the shift from wingless to flying insects was not accompanied by the addition of any new neuronal lineages in the thorax. Instead, selected lineages undergo a proliferative expansion to supply the additional neurons presumably needed for flight. The expansion of specific thoracic lineages was accompanied by the reduction of the terminal abdominal lineages, specifically NB6-3, as flying insects began to de-emphasize their cercal sensory system. This neuroblast is notable in that it is the only neuroblast from the grasshopper set that is missing in Drosophila. A reasonable speculation is that NB 6-3 makes interneurons that deal with ascending information from the cercus (Truman, 1998).

    Although abdominal neurogenesis in Ctenolepisma is roughly equivalent to that in the grasshopper, except in the terminal ganglion where it is higher, in the thorax it falls well below that seen in the grasshopper. In the silverfish, the last thoracic NBs stop dividing by about 70% of the way through embryonic development (70%E), whereas in grasshopper embryos, selected thoracic NBs continue dividing until slightly after 90%E. Comparison of the neurogenic periods between individual grasshopper and silverfish neuroblasts shows that only some of the neuroblasts have participated in this expansion. The greatest differences are seen for NBs 1-1, 5-1, and 6-4; these proliferate for 25%E to more than 30%E longer in the grasshopper as compared to the silverfish. An additional 14 neuroblasts have their proliferative period extended by at least 10%E in grasshopper embryos. The one neuroblast that goes counter to the overall trend is NB6-3, which has a proliferative period that extends for over 20%E longer in the silverfish. Hence, this lineage has become smaller in more advanced insects rather than becoming larger or staying the same. It is argued, however, that an increase in lineage size alone is not sufficient to conclude that that particular lineage produces neurons associated with flight (Truman, 1998).

    Using Fas2 to chart the structure of the neuropile

    Insect neurons are individually identifiable and have been used successfully to study principles of the formation and function of neuronal circuits. In Drosophila, studies on identifiable neurons can be combined with efficient genetic approaches. However, to capitalize on this potential for studies of circuit formation in the CNS of Drosophila embryos or larvae, it is necessary to identify pre- and postsynaptic elements of such circuits and describe the neuropilar territories they occupy. A strategy for neurite mapping is presented, using a set of evenly distributed landmarks labelled by commercially available anti-Fasciclin2 antibodies that remain comparatively constant between specimens and over developmental time. By applying this procedure to neurites labelled by three Gal4 lines, neuritic territories are shown to be established in the embryo and maintained throughout larval life, although the complexity of neuritic arborizations increases during this period. Using additional immunostainings or dye fills, Gal4-targeted neurites can be targetted to individual neurons and they can be characterized further as a reference for future experiments on circuit formation. Using the Fasciclin2-based mapping procedure as a standard (e.g., in a common database) would facilitate studies on the functional architecture of the neuropile and the identification of candiate circuit elements (Landgraf, 2003).

    Working with defined pre- and post-synaptic neurons is a prerequisite for the study of mechanisms that underlie circuit formation. The fact that such neurons establish synaptic contacts with one another requires that some of their neurites project to a common region. Thus, proximity of neurites is a criterion that can be used towards the identification of putative pre- and postsynaptic neurons. In Drosophila (like in other insects), synaptic contacts are restricted to the neuropile, a cell body-free area, which also contains the ascending, descending, and commissural fibers. Unlike the gray matter in the vertebrate spinal cord (where cell bodies and synapses are intermingled), neuronal cell bodies of the Drosophila CNS are restricted to the synapse-free 'cortex' from where they send monopolar projections into the neuropile. These neuropilar accumulations of neurites of CNS neurons (i.e., efferent and interneurons) are joined by projections from peripheral sensory neurons. The functionality of thus established neuronal circuits demands that the spatial arrangements of synapse-bearing neurites in the neuropile are fairly reproducible between different individuals, as has been learned from analyses in larger insects. In order to map these reproducible neurites in the Drosophila neuropile, predominantly anatomical landmarks of the neuropile have been used to date as reference points for the relative positions of neuronal projections. Such landmarks are segmentally repeated nerve roots and commissures, or easily identifiable fiber tracts (so far applied only in the imaginal CNS (Landgraf, 2003).

    This study capitalizes on a set of axon tracts that are labelled by the commercially available antibodies against the intracellular domain of Fasciclin2. These provide a set of standard landmarks that are evenly distributed throughout the neuropile. As shown by double-labellings with presynaptic markers, all Fasciclin2-positive fiber tracts are fully contained within the synaptic neuropile. They can be used in a very easy and efficient way for the charting of neurites in the neuropile. So far, Fasciclin2 fibre tracts have served as one-dimensional (mediolateral) landmarks in younger embryos. This approach has been extended by using the set of Fasciclin2 tracts in three dimensions and at different developmental stages. These analyses were exclusively centered on abdominal neuromeres for two reasons: predominantly, the abdominal motorsystem contributes to larval movement, and abdominal neuromeres face only minor reorganization during larval life (Landgraf, 2003).

    Each Fasciclin2 fascicle has been named according to its relative dorsoventral (D, dorsal; C, central; V, ventral) and mediolateral (M, medial; I, intermediate; L, lateral) position. Such a nomenclature is neutral and can therefore be applied to any set of axon fascicles. The pattern of Fasciclin2 tracts remains relatively constant throughout larval development and thereby permits comparisons and extrapolations across different developmental stages. The main change to the embryonic pattern of Fasciclin2 in the neuropile is the addition of further elements, particularly five transverse projections (TP1-5) per neuromere in larval stages, which provide added reference points for the anteroposterior axis. From their association with different motor axons in the larva (TP1 with RP2 and VUM; TP2 with aCC), it is concluded that TP1 represents the pISN and TP2 the aISN nerve root (Landgraf, 2003).

    In order to facilitate comparisons with published work, attempts were made to relate the Fasciclin2 pattern of the late embryo and larval stages to existing descriptions. For example, the Fasciclin2 pattern has frequently been used for work on the ventral nerve cord of earlier embryos, usually at 13 h of development. At this stage, three tracts can be resolved in the horizontal plane, of which the intermediate Fasciclin2 tract is formed or at least joined by axons of the MP1-interneurons (targeted by C544-Gal4), the medial tract by MP2-interneurons (targeted by 15J2-Gal4;. A split of the three tracts into vertically distinguishable bundles occurs during the next ca. 3 h. During this interval, it is still possible to trace the MP2/pCC- and MP1-axons via the C544- and 15J2-Gal4 lines when visualizing the Gal4-expressing neurons with the Uas-CD8-GFP reporter gene; later their Gal4-expression patterns change dramatically. Thus, despite the highly dynamic changes in the neuropile during this period (i.e., nerve cord condensation, closer apposition of neuropile at the midline and the fact that the intracellular Fasciclin2 domain vanishes from many cell bodies and axons), it is possible to map the MP2/pCC-axons to the DM (dorsointermediate) axons, and the MP1 interneuron axons to the dorsal CI-fascicles (Landgraf, 2003).

    Classical neurobiological work on neuronal circuitry in other insects has been based on mapping strategies that used morphologically distinguishable axonal tracts in the neuropile and relates these to projection patterns of neurons. Similar strategies have been used for the thoracic adult CNS of Drosophila. The DM- and VMd-fascicles serve as reliable landmarks for distinguishing dorsal, intermediate, and ventral commissural tracts. The distinct patterns of sensory projections of different modalities, linked to the classical neurobiological literature, reveal a partitioning of the larval Drosophila neuropile. In an effort to relate the pattern of Fasciclin2 tracts to neuropilar regions, use has been made of three different Gal4 lines that target different subpopulations of sensory neurons (C161-, MJ94-, MzCh-Gal4). Sensory projections are confined to ventral regions, while neurites of motorneurons occupy the very dorsal neuropile. Thus, there is little, if any, physical overlap and contact between afferent sensory projections and central motorneuron neurites during larval stages. However, some overlap might occur lateral to the DM-fascicle, most likely with projections of the dbd and vbd-neurons. Thus, the data suggest that there are few, if any, monosynaptic connections between sensory and motorneurons in the embryonic and abdominal larval ventral nerve cord of Drosophila. However, this is a fairly rough estimation that will have to be tested by more detailed studies in the future (Landgraf, 2003).

    Having described some spatial features of the neuropile with the help of the Fasciclin2 pattern, this charting strategy was next applied to three selected Gal4 driver lines. This effort is intended to identify and characterize neurons that are genetically amenable and that could be used for the investigation of neural circuit formation in the embryonic and larval Drosophila CNS. Three neural Gal4 lines were analyzed with precision. Before presenting detailed characteristics of abdominal Gal4-labelled neurons, an overview of the three Gal4 lines is provided: Per abdominal half-neuromere eve-Gal4RRK expresses Gal4 in two motor-(aCC and RP2) and one interneuron (pCC). DDC-Gal4 displays 9-11 Gal4-neurons, and MzVum-Gal4 12-14 cells plus 3 efferent VUM (Ventral Unpaired Median) neurons located at the ventral midline. In all three lines, Gal4 expression occurs in a defined sequence, and for most cells it is yet unclear to what extent a late onset of expression reflects a late birth and/or differentiation of those cells. Only in the aCC, pCC and VUM neurons is Gal4 expression initiated at the time of their respective births, thus making them amenable to genetic manipulations of axonal pathfinding and differentiation. Next, the relative strengths of Gal4 expression were compared and overall MzVum-Gal4 expresses strongest, followed by DDC-Gal4 and eve-Gal4RRK. However, Gal4 levels of different neuronal subsets in each Gal4 strain can differ significantly (e.g., in MzVum-Gal4, GABAergic interneurons express low levels while VUM and leucokinin-1-positive neurons express high levels). Because of differences in timing and strength of expression, Gal4-based manipulations would not be expected to affect all cells alike (Landgraf, 2003).

    By virtue of the Fasciclin2-positive landmarks, it was possible to work out detailed descriptions of the neuropilar positions of neurites labelled by the three Gal4 lines eve-Gal4RRK, MzVum-Gal4, and DDC-Gal4. These studies clearly show that the Fasciclin2 framework allows spatial relationships between neurites to be pinpointed even across specimens: for example, neurites of the aCC and RP2 neurons (eve-Gal4RRK) are concentrated to form an oval in each hemineuromere that is located at the level of the DL-fascicle, medial to the ascending section of transverse projection 2 and anterior to transverse projection 1. At the same level (of the DL-fascicle), MzVum-Gal4-labelled neurites form whirlwind-like arrangements that have oval holes in their centers. These holes map to the region where aCC and RP2 neurites are concentrated, as indicated by the transverse projection 2. Thus, by using Fasciclin2-positive tracts as landmarks, spatial relationships of neurites are reproducibly revealed in three dimensions (Landgraf, 2003).

    Since the pattern of Fasciclin2-positive axon tracts remains relatively constant from the late embryo to larva, it can also be used to investigate how neuronal projections change during this developmental period. The larval patterns of neurites described above are prefigured in the late embryo. For example, at late embryonic stages, the central arborisations of aCC and RP2 at the level of the DL-fascicle are also concentrated anterior to pISN (equals TP1 in the late larva), which corresponds to the region that lacks neurites in MzVum-Gal4 embryos. Thus, the principle spatial relationships between these sets of neurites (of aCC and RP2 versus those of MzVum-Gal4) appear to be laid down in the late embryo and maintained to larval stages, though the complexity and the spread of neurites increases over developmental time. This is an important observation because it suggests that: (1) By late embryonic stages neuritic arbors define those territories in the neuropile from which they will elaborate and spread during subsequent larval stages. Thus, principle spatial relationships between neurites are laid down during embryogenesis. (2) Data on the distribution of neurites obtained at one stage of development can be extrapolated and used to interpret other stages (Landgraf, 2003).

    As demonstrated so far, using Fasciclin2 stainings significantly improves descriptions of the characteristic patterns of central neurites targeted by different Gal4-lines. However, these patterns of neurites are composites of different neurons. With this in mind, attempts were made to define methods with which to resolve such complex neuritic patterns into their constituent parts (Landgraf, 2003).

    The first approach to tackle this problem is to employ antisera, which would reveal the morphologies of particular subsets of neurons. By using antibodies against the neurotransmitter/neuromodulator Serotonin and the neuropeptides Corazonin and Leucokinin-1 on nerve cords displaying Gal4-driven CD8-GFP expression, it is possible to define these neurites among the composite of Gal4-targeted projections that correspond to Serotonin, Corazonin, and Leucokinin-1 immunoreactive neurons and the regions of the neuropile that these occupy. Anti-Serotonin stains two and anti-Corazonin one neuron per hemineuromere. These three cells are targeted by the DDC-Gal4 line and appear to give rise to most of the DDC-Gal4-labelled neurites in the abdomen. Anti-Leucokinin-1 labels Gal4-targeted efferent projections forming type-3 terminals on the VL1-muscle (type-3v, DDC-Gal4;) and on the segment border muscle (type-3u, MzVum-Gal4). Of these, only the type-3u neuron is revealed by Leucokinin-1-like immunoreactivity and can thereby be traced back to a ventrolateral cell body in the CNS extending side branches toward the VL-fascicle. There are additional Leucokinin-1-positive projections associated with the DM-fascicle that are not targeted by MzVum-Gal4 but seem to originate from 2-4 (Gal4-negative) neurons at the anterior tip of the nerve cord. This has been confirmed by targeting the cytotoxin Ricin to MzVum::CD8-GFP neurons. This selectively abolishes all MzVum-Gal4-specific CD8-staining and the VL- but not the DM-associated Leucokinin-1-like immunoreactivity (Landgraf, 2003).

    In summary, it has been shown that a small range of antisera can readily be used to reveal the projections of particular subsets of neurons. Such specific stainings are well suited to serve as spatial reference points in their own right. Moreover, in this instance, the Serotonin, Corazonin, and Leucokinin-1 immunoreactive neurons were instrumental in revealing some of the constituent parts of the complex projection patterns of the DDC-Gal- and MzVum-Gal4-lines (Landgraf, 2003).

    Next, efferent neurons targeted by the three Gal4 lines were characterized and their axonal projections (nerve root and branch), target muscles, and terminal types were described. Based on morphological, molecular, and ultrastructural characteristics of motor terminals, several types of efferent neurons can be distinguished in Drosophila. It should be emphasised that distinctions between terminal types are not only of importance to studies of the Drosophila motor system but also correlate with differences between the central dendritic arbors of particular efferent neuron types. To classify the Gal4-labelled efferent neurons with respect to terminal type, a range of immunohistochemical stainings was employed: Synaptotagmin, Cysteine string protein, and Synapsin all represent presynaptic proteins involved in regulation of synaptic vesicles; Discs large is a predominantly postsynaptic protein, which labels the subsynaptic reticulum; and anti-Leucokinin-1 antisera detect an insect neuropeptide (Landgraf, 2003).

    While the visualization of Gal4-labelled neurites via immunostaining is efficient, it is at the same time limited to particular subsets of cells, leaving many neurons unidentified. This limitation can be overcome by using standard neuronal tracers. To reveal the morphologies of those Gal4::CD8-GFP neurons, the neural tracer dye Cascade Blue was iontophoretically applied to individual cells. Thus, it was possible to define the positions of somata and central projections of all efferent neurons and a number of interneurons (Landgraf, 2003).

    For the efferent neurons, it was found that their central projections are restricted to the dorsal neuropile (dorsal to the CI-fascicles). The only exception to this was the efferent SBM-neuron (MzVum-Gal4; whose short central arbors reside in the ventral neuropile where they associate with the VL-fascicle (consistent with Leucokinin-1 staining). In addition, it was found that differences in terminal type are reflected by distinctions in the central arbors of efferent neurons: while type-1 motoneurons elaborate extensive dendritic arbors (aCC and RP2; VA), efferent neurons with type-2 and type-3 terminals form comparatively sparse and stunted central arbors (VUM and SBM; VL1). Finally, these analyses suggest that the central projections of the same motoneuron in consecutive neuromeres do not overlap, i.e., they seem to behave in accordance with the tiling principle (Landgraf, 2003).

    The interneurons of two of the Gal4-lines have been identified previously: pCC (eve-Gal4RRK) lies adjacent to the aCC motorneuron; three interneurons of DDC-Gal4 are serotonergic or corazonergic. In addition, two MzVum-Gal4 interneurons were identified via Cascade blue fill. These two intersegmental interneurons seem to contribute to most or all MzVum-Gal4-targeted neurites in the ventral neuropile, ventral to the CI-fascicle (except for intersegmental ascending and descending projections and the leucokinin-1-positive neurites associated with the VL-fascicle). It is possible that additional ventral neurites might be derived from the mVg- and GABAergic neurons of MzVum-Gal4 (Landgraf, 2003).

    In summary, it was found that neurites targeted by MzVum-Gal4 segregate into a dorsal fraction, consisting primarily of motoneuronal side branches, and a ventral fraction derived almost exclusively from interneurons. This pattern simplifies interpretations of experimental results obtained with this Gal4-line (for example, if mutant backgrounds reveal selective impairment of only dorsal or ventral neurites). Having applied a combination of a standardized set of Fasicilin2-positive landmarks, specific antisera, and single cell tracings, it has been possible to (1) assign most neurites of the Gal4-lines to identified neurons, and (2) define the regions of the neuropile that they occupy. Future applications of a standardised mapping strategy to other Gal4 lines will considerably advance the understanding of the functional architecture of the Drosophila neuropile, and it will form a basis with which candidate pre- and postsynaptic circuit elements can be identified (Landgraf, 2003).

    An important aspect of this study is that despite its limited scope it reveals an apparent partitioning of the neuropile into (possibly functionally) distinct regions. Facets of a functional architecture of the neuropile have already been documented such as the modality-specific sensory projections that partition the ventral neuropile. Due to the Fasciclin2-based mapping, these areas can now be named and the regions can be related to projection patterns of other neurons. In accordance with work published for other insects, the dorsal neuropile is predominantly occupied by the central arbors of efferent neurons (with the single exception of the ventral type-3u neuron arbors). There is little overlap with sensory areas so that direct connections between sensory and motor neurons will be the exception. In addition, different efferent neurons elaborate their central arbors in distinct anteroposterior regions of the dorsal neuropile. These territories seem to be defined in the embryo and they are maintained through larval stages, although areas of overlap between formally distinct territories increase as central arbors become more elaborate over time. This relative constancy of the topography of the neuropile over time also exists for Serotonin-, Corazonin-, and Leucokinin-1-positive neurons. An important consequence of such constancy for future research work is that neurites can be compared or descriptions extrapolated across different developmental stages (Landgraf, 2003).

    Interestingly, neuropeptidergic projections seem to cluster in particular areas. Corazonin and Leucokinin-1 (and also Serotonin) are closely associated with the DM-fascicle. Published data suggest that antibodies against FMRF, molluscan neuropeptide SCPB, and Substance-P reveal neural structures that might also be localized in this median area. A second neuropeptide 'hot spot' is the VL-fascicles, where staining with anti-Leucokinin-1 antibodies is found. Also antisera against Allatostatin and Insulin appear to stain in this region. The fascicles are innervated by the posterior ascending cells of MzVum-Gal4 and DDC-Gal4 and curiously are detected with antisera against muscle myosin heavy chain. Interestingly, both of these neuropeptidergic 'hot spot' areas bear very prominent Fasciclin2-labelled neurites (Landgraf, 2003).

    Developmental architecture of adult-specific lineages in the ventral CNS of Drosophila

    In Drosophila most thoracic neuroblasts have two neurogenic periods: an initial brief period during embryogenesis and a second prolonged phase during larval growth. Adult-specific neurons that are born primarily during the second phase of neurogenesis. The fasciculated neurites arising from each cluster of adult-specific neurons express the cell-adhesion protein Neurotactin and they make a complex scaffold of neurite bundles within the thoracic neuropils. Using MARCM clones, the 24 lineages that make up the scaffold of a thoracic hemineuromere were identified. Unlike the early-born neurons that are strikingly diverse in both form and function, the adult specific cells in a given lineage are remarkably similar and typically project to only one or two initial targets, which appear to be the bundled neurites from other lineages. Correlated changes in the contacts between the lineages in different segments suggest that these initial contacts have functional significance in terms of future synaptic partners. This paper provides an overall view of the initial connections that eventually lead to the complex connectivity of the bulk of the thoracic neurons (Truman, 2004).

    In insect embryos, the vast majority of neurons in each segmental ganglion arise from 30 paired and one unpaired neuroblasts. In basal insect groups, these segmental NBs show a single neurogenic period, each producing all of its progeny during embryogenesis. In insects with complete metamorphosis, however, most of the segmental NBs in the thorax have two neurogenic periods, involving a relatively brief phase of neurogenesis during embryonic development followed by a much more prolonged phase during larval life. Mapping of postembryonic NBs in the thoracic neuromeres of Drosophila larvae indicated that 23 out of the 31 segmental NBs showed this second, larval phase of neurogenesis. The count from the present study is that there are 24 such clusters per hemisegment (Truman, 2004).

    The MARCM clones analyzed in this study were induced early in embryogenesis, and should include both the embryonic and postembryonic progeny from a given neuroblast. This, indeed, was seen when Actin-GAL4 or tub-GAL4 was used as a driver to make the MARCM clones. The diversity of morphologies and strength of GFP expression in the larval neurons, however, sometimes obscured some of the neurites arising from the associated adult-specific cluster. When similar clones were generated using the purported pan-neuronal driver line, elav [C155], the fully differentiated larval neurons in the clones typically failed to show GFP expression but expression was strong in the arrested, adult-specific cells. Although the reason that mature larval neurons fail to express under these conditions is not known, elav-based clones are invaluable for determining the exact projection patterns of the clusters of adult-specific neurons and how each contributed to the overall Neurotactin scaffold. Having established the morphology of the adult-specific region of the lineage, it was then possible to return to MARCM clones generated using tub-GAL4 and Actin-GAL4 drivers to associate the neurons of adult-specific clusters with their larval siblings. Since the larval progeny of all of the embryonic neuroblasts have been described, the larval neurons aided in identification of the embryonic neuroblast responsible for many of the adult-specific clusters (Truman, 2004).

    The early neurons generated by a given NB typically show a great diversity in terms of their type and their axonal projections. Indeed, the projection patterns of the daughter cells can change dramatically from one GMC to the next. Later born cells, though, appear to be much more similar in their morphologies, transmitters and functions. The present study shows that the similarity in late-born progeny is a general rule for all lineages. Although each NB may show a high degree of diversity in the first few neurons that it produces, the vast majority of their progeny are similar in their pathfinding decisions, with typically only one or two initial targets for the neurites that leave a cluster. Indeed, only 33 major projection patterns were found for the thousands of neurons that are born within a thoracic hemineuromere (Truman, 2004).

    The diversity of phenotypes in the early born cells of a lineage is accomplished through the sequential expression of a series of transcription factors (hunchback, kruppel, pdm and castor) that are passed from the NB to successive GMCs. This molecular specification of unique identities imposed by the neuroblast on the first few neurons in a lineage appears to be lacking in the later born neurons, all of which express grainyhead. It is suspected that the transition from uniquely specified GMCs to ones that express the same transcription factor marks the transition from generating unique individual neurons to generating neuronal classes. For the latter cells, interaction with other neurons, rather than factors supplied by their NB, may then be essential for establishing identity within their neuronal class. It should be noted that the transition between uniquely identified neurons to neuronal classes does not necessarily lie at the dividing line between the embryonic and postembryonic phases of proliferation. By feeding larvae on diet containing bromodeoxyuridine (BUdR) from the time of hatching, all of the neurons that are born during larval growth can be labelled. Analysis of Elav-based MARCM clones in these larvae showed some lineages in which some of the developmentally arrested neurons were unlabeled and, hence, were born prior to hatching. These were always the neurons in the clone that were nearest the neuropil (i.e., the oldest cells). Hence, the NBs do not necessarily stop dividing after they make the neurons that will be used in the larva, and they may depend on an extrinsic signal to terminate their embryonic phase of neurogenesis. These embryonically born cells may serve as pioneers to guide the growth of postembryonic members of their lineage (Truman, 2004).

    An interesting feature of the adult-specific neurons is that each extends an initial neurite to a lineage-specific location but then their development stalls until pupariation. As illustrated in the developing hippocampus, a developing neuron often sends out a single, unbranched process with a growth cone to navigate to an initial target. This is followed by interstitial sprouting, which then enables interactions with secondary targets. Contact with the initial target may persist or it may be lost through stereotyped pruning but connections with final targets are often then refined through local cell-cell interactions. In the adult-specific neurons in Drosophila, the period of developmental arrest separates axon pathfinding and contact with the initial target from the phase of interstitial sprouting to secondary targets. This arrest is terminated at the start of metamorphosis, when the neurons show a profuse sprouting, accompanied by the appearance of the broad-Z3 transcription factor, and the onset of nitric oxide (NO) sensitivity. The latter observation is especially interesting because studies on other insect neurons show that the onset of NO sensitivity occurs as a neuron shifts from pathfinding to interacting with its synaptic targets. The appearance of NO sensitivity at the termination of arrest suggests that the neurons have switched into a new developmental mode in which interactions with future synaptic partners become of prime importance (Truman, 2004).

    Hence, the larval CNS just prior to metamorphosis gives an unprecedented snap-shot of neuronal development. Thousands of neurons are arrested at their initial targets awaiting the hormonal signals that will initiate secondary sprouting. This probably represents a watershed in the development of the CNS. Up to this point in development, the identity of the neurons and their growth decisions may have been relatively 'hard-wired' by genetic information supplied by the NB and the ganglion mother cell. After this point, interactions with their primary and secondary targets probably dominate in shaping the final phenotypes of the cells (Truman, 2004).

    The map of initial contacts depicted in this study is undoubtedly not a complete description of all of these contacts. In addition, at this time the polarity of the contacts is not known, i.e. who will be presynaptic and who will be postsynaptic. Nevertheless, this map probably provides a broad overview of the first step in establishing the connectivity for the bulk of the thoracic neurons. These initial contacts acquire some functional importance when the segmental variation in their pattern is considered. The patterns in neuromeres T1, T3 and A1 are compared with the situation in T2, since this is the only segment that possesses the full complement of 24 postembryonic lineages. Importantly, many of the segmental changes involve coordinated changes in the lineages that project to the same region of the neuropil. The most obvious example involves the lineages associated with the ventrolateral neuropil. These include the motor lineage (lineage 15) that makes motoneurons exclusively and projects to a leg imaginal disc. Lineage 15 is confined to the thoracic neuromeres as are nine other lineages that send their neurite bundles exclusively to the ventrolateral neuropil. With one exception, these lineages show no obvious variation in their projection patterns between the three thoracic neuromeres. The only lineage that shows a variable projection pattern is lineage 1, which also has initial targets in two adjacent neuromeres. Accordingly this lineage retains its homosegmental projection (bundle 1c) in T1 but it lacks the 1i bundle (i.e., no bundle projects to the SEG). All of the lineages that project to the ventrolateral neuropil are absent from A1, with again the exception of lineage 1. The lineage 1 neurons arising in A1, though, all project to the T3 neuropil (via bundle 1i) and the homosegmental 1c bundle is missing. Identification of the lineages in the subesophageal neuromeres is not complete but it appears that most, if not all, of these lineages are also lacking from the subesophageal ganglion. Apart from lineages that project exclusively to the ventrolateral neuropil, there are a few lineages, like lineages 3 and 19 that have one bundle projecting to this neuropil and another projecting into more dorsal regions. This is especially interesting in the case of lineage 19 because its 19i bundle makes contact with the expanded area of the lineage 15 bundle and therefore may represent premotor interneurons. These ventrolateral projections, though, are missing in the A1 version of lineages 3 and 19. The uniformity of projection patterns within the thorax and their absence outside of this region of the body suggests that all of the lineages that project to ventrolateral neuropil make neurons involved with the sensory or motor requirements of the legs. This functional interpretation is supported by the fact that lineage 14 is one of the above lineages and its proposed homologues in grasshoppers (from NB 4-1) process input from leg mechanosensory hairs and integrate locomotor reflexes of the leg (Truman, 2004).

    Although the ventrolateral projections are relatively stable within the thoracic neuromeres, projections to intermediate and dorsal neuropils show striking segmental variation. For example, lineage 11 is absent from T3 and the two lineages that send neurite bundles that terminate next to those of lineage 11 in more anterior segments, have these bundles reduced (bundle 3id) or missing altogether (the 12im and 12id bundles of lineage 12) in this segment. T1 also has its unique set of changes. In T2 and posterior, the 0 bundle from the median NB projects to the aI commissure and appears to terminate between bundle 10c (ventral to it) and bundle 18c (dorsal). The 18c bundle is missing in T1 and we see that bundle 0 is redirected to the pI commissure. T1 also shows a marked reduction in the number of bundles that project to anterior neuromeres; bundle 18c is missing and bundle 19c is greatly reduced to only a few fibers. Thus, the neurons in the 18c and 19c bundles may be involved in coordination within the thorax rather than taking information to higher centers in the head. No obvious glial structures were found at the sites where the neurite bundles terminate. The correlated loss of converging bundles (such as seen for 12id, 3id and 11id in T3), suggest that the initial targets for the neurites in a bundle from one lineage may be bundles from other lineages. The map that is presented is the first attempt to identify the lineage-level rules that are used for establishing the initial connectivity map in the thoracic CNS. Whether these initial contacts are maintained and how they relate to secondary targets remains to be determined (Truman, 2004).

    Preliminary observations of embryonic induced single and double cell clones in lineage 6 show that in single neuron clones there is a single neurite that is either in the 6cm or 6cd bundle. By contrast, two neuron clones (arising from a GMC) show a neurite in both bundles. This suggests that the two bundles are built up by each GMC producing two daughters, one that chooses one pathway and one that chooses the other. While it obviously needs to be tested, it is expected that this pattern will hold for all of the lineages that have bundles projecting to two initial targets. Interestingly, in the cases in which one bundle is lost in a given segment the cell cluster in that segment is markedly smaller that in other segments. A possible mechanism to explain the segmental difference is that cell death shapes the projection pattern by having the inappropriate daughter cell die after its birth. Studies of the median lineage in grasshopper embryos show the importance of divergent sibling fates and cell death in shaping features of that lineage (Truman, 2004).

    The results from this study have developmental, behavioral and evolutionary implications. Previous studies on the ventral ganglia and the brain show that the neuroblasts express a striking diversity of transcription factors and signaling molecules. Some of these molecules are involved in the establishment of the unique identity of the neuroblasts and their early-born progeny. Others, though, may function later in directing patterns of connectivity. It has been difficult to determine the latter, however, because projection patterns and potential targets were unknown for the vast majority of neurons in the lineage. This study indicates that the first step in establishing the extreme complexity of CNS connections involves a rather simple set of rules, with the bulk of the neurons of a given lineage following one or two projection paths. At this time, it is not known if the 33 different projection trajectories seen in the thoracic neuropil are the product of just 33 individual neurons per hemineuromere that pioneer the track for the rest of their lineage or if all of the adult-specific neurons follow the same set of cues to their initial targets. Irrespective of how they navigate their path, the initial connectivity patterns suggest that neurons in one lineage use other lineages as their targets. This information should help gain an understanding of the roles of patterning genes such as wingless and hedgehog in establishing connectivity and neuronal properties within the CNS (Truman, 2004).

    The elegant studies of the neural circuitry underlying sensory to motor coordination in the legs of grasshoppers showed that functionally related neurons were clustered, and some, indeed are siblings that come from the same neuroblast. The uniformity of initial projections that were seen within each of the adult-specific lineages led to a speculation that each neuroblast is devoted to making a very small number of functional neuronal types, with the noted exceptions of the early-born cells that have unique identities sculpted by the expression of hunchback, kruppel, etc. Changes in specific behavioral functions between species might then be reflected in selective alterations in the particular lineages whose neurons participated in that behavior. One possible illustration of this is in the shift from primitively wingless insects to those that can fly. This was accompanied with marked increase in neuronal progeny in only 14 out of the 31 thoracic lineages. Indeed, the later born neurons in some subsets of lineages may co-evolve because these cells are functionally connected. Although there have been only minor differences in the neuroblast arrays when one compares grasshoppers to Drosophila, some of the neuroblasts have changed the blend of transcription factors that they express. It will be interesting to determine if these changes do indeed reflect a change in identity of the neuroblast or whether it reflects an alteration in instructions as to how these neurons should connect (Truman, 2004).

    Lineage and birth date specify motor neuron targeting and dendritic architecture in adult Drosophila

    Locomotion in adult Drosophila depends on motor neurons that target a set of multifibered muscles in the appendages. This study describes the development of motor neurons in adult Drosophila, focusing on those that target the legs. Leg motor neurons are born from at least 11 neuroblast lineages, but two lineages generate the majority of these cells. Using genetic single-cell labeling methods, the birth order, muscle targeting, and dendritic arbors for most of the leg motor neurons were analyze. The results reveal that each leg motor neuron is born at a characteristic time of development, from a specific lineage, and has a stereotyped dendritic architecture. Motor axons that target a particular leg segment or muscle have similar dendritic arbors but can derive from different lineages. Thus, although Drosophila uses a lineage-based method to generate leg motor neurons, individual lineages are not dedicated to generate neurons that target a single leg segment or muscle type (Baek, 2009).

    To study the development of the Drosophila leg motor neurons, a clonal analysis was performed using a modified version of mosaic analysis with a repressible cell marker (MARCM) method. The Vglut-Gal4 (also called OK371-Gal4) driver was used to positively label clones. This Gal4 driver, which is inserted into the Vglut gene, is expressed in all neurons that use glutamate as a neurotransmitter, including all motor neurons. As can be seen in adult leg preparations in which Vglut-Gal4 was used to express a membrane-tagged version of green fluorescent protein (CD8GFP), motor neurons innervating all of the muscles in the coxa (co), trochanter (tr), femur (fe), and tibia (ti) were labeled by this driver. In addition, a subset of sensory neurons, whose cell bodies reside in the tibia and tarsal segments, were labeled by Vglut-Gal4. Except for the tarsus, each leg segment has a stereotyped set of multifibered muscles that are labeled by the MHC-tauGFP reporter gene. This reporter gene was used to identify each of the muscles innervated by the leg motor neurons. In the adult CNS, Vglut-Gal4 labeled groups of neurons in each thoracic hemisegment. In addition to motor neuron cell bodies, the dendritic arbors of these neurons were observed in densely packed neuropils in each thoracic hemisegment. This study focused on the motor neurons innervating the first thoracic (T1) legs. The axons of these motor neurons fasciculate and exit the CNS through a large nerve that extends into the ipsilateral leg (Baek, 2009).

    Drosophila NBs are born during embryogenesis and undergo two waves of neurogenesis, one during embryogenesis and one during larval development. During the first, embryonic wave of NB divisions, the majority of the embryonically born neurons are dedicated to larval motor and sensory functions and die during metamorphosis. To determine how many independent NB lineages give rise to the leg motor neurons, positively labeled MARCM clones were induced during embryogenesis and analyzed in the adult. Because these clones were generated infrequently and early in development, entire NB lineages were labeled. These data revealed that the leg motor neurons are derived from at least 11 independent lineages. Strikingly, two of these lineages, Lin A and Lin B, give rise to the majority of the leg motor neurons. Embryonically induced clones of Lin A innervated the muscles of the femur and tibia but did not include any motor neurons that targeted the coxa or trochanter. Moreover, the tibia is only targeted by Lin A-derived motor neurons. Thus, Lin A motor neurons generally target distal, but not proximal, leg segments (Baek, 2009).

    The second major lineage defined by these experiments is Lin B, which gives rise to seven leg motor neurons. In contrast to Lin A, Lin B motor neurons target the three most proximal leg segments, the coxa, trochanter, and femur, but does not generate any motor neurons that target the tibia. Thus, Lin B motor neurons generally target proximal, rather than distal, leg segments (Baek, 2009).

    Embryonically induced MARCM clones revealed that another 12 Vglut-Gal4+ leg motor neurons are generated from nine additional lineages, Lin C to Lin K. These 12 motor neurons target the coxa (six), the trochanter (one), and the femur (five), but not the tibia. In contrast to Lin A and Lin B, these lineages give rise to only one or two Vglut-Gal4-expressing leg motor neurons. Lin E is distinctive because, in addition to generating a single motor neuron targeting the coxa, it also gives rise to ~25 Vglut-Gal4-expressing interneurons. Five of these lineages (C to G) were labeled frequently, by both embryonic and postembryonic heat shocks. In contrast, four of these lineages, Lin H to Lin K, were labeled infrequently and only by embryonic heat shocks. These findings suggest that these motor neurons, which target the coxa (one) and femur (five), are born during embryogenesis and persist to the adult stage in which they contribute to the adult leg nervous system (Baek, 2009).

    In total, 53 neurons were identified, derived from 11 independent NBs, that innervate the T1 leg. Two of these lineages give rise to 35 of these 53 motor neurons. By characterizing individually labeled motor neurons, the birth dates, muscle targets, and dendritic arbors for most of these motor neurons were determined. These results show that, although each motor neuron is born from a specific lineage, and at a specific time during development, individual lineages give rise to motor neurons that target multiple leg segments and multiple muscles within these leg segments (Baek, 2009).

    Accurate motor neuron development in the fly requires that axons target the correct muscles along the PD axis of the leg. This axis has several levels of refinement. The first level is the global PD axis of the leg. Lin A only generates motor neurons that target the two more distal leg segments, the tibia and the femur. In addition, Lin A is the only lineage that produces motor neurons that target the tibia. In contrast, the seven Lin B motor neurons target all leg segments except the tibia. Thus, there is a PD bias built into these lineages (Baek, 2009).

    A second level of refinement within the PD axis is targeting the correct muscle in individual leg segments. Among the Lin A-derived motor neurons, a PD bias was observed within the tibia and within the femur that correlates with birth date: the first half of the motor neurons born from Lin A have a strong bias for targeting proximal positions in these segments, whereas the later-born half of the motor neurons target distal muscles in these segments (Baek, 2009).

    Third, for muscles that are targeted by multiple motor neurons (e.g., ltm1 in the tibia), it was found that more distal projecting motor neurons are born before those that target more proximal positions in the same muscle. The differential targeting of axons to unique positions within the same muscle suggests the existence of high-resolution topographic maps that match specific motor neurons to specific muscle compartments, as has been observed in mouse skeletal muscles (Baek, 2009).

    Most of the leg motor neurons are born within a narrow window of development. The NB that gives rise to Lin A, for example, switches into a phase that is dedicated to generating leg motor neurons at ~50 h AEL. At that time, this NB begins to produce its 28 motor neurons for the next ~40 h. Presumably, this NB gives rise to nonmotor neuron progeny before this time and possibly after it completes this motor neuron generating phase. This scenario shares some similarities with the lineages that give rise to postembryonic neurons in the fly brain. For example, the entire mushroom body of Drosophila, the portion of the fly brain used in olfactory learning and memory, is derived from only four NBs that each give rise to one of four nearly identical anatomical units. Interestingly, there is a temporal switch in the types of neurons that these NBs generate at specific times of development. Thus, like Lin A, mushroom body NBs switch the type of neuron they generate at specific times. However, unlike the leg motor neuron NBs, those that generate the mushroom body are dedicated to forming this brain structure. In contrast, it was found that functionally related leg motor neurons, for example those that target a specific leg segment, muscle, or muscle type, are often derived from several different NB lineages. This logic is reminiscent of that used to generate olfactory projection neurons in the fly, in which three neuroblasts each give rise to different numbers and types of projection neurons (Baek, 2009).

    The temporal control of NB identity in Drosophila is directed by transcription factors that are sequentially expressed as NBs age. During embryogenesis, progeny postmitotic neurons inherit the transcription factor expressed in the NB at the time it was born. This temporal information works in combination with positional information that makes each NB unique, providing progeny neurons their individual identities. Although the specific factors are not yet known, a similar transcription factor code may exist for leg motor neurons. Two of the temporal control genes that are used during Drosophila embryogenesis, seven-up (svp) and castor (cas), are also important for controlling postembryonic neural fates. Interestingly, some NBs switch from expressing cas to svp at ~50 h AEL, similar to the time that the leg NBs begin to generate their leg motor neuron progeny. It will be interesting to determine whether this or other changes in transcription factors are responsible for initiating the production of leg motor neurons in the lineages defined here (Baek, 2009).

    These results demonstrate that adult motor neurons in the fly come from identifiable lineages that give rise to stereotyped progeny with defined birth dates. Importantly, however, of the 11 lineages that give rise to leg motor neurons in the fly, only one of these, Lin A, appears to be dedicated to producing these neurons. Even this restriction only occurs during the ~50 to ~90 h AEL time window. Although most of the progeny produced by the other lineages were not marked in these experiments (except for Lin E, which generates ~25 Vglut-Gal4+ interneurons), it is likely that these lineages also produce nonmotor neuron progeny. Thus, although seemingly invariant lineages are used in the fly, the closest relatives for many leg motor neurons are not other leg motor neurons. This conclusion is similar to the picture that emerged from lineage analyses performed in the vertebrate spinal cord showing that cell lineages are not dedicated to the production of motor neurons. As in the fly, closely related cells in the spinal cord may have distinct fates. Conversely, although adult fly motor neurons are born from stereotyped lineages, position within the CNS determines NB identities and, consequently, the progeny they generate. Although C. elegans has a more extreme version of a lineage-based mechanism, even in this case cell-cell signaling plays an important role in specifying identities. These considerations blur the distinction between lineage and position-based mechanisms and suggest that both play a role in vertebrates and invertebrates (Baek, 2009).

    Consistent with the idea that lineage may play a role in vertebrates, the transcription factor Coup-TF acts as a temporal switch between neurogenesis and gliogenesis in the vertebrate brain. Interestingly, Coup-TF is a relative of Drosophila svp, which encodes one of the temporal transcription factors used in postembryonic fly neuroblasts. The use of Coup-TF/Svp for executing a temporal switch in both flies and vertebrates suggests the existence of a conserved molecular mechanism for controlling developmental timing in neural lineages (Baek, 2009).

    Because motor neurons receive complex inputs from interneurons and sensory neurons, the architecture of their dendritic arbors is critical for forming the circuitry that is required for locomotion. An initial analysis of the dendritic arbors of the leg motor neurons suggests that, as in other systems, they exhibit a functional organization in the thoracic neuromere. For example, nine leg motor neurons, targeting two different reductor muscles in different leg segments (coxa and femur), have overlapping dendritic arbors. That these nine motor neurons have similar dendritic architectures suggests that they share presynaptic inputs, perhaps allowing these two reductor muscles to contract in synchrony. Similarly, all eight motor neurons that have dendrites that cross the midline of the CNS, and thus probably make contacts with neurons in the contralateral neuromere, send their axons to one of two long tendon muscles, one in the tibia and one in the femur. These two examples suggest that the organization of motor neuron dendrites may be important for muscle synergies as described in vertebrate locomotion (Baek, 2009).

    In vertebrate motor systems, motor neuron cell bodies are organized in columns and pools that correlate with their muscle targets. This organization implies that many of the presynaptic inputs into the motor neurons within individual pools will be similar. Consistently, in some cases, the dendritic arbors of motor neurons have been shown to correlate with motor neuron targeting. In these examples, the arborization patterns are controlled by the transcription factor Pea3, which requires a specific Hox code to be activated, but is only induced after motor axons invade the limb target. In contrast, the myotopic map exhibited by the dendrites of the fly larval motor neurons does not need target muscles to form. In the fly olfactory system, the dendrites of projection neurons form a map in the antennal lobe before the arrival of olfactory receptor neurons (ORNs), suggesting that this map forms independently of ORNs. It remains unclear whether the characteristic dendritic arbors of the fly's leg motor neurons require muscle targeting or whether they form independently of their targets using local cues in the CNS and the identities they acquire at birth (Baek, 2009).

    Lineage mapping identifies molecular and architectural similarities between the larval and adult Drosophila central nervous system

    Neurogenesis in Drosophila occurs in two phases, embryonic and post-embryonic, in which the same set of neuroblasts give rise to the distinct larval and adult nervous systems, respectively. This study identified the embryonic neuroblast origin of the adult neuronal lineages in the ventral nervous system via lineage-specific GAL4 lines and molecular markers. This lineage mapping revealed that neurons born late in the embryonic phase show axonal morphology and transcription factor profiles that are similar to the neurons born post-embryonically from the same neuroblast. Moreover, three thorax-specific neuroblasts not previously characterized were identified, and it was shown that HOX genes confine them to the thoracic segments. Two of these, NB2-3 and NB3-4, generate leg motor neurons. The other neuroblast is novel and appears to have arisen recently during insect evolution. These findings provide a comprehensive view of neurogenesis and show how proliferation of individual neuroblasts is dictated by temporal and spatial cues (Lacin, 2016).

    Neuron hemilineages provide the functional ground plan for the Drosophila ventral nervous system

    Drosophila central neurons arise from neuroblasts that generate neurons in a pair-wise fashion, with the two daughters providing the basis for distinct A and B hemilineage groups. Thirty three postembryonically-born hemilineages contribute over 90% of the neurons in each thoracic hemisegment. This study devised genetic approaches to define the anatomy of most of these hemilineages and to assess their functional roles using the heat-sensitive channel dTRPA1. The simplest hemilineages contain local interneurons and their activation causes tonic or phasic leg movements lacking interlimb coordination. The next level is hemilineages of similar projection cells that drive intersegmentally coordinated behaviors such as walking. The highest level involves hemilineages whose activation elicits complex behaviors such as takeoff. These activation phenotypes indicate that the hemilineages vary in their behavioral roles with some contributing to local networks for sensorimotor processing and others having higher order functions of coordinating these local networks into complex behavior (Harris, 2015).

    Condensation of the central nervous system in embryonic Drosophila is inhibited by blocking hemocyte migration or neural activity: Removing Pvr or disrupting Rac1 function inhibits CNS condensation

    Condensation is a process whereby a tissue undergoes a coordinated decrease in size and increase in cellular density during development. Although it occurs in many developmental contexts, the mechanisms underlying this process are largely unknown. This study investigated condensation in the embryonic Drosophila ventral nerve cord (VNC). Two major events coincide with condensation during embryogenesis: the deposition of extracellular matrix by hemocytes, and the onset of central nervous system activity. Preventing hemocyte migration by removing the function of the Drosophila VEGF receptor homologue, Pvr, or by disrupting Rac1 function in these cells, inhibits condensation. In the absence of hemocytes migrating adjacent to the developing VNC, the extracellular matrix components Collagen IV, Viking and Peroxidasin are not deposited around this tissue. Blocking neural activity by targeted expression of tetanus toxin light chain or an inwardly rectifying potassium channel also inhibits condensation. Disrupting Rac1 function in either glia or neurons, including those located in the nerve cord, causes a similar phenotype. These data suggest that condensation of the VNC during Drosophila embryogenesis depends on both hemocyte-deposited extracellular matrix and neural activity, and suggest a mechanism whereby these processes work together to shape the developing central nervous system (Olofsson, 2005).

    Thus, disrupting hemocyte migration inhibits VNC condensation in the embryo. Lack of hemocyte migration is associated with a severe reduction of ECM components (Collagen IV and Peroxidasin) throughout the embryo and more particularly a loss of these components around the VNC. This leads to a proposal that correct assembly of the ECM depends on hemocytes, and that basement membrane is required for condensation. Supporting a role for ECM in VNC condensation, defects are observed in loss-of-function mutants of integrins, which are ECM receptors and appear themselves to be required for correct assembly of basement membranes. Mutants in integrins or the ECM component Laminin A share at least one other phenotype with embryos in which hemocyte migration has been inhibited: gut morphogenesis is impaired. Thus, a dysfunctional ECM may explain several of the morphogenetic defects seen in embryos with defective hemocyte migration (Olofsson, 2005).

    How might basement membrane contribute to VNC condensation? Basement membrane may serve as a substrate for cellular movements involved in condensation and/or regulate signaling events relevant to condensation. Basement membrane is also required for normal neuromuscular junction development, and might be part of the functional blood-brain barrier in Drosophila. Hence, neural function may be disrupted when basement membrane formation is inhibited. However, condensation phenotypes in embryos with impeded hemocyte migration are more severe than in embryos in which neural activity has been blocked. This argues that the condensation phenotype seen in hemocyte migration-blocked embryos cannot be explained simply by a loss of neural activity (Olofsson, 2005).

    Although animals in which hemocyte migration is blocked fail to deposit Collagen IV appropriately, it has not been demonstrated that Collagen IV function is required for condensation. However, embryos expressing a dominant negative form of Collagen IV under the control of a heatshock promoter fail to condense their nerve cord. While these data point towards a functional role of Collagen IV in condensation, further studies will be necessary to clarify the specific role of Collagen IV during condensation (Olofsson, 2005).

    This study has not investigated whether phagocytosis of cells within the VNC contributes to condensation. pvr mutants show a perdurance of unengulfed cells at the ventral surface of the CNS at stage 14. The majority of these cells seem to disappear later, possibly engulfed by epidermal cells. pvr mutants also maintain some very restricted points of attachment between the epidermis and the VNC. This phenotype is not observed when hemocyte migration is blocked using mutant Rac1 expressed by crq-GAL4. This likely reflects failure of hemocyte migration at a later stage, after the two tissues have separated (Olofsson, 2005).

    The major cell type that engulfs apoptotic corpses within the CNS is the subperineural glia. In the absence of macrophages (in the Bic-D mutants), apoptotic cells are still expelled from the CNS but accumulate at the ventral surface, similar to the observations in the pvr mutant. Hemocytes are required for normal CNS morphogenesis: at stage 16, pvr mutants and Crq RNAi treated embryos have mispositioned glia and minor axon scaffolding defects. These data were interpreted to reflect a failure of engulfment of cell corpses. In the context of these findings, an additional cause for glial mispositioning in pvr mutant embryos could be a loss of basement membrane components and the failure to condense (Olofsson, 2005).

    VNC condensation correlates with the onset of neural activity in the CNS, and it is found that expressing tetanus toxin light chain or the inwardly rectifying K+ channel Kir2.1 pan-neuronally impairs condensation. This suggests that neural activity influences normal condensation. Neural activity could contribute to condensation in multiple ways. It could directly regulate cellular events relevant to condensation, such as adhesion or actin-based motility, or activity could influence the transcription of genes relevant to such events. Alternatively, neural activity could maintain synaptic connectivity among cells necessary for condensation, rather than directing changes in cellular behavior leading to condensation. Some condensation occurs before neural activity begins, and the condensation phenotypes resulting from impeding hemocyte migration are more severe than those resulting from blocking neural activity. This suggests that there may be multiple stages of condensation, including an earlier activity-independent stage and a later stage that is influenced by activity (Olofsson, 2005).

    VNC condensation can be inhibited by expressing mutant Rac1 in lateral glia or neurons. In glia, migration and ensheathing behaviors require cytoskeletal integrity. When mutant Rac1 is expressed in peripheral glia, the formation of cellular extensions is disrupted, and this is accompanied by glia migration and axon ensheathment defects. Similarly, ensheathment of longitudinal axon tracts by longitudinal glia is disrupted in htl loss of function embryos. The VNC condensation phenotype in these embryos is interpreted as indication that glia need dynamic actin cytoskeleton to generate a condensing force. Two types of VNC glia are particularly well placed to generate such a force: longitudinal glia associated with VNC longitudinal connectives, and perineural glia, which ensheath the cortex of the VNC. Cell-cell contacts and cell-ECM contacts among these cells accompanied by remodeling of extracellular matrix could help generate a condensing force within and across neuromeres through changes in cell shape, adhesion or migration. A similar process occurs during mesenchymal condensation (Olofsson, 2005).

    In neurons, neurite extension requires normal Rac GTPase activity. Expressing mutant Rac1 in these cells causes defects in axonal outgrowth. In wild type animals, VNC axons are arranged into longitudinal connectives that extend along the length of the nerve cord, and these are well placed to generate an anteroposterior condensing force. This could happen through differential cell adhesion of neurites within the longitudinal connectives or overall shortening of the axons. The observation that axons in VNC longitudinal connectives loop out during condensation in metamorphic insects is consistent with this idea. It is interesting to note that condensation is inhibited in embryos in which mutant Rac1 is expressed in glia, but longitudinal axon tracts appear normal in these animals. This suggests that if axons help generate a condensing force, they likely do this with the help of glia, possibly using these cells as a substrate (Olofsson, 2005).

    It is also possible that at least part of the force required for condensation may come from outside the VNC. Somatic muscles connect to the VNC during embryogenesis, and embryonic muscle activity toward the end of embryogenesis is well timed for generating such a force. Also, the methods used to manipulate glia or neuron development in this study may affect neuromuscular activity by disrupting blood-brain barrier formation, or by affecting the Rac-dependent formation of synaptic structures. However, the observation that the CNS can condense in mutants in which muscles do not form normally argues against a major contribution from muscle activity (Olofsson, 2005).

    These data identify several areas for further investigation. By following the behavior of small populations of cells in the VNC it may possible to analyze in vivo changes associated with the condensation process and get insight into how changes in organ shape are generated and coordinated. It will also be interesting to examine the contributions made by components of the ECM to normal blood-brain barrier function. Finally, it may be possible to use VNC condensation in embryonic Drosophila to investigate the molecular and cellular basis of how neural activity is translated into a morphogenetic event (Olofsson, 2005).

    Polarity and intracellular compartmentalization of Drosophila neurons

    Proper neuronal function depends on forming three primary subcellular compartments: axons, dendrites, and soma. Each compartment has a specialized function (the axon to send information, dendrites to receive information, and the soma is where most cellular components are produced). In mammalian neurons, each primary compartment has distinctive molecular and morphological features, as well as smaller domains, such as the diffusion barrier marked axon initial segment, that have more specialized functions. How neuronal subcellular compartments are established and maintained is not well understood. Genetic studies in Drosophila have provided insight into other areas of neurobiology, but it is not known whether flies are a good system in which to study neuronal polarity because a comprehensive analysis of Drosophila neuronal subcellular organization has not been performed. This study used new and previously characterized markers to examine Drosophila neuronal compartments. This study found that; (1) axons and dendrites can accumulate different microtubule-binding proteins; (2) protein synthesis machinery is concentrated in the cell body; (3) pre- and post-synaptic sites localize to distinct regions of the neuron, and (4) specializations similar to the initial segment are present. In addition, EB1-GFP dynamics were tracked and it was determined that microtubules in axons and dendrites have opposite polarity. It is concluded that Drosophila will be a powerful system to study the establishment and maintenance of neuronal compartments (Rolls, 2007; full text of article).

    To determine whether microtubule-binding proteins can be preferentially localized to axons and dendrites in flies, exogenous and endogenous microtubule-binding proteins were examined in the Drosophila larval brain. Two exogenous proteins were examined: tau-green fluorescent protein (tau-GFP) and nod-yellow fluorescent protein (nod-YFP). Some reports have suggested that tagged versions of the microtubule binding domain of bovine tau preferentially label axons in flies, although others have also reported dendrite localization. The distribution of one of these tagged bovine tau proteins, tau-myc-GFP (which is call tau-GFP for simplicity) was examined in mushroom body and projection neurons. In both mushroom body and projection neurons, tau-GFP was abundant in the main axon tracts. It was less abundant in distal axons and dendrites. For comparison, mCD8-GFP is present in all neuronal compartments. Thus, tau-GFP preferentially labels proximal axons. Fusion proteins that consist of the nod motor domain, kinesin coiled-coil, and a tag have previously been localized to dendrites. To confirm that nod fusions label dendrites specifically, nod-YFP was expressed in mushroom body and projection neurons. In both cases nod-YFP localized clearly to dendrites but not axons. Thus, two exogenous microtubule-binding proteins, tau-GFP and nod-YFP, localize to different neuronal compartments, indicating that axonal and dendritic microtubules have distinct features in Drosophila (Rolls, 2007).

    The localization was examined of two Drosophila microtubule-binding proteins under control of their own promoters, GFP-Map205 and GFP-Jupiter. Since a difference in microtubule orientation in axons and dendrites is a fundamental aspect of vertebrate neuronal polarity, it was of interest to determine exactly how microtubules are arranged in fly neurons. The dendritic localization of nod fusion proteins, which are believed to act as minus-end directed motors, has been used to argue that Drosophila dendrites are likely to have minus-ends distal to the cell body like vertebrate dendrites. However, direct analysis of the orientation of individual microtubules has not been performed in any invertebrate neuron (Rolls, 2007).

    Analysis of a microtubule plus-end tracking protein was used in mammalian neurons to confirm that axon and dendrite microtubules have different orientations. Since these proteins generally bind only to the growing plus ends of microtubules, microtubule orientation can be inferred from the direction of movement of a tagged plus-end binding protein. The peripheral nervous system of the Drosophila larva is well-suited to live imaging and has been used to study actin dynamics. The plus-end binding protein EB1-GFP was expressed throughout the nervous system using an elav-Gal4 driver, and time lapse imaging of the dorsal cluster of the peripheral nervous system was performed in live, whole, early L2 larvae. EB1-GFP dynamics were analyzed in axons and dendrites of dendritic arborization neurons, which have highly branched dendrites (Rolls, 2007).

    EB1-GFP dots were clearly seen moving in the cell body, axons, and dendrites. Movements of EB1-GFP dots were consistent with the tagged protein binding only to growing microtubule plus ends: an individual dot that could be followed through multiple frames never changed direction, and after a dot tracked through a particular region of an axon or dendrite and disappeared, often a dot with a similar track appeared several frames later. All EB1-GFP dots in axons moved away from the cell body. In dendrites the movements were very different. The vast majority of dots moved toward the cell body. Occasionally, dendrites were observed that had dots moving in opposite directions (Rolls, 2007).

    Thus, in fly dendritic arborization neurons, axonal microtubules were oriented with plus ends distal to the cell body. Most dendritic microtubules were oriented with minus ends distal to the cell body, although dendrite microtubules were sometimes mixed in orientation (Rolls, 2007).

    In mammalian neurons, the bulk of protein synthesis takes place in the cell body. A tagged ribosomal protein, L10-YFP, was generated to determine where protein synthesis takes place in Drosophila neurons. When expressed in both mushroom body and projection neurons, L10-YFP was concentrated in the cell body, with only very faint signal present in neuropil. Within the cell body it was seen in the cytoplasm, and in some cells it was also present in the nucleus, where ribosomes are assembled. Faint signal in dendrites may represent ribosomes or free L10-YFP (Rolls, 2007).

    To confirm that the L10-YFP marker represents the localization of endogenous protein synthesis machinery, its localization was compared to two proteins identified in a protein trap screen. One of the GFP insertions was in the belle gene. Bel is a DEAD-box protein that is likely to function as an RNA-binding protein with a role in translation initiation. The GFP transposon insertion was homozygous viable. Since bel is an essential gene, this means that the GFP-tagged protein that is generated from the insertion is likely to be functional, and thus the localization of the protein trap very likely represents that of the endogenous protein. Bel was broadly expressed, and in neurons it localized to the cell body. The other insertion analyzed was in the Protein disulfide isomerase gene. Pdi is a chaperone that resides in the endoplasmic reticulum (ER) lumen and is involved in processing newly synthesized membrane and secretory proteins. GFP-Pdi was also seen throughout the brain and was highly concentrated in the neuron cell body. Within the cell body, GFP-Pdi was brightest in the perinuclear region, which is consistent with localization to the ER. Thus, an exogenous protein synthesis protein, L10-YFP, and two endogenous ones, GFP-Bel and GFP-Pdi, were all concentrated in the neuronal cell body, suggesting that the bulk of protein synthesis takes place there (Rolls, 2007).

    One of the longest recognized forms of neuronal compartmentalization is concentration of postsynaptic sites to dendrites and presynaptic sites to axons. To determine whether excitatory synaptic inputs are received in dendrites, the distribution of the postsynaptic marker homer-GFP was analyzed in mushroom body neurons. Mammalian homer proteins bind metabotropic glutamate receptors and Shank, which forms a complex with NMDA glutamate receptors. The Drosophila Homer protein also binds Shank and localizes to postsynaptic sites. Homer-GFP localizes similarly to endogenous Homer. In brains that expressed Homer-GFP at low levels in the mushroom body, fluorescence was confined to dendrites and dots in the cell body (likely to be the Golgi), and was not present in axons. The pattern of Homer-GFP fluorescence in the mushroom body dendrite region (calyx) was similar to the strongest regions of staining with anti-Dlg and anti-Scrib immunofluorescence; both of these proteins are concentrated at postsynaptic sites. Thus, a marker of excitatory postsynaptic sites was polarized to dendrites (Rolls, 2007).

    Low expression level n-synaptobrevin-YFP (n-syb-YFP) transgenic flies were generatede to specifically label synaptic vesicles in the mushroom body. These transgenes had either one or two UAS sites upstream of the transcriptional start site, rather than the usual five. Spots of fluorescence were seen in the axons and dendrites; very little was present in the cell body. To confirm that the dots of fluorescence represented synaptic vesicles, brains expressing n-syb-YFP were stained in the mushroom body with cysteine string protein (CSP) and Scrib antibodies. CSP is a synaptic vesicle protein that is abundant in all presynaptic terminals, and Scrib is a synaptic protein that is concentrated in the postsynaptic terminal. Many of the n-syb-YFP dots in the calyx region of the brain, which contains mushroom body dendrites, overlapped with the CSP, but not the Scrib, pattern, indicating that n-syb-YFP is present in synaptic vesicles. Extrinsic neurons, such as olfactory projection neurons, synapse onto mushroom body dendrites in the calyx, and so it is expected that not all synaptic vesicles would be accounted for by n-syb-YFP expressed in mushroom body neurons. Having verified that the low expression n-syb-YFP marker colocalized with synaptic vesicles, it is concluded that synaptic vesicles are present in axons and dendrites of mushroom body neurons (Rolls, 2007).

    Thus far, this study has concentrated on basic differences between axons, dendrites and the cell body. One of the most important further regional specializations is the axon initial segment, which contains specific arrangements of membrane and cytoskeletal proteins. In this survey of marker localization in Drosophila neurons, two proteins were identified that showed very distinctive localization to the proximal neurite and axon (Rolls, 2007).

    NgCAM-YFP expressed at low levels was concentrated at the beginning of the neurite in mushroom body neurons. In projection neurons, NgCAM-YFP was clearly seen in the primary neurite before the dendrites branched off, and in the proximal axon beyond the dendrite branch point. Much fainter fluorescence was present in distal axons and dendrites. NgCAM is a chick neural cell adhesion molecule that is selectively localized to axons when expressed in cultured hippocampal neurons, and can be tethered by ankyrins in the initial segment (Rolls, 2007).

    Another tagged protein, Apc2-GFP, was targeted to the proximal region of Drosophila axons. In both mushroom body and projection neurons, Apc2-GFP was present in the cell body and dendrites. In mushroom body neurons it localized to just one region of the axons, near the beginning of the peduncle. In projection neurons, Apc2-GFP also localized to the proximal axon, but the pattern was not quite as striking as in the mushroom body, probably because Apc2-GFP expression levels were lower in the projection neurons. The region of the proximal axon to which Apc2-GFP was localized in mushroom body neurons was just distal to the stretch of proximal neurites in which NgCAM-YFP was concentrated. Adenomatous polyposis coli (APC) proteins regulate wingless signaling, and they also bind a number of cytoskeletal proteins, including microtubules and the plus-end microtubule binding protein EB1. The localization of two cytoskeleton-interacting proteins to the proximal axon in flies indicates that the Drosophila axon is divided into domains with specialized cytoskeletal properties. It will be interesting to determine whether this region is functionally similar to the vertebrate axon initial segment (Rolls, 2007).

    A pair of interneurons influences the choice between feeding and locomotion in Drosophila

    The decision to engage in one behavior often precludes the selection of others, suggesting cross-inhibition between incompatible behaviors. For example, the likelihood to initiate feeding might be influenced by an animal's commitment to other behaviors. This study examined the modulation of feeding behavior in the fruit fly, Drosophila melanogaster, and identified a pair of interneurons in the ventral nerve cord that is activated by stimulation of mechanosensory neurons and inhibits feeding initiation, suggesting that these neurons suppress feeding while the fly is walking. Conversely, inhibiting activity in these neurons promotes feeding initiation and inhibits locomotion. These studies demonstrate the mutual exclusivity between locomotion and feeding initiation in the fly, isolate interneurons that influence this behavioral choice, and provide a framework for studying the neural basis for behavioral exclusivity in Drosophila (Mann, 2013).

    The neurons that inhibit proboscis extension (which are named PERin) have cell bodies and processes in the first leg neuromeres of the VNC and projections to the SOG, the brain region that contains gustatory sensory axons and proboscis motor neuron dendrites. Labeling with the presynaptic synaptotagmin- GFP marker and the postsynaptic DenMark marker indicated that the dendrites of PERin neurons are restricted to the first leg neuromeres, whereas axons are found in both the SOG and the first leg neuromeres. The anatomy of these neurons suggests that they convey information from the leg neuromeres to a region of the fly brain involved in gustatory processing and proboscis extension. Anatomical studies examining the proximity of PERin fibers to gustatory sensory dendrites or proboscis motor axons revealed that PERin neurons do not come into close contact with known neurons that regulate proboscis extension (Mann, 2013).

    Many behaviors are mutually exclusive, with the decision to commit to one behavior excluding the selection of others. This study shows that feeding initiation and locomotion are mutually exclusive behaviors and that activity in a single pair of interneurons influences this behavioral choice. PERin neurons are activated by stimulation of mechanosensory neurons and activation of PERin inhibits proboscis extension, suggesting that they inhibit feeding while the animal is walking. Consistent with this, leg removal or immobilization enhances proboscis extension probability and this is inhibited by increased PERin activity. The opposite behavior is elicited upon inhibiting activity in PERin neurons: animals show constitutive proboscis extension at the expense of locomotion. This work shows that activity in a single pair of interneurons dramatically influences the choice between feeding initiation and movement (Mann, 2013).

    The precise mechanism of activation of PERin neurons remains to be determined. PERin dendrites reside in the first leg neuromere, suggesting that they process information from the legs. Stimulation of leg chemosensory bristles with sucrose or quinine or activation of sugar, bitter, or water neurons using optogenetic approaches did not activate PERin neurons, nor did satiety state change tonic activity. Stimulation of sensory nerves into the ventral nerve cord and stimulation of mechanosensory neurons, using a nompC driver, activated PERin. In addition, by monitoring activity of PERin while flies moved their legs, it was demonstrated that activity was coincident with movement (Mann, 2013).

    These studies argue that PERin is activated by nongustatory cues in response to movement, likely upon detection of mechanosensory cues. Additional cues may also activate PERin. Studies of behavioral exclusivity in other invertebrate species suggest two mechanisms by which one behavior suppresses others. One strategy is by competition between command neurons that activate dedicated circuits for different behaviors. More common is a strategy in which decision- making occurs by distributed activity changes across neural populations. Although this studies are a starting point to begin to examine these models in Drosophila, the circuits for proboscis extension and locomotion drive different motor neurons, muscles, and behaviors, suggesting that they may be connected by a few links rather than largely overlapping circuitry. PERin is likely to inhibit feeding initiation while the animal is moving and is one critical link. The observation that simply gluing the proboscis in an extended state, but not in a retracted state, inhibits locomotion suggests that motor activity or proprioceptive feedback from the proboscis acts as a reciprocal link to locomotor circuits (Mann, 2013).

    Neurons act over different timescales and in response to different sensory cues to influence behavior. The powerful molecular genetic approaches available in Drosophila enable the precise manipulation of individual neurons and allow for the examination of their function in awake, behaving animals. Modulatory neurons such as PERin are difficult to identify by calcium imaging or electrophysiological approaches because they influence gustatory-driven behavior but are not activated by gustatory stimulation. The ability to probe the function of neurons in unbiased behavioral screens facilitates the identification of neurons that act as critical nodes to influence behavior. The identification and characterization of PERin as a significant modulator of feeding initiation provides a foundation for future studies determining how PERin influences proboscis extension circuits to alter behavioral probability and how mechanosensory inputs activate PERin. In addition, examining how proboscis extension suppresses locomotion will provide important insight into the links between different behaviors (Mann, 2013).

    Neural circuits for a given behavior do not work in isolation. Information from multiple sensory cues, physiological state, and experience must be integrated to guide behavioral decisions. This work uncovers a pair of interneurons that influences the choice between feeding initiation and locomotion. The discovery of the PERin neurons will aid in examining the neural basis of innate behaviors and the decision-making processes that produce them (Mann, 2013).

    A group of segmental premotor interneurons regulates the speed of axial locomotion in Drosophila larvae

    Animals control the speed of motion to meet behavioral demands. Yet, the underlying neuronal mechanisms remain poorly understood. This study shows that a class of segmentally arrayed local interneurons (period-positive median segmental interneurons, or PMSIs) regulates the speed of peristaltic locomotion in Drosophila larvae. PMSIs formed glutamatergic synapses on motor neurons and, when optogenetically activated, inhibited motor activity, indicating that they are inhibitory premotor interneurons. Calcium imaging showed that PMSIs are rhythmically active during peristalsis with a short time delay in relation to motor neurons. Optogenetic silencing of these neurons elongated the duration of motor bursting and greatly reduced the speed of larval locomotion. These results suggest that PMSIs control the speed of axial locomotion by limiting, via inhibition, the duration of motor outputs in each segment. Similar mechanisms are found in the regulation of mammalian limb locomotion, suggesting that common strategies may be used to control the speed of animal movements in a diversity of species (Kohsaka, 2014).

    Even-skipped+ interneurons are core components of a sensorimotor circuit that maintains left-right symmetric muscle contraction amplitude

    Bilaterally symmetric motor patterns--those in which left-right pairs of muscles contract synchronously and with equal amplitude (such as breathing, smiling, whisking, and locomotion)--are widespread throughout the animal kingdom. Yet, surprisingly little is known about the underlying neural circuits. A thermogenetic screen was performed to identify neurons required for bilaterally symmetric locomotion in Drosophila larvae, and the evolutionarily conserved Even-skipped+ interneurons (Eve/Evx) were discovered. Activation or ablation of Eve+ interneurons disruptes bilaterally symmetric muscle contraction amplitude, without affecting the timing of motor output. Eve+ interneurons are not rhythmically active and thus function independently of the locomotor central pattern generator. GCaMP6 calcium imaging of Eve+ interneurons in freely moving larvae showed left-right asymmetric activation that correlated with larval behavior. TEM reconstruction of Eve+ interneuron inputs and outputs showed that the Eve+ interneurons are at the core of a sensorimotor circuit capable of detecting and modifying body wall muscle contraction (Heckscher, 2015).

    Bilaterally symmetric motor patterns have broad and essential functions. Despite the nearly ubiquitous use of bilaterally symmetric motor patterns throughout the animal kingdom, little is understood about the relevant neural circuitry. This study identified an anatomical sensorimotor circuit containing an evolutionarily conserved population of Eve/Evx+ interneurons that is required to maintain left-right symmetric muscle contraction amplitude both during active muscle contraction and at rest. These interneurons are the first known to regulate bilaterally symmetric muscle contraction amplitude. In mouse, Sim1+ V3 interneurons have a related function during alternating gait. In the future, it will be interesting to directly examine muscle contraction amplitude in 'V3 defective' mice to determine whether this class of interneuron is responsible for balancing amplitude of left-right muscle contraction during alternating motor patterns. Similarly, it will be interesting to determine the role of Drosophila Single-minded (Sim)+ interneurons during left-right symmetric motor output (Heckscher, 2015).

    EL interneurons act in a sensorimotor circuit independent of the CPG that generates locomotion. First, in the absence of sensory input, ELs do not show locomotion-like patterns of activity. Second, EL perturbation does not alter left-right timing of muscle contraction. Third, EL perturbation alters muscle contraction amplitude during locomotion and at rest (Heckscher, 2015).

    The data suggest that EL interneurons receive sensory input that is primarily proprioceptive. Because proprioceptive neurons can detect muscle length and movement, they are well suited to convey muscle amplitude information to the ELs. Closer inspection of the proprioceptor to EL connectivity generates interesting hypotheses. First, proprioceptors are presynaptic to both projection and local EL interneurons; the former may send body posture information to the brain, while the latter may act locally to maintain left-right symmetric muscle length in each segment. Second, Jaam interneurons, which are presynaptic to EL neurons. are well positioned to process sensory information (e.g., from dorsal or ventral regions of the body wall) prior to transmitting information to the ELs. Although little is currently known about Jaam neurotransmitter expression or function, their position in the circuit raises the question of whether EL interneurons show state-dependent responses to proprioceptive inputs (Heckscher, 2015).

    The data demonstrate that EL interneurons are presynaptic to motor neurons and can modify motor output. EL perturbation results in slow crawling and asymmetric left-right muscle contraction amplitude, while optogenetic stimulation of ELs induces motor neuron activity. The majority of ELs are cholinergic and likely excitatory, they provide direct input to contralateral motor neurons, and motor neurons are glutamatergic and excitatory. Thus, EL activity on one side of the body should result in increased contralateral motor neuron activity and contralateral muscle contraction. This may be reinforced by the disynaptic (EL-SA-MN) pathway, in which EL activity would prevent ipsilateral motor neuron activity if the SA neurons were inhibitory. This model awaits future characterization of SA neurotransmitter expression and function. It is proposed that ipsilateral muscle relaxation (via the EL-SA-MN pathway) and contralateral muscle contraction (via the direct EL-MN pathway) are used for dynamic adjustment of body posture (Heckscher, 2015).

    Left-right differences in muscle contraction amplitude inevitably arise due to stochastic external (environmental) or internal (CNS/muscle) asymmetries. Without proper compensation, these perturbations would result in mismatched muscle contraction amplitude on left-right sides of the body. It is hypothesized that sensory input generates a representation of body wall curvature that is delivered to the EL interneurons. Left-right interactions among ELs would allow them to compare left versus the right sides of the body, followed by EL stimulation of motor output to restore left-right symmetric muscle length (Heckscher, 2015).

    How does EL interneuron ablation and activation generate the same phenotype? A model is favored in which ELs are part of a 'perturbation-compensation' circuit. A larva that experiences an asymmetrical perturbation from an external or internal source would generate left-right mismatched muscle contraction amplitudes in the absence of any compensation. It is proposed that the EL circuit detects and compensates for these asymmetries. When the ELs are absent or constitutively active, they lose the ability to perform the left-right comparison and the asymmetries persist. In this way, two 'opposite' manipulations yield the 'same' phenotype (Heckscher, 2015).

    There is deep conservation of genetic programs that specify neuronal fate. This is particularly true for the Eve/Evx+ interneurons, which have been found in all bilateral animals examined to date except C. elegans. Annelids, chordates, insects, fish, birds, and mammals -- as well as the presumed last common ancestor between invertebrates and vertebrates, Platynereis dumerilii -- all contain Eve/Evx+ interneurons. Evx+ neurons in mice are commissural, excitatory, and directly contact motor neurons; this study shows that fly Eve+ interneurons are commissural, likely excitatory, and directly contact motor neurons. A hypothesis to explain the remarkable parallels between Eve/Evx+ interneurons is that the last common ancestor between vertebrates and invertebrates was segmented and motile; and thus the genetic programs used to create locomotor circuitry may be evolutionarily ancient (Heckscher, 2015).

    This study has shown that the Drosophila Eve+ lateral interneurons are required to maintain left-right symmetrical motor output in the larva. Do Evx+ interneurons have a similar function in other organisms? Genetic removal of Evx1+ interneurons in mice did not reveal any specific function in either gross motor patterns or in the timing of left-right alternating motor neuronal activity as assayed by nerve root recordings. Subsequently, a broader genetic manipulation which reduced the number of Evx1+ interneurons to 25% of wild-type levels, as well as ablating a large, but unspecified number of Evx1- neurons, resulted in a hind limb hopping phenotype during fast locomotion (Talpalar, 2013). This study raised the possibility that Evx1+ interneurons regulate locomotion in mice. This study has shown that highly specific ablation or activation of Eve+ lateral interneurons disrupts larval crawling. It will be interesting to determine whether Evx1+ interneurons regulate bilaterally symmetric or alternating gait in other organisms, as well as whether Eve+ interneurons regulate alternating gait or symmetric flight in adult flies (Heckscher, 2015).

    A circuit mechanism for the propagation of waves of muscle contraction in Drosophila

    Animals move by adaptively coordinating the sequential activation of muscles. The circuit mechanisms underlying coordinated locomotion are poorly understood. This study reports on a novel circuit for propagation of waves of muscle contraction, using the peristaltic locomotion of Drosophila larvae as a model system. An intersegmental chain of synaptically connected neurons, alternating excitatory and inhibitory, was found to be necessary for wave propagation and active in phase with the wave. The excitatory neurons (A27h) are premotor and necessary only for forward locomotion, and are modulated by stretch receptors and descending inputs. The inhibitory neurons (GDL) are necessary for both forward and backward locomotion, suggestive of different yet coupled central pattern generators, and its inhibition is necessary for wave propagation. The circuit structure and functional imaging indicated that the commands to contract one segment promote the relaxation of the next segment, revealing a mechanism for wave propagation in peristaltic locomotion (Fushiki, 2016).

    Postmating circuitry modulates salt taste processing to increase reproductive output in Drosophila

    To optimize survival and reproduction, animals must match their nutrient intake to their current needs. Reproduction profoundly changes nutritional requirements, with many species showing an appetite for sodium during reproductive periods. How this internal state modifies neuronal information processing to ensure homeostasis is not understood. This study shows that dietary sodium levels positively affect reproductive output in Drosophila melanogaster; to satisfy this requirement, females develop a strong, specific appetite for sodium following mating. It was shown that mating modulates gustatory processing to increase the probability of initiating feeding on salt. This postmating effect is not due to salt depletion by egg production, since abolishing egg production leaves the sodium appetite intact. Rather, the salt appetite is induced need-independently by male-derived Sex Peptide acting on the Sex Peptide Receptor in female reproductive tract neurons. Further, postmating appetites for both salt and yeast are driven by the resultant silencing of downstream SAG neurons. Surprisingly, unlike the postmating yeast appetite, the salt appetite does not require octopamine, suggesting a divergence in the postmating circuitry. These findings demonstrate that the postmating circuit supports reproduction by increasing the palatability of specific nutrients. Such a feedforward regulation of sensory processing may represent a common mechanism through which reproductive state-sensitive circuits modify complex behaviors across species (Walker, 2015).

    The nutritional requirements of animals vary over their life cycle, and this necessitates specific behavioral mechanisms to adapt their food choices to their current internal state. This study shows that similarly to the previously characterized switch in feeding preference toward high-protein yeast, Drosophila also develop a specific appetite for sodium following mating. This appetite is adaptive for the female since, like protein, salt is important for reproductive success. This study demonstrates that dietary sodium levels positively impact the rate of offspring production. Salt could increase reproductive output in two ways: it could support egg production by providing ions required for the osmotic balance within the newly created eggs, or the phagostimulatory power of sodium could result in increased total food intake and hence an increase in egg production. Irrespective of the exact mechanisms, the current results show that dietary sodium clearly affects the rate of offspring production. The postmating salt appetite is due primarily to an increase in the probability of initiating feeding from salt, which can be attributed to an increased gustatory attraction to sodium. Mating not only elevates the gustatory response to all concentrations of salt, but also results in a shift in the peak response toward higher concentrations. This shift would allow mated females to regulate their salt consumption to a different intake target from virgins, without requiring nutrient-specific feedback to operate within the fly. Indeed, neither the postmating salt nor yeast appetites are driven by feedback from depletion of internal nutrient stores by egg production. While it is not possible to exclude the possibility that physiological processes induced by mating, other than egg production, could consume salt or protein, the data indicate that a feedforward signal in the male seminal fluid, Sex Peptide, directly drives salt and yeast appetites. Sex Peptide binds to SPR in SPSNs, whose silencing results in silencing of SAG neurons. This leads to appetites for both salt and yeast, in addition to the previously described changes in receptivity and egg laying. These results suggest that the intake of reproductive nutritional resources is controlled by a common regulatory logic, whereby the signal of mating is detected by local uterine neurons and changes nutrition in a feedforward, anticipatory manner. It will be interesting to explore to what extent feedforward regulation is employed to control specific behavioral strategies used to acquire nutrients depending on different internal state signals (Walker, 2015).

    The data are consistent with the current view that the signal of mating status is brought into the central brain through a common pathway, the SPSN-SAG axis (SPSN refers to sensory neurons of the female reproductive tract and SAG refers to targets of the SPSN in the abdominal ganglion), to regulate the full set of postmating responses including egg laying, remating, and nutrition. Given the diverse set of behaviors regulated by mating, one would expect the circuit to diverge downstream. However, the point of divergence is currently unknown. Octopamine is known to be required for ovulation and is required for the full reduction in receptivity that normally follows mating. In agreement with these results, this study found that octopamine is also required for the postmating increase in yeast intake in protein-deprived females, while it is dispensable for sensing internal amino acid deficiency. However, while octopamine does influence the overall level of salt responses, the results show that it is not necessary for the postmating change in salt response. These data suggest that octopamine may represent such a divergence point in the postmating circuit, with the previously characterized dsx+Tdc2+ neurons being likely neuronal candidates mediating this divergence. It has, however, been proposed that octopamine may act genetically upstream of SP; this could be compatible with the current results if the salt appetite is relatively insensitive to small changes in SP levels. Regardless, this result hints at distinct circuitry controlling the different behavioral changes elicited by mating, which could aid in the future elucidation of how a specific internal state signal could coordinate changes in a wide range of different behaviors (Walker, 2015).

    Salt has been shown to be one of the most limiting nutritional resources in many ecosystems. The results provide insights into the physiological regulation of salt intake, which until now has remained unexplored in Drosophila. The postmating sodium appetite demonstrated in this study is intriguing in the light of the specific sodium appetite seen during pregnancy and lactation in various mammalian herbivores, and even humans. As in Drosophila, these species show an increased gustatory attraction to salt following mating. While the mechanism used to detect mating in these species is different, the feedforward, need-independent nature of the salt appetite is likely to be similar. In rats, this appetite is induced within a few days after mating and is present even if the animal has access to sufficient salt in its diet; furthermore, a salt appetite can be induced in rabbits by administration of a mixture of reproductive hormones in the absence of mating. Thus, the detection of mating by the nervous system and the subsequent feedforward increase in response to salt taste is likely to be a common feature of many non-carnivorous species, making alliesthesia a likely universal mechanism driving reproductive salt appetites. While much is known about the regulation of salt intake in mammals, the mechanisms through which reproduction affects salt appetite remain poorly understood in any species. Functional genetic circuit analysis combined with activity imaging in Drosophila offer the unique opportunity to understand the circuit mechanisms through which this internal state signal can modulate taste processing in the brain, and thus bring about an adaptive change in food preference. To achieve this, three possibilities exist. Mating could modulate the response of sensory neurons to salt taste, as demonstrated in the olfactory pheromone system of moths. In a similar way, GRN responses are modulated by starvation, and the sensitivity of pheromone-sensitive olfactory receptor neurons in mice is modulated across the estrus cycle. Alternatively, mating could alter higher-order taste processing. Finally, mating state could lead to a combination of modulation at the receptor neuron level and modification of higher-order processing. Identifying how alliesthesia is implemented at the circuit level will represent a unique opportunity to understand how internal state changes affect sensory processing to mediate adaptive behaviors (Walker, 2015).

    MicroRNA-encoded behavior in Drosophila

    The relationship between microRNA regulation and the specification of behavior is only beginning to be explored. This study finds that mutation of a single microRNA locus (miR-iab4/8 - (miR-iab4/iab8)) in Drosophila larvae affects the animal's capacity to correct its orientation if turned upside-down (self-righting). One of the microRNA targets involved in this behavior is the Hox gene Ultrabithorax whose derepression in two metameric neurons leads to self-righting defects. In vivo neural activity analysis reveals that these neurons, the self-righting node (SRN), have different activity patterns in wild type and miRNA mutants while thermogenetic manipulation of SRN activity results in changes in self-righting behavior. These data thus reveal a microRNA-encoded behavior and suggests that other microRNAs might also be involved in behavioral control in Drosophila and other species (Picao-Osorio, 2015).

    The regulation of RNA expression and function is emerging as a hub for gene expression control across a variety of cellular and physiological contexts, including neural development and specification. Small RNAs such as microRNAs (miRNAs) have been shown to affect neural differentiation, but their roles in the control of behavior are only beginning to be explored (Picao-Osorio, 2015).

    Previous work has focused on the mechanisms and impact of RNA regulation on the expression and neural function of the Drosophila Hox genes. These genes encode a family of evolutionarily conserved transcription factors that control specific programs of neural differentiation along the body axis, offering an opportunity to investigate how RNA regulation relates to the formation of complex tissues such as the nervous system (Picao-Osorio, 2015).

    This study used the Hox gene system to investigate the roles played by a single miRNA locus (miR-iab4/iab8) on the specification of the nervous system during early Drosophila development. This miRNA locus controls the embryonic expression of posterior Hox genes. Given that no detectable differences were found in the morphological layout of the main components of the nervous system in late Drosophila embryos of wild type and miR-iab4/iab8-null mutants [herein ΔmiR], this study analyzed early larval behavior as a stratagem to probe the functional integrity of the late embryonic nervous system (Picao-Osorio, 2015).

    Most behaviors in early larva were unaffected by the miRNA mutation, except self-righting (SR) behavior: miRNA mutant larvae were unable to return to their normal orientation at the same speed as their wild-type counterparts (Picao-Osorio, 2015).

    By means of selective target overexpression followed by SR phenotype analyses, this study identified the Drosophila Hox gene Ultrabithorax (Ubx) as a miRNA target implicated in the genetic control of SR behavior. Overexpression of Ubx within its expression domain did not affect any larval behavior tested except SR, which is in agreement with the effects observed in miRNA mutants. Analysis of Ubx 3' untranslated region (3'UTR) fluorescent reporter constructs expressed in the Drosophila central nervous system (CNS) indicates that the interaction between miR-iab4/iab8 and Ubx is direct, which is in line with prior observations in other cellular contexts (Picao-Osorio, 2015).

    To identify the cellular basis for SR control, Ubx was systematically overexpressed within subpopulations of neurons. Increased levels of Ubx within the pattern of Cha(7.4kb)-Gal4, which largely targets cholinergic sensory and interneurons, phenocopied the miRNA SR anomalies. Further overexpression analysis identified two metameric neurons as the minimal node required for the SR behavior [self-righting node (SRN)] (Picao-Osorio, 2015).

    Several lines of evidence confirm the role of miRNA-dependent Ubx regulation within the SRN as a determinant of SR. First, both Ubx and miRNA transcripts (miR-iab4) derived from the miR-iab4/iab8 locus were detected within the SRN. Second, in the context of miRNA mutation, Ubx protein expression is increased within the SRN. Third, reduction of Ubx (Ubx RNAi) specifically enforced within SRN cells is able to ameliorate or even rescue the SR phenotype observed in miRNA mutants (Picao-Osorio, 2015).

    Two plausible scenarios arise to explain the effects of miR-iab4/iab8 in regard to SR behavior. One is that miRNA input is required for the late embryonic development of the neural networks underlying SR, arguing for a 'developmental' role of the miRNA; another is that miRNA repression affects normal physiological/behavioral functions largely without disrupting neural development in line with a 'behavioral' role. Two independent experiments support that the primary roles of miR-iab4/8 are behavioral. First, anatomical analysis of SRN cells in wild type (wt), ΔmiR, and R54503>Ubx [SRN-driver line] show no significant differences in total numbers of SRN cells or in SRN cell body size; furthermore, analysis of wt, ΔmiR, and R54503>Ubx show indistinguishable SRN-projection patterns. Second, Gal-80ts-mediated conditional expression experiments show that SRN-specific Ubx overexpression after embryogenesis is sufficient to trigger the SR behavior (Picao-Osorio, 2015).

    These results suggest that miRNA-dependent Hox regulation within the SRN must somehow modify the normal physiology of SRN cells so that when the miRNA is mutated, these neurons perform functions different from those in wild-type animals. To test this hypothesis, genetically encoded calcium sensors [GCaMP6] specifically expressed in SRN cells were used, and spontaneous profiles of neural activity were tracked down. SRN cells in miRNA mutants produce activity traces that are significantly different from those observed in wild-type SRN cells. Quantification of maximal amplitude and proportion of active cells in each genotype also reveal significant differences in SRN function across the genotypes, but no change in cell viability is observed. Neural activity differences across genotypes are significant within regions of expression of miR-iab4, suggesting that this miRNA (and not miR-iab8) might be the main contributor to SR control. Analysis of mutations that selectively affect miR-iab4 or miR-iab8 strongly suggests that miR-iab4 is the key regulator of SR (Picao-Osorio, 2015).

    To demonstrate that the changes in SRN neural activity were causal to SR behavior, SRN cells were artificially activated or inhibited this was shown to trigger the aberrant SR phenotype. This suggested that activation of SRN cells in larvae placed 'right side up' might be sufficient to 'evoke' actions reminiscent of a self-righting response. An optogenetic system was developed in which SRN cells were activated by means of R54F03-driven channelrhodopsin 2 (ChR2) in trans-retinal fed larvae. Under blue light stimulation, larvae performed an atypical bending movement, frequently adopting a 'lunette' position. Neither parental line R54F03-Gal4 nor UAS-Ch2R showed similar reactions to stimulation, confirming the specificity of this effect (Picao-Osorio, 2015).

    To study the links between SRN neurons and the SR movement, SRN projections were labeled with myr-GFP and SRN cells were discovered to innervate two of the lateral transverse (LT) muscles and can be colabeled antibodies against Fasciclin 2 (Fas2), demonstrating these to be motorneurons. LT muscles are innervated by Bar-H1+ motorneurons, so Bar-H1-Gal4 was used as a second driver to demonstrate that appropriate Ubx levels in these cells are required for normal SR behavior, establishing the SRN cells as the LT-MNs (Picao-Osorio, 2015).

    This study has therefore shown that miRNA-dependent Hox gene repression within a distinct group of motorneurons (SRN/LT-MNs) is required for the control of a specific locomotor behavior in the early Drosophila larva. The finding that Hox gene posttranscriptional regulation is involved in SR control suggests that other RNA-based regulatory processes affecting Hox gene expression might also impinge on specific neural outputs; this possibility is currently being investigated, with special regard to the roles of the Hox genes in the specification of neural lineages with axial-specific architectures, and the roles of other miRNAs on behavior are being systematically tested (Picao-Osorio, 2015).

    That no obvious neuro-anatomical changes in miRNA mutant embryos could be detected suggests that these are either very subtle or that the role of miRNA regulation may be primarily behavioral, in the sense of affecting the performance of a correctly wired neural system, rather than developmental, contributing to the development of the network. Given that miR-iab4/iab8 is involved in adult ovary innervation, it seems that miRNAs -- much like ordinary protein-coding genes -- can be involved in several distinct roles within the organism (Picao-Osorio, 2015).

    The results of this study contribute to the understanding of how complex innate behaviors are represented in the genetic program. The data lead to a proposal that other miRNAs might also be involved in the control of behavior in Drosophila and other species (Picao-Osorio, 2015).

    Anterior-posterior gradient in neural stem and daughter cell proliferation governed by spatial and temporal Hox control

    A readily evident feature of animal central nervous systems (CNSs), apparent in all vertebrates and many invertebrates alike, is its "wedge-like" appearance, with more cells generated in anterior than posterior regions. This wedge could conceivably be established by an antero-posterior (A-P) gradient in the number of neural progenitor cells, their proliferation behaviors, and/or programmed cell death (PCD). However, the contribution of each of these mechanisms, and the underlying genetic programs, are not well understood. Building upon recent progress in the Drosophila melanogaster (Drosophila) ventral nerve cord (VNC), this study addressed these issues in a comprehensive manner. Although PCD plays a role in controlling cell numbers along the A-P axis, the main driver of the wedge is a gradient of daughter proliferation, with divisions directly generating neurons (type 0) being more prevalent posteriorly and dividing daughters (type I) more prevalent anteriorly. In addition, neural progenitor (NB) cell-cycle exit occurs earlier posteriorly. The gradient of type I > 0 daughter proliferation switch and NB exit combine to generate radically different average lineage sizes along the A-P axis, differing by more than 3-fold in cell number. The Hox homeotic genes, expressed in overlapping A-P gradients and with a late temporal onset in NBs, trigger the type I > 0 daughter proliferation switch and NB exit. Given the highly evolutionarily conserved expression of overlapping Hox homeotic genes in the CNS, these results point to a common mechanism for generating the CNS wedge (Monedero Cobeta, 2017).

    Temporal cohorts of lineage-related neurons perform analogous functions in distinct sensorimotor circuits

    An important, but unaddressed question is whether temporal information that diversifies neuronal progeny within a single lineage also impacts circuit assembly. Circuits in the sensorimotor system (e.g., spinal cord) are thought to be assembled sequentially, making this an ideal brain region for investigating the circuit-level impact of temporal patterning within a lineage. This study used intersectional genetics, optogenetics, high-throughput behavioral analysis, single-neuron labeling, connectomics, and calcium imaging to determine how a set of bona fide lineage-related interneurons in the ventral cord contribute to sensorimotor circuitry in the Drosophila larva. Even-skipped lateral interneurons (ELs) are sensory processing interneurons. Late-born ELs contribute to a proprioceptive body posture circuit, whereas early-born ELs contribute to a mechanosensitive escape circuit. These data support a model in which a single neuronal stem cell can produce a large number of interneurons with similar functional capacity that are distributed into different circuits based on birth timing. In summary, these data establish a link between temporal specification of neuronal identity and circuit assembly at the single-cell level (Wreden, 2017).

    This study took advantage of the extremely well-characterized neuronal stem cells (neuroblasts) and their lineages in the Drosophila larval nerve cord to study lineage-circuitry relationships in a sensorimotor system. The Drosophila larval nerve cord is subdivided into a series of bilaterally symmetric segments, each of which contains 30 pairs of neuroblasts that give rise to all nerve cord neurons. This study focused on one class of bona fide sibling neurons-Even-skipped (Eve)-expressing interneurons with lateral cell body positions (ELs), a morphologically diverse set of excitatory interneurons from Neuroblast 3-3 (NB3-3) (Wreden, 2017).

    The first abdominal segment consists of left/right clusters of ten ELs that can be subdivided into two groups based on the expression of the enhancer 'R11F02'. R11F02 expresses in the lateral-most ELs and a few other cells. During neurogenesis, newly born neurons displace their older siblings away from the parent neuroblast-generating an early-to-late, medial-to-lateral spatial pattern. Thus, it was hypothesized that R11F02(+) ELs were late born. Using a panel of transcription factors to assess birth order, R11F02(+) ELs were found to express the late-born marker Nab, but not early-born markers Kruppel/Pdm2. Thus, expression of R11F02 subdivides ELs into early-born and late-born temporal cohorts. However, the functional significance of this subdivision is unknown (Wreden, 2017).

    To investigate early-born and late-born ELs, lines were used that specifically target each temporal cohort of neurons. For this study, R11F02-GAL80 were generated, that, when used with EL-GAL4, allows GAL4 to remain functional only in early-born, medial ELs. In addition, the split GAL4 lines R11F02-DBD and EL-AD was used to generate functional GAL4 in late-born, lateral ELs. Thus, the activity of each temporal cohort can be selectively manipulated (Wreden, 2017).

    The behavioral response to acute stimulation of late-born ELs was examined. Previous work (Heckscher, 2015) showed that chronic stimulation of R11F02(+) ELs caused larvae to crawl with abnormal left/right body posture, but that study did not monitor initial responses of larvae to activation. Thus, it was unknown to what extent acute activation of late-born ELs slows larval motion, as would be expected if late-born ELs process proprioceptive information. In this study a behavior rig was build to monitor behavior before, during, and after optogenetic stimulation. Larval speed was measured by calculating the distance traveled by the larval centroid over time without regard to whether the direction of movement aligned with the body axis. Immediately upon stimulation of late-born ELs, body movements became left/right uncoordinated and speed was significantly reduced. Thus, the normal activity of late-born ELs is required for normal crawling, consistent with the idea that late-born ELs process proprioceptive information (Wreden, 2017).

    It was asked whether stimulation of early-born and late-born ELs elicit similar or distinctive behavioral responses. Surprisingly, during optogenetic stimulation of early-born ELs speed transiently increased. Furthermore, all ELs were simultaneously stimulated and a transient increase was found followed by a sustained reduction in speed, which extended a previous finding that measured the later, but not initial, responses of larvae to activation of all ELs (Heckscher, 2015). Thus, it is likely that late-born and early-born ELs operate in distinct circuits (Wreden, 2017).

    Increases in speed upon stimulation of early-born ELs could be due either to faster crawling or to larvae initiating a distinct movement-escape rolling. Escape rolling is the fastest larval movement and can be identified both because trachea on the dorsal side of the larva disappear beneath the body and because the direction of movement is lateral to the body axis. Higher resolution imaging showed that stimulation of early-born ELs frequently elicited multiple rolls, whereas stimulation of late-born ELs rarely elicited rolling. It was asked whether stimulation of early-born ELs triggered other escape-related behaviors -- hunching, fast crawling, reversals, body bending -- and an increase in body bending was found. Thus, activation of early-born ELs robustly triggers some, but not all, escape-related behaviors (Wreden, 2017).

    Next, it was asked whether any early-born EL could be part of an escape circuit. Recently, an escape circuit has been characterized, which contains a set of roll-inducing 'Basin' interneurons (Ohyama, 2015). Furthermore, the neurons downstream of Basins have been identified in a transmission electron microscopic (TEM) volume that contains the entire larval CNS. In the current study it was asked whether any neurons that receive synapses from Basins are early-born ELs. Single-cell clones were generated of early-born ELs, and single-neuron morphology was imaged with fluorescent microscopy. Then, collection of early-born EL morphologies, as determined by light microscopy, was compared to the morphologies of neurons downstream of Basins, as determined by TEM. Three early-born ELs were found receive inputs from Basins. Thus, early-born ELs contribute functionally, and anatomically, to an escape circuit (Wreden, 2017).

    This is the first time that single-cell morphology and connectivity have been identified for a majority of lineage-related interneurons within a Drosophila larval segment. This was accomplished by determining the spatiotemporal origin of neurons that were recently annotated in a Drosophila larval brain TEM volume. Within each segment, each neuroblast gives rise to a unique set of neurons, so this study asked what features are shared among ELs because these features are excellent candidates to be encoded at the stem cell level. First, late-born ELs contribute to a proprioceptive processing circuit. All TEM-annotated, late-born ELs in segment A1 receive direct synaptic input from proprioceptors, and some also receive direct synaptic inputs from Jaam interneurons, which themselves receive a large amount of direct proprioceptive input (Heckscher, 2015). Late-born ELs and Jaams get little input from other sensory neurons (Heckscher, 2015). Second, early-born ELs contribute to a mechanosensitive circuit. The TEM-annotated, early-born ELs in segment A1 receive direct synaptic input from mechanosensitive chordotonal sensory neurons and receive direct synaptic input from Basins 1 and 3, which themselves receive a large amount of direct mechanosensory chordotonal input. These early-born ELs, Basin1, and Basin 3 receive little known input from other sensory neurons. Currently, the inputs on to the remaining early-born ELs in segment A1 are unknown. Nonetheless, a majority of ELs in segment A1 are first-order sensory processing interneurons, directly receiving sensory neuron input, and many ELs are second-order sensory processing interneurons, indirectly receiving sensory neuron input. Notably, ELs are largely silent in the absence of sensory input and are therefore likely to encode sensory information. Taken together, these functional and anatomical data suggest that NB3-3 produces many sensory processing neurons (Wreden, 2017).

    Anatomically, early-born ELs receive synapses from mechanosensitive, chordotonal sensory neurons (Mechano CHOs), whereas late-born ELs receive synapses from other sensory neurons. Thus, early-born versus late-born ELs are likely to process different stimuli. This study tested this idea by monitoring EL responses to a sound that activates chordotonals (Wreden, 2017).

    First, it was shown that sound/vibration stimulus specifically activates chordotonals. In response to sound/vibration, Drosophila larvae perform an avoidance hunch. Hunching can be identified because larvae rapidly reduce crawling speed and shorten their body. A new sound stimulus was generated using a composite of known stimuli. To validate the stimulus, the behavioral rig was adapted by adding a speaker and amplifier, the stimulus was played to larvae, and speed and body perimeter was measured over time. In response to stimulation, control larvae robustly hunched, whereas larvae lacking chordotonals did not hunch. Thus, the sound/vibration stimulus can be sensed by larvae, and the response depends on mechanosensitive chordotonal sensory neurons (Wreden, 2017).

    Next, it was asked to what extent do chordotonals and early-born and late-born ELs respond to sound/vibration. A previously described, head-fixed preparation was adapted, in which the anterior portion of the larva that contains the CNS is flattened and nearly immobilized, but the posterior is untouched. Calcium imaging monitored neuronal activity before, during, and after stimulation, and ΔF/F measured fluorescence intensity. As expected, chordotonals robustly responded to stimulation. Early-born ELs responded to stimulation with a smaller amplitude, but with a similar percentage responding in comparison to chordotonals. In contrast, late-born ELs showed little to no response. Thus, early-born versus late-born ELs differentially respond to sensory input. Furthermore, these data strongly suggest that the chordotonal-to-early-born EL connections seen in the TEM are functional, present in multiple larvae, and present in multiple segments along the anterior-posterior axis of the nerve cord (Wreden, 2017).

    This work contributes an additional concept, showing that within a temporal cohort interneurons are similar. Furthermore, in the Drosophila motor system there may be many temporal cohorts-for example, Basins, Jaams, as well as another group of neurons that contact ELs, Saaghis, may be temporal cohorts. These interneurons are morphologically similar to each other, and Basins have been explicitly hypothesized to be lineage related. Thus, the observed link between temporal patterning and functional circuit assembly may be representative of a widely occurring phenomenon (Wreden, 2017).

    How lineages contribute to neuronal circuits has been investigated in a few brain regions, none of which are sensorimotor. These studies have demonstrated that different brain regions have different lineage-circuitry relationships, which are likely to be critical for establishing region-specific functional differences. Sensorimotor systems perform a unique series of computations-sensing multiple kinds of stimuli, such as self-movement or pain, and producing adaptive motor outputs, such as locomotion or escape. This study used the Drosophila sensorimotor system to show that late-born, lineage-related ELs contribute to a proprioceptive circuit and that early-born, lineage-related ELs contribute to a mechanosensitive circuit. In both circuits, ELs are sensory processing interneurons. Thus, it appears that the NB3-3 lineage endows ELs with the capacity to processes sensory information regardless of circuit identity, and birth-timing segregates ELs into different circuits. Assembling circuitry according to these rules elucidates the developmental mechanisms that generate sensorimotor systems with the ability to process different types of sensory information in parallel (Wreden, 2017).

    Depending on context, Basin interneurons can promote multiple types of escape responses, such as rolling, hunching, and bending. Some, but not all, of these escape behaviors occur upon stimulation of early-born ELs, which are downstream of Basins. These findings raise the questions: do other members of the NB3-3 lineage promote other escape responses? Do early-born neurons from other lineages promote other escape responses? Addressing these questions will be important for the field (Wreden, 2017).

    The data support the idea that a developmental strategy for assembling sensorimotor circuits is as follows: a given neuronal stem cell can produce many neurons with similar functional capacity, which are distributed into different circuits based on birth timing. This developmental strategy may be used in other sensorimotor systems. Although the exact lineage-circuit relationship is unclear, the mammalian spinal cord provides additional examples of temporal cohorts of developmentally related neurons performing analogous functions in different circuits. For example, Renshaw cells and Ia interneurons are sequentially produced by p1 progenitors. Renshaw cells contribute to a motor neuron feedback circuit, whereas Ia interneurons contribute to a stretch reflex circuit. Despite participation in distinct circuits, Renshaw cells and Ia interneurons perform analogous functions-directly synapsing onto motor neurons and terminating firing. In addition, for extensor and flexor premotor interneurons, many of which originate from the same progenitor domain, time of neurogenesis is correlated with spatial, and inferred functional, segregation. Thus, temporal segregation of lineage-related neurons with similar functional capacities is likely to occur in evolutionarily distant species, suggesting the fundamental importance of this developmental strategy (Wreden, 2017).

    Furthermore, the data reveal a correspondence between vertebrate and Drosophila sensorimotor development. In zebrafish, early-born neurons contribute to circuits for fast escape, whereas later-born neurons contribute to circuits for refined movements. These observations led the hypothesis-circuits for fast/gross movements and neurons in these circuits develop early, whereas circuits for slow/refined movements and neurons in these circuits develop later. However, it is unclear how broadly this hypothesis applies. This study shows that similar to zebrafish, in Drosophila, early-born neurons contribute to circuits for fast escape, whereas later-born neurons contribute to circuits for proprioceptive refinement of movements. Thus, this developmental principle guiding sensorimotor circuit assembly may be conserved across species despite separation by hundreds of millions of years of evolution (Wreden, 2017).

    Neural circuitry that evokes escape behavior upon activation of nociceptive sensory neurons in Drosophila larvae

    Noxious stimuli trigger a stereotyped escape response in animals. In Drosophila larvae, class IV dendrite arborization (C4 da) sensory neurons in the peripheral nervous system are responsible for perception of multiple nociceptive modalities, including noxious heat and harsh mechanical stimulation, through distinct receptors. Silencing or ablation of C4 da neurons largely eliminates larval responses to noxious stimuli, whereas optogenetic activation of C4 da neurons is sufficient to provoke corkscrew-like rolling behavior similar to what is observed when larvae receive noxious stimuli, such as high temperature or harsh mechanical stimulation. How C4 da activation triggers the escape behavior in the circuit level is still incompletely understood. This study identified segmentally arrayed local interneurons (medial clusters of C4 da second-order interneurons [mCSIs]) in the ventral nerve cord that are necessary and sufficient to trigger rolling behavior. GFP reconstitution across synaptic partners (GRASP) analysis indicates that C4 da axons form synapses with mCSI dendrites. Optogenetic activation of mCSIs induces the rolling behavior, whereas silencing mCSIs reduces the probability of rolling behavior upon C4 da activation. Further anatomical and functional studies suggest that the C4 da-mCSI nociceptive circuit evokes rolling behavior at least in part through segmental nerve a (SNa) motor neurons. These findings thus uncover a local circuit that promotes escape behavior upon noxious stimuli in Drosophila larvae and provide mechanistic insights into how noxious stimuli are transduced into the stereotyped escape behavior in the circuit level (Yoshino, 2017).

    Neuronal cell fate specification by the molecular convergence of different spatio-temporal cues on a common initiator terminal selector gene

    The extensive genetic regulatory flows underlying specification of different neuronal subtypes are not well understood at the molecular level. The Nplp1 neuropeptide neurons in the developing Drosophila nerve cord belong to two sub-classes; Tv1 and dAp neurons, generated by two distinct progenitors. Nplp1 neurons are specified by spatial cues; the Hox homeotic network and GATA factor grn, and temporal cues; the hb -> Kr -> Pdm -> cas -> grh temporal cascade. These spatio-temporal cues combine into two distinct codes; one for Tv1 and one for dAp neurons that activate a common terminal selector feedforward cascade of col -> ap/eya -> dimm -> Nplp1. This study molecularly decodes the specification of Nplp1 neurons, and finds that the cis-regulatory organization of col functions as an integratory node for the different spatio-temporal combinatorial codes. These findings may provide a logical framework for addressing spatio-temporal control of neuronal sub-type specification in other systems (Stratmann, 2017).

    The Drosophila ventral nerve cord (VNC; defined here as thoracic segments T1-T3 and abdominal A1-A10) contains ~10,000 cells at the end of embryogenesis, which are generated by a defined set of ~800 neuroblasts (NBs). The Apterous neurons constitute a small sub-group of interneurons, identifiable by the selective expression of the Apterous (Ap) LIM-homeodomain factor, as well as the Eyes absent (Eya) transcriptional co-factor and nuclear phosphatase. A subset of Ap neurons express the Nplp1 neuropeptide, but can be sub-divided into the lateral thoracic Tv1 neurons, part of the thoracic Ap cluster of four cells, and the dorsal medial row of dAp neurons. In line with the distinct location of the Tv1 and dAp neurons, studies have revealed that they are generated by distinct NBs; NB5-6T and NB4-3, respectively. A number of studies have addressed the genetic mechanisms underlying the specification of the Tv1 and dAp neurons, and the regulation of the Nplp1 neuropeptide. These have revealed that two distinct spatio-temporal combinatorial transcription factor codes, one acting in NB5-6T and the other in NB4-3, converge on a common initiator terminal selector gene; collier, encoding a COE/EBF transcription factor. Col in turn is necessary and sufficient to trigger a feed forward loop (FFL) consisting of Ap, Eya and the Dimmed (Dimm) bHLH transcription factor, which ultimately activates the Nplp1 gene. Strikingly, the combinatorial coding selectivity of the spatio-temporal cues combined with the information-coding capacity of the FFL results in the selective activation of Nplp1 in only 28 out of the ~10,000 cells within the VNC. While these genetic studies have helped resolve the regulatory logic of this cell specification event, they have not addressed the molecular mechanisms by which the two different spatio-temporal combinatorial codes intersect upon the col initiator terminal selector, to trigger a common terminal FFL, or the molecular nature of the FFL (Stratmann, 2017).

    To address this issue, this study has identified enhancers for Tv and dAp neuron expression for the genes in the common Tv1/dAp FFL: col, ap, eya, dimm and Nplp1. Transgenic reporters were generated for these enhancers, both wildtype and mutant for specific transcription factor binding sites, to test their regulation in mutant and misexpression backgrounds. CRISPR/Cas9 technology was used to delete these enhancers in their normal genomic location to test their necessity for gene regulation. Strikingly, this study found that the distinct upstream spatio-temporal combinatorial codes, which trigger col expression in Tv1 versus dAp neurons, converge onto different enhancer elements in the col gene. Hence, the col Tv1 neuron enhancer is triggered by Antp, hth, exd, lbe and cas, while the dAp enhancer is triggered by Kr, pdm and grn. In contrast to this subset-specific enhancer set-up for col activation, the subsequent, col-driven Nplp1 FFL feeds onto common enhancers in each downstream gene. These findings reveal that distinct spatio-temporal cues, acting in different neural progenitors, can trigger the same FFL by converging on discrete enhancer elements in an initiator terminal selector, to thereby dictate the same ultimate neuronal subtype cell fate (Stratmann, 2017).

    This study has been able to molecularly decode the Tv1/dAp genetic FFL cascades, bolstering evidence for a complex molecular FFL, based upon sequential transcription factor binding to the downstream genes. The NB4-3 and NB5-6T neuroblasts are born in different regions of the VNC, and express different spatial determinants i.e., Antp, Lbe, Hth, Exd and Gr. As lineage progression commences, they undergo a programmed cascade of transcription factor expression; the temporal cascade. Early temporal factors Kr and Pdm integrate with Grn in NB4-3, while the late temporal factor Cas integrates with Antp, Lbe, Hth and Exd in NB5-6T, to create two distinct combinatorial spatio-temporal codes. These two codes converge on two different enhancers in the col gene, triggering Col expression, and hence the Nplp1 FFL. The FFL, in this case a so-called coherent FFL, where regulators act positively at one or several steps of a cascade, was first identified in E.coli and yeast regulatory networks, but have also been identified in C.elegans and Drosophila. Coherent FFLs can act as regulatory timing devices, exemplified by the action of col in NB5-6T: The initial expression of col in Ap cluster cells triggers a generic Ap/Eya interneuron fate in all four cells, while its downregulation in Tv2-4 and maintenance in Tv1 helps propagate the FFL leading to Nplp1 expression (Stratmann, 2017).

    This study has found that the two different spatio-temporal programs converge on col, but on different enhancer elements. However, neither enhancer element gave complete null effects when deleted. Specifically, the 6.3kb col-Tv-CRM shows robust reporter expression, overlaps with endogenous col expression, responds to the upstream mutants, and is affected by TFBS mutations. However, when deleted (generating the colΔTv-CRM mutant), it had weak effects upon endogenous col expression in NB5-6T, and no effect upon Eya and Nplp1 expression. Deletion of the col-dAp-CRM (generating the colΔdAp-CRM mutant), gave more robust effects with reduction of Col, Eya and Nplp1 in dAp cells, although the expression was not lost completely (Stratmann, 2017).

    Early developmental genes, which often are dynamically expressed, may be controlled by multiple enhancer modules, to thereby ensure robust onset of gene expression. This has been reported previously in studies of early mesodermal and neuro-ectodermal development, in which several genes i.e., twist, sog, snail are controlled by multiple distal enhancer fragments, so called 'shadow enhancers', in order to ensure reliable onset of gene expression. The shadow enhancer principle is also supported by recent findings on the Kr gene. Moreover, extensive CRM transgenic analysis, scoring thousands of fragments in transgenic flies, has also supported the shadow enhancer idea, revealing that a number of early regulators, several of which encode for transcription factors, indeed have shadow enhancers. The dichotomy between the col transgenic reporter results and the partial impact on col expression upon deletion of its Tv1 and dAp enhancers, gives reason to speculate that col may be under control of additional enhancers, some of which may be referred to as shadow enhancers (Stratmann, 2017).

    The results on the eya, ap, dimm and Nplp1 enhancer mutants stand in stark contrast to the col CRMs findings. For these four genes, the enhancer deletion resulted in robust, near null effects, on expression. It is tempting to speculate that the current findings, combined with previous studies, points to a different logic for early regulators, with highly dynamic patterns, requiring several functionally overlapping enhancers for fidelity, and late regulators and terminal differentiation genes, which may operate with one enhancer that is inactive until the pertinent combinatorial TF codes have been established (Stratmann, 2017).

    Analysis of the ap and eya enhancers indicates that Col directly interacts with these enhancers. Both of these enhancer-reporter transgenes are affected in col mutants, and can be activated by ectopic col. Moreover, mutation of one Col binding site in the ap enhancer and two sites in the eya enhancer, was enough to dramatically reduce enhancer activity. Direct action of Col on ap and eya is furthermore supported by recent data on Col genome-wide binding, using ChIP, which demonstrated direct binding of Col to these regions of ap and eya in the embryo. The regulation of ap is an excellent example of the complexity of gene regulation, and studies have identified additional enhancers controlling ap expression in the wing, muscle and brain (Stratmann, 2017).

    In contrast to regulation of ap and eya, a direct action of Col on dimm and Nplp1 is less clear. Analysis of the dimm and Nplp1 enhancers did not reveal perfectly conserved Col binding sites. Mutation of multiple non-perfect Col binding sites in the dimm enhancer did not affect reporter expression in the Ap cluster, but did however reduce levels in the dorsal Ap cells. Mutation of non-perfect Col binding sites in the Nplp1 enhancer had no impact on enhancer activity, neither in Tv1 nor dAp. These findings support a model where Col is crucial for directly activating ap and eya, which in turn directly activate dimm and Nplp1, with some involvement of Col on dimm. However, support for a direct role for Col on Nplp1 comes from RNAi studies in larvae or adult flies, showing that knockdown of col resulted in loss of Nplp1, while Ap, Eya and Dimm expression was unaffected (Stratmann, 2017).

    It is tempting to speculate that Col regulates Nplp1 not via direct interaction with its enhancer, but rather as a chromatin state modulator, keeping the chromatin around the Nplp1 locus in an accessible state, in order for Dimm, Ap and Eya to be able to access the Nplp1 gene. Support for this notion comes from studies on the mammalian Col orthologue EBF, which is connected to the chromatin remodeling complex SWI/SNF during EBF-mediated gene regulation in lymphocytes (Gao, 2009). Moreover, the central SWI/SNF component Brahma was recently identified in a genetic screen for Ap cluster neurons, and found to affect FMRFa neuropeptide expression in Tv4 without affecting Eya expression, indicating a late role in Ap cluster differentiation. Alternatively, Col may activate Nplp1 via unidentified, low affinity sites, similar to the mechanism by which Ubx regulates some of its embryonic target genes (Stratmann, 2017).

    ap encodes a LIM-HD protein, a family of transcription factors well known to control multiple aspects of terminal neuronal subtype fate, including neurotransmitter identity, axon pathfinding and ion channel expression. The current results indicate that Ap in turn acts upon dimm, and subsequently with Dimm on Nplp1. eya encodes an evolutionary well-conserved phosphatase and does not bind DNA directly, instead acting as a transcriptional co-factor. Eya (and its orthologues) have been found to interact with several transcription factors in different systems, but whether it forms complexes with Col and Ap is not known (Stratmann, 2017).

    The final transcription factor in the FFL is Dimm, a bHLH protein. Dimm is selectively expressed by the majority of neuropeptide neurons in Drosophila, and is important for expression of many neuropeptides. Intriguingly, Dimm is also both necessary and sufficient to establish the dense-core secretory machinery, found in neuropeptide neurons. Based upon these findings Dimm has been viewed as a cell type selector gene, acting to up-regulate the secretory machinery. This study found evidence for that Dimm acts directly on the Nplp1 enhancer, and this raises the possibility that Dimm is both a selector gene for the dense-core secretory machinery, and can act in some neuropeptide neurons to directly regulate specific neuropeptide gene expression (Stratmann, 2017).

    Gap junction-mediated signaling from motor neurons regulates motor generation in the central circuits of larval Drosophila

    This study used the peristaltic crawling of Drosophila larvae as a model to study how motor patterns are regulated by central circuits. An experimental system was constructed that allows simultaneous application of optogenetics and calcium imaging to the isolated ventral nerve cord (VNC). Next, the effects of manipulating local activity of motor neurons (MNs) on fictive locomotion were observed as waves of MN activity propagating along neuromeres. Optical inhibition of MNs with halorhodopsin3 (NpHR3) in a middle segment (A4, A5 or A6), but not other segments, dramatically decreases the frequency of the motor waves. Conversely, local activation of MNs with channelrhodopsin2 (ChR2) in a posterior segment (A6 or A7) increases the frequency of the motor waves. Since peripheral nerves mediating sensory feedback are severed in the VNC preparation, these results indicate that MNs send signals to the central circuits to regulate motor pattern generation. These results also indicate segmental specificity in the roles of MNs in motor control. The effects of the local MN activity manipulation are lost in shakB2 or ogre2, gap-junction mutations in Drosophila, or upon acute application of the gap junction blocker CBX, implicating electrical synapses in the signaling from MNs. Cell-type specific RNAi suggests shakB and ogre function in MNs and interneurons, respectively, during the signaling. These results not only reveal an unexpected role for MNs in motor pattern regulation but also introduce a powerful experimental system that enables examination of the input-output relationship among the component neurons in this system (Matsunaga, 2017).

    Animal movement is accomplished by spatially and temporally coordinated contraction of various muscles throughout the body. It is generally thought that a neuronal network composed of premotor interneurons generates a motor pattern, and this network sequentially activates different classes of motor neurons (MNs). In this view, MNs play only passive roles in pattern generation, relaying the information they receive from upstream interneuronal networks to muscles. By contrast, there is some evidence that MNs themselves contribute to the motor pattern generation. In the crustacean stomatogastric ganglion and in leech swimming circuits, MNs are part of the pattern-forming network. In mammalian spinal cords, MNs send a collateral to innervate Renshaw cells, which in turn convey feedback signals to MNs. However, whether and how MNs regulate motor pattern generation during animal movements remains largely unexplored (Matsunaga, 2017).

    Larval Drosophila is emerging as an excellent model system for studying motor pattern generation since one can apply powerful genetic tools including a large collection of Gal4-drivers to study the function of individual component neurons in a numerically simple nervous system. Furthermore, previous development of a platform for electronic microscope (EM) image data reconstruction of the entire nervous system of the larval CNS now allows mapping of the circuit structure that mediates specific behaviors. The larval ventral nerve cord (VNC) consists of three thoracic neuromeres (T1, T2, and T3) and eight abdominal neuromeres (A1-A8). Larval peristaltic crawling is accomplished by successive bilateral muscle contraction that propagates from tail to head. Muscle contraction in each segment is in turn regulated by sequential activation of MNs in the corresponding neuromere of the VNC. Although recent studies have begun to identify several types of premotor interneurons that regulate aspects of movement such as the speed of locomotion and left-right or intersegmental coordination, how a motor pattern is generated by the neural circuits remains largely unknown (Matsunaga, 2017).

    In a previous study, halorhodopsin (NpHR) was used to locally and transiently inhibit MN activity in one or a few segments; local activity perturbation was found to halt the propagation of the peristaltic wave at the site of manipulation. This suggests that MNs are part of the neural circuits that generate the peristaltic wave. However, how information is retrogradely transmitted from MNs to the central circuits remained unknown. Furthermore, since muscle contraction was usedt as a measure of the motor outputs, changes in the activity dynamics in the CNS could not be studied. That study was extended by constructing a new experimental system in which the effects of local optogenetic manipulation of MNs on global motor activity could be studied in the VNC. Optical inhibition of MNs in a middle segment (A4, A5, or A6) decreased the motor frequency. Conversely, photoactivation of MNs in a posterior segment (A6 or A7) increased the frequency of the motor wave. These results indicate that the local activity level of MNs impacts the global outputs of the motor circuits in a segment-specific manner. It was also show that gap junctions are involved in this process. While this manuscript was in preparation, a study in zebrafish reported that motor neurons retrogradely influence the activity level of the premotor V2a interneurons via gap junctions and regulate motor generation. Thus, regulation by gap junction-mediated retrograde MN signaling appears to be a common mechanism of motor control (Matsunaga, 2017).

    In a previous study, MN activity was locally and transiently inhibited in one or a few segments during peristalsis of dissected larvae, and activity manipulation was shown to halt peristalsis. This indicates that MN activity is required for the motor activity wave to propagate along the VNC and suggests the presence of retrograde signaling from MNs to the central circuits. However, since dissected larvae were used, the possibility that the signaling was instead mediated via the sensory feedback of muscular contraction could not be excluded. Furthermore, the mechanism of the retrograde signaling remained unknown (Matsunaga, 2017).

    A new experimental system was built that allow studying the direct causal relationship between the manipulation of MN activity and changes in neural dynamics in the motor circuits with superior spatial and temporal resolution. Optical perturbations were applied for a longer period and in a more systematic manner than in the previous study and their effects on the global circuit activity was analyzed. Using the new experimental system, the previous study was extended by showing that (1) MN outputs within the CNS, not mediated by sensory feedback, are critical for motor wave regulation, (2) there is segmental difference in the role of the MN outputs, and (3) the MN signaling is mediated by gap junctions (Matsunaga, 2017).

    This study shows that manipulation of motor neuronal activity in just one segment robustly affects the output of the entire motor network in Drosophila larvae. Optical inhibition or activation of MNs in a single segment decreased or increased, respectively, the calcium level of MNs in distant neuromeres. Furthermore, these perturbations strongly affected the frequency of motor waves. Thus, changes in MN activity in one segment affect the activity level and wave generation of the entire motor system (Matsunaga, 2017).

    It should be noted that in the isolated VNC preparation used in this study, peripheral nerves with motor activity output and sensory feedback input were severed. Local changes in MN activity therefore influenced the activity of distant MNs through intersegmental neural connections within the CNS, not via sensory feedback. Thus, the results establish the presence of retrograde signaling from MNs that is critical for motor pattern regulation. The identity of the synaptic connections mediating the signaling is currently unknown. They could be direct MN-MN connections, or they may also involve coupling between MNs and interneurons (Matsunaga, 2017).

    Electrical synapses are commonly found in the nervous systems of vertebrates and invertebrates. In particular, electrical coupling mediated by gap junctions has been implicated in motor pattern control in various systems. This study has showed that gap junctions are involved in the retrograde MN signaling controlling motor wave frequency in Drosophila larvae. Local photomanipulation of MNs that would normally increase or decrease wave frequency had no effect in shakB2 and ogre2 mutants. This suggests that electrical synapses including ShakB and Ogre mediate the MN signaling controlling motor frequency. In contrast, CBX administration but not shakB2 or ogre2 mutation abolished the calcium level changes of distant MNs induced by the activity manipulation, suggesting that innexins other than those deleted in shakB2 or ogre2 mediate this aspect of motoneuronal communication (eight innexin genes are present in the Drosophila genome). It should also be noted that wave generation normally occurred in the isolated VNCs of shakB2 and ogre2 mutants. There was also no obvious abnormality in the locomotion of the shakB2 or ogre2 larvae. These observations suggest that ShakB and Ogre-mediated MN signaling is part of redundant pathway(s) regulating motor waves. Only upon optical perturbation are the role of MN signaling in wave generation and involvement of ShakB and Ogre manifested (Matsunaga, 2017).

    Previous work has reported the existence of electrical coupling between MNs and the premotor excitatory V2a interneurons, a neuronal class that provides a major drive for MNs during locomotion in zebrafish. Hyperpolarizing or depolarizing MNs decreased or increased the firing activity of V2a interneurons. Furthermore, selective inhibition of MNs during locomotion interrupted the recruitment of V2a interneurons and decreased the frequency of locomotion. Thus, control of locomotor circuits by gap junction-mediated retrograde MN signaling may be an evolutionarily conserved mechanism used in both invertebrates and vertebrates (Matsunaga, 2017).

    An interesting feature of MN signaling revealed in this study is segment specificity. On the one hand, inhibition of MNs in A4, A5, or A6, but not other segments, reduced the motor wave frequency. On the other hand, activation of MNs in A6 or A7, but not other segments, increased the frequency of the wave. This segmental discord with regard to the MN signaling may contribute to the regulation of the wave initiation. How can gap junctions selectively mediate one type of activity change but not another? For example, how can the decline but not the elevation in activity level of MNs in the A4 or A5 segment affect the wave frequency? One possibility is the involvement of rectifying electrical synapses. Rectifying electrical synapses have been found in both vertebrates and invertebrates and can mediate unidirectional synaptic transmission in a voltage-dependent manner. Rectifying electrical synapses are often composed of a heteromeric assembly of gap junction proteins on each side of the apposing neurons. A role of ShakB in rectification has been shown in the giant fiber system of adult Drosophila. Two splicing forms of ShakB, ShakB(N) and ShakB(N+16), are expressed in the presynaptic and postsynaptic sites of the giant synapse, respectively. When expressed in neighboring oocytes, these two ShakB variants form heterotypic channels that are asymmetrically gated by voltage. Since RNAi knockdown experiments showed that shakB, but not ogre, is required in MNs to mediate the retrograde signaling, an interesting possibility is the involvement of heterotypic channels composed of ShakB in MNs and Ogre in interneurons. Future studies are necessary first to identify the target neurons that receive the retrograde MN signaling and then to study whether the relevant electrical synapses are indeed rectified. Revealing the information flow mediating the MN retrograde signaling will provide valuable insights on how intersegmentally coordinated motor patterns are generated in this and other systems. The experimental system established in this study can also be applied more generally to study the input-output relationship among the component neurons in this system. While the GAL4/UAS system alone was sufficient to express both GCaMP/RGECO and NpHR/ChR2 in MNs in this study, the introduction of another expression system such as the LexA system will allow expression of GCaMP/RGECO and NpHR/ChR2 in different classes of neurons, including interneurons. This will allow study of the the influence of the optogenetic manipulation of one class of neurons on the activity of others. The functional analyses may also be combined with the circuit diagram elucidated by ongoing EM reconstruction. It is anticipated that such systematic analyses will elucidate fundamental mechanisms of how central circuits coordinate intersegmental movements (Matsunaga, 2017).


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    genes expressed in the CNS

    Genes involved in organ development

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