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).

Nuclear Prospero allows one-division potential to neural precursors and post-mitotic status to neurons via opposite regulation of Cyclin E

In Drosophila embryonic CNS, the multipotential stem cells called neuroblasts (NBs) divide by self-renewing asymmetric division and generate bipotential precursors called ganglion mother cells (GMCs). GMCs divide only once to generate two distinct post-mitotic neurons. The genes and the pathways that confer a single division potential to precursor cells or how neurons become post-mitotic are unknown. It has been suggested that the homeodomain protein Prospero (Pros) when localized to the nucleus, limits the stem-cell potential of precursors. This study shows that nuclear Prospero is phosphorylated, where it binds to chromatin. In NB lineages such as MP2, or GMC lineages such as GMC4-2a, Pros allows the one-division potential, as well as the post-mitotic status of progeny neurons. These events are mediated by augmenting the expression of Cyclin E in the precursor and repressing the expression in post-mitotic neurons. Thus, in the absence of Pros, Cyclin E is downregulated in the MP2 cell. Consequently, MP2 fails to divide, instead, it differentiates into one of the two progeny neurons. In progeny cells, Pros reverses its role and augments the downregulation of Cyclin E, allowing neurons to exit the cell cycle. Thus, in older pros mutant embryos Cyclin E is upregulated in progeny cells. These results elucidate a long-standing problem of division potential of precursors and post-mitotic status of progeny cells and how fine-tuning cyclin E expression in the opposite direction controls these fundamental cellular events. This work also sheds light on the post-translational modification of Pros that determines its cytoplasmic versus nuclear localization (Mar, 2022).

What makes precursor cells divide a certain number of times and how the progeny cells become post-mitotic has remained enigmatic. These questions are among some of the most fundamental questions in neurobiology. The work described in this study provides a clue and indicates that Pros and Cyclin E may be some of the key players in these processes. The results indicate that the cytoplasmic, non-phosphorylated Pros becomes phosphorylated and nuclear and binds to chromatin in cells destined to divide once. It must directly or indirectly regulate gene expression in the nucleus. One such gene regulated by Pros appears to be Cyclin E. Ample data indicates that Cyclin E is essential but also sufficient to drive entry of precursor cells to the cell cycle, although within a temporal window of developmental time. The data presented in this study show that Pros regulates Cyclin E levels in opposing directions between precursor cells and their progeny. This is an elegant, yet simple mechanism by which Pros through Cyclin E confers the one-division potential to MP2, GMC4-2a, or GMCs from NB7-3, and then helps commit their progeny to a post-mitotic state. How many lineages in the CNS also utilize this mechanism remains unknown (Mar, 2022).

The situation is not an ON/OFF scenario. A clear ON/OFF scenario will also be evolutionarily prohibitive as it would negatively affect the neuronal number, plasticity, and diversity. Instead, Pros appears to augment the upregulation of Cyclin E level in the precursor enough to commit that cell to divide once. Once it divides to generate two daughters, Pros augments the downregulation of levels of Cyclin E such that progeny cells do not enter the cell cycle, but instead become post-mitotic. The evidence to support this model comes from the fact that in pros loss of function mutants, cells such as MP2 and GMC4-2a fail to divide. The levels of Cyclin E were also downregulated in MP2 in pros mutant embryos, indicating a positive role for Pros via augmenting Cyclin E expression in MP2 division. However, in pros loss of function mutants at a later developmental stage, the level of Cyclin E was upregulated, indicating a repression role for Pros in older stage embryos. Thus, a repression of Cyclin E by Pros could lead to the post-mitotic status of progeny neurons. A switch from an activator to a repressor can be achieved by partnering with different transcription regulators before and after cell division. These results are further supported by the finding that MP2 fails to divide in loss of function for cyclin E, and gain of function for cyclin E, or gain of function for pros leads to extra divisions. The penetrance of these defects is not very high, but it is thought that this is to be expected since players such as Cyclin E will be tightly regulated during development. There is also the issue of maternal deposition when loss of function mutations is in question (Mar, 2022).

The opposing role of Pros in cyclin E regulation, depending upon the developmental stage, is meant to switch from facilitating a single division of precursors to facilitating a post-mitotic commitment of progeny cells. De-repression of Cyclin E alone in progeny cells in late-stage embryos either in the pros mutant or by over-expression of Cyclin E in post-mitotic progeny cells does not appear to be sufficient to make them re-enter the cell cycle. It may be that the Cyclin E level was not high enough in those stages of development, or the process that makes cells post-mitotic involves additional players. Thus, elevating Cyclin E alone at the 'Cyclin E-insensitive' stage may not be enough to make them re-enter cell cycle. Additionally, differentiation genes also begin to express in progeny cells, and then there is the physical process of differentiation that gets underway with neurites sprouting and axon forming. These structural changes in post-mitotic neurons could also prevent them, in addition to new gene expression programs, from re-entering the cell cycle despite elevated levels of Cyclin E. Furthermore, in 12 hpf or older pros mutant embryos, there is a general up-regulation of Cyclin E, not only in the MP2 lineage, but also in other cells in the nerve cord. The consequence of this upregulation, such as if those lineages produce extra cells, is not known. This question is currently being examined. It is also not known if Pros plays a similar role in type II NBs in the embryonic nervous system or during neurogenesis of the adult brain (Mar, 2022).

These results also argue that there may not be a dedifferentiation of cells in pros mutants as previously thought, at least not in every lineage. Pros, with its chromatin localization in cells committed to a differentiation pathway, appears to control many genes. Cyclin E alone, at least in earlier stages of development, is sufficient to make cells divide or not divide depending on the levels, and Pros fits in this Cyclin-E-mediated model by its ability to regulate cyclin E expression. In how many lineages Pros confers the single-division potential to precursor cells is not clear. It is not known if the asymmetrically localized cytoplasmic Pros in NBs has any role in cell division or if it simply is a mechanism to segregate Pros to GMCs. In any event, Pros is unlikely to regulate Cyclin E in NBs (other than MP2/NBs that have single division potential) (Mar, 2022).

Finally, these results are consistent with previous finding that in embryos mutant for a gene called midline, MP2 undergoes multiple self-renewing asymmetric divisions. Pros was cytoplasmic in MP2 in midline mutants [24], which further indicates that the single division potential of MP2 correlates with a nuclear/chromatin-bound Pros. A recent paper indicated that Pros remodels H3K9me3+ pericentromeric heterochromatin by recruiting Heterochromatin Protein 1 during neuronal differentiation (Liu, 2020). This conclusion is consistent with the supposition that Pros augments post-mitotic commitment and neuronal differentiation of progeny cells, and regulation of Cyclin E and modulating heterochromatin are essential to these developmental events. Neuronal differentiation is a complex and evolutionarily crucial process for survival; therefore, it is not surprising that various mechanisms will augment the process as a shared phenomenon. A partial redundancy for a gene or a pathway is a common theme during neurogenesis or development (Mar, 2022).

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 candidate 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-Gal4^RRK 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-Gal4^RRK. 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-Gal4^RRK, 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-Gal4^RRK) 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-Gal4^RRK) 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 SCP_B, 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).

Transcription factor expression uniquely identifies most postembryonic neuronal lineages in the Drosophila thoracic central nervous system

Most neurons of the adult Drosophila ventral nerve cord arise from a burst of neurogenesis during the third larval instar stage. Most of this growth occurs in thoracic neuromeres, which contain 25 individually identifiable postembryonic neuronal lineages. Initially, each lineage consists of two hemilineages--'A' (Notch(On)) and 'B' (Notch(Off))--that exhibit distinct axonal trajectories or fates. No reliable method presently exists to identify these lineages or hemilineages unambiguously other than labor-intensive lineage-tracing methods. By combining mosaic analysis with a repressible cell marker (MARCM) analysis with gene expression studies, a gene expression map was constructed that enables the rapid, unambiguous identification of 23 of the 25 postembryonic lineages based on the expression of 15 transcription factors. Pilot genetic studies reveal that these transcription factors regulate the specification and differentiation of postembryonic neurons: for example, Nkx6 is necessary and sufficient to direct axonal pathway selection in lineage 3. The gene expression map thus provides a descriptive foundation for the genetic and molecular dissection of adult-specific neurogenesis and identifies many transcription factors that are likely to regulate the development and differentiation of discrete subsets of postembryonic neurons (Lacin, 2014).

Understanding how cell-type diversity in nervous systems arises remains a key goal in developmental biology. Even simple nervous systems, such as those in insects, involve hundreds of different subtypes of cells. Over the last several decades, research using the Drosophila embryonic CNS as a model system has unveiled basic principles that underlie nervous system development in invertebrates and vertebrates. Drosophila and other holometabolous insects, however, undergo two distinct waves of neurogenesis: embryonic neurogenesis creates the larval nervous system; postembryonic neurogenesis creates the adult nervous system. Relative to embryonic neurogenesis, little is known about the genetic and molecular control of postembryonic neurogenesis (Lacin, 2014).

Within each hemisegment of the segmented embryonic nerve cord, 30 neuroblasts (NBs) divide in a stem cell manner to produce ~400 neurons and glia that interconnect to form a functional CNS. Towards the end of embryogenesis, NBs become quiescent or undergo apoptosis: in abdominal segments, most NBs die; in thoracic segments, 25 of the 30 NBs become quiescent and persist into larval stages. This study focused on the postembryonic neuronal lineages produced by these 25 NBs. During the second larval-instar stage, in response to glia-derived insulin signaling, thoracic NBs regain their proliferative activity. Initially, NBs divide slowly to produce a small number of large, Chinmo-positive (Chinmo+) neurons (termed early-born neurons). Shortly after larvae enter the third (last) instar stage, NBs divide more quickly and produce many, small Broad+ neurons (termed late-born neurons), ceasing their proliferation in the early pupal stage (Lacin, 2014).

Elegant mosaic analysis with a repressible cell marker (MARCM)-based lineage-tracing studies revealed that each neuronal lineage in the thoracic CNS is uniquely identifiable based on its relative position size and neuronal projection patterns. Each postembryonic NB, which resides in the ventral-most region of a lineage, divides in a stem cell manner to self-renew and produce a chain of secondary precursor cells, called ganglion mother cells (GMCs). Typically, each GMC divides to produce sibling post-mitotic neurons that adopt distinct fates based on the state of Notch signaling -- 'A' (NotchON), 'B' (NotchOFF). In contrast to the embryo, in which sequentially born 'A' (or 'B') daughter cells often adopt distinct identities, most A (or B) cells within a given postembryonic lineage manifest the same cellular phenotype, extending projections along a common path to a similar target region. Thus, initially, each postembryonic lineage consists of a NB and some GMCs in the ventral region of the clone and two major subtypes of neurons (A and B) more dorsally. In some lineages, most or all cells of the A (or B) hemilineage undergo apoptosis, resulting in a monotypic lineage that consists largely, if not exclusively, of cells from the A or B hemilineage (Lacin, 2014).

At present, the only reliable way to identify which lineage a group of postembryonic neurons belongs to is through labor-intensive MARCM-based lineage tracing methods. In the work reported in this study, by combining gene expression studies of 14 transcription factors with MARCM-based lineage tracing methods, a gene expression map was created that unambiguously identifies 23 of the 25 postembryonic neuronal lineages and 29 of the 34 major neuronal hemilineages (See: Schematic models of transcription factor expression in postembryonic neuronal lineages). Pilot functional studies reveal that the identified transcription factors direct the development and differentiation of the postembryonic neurons expressing them (Lacin, 2014).

By a ten-to-one ratio, postembryonic neurons outnumber embryonic neurons, and the adult fly CNS is composed almost entirely of postembryonic neurons. Yet much less is known about postembryonic neurogenesis than embryonic neurogenesis. The molecular marker map of postembryonic thoracic neuronal lineages presented in this study helps bridge this gap by extending the work of Truman who characterized these lineages on the basis of morphology. The map enables the identification of 23 of 25 postembryonic neuronal lineages based on gene expression alone, buttressing the descriptive foundation of postembryonic neurogenesis and illustrating that the combinatorial code of neuronal specification extends to the postembryonic thoracic CNS. The apparent lack of cell-type diversity in postembryonic neuronal lineages, the utility of the gene expression map in matching postembryonic lineages to their cognate embryonic lineages, and the similarity of hemilineages in flies to pools of neurons in vertebrates are discussed (Lacin, 2014).

Despite their larger size, postembryonic lineages appear less complex than their embryonic counterparts. For example, embryonic NB 3-1 produces four motor neurons and a variable number of intersegmental and local interneurons. Postembryonic lineage 4, which appears to derive from NB 3-1, generates almost 50 cells, but based on morphology and gene expression, neurons in this lineage can be grouped into at most two subtypes of neurons. Are thoracic postembryonic neuronal lineages less complex than their embryonic counterparts? The jury remains out. Studies of postembryonic neurogenesis have not reached the resolution of those in the embryo, and most have assessed postembryonic neuronal lineages at the end of larval life, when the vast majority of neurons have arisen, but still days away from their final differentiation. Thus, even though neurons in a given postembryonic lineage display simple gene expression profiles and extend axons along only one or two paths before metamorphosis, they may manifest complex patterns of target innervation and gene expression after metamorphosis. In this context, the molecular marker map is a key antecedent for studies that dissect the cellular and molecular complexity of postembryonic lineages at single-cell resolution at later stages of development (Lacin, 2014).

Within the thoracic nerve cord, postembryonic neurons derive from the same NBs that generate embryonic neurons. With few exceptions, it has proved difficult to pair postembryonic neuronal lineages with their cognate embryonic lineages and NBs. To do so would provide a continuum of knowledge from the genetic mechanisms that drive the NB formation and specification in the embryo to those that govern the development and differentiation of postembryonic neurons (Lacin, 2014).

In those cases in which the common ancestry of embryonic and postembryonic neuronal lineages is known, the two lineages share similar gene expression profiles. For example, postembryonic lineages 3 and 4 derive from embryonic NBs 7-1 and 3-1, respectively. In embryos, NB 7-1 expresses Nkx6 and generates Eve+ A-type motor neurons and Dbx+ B-type interneurons. In larvae, NB 7-1 continues to produce Dbx+ B-type interneurons and generates Nkx6+, rather than Eve+, A-type neurons. In embryos, NB 3-1 produces B-type Hb9+, Nkx6+, Lim3+ and Islet+ RP1, three to five motor neurons. In larvae, NB 3-1 produces B-type Hb9+ and Nkx6+, but Lim3- and Islet-, interneurons. Thus, lineally related embryonic and postembryonic neurons share similar gene expression profiles and are likely to share functional attributes (Lacin, 2014).

The shared gene expression profiles of lineally related postembryonic and embryonic neurons suggest the gene expression map will help match postembryonic lineages to their cognate embryonic lineages. For example, in embryos NB 2-2 generates six B-type neurons that express Hb9, Nkx6 and Lim3 (Lacin, 2009). In larvae, lineage 10, a monotypic B-type lineage, is the only postembryonic lineage that expresses this combination of transcription factors, and similar to the embryonic neurons produced by NB 2-2, lineage 10 neurons extend axons across the midline as part of the anterior commissure. A systematic pairing of embryonic and postembryonic lineages will still require sophisticated lineage tracing methods that induce clones in the early embryo and analyze the embryonic and postembryonic lineages of single NBs in the CNS of late third instar larvae. Here, the simultaneous use of molecular markers that identify defined embryonic and/or postembryonic neuronal lineages will enable the matching of individual embryonic and postembryonic lineages. Only through such studies will it be possible to follow CNS development uninterrupted from the embryo to the adult (Lacin, 2014).

The hemilineage has been identified as the developmental unit of the postembryonic CNS: most neurons within an individual hemilineage project axons within the same bundle to similar targets (Truman, 2010). This study extends these findings by showing that within a given lineage, most transcription factors are expressed in A- or B-type neurons, but not both. Thus, hemilineages are composed of tightly clustered groups of neurons that share common transcription factor expression profiles and extend axons in the same bundle to innervate similar targets (Lacin, 2014).

At the morphological and molecular level, neuronal hemilineages in flies resemble pools of neurons in vertebrates. Individual motor or inter-neuron pools are composed of clustered groups of neurons that share common transcription factor expression profiles and extend axons in the same bundle to innervate similar targets. For example, motor neurons with cell bodies located medially within the lateral motor column (LMC) express Islet and project axons to ventrally derived limb muscle; motor neurons with cell bodies located laterally in the LMC express Lim1 and project axons to dorsally derived limb muscles. These parallels between neuronal hemilineages in flies and pools of neurons in vertebrates suggest that individual pools of vertebrate neurons share a common lineage and state of Notch activation (Lacin, 2014).

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).

Neurotransmitter identity is acquired in a lineage-restricted manner in the Drosophila CNS

The vast majority of the adult fly ventral nerve cord is composed of 34 hemilineages, which are clusters of lineally related neurons. Neurons in these hemilineages use one of the three fast-acting neurotransmitters (acetylcholine, GABA, or glutamate) for communication. This study generated a comprehensive neurotransmitter usage map for the entire ventral nerve cord. No cases were found of neurons using more than one neurotransmitter, but it was found that the acetylcholine specific gene ChAT is transcribed in many glutamatergic and GABAergic neurons, but these transcripts typically do not leave the nucleus and are not translated. Importantly, this work uncovered a simple rule: All neurons within a hemilineage use the same neurotransmitter. Thus, neurotransmitter identity is acquired at the stem cell level. This detailed transmitter usage/lineage identity map will be a great resource for studying the developmental basis of behavior and deciphering how neuronal circuits function to regulate behavior (Lacin, 2019).

The ventral nerve cord (VNC) of Drosophila melanogaster is home to circuits coding for vital behaviors, such as walking, jumping, and flight. It is composed of about 16,000 neurons, all of which arise from a set of segmentally repeated 30 paired and one unpaired neural stem cells (Neuroblasts [NBs]). NBs generate unique progeny via undergoing two rounds of proliferation: a brief embryonic and an extended postembryonic phase. Embryonic neurogenesis generates the neurons of the larval CNS and many of these cells are then remodeled to function in the adult CNS. 90-95% of the adult neurons, however, are adult-specific and arise during the post-embryonic phase of neurogenesis (Lacin, 2019).

The VNC contains only NBs that show a Type I pattern of proliferation. Each NB divides repeatedly via asymmetric cell division to renew itself and to generate a secondary precursor cell, called a ganglion mother cell (GMC). Each GMC, in turn, divides terminally to form two neurons, each of which acquires a unique identity due to the presence or absence of active Notch signaling. Within a NB progeny, the Notch-ON neurons are called the 'A' hemilineage; their Notch-OFF siblings are called the 'B' hemilineage (Lacin, 2019).

By progressing through the temporal transcriptional cascade, Hunchback -> Kruppel -> Pdm, NBs generate diverse 'A' and 'B' neurons during the early embryonic phase. Subsequently, NBs express Castor and Grainyhead in late embryonic stages and many NBs maintain this Castor/Grainyhead expression into the postembryonic stages. Correlated with this shared gene expression, neurons of a hemilineage ('A' or' B') that are born during the late embryonic and early postembyonic stages often adopt similar fates. Recent studies characterized the morphologies of postembryonic hemilineages in their immature states in the larva and mature states in the adult. These studies revealed that in the larva, the immature neurons of each hemilineage cluster together and extend their initial processes as a bundle to the same region and that after metamorphosis, in the adult, they continue to be clustered and share common anatomical and functional features (Lacin, 2019).

In addition to similar morphology, neurons within a postembryonic hemilineage share patterns of transcription factor expression. In the larval VNC, each hemilineage cluster can be identified with a specific combination of transcription factor expression (Lacin, 2014). Interestingly, vertebrate homologs of many of these hemilineage-specific transcription factors are expressed in the spinal cord and are required for fate determination. For example, in flies, the combinatorial expression of Lim3, Islet, and Nkx6 is observed uniquely in hemilineage 15B, which is composed of leg motor neurons (Lacin, 2014). In mice, homologs of these three factors are essential for the identity of spinal motor neurons. Indeed, interneurons in the vertebrate spinal cord are also organized into discrete cardinal classes that share developmental origins, transcription factor and neurotransmitter expression, and functional roles. The fly VNC appears to be organized in an analogous manner, with the developmental origins of neuronal clusters providing the basis for their transcription factor expression and functional properties. The relationship between specific stem cells and neurotransmitter expression in their progeny, though, has yet to be resolved (Lacin, 2019).

Studies on grasshoppers and Manduca sexta showed that clusters of GABAergic interneurons were based on their lineage of origin. Likewise, cholinergic and glutamatergic neurons are also typically found as clusters in the VNC and the brain consistent with a shared lineage. In the fly, as a prelude to studies that seek to dissect the developmental basis of behavior, neurotransmitter usage was comprehensively mapped across the VNC to determine how neurotransmitter selection relates to lineage identity. A similar comprehensive neurotransmitter map was generated for the C. elegans nervous system and proved to be beneficial in identifying regulatory mechanisms that control neurotransmitter identity and circuit assembly. By using molecular and genetic tools, this study mapped neurotransmitter usage in all hemilineages in the adult fly VNC. The results revealed that, as found in the neurons of the vertebrate cardinal classes, all neurons within a fly hemilineage use the same neurotransmitter. In agreement with earlier findings, this study further shows that hemilineages are not just developmental units but also functional units that drive animal behavior (Lacin, 2019).

The adult VNC is made up primarily from 34 hemilineages -- clusters of lineally related, segmentally repeated, postembryonic-born neurons. Previous work systematically characterized the development and neuronal morphologies of these hemilineages, and showed that neurons within a hemilineage adopt similar fates, evident from their immature axonal projection and transcription factor expression. This study has systematically mapped the neurotransmitter choice of most VNC neurons by studying all of the postembryonic hemilineages. Surprisingly, it was found that neurotransmitter code in the VNC is simple in that all neurons within a hemilineage use the same neurotransmitter, thus transmitter identity is determined at the stem cell level. These results further support earlier findings that hemilineages represent functional units, both in terms of anatomy and now neurotransmitter chemistry (Lacin, 2019).

The statement that all neurons within a hemilineage use the same neurotransmitter excludes the neurons that are born during early embryonic neurogenesis. As mentioned earlier, neurons born during this time are highly diverse and might use a different neurotransmitter than the rest of the neurons in the hemilineage. For example, both NB4-2 and NB5-2 generate glutamatergic motor neurons from their early embryonic divisions, but their postembryonic progenies (both A and B hemilineages) are purely GABAergic. Similarly, glutamatergic U/CQ motor neurons, which are born in early embryonic stages share the same hemilineage with the cholinergic 3A neurons. Thus, neuronal fates within a hemilineage can be dramatically different when embryonic and postembryonic neurons are compared. It is believed that the reason why postembryonic hemilineages in the VNC are homogenous in terms of neuronal fate is due to the expansion of particular, later-born neuronal classes as neuronal lineages became larger during evolution of more derived insects to accommodate more complex behaviors such as flight. Since all insect species have similar sets of NBs, new behaviors (e.g., flight) appear to have evolved via changes in the number of neurons generated by each NB, but not changes in the number of NBs. Moreover, the Notch mediated asymmetric division enabled the insect to have two distinct clonal populations of neurons (hemilineages) from a single NB. Interestingly, only 34 of 50 potential hemilineages are used in the adult fly VNC while 16 of them are eliminated by apoptosis. Thus, flies have the potential to acquire novel behaviors by simply resurrecting hemilineages that are fated to die (Lacin, 2019).

At least within the thorax, the hemilineages express the same transmitter regardless of their segment of residence. This conservation was expected for the hemilineages that contribute to the leg neuropils, since neuron numbers and the projections of these cells appear similar across the different thoracic segments. On the other hand, the hemilineages innervating the dorsal, flight-related neuropils have segment specific organization and show dramatically different axonal projections depending on their segmental location. For example, 7B neurons in each segment have unique projection and appear to execute distinct behaviors. Despite these differences, 7B neurons use acetylcholine in every segment. These results show that the neurotransmitter fate is tightly linked to the lineage origin and that the segmental diversification of the 7B neurons with the evolution of the derived flight system of flies may have had to occur with this transmitter constraint (Lacin, 2019).

It is expected that most hemilineages in the fly brain are also homogenous in terms of neurotransmitter expression as large neuronal clusters were observed expressing the same neurotransmitter. However, some complex brain hemilineages that have diverse neuronal populations might have different neurotransmitter expression as it was shown for the lAL lineage (Lacin, 2019).

This study has also extended earlier transcription factor expression studies in immature neurons of larval stages into the mature neurons of the adult. The expression of many transcription factors is maintained into adult stages and can be used to mark specific hemilineages in the adult. However, some transcription factors are expressed transiently during development. For example, Dbx marks many immature 3B neurons in the larva, but its expression disappears in these neurons after pupa formation. From the expression analysis of the limited number of transcription factors examined in this study, no factor was found that specifically marked all neurons of a specific neurotransmitter type. However, a few transcription factors, whose expression tightly correlated with the neurotransmitter fate, were found. For example, Dbx expression is restricted to GABAergic neurons, even though Dbx does not appear to promote the GABA fate by itself as GABAergic fate is unaltered in response to Dbx loss or misexpression. Similarly, it was found that Unc-4 expression is restricted to cholinergic lineages among the postembryonic lineages in the VNC; however, in the brain Unc-4 is expressed in glutamatergic lineages in addition to cholinergic lineages, suggesting that different parts of the CNS might use the same transcription factor for different fates via utilizing different cofactors. Supporting this, it was found that none of the GABAergic lineages in the VNC are marked with Lim3, which was shown to be expressed in most GABAergic neurons of the fly optic lobe and required for their GABAergic identity. The reverse scenario where neurons acquire the same neurotransmitter identity via different transcriptional regulatory networks is also commonly observed. For example in the C. elegans nervous system, distinct combinations of 13 transcription factors are responsible for VGlut expression in 25 different glutamatergic neuron classes . Thus, transcription factors act together combinatorially rather than individually to specify neurotransmitter fate (Lacin, 2019).

This study did not find any correlation between the Notch state of the neurons and neurotransmitter identity, an observation made in the optic lobe. Any neurotransmitter type can be observed in both 'A' and 'B' hemilineages. It is also noted that NB4-2 (progenitor of 13A/B) and NB5-2 (progenitor of 6A/B) are the only two NBs in which both the 'A' and 'B' hemilineages use the same neurotransmitter, GABA (Lacin, 2019).

Interestingly, the neurotransmitter pattern of Drosophila hemilineages appears to be conserved in other insect species. For example, based on location and morphology, it was deduced that sibling 'Kl' and 'Km' GABAergic clusters of the moth, Manduca Sexta, are homologous to the 13A and 13B hemilineages, respectively, and the 'M' cluster is likely homologous to the 6A, 6B, and 5B neurons, which form a large cluster GABAergic in the posterior thoracic ganglia of the fly. Similar GABAergic clusters were also observed in the nerve cords of grasshopper and silverfish. Interestingly, like the Drosophila VNC, the grasshopper nerve cord contains two clusters of En+GABA+ neurons, named 'A' and 'B' groups, which are likely homologous to 0A and 6B neurons, respectively (Lacin, 2019).

Unexpectedly, this study detected ChAT transcripts in many GABAergic and glutamatergic neurons, most of which are members of lineages 5B and 11B (GABAergic) and 14A and 15B (glutamatergic). The low levels of ChAT transcripts and the lack of ChAT immunostainings in these cells suggested that ChAT transcripts are actively degraded and not translated. It is possible that these neurons produce acetylcholine but only in certain conditions for example during development or under stress. Indeed, neurotransmitter switching has been observed in many neurons of vertebrates, however, most of these switches involve aminergic neurotransmitters (Lacin, 2019 and references therein).

Another possibility for the presence of ChAT transcripts in noncholinergic neurons is that it is a remnant of a neurotransmitter switch that might have happened during evolution. 15B neurons are a good candidate for such a possibility. 15B motor neurons are glutamatergic like all other fly motor neurons, while all vertebrate and some invertebrate (e.g, C. elegans and Aplysia) motor neurons are cholinergic, raising the possibility that motor neurons of the common ancestor used acetylcholine. Thus, the ChAT expression in 15B motor neurons might be a vestige from the cholinergic ancestry of motor neurons (Lacin, 2019).

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).

Condensation of the Drosophila nerve cord is oscillatory and depends on coordinated mechanical interactions

During development, organs reach precise shapes and sizes. Organ morphology is not always obtained through growth; a classic counterexample is the condensation of the nervous system during Drosophila embryogenesis. The mechanics underlying such condensation remain poorly understood. This study characterized the condensation of the embryonic ventral nerve cord (VNC) at both subcellular and tissue scales. This analysis reveals that condensation is not a unidirectional continuous process but instead occurs through oscillatory contractions. The VNC mechanical properties spatially and temporally vary, and forces along its longitudinal axis are spatially heterogeneous. The process of VNC condensation is dependent on the coordinated mechanical activities of neurons and glia. These outcomes are consistent with a viscoelastic model of condensation, which incorporates time delays and effective frictional interactions. In summary, this study has defined the progressive mechanics driving VNC condensation, providing insights into how a highly viscous tissue can autonomously change shape and size (Karkali, 2022).

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).

Drosophila neuroblast selection is gated by Notch, Snail, SoxB, and EMT gene interplay

In the developing Drosophila central nervous system (CNS), neural progenitor (neuroblast [NB]) selection is gated by lateral inhibition, controlled by Notch signaling and proneural genes. However, proneural mutants still generate many NBs, indicating the existence of additional proneural genes. Moreover, recent studies reveal involvement of key epithelial-mesenchymal transition (EMT) genes in NB selection, but the regulatory interplay between Notch signaling and the EMT machinery is unclear. This study finds that SoxNeuro (SoxB family) and worniu (Snail family) are integrated with the Notch pathway, and constitute the missing proneural genes. Notch signaling, the proneural, SoxNeuro, and worniu genes regulate key EMT genes to orchestrate the NB selection process. Hence, this study has uncovered an expanded lateral inhibition network for NB selection and demonstrate its link to key players in the EMT machinery. The evolutionary conservation of the genes involved suggests that the Notch-SoxB-Snail-EMT network may control neural progenitor selection in many other systems (Arefin, 2019).

Based upon previous studies, and the findings in this study, SoxN is necessary for NB generation. Regarding the Snail family, previous studies did not find apparent reductions of NBs numbers in sna, esg, and wor triple mutants. However, these studies focused on early stages of neurogenesis and may not have covered the complete span of NB formation. Analyzing embryos at Stage 11, after most, if not all, NBs have formed, this study found that wor mutants simultaneously removing one gene copy of sna and esg do indeed display a significant reduction in NB numbers. In addition, while AS-C and wor mutants both show a partial loss of NBs, this study found that AS-C;wor double mutants display significantly more severe loss than either single mutant alone. Moreover, misexpression of SoxN or wor, from optimized transgenes, reveals sufficiency for these genes in generating ectopic NBs in the ectoderm. Strikingly, SoxN/ wor co-misexpression can generate extensive numbers of ectopic NBs even in a genetic background lacking any AS-C proneural gene activity. Finally, both SoxN and wor are regulated by, and regulate, the Notch pathway. Based upon these findings, it is proposed that SoxN and wor constitute the missing proneural genes (Arefin, 2019).

What are the connections between the Notch pathway, SoxN, and wor? Logically, in NICD and m8 misexpression, which result in fewer NBs, a reduction was observed in SoxN and wor expression. However, counterintuitively, Notch and neur mutants, which display more NBs, also displayed reductions of SoxN and wor expression. One reason for this effect may pertain to the Notch signaling being important for Crb expression, and that crb is important for both SoxN and wor expression. The reciprocal connection, i.e., between SoxN/wor and the Notch pathway, as measured by m8-GFP expression, is also partly dichotomous. Specifically, SoxN and wor mutants display fewer NBs, a NotchON effect, but reduced m8-GFP expression, a NotchOFF effect. Similarly, misexpressions of SoxN^D and wor^D trigger more NBs, a NotchOFF effect, but increased m8-GFP expression, a NotchON effect. However, these findings mirror the activation of E(spl) genes by the proneural genes and point to a balancing act between the proneural, SoxN, and wor genes, as well as the Notch pathway (Arefin, 2019).

Previous studies found that the proneural genes, i.e., ac, sc, and l'sc, are upstream of wor. In line with this, in AS-C mutants loss of wor cells (NBs) and reduction of wor expression levels were observed in the NBs still generated. Similarly, it was previously found that ase misexpression increased wor expression in NBs. Previous studies revealed that SoxN mutants show reduced Ase expression in NBs, and that misexpression of SoxN could activate wor expression, whereas neither wor nor ase GoF or LoF affected SoxN expression in NBs (Bahrampour et al., 2017). In addition, SoxN mutants were found to display loss of ac, l'sc, and wor expression. This indicates that SoxN, which is expressed in the entire early neuroectoderm, acts upstream of the proneural genes, while proneural genes act upstream of wor. However, this SoxN->proneural->wor regulatory flow is complex: while both SoxN and wor misexpression can trigger NB generation, l'sc does not have this potency. Moreover, while SoxN expression may occur first, both AS-C and wor appear to be important for maintained and elevated SoxN expression in NBs. Hence, SoxN, AS-C, and wor appear to be involved in a mutually reinforcing interplay, which ensures robust NB selection once the Notch pathway balanced is tipped (Arefin, 2019).

DNA-binding studies for the factors studied herein, and analysis of related family members (D, Sna, esg, and Ase), suggest that the elaborate transcriptional interplay between all of the aforementioned TDs/co-factors, i.e., NICD/Su(H)/Mam, E(spl), proneural, SoxN, wor, and their respective genes, may result from direct transcriptional regulation (Arefin, 2019).

In Drosophila, crb and sdt control the epithelial polarity in a number of tissues. Recent studies revealed that Crb stabilizes Notch, and accordingly crb mutants show more NBs. This study furthermore found that crb overexpression results in fewer NBs and, interestingly, that crb overexpression can partly rescue Notch mutants. The reduction of NB numbers in crb mutants is logically accompanied by reduced m8-GFP expression, while, surprisingly, crb misexpression also triggered reduced m8-GFP expression. Notch signaling was found to activate Crb expression, evident by downregulation of Crb in Notch and neur mutants, and upregulation of Crb in NICD and m8 misexpression. Hence, with the exception of crb misexpression on m8-GFP, a clear-cut interplay between canonical Notch signaling and crb/Crb emerges. However, this interplay would constitute a runaway loop, with Notch activating crb, and Crb supporting Notch activation. Perhaps the finding that crb overexpression reduces m8-GFP points to a nebulous brake pedal in this loop (Arefin, 2019).

Regarding the connection between crb with SoxN, wor, and the proneural genes, clear interplay, albeit with a reverse logic was found for mutants versus misexpression. Specifically, misexpression of SoxN, wor, or proneural genes, which generates more NBs, with encompassing delamination, also results in reduced Crb expression. However, surprisingly, SoxN, wor, and proneural mutants, which display fewer NBs, also show reduced Crb expression, underscoring the balancing act of these gene regulations. It is envisioned that this may reflect a role for SoxN, wor, and AS-C in the proneural clusters (prepattern), where they may act to ensure Crb expression in the equivalence regions, thereby ensuring an efficient lateral inhibition process (Arefin, 2019).

In addition, further complexity regarding the role of crb stems from recent findings revealing that Neur, an E3 ligase critical for Dl endocytosis, also controls the stability of Sdt, and thereby affects Crb protein levels. It is tempting to speculate that NotchOFF cells (NBs), which maintain proneural gene expression and hence activate neur expression, will have increased Neur, and hence increased endocytosis of Sdt, and thereby decreased Crb levels, leading to reduced Notch receptor activation. Because Neur also increases endocytosis of Dl, high Neur levels would help drive Notch activation in the neighboring cells (epidermal cells). Moreover, since Notch (NICD and m8^CK2) activates Crb expression and represses neur expression, this should ensure more Crb in the NotchON cells (epidermal cells), thereby further supporting Notch activation. By these mechanisms, the transcriptional regulation of crb/neur gene expression and the stability/endocytosis control of Crb/Sdt/Dl levels and localization, and thereby Notch activation, act as a hitherto undiscovered loop providing additional thrust to the lateral inhibition decision (Arefin, 2019).

Similar to the TF interplay described above, the gene-specific and/or genome-wide DNA-binding studies indicate that the gene regulation of crb and neur may be mediated by direct transcriptional regulation of the Notch pathway TFs (NICD/Su(H)/Mam, E(spl), proneural), as well as the SoxB and Snail family TFs (Arefin, 2019).

EMT has been extensively studied in mammals and has revealed roles for the Crb, Scribble (Scrib), and Par complexes, as well as for Notch signaling and the Snail and SoxB families. Previous studies, and the current findings, demonstrate that the majority of these genes also play key roles during Drosophila NB selection and delamination. This supports the notion that NB selection and delamination could be viewed as an EMT-like process. However, the NB-type EMT differs from canonical EMT in several aspects. In canonical EMT, all apical polarity complexes (Crb, Par, and Scrib complexes) and Cadherins are turned off, and there is no activation of asymmetry genes. Hence, delamination is followed by symmetric cell division and cell migration. In contrast, in 'NB-type EMT,' while, similarly, the Crb complex and Cadherins are turned off, the Par complex (baz/par-3, par-6, and aPKC) and the Scribble complex (scrib, dlg, and lgl) remain expressed, and asymmetric genes, e.g., mira, insc, and pros, are turned on. In addition, within NBs, SoxN, wor, and ase activate key cell-cycle driver genes, i.e., Cyclin E and stg, and repress expression of the cell-cycle inhibitor dacapo. These gene expression changes result in NB delamination, but retain apico-basal polarity in the NB, and ensure repetitive rounds of asymmetric cell divisions, generating the unique features of CNS lineages (Arefin, 2019).

Based upon these findings and those previously published, a model emerges wherein SoxB acts early to govern neuroectodermal competence, intersecting with the early transient wave of proneural gene expression in the proneural clusters. SoxN and proneural genes engage in interplay with the Notch-mediated lateral inhibition process, which is also gated by Crb-Sdt-Neur membrane-localized control of Notch receptor activity and Dl ligand endocytosis. The outcome of these interactions is that early NBs become NotchOFF and elevate their SoxN and proneural expressions, as well as activate wor and Ase expression. This results in the downregulation of a subset of EMT genes (i.e., Crb complex and Cadherins), while the Scrib and Par complexes are maintained. The combined action of SoxN, proneural, wor, and Ase triggers activation of asymmetric cell division genes and cell-cycle driver genes, the outcome of which is NB delamination, followed by asymmetric cell divisions and lineage generation. In contrast, the surrounding NotchON cells continue expressing E(spl) genes, downregulating the SoxN, proneural, neur, and Dl genes. This results in the continued expression of the Crb, Scrib, and Par complexes, as well as failure to activate wor, ase, asymmetric, and cell-cycle genes. The combined effect of these regulatory decisions is that these cells remain in the ectoderm and do not divide (Arefin, 2019).

The process of NB selection bears many similarities to the process of peripheral SOP selection. However, while one Snail family gene, esg, is indeed important also for PNS precursor development, there is no study linking sna or wor, nor the SoxB genes SoxN and D, to SOP selection. Hence, while both SOP and NB formation requires AS-C and esg, NB formation additionally requires Sna, wor, SoxN, and Dichaete. It is tempting to speculate that this may relate to two clear differences between SOPs and NBs: EMT and proliferation. Specifically, while NBs undergo a complete EMT-like process, SOPs remain associated with the ectoderm. Moreover, NBs can divide up to 20 times, making 40-cell lineages, while most, if not all, SOPs make 5-cell lineages. The connections between the SoxB and Snail families with NB and GMC proliferation and the EMT pathway (this paper as well as Bahrampour et al., 2017, 2019) suggest that both of these NB-specific properties are driven by the expanded TF code specific to NBs (Arefin, 2019).

In mammals, the neuroepithelial-to-radial glia cell (NE-RGC) transition is in many aspects analogous to the NB selection and delamination process in Drosophila. Intriguingly, recent studies suggest that NE-RGC can perhaps also be viewed as an EMT-like process, although in this case the process has been modified even further, and the RGC retains an apical connection throughout neurogenesis and undergoes interkinetic nuclear migrations. Strikingly, in two recent studies the NE-RGC transition was found to involve the mouse Snail and Scratch factors, both of which are members of the Snail family. Other players in the NB-selection program outlined above also play key roles in the early development of the mammalian CNS and in the NE-RGC transition, although the direct comparison of gene function and cell behavior becomes nebulous. Hence, it would appear that the neuroectoderm->neural progenitor selection and delamination process has undergone several evolutionary modifications, perhaps becoming less and less akin to a canonical EMT process in more derived animals. Nevertheless, it is tempting to speculate that several of the basic principles of EMT are utilized in the mammalian NE-RGC process and that viewing it as such may be helpful for future studies (Arefin, 2019).

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 PER_in) 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 PER_in 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 PER_in fibers to gustatory sensory dendrites or proboscis motor axons revealed that PER_in 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. PER_in neurons are activated by stimulation of mechanosensory neurons and activation of PER_in 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 PER_in activity. The opposite behavior is elicited upon inhibiting activity in PER_in 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 PER_in neurons remains to be determined. PER_in 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 PER_in 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 PER_in. In addition, by monitoring activity of PER_in while flies moved their legs, it was demonstrated that activity was coincident with movement (Mann, 2013).

These studies argue that PER_in is activated by nongustatory cues in response to movement, likely upon detection of mechanosensory cues. Additional cues may also activate PER_in. 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. PER_in 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 PER_in 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 PER_in as a significant modulator of feeding initiation provides a foundation for future studies determining how PER_in influences proboscis extension circuits to alter behavioral probability and how mechanosensory inputs activate PER_in. 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 PER_in 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).

PMSIs are the first interneuronal population shown to be involved in Drosophila larval locomotion. Anatomical and functional analyses strongly suggest that PMSIs are premotor local interneurons that inhibit motor neurons in the same or a neighboring segment. Previous electrophysiological analyses showed that GABA or glutamate application elicits inhibitory responses in motor neurons that reverse at near resting potential and are blocked by the chloride channel blocker picrotoxin. Based on these observations, it has been suggested that motor neurons express Cl−-permeable GABA and glutamate receptors. Glutamate-gated inhibitory channels have been identified and well characterized in arthropods and other invertebrates including C. elegans. Although no such receptors are known in vertebrates, previous structural and pharmacological analyses suggest that invertebrate glutamate-gated chloride channels are orthologous to vertebrate glycine channels. Drosophila homologs of the receptors have been cloned and shown to produce a glutamate-gated chloride current when expressed in Xenopus oocytes (Cully, 1996) and exhibit inhibitory action in Drosophila adult brain (Liu, 2013). Thus, it is likely that PMSIs inhibit motor neurons through glutamate-gated chloride channels. The motor neurons are also glutamatergic but send excitatory input to the muscles. Previous studies report that there are 40 putative vGluT-positive glutamatergic neurons in each hemisegment, of which 34 are motor neurons and six are interneurons. Since the number of PMSIs is comparable to that of the estimated glutamatagic interneurons, PMSIs most likely represent a majority of the glutamatergic interneurons in the ventral nerve cord (Kohsaka, 2014).

This study demonstrated that the duration of motor bursting and segmental muscle contraction is elongated when PMSIs are inhibited. The results indicate that PMSIs regulate the duration of motor output in each segment by terminating motor bursting. Consistent with this idea, dual-color Ca2+ imaging showed that activation of PMSIs is delayed with respect to that of the postsynaptic motor neurons. This temporal pattern allows PMSIs to regulate the time window of motor firing via inhibition. Thus, a main function of PMSIs seems to be to limit the duration of motor output (Kohsaka, 2014).

Similar roles in shaping motor outputs have been proposed for V1 neurons in mice and aIN neurons in Xenopus, both of which are inhibitory interneurons expressing Engrailed and have been proposed to share evolutionarily conserved roles. Loss or acute inactivation of V1 neurons elongates the duration of motor bursting during fictive locomotion in isolated mouse spinal cord. Xenopus aIN neurons provide early-cycle inhibition to motor neurons and other CPG interneurons during swimming. Thus, regulation by on-cycle inhibition seems to be a common mechanism for shaping the duration of motor outputs in vertebrates and in Drosophila larvae. Interestingly, PMSIs share several cellular properties with vertebrate V1 and aIN neurons. The three classes of neurons are all inhibitory premotor interneurons that are rhythmically activated during motor cycles. They are unipolar and send their axons first toward motor neurons and then extend an ascending ipsilateral axon longitudinally. Whereas V1 and aIN use glycine as the inhibitory neurotransmitter, PMSIs use glutamate, which is considered to be the invertebrate counterpart of glycine. These shared features may underlie the common function in motor control (Kohsaka, 2014).

Several mechanisms have been proposed for speed control of animal locomotion, including the recruitment of different motor neurons and change in electrophysiological properties of motor and other CPG neurons. The current results on PMSIs and previous studies on V1 and aIN neurons suggest that limiting the duration of motor firing by inhibition might be a phylogenetically conserved mechanism for speed control. In mice lacking V1 neurons, not only the duration of motor firing but also that of motor cycles is elongated, and thus the speed of locomotion is reduced. Although the role of aIN neurons in speed control has not been directly examined, close correlations have been observed between the activity of these neurons and the frequency of the tadpole swimming. This study demonstrates that blocking activities of PMSIs elongates the duration of motor bursting and reduces the speed of axial locomotion in Drosophila larvae. Taken together, these results suggest that evolutionarily distant organisms with anatomically and functionally distinct motor systems may adopt similar strategies for speed control of locomotion. It is important to note that both activation and inhibition of PMSIs activity lead to a decrease in locomotor speed (paralysis upon activation with ChR2 and slowed locomotion upon inhibition with Shits or NpHR). Thus, these neurons need to be activated at an optimum level and timing to output locomotion with appropriate speed (Kohsaka, 2014).

It still remains to be determined how the change in the duration of motor bursting affects the speed of locomotion. A simple model would be that since motor bursting in each segment is elongated in the absence of PMSI activity, it takes longer for the motor wave to propagate along the segments. In many undulatory movements, such as lamprey and leech swimming and Drosophila larval crawling, intersegmental phase lag (not intersegmental time lag) remains constant at different speeds. This is because the phase of muscle contraction in different segments must remain constant in order to maintain the same motor output pattern (e.g., forming approximately one full wave at a given time during lamprey swimming). Because of this intersegmental coordination, segmental lag of motor activity may have to be prolonged in the absence of PMSI activity to match up with the elongation of segmental motor bursting; otherwise, too many muscle segments would contract at the same time during peristalsis. Indeed, electrophysiological recordings showed that intersegmental time lag of motor firing was prolonged to a similar extent as the motor bursting (~2 fold) when PMSI activity was silenced. Likewise, in mice lacking V1 neurons, while the left-right and flexor-extensor coordination is maintained, both motor bursting and step cycles are elongated to a similar extent (2- to 3-fold). Thus, a common strategy, limiting the duration of motor bursting, may be used to regulate the speed of diverse animal locomotion such as larval locomotion and mammalian limb movements because it leads to changes in the most critical parameters of the speed, intersegmental time delay in axial locomotion, and left-right/flexor-extensor step cycle in limb locomotion. Understanding how intersegmental coordination is regulated in Drosophila larvae is an important future goal (Kohsaka, 2014).

It is also important to explore what might be the upstream neural circuits that activate PMSIs. Good candidates are multidendritic neurons, which are known to be required for fast larval locomotion and believed to feedback muscle contraction status. Another interesting possibility is that PMSIs control the speed of locomotion in response to environmental changes such as temperature or to meet internal demands such as hunger. Preliminary data using the GRASP technique suggest that PMSIs indeed receive afferent projections from sensory neurons. Once the upstream neurons are identified, the input-output relationship between these neurons and PMSIs can be systematically studied using optogenetics and other methods. It is anticipated that such analyses will not only clarify the roles of PMSIs in local neural circuits, but also shed light on conserved mechanisms by which inhibitory interneurons regulate animal locomotion (Kohsaka, 2014).

Reconstruction of motor control circuits in adult Drosophila using automated transmission electron microscopy

To investigate circuit mechanisms underlying locomotor behavior, this study used serial-section electron microscopy (EM) to acquire a synapse-resolution dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM, was developed. Using this dataset, neuronal networks were studied that control leg and wing movements by reconstructing all 507 motor neurons that control the limbs. A specific class of leg sensory neurons was shown to synapse directly onto motor neurons with the largest-caliber axons on both sides of the body, representing a unique pathway for fast limb control. Open access is provided to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. GridTape instrumentation designs and software are provided to make large-scale EM more accessible and affordable to the scientific community (Phelps, 2021).

Large-scale neuronal wiring diagrams at synapse resolution will be a crucial element of future progress in neuroscience. This paper presents GridTape, a technology for accelerating large-scale electron microscopy (EM) data acquisition. The power of this approach is demonstrated by acquiring a dataset encompassing an adult female Drosophila ventral nerve cord (VNC). This dataset was used to identify a monosynaptic circuit that directly links a specialized proprioceptive cell type, the bilateral campaniform sensillum (bCS) neurons, with specific motor neurons (MNs). The results highlight how EM datasets can be used to characterize cell types and guide development of cell type-specific driver lines. The public release of this dataset provides a resource for studying the circuit connectivity underlying motor control and demonstrates the rapid advances that can be powered by the GridTape approach (Phelps, 2021).

Data acquisition remains a rate-limiting step in generating EM connectomics datasets. Manual sectioning for TEM is slow, imprecise, and unreliable. Meanwhile, SEM approaches that circumvent the need for manual sectioning have slow imaging speeds or require massive parallelization of expensive electron optics to acquire comparable datasets. GridTape builds on previous efforts toward TEM parallelization and automation, but overcomes the need for manual sectioning, allowing faster and more consistent section collection and imaging. Because imaging is nondestructive, GridTape is compatible with enhancement by post-section labeling and allows for re-imaging. By eliminating the need to separately handle thousands of fragile sections, GridTape reduces data loss and artifact frequency. This results in better alignment of sections into a coherent, high signal-to-noise image volume, leading to efficient and accurate reconstructions (Phelps, 2021).

GridTape is also less expensive than high-throughput SEM platforms. For the current price of one commercial multi-beam SEM system, ten TEMCA-GTs can be built, and samples collected on GridTape can be distributed across microscopes for simultaneous imaging. The fixed microscope hardware costs are accompanied by consumable costs associated with support film coating (∼USD$4 per slot, or ∼$18,000 for this study), but this cost is expected to decrease due to technological improvements and economies of scale (Phelps, 2021).

In the future, GridTape acquisition rates will increase as cameras and imaging sensors improve. Because TEM imaging is a widefield technique, imaging throughput can be increased by using larger camera arrays and brighter electron sources. Moreover, sections larger than current slot dimensions could be accommodated with wider tape and larger slots, although custom microscopes may be necessary for very large samples and slot size will depend on material properties of the support film (Phelps, 2021).

The EM dataset presented in this study provides a public resource for understanding how the Drosophila nervous system generates behavior. An adult Drosophila VNC was chosen because it is an ideal test case for generating and validating a connectomic dataset. The circuit is genetically and electrophysiologically accessible and neurons are identifiable across individuals. The VNC is compact, containing approximately a third of the neurons in the adult CNS, but contains neuronal networks for executing complex motor behaviors. Because the brain controls behavior via descending projections to the VNC, it is critical to be able to study neuronal circuits in both the brain and the VNC at synaptic resolution. Notably, this VNC dataset complements the recent release of an EM dataset comprising the complete adult female Drosophila brain (Phelps, 2021).

The VNC dataset was validated by automatically mapping its synapses with high accuracy, successfully registering the predicted synapse density map to a standard atlas and finding a high degree of similarity between EM and LM reconstructed neurons. A pipeline is demonstrated for identifying cells of interest in the dataset by comparing EM reconstructions to LM data. Finally, as a foundation for future work, >1,000 neuron reconstructions and their connectivity are made publicly available. Although these reconstructions were generated manually, advances in automated segmentation approaches are dramatically accelerating analysis of serial-section TEM data (Phelps, 2021).

Flexible motor control relies heavily on feedback from proprioceptors, a class of sensory neurons that measure body position, velocity, and load. In both vertebrates and invertebrates, proprioceptive feedback is processed by the central nervous system to tune motor output. In insects, morphologically distinct subclasses of chordotonal neurons encode different features of leg movement such as position, velocity, and vibration. Campaniform sensilla encode load signals similar to mammalian Golgi tendon organs. Althoughthe main proprioceptor types are known and the signals they encode, it is now an opportune time to understand how motor circuits integrate proprioceptive inputs to control the body by mapping the complete wiring diagram of an adult Drosophila VNC (Phelps, 2021).

EM datasets also enable the discovery of cell types and synaptic connections that may be overlooked by other methods. For instance, targeted reconstruction of sensory afferents revealed that the leg sensory neurons with the largest-caliber axons are the bCS neurons, which make direct synapses onto large-caliber leg MNs. This connection is monosynaptic and bCS inputs are specifically located near the putative MN spike initiation zone, suggesting that speed and reliability are essential for the function of these connections (Phelps, 2021).

The unique bilateral and intersegmental projections of bCS neurons suggests that they directly influence multiple limbs on both sides of the body. This leads to several hypotheses about their function. Prior work suggested that campaniform sensilla encode information about step timing that could drive the transition between stance and swing phases of walking. However, this study observed that bCS neurons synapse onto the same MNs on both sides of the bod, suggesting they drive symmetric movements of left and right legs. This makes it unlikely that bCS neurons contribute to walking, which involves antiphase movement of contralateral legs. Instead, bCS neurons may underlie a fast reflex where multiple legs flex in response to bCS activation. CS can signal either increases or decreases in load, depending on the sensillum's placement and orientation on the leg. Therefore, bCS neuron activation could forcefully stabilize posture in response to additional weight (e.g., to prevent the body from being crushed) or to grip a surface in response to a loss of load (e.g., to prevent being blown away by a gust of wind). The genetic tools that were created to target bCS neurons will enable future analyses of their function (Phelps, 2021).

Monosynaptic sensory-to-motor neuron connectivity is infrequent in larval Drosophila, but has been observed in other adult insects. Direct sensory feedback may be key in adults for precise control of their segmented limbs. The absence of such connections in larvae may indicate that controlling a limbless body relies less on sensory feedback and more on feedforward processing. As adult flies move much faster than larvae, another possibility is that fast monosynaptic sensory feedback is crucial for fast-moving animals. Indeed, research on escape responses has demonstrated that high-velocity movements are often controlled by the fastest neuronal pathways (Phelps, 2021).

MNs have diverse but stereotyped functions, reflecting the array of muscles and muscle fibers they innervate. Some MNs have unique and reproducible transcription factor signatures that underlie their physiological properties and axonal morphology. These unique transcription factor patterns specify morphologies that are fairly stereotyped across animals. The results extend these findings by quantitatively demonstrating that most dendritic arborizations of leg MNs are sufficiently stereotyped to be individually identifiable by structure alone. Because the complete population of MNs controlling the two front legs was reconstructed, it was possible to show that mirror symmetry in primary neurite number and position is a systematic principle of MN populations. In contrast, sensory neurons have more redundant copies and variable copy numbers (Phelps, 2021).

Previously, comprehensive neuronal connectivity maps were acquired for the nerve cords of other organisms including C. elegans, leeches, lampreys, and Drosophila larvae. These maps enabled a more complete understanding of how the nervous system controls locomotor rhythms underlying swimming and crawling. Less is known about the connectivity underlying motor control in limbed animals. The EM dataset presented in this study as a public resource will enable complete connectivity mapping for the circuits that control the legs and wings of an adult Drosophila. Combined with recent advances in recording activity from genetically identified VNC neurons during behavior, adult Drosophila is emerging as a powerful system for studying motor control. With these tools, it is expected that a deeper understanding of the circuit basis for complex motor control is within reach (Phelps, 2021).

Development of connectivity in a motoneuronal network in Drosophila larvae

Much of the understanding of how neural networks develop is based on studies of sensory systems, revealing often highly stereotyped patterns of connections, particularly as these diverge from the presynaptic terminals of sensory neurons. Considerably less is known about the wiring strategies of motor networks, where connections converge onto the dendrites of motoneurons. This study investigated patterns of synaptic connections between identified motoneurons with sensory neurons and interneurons in the motor network of the Drosophila larva and how these change as it develops. As animals grow, motoneurons were found to increase the number of synapses with existing presynaptic partners. Different motoneurons form characteristic cell-type-specific patterns of connections. At the same time, there is considerable variability in the number of synapses formed on motoneuron dendrites, which contrasts with the stereotypy reported for presynaptic terminals of sensory neurons. Where two motoneurons of the same cell type contact a common interneuron partner, each postsynaptic cell can arrive at a different connectivity outcome. Experimentally changing the positioning of motoneuron dendrites shows that the geography of dendritic arbors in relation to presynaptic partner terminals is an important determinant in shaping patterns of connectivity. It is concluded that in the Drosophila larval motor network, the sets of connections that form between identified neurons manifest an unexpected level of variability. Synapse number and the likelihood of forming connections appear to be regulated on a cell-by-cell basis, determined primarily by the postsynaptic dendrites of motoneuron terminals (Couton, 2015).

Much of the current view of how sets of synaptic connections form and change during nervous system development is derived from studies of sensory systems. The connections that sensory neurons form are often tightly constrained, enabling the formation of accurate sensory maps, with numbers and distributions of synapses appropriate for network operation. Connectivity at lower-order synapses of the network can be almost invariant and cell autonomously specified. For example, Drosophila photoreceptor neurons reproducibly form ~50 synapses with specific postsynaptic lamina cells, irrespective of photoreceptor function or visual system defects. At higher-order synapses, in contrast, connectivity can be rather variable, reflecting both experience-dependent plasticity and distinct wiring strategies. For example, randomized connections in the mushroom body are thought to maximize coding space (Couton, 2015).

This study focused on the much less well-explored development of connectivity within a motor network. Motor systems manifest a great deal of flexibility, including their ability to adjust to changes in muscle size with growth and exercise, thus maintaining the capacity to trigger effective muscle contractions. This has been most extensively studied at the neuromuscular junction where the growth of the presynaptic terminal is matched with that of the postsynaptic muscle, regulated by muscle-derived retrograde signals. In addition, motoneurons also adjust centrally through changes in the size and connectivity of their dendritic arbors (Couton, 2015).

To investigate patterns of connectivity in a motor network and how these change as the animal develops and grows, this study used the Drosophila larva as a model. A paradigm was developed for studying identified partner neurons at the level of individual synaptic sites across different developmental stages. The following questions were asked: (1) How does connectivity change as the motor network develops? (2) How reproducible or variable are the sets of connections that form? (3) Is there evidence of synaptic patterning information residing with the presynaptic or postsynaptic partner? This study shows that from hatching to later larval stages, existing connections are progressively consolidated by addition of synapses. While patterns of connections are specific to each motoneuron type, considerable variability remains. Moreover, connectivity appears to be set on a cell-by-cell basis by the dendritic arbors of motoneurons, and dendritic positioning is a determinant of the connections that motoneurons make. Together, these findings argue in favor of a flexible regulation of connectivity in the assembly of the larval crawling circuit (Couton, 2015).

To study the emergence of synaptic connectivity in a motor network as it develops, genetic tools were developed for reliably visualizing and manipulating identified, connecting neurons in the Drosophila larval nerve cord. For pre-motor partner neurons, an intersectional 'split-Gal4' enhancer trap screen was fractionated through the set of cholinergic interneurons and sensory neurons, which provide the synaptic drive to motoneurons in this system. From >3,000 lines, those with sparse expression and terminations in the motor neuropile were identified. Single motoneurons ('aCC' and 'RP2') were visualized via a LexA/LexAOp and FLP recombinase-based quaternary system (Singh, 2013). To resolve synaptic sites, the presynaptic active zone marker UAS-brp::mRFP was combined with the GFP reconstitution across synaptic partners (GRASP)-based reporter for cell-cell contacts. Brp::mRFP-positive presynaptic specializations that coincide with physical appositions of presynaptic and postsynaptic membranes, as reported by GRASP, were scored as putative synapses. Thus patterns of connectivity during larval development, from 0 hr after larval hatching (ALH) to the third instar stage (48 hr ALH), were charted between the aCC and RP2 motoneurons and some of their presynaptic partners, made accessible to analysis by the Split-Gal4 line BF29^VP16.AD: two intersegmental descending interneurons and the ddaD and ddaE proprioceptive sensory neurons (Couton, 2015).

Focus was placed on the lateral interneuron (IN_lateral) within the BF29^VP16.AD expression pattern; its axon descends contralaterally from the sub-esophageal ganglion to segment A8 and forms putative en passant synapses with intersegmental nerve motoneurons. In mid-abdominal segments (A2-A6), the number of putative synaptic connections between this IN_lateral and the RP2 motoneuron increases steadily with developmental time from an average of 0.86 ± 0.26 at 0 hr ALH to 6.73 ± 0.78 at 24 hr ALH to 11.09 ± 0.97 at 48 hr ALH. This developmental increase in synapse number is compatible with electrophysiological recordings from these motoneurons. IN_lateral axons also form putative synapses with the two dendritic sub-arbors of the aCC motoneuron. The larger ipsilateral arbor, located on the same side as the aCC soma, receives more putative synapses from the IN_lateral than the smaller sub-arbor on the contralateral side. Both RP2 and aCC project to dorsal body wall muscles. To extend these observations to motoneurons that innervate ventral muscles, RP3 motoneurons were manually labeled with the lipophilic tracer dye DiD, and co-localization with IN_lateral Brp::mRFP sites were charted as putative connections. Here, too, it was found that the number of putative connections between this pair of neurons increases with developmental time, from 1 synapse (±0, n = 3) at 0 hr ALH to an average of 3.6 synapses (±0.4, n = 5) at 24 hr ALH (Couton, 2015).

Cell-type-specific differences in connectivity were documented. These are most evident in the likelihood with which the RP2 and aCC motoneurons receive putative synapses from the ddaD and ddaE sensory terminals (the high density of Brp::mRFP puncta in these sensory terminals prevents resolution of individual puncta). As larvae develop, this sensory-motor connection becomes increasingly frequent, although throughout aCC, motoneurons have a significantly lower probability than RP2 of forming putative synapses with these dda sensory terminals. In addition, it was found that motoneurons such as RP3, which are similar in operation to RP2 and aCC, i.e., in innervating longitudinal body wall muscles, also form putative synapses with the presynaptic IN_lateral, while motoneurons innervating antagonistic transverse muscles do not, even though their dendrites arborize within reach of the IN_lateral axon. For another pre-motor interneuron, INBF59, labeled with the BF59VP16.AD expression line, single cells were resolved by injecting INBF59 interneurons expressing UAS-brp::mRFP with the lipophilic tracer dye, Neuro-DiO, and different motoneurons with the spectrally distinct DiD. Co-localization of these three markers (Neuro-DiO, Brp::mRFP, and DiD) was taken as indicative of a putative synapse. The data suggest that different motoneurons, projecting to dorsal (aCC, RP2), lateral (MN-LL1), and ventral (RP3) muscles, may have different likelihoods of contacting the INBF59 (Couton, 2015).

In summary, in this motor network, the number of putative synapses between partner neurons generally increases as the network matures and the animal grows. Different motoneurons have different likelihoods of forming synapses with the same sets of presynaptic sensory neurons. Such qualitative differences are suggestive of motoneuron-type-specific regulation of connectivity (Couton, 2015).

It was striking by how variable connectivity between identified neurons seemed to be. For example, the number of putative synapses between IN_lateral and RP2 motoneurons ranged from 0 to 3 at 0 hr ALH and 6 to 16 at 48 hr ALH. Similarly, for the sensory-motor connection, only a fraction of RP2 and aCC motoneurons receive putative synaptic contacts from dda sensory terminals. Here, differences in connectivity are mirrored by the diverse routes by which individual neurons attain their connections. For instance, aCC motoneurons form putative synaptic connections with dda sensory axon terminals in every possible way: with contralateral, ipsilateral, or both groups of sensory projections, established by different routes, with dendrites from the main arbor or the soma. This shows that postsynaptic dendritic arbors of motoneurons are quite flexible in how they attain connections with presynaptic terminals (Couton, 2015).

Next, causes for the variable connectivity were explored. There is no clear indication that the connectivity that was measured becomes progressively more reproducible as the network matures. It was then asked whether differences in segmental identity contributed to the variability that was seen. Regression analyses show no statistically significant link between the segmental identity of RP2 and aCC motoneurons and the number of putative synapses that these receive from the IN_lateral at 0 hr ALH, 24 hr ALH, or 48 hr ALH (Couton, 2015).

Next, the effects that local and global network adjustments might have on connectivity were considered. To this end, focus was placed on pairs of RP2 and aCC neurons located in the same nerve cord and connected to the same IN_lateral, and it was asked whether having a common presynaptic partner leads to more similar numbers of synapses formed with the same axon. it was found that RP2 and aCC motoneurons can vary substantially in the number of putative connections they receive from the same presynaptic partner. These data imply that local interactions between individual pairs of neurons, rather than global network effects, might determine the outcome of connectivity (Couton, 2015).

In summary, these observations suggest that variability in connectivity might be an inherent feature of this motor network, at least for the cells analyzed in this study (Couton, 2015).

Since synapses are the product of interactions between presynaptic and postsynaptic terminals, it was asked whether the variability that was observe arises from one or the other synaptic partner. Testing the potential for an instructive role by the presynaptic interneuron, it was asked whether there was any pattern to the distribution of presynaptic sites along the IN_lateral axon. Along the IN_lateral axon (segments A2 to A8), the number of presynaptic sites per neuron was found to be highly variable, ranging from 48 to 107 (85 ± 16.8, SD, n = 17). At the same time, the distribution of presynaptic sites and the spacing between these are indistinguishable from random. Thus, no evidence was seen of positional patterning of en passant presynaptic sites along IN_lateral axons, which has been observed in other systems (Couton, 2015).

It was then asked whether differences in presynapse number could explain the variability in connectivity between different IN_lateral-motoneuron pairs. To this end, each IN_lateral-motoneuron pair the number of putative synapses formed was correlated with the local density of 'available' presynaptic Brp::mRFP puncta located within the IN_lateral axon along the span of the motoneuron dendritic tree. No significant correlation was found. This suggests that, at least in this system, the density of available presynaptic sites is not predictive of how many synaptic connections are formed with the postsynaptic motoneuron. Instead, these data are compatible with a model where the postsynaptic dendritic arbor regulates the number of connections that it forms (Couton, 2015).

Next,the role of postsynaptic motoneuron dendrites in determining connectivity was investigated. Previously, it was shown that postsynaptic dendritic arbors regulate the number of inputs they receive by adjusting dendritic growth. In motor networks, dendritic positioning has been suggested to be important in determining partner choice. To investigate the role of dendritic arbor positioning in shaping connectivity, the medio-lateral territories of motoneuron dendrites was changed. Increasing dendritic sensitivity to the midline attractant Netrin, by targeted overexpression of the cognate receptor Frazzled/DCC, shifts RP2 dendrites from principally lateral to more medial neuropil regions. This shift leads to a reduction of laterally positioned dendrites, so that fewer are in proximity to the IN_lateral axon, and a concomitant increase of dendrites in the medial neuropil, which is innervated by another interneuron with a medial descending projection (IN_medial). As a result, the proportion of synapses between motoneurons and the IN_lateral is drastically reduced, whereas the proportion of synapses with the IN_medial is greatly increased, as compared to controls (Figure 5C; t test, p = 0.0005 and p = 0.0194 for RP2 and aCCi, respectively). Although these observations do not assay for changes in partner choice (RP2 and aCC receive connections from both IN_lateral and IN_medial), these findings are compatible with a model where connections in motor systems emerge, to an extent, as a consequence of geographical overlap between presynaptic and postsynaptic terminals (Couton, 2015).

In summary, the data point to the existence of mechanisms that allow postsynaptic neurons to determine in a cell-type-specific fashion the number of presynaptic synapses they accept. Clearly, geographical overlap between presynaptic and postsynaptic terminals is necessary for synaptic connections to form, and the experiments suggest that dendritic positioning mechanisms contribute to the emergence of connectivity (Couton, 2015).

There is currently no consensus among views on how patterns of connections develop in a motor network. On the one hand, a great deal of genetically encoded specificity is evident in parts of the mouse spinal cord. For example, group 1a afferents target motoneuron pools with accuracy, and their connectivity is buffered, so that normal information flow is largely maintained in the face of considerable disturbances. Precision of wiring is perhaps most explicit in the selective positioning of inhibitory synapses by the so-called GABA pre-interneurons onto terminals of proprioceptive 1a sensory afferents. This precise and apparently invariant wiring is mediated by the expression of at least two sets of complementary heterophilic transsynaptic cell adhesion molecules. Contrasting with this view are studies from Xenopus tadpoles, where two-electrode recordings unequivocally demonstrated a surprising lack of specificity in synaptic connections during early stages of motor network development. Modeling based on these observations further suggests that such rather non-specific wiring patterns are able to generate swimming like motor outputs and that those patterns of connectivity could be formed simply through geographical overlap of coarsely defined presynaptic and postsynaptic termination zones. A limitation in those studies is that they look at groups of similar cells; this has precluded detailed insights at the level of individual synapses over developmental time. This study worked with identified partner neurons and studied how synaptic patterns in a motor network change, as the animal develops and grows (Couton, 2015).

A striking observation from this study is that at the output face of the network, motoneurons increase synaptic contacts with existing presynaptic partners over time. This correlates with previous observations that synaptic drive also increases during this period of larval development, although there is as yet no physiological readout for the specific anatomical changes detailed in this study. For motoneurons, the observed strengthening of existing connections is likely an adaptive mechanism that maintains the ability to effectively depolarize muscles as they enlarge during development. Although it has not been possible to assay for addition of new presynaptic partners during development, this wiring strategy contrasts with those proposed for cortical neurons, where pyramidal cells are thought to maximize the diversity of presynaptic inputs while keeping synapse number with each partner at a minimum (Couton, 2015).

Remarkably, reproducible cell-cell interactions during nervous system development can be genetically encoded, and this has been most clearly demonstrated with identified nerve cells of invertebrates—from highly specific substrate choices during axon path finding to the selection of synaptic partners and the number of synapses formed. In the Drosophila larval motor system, it was found that different motoneuron types have characteristic patterns of connections. For example, the likelihood of forming connections with the proprioceptive dda sensory neurons differs between the RP2 and aCC motoneurons. Qualitative differences in the specificity of partner choice are also present in that the IN_lateral forms connections with motoneurons that innervate longitudinal body wall muscles (e.g., aCC, RP2, and RP3), but not with motoneurons thought to be antagonistic in operation, despite close proximity of their dendrites (Couton, 2015).

At the same time, this motor system also manifests a considerable degree of variability, both in the likelihood and the number of connections that form between motor and pre-motor interneurons. Although some connection patterns seem to become more reproducible during early phases of network maturation, such as those between the RP2 motoneuron and dda sensory terminals, by and large, the observations suggest that connectivity is inherently flexible and that it is the outcome of local cell-cell interactions, at least between most cells that we have been able to study. For example, two identical motoneurons (in different neuromeres) contacting the same IN_lateral axon can form quite different numbers of putative connections with the same presynaptic cell. It is conceivable that these connections are variable because they are not critical to motor system operation, and it remains to be seen to what extent the observations of this study are representative of connectivity elsewhere in this network (Couton, 2015).

Where does the information that determines these connectivity outcomes reside? No correlation was found with segmental identity or evidence for presynaptic patterning information: the number of presynaptic release sites that any one IN_lateral makes varies considerably, both between and within animals (left versus right homolog), and their distribution along the axon appears to be random, yet fairly even, with similar numbers of presynaptic sites per neuromere. Most compatible with the current data is the notion that patterns of connectivity are predominantly determined by the postsynaptic dendrites of motoneurons (Couton, 2015).

It has been previously shown that motoneurons achieve a specific range of synaptic input by adjusting the growth of their dendritic arbors. These structural adjustments mirror and complement homeostatic changes of neuronal excitable properties. This study shows that different dendritic growth patterns lead to different connectivity outcomes. For example, aCC motoneurons are capable of initiating growth of dendritic branches from different parts of the cell, which can form connections with the ipsilateral and/or contralateral dda terminals, or neither. In an analogous situation, in the mouse retina, differences in dendritic growth lead to distinct connection patterns between different bipolar cells and presynaptic photoreceptor terminals. This study experimentally tested how dendritic positioning impacts connectivity. Changing the bias so that motoneurons preferentially elaborate their dendrites toward the ventral midline results in changes in connectivity, namely reductions in the proportion of synapses with the lateral IN_lateral and concomitant increases in connections with the medially located IN_medial axon. Although this experiment does not inform about partner choice, since both the IN_lateral and IN_medial are normally contacted by these motoneurons, it suggests that the number of connections is determined by the extent to which presynaptic and postsynaptic terminal arbors are targeted to common regions. These experiments in the Drosophila larva support observations and models on connectivity in the motor network of Xenopus tadpoles, which suggest that the connectivity matrix might be determined in considerable part by geographical overlap of coarsely defined presynaptic and postsynaptic territories. There is evidence that the conserved Slit-Robo and Netrin-Frazzled/DCC guidance cue systems define such territories for positioning axon tracts and regions of dendritic arborization in the CNS and that these can contribute to shaping synaptic connectivity. That said, it remains to be established how the promiscuity of connections apparent in early Xenopus tadpoles changes over developmental time and to what extent hardwired specificity is genetically encoded elsewhere in the Drosophila or indeed in other motor networks (Couton, 2015).

Comparative connectomics reveals how partner identity, location, and activity specify synaptic connectivity in Drosophila

The mechanisms by which synaptic partners recognize each other and establish appropriate numbers of connections during embryonic development to form functional neural circuits are poorly understood. This study combined electron microscopy reconstruction, functional imaging of neural activity, and behavioral experiments to elucidate the roles of (1) partner identity, (2) location, and (3) activity in circuit assembly in the embryonic nerve cord of Drosophila. Postsynaptic partners were found to be able to find and connect to their presynaptic partners even when these have been shifted to ectopic locations or silenced. However, orderly positioning of axon terminals by positional cues and synaptic activity is required for appropriate numbers of connections between specific partners, for appropriate balance between excitatory and inhibitory connections, and for appropriate functional connectivity and behavior. This study reveals with unprecedented resolution the fine connectivity effects of multiple factors that work together to control the assembly of neural circuits (Valdes-Aleman, 2020).

The human nervous system is organized into circuits with specifically matched and tuned cell-to-cell connections essential for proper function. During development, neurons navigate through the nervous system to reach their target location. Surrounded by numerous cells along their trajectories and in their target areas, developing neurons ignore most cells and connect only to specific partners (Valdes-Aleman, 2020).

The absolute numbers of synapses between specific partners can vary across individuals, hemispheres, or repeated network modules in the same individual. However, recent electron microscopy (EM) reconstructions in multiple Drosophila larvae suggest that, at least in some circuits, the relative numbers of synapses between partners are precisely regulated. Thus, the fraction of inputs a neuron receives from a specific partner, relative to its total number of inputs, is remarkably conserved across individuals, across larval stages, and even between larva and adult. For example, the fraction of input varied by an average factor (fold change; i.e., the ratio of two fractions) of 1.07 ± 0.22 between different first instar larvae (n = 13 homologous connections) and 1.09 ± 0.20 from first to third instar (n = 12 homologous connections). Similarly, the average input a mushroom body output neuron receives from a modulatory neuron in the larva and adult is 3.4% and 3.3%, respectivelyh. These examples of conserved fractions of synaptic input across individuals and life stages raise several key questions: (1) How important are the precise numbers of connections between neurons for normal behavior? (2) How are the precise numbers of connections between partners specified? and (3) How is the appropriate balance between excitatory and inhibitory connections in the circuit achieved (Valdes-Aleman, 2020)?

The chemoaffinity hypothesis proposes that pre- and postsynaptic partners express specific matching combinations of cell surface molecules that enable them to seek out and recognize each other during development. However, relatively few examples of partner-recognition molecules have been identified, so it is unclear whether their use is a general principle or if they are used only in some systems. It is also unknown if these partner-recognition mechanisms specify precise numbers of synapses between partners, or only instruct two neurons to form synapses, but not how many (Valdes-Aleman, 2020).

Alternative hypotheses propose that neurons seek out specific locations in the nervous system, rather than specific partners, indiscriminately connecting to whichever neurons are present there. Consistent with this, neurons have been shown to use non-partner-derived positional cues, such as third-party guidepost cells or gradients of repellents, to select their termination and synaptogenesis area independently of their partners. Additionally, activity-dependent mechanisms are thought to refine connections through Hebbian and/or homeostatic plasticity mechanisms. Neurons that fire together preferentially wire together in many areas of the vertebrate nervous system through positive feedback. At the same time, homeostatic mechanisms restore activity toward a specific set point through negative feedback, imposing competition and preventing runaway excitation or complete silencing of the circuit. However, the extent to which activity modulates numbers versus the strength of existing synapses is still an open question (Valdes-Aleman, 2020).

These questions have been difficult to address because they require manipulating candidate factors that could influence connectivity, visualizing synapses between uniquely identified partners, and relating observed structural changes to effects on functional connectivity and behavior. This study therefore used the tractable Drosophila larva as a model system with the following advantages: (1) excellent genetic tools for selective manipulation of uniquely identified neurons; (2) a compact nervous system amenable to rapid imaging with synaptic resolution, and (3) a rich behavioral repertoire with well-established quantitative assay (Valdes-Aleman, 2020).

Recently, comprehensive synaptic-resolution connectivity maps of the circuitry downstream of the mechanosensory Chordotonal (hereafter 'mechanosensory') neurons and nociceptive multidendritic class IV (hereafter 'nociceptive') neurons in an abdominal segment of a first instar larva have been generated. Portions of this circuit were also reconstructed in two different abdominal segments (A1 and A3) of two different first instar individuals and at two different life stages: first (A1) and third instar (A3) (Valdes-Aleman, 2020).

This study selectively altered the location or activity of the mechanosensory neurons and generated new EM volumes of the manipulated samples to investigate the effects on connectivity. These anatomical studies were completed with functional connectivity and behavioral assays. This study reveals that proper location, partner identity, and activity are all required to achieve appropriate connectivity and behavior (Valdes-Aleman, 2020).

In some systems the position of pre- or postsynaptic terminals is specified by non-partner-derived positional cues. In other systems, molecules have been identified that mediate partner matching. However, it was unclear whether both mechanisms could operate in the same system and whether either mechanism specifies numbers of connections between partners (Valdes-Aleman, 2020).

Although developing sensory axons use non-partner-derived positional cues to select their final termination area in the Drosophila nerve cord, the current results suggest that position alone does not specify connectivity and that partner recognition also exists. When the location of sensory axons was altered, their postsynaptic partners extended ectopic branches and formed synaptic connections with them. The shifted axons did not gain any new strongly connected partners at their ectopic location, providing further evidence of remarkable partner selectivity. It is hard to imagine which cue, other than the mechanosensory axons themselves, instructed partner dendrites to form these ectopic branches and synapses. Nevertheless, the final proof of the existence of the partner-derived cues will be their identification in the future (Valdes-Aleman, 2020).

If partner-recognition molecules are sufficient for selective synaptogenesis irrespective of the location of partners, why is the precise location of sensory neuron axon terminals so tightly regulated by non-partner-derived positional cues? Despite partner neurons' connecting in ectopic locations, they did not establish appropriate numbers of synapses, resulting in defective responses to mechanosensory stimuli. This indicates that precise positioning of presynaptic mechanosensory axons is necessary for the formation of appropriate number of synapses (Valdes-Aleman, 2020).

It is not known why some partners received more synapses from shifted mechanosensory axons and others fewer than in wild-type. One possibility could be the involvement of third-party guidepost cells in synaptogenesis which would not be present in the aberrant location. Another speculation is that some neurons are better than others at finding their misplaced partners. Yet another possibility could be that shifting mechanosensory neurons initially resulted in fewer or weaker synaptic connections. This could have triggered compensatory homeostatic changes in the balance of excitation and inhibition within the circuit by increasing mechanosensory connections onto excitatory interneurons and reducing those onto inhibitory interneurons. This latter possibility could explain why similar connectivity effects were observed when sensory neurons were shifted and when they were inactivated during development (Valdes-Aleman, 2020).

Finally, in addition to changes in synapse numbers, silencing or shifting presynaptic partners could have also induced changes in synaptic strength and electrical properties (e.g., through changes in ion channel composition) that could account for some of the observed effects in behavior and functional connectivity. Furthermore, changes in the shapes of arbors could potentially affect electrical signal propagation. Future patch-clamp recordings following the same experimental manipulations could reveal the extent to which this occurs (Valdes-Aleman, 2020).

Activity plays a major role in refining the patterns of neuronal connections during development, especially in vertebrates. However, the effects induced within the network in response to selective silencing of specific neuron types are not fully understood (Valdes-Aleman, 2020).

The role activity plays in the development of the insect central nervous system is less clear. Some studies have shown that a lack of sensory activity during development does not affect neuron morphology or the capacity to form connections. Other studies have reported neural circuits can adapt their morphology, connectivity, or behavior in response to changes in developmental activity. However, a comprehensive synaptic-resolution analysis of the effects of silencing a specific neuron type on the numbers of connections between partners was lacking (Valdes-Aleman, 2020).

EM reconstructions revealed that silenced mechanosensory neurons connected to the appropriate partners, but with inappropriate numbers of synapses. Interestingly, excitatory multisensory interneurons (Basin) received a higher fraction of input from silenced mechanosensory neurons than in controls, while inhibitory interneurons (Ladder and Griddle) received a lower fraction. Selective silencing of mechanosensory neurons also increased input from a different sensory modality (nociceptive) onto Basin interneurons and decreased their input from inhibitory interneurons. This overall effect is similar to observations in the cortex, where sensory deprivation induces network-level homeostasis that alters the balance of excitation and inhibition. Synaptic scaling in the cortex is thought to be multiplicative, such that all excitatory connections onto an excitatory neuron are scaled equally when excitatory drive onto that neuron is reduced. In contrast, the inhibitory connections onto excitatory neurons are reduced. Although the majority of studies in the cortex focus on homeostatic plasticity of functional connections, this study demonstrated a drastic plasticity in the number of synaptic connections between partners. This apparent homeostasis of synapse numbers may follow similar multiplicative rules, because this study found that both mechanosensory and nociceptive inputs onto Basin interneurons were increased when mechanosensory neurons were silenced (Valdes-Aleman, 2020).

It was found that larvae with permanently silenced mechanosensory neurons not only had increased structural connections between nociceptive and Basin neurons but also stronger functional connections and behavioral responses to nociceptive stimuli. This structural and behavioral compensation is reminiscent of findings in mammals, in which if one sensory modality is removed, another modality is restructured and improved (Valdes-Aleman, 2020).

Interestingly, silencing mechanosensory neurons during development permanently decreased responses to mechanosensory stimuli, even days after restoring activity. This is also reminiscent of findings in mammals, in which deprivation of visual input during an early critical period permanently impairs vision. However, this result appears at odds with the increased structural and functional connections from silenced mechanosensory neurons onto the excitatory Basins. A possible explanation is the reduction of mechanosensory connections onto inhibitory neurons under the same conditions. Unlike nociceptive neurons, the mechanosensory neurons have more inhibitory than excitatory postsynaptic partners, and these inhibitory interneurons play a role in triggering mechanosensory behaviors through disinhibition. Silencing the mechanosensory neurons may therefore result in a permanent reduction in disinhibition in the circuit with permanent consequences on behavior (Valdes-Aleman, 2020).

In summary, although partner-recognition molecules can ensure neurons recognize and connect only with appropriate partners, they are not sufficient to robustly specify appropriate numbers of synapses. Conversely, although neither precise location of presynaptic terminals nor neuronal activity in presynaptic partners directly instructs partner specificity, both are crucial to achieve appropriate numbers of connections, appropriate strengths of functional connections, appropriate balance of excitation and inhibition, and appropriate behavior. This study reveals with unprecedented resolution how location, identity, and activity work together to give rise to appropriately wired neural circuits and appropriate behaviors (Valdes-Aleman, 2020).

Neuroarchitecture of peptidergic systems in the larval ventral ganglion of Drosophila melanogaster

Recent studies on Drosophila melanogaster and other insects have revealed important insights into the functions and evolution of neuropeptide signaling. In contrast, in- and output connections of insect peptidergic circuits are largely unexplored. Existing morphological descriptions typically do not determine the exact spatial location of peptidergic axonal pathways and arborizations within the neuropil, and do not identify peptidergic input and output compartments. Such information is however fundamental to screen for possible peptidergic network connections, a prerequisite to understand how the CNS controls the activity of peptidergic neurons at the synaptic level. This study provide a precise 3D morphological description of peptidergic neurons in the thoracic and abdominal neuromeres of the Drosophila larva based on fasciclin-2 (Fas2) immunopositive tracts as landmarks. Comparing the Fas2 "coordinates" of projections of sensory or other neurons with those of peptidergic neurons, it is possible to identify candidate input and output connections of specific peptidergic systems. These connections can subsequently be more rigorously tested. By immunolabeling and GAL4-directed expression of marker proteins, this study analyzed the projections and compartmentalization of neurons expressing 12 different peptide genes, encoding approximately 75% of the neuropeptides chemically identified within the Drosophila CNS. Results are assembled into standardized plates which provide a guide to identify candidate afferent or target neurons with overlapping projections. In general, this study found that putative dendritic compartments of peptidergic neurons are concentrated around the median Fas2 tracts and the terminal plexus. Putative peptide release sites in the ventral nerve cord were also more laterally situated. The results suggest that peptidergic neurons in the Drosophila ventral nerve cord have separated in- and output compartment. The lack of a strict segmentally reiterated pattern throughout the thoracic and abdominal neuromeres suggests that the restricted and differential distribution of peptidergic neurons reflects neuromere-specific functional connections. Other larval neuron types or circuits that match the observed peptidergic distribution patterns have not been characterized (Santos, 2007).

The last two abdominal neuromeres a8/9 have a unique pattern of peptidergic somata and projections (e.g. FMRFa, MIP or PDF neurons, and show the least serial homology to the more anterior neuromeres of the ventral ganglion. This finding also extends to descending processes. Descending axons may stop before or when reaching the border to a8 (HUG and DTK neurons), form extensive varicose ramifications within the neuropil of a8 (AST, corazonin or branch extensively in the terminal plexus of a9 (FMRFa-, leucokinin-, MIP and PDF-neurons. Belonging to the tail region, the segments a8/9 differ from the homomeric segments a1-7 with respect to the organization of muscles and sensory neurons. Furthermore, several unique structures such as the spiracles or the anal pads belong to these terminal segments. Unlike other segmental nerves, the segmental nerve of a9 innervates the hindgut musculature. The unique pattern of peptidergic neurons in a8/9 might thus, at least partially, reflect a segment-specific function related to e.g. control of spiracles or intestinal functions. For example, the PDF neurons innervate the hindgut, but their exact function is so far unknown. Similar segmental differences between a8 and the rest of the abdominal neuromeres have been found for neurons expressing biogenic amines (Santos, 2007).

The fusion construct syb.egfp has been developed as a presynaptic marker. Since synaptobrevin (SYB) is an integral membrane protein of small synaptic vesicles and large peptide-containing vesicles alike, SYB.EGFP also labels peptide vesicles and hence peptide accumulation and release sites (varicosities), which typically do not spatially coincide with synapses. Concomitantly, it is assumed that purely dendritic compartments of peptidergic neurons do not contain vesicles and show no or only weak SYB.EGFP labeling. These assumptions are supported by results obtained for PDF neurons in the brain, and the Tv and Va neurons that innervate neurohemal organs. SYB.EGFP was only found in the cell bodies (where the protein is made) and in the terminals in the neurohemal organs. The axonal projections as well as the arborizations within the VNC were unlabeled. Nevertheless, when interpreting the SYB.EGFP distribution, it has to be kept in mind that SYB.EGFP might also label presynaptic sites if the peptidergic neurons contain colocalized classical neurotransmitters (Santos, 2007).

The haemagglutinin-tagged GABAA receptor subunit RDL.HA has been shown to be a useful specific postsynaptic marker in motor neurons. Since The GABAA receptor subunit RDL is involved in mediating GABAergic postsynaptic currents, attempts were made to see whether ectopic RDL.HA expression indicates postsynaptic sites (dendrites) of peptidergic neurons also. The general expression level of RDL.HA was very weak, and only discernible labeling intensities were obtained with two different GAL4-drivers: Ccap- and c929-GAL4. Nevertheless, the labeling was spatially very confined to neuron compartments that showed no varicosities or only weak SYB.EGFP fluorescence. This suggests that RDL.HA labeled postsynaptic sites in peptidergic neurons (Santos, 2007).

Arborizations around the median DM and VM tracts turned out to be a prominent feature of most characterized peptidergic neurons with somata in the ventral ganglion, including the AST, CCAP, corazonin, FMRFa, MIP and PDF neurons. In contrast to motor neurons, the prominent midline arborizations of peptidergic neurons were rather short, and did not occupy large areas in the more lateral neuropils between the median and lateral tracts. For the CCAP neurons, ectopically expressed RDL.HA localized exclusively to these median arborizations. In contrast, SYB.EGFP as well as peptide-immunoreactivity was absent or relatively low in these arborizations. Also in the general peptidergic c929-GAL4-line, SYB.EGFP expression was low in the median compared to lateral fascicles. This might suggest that the median arborizations represent peptidergic dendrites. Descending processes of CCAP, EH, HUG and leucokinin neurons (originating from somata in the suboesophageal ganglion or in the brain) all have putative release sites around the DM and VM tracts. Of the peptidergic neurons with cell bodies in the VNC, only those expressing corazonin were found to have varicosities indicative of release sites around the DM and VM tracts (Santos, 2007).

Taken together, these findings suggest that the arborizations around the dorsomedial (DM) and ventromedial (VM) tracts are mainly input compartments for peptidergic VNC neurons, and point to this midline region as a main site for synaptic inputs onto peptidergic neurons including the CCAP neurons. The different putative sites of in- and outputs to peptidergic neurons in the VNC are summarized (see Assignment of putative main compartment identities as suggested by morphology, immunolabeling intensities and distribution of synaptic markers). Peptides released from varicosities of leucokinin, CCAP, HUG-, EH and corazonin neurites along the DM tract may modulate synaptic transmission around the DM tracts, or might represent direct input signals to peptidergic neurons. Also, the dorsal ap-let neurons with somata in the ventral ganglion expressing the peptide precursor Nplp1 appear to have their output sites along the DM tracts as indicated by strong peptide immunoreactivity. Unlike any of the peptidergic neurons characterized here, the dorsal ap-let neurons seem to have extensive arborizations within the neuropil of each hemineuromere, which appear to contain no or only little peptide immunoreactive material and hence might represent dendritic regions. Also the leucokinin neurons with somata in the ventral ganglion do not send projections towards the midline. Since leucokinin release is likely to occur at peripheral release sites on body wall muscles, it is possible that a synaptic input region is located along the VL tract, the only projection site of abdominal leucokinin neurons within the CNS neuropil (Santos, 2007).

A developmental framework linking neurogenesis and circuit formation in the Drosophila CNS

The mechanisms specifying neuronal diversity are well-characterized, yet it remains unclear how or if these mechanisms regulate neural circuit assembly. To address this, the developmental origin was mapped of 160 interneurons from seven bilateral neural progenitors (neuroblasts), and they were identified in a synapse-scale TEM reconstruction of the Drosophila larval CNS. Lineages were found to concurrently build the sensory and motor neuropils by generating sensory and motor hemilineages in a Notch-dependent manner. Neurons in a hemilineage share common synaptic targeting within the neuropil, which is further refined based on neuronal temporal identity. Connectome analysis shows that hemilineage-temporal cohorts share common connectivity. Finally, this study showed that proximity alone cannot explain the observed connectivity structure, suggesting hemilineage/temporal identity confers an added layer of specificity. Thus, this study demonstrated that the mechanisms specifying neuronal diversity also govern circuit formation and function, and that these principles are broadly applicable throughout the nervous system (Mark, 2021).

Tremendous progress has been made in understanding the molecular mechanisms generating neuronal diversity in both vertebrate and invertebrate model systems. In mammals, spatial cues generate distinct pools of progenitors, which generate neuronal diversity in each spatial domain. The same process occurs in invertebrates like Drosophila, but with a smaller number of cells, and this process is particularly well understood. The first step occurs when spatial patterning genes act combinatorially to establish single, unique progenitor (neuroblast) identities. These patterning genes endow each neuroblast with a unique spatial identity (Mark, 2021).

The second step is temporal patterning -- the specification of neuronal identity based on birth-order, an evolutionarily conserved mechanism for generating neuronal diversity. This study focused on Drosophila embryonic neuroblasts, which undergo a cascade of temporal transcription factors: Hunchback (Hb), Krüppel (Kr), Pdm, and Castor (Cas). Each temporal transcription factor is inherited by ganglion mother cells (GMCs) born during each expression window. The combination of spatial and temporal factors endows each GMC with a unique identity (Mark, 2021).

The third step is hemilineage specification, which was initially characterized in Drosophila larval and adult neurogenesis, and may also be used in vertebrate neurogenesis. Hemilineages are formed by GMC asymmetric division into a pair of post-mitotic neurons; during this division, the Notch inhibitor Numb (Nb) is partitioned into one neuron (NotchOFF neuron), whereas the other sibling neuron receives active Notch signaling (NotchON neuron), thereby establishing NotchON and NotchOFF hemilineages. In summary, three mechanisms generate neuronal diversity within the embryonic central nervous system (CNS): neuroblast spatial identity, GMC temporal identity, and neuronal hemilineage identity (Mark, 2021).

A great deal of progress has also been made in understanding neural circuit formation in both vertebrates and invertebrate model systems, revealing a multi-step mechanism. Neurons initially target their axons to broad regions (e.g., thalamus/cortex), followed by targeting to a neuropil domain (glomeruli/layer), and finally forming highly specific synapses within the targeted domain (Mark, 2021).

Despite the progress in understanding the generation of neuronal diversity and the mechanisms governing axon guidance and neuropil targeting, how these two developmental processes are coordinated remains largely unknown. While it is accepted that the identity of a neuron is linked to its connectivity, the developmental mechanisms involved are unclear. For example, do clonally related neurons target similar regions of the neuropil due to the expression of similar guidance cues? Do temporal cohorts born at similar times show preferential connectivity? This study addressed the question of whether any of the three developmental mechanisms (spatial, temporal, hemilineage identity) are correlated with any of the three circuit-wiring mechanisms (neurite targeting, synapse localization, connectivity). This study mapped the developmental origin for 80 bilateral pairs of interneurons in abdominal segment 1 (A1) by identifying and reconstructing these neurons within a full CNS TEM volume -- this is over a quarter of the ~300 neurons per hemisegment. The unexpected observation was made that hemilineage identity determines neuronal projection to sensory or motor neuropils; thus, neuroblast lineages coordinately produce sensory and motor circuitry. In addition, it was shown that neurons with shared hemilineage-temporal identity target pre- and post-synapse localization to similar positions in the neuropil, and that hemilineage-temporal cohorts share more common synaptic partners than that produced by neuropil proximity alone. Thus, temporal and hemilineage identity plays essential roles in establishing neuronal connectivity (Mark, 2021).

This study determined the relationship between developmental mechanisms (spatial, temporal, and hemilineage identity) and circuit assembly mechanisms (projections, synapse localization, and connectivity). To do this, both developmental and circuit features were mapped for 160 neuronal progeny of 14 neuroblast lineages in a serial section TEM reconstruction - this allows characterization neurons that share a developmental feature at single synapse resolution. It is important to note that the seven neuroblasts in this study were chosen based on successful clone generation and availability of single neuroblast Gal4 lines, and thus there should be no bias towards a particular pattern of neurite projections, synapse localization, or connectivity. The results show that individual neuroblast lineages have unique but broad axon and dendrite projections to both motor and sensory neuropil; hemilineages restrict projections and synapse localization to either motor or sensory neuropil; and distinct temporal identities within hemilineages provide additional specificity in synapse localization and connectivity. Thus, all three developmental mechanisms act combinatorially to progressively refine neurite projections, synapse localization, and connectivity (Mark, 2021).

In mammals, clonally related neurons often have a similar location, morphology, and connectivity. In contrast, this study found that clonally related neurons project widely in the neuropil, to both sensory and motor domains, and thus lack shared morphology. Perhaps as brain size expands to contain an increasing number of progenitors, each clone takes on a more uniform structure and function. Yet the observation that each neuroblast clone had highly stereotyped projections suggests that neuroblast identity (determined by the spatial position of the neuroblast) determines neuroblast-specific projection patterns. Testing this functionally would require manipulating spatial patterning cues to duplicate a neuroblast and assay both duplicate lineages for similar projections and connectivity (Mark, 2021).

This study found that hemilineages produce sensory and motor processing units via a Notch-dependent mechanism. Pioneering work on Drosophila third instar larval neuroblast lineages showed that each neuroblast lineage is composed of two hemilineages with different projection patterns and neurotransmitter expression. These studies were extended to embryonic neuroblasts and showed that Notch signaling determines motor versus sensory neuropil projections in all lineages examined. Surprisingly, the NotchON hemilineage always projected to the dorsal/motor neuropil, whereas the NotchOFF hemilineage always projected to the ventral/sensory neuropil. The relationship between the NotchON hemilineage projecting to the motor neuropil may be a common feature of all 30 segmental neuroblasts or it could be that the NotchON/NotchOFF provides a switch to allow each hemilineage to respond differently to dorsoventral guidance cues, with some projecting dorsally and some projecting ventrally. Analysis of additional neuroblast lineages will resolve this question. Another point to consider is the potential role of Notch in post-mitotic neurons as these experiments generated Notchintra misexpression in both newborn sibling neurons as well as mature post-mitotic neurons. Future work manipulating Notch levels specifically in mature post-mitotic neurons undergoing process outgrowth will be needed to identify the role of Notch in mature neurons, if any (Mark, 2021).

Elegant work has identified neuropil gradients of Slit and Netrin along the mediolateral axis, Semaphorins along the dorsoventral axis, and Wnt5 along the anteroposterior axis. The finding that neurons in a hemilineage project to a common region of the neuropil strongly suggests that all neurons within a hemilineage respond in the same way to these global pathfinding cues. Conversely, the finding that neurons in different hemilineages target distinct regions of the neuropil suggests that each hemilineage expresses a different palette of guidance receptors, which enable them to respond differentially to the same global cues. For example, neurons in ventral hemilineages may express Plexin receptors to repel them from high Semaphorins in the dorsal neuropil (Mark, 2021).

Hemilineages have not been well described in vertebrate neurogenesis. Notch signaling within the Vsx1 + V2 progenitor lineage generates NotchOFF V2a excitatory interneurons and NotchON V2b inhibitory interneurons, which may be distinct hemilineages. Interestingly, both V2a and V2b putative hemilineages contain molecularly distinct subclasses; this study raises the possibility that these subtypes arise from temporal patterning within the V2 lineage. In addition, NotchON/NotchOFF hemilineages may exist in the pineal photoreceptor lineage, where NotchON and NotchOFF populations specify cell-type identity (Mark, 2021).

Only recently have the role of hemilineages been tested for their functional properties. In adults, activation of each larval hemilineage from NB5-2 showed similar behavioral output, whereas each hemilineage from NB6-1 elicited different behaviors. Previous work showed that the Eve+, Saaghi, and Jaam neurons are part of a proprioceptive circuit (Heckscher, 2015); this study shows that each class of neurons represents a hemilineage-temporal cohort. Note that the Jaam neurons process sensory input and are in a NotchOFF hemilineage, supporting the conclusion that NotchOFF hemilineages are devoted to sensory processing; the Saaghi premotor neurons are in a NotchON hemilineage consistent with their role in motor processing. Interestingly, both input and output neurons in this circuit arise from a common progenitor (NB5-2), which may generate late-born Jaam/Saaghi sibling neurons. In the future, it would be interesting to determine if other sibling hemilineages are in a common circuit to generate a specific behavior (Mark, 2021).

The hemilineage results have several implications. First, the results reveal that sensory and motor processing components of the neuropil are being built in parallel, with one half of every GMC division contributing to either sensory or motor networks. This would be an efficient mechanism to maintain sensory/motor balance as lineage lengths are modified over evolutionary time. Second, the results suggest that looking for molecular or morphological similarities in full neuroblast clones may be misleading due to the full neuroblast clone comprising two different hemilineages. For example, performing bulk RNAseq on all neurons in a neuroblast lineage is unlikely to reveal key regulators of pathfinding or synaptic connectivity due to the mixture of disparate neurons from the two hemilineages (Mark, 2021).

The cortex neurite length of neurons was used as a proxy for birth-order and shared temporal identity. This is thought to be a good approximation, but it clearly does not precisely identify neurons born during each of the Hb, Kr, Pdm, Cas temporal transcription factor windows. Nevertheless, there was sufficient resolution to observe that neurons with the same temporal identity clustered their pre- or postsynapses, rather than localizing them uniformly through the hemilineage neuropil domain. Interestingly, the three-dimensional location of each hemilineage temporal cohort synaptic cluster is identical on the left and right side of A1, ruling out the mechanism of stochastic self-avoidance. Other possible mechanisms include hemilineage-temporal cohorts expressing different levels of the presynapse spacing cue Sequoia or hemilineage-temporal cohorts exhibiting different responses to global patterning cues. Testing the function of temporal identity factors in synaptic tiling will require hemilineage-specific alteration of temporal identity, followed by assaying synapse localization within the neuropil (Mark, 2021).

The results strongly suggest that hemilineage identity and temporal identity act combinatorially to allow small pools of neurons to target pre- and postsynapses to highly precise regions of the neuropil, thereby restricting synaptic partner choice. Yet precise neuropil targeting is not sufficient to explain connectivity as many similarly positioned axons and dendrites fail to form connections. The model is favored that hemilineages direct gross neurite targeting to motor or sensory neuropil, whereas temporal identity acts combinatorially with each hemilineage to direct more precise neurite targeting and synaptic connectivity. Thus, the same temporal cue (e.g., Hb) could promote targeting of one pool of neurons in one hemilineage and another pool of neurons in an adjacent hemilineage. This limits the number of regulatory mechanisms needed to generate precise neuropil targeting and connectivity for all ~600 neurons in a segment of the larval CNS (Mark, 2021).

In conclusion, this study demonstrates how developmental information can be integrated with connectomic data. Lineage information, hemilineage identity, and temporal identity can all be accurately predicted using morphological features (e.g., number of fascicles entering the neuropil for neuroblast clones and radial position for temporal cohorts). This both greatly accelerates the ability to identify neurons in a large EM volume as well as sets up a framework in which to study development using data typically intended for studying connectivity and function. This framework is used to relate developmental mechanism to neuronal projections, synapse localization, and connectivity. Lineage, hemilineage, and temporal identity were found act sequentially to progressively refine neuronal projections, synapse localization, and connectivity, and the data supports a model where hemilineage-temporal cohorts are units of connectivity for assembling motor circuits (Mark, 2021).

Astrocytes close a motor circuit critical period

Critical periods (brief intervals during which neural circuits can be modified by activity) are necessary for proper neural circuit assembly. Extended critical periods are associated with neurodevelopmental disorders; however, the mechanisms that ensure timely critical period closure remain poorly understood. This study defined a critical period in a developing Drosophila motor circuit and identified astrocytes as essential for proper critical period termination. During the critical period, changes in activity regulate dendrite length, complexity and connectivity of motor neurons. Astrocytes invaded the neuropil just before critical period closure, and astrocyte ablation prolonged the critical period. Finally, a genetic screen was used to identify astrocyte-motor neuron signalling pathways that close the critical period, including Neuroligin-Neurexin signalling. Reduced signalling destabilized dendritic microtubules, increased dendrite dynamicity and impaired locomotor behaviour, underscoring the importance of critical period closure. Previous work defined astroglia as regulators of plasticity at individual synapses. This study shows that astrocytes also regulate motor circuit critical period closure to ensure proper locomotor behaviour (Ackerman, 2021).

Critical periods are brief windows during which neural circuit activity can modify the morphological properties of neurons, producing permanent changes to circuit structure and function. Critical periods integrate multiple forms of plasticity to modify neural circuits. 'Homeostatic plasticity' encompasses changes to synapse number, structure and function across an entire neuron, as well as changes to long-range connectivity. Whereas homeostatic plasticity can occur in the adult brain, substantial activity-dependent remodelling peaks in early development. Indeed, failure to terminate critical period plasticity is linked to neurodevelopmental disorders such as autism and epilepsy. Although putative critical period disorders present with motor defects, the field has largely focused on sensory circuits. To that end, this study developed a novel critical period model in a developing motor circuit (Ackerman, 2021).

This study focused on two well-characterized Drosophila motor neurons, aCC and RP2, which are segmentally repeated in the central nervous system. These motor neurons are susceptible to activity-induced remodelling, although pioneering studies used chronic activity manipulations and did not define an end point for homeostatic plasticity. This study expressed the anion channelrhodopsin GtACR215 specifically in the aCC-RP2 motor neurons using the Gal4-upstream activation system (UAS) system and delivered acute 1-h windows of silencing, terminating at progressively later times in development. Silencing motor neurons for the last hour of embryogenesis (stage 17) increased aCC-RP2 dendritic volume at 0 h after larval hatching (ALH), whereas silencing for 1 h at later stages showed progressively less of an effect, with no remodelling occurring at 8 h ALH or beyond. By contrast, acute 1-h windows of activation using the channelrhodopsin Chrimson resulted in significant loss of motor neuron dendrites at 0 h ALH; activation at 8 h ALH and beyond had little or no effect. Activity-induced changes to dendrite length for single-cell RP2 clones [using the MultiColor FlpOut (MCFO) system] showed similar results Note that these experiments used far shorter periods of tonic activation than past studies. Although Tonic activity manipulations were primarily used, identical results were observed using 600 ms:400 ms pulses of activation or silencing, as well as thermogenetics to activate (via TrpA1) or silence [using the temperature-sensitive shibire gene (shibire^ts)] motor neurons. Notably, dendrite loss following acute activation could be rescued by a 22-h period of dark rearing, indicating that activity induces dendrite plasticity and not excitotoxicity. Together, these experiments define a critical period for activity-dependent motor dendrite plasticity represent the first analyses of motor circuit critical period closure within the central nervous system (Ackerman, 2021).

In vertebrates, homeostatic plasticity functions on a slow timescale, from hours to days. To determine the timescale for motor neuron dendrite expansion following GtACR2 silencing, aCC-RP2 motor neurons were silenced for 15 min, 1 h or 4 h in stage 17 embryos, terminating silencing at 0 h ALH. Larvae were then immediately dissected and dendritic morphology was assessed in single, well-spaced RP2 neurons using MCFO17. Increased dendritic arbor size and complexity following 1 h and 4 h of silencing were used. These results were confirmed using shibire^ts. By contrast, embryonic Chrimson activation resulted in decreased dendrite length and complexity at 0 h ALH after as little as 15 min of activation. Furthermore, using live imaging, significant dendrite retraction was observed within 12 min of Chrimson activation. The fact that silencing required more time to show an effect is not surprising, as extension requires generation of new membrane. It is concluded that activity-induced remodelling of Drosophila motor neurons occurs within minutes, much more quickly than previously documented for homeostatic plasticity in mammals (Ackerman, 2021).

This study showed that motor neurons scale dendrite length according to activity. An important question is whether these morphological changes are accompanied by changes in excitatory or inhibitory synaptic inputs. The excitatory cholinergic neuron A18b and inhibitory GABAergic neuron A23a were examined that are synaptically coupled to aCC-RP2 dendrites in a larval transmission electron microscopy (TEM) reconstruction. To quantify excitatory and inhibitory synapse number by light microscopy, a functionally inactive pre-synaptic marker, Bruchpilotshort::Cherry (Brp), was expressed in excitatory cholinergic neuron A18b or inhibitory GABAergic neuron A23a using the complementary LexA-LexAop binary expression system. A23a-inhibitory GABAergic synapses onto aCC-RP2 dendrites were examined, quantifying cell-type specific Brp puncta overlapping with aCC-RP2 dendritic membrane (putative synapses) using published standards. All critical period manipulations terminated at 4 h ALH (stage matched to the TEM data). It was found that 1 h of motor neuron silencing reduced the number of inhibitory synapses between A23a and aCC-RP2 dendrites. Silencing for a longer period (4 h) also yielded a significant increase in A18b excitatory synapses. Decreasing motor neuron activity thus leads to a compensatory reduction of inhibitory inputs and a corresponding increase in excitatory inputs to rebalance network activity. A18b excitatory cholinergic synapse numbers onto aCC-RP2 dendrites were quantified after activation or silencing. Motor neuron activation was found to significantly decreased numbers of A18b excitatory synapses onto aCC-RP2 dendrites following 1 h and 4 h manipulations. A significant increase in inhibitory synapse number following extended motor neuron activation was observed, possibly owing to insufficient dendritic membrane after activity-induced dendrite retraction. Increasing motor neuron activity thus leads to a compensatory reduction of excitatory pre-synaptic inputs. Finally, a functionally inactive reporter of excitatory post-synaptic densities (Drep2::GFP or Drep2::mStrawberry) was observed, specifically in aCC-RP2, and scaling of synapses was observed accross the entire dendritic arbor in response to altered activity—reduced excitatory post-synapses followed motor neuron activation, whereas increased excitatory post-synapses followed motor neuron silencing during the critical period. Of note, homeostatic scaling of motor neuron synapses did not occur after critical period closure. In sum, motor neurons scale excitatory and inhibitory inputs relative to their level of activity during the critical period (Ackerman, 2021).

The mechanisms that close critical periods remain poorly defined. Drosophila astrocytes infiltrate the neuropil at late embryogenesis and progressively envelop motor neuron synapses as the critical period closes. To test whether astrocytes promote critical period closure, all astrocytes were genetically ablated and optogenetics was used to assay for extension of critical period plasticity at 8 h ALH. Astrocyte elimination was confirmed by loss of the astrocyte marker Gat3. As expected, controls closed the critical period by 8 h ALH. By contrast, astrocyte ablation extended dendrite plasticity following Chrimson activation or GtACR2 silencing up to 8 h ALH. This effect was not observed at earlier stages, indicating that astrocytes do not constitutively dampen plasticity. Additionally, it was found that control motor dendrites were less dynamic after critical period closure, but that astrocyte ablation extends dendrite filopodial dynamicity. It is concluded that astrocytes are required for the transition from dynamic to stable filopodia and concurrent critical period closure (Ackerman, 2021).

To determine how astrocytes close the critical period, the astrocyte-specific alrm-gal4 was used to perform a targeted UAS RNA-mediated interference (RNAi) knockdown screen. Flies were assayed for critical period extension following 1 h of Chrimson activation from 7-8 h ALH. Four genes were identified that were required in astrocytes for timely critical period closure: gat (regulates excitatory-inhibitory balance), chpf [synthesizes chondroitin sulfate proteoglycans (CSPGs)] and the Neuroligins (Nlg) 4 and 2 (Ackerman, 2021).

Neuroligins are cell-adhesion proteins that are known to regulate astrocyte morphogenesis. In Drosophila, astrocyte-specific knockdown of nlg2 (the mouse orthologue is known as Nlgn1) had no effect on astrocyte volume or tiling, suggesting a more specific defect in astrocyte-motor neuron signalling. Knockdown of the remaining critical period regulators had variable effects on astrocyte morphology but all extended the critical period. Neuroligins bind cell adhesion proteins called Neurexins. RNAi against nrx-1 was used, which is known to bind both Nlg2 and Nlg4, specifically in aCC-RP2 motor neurons, and critical period extension was observed; this is consistent with astrocyte Nlg2 and motor neuron Nrx-1 acting in a common pathway to close the critical period. Motor neuron-specific RNAi knockdown of the CSPG receptor Lar also extended critical period plasticity. Notably, while Nrx-1 is often pre-synaptic, there is evidence for dendritic localization of these receptors. Furthermore, antibody staining for endogenous Nrx-1 and Nlg2 revealed localization of this receptor-ligand pair on motor dendrites and astrocytes, respectively. Finally, cell-type-specific overexpression of Nrx-1 and Nlg2 could induce precocious critical period closure (assayed by Chrimson activation from 3-4 h ALH). It is concluded that Nlg2-Nrx-1 ligand-receptor signalling between astrocytes and motor neurons is required for timely critical period closure (Ackerman, 2021).

How does Nlg2-Nrx-1 signalling close the critical period? The balance of excitatory to inhibitory synapses in neural circuits can instruct critical period timing. Additionally, numbers of excitatory synapses are decreased following astrocyte-specific knockout of neuroligins in mouse. This study observed no significant changes in excitatory-inhibitory balance following knockdown of nlg2 in astrocytes, suggesting that critical period closure is not dependent on Nlg2-mediated excitatory-inhibitory synapse balance (Ackerman, 2021).

Alternatively, Nrx-1 can promote microtubule stability in axons of motor neurons, suggesting a mechanism for critical period closure involving microtubule stabilization. To test this hypothesis, Chrimson::mVenus was used to activate and visualize aCC-RP2 dendrite membranes at 0 h ALH (peak critical period), and Cherry::Zeus to visualize stable microtubules during and after dendritic retraction. In live preparations, dendrites showed a reduction in Cherry::Zeus intensity immediately preceding activity-dependent retraction, suggesting that microtubule collapse in distal branches can induce dendrite retraction. In fixed preparations, this study found that proximal dendrites with the highest levels of stable microtubules were protected from activity-dependent retraction. Of note, overexpression of Nrx-1 was sufficient to increase both stable microtubules and stable dendrites at 4 h ALH. It is proposed that Nlg2 in astrocytes binds Nrx-1 in motor neurons to stabilize dendritic microtubules and close the critical period (Ackerman, 2021).

In mammals, inappropriate critical period extension has long-term effects on nervous system function. Indeed, this study observed persistent changes in motor neuron connectivity at least 24 h following acute motor neuron activation at the end of the critical period, which lead to an assay for long-term effects on behaviour. The critical period was transiently extended until 12 h ALH (4 h beyond control critical period closure), and then behaviour was assayed 1.5 days later. Control larvae showed persistent linear locomotion; by contrast, larvae with extended critical periods due to transient knockdown of motor neuron genes showed excessive turning, leading to abnormal spiralling behaviour. Similar but less severe effects were seen in larvae following knockdown of astrocyte genes. It is concluded that a modest extension of the critical period can, in some cases, lead to long-lasting alteration in locomotor behaviour (Ackerman, 2021).

Astrocytes regulate synaptogenesis, synaptic pruning and synaptic efficacy. Within critical periods, astrocyte signalling can tune neuronal plasticity, but its role in critical period closure was not known. This study identified astrocytes as promoting closure of a motor critical period, and defined a series of astrocyte-motor neuron signalling pathways required to close the critical period. Based on previous literature, it is hypothesized that astrocytes could modify critical period closure through regulation of excitatory-inhibitory balance or extracellular matrix composition. Consistent with mammalian studies, it was found that perturbing excitatory-inhibitory balance through astrocyte-specific RNAi of the sole GABA transporter gat was sufficient to extend critical period plasticity. Furthermore, it was found that decreasing signalling from inhibitory extracellular matrix CSPGs through RNAi knockdown of Chondroitin polymerizing factor (Chpf) in astrocytes extended critical period plasticity. Thus, astrocytes use similar strategies in Drosophila and mammals to regulate critical period timing. Unexpectedly, this study also identified astrocyte-derived Neuroligins and their neuronal partner Nrx-1 as instrumental for critical period closure. In sum, this study have identified a key role of astrocytes in closure of a motor critical period required for locomotor function (Ackerman, 2021).

Lipin knockdown in pan-neuron of Drosophila induces reduction of lifespan, deficient locomotive behavior, and abnormal morphology of motor neuron

The Lipin family is evolutionarily conserved among insects and mammals, and its crucial roles in lipid synthesis and homeostatic control of energy balance have been well documented. This study investigated the function of Lipin in neuronal function and neurodegeneration. The GAL4/UAS system was used to knock down Lipin in the nervous system of Drosophila and investigate its behavioral and cellular phenotypes. The neuromuscular junction (NMJ) morphology was detected by immunostaining. Moreover, triacylglycerol and ATP levels were analyzed by using assay Kit. This study found that Lipin is localized almost in the cytoplasm of neurons in the brain lobe and ventral nerve cord, which are part of the central nervous system (CNS) of Drosophila melanogaster. Lipin knockdown larvae exhibit decreased locomotor activity, aberrant morphology of motor nerve terminals at NMJs, and reduced number and size of lipid droplets in the CNS. Furthermore, neuron-specific knockdown of Lipin leads to locomotor defects and a shortened lifespan, accompanied by a reduction in ATP levels in the adult stage. These results indicate that Lipin plays a crucial role in the CNS of Drosophila (Nguyen, 2023).

MDN brain descending neurons coordinately activate backward and inhibit forward locomotion

Command-like descending neurons can induce many behaviors, such as backward locomotion, escape, feeding, courtship, egg-laying, or grooming ('command-like neuron' is defined as a neuron whose activation elicits or 'commands' a specific behavior). In most animals, it remains unknown how neural circuits switch between antagonistic behaviors: via top-down activation/inhibition of antagonistic circuits or via reciprocal inhibition between antagonistic circuits. This study used genetic screens, intersectional genetics, circuit reconstruction by electron microscopy, and functional optogenetics to identify a bilateral pair of Drosophila larval 'mooncrawler descending neurons' (MDNs) with command-like ability to coordinately induce backward locomotion and block forward locomotion; the former by stimulating a backward-active premotor neuron, and the latter by disynaptic inhibition of a forward-specific premotor neuron. In contrast, direct monosynaptic reciprocal inhibition between forward and backward circuits was not observed. Thus, MDNs coordinate a transition between antagonistic larval locomotor behaviors. Interestingly, larval MDNs persist into adulthood, where they can trigger backward walking. Thus, MDNs induce backward locomotion in both limbless and limbed animals (Carreira-Rosario, 2018).

The role of Even-skipped in Drosophila larval somatosensory circuit assembly

Proper somatosensory circuit assembly is critical for processing somatosensory stimuli and for responding accordingly. In comparison to other sensory circuits (e.g., olfactory and visual), somatosensory circuits have unique anatomy and function. However, understanding of somatosensory circuit development lags far behind that of other sensory systems. For example, there are few identified transcription factors required for integration of interneurons into functional somatosensory circuits. This study examined one type of somatosensory interneuron, Even-skipped expressing Laterally placed interneurons (ELs) of the Drosophila larval nerve cord. Even-skipped (Eve) is a highly conserved, homeodomain transcription factor known to play a role in cell fate specification and neuronal axon guidance. Because marker genes are often functionally important in the cell types they define, this study deleted eve specifically from EL interneurons. On the cell biological level, using single neuron labeling, this study found eve plays several previously undescribed roles in refinement of neuron morphogenesis. Eve suppresses aberrant neurite branching, promotes axon elongation, and regulates dorsal-ventral dendrite position. On the circuit level, using optogenetics, calcium imaging, and behavioral analysis, it was found that eve is required in EL interneurons for the normal encoding of somatosensory stimuli and for normal mapping of outputs to behavior. It is concluded that eve coordinately regulates multiple aspects of EL interneuron morphogenesis and is critically required to properly integrate EL interneurons into somatosensory circuits. These data shed light on the genetic regulation of somatosensory circuit assembly (Marshall, 2022).

This study shows that eve expression is required for positioning EL interneuron neurites in all three axes (i.e., medial-lateral, anterior-posterior, and dorsal-ventral). In Drosophila, each axis is patterned by a separate ligand/receptor signaling system. However, how individual interneurons read and interpret each signal is not well understood. The data suggest eve is important for ELs to simultaneously read and/or interpret multiple ligand gradients simultaneously (Marshall, 2022).

Generally, eve is considered a cell fate determinant. For example, in mouse V0v interneurons, evx1 represses expression of en1, a marker of V1 interneuron identities. In V0v interneurons that lack evx1, en1 expression is derepressed and take on V1-like axonal projections. Similar fate changes are seen in Drosophila and C. elegans motor neurons when eve is disrupted. The current data are more consistent with the idea that eve plays a role in the refinement of EL morphogenesis. In support for the morphogenetic refinement model is, first, in wild-type, there are no neurons with morphology that matches the morphology of Eve^- ELs, as would be expected by a cell fate switching model. Second, there are no obvious large-scale changes in gene expression, which are typically associated with cell fate changes. Third, eve expression in ELs is not playing a role in initial morphogenesis (Marshall, 2022).

Both Eve^- and Eve⁺ ELs cross the midline at embryonic stage 15. Thus, eve expression is either dispensable for initial morphogenesis, or in EL eve mutants there is an undetectable pulse of early eve expression in ELs. But, no Eve protein expression was found in ELs in EL eve mutants at any stage of development. In later stage embryos and larvae, morphologic defects were observed in Eve^- ELs. This raises the possibility that, in general, eve genes may play a later role in morphogenesis. This is consistent with the observation that, in mouse V0v interneurons, there is early evx1 expression and later evx2 expression. However, the later role of evx2 is unknown (Marshall, 2022).

In general, eve genes are known to regulate axon morphogenesis. This study shows that late-born Eve^- ELs have axonal defects. Notably, the role of eve in dendrite morphogenesis is extremely poorly characterized. The distinction between dendrite and axon is important because these two compartments carry out different functions. Further, in Drosophila, interneuron axons and dendrites are structurally different. Dendrites are often highly branched, and lack mitochondria and postsynaptic machinery. Whereas, axon terminals (boutons) are full of mitochondria, pools of synaptic vesicles, microtubules, and vesicle release sites, each part of the arbor (axon or dendrite) can be independently controlled by different transcription factors. For example, in Drosophila sensory neurons, the transcription factors Knot and Cut specifically regulate dendrite morphogenesis, but not axonal morphology. Thus, in Drosophila, axon and dendrite morphology can be controlled as independent modules. This study has shown that in addition to regulating axon morphology, eve regulates dorsal-ventral dendrite positioning. eve expression is also required for dendrite morphogenesis in RP motor neurons. Taken together, these data show that eve coordinately regulates multiple aspects of neuronal morphogenesis, and that coordinate control may be a widely-occurring role for neuronal eve (Marshall, 2022).

Neuronal circuits are functional units of the nervous system. Sensorimotor circuits, specifically, transform somatosensory stimuli into motor output. Therefore, functional assays are required for the study of somatosensory circuit assembly. However, because the circuit context of individual interneurons is not well characterized, often researchers rely on anatomic assays to infer changes at the circuit level. One reason an anatomic approach can be flawed is the existence of compensatory mechanisms that allow for relatively normal circuit wiring despite changes in neuron morphology. This study links defects in neuronal morphology to changes in circuit function, thereby explicitly demonstrating the role of eve expression in somatosensory circuit assembly (Marshall, 2022).

It was shown that eve is required for somatosensory stimulus encoding by ELs. Based on known connectivity of ELs with other neurons, it is inferred that in ELs, eve is required for the formation of at least four types of functional input synapses: those from vibration (chordotonal) sensory neurons to early-born ELs, from vibration-sensitive interneurons (Basins) to early-born ELs, from proprioceptive sensory neurons to late-born ELs, and from proprioceptive-sensitive interneurons (Jaams) to late-born ELs. The likely cell biological underpinning, at least for late-born ELs, is that axons from input sensory neurons are not in close enough proximity to make synaptic contact with Eve^- ELs. Because of technical limitations, dendrite morphology of early-born ELs could not be visualized (Marshall, 2022).

In the Drosophila nerve cord, there is unidirectional compensatory growth from interneurons to genetically misplaced sensory neurons. Thus, Drosophila sensory neuron-to-interneuron wiring can be robust to morphologic alterations to circuit components. The observation that sensory neurons do not grow to reach mispositioned Eve^- EL dendrites raises two possibilities: (1) in this system, compensatory growth is unidirectional (i.e., interneurons grow to misplaced sensory neurons, but not vice versa); and (2) alternatively, compensatory growth is bidirectional, however, eve expression is required for this process. Future experiments will be needed to distinguish between these models (Marshall, 2022).

The data show Eve^- EL output synapses are functional, but remapped. Spontaneously-occurring crawling behavior is disrupted in EL eve mutants, and that this disruption is significantly worse than in larvae which lack EL neurons altogether. This could be explained by requirement for ELs during early circuit development (e.g., acting as a scaffold for normal axonal pathfinding for other neurons). Alternatively, mature Eve^- ELs could exert a dominant negative effect at the level of circuit function. The latter idea is favored because it is consistent with optogenetic experiments, and anatomic data. In controls, EL output synapses are excluded from many zones of the neuropile including the dorsal lateral zone, which houses the dendrites of dorsally-projecting motor neurons. However, Eve^- ELs are likely to form output synapses in this region. This specific re-distribution of output synapses is notable because it raises the possibility that Eve^- ELs output synapses (ELs are excitatory) could be directly re-mapped to dorsal motor neurons. Such a re-mapping could explain the novel behavioral phenotype, dorsal body bending phenotype seen on optogenetic activation of Eve^- ELs. Regardless of the exact anatomic changes, the data show that output synapses of Eve^- ELs are functional, but are functionally re-mapped to new output circuits (Marshall, 2022).

In conclusion, this study has provided an updated understanding of the role of eve expression in neurons. The data provide understanding of the role of neuronal eve at the levels of circuit physiology and animal behavior. Further they provide insight into the genetic logic of somatosensory circuit assembly, demonstrating that multiple terminal neuronal features can be coordinately regulated by the activity of a single postmitotic transcription factor. Finally, the data raises new questions about the role of eve expression in other neuron types and enable future experimental inquiry into somatosensory circuit assembly in Drosophila (Marshall, 2022).

A population of descending neurons that regulates the flight motor of Drosophila

Similar to many insect species, Drosophila melanogaster is capable of maintaining a stable flight trajectory for periods lasting up to several hours. Because aerodynamic torque is roughly proportional to the fifth power of wing length, even small asymmetries in wing size require the maintenance of subtle bilateral differences in flapping motion to maintain a stable path. Flies can even fly straight after losing half of a wing, a feat they accomplish via very large, sustained kinematic changes to both the damaged and intact wings. This study describes an unusual type of descending neuron (DNg02) that projects directly from visual output regions of the brain to the dorsal flight neuropil of the ventral nerve cord. Unlike many descending neurons, which exist as single bilateral pairs with unique morphology, there is a population of at least 15 DNg02 cell pairs with nearly identical shape. By optogenetically activating different numbers of DNg02 cells, this study demonstrated that these neurons regulate wingbeat amplitude over a wide dynamic range via a population code. Using two-photon functional imaging, it was shown that DNg02 cells are responsive to visual motion during flight in a manner that would make them well suited to continuously regulate bilateral changes in wing kinematics (Namiki, 2022).

This study describes a class of DNs in Drosophila (DNg02) that are unusual in that instead of existing as a unique bilateral pair, they constitute a large, nearly homomorphic population. By optogenetically driving different numbers of cells, it was demonstrated that DNg02 cells can regulate wingbeat amplitude over a wide dynamic range and can elicit maximum power output from the flight motor. Using two-photon functional imaging, it was also shown that at least some DNg02 cells are responsive to large field visual motion during flight in a manner that would make them well suited for continuously regulating wing motion in response to both bilaterally symmetrical and bilaterally asymmetrical patterns of optic flow (Namiki, 2022).

Compared with birds, bats, and pterosaurs-the three other groups of organisms capable of sustained active flight-a unique feature of insects is that their wings are novel structures that are not modified from prior ambulatory appendages. Insects retained the six legs of their apterogote ancestors but added two pairs of more dorsally positioned wings. This evolutionary quirk has profound consequences for the underlying neuroanatomy of the insect flight system. Within their thoracic ganglia, the sensory-motor neuropil associated with the wings constitutes a thin, dorsal layer sitting atop the larger ventral regions that control leg motion. Numerically, however, there appear to be comparable numbers of DNs targeting the wing and leg neuropils. This is a bit surprising, given the more ancient status of the leg motor system and the importance of legs in so many essential behaviors. However, the relatively large number of flight DNs may reflect the fact that the control of flight requires greater motion precision because even minute changes in wing motion have large consequences on the resulting aerodynamics (Namiki, 2022).

Straight flight in Drosophila is only possible because of the maintenance of subtle and constant bilateral differences in wing motion, carefully regulated by feedback from sensory structures such as the eyes, antennae, and halteres. The control system necessary for straight flight must permit the maintenance of very large, yet finely regulated, distortions of wing motion in order to produce perfectly balanced forces and moments. One means of controlling fine-scaled sensitivity over a large dynamic range is through the use of a population code with range fractionation, a phenomenon that bears similarity to the size principle of spinal motor neuron recruitment. The use of a population code to specify motor output is a general principle that has been observed in a wide array of species including leeches, crickets, cockroaches, and monkeys. In dragonflies, 8 pairs of DNs-a group of cells roughly comparable in number to the DNg02 cells-project to the flight neuropil and encode the direction to small visual targets. In the case of the DNg02 cells, it is hypothesized that the population activity serves to trim out rotational torques and translational forces, allowing the animal to fly straight. Although the DNg02 neurons are morphologically similar, it is strongly suspected that the population is not functionally homogeneous. To fly straight with perfect aerodynamic trim, an animal needs to zero its angular velocity about the yaw, pitch, and roll axes, in addition to regulating its forward flight speed, side slip, and elevation. Thus, if the DNg02 cells are the main means by which flies achieve flight trim, one would expect that they would be organized into several functional subpopulations, with each set of cells controlling a different degree of freedom of the flight motor system. For example, one subpopulation of DNg02 cells might be primarily responsible for regulating roll, whereas another is responsible for regulating pitch, and yet, another regulates forward thrust. Such subpopulations need not constitute exclusive sets but rather might overlap in function, collectively operating similar to a joystick to regulate flight pose. If this hypothesis is correct, it would be expected that the DNg02 neurons differ with respect to both upstream inputs from directionally tuned visual interneurons as well as downstream outputs to power and steer muscle motor neurons. Unfortunately, this study could not distinguish individual cell types across the different driver lines used at the level of light-based microscopy. If DNg02 cells are further stratified into subclasses, it is likely that each driver line targets a different mixture of cell types. Indeed, the variation observed in changes in wingbeat amplitude as a function of the number of DNg02 cells activated might reflect this variation in the exact complement of cells targeted by the different driver lines. Furthermore, although one driver line (R42B02) targets 15 DNg02 neurons, it is likely that this number underestimates the size of the entire population, and it is speculate that there may be a small set of neurons dedicated to regulating each output degree of freedom. Collectively, these results indicate that this study haa identified a critical component of the sensory-motor pathway for flight control in Drosophila, the precise organization of which is now available for further study using a combination of genetic, physiological, and connectomic approaches (Namiki, 2022).

Synaptic architecture of leg and wing motor control networks in Drosophila

Animal movement is controlled by motor neurons (MNs), which project out of the central nervous system to activate muscles. Because individual muscles may be used in many different behaviors, MN activity must be flexibly coordinated by dedicated premotor circuitry, the organization of which remains largely unknown. This study used comprehensive reconstruction of neuron anatomy and synaptic connectivity from volumetric electron microscopy (i.e., connectomics) to analyze the wiring logic of motor circuits controlling the Drosophila leg and wing. Both leg and wing premotor networks were found to be organized into modules that link MNs innervating muscles with related functions. However, the connectivity patterns within leg and wing motor modules are distinct. Leg premotor neurons exhibit proportional gradients of synaptic input onto MNs within each module, revealing a novel circuit basis for hierarchical MN recruitment. In comparison, wing premotor neurons lack proportional synaptic connectivity, which may allow muscles to be recruited in different combinations or with different relative timing. By comparing the architecture of distinct limb motor control systems within the same animal, this study identified common principles of premotor network organization and specializations that reflect the unique biomechanical constraints and evolutionary origins of leg and wing motor control (Lesser, 2023).

Single-cell RNA sequencing of motoneurons identifies regulators of synaptic wiring in Drosophila embryos

The correct wiring of neuronal circuits is one of the most complex processes in development, since axons form highly specific connections out of a vast number of possibilities. This study investigated Drosophila embryonic motoneurons using single-cell genomics, imaging, and genetics. A cell-specific combination of homeodomain transcription factors and downstream immunoglobulin domain proteins is expressed in individual cells and plays an important role in determining cell-specific connections between differentiated motoneurons and target muscles. Genetic evidence is provided for a functional role of five homeodomain transcription factors and four immunoglobulins in the neuromuscular wiring. Knockdown and ectopic expression of these homeodomain transcription factors induces cell-specific synaptic wiring defects that are partly phenocopied by genetic modulations of their immunoglobulin targets. Taken together, these data suggest that homeodomain transcription factor and immunoglobulin molecule expression could be directly linked and function as a crucial determinant of neuronal circuit structure (Velten, 2022).

The matricellular protein Drosophila CCN is required for synaptic transmission and female fertility

Within the extracellular matrix, matricellular proteins (MCPs) are dynamically expressed non-structural proteins that interact with cell surface receptors, growth factors, and proteases, as well as with structural matrix proteins. The CCN (Cellular Communication Network Factors) family of MCPs serve regulatory roles to regulate cell function and are defined by their conserved multi-modular organization. This study characterize the expression and neuronal requirement for the Drosophila CCN family member. Drosophila CCN (dCCN) is expressed in the nervous system throughout development including in subsets of monoamine-expressing neurons. dCCN-expressing abdominal ganglion neurons innervate the ovaries and uterus and the loss of dCCN results in reduced female fertility. In addition, dCCN accumulates at the synaptic cleft and is required for neurotransmission at the larval neuromuscular junction. Analyzing the function of the single Drosophila CCN family member will enhance the ability to understand how the microenvironment impacts neurotransmitter release in distinct cellular contexts and in response to activity (Garrett, 2023).

Drosophila Laser Axotomy Injury Model to Investigate RNA Repair and Splicing in Axon Regeneration

The limited axon regeneration capacity of mature neurons often leads to insufficient functional recovery after damage to the central nervous system (CNS). To promote CNS nerve repair, there is an urgent need to understand the regeneration machinery in order to develop effective clinical therapies. To this aim, this study developed a Drosophila sensory neuron injury model and the accompanying behavioral assay to examine axon regeneration competence and functional recovery after injury in the peripheral and central nervous systems. Specifically, a two-photon laser was used to induce axotomy and performed live imaging to assess axon regeneration, combined with the analysis of the thermonociceptive behavior as a readout of functional recovery. Using this model, it was found that the RNA 3'-terminal phosphate cyclase (Rtca), which acts as a regulator for RNA repair and splicing, responds to injury-induced cellular stress and impedes axon regeneration after axon breakage. How the Drosophila model is used to assess the role of Rtca during neuroregeneration is described (Wang, 2023).

Astrocyte store-operated calcium entry is required for centrally mediated neuropathic pain

Central sensitization is defined by nociceptive and somatosensory circuitry changes in the spinal cord leading to dysfunction of antinociceptive gamma-aminobutyric acid (GABA)ergic cells, amplification of ascending nociceptive signals, and hypersensitivity. Astrocytes are key mediators of the neurocircuitry changes that underlie central sensitization and neuropathic pain, and astrocytes respond to and regulate neuronal function through complex Ca (2+) signaling mechanisms. Ca (2+) release from astrocyte endoplasmic reticulum (ER) Ca (2+) stores via the inositol 1,4,5-trisphosphate receptor (IP(3)]R) is required for centrally mediated neuropathic pain; however recent evidence suggests the involvement of additional astrocyte Ca (2+) signaling mechanisms. Therefore this study investigated the role of astrocyte store-operated Ca (2+) entry (SOCE), which mediates Ca (2+) influx in response to ER Ca (2+) store depletion. Using an adult Drosophila melanogaster model of central sensitization based on thermal allodynia in response to leg amputation nerve injury, it was shown that astrocytes exhibit SOCE-dependent Ca (2+) signaling events three to four days following nerve injury. Astrocyte-specific suppression of Stim and Orai, the key mediators of SOCE Ca (2+) influx, completely inhibited the development of thermal allodynia seven days following injury, and also inhibited the loss of ventral nerve cord (VNC) GABAergic neurons that is required for central sensitization in flies. It was lastly shown that constitutive SOCE in astrocytes results in thermal allodynia even in the absence of nerve injury. These results collectively demonstrate that astrocyte SOCE is necessary and sufficient for central sensitization and development of hypersensitivity in Drosophila, adding key new understanding to the astrocyte Ca (2+) signaling mechanisms involved in chronic pain (Prokhorenko, 2023).

Identification of inhibitory premotor interneurons activated at a late phase in a motor cycle during Drosophila larval locomotion

Rhythmic motor patterns underlying many types of locomotion are thought to be produced by central pattern generators (CPGs). This study used the motor circuitry underlying crawling in larval Drosophila as a model to try to understand how segmentally coordinated rhythmic motor patterns are generated. Whereas muscles, motoneurons and sensory neurons have been well investigated in this system, far less is known about the identities and function of interneurons. A recent study identified a class of glutamatergic premotor interneurons, PMSIs (period-positive median segmental interneurons), that regulate the speed of locomotion. This study reports on the identification of a distinct class of glutamatergic premotor interneurons called Glutamatergic Ventro-Lateral Interneurons (GVLIs). Calcium imaging was used to search for interneurons that show rhythmic activity, and GVLIs were identified as interneurons showing wave-like activity during peristalsis. Paired GVLIs were present in each abdominal segment A1-A7 and locally extended an axon towards a dorsal neuropile region, where they formed GRASP-positive putative synaptic contacts with motoneurons. The interneurons expressed vesicular glutamate transporter (vGluT) and thus likely secrete glutamate, a neurotransmitter known to inhibit motoneurons. These anatomical results suggest that GVLIs are premotor interneurons that locally inhibit motoneurons in the same segment. Consistent with this, optogenetic activation of GVLIs with the red-shifted channelrhodopsin, CsChrimson ceased ongoing peristalsis in crawling larvae. Simultaneous calcium imaging of the activity of GVLIs and motoneurons showed that GVLIs' wave-like activity lagged behind that of motoneurons by several segments. Thus, GVLIs are activated when the front of a forward motor wave reaches the second or third anterior segment. It is proposed that GVLIs are part of the feedback inhibition system that terminates motor activity once the front of the motor wave proceeds to anterior segments (Itakura, 2015).

The motoneurons involved in Drosophila larval peristaltic locomotion are known to be responsive to at least three neurotransmitters, excitatory acetylcholine and inhibitory GABA and glutamate. Therefore, motoneurons likely generate rhythmic motor outputs by integrating multiple inputs. In order to clarify how interneurons contribute to the generation of motoneuronal rhythmic activity, it is essential to identify premotor interneurons and determine how they control the activity of motoneurons. This study identified GVLIs as putative premotor interneurons in this system (Itakura, 2015).

Four lines of evidence suggest that GVLIs are inhibitory premotor interneurons. First, GVLIs express vGluT, a vesicular transporter of glutamate, and thus likely secrete glutamate, a neurotransmitter known to elicit inhibitory responses in motoneurons. Second, vGluT-positive GVLI axon terminals are present in the dorsal region of the neuropile in the vicinity of motoneurons' dendrites in the same segment. Third, GVLIs form GRASP-positive putative synaptic contacts with motoneurons, although uncertainty remains as to the identity of the target motoneurons. The contact sites express the presynaptic markers Synaptotagmin and vGluT and show robust increases in calcium concentration during peristaltic waves, strongly suggesting that they are presynaptic terminals. Fourth, optogenetic activation of GVLIs inhibited motor function. Activation of GVLIs in crawling larvae disrupted ongoing peristaltic waves. Local activation of GVLIs in dissected larvae halted peristaltic waves in the corresponding region in the body wall. These results are consistent with the idea that GVLIs send inhibitory inputs locally to motoneurons. Taken together, anatomical and functional analyses strongly suggest that GVLIs are premotor local interneurons that inhibit motoneurons in the same segment. It should be noted, however, that this study has not examined whether GVLIs form synaptic connections with interneurons. Thus, it remains possible that GVLIs innervate some interneurons in addition to motoneurons. It is also important to note that axon terminals of GVLIs cover only a small portion of the dendritic region of motoneurons and thus likely innervate only a small subset of motoneurons. Considering the strong effect of GVLIs activation, GVLIs may well inhibit a large number of motoneurons via other interneurons (Itakura, 2015).

In Drosophila, several glutamate receptors (GluR) have been identified, such as metabotropic GluRs (DmGluR), AMPA/kinate receptor homologues, N-methyl-D-aspartate (NMDA) receptor homologues [56], and glutamate-gated chloride channels (GluCl). Thus Glu can have various effects on postsynaptic cells depending on the receptors expressed. For instance, Glu causes excitatory junction currents (EJCs) when released at neuromuscular junction (NMJ) and induces hyperpolarizing responses in antennal lobe neurons. Glutamate application elicits inhibitory responses in larval motoneurons. The effect is blocked by the chloride channel blocker picrotoxin, suggesting the existence of GluCl on motoneurons. Thus it is most likely that GVLIs inhibit motoneurons via GluCl. It should be noted, however, that the inhibitory effects of glutamate via GluCl has only been examined in subsets of motoneurons. It should also be noted that GVLIs may secrete other neurotransmitters in addition to Glu and/or transmit information through gap junctions. Future identification of the postsynaptic partners of GVLIs and the receptors expressed on the cells will provide more information on how GVLIs regulate the activity of downstream motoneurons (Itakura, 2015).

This study used calcium imaging to characterize the activity of GVLIs and aCCs in T3-A7 segments and the activity timing relationships among them. During forward locomotor waves, GVLIs are activated at a similar timing as are aCC neurons in the second or third more anterior neuromeres and later than aCC neurons in the same segment. The phase delay between GVLI and aCC activity remained relatively constant over wide range of wave durations. The identity of the postsynaptic motoneuron(s) of GVLIs remains to be determined. However, the axon terminals of GVLIs are located in a neuropile region occupied by dendrites of motoneurons that innervate dorsal/ventral muscles and are activated at the same timing as aCCs. GVLIs therefore are likely to be activated with a delay of 2-3 segments to their target motoneurons. It should be noted, however, the delay would be shorter if the target motoneurons are those innervating lateral muscles since they are known to be activated later than those innervating ventral/dorsal muscles (Itakura, 2015).

By studying the activity of aCCs and GVLIs during peristalsis at varying speeds, this study showed that phase delays between the two neurons remain relatively constant over a range of wave durations as in many undulatory movements spanning multiple body segments. The current results conform to a previous study that showed phase constancy based on the observation of muscle movements. The phase representation of the activation of aCCs and GVLIs, consisting of composite data derived from multiple larvae undergoing peristalsis at different speeds, well recapitulated the sequential activation from posterior to anterior segments observed in a single larva. Thus, use of the phase representation is adequate in the analyses of neural activity in this system. The phase delay data indicates that GVLIs, like motoneurons, are regulated by intersegmental networks that maintain phase constancy over different speeds of peristalsis. Although GVLIs were activated at a similar time as aCCs in the second or third anterior neuromere, they were not active at exactly the same time as aCC neurons. This suggests that upstream partners of GVLIs are different from those of motoneurons (Itakura, 2015).

The onset and termination of muscle contraction must be finely regulated to generate efficient forward movement during larval locomotion. Excitatory and inhibitory premotor neurons active at distinct phases of larval locomotion are likely to be involved in this regulation. During forward locomotion, muscles in three or more segments are simultaneously contracted at a given time. This indicates that muscle activity is shut down when the front of a muscle contraction wave reaches the third or more anterior segment. The activity pattern of GVLIs revealed by calcium imaging (phasic activation with a two-to-three segment delay compared to aCC motoneurons) is consistent with a role for GVLIs in this process. The anatomy of GVLIs is also consistent with a role in feedback inhibition: each GVLIs extend their putative dendritic processes to anterior neuromeres and their axonal processes to motoneurons in the same segment. GVLIs may thus inhibit motoneurons and help to terminate muscle contraction when the motor wave reaches the anterior segments, by integrating information from anterior segments and transmitting the signal to motoneurons in the same segment. Whether GVLIs indeed play essential roles in this process remains to be determined since functional analyses with currently available neural silencers failed to show any obvious phenotypes. It should also be noted that if GVLIs do play such a role, they should only be part of the system since their axonal terminals do not cover the entire dendritic field of motoneurons and thus likely innervate only a subset of motoneurons (Itakura, 2015).

In an independent study, another class of premotor inhibitory neurons PMSIs (period-positive median segmental interneurons) were identified. Like GVLIs, PMSIs are glutamatergic and inhibit motor function when activated, and show wave-like activity during peristalsis. However, they are activated at a different phase from that of GVLIs. They are activated much earlier than GVLIs, shortly after the activation of the postsynaptic motoneurons with a time delay of ~0.5 neuromere, and control the duration of motor bursting and the speed of locomotion. Thus, PMSIs appear to provide early-cycle inhibition that is critical for determining the duration of motor bursting. In contrast, GVLIs may contribute to late-cycle inhibition that terminates motor bursting. Future studies will elucidate how GVLI, PMSI and other premotor interneurons, active at distinct phases of a motor cycle, shape the motor pattern. For example, optogenetic activation of the interneurons can be combined with patch-clamp recordings in motoneurons to study how the activity manipulation changes the pattern of motor activity. Such analyses will pave the way for understanding how rhythm is generated during larval locomotion (Itakura, 2015).

Identification of excitatory premotor interneurons which regulate local muscle contraction during Drosophila larval locomotion

Drosophila larval locomotion was used as a model to elucidate the working principles of motor circuits. Larval locomotion is generated by rhythmic and sequential contractions of body-wall muscles from the posterior to anterior segments, which in turn are regulated by motor neurons present in the corresponding neuromeres. Motor neurons are known to receive both excitatory and inhibitory inputs, combined action of which likely regulates patterned motor activity during locomotion. Although recent studies identified candidate inhibitory premotor interneurons, the identity of premotor interneurons that provide excitatory drive to motor neurons during locomotion remains unknown. This study searched for and identified two putative excitatory premotor interneurons in this system, termed CLI1 and CLI2 (cholinergic lateral interneuron 1 and 2). These neurons were segmentally arrayed and activated sequentially from the posterior to anterior segments during peristalsis. Consistent with their being excitatory premotor interneurons, the CLIs formed (GFP Reconstruction Across Synaptic Partners) (GRASP)- and ChAT-positive putative synapses with motoneurons and were active just prior to motoneuronal firing in each segment. Moreover, local activation of CLI1s induced contraction of muscles in the corresponding body segments. Taken together, these results suggest that the CLIs directly activate motoneurons sequentially along the segments during larval locomotion (Hasegawa, 2016).

Animals perform various types of rhythmic movements such as respiration, chewing and locomotion for their survival. These rhythmic movements are thought to be regulated by neuronal circuits termed central pattern generators (CPGs). CPGs consist of interneurons and motoneurons whose rhythmic activities induce coordinated patterns of muscle contraction. Although CPGs are regulated by descending and sensory inputs, rhythms very similar to those seen in the intact animal can be generated without these inputs. Because CPGs of invertebrates and vertebrates share many characteristics, CPGs in one animal could be a model for other animals. Moreover, because CPGs show many characteristics common to other neuronal systems, CPGs could be a general model linking neuronal circuits to behaviour. Despite the efforts to elucidate the function of CPGs, their identities and functional mechanisms are not completely understood, in particular in animals with a large central nervous system (CNS). This is partly because manipulating the function of specific neurons in the neural circuits is often difficult, especially in animals with vast numbers of neurons such as mammals (Hasegawa, 2016).

The Drosophila larva is emerging as an excellent model system for studies of CPGs because one can use sophisticated genetic methods, such as the Gal4-UAS system, to manipulate and visualize the activity of specific component neurons in a moderately sized CNS consisting of ~10,000 neurons. Larval forward locomotion is executed by the sequential contraction of muscles from the posterior to the anterior segments. Motoneurons in the ventral nerve cord (VNC) actualize the sequential muscle contraction by being activated from the posterior to the anterior segments during forward locomotion. CPGs responsible for the locomotion seem to be present in the VNC, since neuronal circuits in the thoracic and abdominal segments have been shown to be sufficient for generating the behavior (Berni, 2012; Berni, 2015). Calcium imaging of the entire CNS has visualized neurons that are active during larval locomotion including those in the brain, sub-oesophageal zone (SEZ), and the VNC16. However, the identities of these neurons are only beginning to be characterized (Hasegawa, 2016).

Previous studies showed that motor neurons in the VNC receive both excitatory and inhibitory inputs. It is therefore likely that specific patterns of motoneuron activation are regulated by the balance and the timing of excitatory and inhibitory inputs as shown in other systems. Recently, two types of inhibitory premotor interneurons that regulate larval locomotion have been identified. PMSIs (period-positive median segmental interneurons) are glutamatergic inhibitory premotor interneurons that regulate the speed of larval locomotion (Kohsaka, 2014). Another glutamatergic interneuron, GVLIs (glutamatergic ventro-lateral interneurons) seem to function as premotor inhibitory neurons to terminate motor bursting (Itakura, 2015). In contrast, premotor interneurons that provide excitatory inputs to motor neurons during locomotion remain to be identified, although they are known to be cholinergic. A recent study identified two cholinergic descending interneurons that form putative synaptic contacts with segmental motoneurons (Couton, 2015). However, whether they are active and play roles during locomotion remains unknown (Hasegawa, 2016).

This study sought and identified putative excitatory premotor interneurons that activate motoneurons during locomotion. These neurons, termed CLI1 and CLI2 (cholinergic lateral interneuron), are segmental interneurons that show wave-like activity during locomotion concurrent with the activity propagation of motoneurons. Consistent with CLIs being excitatory premotor neurons, these neurons form GRASP- and ChAT-positive synaptic contacts with motor neurons and are activated just before the activation of motoneurons in each segment. In addition, forced activation of these neurons locally induces the contraction of muscles. These results suggest that wave-like activity of CLIs activates motoneurons sequentially along the segments during forward locomotion (Hasegawa, 2016).

What are the circuit mechanisms that regulate Drosophila larval locomotion? To answer this question, it is necessary first to identify the neuronal components of the circuits. Excitatory inputs are critical for the generation of locomotor rhythms in various animals. However, identities and roles of excitatory interneurons that regulate Drosophila larval locomotion are unknown. The present study sought such excitatory interneurons using calcium imaging and identified CLI1s and CLI2s as candidate interneurons that excite motor neurons. Anatomical and behavioural studies suggest that these neurons directly activate motoneurons locally in each segment during larval locomotion (Hasegawa, 2016).

The following four lines of evidence suggest that CLIs are excitatory premotor interneurons: (i) CLIs are activated just before the activation of motoneurons in each segment during fictive locomotion, consistent with their providing excitatory drive to motoneurons. (ii) CLIs express ChAT, which synthesizes acetylcholine, a neurotransmitter known to excite motor neurons in this system. (iii) CLIs form GRASP-positive contacts with motoneurons. (iv) Local activation of CLIs results in the contraction of muscles in the corresponding body segments. Although these data are consistent with direct connection between CLIs and motoneurons, it remains possible that CLIs also excite motor neurons indirectly via other interneurons (Hasegawa, 2016).

CLI1s and CLI2s share many morphological and functional characteristics. i) They are neighboring neurons that send axons along a common path to reach the neuropile. This suggests that they are sibling neurons derived from the same neuroblast. Consistent with this notion, they also share the expression of R47E12-Gal4. ii) They both project axons along the same fascicle in the anterior commissure and locally innervate motor neurons in the contralateral side of the CNS. iii) They both are cholinergic premotor interneurons and are activated simultaneously during forward locomotion. iv) Activation of these neurons elicits muscle contraction. Taken together, these observations suggest that CLI1s and CLI2s belong to a class of interneurons that fulfill common function(s). There are also distinct features between these two neurons. i) CLI1s innervate the medial neuropile while CLI2s innervate a lateral region, suggesting that they target distinct neurons. ii) CLI1s but not CLI2s project to the next anterior segment. iii) CLI2s are active both during forward and backward locomotion, whereas CLI1s are active only during forward locomotion. Thus, CLI1s only participate in forward locomotion and may activate motor neurons not only in the same segment but also in the next anterior segment, and thus contribute to feed-forward propagation of motor excitation. In contrast, CLI2s may act locally to excite motoneurons only in the same segment and do so both during forward and backward locomotion (Hasegawa, 2016).

It is currently unknown what motor neurons are the targets of CLI1/2s. Dendrites of motoneurons that innervate different muscle domains form myotopic map along both antero-posterior and medio-lateral axes. The axon terminals of CLI1s are located in the medial neuropile, a region occupied by the dendrites of motoneurons innervating ventral muscles. Thus, CLI1s may form synaptic contacts with the ventral motoneurons. Similarly, candidate targets of CLI2s are dorsal motoneurons, since axon terminals of CLI2s are located in a lateral region occupied by these motoneurons. Consistent with this, it was observed that lifting of the tail, which is likely caused by dorsal muscle contraction when CLI2s but not CLI1s is activated. Moreover, CLI1s and CLI2s are activated at a similar timing as aCC in the same segment, a motor neuron that innervates a dorsal muscle and is activated simultaneously with other motor neurons innervating dorsal/ventral internal muscles. Future studies such as connectomic analyses using serial EM will determine more precisely the downstream circuits of the CLIs (Hasegawa, 2016).

It is also important to determine in the future the upstream circuits of the CLIs. Since dendritic region of CLI1s and CLI2s partially overlap, these neurons may share common upstream neurons. In particular, because the wave-like activity of CLIs was observed in the isolated CNS that receives no sensory inputs, the activity of CLIs must be regulated by the central circuits that generate a rhythm in an autonomous manner. However, it is also possible that CLIs are activated in response to specific sensory stimulation. Recently, neuronal circuits regulating larval behavior in response to specific sensory stimuli have been identified. It will be interesting to study the link between these circuits and CLIs (Hasegawa, 2016).

The wave-like activity of CLIs that occurs concomitant with motor activation strongly suggests that these neurons contribute to sequential activation of motor neurons along the segments during locomotion. Since these neurons are commissural neurons, they may also play a role in left-right coordination, as has been proposed for Dbx1-positive neurons in vertebrates and recently identified EL neurons in Drosophila. However, loss-of-function analyses thus far failed to reveal roles of CLIs in larval behaviors. Shibire^ts, tetanus toxin light chain, Kir2.1, hid and reaper, and ChAT-RNAi were used to inhibit the function of CLIs but no obvious phenotypes were observed. This could be due to insufficient silencing of these neurons by the activity manipulations. It could also be due to the redundancy in the circuit function. It should be noted in this regard that there are likely more CLIs-like neurons present in each segment. The axon terminals of CLI1 and CLI2 only cover part of the motor dendritic region, suggesting other neurons excite motor neurons not targeted by CLIs. Indeed, preliminary results obtained by the ongoing EM reconstruction of the larval CNS suggest that about 10 neurons, in the same neuroblast lineage as CLIs, send their axons locally and contralaterally to the motor region along the common path as CLI1s and CLI2s. It is likely that a group of CLIs-like neurons function in a similar manner and together excite the entire motor system. Unfortunately, direct testing of this possibility is not currently feasible due to the unavailability of Gal4 lines specific to this lineage (Hasegawa, 2016).

Recently, research has identified two classes of segmental premotor inhibitory interneurons PMSIs and GVLIs. These neurons are activated slightly later than the motor neurons and appear to inhibit the activity of motoneurons at distinct timings during the motor cycle: PMSIs at an early phase and GVLIs at a final phase of motoneuronal activation (Kohsaka, 2014; Itakura, 2015). This study identified CLIs that are activated prior to motor neurons and appear to provide an excitatory drive to the motoneurons. These three classes of premotor interneurons likely help shape the pattern of motor activity by providing excitatory and inhibitory inputs to motoneurons at distinct phases of the motor cycle. Since there are only ~400 interneurons per hemisegment in the larval ventral nerve cord, whose connectivity is being reconstructed, it is hoped that all major classes of premotor interneurons in this system will be identified in the near future. Systematic analyses of CLIs, PMSIs, GVLIs and other premotor neurons will elucidate how the motor patterns generating distinct behaviors are shaped by the combinatorial action of premotor interneurons (Hasegawa, 2016).

Sequential addition of neuronal stem cell temporal cohorts generates a feed-forward circuit in the Drosophila larval nerve cord

How circuits self-assemble starting from neuronal stem cells is a fundamental question in developmental neurobiology. This study addressed how neurons from different stem cell lineages wire with each other to form a specific circuit motif. In Drosophila larvae, developmental genetics (Twin spot MARCM, Multi-color Flip Out, permanent labeling) was combined with circuit analysis (calcium imaging, connectomics, network science). For many lineages, neuronal progeny are organized into subunits called temporal cohorts. Temporal cohorts are subsets of neurons born within a tight time window that have shared circuit level function. Sharp transitions in patterns of input connectivity were found at temporal cohort boundaries. In addition, a feed-forward circuit was identified that encodes the onset of vibration stimuli. This feed-forward circuit is assembled by preferential connectivity between temporal cohorts from different lineages. Connectivity does not follow the often-cited early-to-early, late-to-late model. Instead, the circuit is formed by sequential addition of temporal cohorts from different lineages, with circuit output neurons born before circuit input neurons. Further, this study generated new tools for the fly community. These data raise the possibility that sequential addition of neurons (with outputs oldest and inputs youngest) could be one fundamental strategy for assembling feed-forward circuits (Wang, 2022).

This study addressed two questions about the stem cell-based assembly of neuronal circuits. First, what is the relationship between neuronal birth order within a lineage and patterns of synaptic connectivity at the single-neuron level? Second, how do neurons from different lineages wire with each other? This study characterized the birth order, morphology, and input connectivity of all neurons in the NB3-3A1L/R lineage at single-neuron and single-synapse resolution. A feed-forward circuit was identified that encodes the onset of vibrational stimuli. And, for a majority of nerve cord interneurons within this circuit, their stem cell parent, birth order within their lineage, and birth timing relative to each other were identified. Together, this identifies four temporal cohorts, all of which have sharp connectivity boundaries. For most, but not all, there is inter-segmental connectivity between segmentally homologous temporal cohorts (e.g., early-born ELs in other segments connect to early-born ELs in A1). Further, neurons of different temporal cohorts from different lineages assemble sequentially, with circuit output neurons born before circuit input neurons (Wang, 2022).

The Drosophila larval connectome is a resource that can be used to understand circuit assembly. However, because the connectome is an anatomical dataset, a major challenge is to develop approaches that connect anatomy to development. In this article, several approaches were developed. For example, ts-MARCM was optimized for use in Drosophila embryos. ts-MARCM is the gold standard for determining birth order in Drosophila. However, for technical reasons, ts-MARCM has been used only in adults. This study discovered ts-MARCM clones can be robustly generated in early stage larvae with the addition of an amplifying and immortalizing gene cassette. In addition, several recently developed methods for inferring developmental origin were independently validated based on anatomical features in the Drosophila larval connectome. Specifically, the validations include use of neurite bundles as a proxy for lineage-relatedness and use of cortex neurite length as a rough proxy for birth order within a lineage. Finally, network science methods (distance analysis) were adapted to characterize the patterns of connectivity in connectome data. These approaches should be useful for Drosophila neurobiologists and beyond (Wang, 2022).

This study also developed NB3-3A1L/R as the first entire lineage for which birth order, morphology, and input connectivity is known at single neuron and synapse precision. One reason this is important is because NB3-3 has been extensively studied in embryos and much is known about molecular marker expression of NB3-3 progeny at the single-neuron level. Because the current dataset achieves cellular resolution, good guesses about the embryonic molecular-larval morphological pairings can be made using single-neuron birth order as a point of cross-reference. Such integrated data generates detailed and testable predictions. For example, the data predict that Castor expression in the late-born ELs promotes projection neuron morphology. Additionally, this study generated a new NB3-3-GAL4 line that can be used to manipulate gene expression in NB3-3. Thus, NB3-3A1L/R is a model lineage in which transcription factor expression in neuronal stem cells can be linked to circuit assembly and tested for function, and this study has generated tools that will enable hypothesis testing in the future (Wang, 2022).

A circuit motif is a pattern of synaptic connections between a set of specific neuron types that can be found across brain areas and across species. Circuit motifs have been suggested to represent the physical substrates of 'computational primitives'. There a are small number of fundamental, recurring circuit motifs (e.g., feed-forward, feedback, lateral inhibition, etc.). And so, a new conceptual approach in this article is to ask how are specific circuit motifs assembled during development. This study identified a new feed-forward circuit motif. Generally, feed-forward motifs are characterized as a pattern of connectivity in which one neuron (or neuron type) provides both direct and indirect input onto a second neuron (or neuron type). Feed-forward circuit motifs are common, found in many animals (e.g., nematodes, insects, mouse) and in many brain regions (e.g., somatosensory systems, olfactory systems, neocortex). Thus, feed-forward motifs are fundamental to neural signal processing. Feed-forward motifs can be further subdivided into feed-forward inhibitory or feed-forward excitatory circuit motifs, depending on the transmitter types of the neurons involved. Notably, anatomically the pattern of synaptic connectivity between neurons is the same in either motif subtype, and so this study does not distinguish between the two (Wang, 2022).

This study found that, in general, early-born ELs get direct excitatory synapses from CHOs and indirect (excitatory or inhibitory) input from chordotonals via Basins and Ladder interneurons (see Early-born ELs are embedded in a feed-forward motif and encode the onset of vibrational stimuli). Notably, there are differences in connectivity patterns between early-born ELs. For example, although all early-born ELs get direct input from chordotonals and Ladders, A08j1-3s get left-right symmetrical inputs, whereas A08x and A08m get asymmetrical inputs. This could correspond to functional differences. For example, A08x and A08m may be involved in left-right asymmetrical signal detection. Teasing apart the diversity of computations performed by each individual EL interneurons is the domain of future studies (Wang, 2022).

Because this study characterized the development of a feed-forward circuit in Drosophila, information is available that allows searching for similar circuits in other insect species. For example, in Drosophila, ELs express the transcription factor, Even-skipped (Eve). In Locust and other insects, lateral interneurons also express Eve. In Drosophila, MNB generates H-shaped, inhibitory interneurons (Ladders), which get input from sound/vibration sensitive, CHOs. In Locust, MNB generates H-shaped, inhibitory interneurons, which encode sound stimuli. Further, in both Drosophila and Locusts, MNB interneuron progeny express the transcription factor Engrailed. Thus, the neuronal components of the Ladder to early-born EL circuit are conserved, which raises the possibility of circuit-level conservation (Wang, 2022).

A major unanswered question in developmental neuroscience is how the mechanisms that generate neuronal diversity contribute to the formation of functional neuronal circuits. Part of the answer lies in the observation that temporal cohorts are subunits of lineages, linked both to larval circuit anatomy/function as well as to the embryonic gene expression programs that generate neuronal diversity. Thus far, temporal cohorts had been looked for and found in 8 out of 30 lineages in the nerve cord. This study identified three additional temporal cohorts-Basins, Ladders, and Notch OFF NB7-1 interneurons-in three lineages-NB3-5, MNB, and NB7-1, bringing the number to 11. This underscores the idea that temporal cohorts are common (Wang, 2022).

One open question about temporal cohorts was to what extent are temporal cohort borders associated with sharp changes in connectivity. Previous studies had identified temporal cohorts and linked them with function and connectivity. However, these studies lacked the resolution to distinguish between a 'graded' or 'sharp' wiring transition models. This study identified four temporal cohorts (early-born ELs, late-born ELs, Basins, Ladders) in three lineages (NB3-3, NB3-5, MNB), all of which have sharp changes in connectivity correlated with temporal cohort borders. For the Basin temporal cohort, it is noted that Basins have similar connectivity patterns with two additional NB3-5 progeny, 'Down and Back' and 'Crescent' neurons. Crescent is adjacent to Basins in birth order, whereas Down and Back is born much earlier. From this, two things were learned: (1) there can be significantly similar connectivity between neurons in one hemilineage that have nonadjacent birth times. (2) There can be significantly similar connectivity between neurons with adjacent birth times, but of different morphological classes. Another example of morphological variants with similar connectivity are late-born ELs, which can be either local or projection neurons. This underscores the idea that temporal cohorts are subunits of hemilineages defined by birth within a tight time window, rather than defined by similar neuronal morphology per se. Further, it is noted that within a temporal cohort, sequentially born neurons are often, but not always, the most similar in terms of connectivity. For example, within the early-born EL temporal cohort, the fifth-born neuron (A08m) is more similar to the first-born neuron (A08x) than its temporal neighbor (A08j2), the fourth-born EL. Thus, the data, combined with previous studies, show how temporal cohorts are developmental units related to circuits both at the functional and anatomical levels (Wang, 2022).

A second open question about temporal cohorts was the extent to which they are copies of each other. One reason this is an interesting question relates to circuit evolution. For the evolution of gene function, a popular model is a 'duplicate and diverge' model. Similarly, temporal cohorts could be duplications within a lineage whose function could then diverge, thereby driving circuit evolution. Such an idea motivated asking the question to what extent are temporal cohort copies. For example, do early-born ELs process chordotonal stimuli in a manner identical to how late-born ELs process proprioceptive stimuli? These data suggest a more complex picture. Early-born and late-born ELs differ in their connectivity patterns-including left-right and following pair similarities and inter-and intra-segmental connectivity patterns, suggesting that early-born and late-born ELs are likely to process information differently. Independent of the evolutionary implications, the data reveal previously unknown diversity in the structure of connectivity between neurons within adjacent temporal cohorts, which may indicate a diversity in underlying circuit assembly mechanisms (Wang, 2022).

A third open question is what sets up the borders of temporal cohorts. Previous work used motor neuron temporal cohorts of NB7-1 and NB3-1 as a model to address this question. In these stem cells, mis-expression of temporal transcription factors, Hunchback, Pdm, and Castor, modulates the number of motor neurons in a temporal cohort, without changing the size of the lineage. Thus, temporal transcription factors are able to regulate motor neuron temporal cohort borders. But it remains unclear how temporal transcription factors do so. Two, not mutually exclusive possibilities are that they act as transcriptional co-factors to induce differential gene expression programs and/or that they act as pioneer factors to alter the chromatin landscape. For NB3-3, it is noted that during the 7th to 11th divisions, which generate late-born ELs, NB3-3 expresses Grainyhead, which raises the possibility that Grainyhead may define the late-born EL cohort. Testing this idea is an important future direction (Wang, 2022).

It has been hypothesized that nerve cord circuits are assembled by preferential connectivity between distinct temporal cohorts. The current data provide experimental support for this hypothesis. Specifically, this study found that interneurons from three temporal cohorts wire together to form a feed-forward circuit-early-born ELs from NB3-3, mid-to-late-born Basins from NB3-5, and late-born Ladders from MNB. Although a vast majority of both the total synaptic input and the strongly connected individual neurons come from these lineages, there are also neurons from other lineages that synapse on early-born ELs (Wang, 2022).

Notably, one other study provided limited supported for the hypothesis that Drosophila nerve cord circuits are assembled by preferential connectivity between distinct temporal cohorts (Mark, 2021). This study focused on the Jaam-to-late-born EL-to-Saaghi circuit (Heckscher, 2015). Jaams are later-born interneurons in the NB5-2, Notch OFF hemilineage, and Saaghis are later-born interneurons in the NB5-2 Notch ON hemilineage (Mark, 2021). These data raised several possibilities: (1) there could be global alignment between lineages (e.g., all NB3-3 neurons get synapses from NB5-2 neurons). (2) Notch ON/OFF pairs of neurons might be pre-/postsynaptic partners of neurons within a temporal cohort. (3) Birth order-matched temporal cohorts from different lineages might selectively wire together (e.g., early-to-early and late-to-late connectivity). However, the current data demonstrate that none of these possibilities are generally true. Instead, a diversity was found in the manner in which temporal cohorts associate. The one consistent theme is that a limited number of temporal cohorts highly interconnect (Wang, 2022).

For most of this discussion, neurons were labelled as 'early-born' and 'late-born.' These labels refer to the birth order of neurons within a lineage. However, these labels do not refer to the absolute time at which a neuron is born. This is because in the Drosophila nerve cord neuroblasts are generated over a large span of embryogenesis. Thus, early-born neurons from one lineage can be generated at the same time as later-born neurons from a different lineage. This study is unique in that it determined both neuronal birth order and birth time (Wang, 2022).

One unexpected findings is that circuit outputs in one lineage are most often born before circuit inputs from other lineages. Broadly speaking, nerve cord and spinal cord contain many local circuits, which output to the brain (e.g., circuits processing somatosensory stimuli) or which output to muscles (e.g., circuits that generate motor patterns). Here, early-born ELs are the outputs of a somatosensory processing circuit and transmit information to the brain. Early-born ELs are born before their local, nerve cord inputs-Ladders, Basins, NB7-1 Notch OFF interneurons. However, from a first principles perspective, the opposite would be expected. This is because 22 of 30 neuroblasts in the nerve cord are born before NB3-3, and many of them divide multiple times to produce neurons before NB3-3 begins to generate early-born neurons. Therefore, there should be more neurons born before early-born ELs compared to those born after early-born ELs. And so, by chance alone one would expect early-born ELs to get more input from neurons born earlier or at the same time (Wang, 2022).

What are the hypotheses about the importance of birth order of output versus input neurons? In Drosophila, birth order is linked to two things: (1) lineage-intrinsic factors such as dynamically changing programs of gene expression in the stem cell, and (2) lineage-extrinsic factors or the dynamic environmental context into which neurons are born. Intrinsic factors, or extrinsic factors, or both may be playing a role in the assembly of this feed-forward circuit. A potential intrinsic mechanism is that of a temporal transcription factor 'matching code,' in which early-born ELs, Ladders, and Basins would all be derived from the same temporal transcription factor window. For NB3-5 and MNB, temporal transcription factor expression is only partially characterized. But tantalizingly both early-born ELs and a subset of Ladders are born during a period in which their respective neuroblasts express the temporal transcription factor, Castor. A potential extrinsic mechanism is that early-born EL dendrites may provide some type of signal that promotes later born neurons to synapse. There is evidence for such communication among Drosophila nerve cord neurons. Finally, it will be interesting to understand if sequential assembly is an absolute requirement, or if instead it facilitates rapid, efficient, or robust circuit assembly (Wang, 2022).

Studies of circuit assembly are still in their infancy. It is known that lineage-circuit relationships differ depending on circuit anatomy. But the converse is not known. That is, do all circuits of the same anatomy have a common lineage-circuit relationship? In the case of this study, focus was place on a single feed-forward circuit in the Drosophila larval nerve cord. And the specific question it raises is: To what extent is sequential addition of temporal cohorts from different lineages the only mechanism used to assembly all feed-forward motifs? Currently, the current answers are only partial and speculative. It is noted that there are so many connections made among neurons, it is unlikely that just one simple phenomenon that can explain the full complexity. The goals of the current research must be to identify rules and the circumstances where those rules apply. For example, these data rule out a 'strict' early-to-early, late-to-late model, meaning that this model alone cannot explain the wiring observe in this circuit. And yet, an early-to-early model does apply to inter-segmental wiring among temporal cohorts of the same lineage. Further, this early-to-early wiring phenomenon occurs alongside a sequential addition phenomenon. It speculate there could be additional phenomena underlying assembly of this simple motif. For example, beyond local interneurons, the birth date of sensory neurons was not determined, which provide the initial input to the circuit, nor did this study investigate the developmental origins of central brain neurons, which receive the output from the circuit. It is noted there is some evidence to support the generality that for assembly of feed-forward circuit motifs, presynaptic interneurons are born after their postsynaptic partners. Specifically, motor neurons are always circuit outputs (to muscle), and, in general, they are among the first neurons to be born during neurogenesis. Moreover, this pattern holds true in both Drosophila nerve cord and spinal cord. Therefore, the current data raise the possibility that one of many fundamental rules for circuit assembly is that feed-forward circuits are assembled sequentially from circuit output to circuit input (Wang, 2022).

Topological and modality-specific representation of somatosensory information in the fly brain

Insects and mammals share similarities of neural organization underlying the perception of odors, taste, vision, sound, and gravity. This study observed that insect somatosensation also corresponds to that of mammals. In Drosophila, the projections of all the somatosensory neuron types to the insect's equivalent of the spinal cord segregated into modality-specific layers comparable to those in mammals. Some sensory neurons innervate the ventral brain directly to form modality-specific and topological somatosensory maps. Ascending interneurons with dendrites in matching layers of the nerve cord send axons that converge to respective brain regions. Pathways arising from leg somatosensory neurons encode distinct qualities of leg movement information and play different roles in ground detection. Establishment of the ground pattern and genetic tools for neuronal manipulation should provide the basis for elucidating the mechanisms underlying somatosensation (Tsubouchi, 2017).

Only three distinct types of sensory information are transmitted directly to the brain by primary neurons [leg gustatory sensilla (gs), chordotonal organs (co), and wing and haltere campaniform sensilla (cs)]. Such connection has also been reported in other insects, suggesting that this might be a general feature across insecta. Whereas only a small portion of leg co neurons project directly to the brain, most wing and haltere cs neurons innervate the brain; these cs neurons are known to detect various aspects of wing-beat force during flight to provide feedback control. Direct projections to the brain would be important for these neurons to enable fast transmission of information about rapidly changing sensory parameters during flight (Tsubouchi, 2017).

It was found that ground detection for wind-induced suppression of locomotion (WISL), which would require slower temporal resolution than flight control, is mediated by both direct and indirect pathways. Primary neurons and secondary interneurons of the same sensory modality tend to converge in specific subregions of the brain, forming modality-specific somatosensory representation. In spite of the similar axon trajectory in the brain, these neurons convey information about leg movement in different ways (Tsubouchi, 2017).

Interneurons associated with the leg co and es terminate in neighboring but different regions of the lateral brain, yet some of them have shared roles in WISL control. Because their signals are transmitted to distinct parts of the brain, yet-unidentified higher-order neurons in the brain should converge those signals to the motor control circuitry (Tsubouchi, 2017).

In this respect, it is important to note that most ascending secondary interneurons identified in this study have presynaptic output sites, not only in the brain but also in the VNC. Local circuitry in the leg neuropil is important for controlling leg movement. Those local neurons are likely candidates that receive output from the ascending interneurons, because axon terminals of sensory neurons hardly have postsynaptic sites. Similar local output has also been found in other sensory modalities; many olfactory and visual projection interneurons have collateral output synapses in the antennal lobe and optic lobes (Tsubouchi, 2017).

There are three pairs of leg neuropils. Among them, the foreleg neuropil has specialized arborization of the gs neurons that exist only in the foreleg. Other than this, no substantial differences of arborization patterns were found between the fore-, mid-, and hindleg neuropils (Tsubouchi, 2017).

The present results provide data for a systematic comparison of the insect somatosensory system with its mammalian counterparts. Insects and mammals share similarities of neural organization underlying the perception of odors, taste, vision, sound, and gravity, and the current data also reveal marked similarity for the mechanosensory system. In insects, some primary neurons project directly to distinct parts of the ventral and lateral brain, whereas others terminate within the VNC. Likewise, in mammals, some neurons project directly to the ventral brain at the medulla oblongata, whereas others terminate within the spinal cord. Modality-specific pathways tend to converge in different subregions of the medulla, as well as in the thalamus of the mammalian brain. Similarly, direct and indirect pathways tend to converge in common subregions of the insect brain, and neurons conveying information about different somatosensory modalities tend to terminate in different subregions. As in mammals, these subregions often lie adjacent to each other in certain parts of the brain; for example, the entire terminal arborizations of the leg co and es secondary interneurons are confined in a 40-μm-wide, 150-μm-tall cylindrical volume in the lateral brain (Tsubouchi, 2017).

Somatosensory signals are sent predominantly to the ipsilateral brain side in insects and contralateral in mammals. Considering that descending neurons tend to project ipsilaterally in insects but contralaterally in mammals, however, somatosensory signals and motor control computation are processed primarily in the same side of the brain in both cases (Tsubouchi, 2017).

Layers of sensory axon terminals in the insect VNC and mammalian spinal cord are also organized in a similar order. Insect multidendritic neurons and mammalian free nerve endings share various characteristics in common: Their dendrites both have free endings without forming particular sense organs to detect pain, temperature, and other submodalities. The md neurons project to the most ventral layer of the VNC, whereas free nerve endings innervate the most dorsal layer of the spinal cord. Axons from the insect external sensilla and mammalian hair receptors, both of which detect haptic contact to the tips of the bristles and hairs, terminate in the second-ventral and second-dorsal layers, respectively. Insect chordotonal organ and mammalian muscle spindle, as well as insect campaniform sensilla and mammalian Golgi tendon organ, also show similarity with respect to their functions in motor control. These receptor systems supply afferents to the most dorsal and most ventral layers in insects and mammals, respectively. A fly's stretch receptors and mammalian Merkel cell neurites-as well as Meissner, Ruffini, and Pacinian corpuscles-terminate in the third-ventral and third-dorsal layer, respectively. Although correspondence between them is less obvious, they similarly detect deformation of the exoskeleton and skin. Thus, functionally comparable somatosensory terminals are layered in reverse order between the two systems. Considering that the dorsoventral axis of the mammalian body is developmentally upside down compared with the insect one, the corresponding order of sensory arrangements is actually conserved exactly between the two systems (Tsubouchi, 2017).

Do corresponding somatosensory cell types express common genes? Modality-dependent molecular specialization is not apparent even within insects or mammals, because the same genes are often expressed in multiple cell types and only a few genes share expression in the corresponding cell types across taxa. This might be a rather general feature; receptor molecules as well as developmental origins of the sensory organs are not identical between insects and mammals also in olfactory and auditory systems, yet sensory centers in the brain share architectural similarities (Tsubouchi, 2017).

With this somatosensory analysis, transphyletic correspondence of neuronal circuitry has been found in all of the sensory modalities. Corresponding organization has been suggested also for associative centers and motor systems. The fact that essentially all important components of the brain system share conserved features across the two evolutionary clades, which have been separated since at least the end of the Ediacaran period more than 550 million years ago, would suggest that basic development programs for the orderly and secrete segregation of those circuits may have evolved before deuterostome-protostome or deuterostomia-ecdysozoa divergence (Tsubouchi, 2017).

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).

This study discovered a circuit whose structure and function provides a mechanism for understanding forward wave propagation in peristaltic locomotion. This circuit consists of a chain of alternating excitatory and inhibitory neurons spanning all abdominal segments. The core elements of the chain include just one excitatory and one inhibitory neuron per hemisegment. The inhibitory neuron (GDL) is demonstrated to be sufficient to halt the peristalsis and to relax muscles in all segments, suggesting it is a point of coordination between forward and backward locomotion. It was further demonstrated that the excitatory neuron (A27h) is active during forward but not backward peristalsis, suggesting the existence of another excitatory circuit component critical for backward peristalsis among the synaptic partners of the GDL inhibitory neuron. This circuit defines a backbone of repeating, connected, modules for excitation and inhibition similar to those postulated in a computational model for peristalsis on the basis of behavioral observations that predicted the existence of central pattern generators (Fushiki, 2016).

This study found that the excitatory neuron (A27h) is premotor, directly synapsing onto motor neurons of its own segment only and that control both dorsal and ventral longitudinal muscles. This suggests an explanation for the observation that in forward crawling, dorsal and ventral longitudinal muscles contract simultaneously. In backward peristalsis, however, a phase gap has been observed in the timing of dorsal and ventral muscle contraction. This decoupling could require a more complex circuit structure for backward wave propagation, and therefore suggests an explanation for the lack of an equivalent excitatory neuron in the circuit chain for backward peristalsis. This study found, however, neurons postsynaptic to the inhibitory neuron (GDL) whose anatomy and position in the circuit suggest a role in backward peristalsis. In contrast, the inhibitory neuron (GDL) itself does not synapse onto motor neurons, and therefore occupies a higher-order position in the circuit that allows its participation in both forward and backward wave propagation in peristalsis. Furthermore, the GDL axon targets the intermediate lateral neuropil, which is neither in the domain of motor neuron dendrites nor in the somatosensory domain, suggestive of a role higher-order motor coordination. Relevant for forward peristalsis, GDL disinhibits the excitation of its anterior homologs, by removing inhibition from a glutamatergic interneurons (A02j) implicated in the regulation of peristaltic speed (one of the PMSIs). A02j is presynaptic to GDLs in anterior segments (Fushiki, 2016).

A model of peristaltic locomotion must consider the coordination of left and right hemisegments. Though this study found that the chain of alternating inhibitory and excitatory neurons runs independently on the left and right sides of the body, the excitatory neuron (A27h) presents a bilateral arbor and drives motor neurons bilaterally. The wiring diagram best supports a model of left-right coordination where excitatory neurons communicate with each other, but with the caveat that this synergy takes place by the simultaneous co-activation of the target motor neurons rather than reciprocal excitation. This model has been shown to support longer contraction episodes at the front of the wave, consistent with observations of muscle contraction in peristalsis. Independently of the timing, the fine-tuning in the intensity of left-right contractions has been shown to be under control of Even-skipped+ evolutionarily conserved neurons, which integrate both proprioceptive inputs and motor commands (Fushiki, 2016).

The dissected larval CNS undergoes spontaneous waves of motor neuron activation at about 1/10th the normal speed. These waves occur in the absence of sensory feedback, indicating the presence of CPGs and also suggesting a role for sensory feedback in speeding up the peristaltic wave. The circuit chain of excitatory and inhibitory neurons described in this study could be a part of the CPG, and this study additionally found these neurons are modulated by proprioceptive inputs (from vpda class I dendritic arborization neuron). Given that the vpda is a stretch receptor, it would be active in the segment ahead of the wave of contraction, which is being stretched by the pull exerted by the contracting segment. Proprioceptive feedback action onto the excitatory neuron of the circuit chain could then have two simultaneous effects: promotion of the contraction in the segment ahead of the wave (via activation of A27h), and relaxation of the segment twice removed (via activation of GDL, which acts on the segment anterior to it). Two somatosensory neurons (vdaA and vdaC) were found to synapse axo-dendritically onto the premotor excitatory neuron (A27h) and axo-axonically onto the inhibitory neuron (GDL) in their own segment. Although the function of these two sensory neurons remains unclear, it is speculated that this axo-axonic, likely depolarizing, connection onto GDL reduces the membrane action potential of its axon, reducing synaptic release of GABA onto A27h in the same segment. This model refines a previous model where the proprioceptive feedback was thought to signal the successful contraction of a segment. It is suggested that, in addition, at least some of the proprioceptive feedback (vpda) facilitates wave propagation and, therefore, may underlie the reduction in speed observed in fictive crawling (Fushiki, 2016).

In addition to the excitatory premotor interneuron A27h, this study found two other interneurons that receive direct synaptic inputs from a GDL (A02d and A08e3) and that, like A27h, also integrate inputs from stretch receptors (vpda, dbd and vbd). One interneuron (A08e3) is an Even-Skipped+ neuron that maintains left-right symmetric muscle contraction amplitude (Heckscher, 2015). The other (A02d) is a glutamatergic interneuron that belongs to a lineage of neurons thought to mediate speed of locomotion (one of the PMSIs). While A02d is a segment-local interneuron, proprioceptive axons span multiple segments, suggesting that a GDL can suppresses the effect of proprioceptive feedback specifically within its own segment without affecting the relay of proprioception to adjacent segments. Furthermore, A02d synapses onto a glutamatergic interneuron (A08a) thought to contribute to muscle relaxation in the wake of the peristaltic wave, which could be mediated via putative GABAergic premotor neurons (A31d). Taken together, it is suggested that one of the functions of the inhibitory neuron GDL is to gate proprioceptive feedback within its segment which has implications for the control of both speed and posture (Fushiki, 2016).

Finally, a descending neuron from the SEZ was observed that synapses onto the excitatory neuron (A27h) of the circuit chain in all segments. This motif has been observed and modeled in the leech and crayfish, where it enables the modulation of wave propagation speed. The brain and SEZ have been deemed non-essential for wave propagation. Speed of wave propagation, therefore, may be controlled in at least two ways: by proprioceptive feedback and by descending inputs. The existence of a circuit chain formed by excitatory and inhibitory neurons might be all that remains when both sensory feedback and the brain are absent, explaining the existence of wave propagation in decerebrated animals, and even for a small set of isolated abdominal segments (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).

GABAergic signaling shapes multiple aspects of Drosophila courtship motor behavior

Inhibitory neurons are essential for orchestrating and structuring behavior. This study used one of the best studied behaviors in Drosophila, male courtship, to analyze how inhibitory, GABAergic neurons shape the different steps of this multifaceted motor sequence. RNAi-mediated knockdown of the GABA-producing enzyme GAD1 and the ionotropic receptor Rdl in sex specific, fruitless expressing neurons in the ventral nerve cord causes uncoordinated and futile copulation attempts, defects in wing extension choice and severe alterations of courtship song. Altered song of GABA depleted males fails to stimulate female receptivity, but rescue of song patterning alone is not sufficient to rescue male mating success. Knockdown of GAD1 and Rdl in male brain circuits abolishes courtship conditioning. The around 220 neurons coexpressing GAD1 and Fruitless in the Drosophila male nervous system were characterized, and inhibitory circuit motifs underlying key features of courtship behavior were proposed based on the observed phenotypes (Amin, 2023).

Flexible circuit mechanisms for context-dependent song sequencing

Sequenced behaviours, including locomotion, reaching and vocalization, are patterned differently in different contexts, enabling animals to adjust to their environments. How contextual information shapes neural activity to flexibly alter the patterning of actions is not fully understood. Previous work has indicated that this could be achieved via parallel motor circuits, with differing sensitivities to context. This study demonstrated that a single pathway operates in two regimes dependent on recent sensory history. The Drosophila song production system was leveraged to investigate the role of several neuron types in song patterning near versus far from the female fly. Male flies sing 'simple' trains of only one mode far from the female fly but complex song sequences comprising alternations between modes when near her. Ventral nerve cord (VNC) circuits are shaped by mutual inhibition and rebound excitability between nodes driving the two song modes. Brief sensory input to a direct brain-to-VNC excitatory pathway drives simple song far from the female, whereas prolonged input enables complex song production via simultaneous recruitment of functional disinhibition of VNC circuitry. Thus, female proximity unlocks motor circuit dynamics in the correct context. A compact circuit model was constructed to demonstrate that the identified mechanisms suffice to replicate natural song dynamics. These results highlight how canonical circuit motifs can be combined to enable circuit flexibility required for dynamic communication (Roemschied, 2023).

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).

Nociceptive interneurons control modular motor pathways to promote escape behavior in Drosophila

Rapid and efficient escape behaviors in response to noxious sensory stimuli are essential for protection and survival. Yet, how noxious stimuli are transformed to coordinated escape behaviors remains poorly understood. In Drosophila larvae, noxious stimuli trigger sequential body bending and corkscrew-like rolling behavior. A population of interneurons in the nerve cord of Drosophila, termed Down-and-Back (DnB) neurons, was identified that are activated by noxious heat, promote nociceptive behavior, and are required for robust escape responses to noxious stimuli. Electron microscopic circuit reconstruction shows that DnBs are targets of nociceptive and mechanosensory neurons, are directly presynaptic to pre-motor circuits, and link indirectly to Goro rolling command-like neurons. DnB activation promotes activity in Goro neurons, and coincident inactivation of Goro neurons prevents the rolling sequence but leaves intact body bending motor responses. Thus, activity from nociceptors to DnB interneurons coordinates modular elements of nociceptive escape behavior (Burgos, 2018).

Nocifensive escape behavior in Drosophila larvae consists of C-shaped body bending and rolling, followed by rapid forward crawling. Recent studies have begun to identify circuits that mediate nocifensive behaviors (Kaneko, 2017; Ohyama, 2015; Yoshino, 2017). Prior work identified Basin neurons as multisensory interneurons that drive rolling behavior in response to vibration and noxious stimuli, and identified downstream Goro as command-like neurons for rolling. This study has identified and characterized DnB interneurons that are essential for nocifensive behavior in Drosophila larvae (see Summary model for DnB neurons controlling nocifensive escape). DnB neurons are direct targets of nociceptive cIV neurons and multiple mechanosensory cell types, including cII and cIII gentle touch da neurons and es neurons. Thus, DnBs provide a potential node for multisensory integration of tactile and noxious stimuli. The convergence of input from cIII gentle-touch receptors and cIV nociceptors onto DnB neurons is reminiscent of vertebrate interneurons that receive direct excitatory input from C-fiber/A∂ nociceptors and Aβ mechanoreceptors. Based on these studies nociceptive inputs appear to be integrated with multiple mechanosensory submodalities by Basin and DnB interneurons (Burgos, 2018).

EM reconstruction of DnB targets supported divergent major downstream circuitry. Output synapses on DnB axons converge on premotor neurons, at least some of which promote peristaltic wave propagation during locomotion. Other downstream neurons receive input from presynaptic sites on the DnB dendrite, and lead to Goro rolling command-like neurons. The spatial segregation of DnB output sites may mirror a functional segregation of downstream circuitry into bending and rolling modules. It is still unclear which muscle groups are recruited and how segments coordinate during body bending and rolling. This study provides evidence that silencing the period-positive median segmental interneuron (PMSI) cohort, which includes direct DnB targets A02g and A02e, reduces rolling behavior. PMSIs are glutamatergic inhibitory premotor neurons that terminate motor neuron bursting to regulate crawling speed (Kohsaka, 2014). Future work to selectively silence groups of premotor neurons will help to elucidate their role in nocifensive escape downstream of DnBs. Although silencing DnB neurons slightly increased the speed of forward locomotion, overall, forward crawling remained intact. Given that peristaltic waves also consist of segmental contractions, links to premotor neurons provide candidate neurons for dual control of crawling and C-shape bending behavior. Notably, DnB neurons target motor neurons innervating LT1 muscles, which have been implicated in larval self-righting behaviors. Self-righting consists of a C-shape type body bend, and 180° turn, so it is possible that LT1 muscles facilitate curved body bends that underlie both self-righting and rolling behavior. It is noted that the impact of DnB neurons on nociceptive circuits is likely to be more broad than indicated by synaptic connections, since EM and marker expression suggest that DnB neurons are peptidergic. Identification of the putative neuropeptide expressed by DnB neurons, and physiological effects, will be an important future direction, particularly given the important role of neuropeptides in vertebrate pain pathways, and recent evidence that mechanical nociception in larvae is under peptidergic control (Burgos, 2018).

Prior data showed that rolling is directional and is advantageous for dislodging attacking parasitoid wasps. Efficient rolling occurs coincident with deep C-shaped body bends, but the significance of these body bends for escape behavior has not been determined. DnB neural circuitry appears to be critically important for evoking body bend behavior prior to and during nocifensive rolling. Bending may provide the initial, most rapid, form of withdrawal from a noxious stimulus, and may subsequently support rolling locomotion by orienting and focusing the energy of muscle contraction into lateral thrusts. Re-orientation of denticle belts, triangle-shaped extensions of the cuticle, may also aid rapid lateral locomotion by providing substrate traction. Compromised escape rolling upon DnB inactivation may therefore arise both from weakened Goro activation and decreases in body bend angle. Understanding the circuit mechanisms that promote bending downstream of DnB neurons, and the muscle activities and physical mechanisms that underlie rolling behavior are important future aims (Burgos, 2018).

Analysis of DnB function revealed modular control of nocifensive escape behavior, consistent with EM reconstruction data. When DnB neurons were ectopically activated C-shaped body bending was observed that was often, but not always, associated with rolling. Other, non-rolling, animals bent with minimal crawling, or bent persistently while attempting to crawl forward. These observations provided initial evidence that C-shaped bending and rolling control circuits are separable, and that nocifensive bending could be combined with other behaviors, like pausing or crawling. Loss of function data supported bending as a primary motor output of DnB activity, with probabilistic activation of rolling motor programs. These behaviors could conceivably be linked, such that reduction in bending compromises rolling ability, or could arise from parallel influence of DnB activity on bending and rolling as suggested by EM reconstruction. Consistent with an important role for DnBs in promoting rolling, silencing Goro while activating DnB neurons promoted persistent bending without rolling, and uncoordinated snake-like forward crawling. This result further implicates a separate premotor circuitry in nocifensive body bending. These data further suggest that the bend-roll sequence must be tightly regulated by interactions between the parallel bend-roll premotor circuits, such that bending occurs first to facilitate rolling, which occurs second. However, bending can occur without being followed by rolling, indicating C-shaped bending itself is not sufficient to trigger rolling. Such independent, but sequentially regulated behavioral modules are consistent with hierarchical models of sequence generation as in fly grooming, human speech, roll-crawl sequence, and hunch-bend sequence. It is noted however, that although bending and rolling are sequential, they co-occur for much of the defensive behavior sequence, in contrast to such sequential and non-overlapping behavioral sequences. Elucidating the mechanisms of timing and interaction between the different circuit modules (bend vs roll) identified therefore promises to shed light on the general mechanisms of circuit implementation of sequence generation and co-ordination between different motor modules (Burgos, 2018).

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 col^ΔTv-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 col^ΔdAp-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).

The mTOR pathway component Unkempt regulates neural stem cell and neural progenitor cell cycle in the Drosophila central nervous system

The formation of a complex nervous system requires the coordinated action of progenitor cell proliferation, differentiation and maturation. The Drosophila postembryonic central nervous system provides a powerful model for dissecting the cellular and molecular mechanisms underpinning neurogenesis. Previous work has identified the conserved zinc finger/RING protein Unkempt (Unk) as a key temporal regulator of neuronal differentiation in the Drosophila developing eye and showed that Unk acts downstream of the mechanistic target of rapamycin (mTOR) pathway together with its binding partner Headcase (Hdc). This study investigated the role of Unk in Drosophila postembryonic thoracic neurogenesis. The Drosophila central nervous system contains neural stem cells, called neuroblasts, and neural progenitors, known as ganglion mother cells (GMCs). Unk is highly expressed in the central brain and ventral nerve cord but is not required to maintain neuroblast numbers or for the regulation of temporal series factor expression in neuroblasts. However, loss of Unk increases the number of neuroblasts and GMCs in S-phase of the cell cycle, resulting in the overproduction of neurons. This study also showed that Unk interacts with Hdc through its zinc finger domain. The zinc finger domain is required for the synergistic activity of Unk with Hdc during eye development but is not necessary for the activity of Unk in thoracic neurogenesis. Overall, this study shows that Unk and Hdc are novel negative regulators of neurogenesis in Drosophila and indicates a conserved role of mTOR signalling in nervous system development (Maierbrugger, 2020).

A fundamental challenge during neural development is the correct coordination of cell proliferation and differentiation. This is of particular importance in complex tissues, such as the central nervous system (CNS). During nervous system development, secreted ligands bind specific target receptors on neural stem cells and neural progenitor cells, causing them to exit the cell cycle and undergo a complex program of gene expression and morphological changes resulting in neuronal differentiation. Neural development is dependent on progenitor cell proliferation to provide enough cells to generate the mature CNS. This is controlled in a complex spatiotemporal manner and the rate of proliferation and differentiation varies at different stages of development (Maierbrugger, 2020).

Drosophila CNS development and neural stem cell proliferation has proven a powerful model to identify regulatory genes and concepts in neurogenesis. CNS development in Drosophila is characterized by two neurogenic phases, embryonic and postembryonic. The original pool of central brain and ventral nerve cord (VNC) neural stem cells, called neuroblasts in Drosophila, is generated early on during embryogenesis by delamination from the neuroepithelium. Shortly after, embryonic neuroblasts start dividing asymmetrically to generate neural progenitors called GMCs, that produce the differentiated neurons and glia necessary for larval life. After extensive proliferation in the embryo, neuroblasts undergo a period of quiescence (defined as reversible cell cycle arrest accompanied by low biosynthetic activity), after which proliferation is reactivated in early larval life. This second phase of neurogenesis will generate 90% of neurons that comprise the central brain and VNC of the adult (Maierbrugger, 2020).

Neuroblast lineages divide asymmetrically in a self-renewable manner. Notch signalling, originally deployed via lateral inhibition to specify neuroblasts, is redeployed along with asymmetric protein complexes to regulate asymmetric neuroblast divisions. The vast majority, so-called type I neuroblasts, generate a daughter neuroblast and a single GMC, which divides once to give rise to two post-mitotic neurons/glia. Eight lineages in the central brain, so-called type II neuroblasts, generate a daughter neuroblast and two types of intermediate neural progenitors (INPs), which also divide asymmetrically in turn to produce around 6 ​GMCs and 12 neurons/glia (Maierbrugger, 2020).

One key feature of neuroblasts and INPs is their ability to generate different types of neuronal progeny over time. This is regulated by a transcriptional cascade called the 'temporal series'. The temporal series was first identified in the Drosophila embryo and consists of sequential expression of transcription factors (Hb -> Svp -> Kr -> Pdm -> Cas) in the neuroblast and its progeny. More recently evidence has emerged for a temporal series mechanism in postembryonic lineages. Type I postembryonic neuroblasts re-entering the cell cycle still express the last transcription factor of the series, Castor (Cas) and subsequently go on to express the orphan nuclear hormone receptor Seven-up (Svp) a second time. Postembryonic neurons born early express the BTB transcription factor Chinmo, as well as the RNA binding proteins IGF-II mRNA-binding protein (Imp) and Lin-28, whereas those born late express the BTB transcription factor Broad Complex (Br-C), as well as the RNA binding protein Syncrip (Syp) and Ecdysone-induced protein 93F. Cas and Svp, acting upstream of the ecdysone receptor, regulate the early to late born temporal transition, which determines the neuronal identity and eventual post-synaptic targets (Maierbrugger, 2020).

Regional differences exist in the temporal series mechanisms active in postembryonic neurogenesis. Optic lobe neuroblasts are regulated by a temporal series consisting of Homothorax (Hth) -> Klumpfuss (Klu) -> Eyeless (Ey) -> Sloppy paired 1 and 2 (Slp1 and Slp2) -> Dichaete (D) -> Tailless (Tll). Loss-of-function studies have shown that the last four factors are necessary for temporal series progression. Type II neuroblasts express Imp, Lin28, and Chinmo during early larval development and Syp and the Ecdysone receptor β1 (EcRβ1) during the late phase. Interestingly, INPs are regulated by a distinct temporal series consisting of D -> Grh -> Ey. This temporal series causes early born INPs to produce D or brain-specific homeobox (Bsh) expressing neurons, while late born INPs produce Toy expressing neurons or Repo expressing glia (Maierbrugger, 2020).

Unkempt (Unk) is a zinc finger/RING domain protein expressed in the Drosophila nervous system where it plays a role in patterning. Reduced Unk expression results in adult flies with disorganised bristles and rough eyes. In the developing eye imaginal disc Unk, along with its binding partner Headcase (Hdc), is required for the correct timing of photoreceptor differentiation. During eye development Unk acts downstream of the insulin receptor/mechanistic target of rapamycin (mTOR) pathway. mTOR signalling downregulates Unk and loss of Unk, or activation of mTOR signalling, causes precocious photoreceptor differentiation. It is not known whether Unk plays a role in neurogenesis in the Drosophila CNS (Maierbrugger, 2020).

This study investigated the requirement for Unk in neurogenesis in the Drosophila CNS. Unk was found to be expressed throughout the Drosophila CNS and is strongly expressed in central brain and VNC neuroblasts and their progeny. Unk does not regulate the postembryonic temporal series, nor the number of postembryonic neuroblasts. However, clonal analysis demonstrates that loss of Unk expression increases the number neuroblasts and GMCs in S-phase of the cell cycle, resulting in increased numbers of neurons. Consistent with the role of Unk as a mediator of mTOR signalling, mTOR pathway activity is also required to maintain correct neuronal numbers. Finally, this study showed that the zinc finger domain of Unk interacts with Hdc but this domain is not necessary for the function of Unk in neurogenesis (Maierbrugger, 2020).

Temporal regulators of neuronal identity are defined by their requirement to establish specific neuronal fates. For example, in type I neuroblasts the orphan nuclear hormone receptor Svp is absolutely required for the switch from early to late born neuronal fates. Svp is similarly required in type II neuroblasts, where it regulates ecdysone receptor (EcR) expression and loss of the EcR also prevents the switch from early to late born fates. In the developing eye imaginal disc loss of Unk does not alter photoreceptor identity but causes precocious differentiation of photoreceptor neurons, resulting in patterning defects in the adult eye. Similarly, in the postembryonic CNS Unk is not required for neurons to acquire their identity, but regulates the numbers of neurons generated through control of the cell cycle in neuroblasts and GMCs. Loss of Unk expression causes increased EdU incorporation in neuroblasts and GMCs during early (48 h ALH) larval development, suggesting that Unk has a particularly important role at this stage. Mis-regulation of the cell cycle is not sufficient to increase the number of neuroblasts or GMCs, but results in an increase in the number of post-mitotic neurons by the end of larval development. The precise mechanism by which this increase occurs requires further investigation, and it cannot be excluded that the loss of Unk causes GMCs to divide more than once. Early and late born postembryonic thoracic motor neurons innervate specific domains of adult leg muscles. Loss of Unk and the resulting deregulation of neurogenesis may therefore affect motor circuits controlling locomotion (Maierbrugger, 2020).

mTOR signalling plays key roles in mammalian neurogenesis. In utero electroporation experiments using overexpression of a constitutively active form of Rheb have revealed the requirements for mTOR signalling in the mammalian subventricular zone. Activation of mTOR signalling caused precocious differentiation of highly proliferative Mash1-expressing transit amplifying cells, at the expense of self-renewal, resulting in increased numbers of neurons. Previous work has shown that loss of Unk or activation of mTOR signalling causes precocious differentiation of photoreceptor neurons. The current study has shown that clones mutant for unk have more EdU-incorporation in neuroblasts and GMCs and increased numbers of neurons. Together these studies point to a conserved role for Unk and mTOR signalling in regulating the cell cycle in neural stem cells and neural progenitors and their differentiation into neurons (Maierbrugger, 2020).

Although Unk acts downstream of the mTOR pathway, unlike other mTOR pathway components Unk does not regulate cell growth in nutrient rich conditions. However, a recent study showed that Unk and Hdc negatively regulate tissue growth under nutrient restriction in Drosophila. Clones mutant for unk or hdc in the eye or wing imaginal disc are larger than controls and have accelerated cell cycle progression only in larvae fed a low protein diet. The ability of Unk and Hdc to regulate mitotic cell proliferation under nutrient restriction requires mTOR pathway activity, consistent with the role of these proteins as mTOR pathway components. The precise role of Unk in the mTOR pathway remains to be determined. Although, Li (2019) found that Unk physically interacts with the mTOR complex 1 (mTORC1) component Raptor. Moreover, a mass spectrometry analysis of the insulin receptor/mTOR proteome in Drosophila showed that Unk physically interacts with Raptor, mTOR and 4E-BP (Glatter, 2011). Therefore, Unk may be a component of mTORC1 (Maierbrugger, 2020).

Physical interaction of Unk and Hdc in Drosophila has been observed in multiple independent studies. In the current study, the first half of the zinc finger domain in Unk was defined as necessary and sufficient for the interaction with Hdc. In keeping with co-immunoprecipitation experiments, using a yeast-2-hydrid screen Li (2019) found that amino acids 94-154 in Unk, which includes part of the second and all the third zinc finger, are necessary for the physical interaction with Hdc. Mammalian UNK also physically interacts with the Hdc ortholog HECA (Li, 2019) and so the interaction of these binding partners is evolutionarily conserved (Maierbrugger, 2020).

Unk is a highly conserved protein and recent studies have provided insight into the function of UNK in mammals. The zinc finger domain of mammalian UNK was shown to bind mRNAs and to negatively regulate the translation of these targets in neuroblastoma cells (Murn, 2015; Murn, 2016). UNK binds the transcripts of several hundred genes with diverse functions, including regulators of translation and S6K signalling. The zinc finger domain of Drosophila Unk is not required to rescue the increase in number of neurons in unk mutant MARCM clones. The role of the zinc finger domain may therefore vary depending on the context or cell type (Maierbrugger, 2020).

UNK is strongly expressed in the murine brain and knock-down of UNK in the developing cortex causes defects in the migration and morphology of neural progenitors (Murn, 2015). Although these phenotypes need to be confirmed using knock-out approaches, they suggest the exciting possibility that Unk plays a conserved role in nervous system development. Further characterisation of Unk using invertebrate and vertebrate models will decipher the role(s) of this highly conserved protein in the nervous system (Maierbrugger, 2020).

A single-cell transcriptomic atlas of the adult Drosophila ventral nerve cord

The Drosophila ventral nerve cord (VNC) receives and processes descending signals from the brain to produce a variety of coordinated locomotor outputs. It also integrates sensory information from the periphery and sends ascending signals to the brain. This study used single-cell transcriptomics to generate an unbiased classification of cellular diversity in the VNC of five-day old adult flies. An atlas was produced of 26,000 high-quality cells, representing more than 100 transcriptionally distinct cell types. The predominant gene signatures defining neuronal cell types reflect shared developmental histories based on the neuroblast from which cells were derived, as well as their birth order. The relative position of cells along the anterior-posterior axis could also be assigned using adult Hox gene expression. This single-cell transcriptional atlas of the adult fly VNC will be a valuable resource for future studies of neurodevelopment and behavior (Allen, 2020).

Selective role of the DNA helicase Mcm5 in BMP retrograde signaling during Drosophila neuronal differentiation

The MCM2-7 complex is a highly conserved hetero-hexameric protein complex, critical for DNA unwinding at the replicative fork during DNA replication. Overexpression or mutation in MCM2-7 genes is linked to and may drive several cancer types in humans. In mice, mutations in MCM2-7 genes result in growth retardation and mortality. All six MCM2-7 genes are also expressed in the developing mouse CNS, but their role in the CNS is not clear. This study used the central nervous system (CNS) of Drosophila melanogaster to begin addressing the role of the MCM complex during development, focusing on the specification of a well-studied neuropeptide expressing neuron: the Tv4/FMRFa neuron. In a search for genes involved in the specification of the Tv4/FMRFa neuron this study identified Mcm5 and found that it plays a highly specific role in the specification of the Tv4/FMRFa neuron. Other components of the MCM2-7 complex phenocopies Mcm5, indicating that the role of Mcm5 in neuronal subtype specification involves the MCM2-7 complex. Surprisingly, no evidence was found of reduced progenitor proliferation, and instead it was found that Mcm5 is required for the expression of the type I BMP receptor Tkv, which is critical for the FMRFa expression. These results suggest that the MCM2-7 complex may play roles during CNS development outside of its well-established role during DNA replication (Rubio-Ferrera, 2022).

Unc-4 acts to promote neuronal identity and development of the take-off circuit in the Drosophila CNS

The Drosophila ventral nerve cord (VNC) is composed of thousands of neurons born from a set of individually identifiable stem cells. The VNC harbors neuronal circuits required to execute key behaviors, such as flying and walking. Leveraging the lineage-based functional organization of the VNC, this study investigated the developmental and molecular basis of behavior by focusing on lineage-specific functions of the homeodomain transcription factor, Unc-4. Unc-4 was found to function in lineage 11A to promote cholinergic neurotransmitter identity and suppress the GABA fate. In lineage 7B, Unc-4 promotes proper neuronal projections to the leg neuropil and a specific flight-related take-off behavior. It was also uncovered that Unc-4 acts peripherally to promote proprioceptive sensory organ development and the execution of specific leg-related behaviors. Through time-dependent conditional knock-out of Unc-4, it was found that its function is required during development, but not in the adult, to regulate the above events (Lacin, 2020).

How does a complex nervous system arise during development? Millions to billions of neurons, each one essentially unique, precisely interconnect to create a functional central nervous system (CNS) that drives animal behavior. Work over several decades shows that developmentally established layers of spatial and temporal organization underlie the genesis of a complex CNS. For example, during spinal cord development in vertebrates, different types of progenitor cells arise across the dorso-ventral axis and generate distinct neuronal lineages in a precise spatial and temporal order. The pMN progenitors are located in a narrow layer in the ventral spinal cord and generate all motor neurons. Similarly, twelve distinct pools of progenitors that arise in distinct dorso-ventral domains generate at least 22 distinct interneuronal lineages. Within each lineage, neurons appear to acquire similar identities: they express similar sets of transcription factors, use the same neurotransmitter, extend processes in a similar manner and participate in circuits executing a specific behavior (Lacin, 2020).

The adult Drosophila ventral nerve cord (VNC), like the vertebrate spinal cord, also manifests a lineage-based organization. The cellular complexity of the VNC arises from a set of segmentally repeated set of 30 paired and one unpaired neural stem cells (Neuroblasts [NBs]), which arise at stereotypic locations during early development. These individually identifiable NBs undergo two major phases of proliferation: the embryonic phase generates the functional neurons of the larval CNS, some of which are remodeled to function in the adult, and the post-embryonic phase generates most of the adult neurons. The division mode within NB lineages adds another layer to the lineage-based organization of the VNC. Each NB generates a secondary precursor cell, which divides via Notch-mediated asymmetric cell division to generate two neurons with distinct identities. After many rounds of such cell divisions, each NB ends up producing two distinct hemilineages of neurons, termed Notch-ON or the 'A' and Notch-OFF or the 'B' hemilineage. This paper focuses only on postembryonic hemilineages, which from this point on in the paper are refered to as hemilineages for simplicity. Within a hemilineage, neurons acquire similar fates based on transcription factor expression, neurotransmitter usage, and axonal projection. Moreover, neurons of each hemilineage appear dedicated for specific behaviors. For example, artificial neuronal activation of the glutamatergic hemilineage 2A neurons elicit specifically high frequency wing beating, while the same treatment of the cholinergic hemilineage 7B neurons leads to a specific take-off behavior. Thus, hemilineages represent the fundamental developmental and functional unit of the VNC (Lacin, 2020).

Previous work has mapped the embryonic origin, axonal projection pattern, transcription factor expression, and neurotransmitter usage of essentially all hemilineages in the adult Drosophila VNC (see Lacin, 2019; Shepherd, 2019). This study leveraged this information to elucidate how a specific transcription factor, Unc-4, acts within individual hemilineages during adult nervous system development to regulate neuronal connectivity and function, and animal behavior. Unc-4, an evolutionarily conserved transcriptional repressor, is expressed post-mitotically in seven of the 14 cholinergic hemilineages in the VNC: three 'A' -Notch-ON- hemilineages (11A, 12A, and 17A) and four 'B' -Notch-OFF- hemilineages (7B, 18B, 19B, and 23B). For four of the Unc-4+ hemilineages (7B, 17A, 18B, and 23B), the neurons of the sibling hemilineage undergo cell death. For the remaining three (11A, 12A, and 19B), the neurons of the sibling hemilineage are GABAergic (Lacin, 2019). Unc-4 expression in these hemilineages is restricted to postmitotic neurons and it appears to mark uniformly all neurons within a hemilineage during development and adult life (Lacin, 2014; Lacin, 2016; Lacin, 2020 and references therein).

This study generated a set of precise genetic tools that allowed uncovering of lineage-specific functions for Unc-4: in the 11A hemilineage, Unc-4 drives the cholinergic identity and suppresses the GABAergic fate; in the 7B hemilineage, Unc-4 promotes correct axonal projection patterns and the ability of flies to execute a stereotyped flight take-off behavior. It was also found that Unc-4 is expressed in the precursors of chordotonal sensory neurons and required for the development of these sensory organs, with functional data indicating Unc-4 functions in this lineage to promote climbing, walking, and grooming activities (Lacin, 2020).

Using precise genetic tools, this study dissected the function of the Unc-4 transcription factor in a lineage-specific manner. Within the PNS, Unc-4 function is needed for the proper development of the leg chordotonal organ and walking behavior; whereas in the CNS, Unc-4 dictates neurotransmitter usage within lineage 11A and regulates axonal projection and flight take-off behavior in lineage 7B. Below, are discussed three themes arising from this work: lineage-specific functions of individual transcription factors, an association of Unc4+ lineages with flight, and the lineage-based functional organization of the CNS in flies and vertebrates (Lacin, 2020).

Seven neuronal hemilineages express Unc-4 in the adult VNC, but the phenotypic studies revealed a function for Unc-4 in only two of them: in the 11A hemilineage, Unc-4 promotes the cholinergic fate and inhibits the GABAergic fate, while in the 7B hemilineage, Unc-4 ensures proper flight take-off behavior likely by promoting the proper projection patterns of the 7B interneurons into the leg neuropil. Why was no loss-of-function phenotype detected for Unc4 in most of the hemilineages in which it is expressed? A few reasons may explain this failure. First, the phenotypic analysis was limited: Neuronal projection patterns and neurotransmitter fate were detected, but not other molecular, cellular, or functional phenotypes. Unc-4 may function in other lineages to regulate other neuronal properties that were not assayed, such as neurotransmitter receptor expression, channel composition, synaptic partner choice, and/or neuronal activity. In addition, as this analysis assayed all cells within the lineage, it would have missed defects that occur in single cells or small groups of cells within the entire hemilineage. Second, Unc-4 may act redundantly with other transcription factors to regulate the differentiation of distinct sets of neurons. Genetic redundancy among transcription factors regulating neuronal differentiation is commonly observed in the fly VNC. Thus, while the research clearly identifies a role for Unc-4 in two hemilineages, it does not exclude Unc-4 regulating more subtle cellular and molecular phenotypes in the other hemilineages in which it is expressed. Similarly, pan-neuronal deletion of Unc-4 specifically in the adult did not lead to any apparent behavioral defect even though Unc-4 expression is maintained in all Unc-4+ lineages throughout adult life, suggesting that Unc-4 function is dispensable in mature neurons after eclosion under standard lab conditions. Future work will be required to ascertain whether Unc-4 functions during adult life or in more than two of its expressing hemilineages during development. Nonetheless, this work shows that Unc-4 executes distinct functions in the 7B and 11A lineages. The Hox transcription factors, Ubx, Dfd, Scr, and Antp, have also been shown to execute distinct functions in different lineages in the fly CNS, suggesting transcription factors may commonly drive distinct cellular outcomes in the context of different lineages. What underlies this ability of one transcription factor to regulate distinct cellular events in different neuronal lineages? The ancient nature of the lineage-specific mode of CNS development likely holds clues to this question. The CNS of all insects arises via the repeated divisions of a segmentally repeated array of neural stem cells whose number, ~30 pairs per hemisegment, has changed little over the course of insect evolution. Within this pattern, each stem cell possessing a unique identity based on its position and time of formation. Each stem cell lineage has then evolved independently of the others since at least the last common ancestor of insects, approximately 500 million years ago. Thus, if during evolution an individual transcription factor became expressed in multiple neuronal lineages after this time, it would not be surprising that it would execute distinct functions in different neuronal lineages. The lineage-specific evolution of the CNS development in flies, worms, and vertebrates may explain why neurons of different lineages that share specific properties, for example, neurotransmitter expression, may employ distinct transcriptional programs to promote this trait (Lacin, 2020).

Although Unc-4 appears to have distinct functions in different lineages, this study found that an association with flight is a unifying feature among most Unc4+ interneuron lineages and motor neurons. All Unc-4+ hemilineages in the adult VNC except the 23B hemilineage heavily innervate the dorsal neuropils of the VNC, which are responsible for flight motor control and wing/haltere related behaviors, including wing-leg coordination. For example, hemilineages 7B, 11A, and 18B regulate flight take-off behavior and 12A neurons control wing-based courtship singing. In addition, most Unc-4+ motor neurons are also involved with flight - these include MN1-5, which innervate the indirect flight muscles, as well as motor neurons that innervate the haltere and neck muscles, which provide flight stabilization. Since Unc-4 is conserved from worms to humans, it is likely that Ametabolous insects, like silverfish, which are primitively wingless, also have unc-4. It has yet to be determined, though, whether in such ametabolous insects the same hemilineages express Unc-4, and hence this pattern was in place prior to the evolution of flight. This would suggest that there was some underlying association amongst this set of hemilineages that may have been exploited in the evolution of flight. Alternatively, Unc-4 may be lacking in these hemilineages prior to the evolution of flight but then its expression may have been acquired by these hemilineages as they were co-opted into a unified set of wing-related behaviors (Lacin, 2020).

The adult fly VNC is composed of 34 segmentally repeated hemilineages, which are groups of lineally related neurons with similar features for example, axonal projection and neurotransmitter expression. These hemilineages also appear to function as modular units, each unit appears responsible for regulating particular behaviors, indicating the VNC is assembled via a lineage-based functional organization. The vertebrate spinal cord exhibits similar organization: lineally-related neurons acquire similar fates ('cardinal classes') and function in the same or parallel circuits. The similarity of the lineage-based organization in insect and vertebrate nerve/spinal cords raises the question whether they evolved from a common ground plan or are an example of convergent evolution. Molecular similarities in CNS development between flies and vertebrates support both CNS's arise from a common ground plan. For example, motor neuron identity in both flies and vertebrates, use the same set of transcription factors: Nkx6, Isl, and Lim3. Moreover, homologs of many transcription factors expressed in fly VNC interneurons, such as Eve and Lim1, also function in interneurons of the vertebrate spinal cord. Whether any functional/molecular homology is present between fly and vertebrate neuronal classes awaits comparative genome-wide transcriptome analysis and functional characterization of neuronal classes in the insect VNC and vertebrate spinal cord (Lacin, 2020).

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 shak^B2 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).

Neuronal gluconeogenesis regulates systemic glucose homeostasis in Drosophila melanogaster

Gluconeogenesis is a well-established metabolic process whereby glucose is generated from small carbon molecules in the liver and kidney to maintain blood glucose levels. Expression of gluconeogenic genes has been reported in other organs of mammals and insects, where their function is not yet known. In the fruit fly, one of the gluconeogenic genes, glucose-6-phosphatase (G6P) is exclusively expressed in the CNS. Using a fluorescence resonance energy transfer (FRET)-based glucose sensor, this study shows that a small subset of neurons in the fly brain is capable of carrying out gluconeogenesis. Moreover, G6P mutant flies exhibit low whole-body glucose levels within 24 h of food deprivation. This phenotype can be mimicked by silencing G6P neurons and rescued by experimentally controlled activation in the absence of G6P. These results indicate that neural activity of G6P neurons, but not glucose production per se, is critical for glucose homeostasis. Lastly, it was observed that neuronal gluconeogenesis promotes anterograde neuropeptide distribution from the soma to axon terminals, suggesting that the generation of glucose facilitates neuropeptide transport. Together, this analysis reveals a novel role for gluconeogenesis in neuronal signaling (Miyamoto, 2019).

A Drosophila larval premotor/motor neuron connectome generating two behaviors via distinct spatio-temporal muscle activity

A multilayer circuit architecture for the generation of distinct locomotor behaviors in Drosophila

Animals generate diverse motor behaviors, yet how the same motor neurons (MNs) generate two distinct or antagonistic behaviors remains an open question. This study characterized Drosophila larval muscle activity patterns and premotor/motor circuits to understand how they generate forward and backward locomotion. All body wall MNs are activated during both behaviors, but a subset of MNs change recruitment timing for each behavior. TEM was used to reconstruct a full segment of all 60 MNs and 236 premotor neurons (PMNs), including differentially-recruited MNs. Analysis of this comprehensive connectome identified PMN-MN ‘labeled line’ connectivity; PMN-MN combinatorial connectivity; asymmetric neuronal morphology; and PMN-MN circuit motifs that could all contribute to generating distinct behaviors. We generated a recurrent network model that reproduced the observed behaviors, and used functional optogenetics to validate selected model predictions. This PMN-MN connectome will provide a foundation for analyzing the full suite of larval behaviors (Zarin, 2019a and b).

This study reports a comprehensive larval proprioceptor-PMN-MN connectome and describes individual muscle/MN phase activity during both forward and backward locomotor behaviors. PMN-MN connectivity motifs were identified that could generate muscle activity phase relationships, and selected experimental validation was performed. Proprioceptor-PMN connectivity was identified that provides an anatomical explanation for the role of proprioception in promoting locomotor velocity, and it identifies a new candidate escape motor circuit. Finally, a recurrent network model was generated that produces the observed sequence of motor activity, showing that the identified pool of premotor neurons is sufficient to generate two distinct larval behaviors. It is concluded that different locomotor behaviors can be generated by a specific group of premotor neurons generating behavior-specific motor rhythms (Zarin, 2019a and b).

Locomotion is a rhythmic and flexible motor behavior that enables animals to explore and interact with their environment. Birds and insects fly, fish swim, limbed animals walk and run, and soft-body invertebrates crawl. In all cases, locomotion results from coordinated activity of muscles with different biomechanical output. This precisely regulated task is mediated by neural circuits composed of motor neurons (MNs), premotor interneurons (PMNs), proprioceptors, and descending command-like neurons. A partial map of neurons and circuits regulating rhythmic locomotion have been made in mouse, cat, fish, tadpole, lamprey, leech, crayfish, and worm. These pioneering studies have provided a wealth of information on motor circuits, but with the exception of C. elegans, there has been no system where all MNs and PMNs have been identified and characterized. Thus, a comprehensive picture of how an ensemble of interconnected neurons generate diverse locomotor behaviors is missing (Zarin, 2019a and b).

How does the Drosophila larva executes multiple behaviors, in particular forward versus backward locomotion (Carreira-Rosario, 2018). Are there different motor neurons used in each behavior? Are the same motor neurons used but with distinct patterns of activity determined by premotor inputs? How does the ensemble of premotor and motor neurons generate additional behaviors, such as escape behavior via lateral rolling? A rigorous answer to these questions requires both comprehensive anatomical information -- i.e., a premotor/motor neuron connectome -- and the ability to measure rhythmic neuronal activity and perform functional experiments. All of these tools are currently available in Drosophila, and this study used them to characterize the neuronal circuitry used to generate forward and backward locomotion, and how proprioception is integrated by the PMN ensemble (Zarin, 2019a and b).

The Drosophila larva is composed of 3 thoracic (T1-T3) and 9 abdominal segments (A1-A9), with sensory neurons extending from the periphery into the CNS, and motor neurons extending out of the CNS to innervate body wall muscles. Most segments contain 30 bilateral body wall muscles that are grouped by spatial location and orientation: dorsal longitudinal (DL; includes previously described DA and some DO muscles), dorsal oblique (DO), ventral longitudinal (VL), ventral oblique (VO), ventral acute (VA) and lateral transverse (LT). Using these muscles, the larval nervous system can generate forward locomotion, backward locomotion, turning, hunching, digging, self-righting, and escape. This study focused on forward and backward locomotion. Forward crawling behavior in larvae involves a peristaltic contraction wave from posterior to anterior segments; backward crawling entails a posterior propagation of the contraction wave (Zarin, 2019a and b).

Body wall muscles are innervated by approximately 60 MNs per segment, consisting of 28 left/right pairs that typically each innervate one muscle, and whose neuromuscular junctions have big boutons, therefore also called type-Ib MNs; two pairs of type-Is (small bouton) MNs that innervate large groups of dorsal or ventral muscles; three type II ventral unpaired median MNs that provide octopaminergic innervation to most muscles; and one or two type III insulinergic MNs innervating muscle 12. All MNs in segment A1 have been identified by backfills from their target muscles, and several have been shown to be rhythmically active during larval locomotion, but only a few of their premotor inputs have been described. Some excitatory PMNs are involved in initiating activity in their target MNs, while some inhibitory PMNs limit the duration of MN activity or produce intrasegmental activity offsets. Interestingly, some PMNs are active specifically during forward locomotion or backward locomotion. In addition, there are six pair of proprioceptor neurons in each abdominal segment (ddaE, ddaD, vpda, dmd1, dbd and vbd). They are important for promoting locomotor velocity and posture, and some of their CNS targets have been identified, but to date little is known about how or if they are directly connected to the PMN/MN circuits (Zarin, 2019a and b).

It is a major goal of neuroscience to comprehensively reconstruct neuronal circuits that generate specific behaviors, but to date this has been done only in C. elegans. Recent studies in mice and zebrafish have shed light on the overall distribution of PMNs and their connections to several well-defined MN pools. However, it remains unknown if there are additional PMNs that have yet to be characterized, nor are their any insights into potential connections between PMNs themselves, which would be important for understanding the network properties that produce coordinated motor output. In the locomotor central pattern generator circuitry of leech, lamprey, and crayfish, the synaptic connectivity between PMNs or between PMNs and other interneurons are known to play critical roles in regulating the swimming behavior. However, it is difficult to be certain that all the neural components and connections of these circuits have been identified. Thus, the comprehensive anatomical circuitry reconstructed in this study provides an anatomical constraint on the functional connectivity used to drive larval locomotion; all synaptically-connected neurons may not be relevant, but at least no highly connected local PMN is absent from this analysis (Zarin, 2019a and b).

The current results confirm and significantly extend previous studies of Drosophila larval locomotion. For example, a recent study has shown that the GABAergic A14a inhibitory PMN (also called iIN1) selectively inhibits MNs innervating muscle 22/LT2 (CMuG F4; CMuG refers to Co-active Muscle Group), thereby delaying muscle contraction relative to muscle 5/LO1 (CMuG F2). This study was extended by showing that A14a also disinhibits MNs in early CMuGs F1/2 via the inhibitory PMN A02e. Thus, A14a both inhibits late CMuGs and disinhibits early CMuGs. In addition, previous work has suggested that all MNs receive simultaneous excitatory inputs from different cholinergic PMNs. However, dual calcium imaging data of the A27h excitatory PMN shows that it is active during CMuG F4 and not earlier. Therefore, MNs may receive temporally distinct excitatory inputs, in addition to the previously reported temporally distinct inhibitory inputs. This study has identified dozens of new PMNs that are candidates for regulating motor rhythms; functional analysis of all of these PMNs is beyond the scope of this paper, particularly due to the additional work required to screen and identify Gal4/LexA lines selectively targeting these PMNs, but the predictions of this paper are clear and testable when reagents become available (Zarin, 2019a and b).

MNs innervating a single Spatial Muscle Group (SMuG) belong to more than one CMuG, therefore SMuGs do not generally match CMuGs. This could be due to the several reasons: (1) MNs in each SMuGs receive inputs from overlapping but not identical array of PMNs. (2) Different MNs in the same SMuG receive a different number of synapses from the same PMN. (3) MNs in the same SMuG vary in overall dendritic size and total number of post-synapses, thereby resulting in MNs of the same SMuGs fall into different CMuGs (Zarin, 2019a and b).

This study demonstrates that during both forward and backward crawling, most of longitudinal and transverse muscles of a given segment contract as early and late groups, respectively. In contrast, muscles with oblique or acute orientation often show different phase relationships during forward and backward crawling. Future studies will be needed to provide a biomechanical explanation for why oblique muscles -- but not longitudinal or transverse muscles -- need to be recruited differentially during forward or backward crawling. Also, it will be interesting to determine which spatial muscle groups (e.g., either or both VOs and VLs) are responsible for elevating cuticular denticles during propagation of the peristaltic wave in forward and backward crawling; if the VOs, it would mean that lifting the denticles occurs at different phases of the crawl cycle in forward and backward locomotion. Finally, understanding how the premotor-motor circuits described in this study are used to generate diverse larval motor behaviors will shed light on mechanisms underlying the multi-functionality of neuronal circuits (Zarin, 2019a and b).

A recent study has reported that proprioceptive sensory neurons (dbd, vbd, vpda, dmd1, ddaE, and ddaD) show sequential activity during forward crawling. dbd responds to stretching and whereas the other five classes are activated by muscle contraction (Vaadia, 2019). All proprioceptors show connectivity to the tier of PMNs described in this study, and this study has identified circuit motifs that are consistent with the observed timing and excitatory function of each proprioceptor neuron. It will be of great interest perform functional experiments to test these anatomical circuit motifs for functional relevance (Zarin, 2019a and b).

A recurrent network model accurately predicts the order of activation of specific PMNs, yet many of its parameters remain unconstrained, and some PMNs may have biological activity inconsistent with activity predicted by this model. Sources of uncertainty in the model include incomplete reconstruction of inter-segmental connectivity and descending command inputs, the potential role of gap junctions (which are not resolved in the TEM reconstruction), as well as incomplete characterization of PMN and MN biophysical properties. Recent studies have suggested that models constrained by TEM reconstructions of neuronal connectivity are capable of predicting features of neuronal activity and function in the Drosophila olfactory and visual systems, despite the unavoidable uncertainty in some model parameters. Similarly, for the locomotor circuit described in this study, it is anticipated that the addition of model constraints from future experiments will lead to progressively more accurate models of PMN and MN dynamics. Despite it's limitations, the ability for the PMN network to generate appropriate muscle timing for two distinct behaviors in the absence of any third-layer or command-like interneurons suggests that a single layer of recurrent circuitry is sufficient to generate multiple behavioral outputs, and provides insight into the network architecture of multifunctional pattern generating circuits (Zarin, 2019a and b).

Previous work in other animal models have identified multifunctional muscles involved in more than one motor behavior: swimming and crawling in C. elegans and leech; walking and flight in locust; respiratory and non-respiratory functions of mammalian diaphragm muscle unifunctional muscles which are only active in one specific behavior in the lobster Homarus americanus; swimming in the marine mollusk Tritonia diomedea; and muscles in different regions of crab and lobster stomach. Single-muscle calcium imaging data indicates that all imaged larval body wall muscles are bifunctional and are activated during both forward and backward locomotion. It will be interesting to determine if all imaged muscles are also involved in other larval behaviors, such as escape rolling, self-righting, turning, or digging. It is likely that there are different CMuGs for each behavior, as this study has \ seen for forward and backward locomotion, raising the question of how different CMuGs are generated for each distinct behavior (Zarin, 2019a and b).

Neural circuitry linking mating and egg laying in Drosophila females

Mating and egg laying are tightly cooordinated events in the reproductive life of all oviparous females. Oviposition is typically rare in virgin females but is initiated after copulation. This study identified the neural circuitry that links egg laying to mating status in Drosophila melanogaster. Activation of female-specific oviposition descending neurons (oviDNs) is necessary and sufficient for egg laying, and is equally potent in virgin and mated females. After mating, sex peptide-a protein from the male seminal fluid-triggers many behavioural and physiological changes in the female, including the onset of egg laying. Sex peptide is detected by sensory neurons in the uterus, and silences these neurons and their postsynaptic ascending neurons in the abdominal ganglion. This study shows that these abdominal ganglion neurons directly activate the female-specific pC1 neurons. GABAergic (gamma-aminobutyric-acid-releasing) oviposition inhibitory neurons (oviINs) mediate feed-forward inhibition from pC1 neurons to both oviDNs and their major excitatory input, the oviposition excitatory neurons (oviENs). By attenuating the abdominal ganglion inputs to pC1 neurons and oviINs, sex peptide disinhibits oviDNs to enable egg laying after mating. This circuitry thus coordinates the two key events in female reproduction: mating and egg laying (Wang, 2020).

It was reasoned that egg laying is likely to depend on cell types that are female-specific and hence express one or both of the sex-determination genes fruitless (fru) and doublesex (dsx). In particular, egg laying is blocked by either silencing or masculinizing all fru+ neurons. Some of these fru+ neurons are descending interneurons, which project from the brain to the ventral nerve cord and are thought to convey high-level motor commands. This study therefore focused on female-specific fru+ descending neurons and used the split-GAL4 technique to obtain two driver lines that label two female-specific fru+dsx- cholinergic descending neurons per brain hemisphere. In optogenetic activation experiments using Chrimson, both split-GAL4 driver lines reliably induced oviposition behaviour in mated females, with most but not all females also depositing an egg (it is presumed that not all females had an egg in the uterus at the time of neuronal activation). Accordingly, these neurons are referred to as oviposition descending neurons (oviDNs), and to the two split-GAL4 driver lines that label them as oviDN-SS1 and oviDN-SS2 (in which SS denotes stable split-GAL4). Stochastic labelling of single neurons resolved two morphologically distinct types of oviDN, which are refered to as oviDNa and oviDNb cells. In an electron microscopy volume of a full adult female brain (FAFB15), two oviDNa-like cells and one oviDNb-like cell were identified in each hemisphere (Wang, 2020).

Egg laying by mated females was completely blocked by genetic ablation of oviDNs, and markedly reduced by their chronic silencing. Virgin females in which oviDNs were ablated were as receptive to mating as control females. Several days after mating, the ovaries of oviDN-ablated females contained many mature eggs, and most carried either a fertilized egg or a first-instar larva in the uterus. It is concluded that oviDNs are essential for oviposition, but dispensable for mating, ovulation and fertilization (Wang, 2020).

It was not possible to generate driver lines that specifically target oviDNa or oviDNb cells. To determine which oviDN subtype is involved in oviposition, a stochastic 'unsilencing' experiment was performed, in which a tdTomato-tagged silencing transgene was targeted to all oviDNs, but stochastically replaced in some of these cells with GFP. Individual females were assayed for egg laying over five days after mating, then dissected and stained to determine their complement of red (tdTomato; silenced) and green (GFP; unsilenced) oviDNs. Females with no unsilenced cells laid no or very few eggs, whereas those with just a single functional oviDN cell generally laid large numbers of eggs. The number of eggs laid per female was variable in these cases, but there was no appreciable difference between females in which an oviDNa cell was unsilenced and those in which an oviDNb cell was unsilenced, nor between females in which either one or two cells of either type were functional. Although the oviDNa and oviDNb subtypes differ in their morphology-and probably their connectivity and physiology-these data suggest that they nonetheless have similar functions in oviposition (Wang, 2020).

Oviposition involves a coordinated and highly stereotyped sequence of motor actions that progresses from abdomen bending to ovipositor extrusion and egg deposition. Abdomen bending, ovipositor extrusion and egg deposition were all eliminated in females in which oviDNs were ablated. Conversely, abdomen bending and ovipositor extrusion were reliably triggered by strong photoactivation of oviDNs in either virgin or mated females. Egg deposition was also induced, but only in mated females (presumably because mating is required to stimulate ovulation). In all of these oviDN activation experiments, the sequence of motor actions was the same as that in natural egg laying. By varying the stimulus intensity, it was found that egg deposition has a higher activation threshold than abdomen bending and ovipositor extrusion, and that action latencies were shorter at higher stimulus intensities. Moreover, at low stimulus intensities, the oviposition sequence was often truncated, but an action was never skipped, and only once was a single action occurring out of order observed (in a total of 38 flies at each of 3 intensities). These data suggest that oviDNs may use a ramp-to-threshold mechanism to elicit the successive motor actions of oviposition. Notably, the activation thresholds and action latencies were indistinguishable between virgins and mated females, indicating that mating status regulates egg laying through the brain circuits upstream of oviDNs rather than through downstream motor circuits (Wang, 2020).

The onset of egg laying after mating is induced by sex peptide, a protein of the male seminal fluid that is detected by sex-peptide sensory neurons (SPSNs) of the uterus. Sex peptide silences both SPSNs and their postsynaptic targets in the abdominal ganglion, the SP abdominal ganglion (SAG) neurons. Artificially activating either SPSNs or SAG neurons suppressed egg laying in mated females. Conversely, ablating or silencing these cells increased the number of eggs laid by virgin females. Virgin egg laying as a result of SPSN or SAG ablation depended on oviDNs, as egg laying was prevented if these cells were co-ablated. SPSN and SAG activity is thus critical in keeping oviDNs inactive until after mating. This inhibition is most likely to be indirect, because the SAGs are cholinergic and hence probably excitatory. This study identified and extensively traced the ascending projections of the two SAG neurons in the FAFB volume and found just a single synapse from SAG neurons to oviDNs (Wang, 2020).

The targets of SAG neurons in the brain have not been identified. Because SAG neurons regulate female receptivity as well as egg laying, it is speculated that their targets could include the female-specific fru-dsx+ pC1 neurons in the protocerebrum, which are known to regulate receptivity. Within the FAFB volume five morphologically distinct pC1 cells were identified in each hemisphere, which are referred to as pC1a-pC1e. Extensive tracing of single pC1a, pC1c and pC1e cells, as well as more limited tracing of pC1b and pC1d cells, suggests that the SAG neurons provide numerous synaptic inputs to the pC1a, pC1b and pC1c cells, with fewer if any direct inputs to pC1d and pC1e cells. Whole-cell recordings were performed from individual pC1 neurons while photoactivating the SAGs; pC1a cells were strongly depolarized, pC1b cells were weakly depolarized and pC1c, pC1d and pC1e cells showed little or no response upon SAG activation. There were numerous synaptic connections amongst all five pC1 subtypes, however, suggesting that any information on mating status that is obtained from SAG neurons by pC1a and pC1b cells is potentially shared across the entire set of pC1 cells (Wang, 2020).

Two split-GAL4 driver lines were obtained for pC1 neurons: pC1-SS1, which labels pC1a, pC1c and pC1e, and pC1-SS2, which labels all five pC1 cells. Ablation of pC1 cells using either driver resulted in an increase in egg laying in virgin females that was dependent on oviDN function, whereas mated females in which pC1 neurons were chronically activated laid fewer eggs. Brief optogenetic silencing of pC1 neurons in virgins did not acutely trigger egg laying, as would be expected if pC1-inactivated virgins (like pC1-intact mated females) rely on additional substrate-borne cues for the induction of egg laying (Wang, 2020).

These behavioural data indicate that-similar to SPSNs and SAG neurons-pC1 neurons suppress the function of oviDNs and therefore suppress egg laying in virgin females. Consistent with this interpretation, it was found by in vivo imaging that basal calcium levels in pC1 neurons, although variable, are generally higher in virgin than mated females. Moreover, whole-cell recordings from oviDNs revealed that both oviDNa and oviDNb cells are hyperpolarized after photoactivation of pC1 neurons, and that this effect is sensitive to picrotoxin, a chloride channel blocker. This inhibition is probably indirect, because pC1 neurons are cholinergic and have very few synapses onto the oviDNs (Wang, 2020).

To look for inhibitory intermediates from pC1 to oviDN cells-as well as excitatory inputs that might stimulate egg laying upon detection of a preferred substrate- the synaptic inputs to oviDNa and oviDNb cells were reconstructed in the Full Adult Fly Brain (FAFB) volume. Sparse split-GAL4 driver lines were obtained for the two cell types with the largest numbers of oviDN input synapses. Whole-cell recordings reliably showed changes in membrane potential in oviDNs after photoactivation of either of these two cell types. The cell type with the most oviDN input synapses is cholinergic, and activation of these cells depolarized oviDNs. These cells were therefore named oviposition excitatory neurons (oviENs). The cell type with the second-highest number of oviDN input synapses is GABAergic, and activation of these cells hyperpolarized oviDNs. Accordingly, these cells were named oviposition inhibitory neurons (oviINs). There is a single oviEN and a single oviIN per hemisphere, and they are reciprocally connected. The oviINs are also reciprocally connected with pC1 cells, and calcium-imaging experiments showed that photoactivation of pC1 cells elicits an excitatory response in oviINs. The pC1 cells have few direct synaptic connections with oviENs, and no connections were detected between SAG neurons and either oviINs or oviENs (Wang, 2020).

Silencing oviENs in mated females strongly suppressed egg laying, similarly to the effect observed when oviDNs were silenced. By contrast, potentiating oviENs in virgin females caused them to lay significantly more eggs than control virgins, albeit not as many as mated females (presumably because ovulation remains infrequent). Manipulating oviIN activity had the opposite effects: silencing oviINs caused virgins to lay significantly more eggs, whereas depolarizing oviINs reduced the number of eggs laid by mated females. Thus, as expected from the sign of their inputs to oviDNs (that is, excitatory for oviENs; inhibitory for oviINs), oviENs promote egg laying, whereas oviINs inhibit it (Wang, 2020).

It was hypothesized that oviENs could mediate the external sensory signals that trigger egg laying in mated females, which are likely to include both gustatory and mechanosensory cues from the substrate. When provided with a choice of substrates, females lay more eggs on agarose medium than on a hard surface or a substrate of agarose and sucrose. Therefore in vivo calcium imaging was performed to determine the responses of oviDNs, oviENs and oviINs to the presentation of each of these substrates to the legs. In oviDNs, an increase was observed in calcium levels only upon contact with the agarose substrate. This response was stronger in mated females than in virgins. The agarose-and-sucrose substrate elicited a small reduction in calcium levels, which was more pronounced in virgin females. The oviENs showed a positive calcium response to agarose but to neither of the other two substrates, and this response was indistinguishable between virgins and mated females. The oviINs responded to all three substrates, but more strongly to agarose and sucrose than to agarose alone, and only weakly to the hard surface. Regardless of substrate, oviIN responses were stronger in virgins than in mated females (Wang, 2020).

In conclusion, these findings support the following model for the neural coordination of mating and egg laying in Drosophila. The oviDNs control the entire oviposition motor programme. They receive excitatory input from oviENs, which respond to stimulatory cues from the substrate, and inhibitory input from oviINs, which convey information about mating status from pC1 cells. In virgins, increased activity of pC1 neurons potentiates oviIN-mediated inhibition of both oviDNs and oviENs, which suppresses egg laying. After mating, sex peptide silences SAG inputs onto pC1 neurons, thereby decreasing the activity of pC1 neurons and oviINs to facilitate egg laying when a preferred substrate is encountered. Reciprocal connections between oviINs and oviENs might ensure that oviDNs respond to oviEN activation with the appropriate temporal pattern and dynamic range, through feed-forward and feedback inhibition, respectively. The oviDNs, oviENs and oviINs all have numerous synaptic inputs in addition to those that have been described in this stduy-all of which remain functionally uncharacterized. These inputs may mediate other controls on the egg-laying process, such as the presence of an egg in the uterus and the nutritional state of the female. The pC1 neurons might also regulate other female behaviours that switch after mating, perhaps through different sets of output neurons. Notably, the male counterparts of pC1 neurons are thought to encode an analogous state of courtship arousal that modulates command pathways for specific motor actions such as courtship song and 'licking'. Thus, functionally analogous but anatomically divergent circuits-shaped during development by fru and dsx-could account for the distinct reproductive behaviours of Drosophila males and females (Wang, 2020).

Parallel transformation of tactile signals in central circuits of Drosophila

To distinguish between complex somatosensory stimuli, central circuits must combine signals from multiple peripheral mechanoreceptor types, as well as mechanoreceptors at different sites in the body. This study investigated the first stages of somatosensory integration in Drosophila using in vivo recordings from genetically labeled central neurons in combination with mechanical and optogenetic stimulation of specific mechanoreceptor types. Three classes of central neurons were identified that process touch: one compares touch signals on different parts of the same limb, one compares touch signals on right and left limbs, and the third compares touch and proprioceptive signals. Each class encodes distinct features of somatosensory stimuli. The axon of an individual touch receptor neuron can diverge to synapse onto all three classes, meaning that these computations occur in parallel, not hierarchically. Representing a stimulus as a set of parallel comparisons is a fast and efficient way to deliver somatosensory signals to motor circuits (Tuthill, 2016).

This study used somatosensory circuits in the Drosophila VNC to investigate the neural computations that occur at the first stages of touch processing. The results suggest a conceptual framework for the central integration of peripheral touch signals. First, signals from peripheral touch receptors are directly transmitted to multiple, parallel processing channels. Within these channels, spatial selectivity is achieved through integration of excitatory and inhibitory inputs from touch receptors in different locations. In parallel, contextual selectivity is achieved by integrating touch signals with information from proprioceptors (Tuthill, 2016).

One idea unites the three CNS cell classes described in this study. Namely, cells within all three classes are performing comparisons-within a limb, across limbs, or between different mechanoreceptor types. These comparisons encode the difference between mechanical stimuli of different types, and/or mechanical stimuli at different sites on the body. In general terms, any neuron with an inhibitory receptive field component is encoding a comparison. What is notable in these results is the observation that different central neurons directly postsynaptic to the same afferent axon are performing a variety of different comparisons. At the very first synapse of the somatosensory system, excitation from a given afferent is being integrated with inhibition from several different sources, with each type of comparison occurring in a distinct parallel processing channel. Collectively, these comparisons span a wide range of spatial scales, even though they are all being performed one synapse from the periphery (Tuthill, 2016).

Encoding sensory information via comparisons brings several advantages. When a neuron computes the difference between two input signals, the shared component of those input signals is suppressed. This arrangement can allow neurons to transmit finer spatial or temporal features of a stimulus and reduce redundancy among the spike trains of different neurons, thereby increasing metabolic efficiency. This strategy may be particularly useful in a system facing an information bottleneck. In this case, the relevant bottleneck is the neck of the fly, which contains only ~3,600 axons. Among the cell classes examined in this study, one projects directly to the brain (the intersegmental neurons), while the others may relay information indirectly to the brain, as well as participating in local VNC reflex circuits (Tuthill, 2016).

This study shows that an individual touch receptor axon diverges to directly contact multiple postsynaptic cell classes, each performing a different parallel computation. Why perform these computations in parallel, rather than hierarchically? One important consideration is the necessity for speed. Speed may be a particularly important constraint in somatosensory processing, because the site of sensory transduction (e.g., the foot) can be relatively distant from the CNS. Because Drosophila axons are unmyelinated and usually narrow, axonal conduction is likely to be slow. Indeed, a consistent delay of about 3 ms was observed from the time of a femur bristle neuron spike in the periphery to the onset of an EPSP in the VNC. This delay is presumably even longer for mechanosensory signals arising from the distal leg, since the axons of tarsus bristle neurons can be over twice as long as the axons of femur bristle neurons (Tuthill, 2016).

Some of the central neurons described in this study - the midline projection neurons - integrate information from the right and left legs. Although considerable receptive field diversity is observed within this neural population, the general receptive field structure consisted of ipsilateral excitation, together with mixed excitation and inhibition from the contralateral leg. This organization is similar to that of some neurons in vertebrate spinal cord and somatosensory cortex, which integrate excitatory input from one side of the body with mixed excitation and inhibition from the opposite side (Tuthill, 2016).

Bilateral tactile integration is clearly important to some behaviors. For example, rats can distinguish the relative distance of two walls using their whiskers, a behavior that requires activity in somatosensory cortices of both hemispheres. In a similar manner, integrating touch signals from the two legs may allow the fly to compare bilateral tactile features. For example, when faced with a small gap, flies reach forward across the void with their front legs and attempt to cross only when both legs have contacted the opposite side. Comparison of bilateral somatosensory signals is also critical for the refinement of rhythmic motor behaviors, such as crawling in Drosophila larvae (Tuthill, 2016).

In vertebrates, different types of peripheral mechanoreceptors have been traditionally considered to be functionally segregated pathways. Different mechanoreceptor types have been thought to independently mediate the perception of specific somatosensory 'submodalities,' such as vibration, stretch, and texture. However, mounting evidence suggests that signals from distinct somatosensory submodalities are in fact combined in the CNS, and most tactile percepts rely on multiple submodalities. For example, a recent study found that all areas of somatosensory cortex receive input from both touch and proprioceptive neurons (Tuthill, 2016).

Where in the somatosensory processing hierarchy are signals from different mechanoreceptor types first integrated? There is some anatomical evidence that this type of integration begins within the dorsal horn of the spinal cord. There is also functional evidence of early submodality integration-for example, some neurons in the cat spinal cord respond to both skin touch and joint movement, while neurons in the cuneate nucleus of the brainstem exhibit tactile feature selectivity that is indicative of submodality integration. In the mouse brainstem, there are neurons that receive direct convergent projections from different mechanoreceptor types that innervate the same whisker on the face . However, despite these examples, little is known about the specific sites and mechanisms of submodality integration in vertebrate somatosensation (Tuthill, 2016).

The current results provide an example of submodality integration at the very first stage of somatosensory processing, immediately postsynaptic to peripheral touch receptors. Specifically, this study found that intersegmental neurons integrate excitatory touch signals from bristle neurons with inhibition from proprioceptive neurons in the femoral chordotonal organ. Studies of the femoral chordotonal organ in larger insects suggest that individual chordotonal neurons encode movements and static positions of the tibia. Thus, inhibitory input to ascending neurons may serve to suppress excitatory touch signals at specific phases of the walking cycle, or during grooming behavior. This reafferent signal may function in a manner analogous to corollary discharge, in which efferent motor commands are used to alter sensory signals that arise from self-generated movements. Interestingly, a recent study in larval Drosophila found that nociceptive inputs and proprioceptive inputs can converge at the level of second-order neurons, and in this case, the interaction is summation rather than suppression. Together, these results suggest that integration across submodalities is widespread and very early in this system, consistent with the evidence in vertebrates (Tuthill, 2016).

Many features of these data are similar to previous observations in larger insects such as the locust, cockroach, and stick insect. For example, a single bristle on the locust leg can provide direct synaptic input to multiple classes of central neurons, and the spatial gradients of tactile sensory input in some of these neurons resemble the receptive fields of the midline spiking neurons in this study. In addition, a study in the locust described second-order somatosensory neurons that integrate touch with signals from leg chordotonal neurons. Another described a central neuron that integrates bristle signals from ipsilateral and contralateral legs. The morphologies of some of the neurons identified in this study resemble the morphologies of previously described locust neurons, including the ascending intersegmental neurons and the midline local neurons (Tuthill, 2016).

By using genetic techniques to identify, target, and manipulate specific neuron populations, this study builds upon these previous results in several ways. First, population-level two-photon calcium imaging allowed estimation of the total number and distribution of central neurons that process touch and these results to be situated within that map. Second, optogenetic tools allowed fully cataloging of the inputs to each central neuron class from different genetically defined mechanoreceptor types and systematically investigating how these inputs are integrated. Third, by recording from the same genetically identified neurons in multiple individuals, it was possible to build up a cumulative picture of each cell class and make explicit comparisons between classes. In the future, because all these neurons are genetically identifiable, it should be possible to trace their output connections, and to identify their functional role within the broader context of sensory and motor circuits in the VNC. By combining the classic advantages of insect neurophysiology with new genetic tools, Drosophila should prove a useful complement to other model organisms in dissecting the fundamental mechanisms of somatosensory processing (Tuthill, 2016).

A neural basis for categorizing sensory stimuli to enhance decision accuracy

Sensory stimuli with graded intensities often lead to yes-or-no decisions on whether to respond to the stimuli. How this graded-to-binary conversion is implemented in the central nervous system (CNS) remains poorly understood. This study shows that graded encodings of noxious stimuli are categorized in a decision-associated CNS region in the ventral cord of Drosophila larvae, and then decoded by a group of peptidergic neurons for executing binary escape decisions. GABAergic inhibition gates weak nociceptive encodings from being decoded, whereas escalated amplification through the recruitment of second-order neurons boosts nociceptive encodings at intermediate intensities. These two modulations increase the detection accuracy by reducing responses to negligible stimuli whereas enhancing responses to intense stimuli. These findings thus unravel a circuit mechanism that underlies accurate detection of harmful stimuli (Hu, 2020).

This study identified a neural network that categorizes noxious stimuli of graded intensities to generate binary escape decisions in Drosophila larvae, and a gated amplification mechanism was unraveled that underlies such binary categorization. In responding to the noxious stimuli, whereas failure in prompt responses may cause harm, excessive escape responses to negligible stimuli would lead to the loss of resources for survival. The gated amplification mechanism could reduce the responses to negligible stimuli whereas enhancing the responses to intense stimuli. In this way, the accuracy in deciding whether to escape from the stimuli is enhanced (Hu, 2020).

Information processing in the nervous system is affected by noise, which may be embedded in external sensory stimuli (e.g., sensory noise) or generated within the nervous system (e.g., electric noise). A recent study in C. elegans shows that activation mediated by electrical synapses and disinhibition mediated by glutamatergic chemical synapses form an AND logic gate to integrate the presentation of the salience of attractive odors (Dobosiewicz, 2019). The AND-gate computation in worm AIA interneurons requires multiple sensory neurons to report the presence of attractive odors and, consequently, filters out the noise embedded in the sensory stimuli. Another study on the olfactory system of adult Drosophila reported a mechanism to address the noise that is produced within the nervous system. A three-layered feedforward network averages the noise to enhance peak detection accuracy and then uses coincidence detection to distinguish real signals arrived synchronously from noise caused by spontaneous neural activities. In the nervous system, the noise can be produced at each stage of the sensori-motor transformation. Compared with the two mechanisms mentioned above, which filter out the existing noise, the graded-to-binary conversion through the gated amplification mechanism reported in this study makes the converted signals less vulnerable to the noise produced at later stages of sensori-motor transformation. This is because after the graded signals become binary, the signals are more separated (either suppressed or amplified) according to stimulus intensities and, consequently, the same level of noise is less likely to cause the binary signals to falsely pass the decision threshold than the graded ones. As a result, the ambiguous encoding range of the stimulus intensity is narrowed and the frequency of false decisions is reduced, as demonstrated by computational modeling (Hu, 2020).

Thresholding of gradually accumulated sensory evidence has been considered to be fundamental for generating yes-or-no decisions. For example, a recent study in mammals has shown that visual evidence of danger can be gradually accumulated by recurrent circuits to overcome the threshold for escape behaviors. Such a mechanism takes time to build up decision-associated activities for decisions with higher accuracy, which leads to the well-known speed-accuracy trade-off in perceptual decision making. However, the current findings add a new dimension to the processing of sensory evidence for perceptual decision making: different from recurrent networks, the recruitment of a number of SONs can instantaneously boost the decision-associated activity to reach the decision threshold, which ensures decision speed. Because the gated amplification mechanism reported here also ensures the detection accuracy, such a mechanism might bypass the speed-accuracy trade-off in sensory signal detection (Hu, 2020).

An electron microscopy connectome study reported 13 types of second-order neurons (SONs) in the Drosophila larval nociceptive system, each of which has distinct connectivity and functions. For example, Basin-4, DnB, and Wave neurons also receive mechanosensory inputs (Burgos, 2018; Takagi, 2017; Ohyama, 2015), whereas A08n does not. Moreover, Wave neurons detect stimulus positions on larval body walls. Furthermore, serotonergic modulation acts on this network during development to adjust the nociceptive responses, providing a mechanism for larvae to adjust the escape threshold according to their developmental environment. However, because at least 5 types of SONs are both required and sufficient for larval escape behaviors, it remains a mystery why there exist so many seemingly redundant neurons at the same level in the network. The nociceptive system is a dedicated protective system that responds to potential tissue-damaging insults, so both speed and accuracy of the perceptual decision-making process are important. This is probably why the nociceptive system uses an amplification network formed by a large number of SONs to dissociate time from accuracy in the perceptual decision-making process and avoid the trade-off between decision speed and accuracy (Hu, 2020).

Novel unbiased computational toolsets were developed for automatically analyzing the functional connectivity of all neural structures, including both somas and neurites in the larval VNC. Using these toolsets, a decision-associated CNS region, the PMC, was identified that covers the neuropil structure TP. The TP is concentrated with large amounts of neurites, especially those of peptidergic neurons. Although this anatomical structure was identified previously, its function is unknown. The finding of this study of its important function in sensori-motor transformation suggests that this region is possibly a hub for information exchange and integration. The detailed anatomical and functional connectivity of the TP could be a fascinating direction for future studies (Hu, 2020).

In summary, this study postulates a neural basis for converting graded sensory inputs to yes-or-no behavioral decisions. A previous study showed that neurons in the rat posterior parietal cortex encode a graded value of accumulating evidence whereas those in the prefrontal cortex have a more categorical encoding that indicates the decisions. Thus, the categorization of sensory evidence by making graded encodings binary in perceptual decision making is likely an evolutionarily conserved process. In this study, advantage was taken of the powerful genetic model Drosophila to unravel how such computation might be implemented at the cellular and molecular level. Finally, because whole-CNS functional imaging analysis is a key approach to decipher the neural basis for sensori-motor integration and perceptual decision making, it is anticipated that the computational tools developed in this study will facilitate investigations in these fields (Hu, 2020).

Vps54 Regulates Lifespan and Locomotor Behavior in Adult Drosophila melanogaster

Vps54 is an integral subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans-Golgi network (TGN). A destabilizing missense mutation in Vps54 causes the age-progressive motor neuron (MN) degeneration, muscle weakness, and muscle atrophy observed in the wobbler mouse, an established animal model for human MN disease. It is currently unclear how the disruption of Vps54, and thereby the GARP complex, leads to MN and muscle phenotypes. To develop a new tool to address this question, this study has created an analogous model in Drosophila by generating novel loss-of-function alleles of the fly Vps54 ortholog (scattered/scat). Null scat mutant adults are viable but have a significantly shortened lifespan. Like phenotypes observed in the wobbler mouse, this study shows that scat mutant adults are male sterile and have significantly reduced body size and muscle area. Moreover, this study demonstrates that scat mutant adults have significant age-progressive defects in locomotor function. Interestingly, sexually dimorphic effects are seen, with scat mutant adult females exhibiting significantly stronger phenotypes. Finally, it was shown that scat interacts genetically with rab11 in MNs to control age-progressive muscle atrophy in adults. Together, these data suggest that scat mutant flies share mutant phenotypes with the wobbler mouse and may serve as a new genetic model system to study the cellular and molecular mechanisms underlying MN disease (Wilkinson, 2021).

Localization of muscarinic acetylcholine receptor-dependent rhythm-generating modules in the Drosophila larval locomotor network

This study explored how muscarinic acetylcholine receptor (mAChR)-modulated rhythm-generating networks are distributed in the central nervous system (CNS) of soft-bodied Drosophila larvae. Fictive motor patterns were measured in isolated CNS preparations, using a combination of Ca(2+) imaging and electrophysiology while manipulating mAChR signaling pharmacologically. Bath application of the mAChR agonist oxotremorine potentiated bilaterally asymmetric activity in anterior thoracic regions and promoted bursting in posterior abdominal regions. Application of the mAChR antagonist scopolamine suppressed rhythm generation in these regions and blocked the effects of oxotremorine. Oxotremorine triggered fictive forward crawling in preparations without brain lobes. Oxotremorine also potentiated rhythmic activity in isolated posterior abdominal CNS segments as well as isolated anterior brain and thoracic regions, but it did not induce rhythmic activity in isolated anterior abdominal segments. Bath application of scopolamine to reduced preparations lowered baseline Ca(2+) levels and abolished rhythmic activity. Overall, these results suggest that mAChR signaling plays a role in enabling rhythm generation at multiple sites in the larval CNS (Jonaitis, 2022).

Mutually exclusive expression of sex-specific and non-sex-specific fruitless gene products in the Drosophila central nervous system

The fruitless gene of Drosophila produces multiple protein isoforms, which are classified into two major classes, sex-specific Fru proteins (FruM) and non-sex specific proteins (FruCOM). Whereas FruM proteins are expressed in ∼2000 neurons to masculinize their structure and function, little is known about FruCOM's roles. As an attempt to obtain clues to the roles of FruCOM, this study compared expression patterns of FruCOM and FruM in the central nervous system at the late larval stage. Nearly all neuroblasts were found to express FruCOM but not FruM, whereas a subset of ganglion mother cells and differentiated neurons express FruM but not FruCOM. It is inferred that FruCOM proteins support fundamental stem cell functions, contrasting to FruM proteins, which play major roles in sex-specific differentiation of neurons (Sato, 2022).

Serotonin distinctly controls behavioral states in restrained and freely moving Drosophila

When trapped in a physical restraint, animals must select an escape strategy to increase their chances of survival. After falling into an inescapable trap, they react with stereotypical behaviors that differ from those displayed in escapable situations. Such behaviors involve either a wriggling response to unlock the trap or feigning death to fend off a predator attack. The neural mechanisms that regulate animal behaviors have been well characterized for escapable situations but not for inescapable traps. This study reports that restrained vinegar flies exhibit alternating flailing and immobility to free themselves from the trap. Optogenetics and intersectional genetic approaches were used to show that, while broader serotonin activation promotes immobility, serotonergic cells in the ventral nerve cord (VNC) regulate immobility states majorly via 5-HT7 receptors. Restrained and freely moving locomotor states are controlled by distinct mechanisms. Taken together, this study has identified serotonergic switches of the VNC that promote environment-specific adaptive behaviors (Gowda, 2023).

Specification of the Drosophila Orcokinin A neurons by combinatorial coding

The central nervous system contains a daunting number of different cell types. Understanding how each cell acquires its fate remains a major challenge for neurobiology. The developing embryonic ventral nerve cord (VNC) of Drosophila melanogaster has been a powerful model system for unraveling the basic principles of cell fate specification. This pertains specifically to neuropeptide neurons, which typically are stereotypically generated in discrete subsets, allowing for unambiguous single-cell resolution in different genetic contexts. The specification of the OrcoA-LA neurons, characterized by the expression of the neuropeptide Orcokinin A and located laterally in the A1-A5 abdominal segments of the VNC, was studied. The progenitor neuroblast (NB; NB5-3) and the temporal window (castor/grainyhead) that generate the OrcoA-LA neurons were identified. The role of the Ubx, abd-A, and Abd-B Hox genes in the segment-specific generation of these neurons was studied. Additionally, these results indicate that the OrcoA-LA neurons are "Notch Off" cells, and neither programmed cell death nor the BMP pathway appears to be involved in their specification. Finally, a targeted genetic screen was performed of 485 genes known to be expressed in the CNS and nab, vg, and tsh were identified as crucial determinists for OrcoA-LA neurons. This work provides a new neuropeptidergic model that will allow for addressing new questions related to neuronal specification mechanisms in the future (Rubio-Ferrera, 2023).

Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila

Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. These genes were found to function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. The results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance (Scott, 2023).

Protein glycosylation, the most common type of posttranslational modification, plays numerous important biological roles, and regulates molecular and cell interactions in animal development, physiology, and disease. The addition of sialic acid (Sia), i.e., sialylation, has prominent effects due to its negative charge, bulky size, and terminal location of Sia on glycan chains. Essential roles of sialylated glycans in cell adhesion, cell signaling, and proliferation have been documented in many studies. Sia is intimately involved in the function of the nervous system. Mutations in genes that affect sialylation are associated with neurological symptoms in human, including intellectual disability, epilepsy, and ataxia due to defects in sialic acid synthase (N-acetylneuraminic acid synthase [NANS]), sialyltransferases (ST3GAL3 and ST3GAL5), the CMP-Sia transporter (SLC35A1), and CMP-Sia synthase (CMAS). Polysialylation (PSA) of NCAM, the neural cell adhesion molecule, one of the best studied cases of sialylation in the nervous system, is involved in the regulation of cell interactions during brain development. Non-PSA-type sialylated glycans are ubiquitously present in the vertebrate nervous system, but their functions are not well defined. Increasing evidence implicates these glycans in essential regulation of neuronal signaling. Indeed, N-glycosylation can affect voltage-gated channels in different ways, ranging from modulation of channel gating to protein trafficking, cell surface expression, and recycling/degradation. Similar effects were shown for several other glycoproteins implicated in synaptic transmission and cell excitability, including neurotransmitter receptors. Glycoprotein sialylation defects were also implicated in neurological diseases, such as Angelman syndrome and epilepsy. However, the in vivo functions of sialylation and the mechanisms that regulate this posttranslational modification in the nervous system remain poorly understood (Scott, 2023).

Drosophila has recently emerged as a model to study neural sialylation in vivo, providing advantages of the decreased complexity of the nervous system and the sialylation pathway, while also showing conservation of the main biosynthetic steps of glycosylation (Koles, 2009; Scott, 2014). The final step in sialylation is mediated by sialyltransferases, enzymes that use CMP-Sia as a sugar donor to attach Sia to glycoconjugates (see Schematic of the sialylation pathways in vertebrate and Drosophila. Unlike mammals that have 20 different sialyltransferases, Drosophila possesses a single sialyltransferase, DSiaT, that has significant homology to mammalian ST6Gal enzymes. The two penultimate steps in the biosynthetic pathway of sialylation are mediated by sialic acid synthase (also known as NANS) and CMP-sialic acid synthetase (CSAS, also known as CMAS), the enzymes that synthesize sialic acid and carry out its activation, respectively. These enzymes have been characterized in Drosophila and found to be closely related to their mammalian counterparts. In vivo analyses of DSiaT and CSAS demonstrated that Drosophila sialylation is a tightly regulated process limited to the nervous system and required for normal neural transmission. Mutations in DSiaT and CSAS phenocopy each other, resulting in similar defects in neuronal excitability, causing locomotor and heat-induced paralysis phenotypes, while showing strong interactions with voltage-gated channels (Repnikova, 2010; Islam, 2013). DSiaT was found to be expressed exclusively in neurons during development and in the adult brain (Repnikova, 2010). Intriguingly, although the expression of CSAS has not been characterized in detail, it was noted that its expression appears to be different from that of DSiaT in the embryonic ventral ganglion (Koles, 2009), suggesting a possibly unusual relationship between the functions of these genes. This study tested the hypothesis that CSAS functions in glial cells, and that the separation of DSiaT and CSAS functions between neurons and glia underlies a novel mechanism of glia-neuron coupling that regulates neuronal function via a bipartite protein sialylation (Scott, 2023).

In vertebrates, phosphorylated sialic acid is produced by N-acetylneuraminic acid synthase (Neu5Ac-9-P synthase, or NANS) from N-acetyl-mannosamine 6-phosphate (ManNAc-6-P), converted to sialic acid (Scott, 2023).

Glial cells have been recognized as key players in neural regulation. Astrocytes participate in synapse formation and synaptic pruning during development, mediate the recycling of neurotransmitters, affect neurons via Ca2+ signaling, and support a number of other essential evolutionarily conserved functions. Studies of Drosophila glia have revealed novel glial functions in vivo. Drosophila astrocytes were found to modulate dopaminergic function through neuromodulatory signaling and activity-regulated Ca2+ increase. Glial cells were also shown to protect neurons and neuroblasts from oxidative stress and promote the proliferation of neuroblasts in the developing Drosophila brain. The metabolic coupling between astrocytes and neurons, which is thought to support and modulate neuronal functions in mammals, is apparently conserved in flies. Indeed, Drosophila glial cells can secrete lactate and alanine to fuel neuronal oxidative phosphorylation. In the current work, a novel mechanism id described of glia-neuron coupling mediated by a unique compartmentalization of different steps in the sialylation pathway between glial cells and neurons in the fly nervous system. This study explored the regulation of this mechanism and demonstrate its requirement for neural functions (Scott, 2023).

Single-cell type analysis of wing premotor circuits in the ventral nerve cord of Drosophila melanogaster

To perform most behaviors, animals must send commands from higher-order processing centers in the brain to premotor circuits that reside in ganglia distinct from the brain, such as the mammalian spinal cord or insect ventral nerve cord. How these circuits are functionally organized to generate the great diversity of animal behavior remains unclear. An important first step in unraveling the organization of premotor circuits is to identify their constituent cell types and create tools to monitor and manipulate these with high specificity to assess their function. This is possible in the tractable ventral nerve cord of the fly. To generate such a toolkit, a combinatorial genetic technique (split-GAL4) was used to create 195 sparse driver lines targeting 198 individual cell types in the ventral nerve cord. These included wing and haltere motoneurons, modulatory neurons, and interneurons. Using a combination of behavioral, developmental, and anatomical analyses, the cell types targeted in this collection was systematically characterized. Taken together, the resources and results presented in this study form a powerful toolkit for future investigations of neural circuits and connectivity of premotor circuits while linking them to behavioral outputs (Ehrhardt, 2023).

A Drosophila glial cell atlas reveals a mismatch between transcriptional and morphological diversity

glia display diverse morphologies, both across and within glial classes. This study explored the relationship between glial morphology and transcriptional signature using the Drosophila CNS, where glia are categorised into 5 main classes (outer and inner surface glia, cortex glia, ensheathing glia, and astrocytes). Single-cell RNA sequencing data of Drosophila glia were analysedin 2 well-characterised tissues from distinct developmental stages containing distinct circuit types: the embryonic ventral nerve cord (VNC) (motor) and the adult optic lobes (sensory). This analysis identified a new morphologically and transcriptionally distinct surface glial population in the VNC. However, many glial morphological categories could not be distinguished transcriptionally, and indeed, embryonic and adult astrocytes were transcriptionally analogous despite differences in developmental stage and circuit type. While extensive within-class transcriptomic diversity was detected for optic lobe glia, this could be explained entirely by glial residence in the most superficial neuropil (lamina) and an associated enrichment for immune-related gene expression. In summary, this study generated a single-cell transcriptomic atlas of glia in Drosophila, and this extensive in vivo validation revealed that glia exhibit more diversity at the morphological level than was detectable at the transcriptional level (Lago-Baldaia, 2023).

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Genes involved in organ development

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