The Interactive Fly

Genes involved in tissue and organ development

Ventral Nervous system (page 1 | page 2)

  • Genes involved in neurogenesis of the ventral nervous system
  • Lateral views of Drosophila CNS
  • Embryonic origins of a motor system: Motor dendrites form a myotopic map in Drosophila
  • Developmental origins and architecture of Drosophila leg motoneurons
  • A size principle for recruitment of Drosophila leg motor neurons
  • Morphological identification and development of neurite in Drosophila ventral nerve cord neuropil
  • Programmed cell death in the embryonic central nervous system of Drosophila melanogaster
  • Extensive and diverse patterns of cell death sculpt neural networks in insects
  • sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system
  • Evolutionarily conserved anterior expansion of the central nervous system promoted by a common PcG-Hox program
  • Drosophila voltage-gated sodium channels are only expressed in active neurons and are localized to distal axonal initial segment-like domains

    Temporal gene expression and CNS development
  • Temporal control of the development of neural sublineages
  • Control the temporal sequence of neuroblast specification
  • The role of Dichaete in transcriptional regulation during Drosophila embryonic development
  • Global programmed switch in neural daughter cell proliferation mode triggered by a temporal gene cascade
  • Drosophila embryonic type II neuroblasts: origin, temporal patterning, and contribution to the adult central complex
  • Chromatin remodeling during in vivo neural stem cells differentiating to neurons in early Drosophila embryos
  • Specification of individual adult motor neuron morphologies by combinatorial transcription factor code
  • Neuronal cell fate diversification controlled by sub-temporal action of Kruppel
  • Neuronal cell fate specification by the convergence of different spatiotemporal cues on a common terminal selector cascade
  • Gene regulatory networks in Drosophila early embryonic development as a model for the study of the temporal identity of neuroblasts
  • A novel temporal identity window generates alternating Eve(+)/Nkx6(+) motor neuron subtypes in a single progenitor lineage

  • Linking neuroblasts to their corresponding lineage, a site carried by Flybrain.
  • Chris Doe's Hyper-neuroblast map.

    Genes involved in neurogenesis of the central nervous system

    *** indicates a special link to information about gene involvement in CNS neuroblast cell fate



    Embryonic origins of a motor system: Motor dendrites form a myotopic map in Drosophila

    The organisational principles of locomotor networks are less well understood than those of many sensory systems, where in-growing axon terminals form a central map of peripheral characteristics. Using the neuromuscular system of the Drosophila embryo as a model and retrograde tracing and genetic methods, principles underlying the organisation of the motor system have been uncovered. Dendritic arbors of motor neurons, rather than their cell bodies, are partitioned into domains to form a myotopic map, which represents centrally the distribution of body wall muscles peripherally. While muscles are segmental, the myotopic map is parasegmental in organisation. It forms by an active process of dendritic growth independent of the presence of target muscles, proper differentiation of glial cells, or (in its initial partitioning) competitive interactions between adjacent dendritic domains. The arrangement of motor neuron dendrites into a myotopic map represents a first layer of organisation in the motor system. This is likely to be mirrored, at least in part, by endings of higher-order neurons from central pattern-generating circuits, which converge onto the motor neuron dendrites. These findings will greatly simplify the task of understanding how a locomotor system is assembled. These results suggest that the cues that organise the myotopic map may be laid down early in development as the embryo subdivides into parasegmental units (Landgraf, 2003).

    The analysis began by correlating the positions of motor neuron dendrites with the distribution of their muscle targets in the periphery. Motor neurons were retrogradely labelled in a pairwise fashion and the positions of their dendritic arbors were mapped. Because of an interest in the mechanisms that underlie the assembly of the motor system, focus was placed on stages when each motor neuron first establishes a characteristic domain of arborisation within the neuropile (early stage 17, 15h after egg-laying [AEL]) (Landgraf, 2003).

    Motor axons project into the muscle field via two main nerves, the intersegmental (ISN) and the segmental nerve (SN). The transverse nerve (TN) runs along the segment border and has few motor axons. Choice of nerve root is one of several features that divide the motor neurons into two principal sets, the ISN and SN. (1) The cell bodies of SN motor neurons are located in the same segment as the muscles that they innervate, whereas ISN motor neuron somata are located in the segment next anterior (with the exception of the RP2 and two neuromodulatory efferent ventral unpaired median [VUM] neurons. (2) ISN motor neurons innervate internal muscles, which span a segment from anterior to posterior, whereas SN (and the TN) motor neurons innervate external muscles. External muscles are distinct from the internal set in several respects: (1) they are generally transverse; (2) unlike internal muscles, they require wingless (wg) signalling for their specification; (3) external (but not internal) muscles and their innervating motor neurons express the cell adhesion molecule (CAM) Connectin, with the single exception of muscle ventral transverse 1 (VT1) (Landgraf, 2003 and references therein).

    In addition, ISN and SN motor neurons elaborate their dendrites in distinct regions of the neuropile. Dendrites of ISN motor neurons occupy a domain extending posteriorly from the posterior part of one neuromere into the anterior part of the next. SN motor neuron dendrites occupy a domain that lies between the domains of ISN motor neuron arbors (Landgraf, 2003).

    Thus, the organisation of the body wall muscles into internal and external sets is reflected centrally in patterns of motor neuron arborisations. The innervating motor neurons project their axons through different nerves and elaborate their dendritic fields in distinct regions of the neuropile. Although dendritic arbors become progressively more elaborate and extensive over developmental time, their separate domains remain clearly recognisable and appear to be maintained at least until the motor system is fully functional (18 h AEL) (Landgraf, 2003).

    Having established that there is a central representation of the muscle field, the organisation of the motor neuron dendrites was analyzed in greater detail. (1) The set of external muscles and their innervating (SN) motor neurons were examined. Muscles of similar anteroposterior positions, such as the ventral acute muscle (VA3) and the segment border muscle (SBM), are innervated by motor neurons whose dendritic arbors lie in a common region of the neuropile. Conversely, motor neurons supplying the anterior (lateral transverse 1-2 [LT1-LT2]) versus the posterior (SBM) muscles have dendritic arbors that are correspondingly separated in the anteroposterior axis of the CNS (Landgraf, 2003).

    To put the idea of a regular map to the test, focus was placed on an unusual external motor neuron-muscle pair. Muscle VT1 is innervated by a TN rather than an SN motor neuron. However, VT1 lies at the same place in the anteroposterior axis as the SBM, although VT1 is ventral and the SBM more dorsal. The VT1 motor neuron dendritic field is found to overlaps with that of the SBM motor neuron. For the external set, it is concluded that differences in target muscle location in the anteroposterior axis are mapped centrally as regular differences in dendritic position, but dorsoventral distinctions are not (Landgraf, 2003).

    It was next asked whether there is a similarly regular representation of the internal muscles in the developing CNS. While most external muscles are transverse and have unique anteroposterior locations, the internal muscles span the width of a segment so that positional distinctions between them are solely in the dorsoventral axis. It was found that the set of internal muscles is represented centrally by three dendritic domains. Motor neurons innervating ventral internal muscles elaborate their dendritic arbors in the anterior half of the ISN dendritic domain. Motor neurons with dorsolateral internal muscle targets (lateral longitudinal [LL] 1, dorsal acute [DA] 3, dorsal oblique 3-5 [DO3-DO5]) put their arbors into the posterior part of the ISN dendritic domain. Finally, dorsal muscles are represented by a motor neuron dendritic domain that lies between those representing ventral (anterior) and dorsolateral (posterior) internal muscle groups. Thus, the internal muscles are represented in the neuropile by three domains of dendritic arborisation that reflect their different dorsoventral locations in the periphery. Once again, it is concluded that there is a regular mapping of muscle position in the neuropile: in this case, it is positions in the dorsoventral axis peripherally that are represented centrally as differences in the anteroposterior locations of dendrites (Landgraf, 2003).

    To test the idea that dendritic arbor positions relate to the distribution of muscles, an atypical motor neuron-muscle pair was examined. The RP2 motor neuron is reported to innervate dorsal muscle DA2, yet its dendrites span the domains that represent both dorsal and dorsolateral internal muscles. However, on careful analysis it was found that DA2 is, in fact, specifically innervated by a U neuron whose dendrites lie in the dorsal internal domain, whereas the RP2 axon forms endings generally on all dorsolateral and dorsal muscles by 19 h AEL. These seem to correspond to the type 1s boutons found in late larvae. Thus, the RP2 neuron puts its dendrites into a region of the neuropile that does indeed represent its targets, namely the dorsolateral and dorsal internal muscles (Landgraf, 2003).

    Like the muscle field itself, the map of motor neuron dendrites is metamerically repeated. However, the boundaries of these two units are out of register with one another, since the dendrites of the motor neurons innervating internal muscles lie in the next anterior neuromere. The anterior border of the dendritic map, as defined by the extent of these anterior dendrites, coincides with the anterior margin of engrailed (en) expression. Thus, while the muscles are segmental in their organisation, the domains occupied by the dendrites of their innervating motor neurons are parasegmental (Landgraf, 2003).

    To test whether genes that implement the parasegmental pattern in the epidermis are also required for the formation of the parasegmental organisation of the neuromuscular system, the formation of SN and ISN dendritic fields was studied in embryos singly mutant for the following segment polarity genes: en/invected (Df(enE)), wg (wgCX4), naked (nkd2), patched (ptc9), hedgehog (hh21), and gooseberry (Df2R(gsb)). Every one of the six different mutants that were analysed has partially aberrant patterns of neuroblasts (NBs). Nevertheless, SN and ISN motor neurons still form and can be identified by their characteristic axonal projections into the periphery. In addition, it was found that the fundamental separation between SN and ISN dendritic domains is present despite often severe perturbations in CNS structure. For example, in gsb mutant embryos, both nerve roots are frequently fused so that the SN and ISN share a common CNS exit point. Nevertheless, SN and ISN axons as well as their dendritic fields do not intermingle but remain separate. These results suggest that the subdivision of the neuropile into the principal ISN and SN dendritic domains is a robust feature of the system, which appears to be specified early in development, since the embryo subdivides into parasegmental units (Landgraf, 2003).

    It was next asked what mechanisms underlie the formation of the myotopic map. Because ISN and SN motor neurons lie at different positions in the CNS and their axons grow out into the muscle field through different nerves, it is reasonable to suppose that at least the major subdivision of dendritic arborisations into internal and external domains could be a byproduct of the locations at which the motor neurons are generated and the paths taken by their growing axons. This ‘passive mapping' explanation can be excluded by considering a single motor neuron-muscle pair, namely dorsal transverse 1 (DT1) and its innervating motor neuron. DT1 is an external muscle (by position, orientation, wg dependence, and Connectin expression), yet its motor neuron is clustered with the internal muscle innervating set and its axon (uniquely for the external muscles) grows out through the ISN. Despite its packing within the ‘internal motor neuron' set, the DT1 motor neuron makes a long posterior projection through the internal muscle domain of the myotopic map to reach the external domain, where it arborises appropriately, reflecting the orientation and external nature of its target muscle. In contrast, motor neurons derived from the same NB as DT1 innervate neighboring internal muscles DO3-DO5 and put their dendrites in a more anterior region characteristic of the dorsolateral muscles. These findings strongly suggest that the mapping of the muscle field within the CNS is an active process of growth and arborisation that partitions dendrites into subdomains of the neuropile that are appropriate to their function, rather than a passive subdivision of available space by position of origin or axon trajectory (Landgraf, 2003).

    Since dendritic arbors form after motor axons have reached their targets, the muscles could be instrumental in dictating the organisation of the central map. To test this idea, the UAS/GAL4 system was used to misexpress an activated form of Notch (Kidd et al. 1998) in the developing mesoderm, suppressing the formation of muscle founder cells while leaving other tissues intact. In such muscleless embryos, the main nerve trunks, SN and ISN, still form and project into the periphery. Retrograde labellings of these nerves show that SN and ISN motor neurons form relatively normal dendritic arbors that consistently conform to the characteristic separation of SN and ISN dendrites. Thus, the neuropile is partitioned into distinct fields of dendritic arborisation independently of the muscles. It is concluded that the mapping process is likely to be an autonomous property of the motor neurons and their neighboring cells (Landgraf, 2003).

    It was next asked whether motor neuron dendritic fields could be patterned by the substrates on which they grow. In the Drosophila ventral nerve cord (VNC), motor neuron dendrites form in the dorsal-most region of the neuropile, sandwiched between longitudinal glia above and the underlying scaffold of axons. Glial cells can act as substrates for supporting and guiding axonal growth. To test whether they might also be required for the growth and spatial patterning of dendritic fields, dendritic arbors were analysed in glial cells missing (gcm) mutant embryos, which are defective in glial cell differentiation. Although the structure of the nervous system is disrupted in gcm mutant embryos and the dendritic arbors are abnormal, they continue to form in their characteristic locations and the fundamental distinction between the ISN and SN motor neuron dendritic fields is maintained. Remarkably, even the long posterior dendritic projection of the DT1 motor neuron forms and reaches its target region, the SN external muscle dendritic domain. These results suggest that the patterning of the neuropile into distinct motor neuron dendritic domains is a process that appears to be intrinsic to the motor neurons and their neighboring neurons, but does not require proper glial cell differentiation (Landgraf, 2003).

    One likely explanation for the division of dendrites into separate domains is that there is a process of mutual exclusion between the arborisations of neighboring cells. Such a process of dendritic ‘tiling' has so far only been documented between particular classes of sensory neurons, but could also occur in the motor system. The idea of tiling was tested by considering two groups of motor neurons whose axons have a common trajectory, but whose dendritic fields form in adjacent territories. The DO3-DO5 and DT1 motor neurons project their dendrites posteriorly, and at their most-anterior point, these dendrites meet the axons and dendrites of the anterior corner cell (aCC) and U/CQ neurons. To show whether the aCC and U/CQ axons and/or dendrites inhibit the growth of DO3-DO5 and DT1 dendrites anteriorly, these neurons (as well as RP2 and the posterior corner cell [pCC] interneuron) were selectively ablated. Using anti-Even-skipped (Eve) staining as a marker for aCC, RP2, and U/CQs (there are an additional two medially located eve-expressing interneurons, pCC and friend of pCC [fpCC], it was found that these neurons can be selectively ablated before they form dendrites (at approximately 11 h AEL): on average, by 10.5 h AEL all but 0.6 and by 12 h AEL all but 0.06 of the seven medially located eve-expressing neurons have been ablated per half-neuromere. In no instance was a concomitant anterior expansion of the DO3-DO5 and DT1 motor neuron dendrites into the regions vacated by the aCC and U/CQ dendrites observed. It is concluded that, at least in this instance, the initial dendritic territory of one set of motor neurons (DO3-DO5 and DT1) is not defined by a process of tiling, in which they are excluded by neighboring (aCC and U/CQ) dendritic arbors. However, it is possible that the elaboration of motor neuron dendritic arbors during later developmental stages may involve interactions between neighboring dendritic territories, activity-dependent processes, or both (Landgraf, 2003).

    Thus, in summary, these results suggest that the mechanisms that subdivide the neuropile into distinct dendritc domains are very robust and refractory to perturbations. They further suggest that the cues that organise the map may be laid down early in development as the embryo subdivides into parasegmental units (Landgraf, 2003).

    The patterning of the motor neuron dendritic arbors in the Drosophila embryo represents a first layer of organisation in the motor system. This is likely in part to be mirrored by the endings of higher-order neurons of central pattern generating circuits, which converge onto the myotopic map. While motor neuron cell body positions may, as has been proposed for vertebrate systems, relate to the ontogeny of target muscles, the operation of mature muscles is reflected by the allegiance of corresponding motor neuron dendrites to a particular territory in the neuropile. Thus, changes in muscle operation could be accommodated by a change of allegiance of the appropriate motor neuron dendrites from one domain to another (e.g., the DT1 motor neuron-muscle pair) without the need for rewiring the underlying higher-order circuitry. Such a model resolves the apparent discrepancy between the distributions of motor neuron cell bodies centrally and target muscles in the periphery. It also implies a considerable degree of flexibility, particularly at the level of motor output, yet suggests that elements of the underlying motor circuitry may have been highly conserved (Landgraf, 2003).

    Developmental origins and architecture of Drosophila leg motoneurons

    Motoneurons are key points of convergence within motor networks, acting as the 'output channels' that directly control sets of muscles to maintain posture and generate movement. This study used genetic mosaic techniques to reveal the origins and architecture of the leg motoneurons of Drosophila. A small number of leg motoneurons are born in the embryo but most are generated during larval life. These postembryonic leg motoneurons are produced by five neuroblasts per hemineuromere, and each lineage generates stereotyped lineage-specific projection patterns. Two of these postembryonic neuroblasts generate solely motoneurons that are the bulk of the leg motoneurons. Within the largest lineage, lineage 15, distinct birth-order differences are seen in projection patterns. A comparison of the central projections of leg motoneurons and the muscles they innervate reveals a stereotyped architecture and the existence of a myotopic map. Timeline analysis of axonal outgrowth reveals that leg motoneurons reach their sites of terminal arborization in the leg at the time when their dendrites are elaborating their subtype-specific shapes. These findings provide a comprehensive description of the origin, development, and architecture of leg motoneurons that will aid future studies exploring the link between the assembly and organization of connectivity within the leg motor system of Drosophila (Brierley, 2012).

    In insects that undergo a complete metamorphosis, like Drosophila, the ventral nerve cord is produced by two distinct phases of neurogenesis. The first wave occurs during embryonic development and produces the components required for the control of larval behavior. Some of the neurons generated at this time remodel and play a role in adult circuits. The bulk of neurons found in the adult fly are produced during the second, more prolonged neurogenic phase during larval and early pupal life. Within this process, this study determined whether the leg motoneurons are produced during the embryonic or postembryonic phases of neurogenesis (Brierley, 2012).

    This work identified two distinct types of motoneuron clones in the third instar larva VNC, generated by embryonic heatshocks. One type has complex, highly branched dendrites with axons that exit the nerve cord and terminate on body wall muscles. This type of neuron in insects is uniquely identifiable and can have one of two different fates during metamorphosis; some remodel and take up a new adult-specific role, whereas others undergo programmed cell death. It was not possible to determine the identity, number, or fate of specific embryonic neurons using their larval morphology alone. However, it is known that in the beetle Tenebrio molitor and the moth Manduca sexta the larval leg motoneurons remodel to become adult leg motoneurons. The second type of motoneuron clones had a simple morphology in the third instar CNS reminiscent of single-cell postembryonic clones born during larval life. This second type of neuron is similar to the flight motoneuron MN5 and the persistent Broad positive neurons seen in the embryonic CNS. These neurons remain in an immature state throughout larval life, before completing their development during metamorphosis (Brierley, 2012).

    To quantify how many types of leg motoneuron are born during the embryonic wave of neurogenesis, MARCM clones were induced in the embryo, and then single-cell motoneuron clones were identified in the adult ventral nervous system with axons in the leg. These data suggest that at least seven different leg motoneuron types are born in the embryo. Using this approach their origins or how many of each type there are could not be determined. These seven types could represent progeny from seven different neuroblasts. A previous study, Baek (2009), predicted that 13 lineages generate leg motoneurons in the embryo, but their data, like the current, cannot definitively answer this question. Although it is believed that these different neurons are bona fide types, it is possible that a single motoneuron could generate two very different terminal arborizations. This seems unlikely, as all the data points toward a high degree of morphological stereotypy in embryonic motoneurons. In future the availability of many more cell type-specific markers should enable identification each of these early born neurons (Brierley, 2012).

    The data reveal that neurons born during larval life make the most significant contribution to the pool of leg motoneurons. These leg motoneurons are generated by five postembryonic Nbs. Of these, two lineages generate exclusively motoneurons: lineage 15, which contains on average 28 motoneurons, and lineage 24 which contains six motoneurons. This confirms the observations of Baek (2009), who also found these lineages. Three postembryonic lineages were found that contain one or two motoneurons along with a large number of interneurons (lineages 20, 21, and 22), whereas Baek (2009) only reported one. The motoneurons within these lineages are born soon after the onset of postembryonic neurogenesis, with the first ganglion mother cell (GMC) generating two siblings, a motoneuron and a local interneuron. Following this, every time a GMC divides the motoneuron sibling undergoes apoptosis, whereas the interneuron survives. Such hemilineage-based programs of cell death play a significant role in determining the type and number of network components in the thoracic nervous system of Drosophila (Brierley, 2012).

    Most knowledge of the origins of Drosophila motoneurons comes from studies in the embryo. Clonal analysis in the embryo revealed that 17 of the 31 Nbs generate motoneurons and all are born early within these lineages, with most Nbs contributing one or two motoneurons, and at most six. If every embryonic born neuron is derived from a different Nb then the maximum number of lineages generating leg motoneurons would be 12, compared with six if all are derived from a single Nb. Baek (2009) concluded that 13 lineages contribute leg motoneurons (Brierley, 2012).

    The general organization of Drosophila leg motoneurons within the CNS shows great similarity with that of the grasshopper Schistocerca americana, with the neurons being clustered into groups. Each of these eight groups are presumably derived from their own single Nb, with the primary neurites inserting into characteristic position in the neuropil (Brierley, 2012).

    The success of holometabolous insects as a group is due largely to their ability to produce radically different body plans at larval and adult stages that allow them to exploit very different ecological niches. Some of the most striking adaptations within the Holometabola are seen in the articulated appendages, particularly the legs. How the developmental programs that control leg motoneuron connectivity have been modified is likely to be very interesting and may provide insights into evolution of neural networks. This census of Drosophila motoneurons is likely to help with comparative studies on leg motoneurons from other insect species (Brierley, 2012).

    Regardless of the exact number of lineages that generate leg motoneurons, it is striking that just two postembryonic lineages contribute the bulk of the leg motoneurons (34 of the 47) and this raises the question of whether there is something fundamentally different about leg motoneuron specification compared with what was already know from studies in the embryo (Brierley, 2012).

    One of the most notable differences between the larval and adult musculoskeletal system is that the muscles of adults are multifiber and often innervated by a number of isomorphic neurons. The dendrites of most leg motoneurons are located ipsilaterally and elaborate into the same neuromere in which they are born, unlike in the embryo where motoneurons can have extensive contralateral dendrites and are parasegmental in their nature. This difference in organization, i.e., segmental vs. parasegmental dendrites, could be an adaptation for larval locomotion, where the control of the next adjacent segment is critical (Brierley, 2012).

    Although there is general agreement between many of the current findings and those of Baek (2009), there are some differences in detail, which may have important implications. Unlike Baek (2009), this study found that the largest leg motoneuron lineage, lineage 15, also innervates muscles in the body wall as well as intrinsic muscles in the femur and the tibia. Lineage 15 therefore has the most extensive coverage along the proximodistal axis of the leg and does not have a distal bias, as previously suggested (Baek, 2009). The extrinsic muscles in the body wall are extremely important, as they control the bodywall/coxal joint, which is in effect a universal joint allowing the leg a near 360 rotation. Although this study has presented a more complete picture of these motoneurons, more work is needed to identify the origins of the other motoneurons that innervate this complex group of muscles. Motoneurons within lineage 24 innervate muscles in the coxa, trochanter, and femur and control the movement of the femur and tibia. In this study no neuron from this lineage was seen innervating the tibia reductor muscle group, or indeed any other glutamatergic leg motoneurons terminating on two distinct muscle targets, as suggested by Baek (2009). The three lineages, lineages 20, 21, and 22, all innervate muscle groups in the coxa (Brierley, 2012).

    What this lineage analysis highlighted is that within the CNS the dendrites of each of the postembryonic lineages each occupy distinct territories along the mediolateral, anteroposterior, and dorsoventral axes. This is important, as it is known from studies on the leg sensory system that the central afferent projections of different classes of sensory neuron occupy distinctive volumes within the dorsoventral axis depending on their modality (Brierley, 2012).

    Lineage 15 has the most medially projecting dendrites located in both the anterior and posterior regions of the neuropil; the dendrites of motoneurons from lineage 24 motoneurons take up more lateral territories, whereas the central projections from lineages 20, 21, and 22 occupy the most lateral neuropil domains and span the anteroposterior axis. These lineage-specific patterns are reproducible, with no obvious variation in the muscles innervated or with a significant difference in the size or morphology of the axonal arborizations. This observation emphasizes that decoding lineage-specific programs of morphogenesis is likely to hold the key for understanding the development and organization of motoneurons within the leg network (Brierley, 2012).

    To explore these motor lineages in more detail, individual neurons were visualized using the MARCM technique to determine how motoneuron birth date is correlated with aspects of morphology. As well as birth-dating, the single-cell clones allowed a close look at the relationship between muscle innervation and dendrite shape (Brierley, 2012).

    In lineage 15, the sequential production was found of at least five distinct motoneuron subtypes during larval life. The first-born neuron innervates a muscle in the bodywall, the next subtype targets a muscle in the proximal femur, with the following subtype targeting a muscle in the proximal tibia. The next subtype innervates targets in the distal femur and then the distal tibia. Thus, there is no simple proximal to distal filling up of the leg, based on the birth-date of neurons; instead, neurons that innervate the most proximal target of a leg segment are born first. The central projections of these motoneuron subtypes were also very stereotyped, with the dendrites of early born cells spanning medial to lateral territories and late-born cells elaborating their dendrites in the lateral and ventral neuropil. Lineage 24 also shows a stereotyped birth-order based pattern of innervation along the proximodistal axis of the leg. It was found that lineage 24 generates three subtypes during larval life with both early and late-born neurons innervating the same muscle group located in the coxa and having dendrites that target lateral regions within the CNS. The second and third subtypes target the trochanter and the femur, respectively (Brierley, 2012).

    It is interesting to speculate how a lineage like 15 may have evolved from an ancestral condition. The first motoneuron subtype innervates a body wall muscle and the next the long tendon muscle located in the femur. The long tendon muscle, also called the unguis retractor, attaches to the apodeme that controls the most distal element in the leg, the pretarsus. Could it be that these early born neuron subtypes are the most 'ancient' within the lineage, while the sequential addition of the later subtypes occurred as new leg segments were introduced? It would be intriguing to look at the homologous neurons in different outgroups (Brierley, 2012).

    The long tendon muscle motoneurons are also unique among the glutamatergic leg motoneurons, as they are the only ones that elaborate dendrites in the contralateral hemineuromere. It is worthy of note that there are more long tendon muscle group (ltm) motoneurons than any other leg motoneuron. This is probably due to the need for the precise control of the pretarsal claw, which is fundamental to all locomotory and nonlocomotory behavior involving the leg. The difference in the birth-order of neuron types between the different lineages is also striking. Rigid birth-order-based rules that control the targeting of terminal processes have been described for other types of secondary neurons, including the antennal lobe projection neurons found in the fly's olfactory system. The sequential production of different neuron subtypes at distinct times during development is a common mechanism for generating the diversity of circuit components in many taxa, including vertebrates. In flies, there is strong evidence that individual Nbs express a sequence of progenitor transcription factors, such as Hunchback, Kruppel, Pdm, and Castor, which in turn regulate the postmitotic transcription factors to specify a distinct identity. The differences observed between neuronal birth-date and the dendritic and axonal arborizations in lineages 15 and 24 could be due to similar transient and sequential expression of temporally controlled transcription factors, like those observed in embryonic lineages or by other transcription factors such as Chinmo and Broad, which are deployed within postembryonic neuron subtypes. Although most studies in insects emphasize stereotyped lineage-specific specification a recent report describes how local interneuron populations within the Drosophila antennal lobe can have great morphological variability. It may be that particular neuronal classes, such as those that transfer information between one part of the nervous system and another, are more developmentally hard-wired than elements that perform mainly local processing (Brierley, 2012).

    The data show that soma location is not an important descriptor of identity, but rather the location of their dendritic terminals. Taken as a whole, this work reveals that, although there is great stereotypy, there is no simple organizing principle that translates birthdate into projection pattern, i.e., that early born neurons innervating proximal leg segments and late born neurons targeting distal ones. Solving lineage-based codes within this system is likely to hold the key to understanding fundamental rules about how networks are assembled (Brierley, 2012).

    Understanding how ordered patterns of synaptic connectivity are established between motoneurons and the rest of the motor network is a fundamental question in neurobiology. Landgraf (2003) revealed that the dendrites of motoneurons in the Drosophila embryo are organized to reflect the innervation of muscles in the periphery. They forwarded the idea that different territories within such a 'myotopic map' reflect patterns of connectivity with premotor elements and that such maps could be a general organizational principle of all motor systems. This study was directed to establishing whether leg motoneurons generate the same kind of myotopic map and thus explore the generality of this compelling idea (Brierley, 2012).

    It was found that the dendrites of leg motoneurons occupy a large volume of the leg neuropil and showed a considerable degree of overlap, even though each occupies a slightly different volume. This is in marked contrast to the myotopic map seen in the embryo, where dendrites appear to generate exclusive, nonoverlapping territories: i.e., the dendrites of motoneurons that innervate internal muscles segregate into a different neuropilar domain from those innervating external muscles, alongside which the motoneurons of the internal muscles organize themselves into a map representing the dorsoventral axis of the body wall. This difference in organization may be due to differences in the skeleton and the biomechanics of the two systems. The adult leg is a complex multijointed appendage with many degrees of freedom, where interjoint coordination between and within legs is of paramount importance. In contrast, the fly larva locomotes using simple peristaltic waves of the abdominal wall and head turns. The organization of Drosophila leg motoneuron dendrites mirrors that of vertebrate somatic motoneurons in the spinal cord, where each motoneuron type has a dendritic arborization that covers a distinctive territory but at the same time has considerable overlap with the dendrites of other types (Brierley, 2012).

    To step back from this, a systematic and unbiased analysis was performed of leg motoneuron dendrite position where the prothoracic neuropil was divided into sectors and the location of the arborizations of 13 different types was measured using single-cell MARCM clones. This approach allowed determination of the volume that each of the different motoneuron types samples within the neuropil and how the different types relate to each other. The dendrogram generated shows how closely related the different subtypes are. Any branch can be reversed around a node point, and the relatedness of different arborizations can be inferred using this. Using five neurons for each type helped provide a robust measure of the similarities/differences between the different motoneurons. It was found the 13 motoneuron types clustered into nine sets. Motoneurons of the same subtype tended to group together in most examples. Some motoneurons that innervate functionally related muscles also clustered together, e.g., the ltm1 and ltm2 muscles located in the femur and the tibia. It was also found that motoneurons that innervate the tibia levator and the tibia reductor muscle also formed a group: these are synergists (Brierley, 2012).

    As a counterpoint to this, a number of motoneurons were seen that innervate antagonistic muscles cluster together, e.g., the trochanter levator and trochanter depressor muscles both located in the coxa being one set, and the tarsal levator and tarsal depressor muscles that control the tarsal segments being another. Baek found four sets that clustered together;they found the motoneurons that innervate the long tendon muscles in both the femur and tibia grouped together, as did antagonistic motoneurons that innervate the coxal segment. They also saw two different types of trochanter neurons cluster, which this study did not have data for. Baek suggested that different reductors clustered together but the neuron they proposed to innervate the tibia reductor does not. This study took the clustering data and remapped this onto the neuropil. It shows clearly the large overlap of the dendrites of many of the different motoneurons. What does this overlap mean functionally? First, it is important to emphasize that just because neurites occupy the same space, judged by light microscopy, it does not mean they make connections with each other or, as in this case, receive the same types of input. In the brachial spinal cord of the bullfrog Rana catesbeiana sensory axons from the triceps brachii muscle make connections with triceps motoneurons but do not innervate subscapularis and pectoralis motoneurons, which are in very close proximity. The monosynaptic connections between triceps brachii motoneurons and sensory neurons appears relatively late in development, after the dendrites have grown into a territory that contains an extant presynaptic terminal field (Brierley, 2012).

    The specific connection occurs then as soon as the motoneuron arrives. Importantly, it says that if the terminals are not within a territory they cannot make connections with inputs there. The occurrence of pre- and postsynaptic elements in space is thus necessary but not sufficient for connectivity. Other examples in the vertebrate spinal cord show that there are considerable similarities in the morphology of somatic motoneuron dendrites within large parts of their arborization, but that key differences in specific regions can occur. A good example of this is seen in the dorsal dendrites in the lumbar motoneurons of the turtle Pseudemys scripta elegans, where such specialized differences in dendrite morphology might reflect a difference in synaptic input or the processing of input. The finding that the dendrites of Drosophila motoneurons that innervate antagonistic muscle pairs are similar is interesting (Brierley, 2012).

    Are such dendritic organizations important for interpreting information from similar inputs? Future work exploring connectivity using physiological approaches should allow us to address whether this is important for function or for determining patterns of connectivity during development. Although there is no simple 'easy to read' map, what the data shows is that there are robust topological relationships between these dendritic arborizations (Brierley, 2012).

    As described above, there exists a diversity of dendritic and axonal projection patterns within neural maps. A key question raised by this is how do neurons within such maps ensure that both the axonal and dendritic terminals execute appropriate programs of morphogenesis. We now know that dendrites, like axons, use conserved molecular cues and various transmembrane receptors to attain their distinct organizations. One possible mechanism for generating a diversity of dendrite shapes could be retrograde signaling from the target muscle. It was of interest to look at the relative timing of axon and dendrite outgrowth in this system to see if this could be possible. The data from lineage 15 reveals that two subtypes, which innervate different long tendon muscle sets, have nearly identical dendritic trees, but their axons target muscles in different segments of the leg. The timeline data shows that motoneuron axon outgrowth in the proximal leg occurs at the same time as dendritic elaboration in the CNS. This opens the possibility that retrograde signals may play a role in the development of neurons that innervate muscle targets in the leg (Brierley, 2012).

    How modular are these programs? These events must be controlled at some level by transcription factor codes regulating blends of guidance receptors. Cell intrinsic temporal transcription factors can control a combinatorial code of postmitotic transcription factors. Feedback from the muscle field could also provide patterning information as seen in the vertebrate spinal cord, where motoneuron dendrite arborizations are controlled in part by the transcription factor Pea3, which is induced by retrograde signaling from target muscles. The respective timing of outgrowth of Drosophila leg motoneuron axons and dendrites opens this up as a possibility. Previously, laser ablation studies revealed that the dendrites of Drosophila flight motoneurons change their growth following the removal of their target muscle. It will be interesting to test this hypothesis experimentally by removing the muscle targets in the leg and quantifying the dendritic arborizations of known motoneurons (Brierley, 2012).

    This study has explored the origins and architecture of the leg motoneurons of Drosophila using genetic mosaic techniques.A small number of leg motoneurons are born in the embryo, but the majority are generated during larval life. These postembryonic leg motoneurons are produced by five Nbs, where the progeny of each lineage generates stereotyped, lineage-specific projection patterns. The dendrites of Drosophila leg motoneurons show similarities with spinal cord motoneurons where different types have a considerable degree of overlap but each has unique regions that it targets. These data reveal that even though there is no simple 'easy-to-read' leg myotopic map, the central projections of leg motoneurons and muscles they innervate manifest robust topological relationships. Understanding the functional relationships within this map and the molecular mechanisms that control its development will provide insights into the way ordered patterns of connectivity are established within neural networks (Brierley, 2012).

    A size principle for recruitment of Drosophila leg motor neurons

    To move the body, the brain must precisely coordinate patterns of activity among diverse populations of motor neurons. This study used in vivo calcium imaging, electrophysiology, and behavior to understand how genetically-identified motor neurons control flexion of the fruit fly tibia. Leg motor neurons exhibit a coordinated gradient of anatomical, physiological, and functional properties. Large, fast motor neurons control high force, ballistic movements while small, slow motor neurons control low force, postural movements. Intermediate neurons fall between these two extremes. This hierarchical organization resembles the size principle, first proposed as a mechanism for establishing recruitment order among vertebrate motor neurons. Recordings in behaving flies confirmed that motor neurons are typically recruited in order from slow to fast. However, this study also found that fast, intermediate, and slow motor neurons receive distinct proprioceptive feedback signals, suggesting that the size principle is not the only mechanism that dictates motor neuron recruitment. Overall, this work reveals the functional organization of the fly leg motor system and establishes Drosophila as a tractable system for investigating neural mechanisms of limb motor control (Azevedo, 2020).

    Dexterous motor behaviors require precise neural control of muscle contraction to coordinate force production and timing across dozens to hundreds of muscles. This coordination is mediated by populations of motor neurons, which translate commands from the central nervous system into dynamic patterns of muscle contraction. Although motor neurons are the final common output of the brain, the scale and complexity of many motor systems have made it challenging to understand how motor neuron populations collectively control muscles and thus generate behavior. For example, a human leg is innervated by over 150,000 motor neurons and a single calf muscle is innervated by over 600 motor neurons. How can the nervous system coordinate the activity of such large motor neuron populations to flexibly control the force, speed and precision of limb movements (Azevedo, 2020)?

    One way to streamline motor control is to establish a hierarchy among neurons controlling a particular movement, such as flexion of a joint. This hierarchy allows premotor circuits to excite different numbers of motor neurons depending on the required force: motor neurons controlling slow or weak movements are recruited first, followed by motor neurons that control progressively stronger, faster movements. A recruitment order for vertebrate motor neurons innervating a single muscle was first postulated over 60 years ago. Subsequent work identified mechanisms associated with the recruitment order and synthesized these findings as the size principle, which states that small motor neurons, with lower spike thresholds, are recruited prior to larger neurons, which have higher spike thresholds. Evidence for the size principle has been provided by electrophysiological analysis of motor neurons in a number of species, from crayfish to humans. These studies have also described systematic relationships between motor neuron electrical excitability, recruitment order, conduction velocity, force production, and strength of sensory feedback and descending input (Azevedo, 2020).

    A simplifying assumption of the size principle is that all motor neurons within a pool receive identical presynaptic input, such that recruitment order is entirely dictated by the gradient of motor neuron excitability. However, recordings in vertebrates have found that presynaptic inputs may vary within a motor pool and that recruitment order can change during specific movements. Are these violations of the size principle a fundamental feature of motor control circuits or rare exceptions to the rule? Answering this question would be greatly aided by a tractable system in which it was possible to genetically target identified motor neurons for recording, manipulation, and mapping of presynaptic inputs (Azevedo, 2020).

    The leg of the fruit fly, Drosophila melanogaster, contains 14 muscles which are innervated by just 53 motor neurons. In spite of this tiny scale, the fly leg supports a variety of fast and flexible behaviors. During forward walking, the fly grips the substrate with the distal segment of its front leg (the tarsus) and flexes the femur-tibia joint, pulling the body forward. The femur-tibia joint of a walking fly flexes and extends 10-20 times per second, reaching swing speeds of several thousand degrees per second. Flies also use their legs to target other body parts during grooming, for social behaviors like aggression and courtship, and to initiate flight take-off and landing. These behaviors require a wide range of muscle force production across multiple timescales. However, little is currently known about the organization and function of leg motor control circuits in the fly. While a great deal of progress has been made on understanding the processing of sensory signals in the Drosophila brain, understanding of how this information is translated into behavior by the fly's ventral nerve cord (VNC) is lacking. Investigating motor control in Drosophila is important because the fly's compact nervous system and identified cell types make it a tractable system for comprehensive circuit analysis (Azevedo, 2020).

    This study has investigated motor control of the Drosophila tibia. First the organization of tibia flexor motor units was mapped using calcium imaging from leg muscles in behaving flies. With electrophysiology, it was then discovered that motor neurons controlling tibia flexion lie along a gradient of anatomical and physiological properties that correlate with muscle force production. Slow motor neurons produce <0.1 μN per spike, while fast motor neurons produce ~10 μN per spike, approximately equal to the fly's weight. Recordings during spontaneous leg movements revealed a recruitment hierarchy: slow motor neurons typically fire first, followed by intermediate, then fast neurons. Interestingly, all tibia flexor motor neurons receive feedback from proprioceptors at the femur/tibia joint, but these sensory signals vary in amplitude, sign, and dynamics across the different motor neuron types. Optogenetic manipulation of each motor neuron type had unique and specific effects on the behavior of walking flies, consistent with their roles in controlling distinct force regimes (Azevedo, 2020).

    Together, these data establish the organization and function of a key motor control module for the fly leg. Overall, this study found that motor neurons controlling the fly tibia exhibit many features consistent with the size principle. However, it was also observed that tibia motor neurons receive distinct proprioceptive feedback signals and that recruitment order is occasionally violated. Thus, in addition to the size principle, heterogeneous input from premotor circuits is likely to play an important role in coordinating neural activity within a motor pool (Azevedo, 2020).

    This study discovered a gradient of properties that co-varies across the motor pool. At one end of the spectrum is the fast motor neuron, which has a larger diameter axon and dendrites, low input resistance, weak sensory input, and produces enough force with each spike to support the fly's entire body weight. At the other end, the slow motor neuron has fine axons and dendrites and high input resistance; each spike in the slow motor neuron produces <1% the force of a fast motor neuron spike. The slow motor neuron also has a high spontaneous firing rate that maintains resting muscle tension, and it receives strong proprioceptive feedback that continuously modulates its firing rate (Azevedo, 2020).

    Leg motor neurons are recruited in a specific order: small, low gain motor neurons that generate weak forces are recruited first, and larger, more powerful motor neurons are recruited later. Consistent with this recruitment order, proprioceptive feedback has the greatest effect on the slow motor neuron: tibia movements of less than 1° significantly impact slow motor neuron firing. By comparison, proprioceptive feedback has a much smaller effect on the firing rate of intermediate and fast motor neurons. Fly behavior is also quite sensitive to aberrant optogenetic recruitment of motor neurons. For example, flies fail to initiate walking if unable to recruit slow motor neurons at the bottom of the recruitment hierarchy (Azevedo, 2020).

    A relationship between force production and recruitment order is a common feature of vertebrate motor systems. This organization has been proposed to confer a number of computational and energetic advantages. Recruitment of additional motor units increases force nonlinearly, overcoming suppressive nonlinearities in spike rates and muscle force production. Furthermore, the relative increase in force does not decrease with successive recruitment, as it would if all motor units produced similar amounts of force. Thus, much like Weber's law describes the constant sensitivity to relative stimulus intensity, a recruitment hierarchy maximizes the resolution of motor unit force while also simplifying the dimensionality of the motor system (Azevedo, 2020).

    A key assumption of the original size principle is that all motor neurons within a pool receive the same presynaptic input; the alternative is that different premotor neurons provide input to different subsets of motor neurons. The results of this study suggest the motor system controlling the fly tibia operates in a middle ground between these two extremes. Although tibia motor neurons generally follow a recruitment order in accordance with the size principle, it was found that the dynamics of proprioceptive feedback vary across motor neurons within the pool. A small but significant proportion of behavioral events were observed in which recruitment order was violated. These data suggest that although the tibia flexor motor neuron pool may share some presynaptic inputs, they are not identical. Thus, motor control of the fly tibia is more complex than a straightforward implementation of the size principle (Azevedo, 2020).

    Similar exceptions to the strict dogma of the size principle have been observed in vertebrate species. For instance, the effect of proprioceptive feedback varies across a pool of motor neurons controlling the cat leg. During rapid escape behaviors in zebrafish, slow motor neurons can completely drop out of the population firing pattern. Muscles controlling human fingers can change recruitment order based on movement direction. These examples suggest that some behaviors exhibit more degrees of freedom than can be supported by recruitment of motor neurons based on their intrinsic excitability alone (Azevedo, 2020).

    Investigations of the vertebrate spinal cord have also identified circuit motifs that support flexible control of motor neurons within a pool. For example, recordings in turtles and zebrafish have shown that motor neurons receive coincident excitation and inhibition, which could underlie selective de-recruitment. Zebrafish spinal cord premotor neurons provide patterned inhibition to speed-specific circuits and can flexibly switch between different motor neuron recruitment patterns (Azevedo, 2020).

    This characterization of identified tibia motor neurons provides a handle to investigate similar premotor circuit motifs for flexible limb motor control in the fly. Little is currently known about the leg premotor circuitry in Drosophila. However, the hypotheses generated by this work should soon be testable using connectomic reconstruction of identified motor neurons and their presynaptic inputs in the Drosophila VNC (Azevedo, 2020).

    The muscles that control flexion of the Drosophila tibia are innervated by approximately 15 motor neurons. This study chose to analyze three identified neurons that span the extremes of this motor pool. Each of these motor neurons was stereotyped across flies in its anatomy, physiology, and function. The fast tibia flexor motor neuron labeled by R81A07-Gal4 (associated with HGTX) is the only motor neuron that innervates the large tibia flexor muscle fibers in the middle of the femur. Because it has the largest axon of any motor neuron in the femur, the fast motor neuron also produces the largest extracellular spikes. The slow tibia flexor motor neuron labeled by R35C09-Gal4 (associated with Zfh1) is one of 8-9 motor neurons that innervates tibia flexor muscle fibers located at the distal tip of the femur. Measurements of force production from other motor neurons that innervated this distal region suggest that the R35C09-Gal4 neuron is among the weakest within this group. Finally, an intermediate motor neuron labeled by R22A08-Gal4 (associated with nAcRalpha-96Aa) was studied that innervates muscle fibers separate from the fast and slow neurons. Previous anatomical studies suggest that 2-5 neurons innervate nearby muscle fibers in the proximal region of the femur. Recordings of other tibia flexor motor neurons were consistent with the gradient of properties that are describe in detail for three identified motor neurons suggesting that the relationship between anatomy, physiology, and force production applies to other neurons in the tibia flexor motor pool (Azevedo, 2020).

    The structure of the leg motor system in Drosophila has several similarities to other well-studied walking insects. In the metathoracic leg of the locust, a single tibia flexor muscle is innervated by nine motor neurons that were also classified into three groups: fast, intermediate, and slow. Different motor neurons within this pool lie along a gradient of intrinsic properties and are sensitive to different types of proprioceptive feedback, such as position vs. velocity or fast vs. slow movements. The femur of the stick insect is innervated by flexor motor neurons that were also described as slow, semi-fast, and fast, based on their intrinsic properties and firing patterns during behavior (Azevedo, 2020).

    Much of the work in bigger insects and crustaceans has focused on the most reliably identifiable neurons, the fast and slow extensor tibiae (FETi and SETi), antagonists to the more diverse tibia flexor motor neurons. Consequently, details of how sensory feedback and local interneurons recruit specific subsets of flexor motor neurons has been relatively understudied. The current work exemplifies the advantage of using Drosophila genetics to identify cell types, and even individual cells, within a diverse motor pool. Genetic markers have also recently been identified for tibia extensor motor neurons in flies, which will enable future investigation of premotor input to antagonist motor neurons (Azevedo, 2020).

    Flies differ from these other invertebrates in one major respect: while most arthropods possess GABAergic motor neurons that directly inhibit leg muscles, holometabolous insects such as Drosophila do not. Indeed, this study found that a transgenic line for GABAergic neurons (Gad1-Gal4) does not label any axons in the Drosophila leg. The presence of inhibitory motor neurons has been proposed as a key underlying reason why insects have been able to achieve flexible motor control with small numbers of motor neurons. That flies lack this capability means that other mechanisms must be at play. Fly legs are innervated by neurons that release neuromodulators, such as octopamine, which could underlie flexible tuning of muscle excitability (Azevedo, 2020).

    The large range of gain, or force production, at the femur-tibia joint implies that different subgroups of flexor neurons will be recruited during distinct behaviors. Activity in slow motor neurons produces small, low force movements, while fast motor neuron activity produces fast, high amplitude movements. Consistent with this division, the output of slow motor neurons is more strongly influenced by feedback from leg proprioceptors. Thus, proprioceptive feedback may continuously modulate the firing rate of slow motor neurons for precise stabilization of posture during standing or grooming. When the slow motor neuron is optogenetically activated, flies stop walking and extend their legs. The reason for this response is unclear but could reflect the fly's reaction to a loss of autonomous motor control (Azevedo, 2020).

    In contrast to the postural movements controlled by slow motor neurons, rapid, stereotyped movements like escape are likely to use fast motor neurons whose activity is less dependent on sensory feedback. This division of labor may also provide energy efficiency: slow contracting muscle fibers use aerobic metabolism and take advantage of energy stores in the form of glycogen, while fast muscle fibers are anaerobic and lack energy stores, leading to more rapid fatigue (Azevedo, 2020).

    Even for a specific behavior, such as walking, different motor neurons may be recruited in different environments and contexts. Optogenetically activating intermediate motor neurons caused stationary flies to start walking and walking flies to walk faster. In a similar manner, stick insects walking on treadmills typically recruit fast motor neurons only during fast walking and, in that case, late in the stance phase. But as friction on the treadmill increases, fast neurons are recruited earlier during stance. Leg kinematics and force production also change as insects walk up or down inclines. When locusts and cockroaches are forced to walk upside-down, fast flexor neurons are recruited to allow the animal to grip the substrate. This context-dependence is not limited to walking: small changes in body posture can have a large effect on which motor neurons are recruited during target reaching movements in locusts (Azevedo, 2020).

    In this study, focus was placed on flexion of the femur-tibia joint of the fly's front leg. How might these results compare to motor neuron pools of antagonist muscles? Tibia flexor motor neurons outnumber the extensor neurons, and thus are likely to possess a shallower gradient of intrinsic and functional properties, as has been found in crayfish. Invertebrate muscles also exhibit polyneural innervation, such that a given muscle fiber may be innervated by multiple motor neurons. Polyneural innervation could allow independent activation of motor neurons in order to use the same muscle fibers during different contexts, or it could make the force produced by one motor neuron dependent on coincident activity in another motor neuron (Azevedo, 2020).

    This study has described an organizing architecture for motor control of tibia movement in Drosophila. These neurons constitute the output of motor circuits that flexibly control a wide range of fly behaviors, from walking to aggression to courtship. Studies of motor control in the fly VNC are now possible thanks to an increasing catalog of genetically-identified cell types, connectomic reconstruction with serial-section EM and the ability to image from VNC neurons in walking flies. The leg motor neurons described in this study also provide valuable targets for understanding the coordinated development of motor neuron and muscle properties. It is anticipated that Drosophila will be a useful complement to other model organisms in understanding the neural basis of flexible motor control. This study characterized the cellular morphology, electrical excitability, force production, and proprioceptive feedback among motor neurons that control flexion of the Drosophila tibia. A gradient of properties was discovered that co-varies across the motor pool. At one end of the spectrum is the fast motor neuron, which has a larger diameter axon and dendrites, low input resistance, weak sensory input, and produces enough force with each spike to support the fly's entire body weight. At the other end, the slow motor neuron has fine axons and dendrites and high input resistance; each spike in the slow motor neuron produces <1% the force of a fast motor neuron spike. The slow motor neuron also has a high spontaneous firing rate that maintains resting muscle tension, and it receives strong proprioceptive feedback that continuously modulates its firing rate (Azevedo, 2020).

    Morphological identification and development of neurite in Drosophila ventral nerve cord neuropil

    In Drosophila, ventral nerve cord (VNC) occupies most of the larval central nervous system (CNS). However, there is little literature elaborating upon the specific types and growth of neurites as defined by their structural appearance in Drosophila larval VNC neuropil. This study reports the ultrastructural development of different types VNC neurites in ten selected time points in embryonic and larval stages utilizing transmission electron microscopy. There are four types of axonal neurites as classified by the type of vesicular content: clear vesicle (CV) neurites have clear vesicles and some T-bar structures; Dense-core vesicle (DV) neurites have dense-core vesicles and without T-bar structures; Mixed vesicle (MV) neurites have mixed vesicles and some T-bar structures; Large vesicle (LV) neurites are dominated by large, translucent spherical vesicles but rarely display T-bar structures. We found dramatic remodeling in CV neurites which can be divided into five developmental phases. The neurite is vacuolated in primary (P) phase, they have mitochondria, microtubules or big dark vesicles in the second (S) phase, and they contain immature synaptic features in the third (T) phase. The subsequent bifurcate (B) phase appears to undergo major remodeling with the appearance of the bifurcation or dendritic growth. In the final mature (M) phase, high density of commensurate synaptic vesicles are distributed around T-bar structures. There are four kinds of morphological elaboration of the CVI neurite sub-types. First, new neurite produces at the end of axon. Second, new neurite bubbles along the axon. Third, the preexisting neurite buds and develops into several neurites. The last, the bundled axons form irregularly shape neurites. Most CVI neurites in M phase have about 1.5-3 microm diameter, they could be suitable to analyze their morphology and subcellular localization of specific proteins by light microscopy, and they could serve as a potential model in CNS in vivo development (Gan, 2014; PubMed).

    Programmed cell death in the embryonic central nervous system of Drosophila melanogaster

    Although programmed cell death (PCD) plays a crucial role throughout Drosophila CNS development, its pattern and incidence remain largely uninvestigated. This study provides a detailed analysis of the occurrence of PCD in the embryonic ventral nerve cord (VNC). The spatio-temporal pattern of PCD was traced and the appearance of, and total cell numbers in, thoracic and abdominal neuromeres of wild-type and PCD-deficient H99 mutant embryos were compared. Furthermore, the clonal origin and fate of superfluous cells in H99 mutants was examined by DiI labeling almost all neuroblasts, with special attention to segment-specific differences within the individually identified neuroblast lineages. These data reveal that although PCD-deficient mutants appear morphologically well-structured, there is significant hyperplasia in the VNC. The majority of neuroblast lineages comprise superfluous cells, and a specific set of these lineages shows segment-specific characteristics. The superfluous cells can be specified as neurons with extended wild-type-like or abnormal axonal projections, but not as glia. The lineage data also provide indications towards the identities of neuroblasts that normally die in the late embryo and of those that become postembryonic and resume proliferation in the larva. Using cell-specific markers it was possible to precisely identify some of the progeny cells, including the GW neuron, the U motoneurons and one of the RP motoneurons, all of which undergo segment-specific cell death. The data obtained in this analysis form the basis for further investigations into the mechanisms involved in the regulation of PCD and its role in segmental patterning in the embryonic CNS (Rogulja-Ortmann, 2007).

    In this analysis of PCD distribution it was found that, macroscopically, the CNS of wt and PCD-deficient (H99) embryos do not show large differences. These observations indicate that the supernumerary cells do not disturb developmental events in the CNS of H99 embryos, such as cell migration and axonal pathfinding. The glial cells mostly find their appropriate positions accurately. The DiI-labeled NB lineages were, in the majority of cases, easily identifiable based on their shape, position and axonal pattern, despite the supernumerary cells. The FasII pattern showed that the axonal projections form and extend along their usual paths. In fact, the supernumerary cells themselves are capable of differentiating i.e. expressing marker genes and extending axons, as shown by clones of several NBs and by cell marker expression analysis in H99 (e.g. NB7-3) (Rogulja-Ortmann, 2007).

    It has been shown that a large number of CNS cells undergo PCD during embryonic development. The distribution of activated Caspase-3-positive cells in wt embryos suggests that the death of some cells is under tight spatial and temporal control, as revealed by their regular, segmentally repeated occurrence. Other dying cells were rather randomly distributed, suggesting a certain amount of developmental plasticity. The overall counts of Caspase-3-positive cells give an estimate of the numbers of dying cells at a given time. They indicate that PCD becomes evident in the CNS at stage 11 and is most abundant in the late embryo (from stage 14). It is however difficult to estimate the total number of apoptotic cells throughout CNS development by anti-Caspase-3 labeling, because the cell corpses are removed fairly quickly. Therefore the total number of cells were counted per thoracic and abdominal hemineuromere in the late embryo. Comparison between stage 16 and stage 17 wt embryos indicates that 25-30 % of all cells are removed in both tagmata after stage 16, which in turn suggests that the total percentage of removed cells must be high, since PCD occurs at high levels already from stage 14 on. In comparison to the developing nervous system of C. elegans, where PCD removes about 10% of cells, and of mammals, where this number can be as high as 50-90%, PCD in the fly CNS appears to show an intermediate prevalence. This lends support to the hypothesis of an increasing contribution of PCD in shaping more advanced nervous systems during evolution (Rogulja-Ortmann, 2007).

    Comparisons between wt and H99 reveal, as expected, a greater number of cells in both tagmata of H99 embryos (151% increase in the thorax and 162% in the abdomen at stage 17). These additional cells in H99 may reflect the total number of cells normally undergoing cell death until stage 17. However, there is a large variability in the total number of cells, especially within the H99 strain. In wt embryos, it seems to be more pronounced in the thorax and at stage 17, which might be a consequence of variable amounts of PCD occurring until this stage. The even higher variability within the H99 strain (both in thorax and abdomen) is likely to reflect variable numbers of additional cell divisions. The great majority of abdominal NBs are normally removed by PCD after they have generated their embryonic progeny, whereas in the thoracic neuromeres most of the NBs enter quiescence at the end of embryogenesis and continue dividing as postembryonic NBs in larval stages. Thus, there are few mitoses occurring in the wt CNS from stage 16 onwards. BrdU labeling experiments revealed a high number of BrdU-positive cells in some H99 embryos injected at early stage 17. It is assumed that these are progeny of mitotic NBs and/or GMCs that survive and continue dividing, generating cells that do not exist in wt. Clones obtained by DiI labeling in H99 confirm this conclusion. The finding that surviving cells divide already in the embryo complement results that showed that, in reaper mutants, NBs in the abdominal neuromeres survive and generate progeny in larval stages (Rogulja-Ortmann, 2007).

    Among the DiI-labeled clones in H99 embryos, very few NB lineages were obtained which did not differ from their wt counterparts. The majority contained, as expected, supernumerary cells. In some cases axons projected by these cells could be identified, showing that they are specified as neurons. In fact, in three cases (NB4-2, NB5-3 and NB7-3), these additional cells were found to be specified as motoneurons. As additional axons within a fascicle were generally difficult to identify, it is possible that these are not the only lineages which make additional motoneurons in H99. Whether these cells are normally born and apoptose, or originate from additional divisions of surviving NBs or GMCs, cannot be determined from these experiments, but similar observations have been made for both cases. It is interesting that none of these cells, regardless of their origin, are specified as glia. No additional glia were observed in the NB clones in H99 embryos, and equal numbers of Repo-expressing glial cells were found in wt and H99. It is concluded that PCD occurs almost exclusively in neurons and/or undifferentiated cells, and that lateral glia are not produced in excess numbers in the embryo. Furthermore, because it is likely that NBs, which normally die, stay in a late temporal window in H99, one could speculate that NBs in this window normally do not give rise to glia. These results are not in agreement with the notion that LG are overproduced, and their numbers adjusted through axon contact. Occasional apoptotic LG have been observed and it is possible that the current method of counting does not allow a resolution fine enough to account for an occasional additional Repo-positive cell in H99 embryos. However, if LG were consistently overproduced, a higher number of glia in would be expected H99 embryos. It is assumed that LG cell death may reflect a small variability in the number of cells needed, and not a general mechanism for adjusting glial cell numbers (Rogulja-Ortmann, 2007).

    Generally, no difference was found between Repo-expressing glia numbers in wt and H99. However, a small difference does become apparent when one separates the total cell counts into those in the CNS and those in the periphery: 25.67±0.45 cells/hs and 28.42±0.64 cells/hs for wt and H99, respectively, were counted in the CNS, whereas 8.50±0.28 cells/hs and 6.35±0.82 cells/hs for wt and H99, respectively, were found in the periphery. The reasons for this difference might be the greater width of the CNS in H99 embryos, and that the cues required for proper migration of the peripheral glia are disturbed by additional cells. Alternatively, the difference might be due to differentiation defects in these cells (Rogulja-Ortmann, 2007).

    In addition to NB clones with too many cells and wild-type-like axon projections in H99, some lineages were obtained whose clones exhibited atypical projection patterns. These projections were found to belong both to motoneurons (e.g. in NB4-2) and interneurons (e.g. NB5-3, NB7-2 and NB-7-4). NB4-2 normally produces two motoneurons (RP2 and 4-2Mar) and 8-14 interneurons. In two out of three NB4-2 clones in H99 two additional motoneurons that project anteriorly were found, similar to RP2. One of the two clones was found in the thorax and had a normal cell number (16), whereas the other was abdominal and had too many cells (25). Thus, the two additional motoneurons are likely to be the progeny of divisions occurring in the wt, and not of an additional NB or GMC mitosis. The fact that the third NB4-2 clone (found in the abdomen and comprising 17 cells) did not show the same motoneuronal projections could be due to these cells not being differentiated at the time of fixation (clones of different ages were occasionally observed in the same embryo), or they may not have differentiated at all. It would be interesting to determine the target(s) of these additional motoneurons and thereby perhaps gain insight into physiological reasons for their death. However, such an experiment has to await tools that allow specifically labeling of the NB4-2 lineage, or these motoneurons, in the H99 mutant background (Rogulja-Ortmann, 2007).

    The other three lineages (NB5-3, NB7-2 and NB7-4) all have atypical interneuronal projections. The cells which these atypical axons belong to may represent evolutionary remnants that are not needed in the Drosophila CNS. Alternatively, they might have a function earlier in development and be removed when this function is fulfilled. Such a role has been shown for the dMP2 and MP1 neurons, which are born in all segments and pioneer the longitudinal axon tracts. At the end of embryogenesis these neurons undergo PCD in all segments except A6 to A8, where their axons innervate the hindgut. It is known that some cells of the NB5-3 lineage express the transcription factor Lbe, and that H99 mutants show about three additional Lbe-positive neurons per hemisegment, which mostly likely belong to NB5-3. The DiI-labeling results complement this finding in that four or more additional neurons were also found in H99 clones. The supernumerary Lbe-positive neurons in H99 could possibly be the ones producing the atypical axonal projections (Rogulja-Ortmann, 2007).

    In the wt embryo, only eight NB lineages show obvious tagma-specific differences in cell number and composition. Tagma-specific differences among serially homologous CNS lineages have been shown to be controlled by homeotic genes. Therefore, these lineages provide useful models for studying homeotic gene function on segment-specific PCD. In H99 embryos, further lineages were observed that were differently affected in the thorax and abdomen. How these tagma-specific differences arise in a PCD-deficient background is an interesting question. For example, NB4-3 shows a wild-type cell number in the thorax (8 and 12-13), but has too many cells in the abdomen (15, 15 and 22). There are a couple of plausible scenarios to explain this observation. (1) The development of the NB4-3 lineage, including the involvement of PCD, could actually differ in the thorax and abdomen of wt embryos, with the final cell number being similar by chance. The DiI-labeled clones allow determination of the final cell number, but do not reveal how this number is achieved. The difference would become obvious in an H99 mutant background, at least regarding the involvement of PCD. (2) This possibility does not exclude the first one, the thoracic NB4-3 could become a postembryonic NB (pNB) and the abdominal NB4-3 might undergo PCD after generating the embryonic lineage. In H99, the abdominal NB would be capable of undergoing a variable number of additional divisions to generate a variable number of progeny. This would easily explain larger discrepancies in cell number between individual clones in H99 (e.g. the abdominal NB4-3 clone with 22 cells), and is in agreement with occasional observations of H99 embryos with a very high CNS cell number per segment, and with the two observed classes of H99 embryos with high and low numbers of BrdU-positive cells (Rogulja-Ortmann, 2007).

    NB6-2 is another lineage whose clones differ in the two tagmata of H99 embryos. In this case, the abdominal clones showed no difference to their wt counterparts, whereas the thoracic clones did (18 and 19 cells). Although no difference in cell number between thoracic and abdominal clones was reported for this lineage, a rather large count range (8-16 cells) was given, which would allow for a thorax-specific PCD of two to three postmitotic progeny. Alternatively, the thoracic NB6-2 might undergo cell death upon generating its progeny, which would make it the first identified apoptotic NB in the thorax. When PCD is prevented, this NB may undergo a few additional rounds of division. The data obtained in these experiments do not counter this notion, but the number of clones obtained in the thorax was not sufficient to draw a definite conclusion. As the abdominal NB6-2 lineage in H99 did not differ from the one in wt, its NB may be one of the few abdominal postembryonic NBs (Rogulja-Ortmann, 2007).

    A specific set of NBs undergoes PCD in the late embryo, whereas surviving NBs resume proliferation in the larva as pNBs, after a period of mitotic quiescence. The identities of the individual NBs undergoing PCD versus those surviving as pNBs are still unknown. The sizes of NB lineages obtained in H99 embryos may provide hints for identifying candidate pNBs in the abdomen [12 NBs/hs in A1, four in A2 and three in A3 to A7, and NBs that undergo PCD in the thorax at the end of embryogenesis [seven NBs/hs in T1 to T3. In the abdomen, NB1-1a and NB6-2 are obvious candidates for pNBs, as they remained consistently unchanged in H99 embryos. Two other NBs, NB1-2 and NB3-2, are also potential abdominal pNBs as they mostly did not differ from their wt counterparts, and only occasionally contained one additional cell. On the other hand, clones which showed more than twice the cell number in H99 (NB2-1, NB5-4a and NB7-3) than in wt, strongly suggest that these NBs normally undergo PCD in the abdomen (but perform additional divisions in H99), because, even if one daughter cell of each GMC undergoes PCD, they still cannot account for all cells found in H99 clones (Rogulja-Ortmann, 2007).

    Regarding thoracic NBs, it can only be speculated on account of low sample numbers. NBs which seem to become pNBs in the thorax, as they showed no difference between wt and H99 clones, are NB3-2, NB4-3 and NB4-4. Potential candidates for NBs which do not become pNBs, but undergo PCD in the thorax, are expected to consistently have a significant increase in cell number in H99. These are NB5-1 and NB5-5. In addition, lineages for which one clone was obtained in H99 but which also showed many more cells in the thorax than normal are NB2-2t, NB5-4t and NB7-3 (Rogulja-Ortmann, 2007).

    In order to investigate the developmental signals and mechanisms involved in the regulation of PCD in the embryonic CNS, some of the apoptotic cells were identified which will be used as single-cell PCD models. These are the dHb9-positive RP neuron from NB3-1, Lbe-positive neurons from NB5-3, the Eg-positive GW neuron from NB7-3 and the Eve-positive U neurons from NB7-1. As not much is known about the dying RP motoneuron or the Lbe-positive neurons, the first goal will be to characterize each of these cells more closely, based on the combination of expressed molecular markers (Rogulja-Ortmann, 2007).

    Some of the dying NB7-3 cells are already known to be undifferentiated daughter cells of the second and third GMC, which undergo PCD shortly after birth. Notch has been identified as the signal initiating PCD. The surviving daughters receive the asymmetrically distributed protein Numb, which counteracts the PCD-inducing Notch signal. The same had been shown in a sensory organ lineage of the embryonic peripheral nervous system, where cells produced in two subsequent divisions undergo Notch-dependent PCD. Both the PCD in the NB7-3 lineage and in the sensory organ lineage require the Hid, rpr and grim genes. It will be interesting to see whether the Notch-Numb interaction also plays a role in the segment-specific PCD of the differentiated GW motoneuron, or if another signal is used for the removal of this, and possibly other, differentiated cells (Rogulja-Ortmann, 2007).

    The U motoneurons also show a segment-specific cell death pattern (they apoptose in A6 to A8), thus somewhat resembling the MP1 and dMP2 neurons. However, in contrast to MP1 and dMP2, the U neurons survive in the anterior segments and undergo PCD in the posterior ones. Whether homeotic genes play any role in the survival or death of these cells remains to be investigated (Rogulja-Ortmann, 2007).

    In summary, this study has presented descriptions of PCD in the developing CNS of the wt Drosophila embryo, and of the CNS of PCD-deficient embryos. The pattern of Caspase-dependent PCD is partly very orderly, suggesting tight spatio-temporal control of cell death, and partly random, which suggests a certain amount of plasticity already in the embryo. The CNS of PCD-deficient embryos is nevertheless well organized, despite the presence of too many cells. These superfluous cells come from both a block in PCD and from additional divisions that surviving NBs go through. It was possible to link the occurence of cell death to identified NB lineages by clonal analysis in PCD-deficient embryos, to uncover segment-specific differences, and to establish single-cell PCD models that will be used in further studies to investigate mechanisms responsible for controlling PCD in the embryonic CNS (Rogulja-Ortmann, 2007).

    Extensive and diverse patterns of cell death sculpt neural networks in insects

    Changes to the structure and function of neural networks are thought to underlie the evolutionary adaptation of animal behaviours. Among the many developmental phenomena that generate change programmed cell death appears to play a key role. This study shows that cell death occurs continuously throughout insect neurogenesis and happens soon after neurons are born. Mimicking an evolutionary role for increasing cell numbers, programmed cell death was artificially blocked in the medial neuroblast lineage in Drosophila melanogaster, which results in the production of 'undead' neurons with complex arborisations and distinct neurotransmitter identities. Activation of these 'undead' neurons and recordings of neural activity in behaving animals demonstrate that they are functional. Focusing on two dipterans which have lost flight during evolution this study revealed that reductions in populations of flight interneurons are likely caused by increased cell death during development. These findings suggest that the evolutionary modulation of death-based patterning could generate novel network configurations (Pop, 2020).

    sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system

    Neural progenitors typically divide asymmetrically to renew themselves, while producing daughters with more limited potential. In the Drosophila embryonic ventral nerve cord, neuroblasts initially produce daughters that divide once to generate two neurons/glia (type I proliferation mode). Subsequently, many neuroblasts switch to generating daughters that differentiate directly (type 0). This programmed type I>0 switch is controlled by Notch signaling, triggered at a distinct point of lineage progression in each neuroblast. However, how Notch signaling onset is gated was unclear. Sequoia (Seq), a C2H2 zinc finger transcription factor with homology to Drosophila Tramtrack and the positive regulatory domain (PRDM) family, has been identified as important for lineage progression. This study found that seq mutants fail to execute the type I>0 daughter proliferation switch, and also display increased neuroblast proliferation. Genetic interaction studies reveal that seq interacts with the Notch pathway, and seq furthermore affects expression of a Notch pathway reporter. These findings suggest that seq may act as a context-dependent regulator of Notch signaling, and underscore the growing connection between Seq, Ttk, the PRDM family and Notch signaling (Gunnar, 2016).

    In seq mutants, an increase was found in the number of cells in the NB5-6T and NB3-3A lineages, as well as aberrant daughter divisions in both lineages. This effect is mirrored globally, with elevated daughter divisions in both the thorax and abdomen. From these results it is concluded that seq plays a key role in promoting the type I>0 daughter proliferation switch. Surprisingly, it was found that seq overexpression also triggers aberrant type I>0 switches. These results could indicate that Seq expression levels are instructive, with high levels promoting the type I proliferation mode and lower levels promoting type 0. On that note, there is precedence for transcription factors switching between repressor and activator function in a concentration-dependent manner. Elevated NB proliferation was also observed in seq mutants and with seq overexpression, in NB3-3A and globally, indicating that the precise NB cell cycle exit at the end of lineage progression is affected by seq function and levels. It was found that seq represses CycE and E2f1, both in thoracic NBs in general and specifically in NB5-6T. Dap expression was weakly increased globally, but weakly reduced in NB5-6T, suggesting an aspect of context dependency with respect to seq regulation of Dap (Gunnar, 2016).

    Notch signaling also plays a key role in triggering the type I>0 switch, and Notch reporter [E(spl)HLHm8-GFP] expression is turned on in NBs during the latter stages of lineage progression (Ulvklo, 2012; Bivik, 2016). This indicates that the timing of Notch signaling onset is important for the precision of the type I>0 switch. Notch activation results in the formation of a tripartite protein complex comprising the Notch intracellular domain (NICD), the DNA-binding factor Su(H) and the co-factor Mastermind (Mam). Molecular and genetic in-depth analysis of how Notch signaling controls the type I>0 switch points to a multi-level model whereby NICD/Su(H)/Mam activates E(spl)HLH and dap, and E(spl)HLH subsequently represses CycE, E2f1 and stg (Bivik, 2016) (Gunnar, 2016).

    The findings point to a complex balancing interplay between seq, the Notch pathway and the cell cycle. Premature and elevated Notch reporter [E(spl)HLHm8-GFP] expression was observed in seq mutants, indicating that seq represses Notch signaling in NBs. However, seq mutants phenocopy Notch pathway perturbation, and both result in a failure to execute the type I>0 switch. Moreover, seq and E(spl)HLH interact strongly genetically in transheterozygotes, as is evident by increased Ap cell numbers. Based on these findings, it is proposed that Seq acts at several steps of the Notch type I>0 cascade by repressing not only E(spl)HLH but also CycE and E2f1, and that the balance in regulation of these different targets is sensitive to the levels of Seq. Specifically, it is proposed that Seq is a stronger repressor of CycE and E2f1 than of E(spl)HLH. Combined with the repressive role of E(spl)HLH on CycE and E2f1, this model might also help to explain why seq mutants phenocopy seq overexpression (Gunnar, 2016).

    Notch signaling plays a key role during development of the external sensory organs (ESOs) in Drosophila. Notch acts at multiple steps of ESO development: in the process of lateral inhibition, to select the sensory organ precursor (SOP), and during subsequent asymmetric cell division events in the SOP lineage. Previous studies of seq in the developing ESOs revealed defects both in the external lineage, with a conversion of shaft cells into socket cells, and the internal lineage, with loss of both neuron and glia specification. These phenotypes could to some extent be explained by the loss of expression of three Notch target genes: D-Pax2 (shaven - FlyBase), pros and hamlet (ham) . Interestingly, of these three seq targets, only ham was affected in the embryonic PNS. Comparing the role of Notch signaling with that of seq during ESO development, in spite of sharing several target genes seq only partly phenocopies Notch pathway mutants: Notch clones show supernumerary SOP cells and conversion of the entire SOP lineage into neurons, whereas seq clones do not show extra SOP cells but instead shaft-to-socket conversion and neuron/glia specification defects (Gunnar, 2016).

    Similar to the lack of SOP selection effects in ESOs in seq mutants, no effects were observed upon NB selection in the embryonic neuroectoderm, a process also controlled by Notch-mediated lateral inhibition. Instead, seq acts at a later stage to modulate expression of the Notch targets E(spl)HLH and CycE, during the subsequent type I>0 daughter proliferation switch. These studies reveal that the interplay between seq and the Notch pathway is highly context dependent, and seq appears to act on different Notch subroutines in different settings. Elevated NB divisions were also observed in seq mutants, in NB3-3A and globally. This is in contrast to Notch signaling, which does not appear to affect NB cell cycle exit in the VNC. Hence, seq can also play roles that are independent of Notch signaling during nervous system development (Gunnar, 2016).

    The C2H2 zinc fingers of Seq are highly homologous to the zinc fingers in Ttk. Seq and Ttk furthermore share homology in their zinc fingers with members of the PRDM family. In addition to related zinc fingers, Seq, Ttk and the PRDM family also share an involvement in Notch pathway signaling. ttk has been identified as a Notch pathway target gene and effector, both during oogenesis and ESO development. The PRDM family plays important roles during development and is also intimately linked to Notch signaling. The Drosophila ham gene, a PRDM family member, was found to control ESO development, chiefly by modulating Notch signaling. Similarly, in olfactory sensory lineages, ham also modifies Notch signaling, intriguingly by repressing E(spl)HLH gene expression via direct binding of Ham to this complex. In the developing mammalian CNS, Prdm8 and Prdm16 were found to be regulated by the bHLH HES genes Hes1, Hes3 and Hes5 (Gunnar, 2016).

    Dynamic protein expression is another common denominator. Seq, Ttk and Ham were all found to be dynamically expressed in the developing SOPs In addition, dynamic Seq protein expression levels govern photoreceptor axon targeting to the optic lobe (Gunnar, 2016).

    In summary, Seq, Ttk and the PRDM family have in common their intimate connection to the Notch pathway, acting to regulate the pathway, and/or being regulated by it, and/or regulating Notch downstream targets. They also share the property of controlling cell fate and proliferation in the developing nervous system, in some cases acting on the same targets (e.g., CycE). Finally, they are highly dynamic in their expression, and the expression levels can act in instructive and temporal manners. These results for seq support the role of the extended PRDM family as context-dependent, temporally controlled and level-sensitive modifiers of Notch signaling during nervous system development (Gunnar, 2016).

    Evolutionarily conserved anterior expansion of the central nervous system promoted by a common PcG-Hox program

    A conserved feature of the central nervous system (CNS) is the prominent expansion of anterior regions (brain) when compared to posterior (nerve cord). The cellular and regulatory processes driving anterior CNS expansion are not well understood in any bilaterian species. This expansion was addressed in Drosophila and mouse. Compared to the nerve cord, the brain, in both Drosophila and mouse, displays extended progenitor proliferation, more elaborate daughter cell proliferation and more rapid cell cycle speed. These features contribute to anterior CNS expansion in both species. With respect to genetic control, enhanced brain proliferation is severely reduced by ectopic Hox gene expression, by either Hox misexpression or by loss of Polycomb Group (PcG) function. Specifically, mutating maternal and zygotic esc (extra sex-combs) expression leads to loss of H3K27me3 and affects Hox-patterning along the AP-axis in neurogenesis. Strikingly, in PcG mutants, early CNS proliferation appears unaffected, whereas subsequently, brain proliferation is severely reduced. Hence, a conserved PcG-Hox program promotes the anterior expansion of the CNS. The profound differences in proliferation and in the underlying genetic mechanisms between brain and nerve cord lend support to the emerging concept of separate evolutionary origins of these two CNS regions (Yaghmaeian, 2018).

    Temporal control of the development of neural sublineages

    What mechanisms control the sequential generation of neurons -- that is, how are unique fates acquired by the successive daughters of a neuroblast? After a neuroblast has been specified positionally, by the actions of segment polarity genes and a network of homeodomain genes, it delaminates from the ectoderm and begins to divide unequally into one large and one small daughter cell. The large cell (still called a neuroblast) continues to go on this way for a variable number of rounds. The small cell, called a ganglion mother cell (GMC), typically divides equally one more time to generate a pair of postmitotic neurons. Often these neurons form a stack on top of the neuroblast from which they originated. As postmitotic neurons in the insect CNS do not generally migrate, the position of a neuron in the CNS depends on whether it was generated early or late. In this way, a histogenetic order is built into the cellular cortex of the insect CNS, with early neurons deep and close to the neuropil and late neurons next to the surface of the brain (Kambadur, 1998; Harris, 2001).

    This arrangement of cells according to relative birth date is also observed in laminated structures in the vertebrate CNS, the best example being the cerebral cortex. In the mammalian cortex, cells acquire their fates at the ventricular surface at the time they are born, and these postmitotic neurons cells then migrate to their specified laminar destinations (McConnell, 1995). In both the cortex and the retina, it is thought that progenitors are pluripotent and realize their particular fates by being exposed to an extracellular environment that changes with time (Harris, 1997). Whether intrinsically or extrinsically controlled, particular combinations of transcription factors are expressed in the neuroblasts of both the vertebrate retina and fly CNS over the course of development, and these factors appear to restrict the competence of neuroblasts to the fates that are appropriate (Harris, 2001 and references therein).

    The first insights into this problem in the fly CNS were made in Odenwald's laboratory at the NIH. They showed that expression of the transcription factor genes hunchback (hb), pdm, and castor (cas) occur sequentially in the embryonic CNS of Drosophila (Kambadur, 1998). Furthermore, the CNS neuroblasts themselves sequentially express these three genes in a conserved order (Brody, 2000), and whichever of the three genes is expressed in the neuroblast when it divides continues to be expressed in the progeny. The transcription factor Grainyhead (Gh) appears to mark the NB after it has generated lineages marked by Hb, Pdm and Cas, and the Gh positive NB also generates a fourth lineage (Brody, 2000). Thus, the earliest generated neurons in the fly CNS tend to express hb, while later generated neurons express pdm, and still later generated neurons express cas followed by gh. By analogy to the spatial coordinate genes, these can be called 'temporal coordinate genes (Harris, 2001).

    Isshiki (2001), working in Doe's laboratory, followed individual neuroblasts and their progeny. It appears that each neuroblast examined express four temporal coordinate genes -- hb, Kruppel (Kr), pdm, and cas, in that invariant order. By following the GMCs and their daughter neurons, Isshiki confirmed on a cellular level that each GMC maintains the expression profile of the temporal coordinate genes that its parent neuroblast displayed at the time the GMC was generated. The relevance of these temporal coordinates to neuronal fate was addressed with misexpression constructs and loss of function mutants in hb and Kr. These experiments led to respecification of GMCs and their progeny to earlier or later fates, as expected if these genes really are important to fate. Thus, these temporal coordinate genes play a similar role in fate specification along a histogenetic axis as the spatial coordinate genes play in the positional axes (Harris, 2001).

    What, one might wonder, are the mechanisms that could generate these temporal transitions in the parent neuroblast? The first possibility is that global temporal cues, such as circulating hormones, or intracellular signaling such as Notch, Egf, Wingless, or Hedgehog, trigger these transitions. Since transitions in transcription factor expression take place in isolation, in cultured clones, it is concluded that once NBs initiate lineage development no additional signaling between NBs and the neuroectoderm and/or mesoderm is required to trigger the temporal progression of transcription factor expression during NB outgrowth (Brody, 2000; Harris, 2001).

    Second, the cascade mechanism itself, in which each of these genes is responsible for turning on the next in the series, functions to regulate the transcription factor transitions. Overexpression of Hb activates Kr and represses Pdm and Cas; overexpression of Kr activates Pdm, represses Cas, but has no effect on Hb expression; Pdm positively regulates Cas expression; and Cas repressed Pdm expression (Kambdadur, 1998 and Isshiki, 2001). Although there are appropriate sequential regulatory interactions of this kind, mutations in any of the earlier genes only subtly affect the temporal expression of subsequently expressed genes. Thus, although these interactions refine sequential expression, there must be additional elements to temporal regulation. Isshiki (2001) has shown that individual NBs go through the same sequence of expression in their own sweet time, independent of the developmental stages at which they delaminate. A third possibility is therefore suggested, a more mysterious clock mechanism may also be responsible for generating the order. The clock in this case appears to be directly related to the cell cycle, since arresting cell division with the Cdc25 mutant, string, freezes the pattern in time (Cui, 1995; Weigmann, 1995; Harris, 2001 and references therein).

    The addition of a time axis adds to understanding of how different neuronal types arise in the Drosophila CNS, and it also raises the intriguing problem of how multiple inputs regulate the expression of the temporal coordinate genes (Harris, 2001).

    Drosophila voltage-gated sodium channels are only expressed in active neurons and are localized to distal axonal initial segment-like domains

    In multipolar vertebrate neurons, action potentials (APs) initiate close to the soma, at the axonal initial segment. Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (NaV) channels are a functional hallmark of the axonal initial segment in vertebrates. An intronic Minos-Mediated Integration Cassette was used to determine the endogenous gene expression and subcellular localization of the sole NaV channel in both male and female Drosophila, para. Despite being the only NaV channel in the fly, this study shows that only 23 ± 1% of neurons in the embryonic and larval CNS express para, while in the adult CNS para is broadly expressed. A single-cell transcriptomic atlas of the whole third instar larval brain was generated to identify para expressing neurons; it positively correlates with markers of differentiated, actively firing neurons. Therefore, only 23&177; 1% of larval neurons may be capable of firing NaV-dependent APs. Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon, which is named the distal axonal segment (DAS). The DAS is present in multiple neuron classes in both the third instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Nav-dependent APs originate in the DAS. Identification of the distal NaV localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates (Ravenscroft, 2020).

    Action potentials (APs) are generated by the sequential opening of voltage-gated sodium (NaV) and potassium channels (KV) in the axons of neurons. Mammalian CNS neurons are typically multipolar; and APs initiate at the dense concentration of NaV channels in the axonal initial segment (AIS) close to the soma, and propagate along the axon via the nodes of Ranvier. In addition to AP initiation, the AIS forms a barrier between the soma and the axon, preventing the free diffusion of organelles, proteins, and lipids between the two compartments. Invertebrate neurons are typically unipolar with the dendrites impinging on the axon distal to the cell body. Whether an AIS is present in these neurons, and where it is located along the axon, is unresolved (Ravenscroft, 2020).

    In order to determine the site of AP initiation, and if and where the AIS is in invertebrate neurons, the location was examined of the sole NaV channel gene in Drosophila melanogaster, paralytic (para). Unlike mammals, which have multiple NaV encoding genes (SCN1-5A, 8-11A), the genome of D. melanogaster encodes only two genes predicted to encode NaV proteins para and Na channel protein 60E (NaCP60E). para is the putative NaV channel as NaCP60E null animals are viable with no loss of inward sodium currents detected in neurons using patch clamp. In contrast, para null animals die as first instar larvae with no detectable inward sodium current in neurons using patch clamp. Despite having one NaV gene, compared with nine in mammals, it is possible that a similar degree of channel protein diversity is achieved via alternate splicing. para has 60 predicted isoforms, some of which have different developmental expression. Very little is known about the expression pattern or subcellular localization of Para. ISH studies determined that para is expressed in the nervous system from embryos to adults. Whether para is expressed in all or just some cells in the nervous system, and where it is subcellularly localized, remains to be established (Ravenscroft, 2020).

    To determine the expression pattern and protein localization of NaV channels in Drosophila neurons, previously established tools were used to develop two novel fly models: a model where the endogenous Para is tagged with GFP to determine Para subcellular localization and another with para replaced with GAL4 to determine para gene expression. Surprisingly, it was found that para is present in a small fraction of CNS neurons in embryos and third instar larvae, while it is broadly expressed in neurons in the adult CNS. A single-cell transcriptomic atlas was generated of the whole third instar larval brain to identify that para correlates with RNAs of active zone proteins and mature neuron markers; hence, para expression is restricted to active, differentiated neurons in larvae. Neurons that coexpress para and active zone protein RNAs are abundant in the adult CNS but only represent 23 ± 1% of neurons in third instar larvae. In neurons where para is expressed, Para protein is enriched at an AIS-like region in axons distal to where the dendritic tree connects to the axons in a distal axonal segment (DAS). Para localized far from the soma is functionally verified electrophysiologically. In longer neurons, Para is expressed throughout the axon, likely to maintain AP propagation to the synapses (Ravenscroft, 2020).

    The site of AP initiation in invertebrate neurons is unclear. In vertebrates, APs are generated at the AIS. The major functional hallmark of vertebrate AIS is clustering of NaV channels. Therefore, the gene expression and subcellular localization of the sole NaV channel in Drosophila, (para) was examined. Despite being the only NaV channel in flies, this study found that para is only expressed in 23 ± 1% of neurons in the third instar larval CNS. In contrast, in the adult, para expression is far broader. By generating a single-cell transcriptomic atlas of the third instar larval brain, it was determined that para expression correlates with the expression of ARGs, genes expressed in active zones, and markers of mature neurons. This implies that cells expressing para, while proportionally small, likely represent the active population of neurons in the third instar larval CNS. This correlation occurs also in the adult CNS, indicating that the number of active neurons is likely much higher in the adult than in the third instar larvae. In the neurons expressing para, it was found that Para distributed into axonal segments that are far removed from the soma, distal to where dendrites integrate into the axon. This structure shares some features of the mammalian AIS, and whole-cell patch-clamp electrophysiological recordings are consistent with the interpretation that Nav-dependent APs initiate there; however, because of its distal location to the cell soma, this region was named the DAS (Ravenscroft, 2020).

    The AIS identifies the origin of AP propagation through the high density of NaV channels. The clustering of NaV channels at the DAS suggests that this is the site of AP initiation in fly neurons. Identification that TTX-sensitive, inward sodium currents that occur after long delays in response to somatic depolarization are also suggestive of a distal site of AP initiation. In vertebrate neurons, TTX-sensitive inward sodium currents are measured immediately after somatic depolarization as the AIS is so close to the soma. Single-cell electrophysiological recordings performed on fly neurons in the CNS are a challenge because of their small size. Recordings performed in the neurons of larger invertebrates, Aplysia, crab, and leeches also exhibit delayed activation and poor space clamp, indicating that APs are initiated at a site distal relative to the cell body. This suggests that AP initiation at the DAS is conserved across invertebrate species. Because of their small size, patch-clamp recordings are made on the soma. Therefore, the soma receives back-propagating APs from the DAS, and passive fluctuations in membrane potential are readily measured in the soma as well in Drosophila CNS neurons. While APs appear to initiate from the DAS, the sources and ionic mechanisms of passive membrane potentials are not clear; however, fluctuations in membrane potential clearly influence the pattern of AP firing in Drosophila neurons. For example, large fluctuations in membrane potential drive burst firing in lLNv, whereas stable membrane potential is associated with regular tonic AP firing. Further, transition between burst and tonic firing AP firing pattern is CaV-dependent as it is modulated by cobalt block of CaV channels. Surprisingly, either TTX or cobalt abolishes lLNv APs altogether, suggesting complex interactions between Para and CaVs in Drosophila neurons. Therefore, the electrical activity recorded at the soma may not be representative of the neuron's actual firing activity, as in some cases the DAS is far away from the soma and these cell bodies may not undergo any depolarization. This emphasizes the need to establish voltage-sensing reporters to simultaneously enable accurate reporting of APs and calcium events at high spatiotemporal resolution. While genetically encoded calcium indicators are often used for a proxy for inferring APs, intracellular calcium sources are highly diverse (CaVs, TRP channels, nonselective cation channels, multiple intracellular stores, etc.) (Ravenscroft, 2020).

    As an alternative to electrophysiological recordings, computational models of compartmentalized fly neurons have been used to model their electrophysiological properties. Models of different neurons in the fly CNS predict that the site of AP propagation occurs, not proximal to the cell body as in mammals, but distally in the axon after the last dendrite innervates the axon. The mapping of the DAS and the ability to visualize it in any fly neuron using Para-GFSTF endorse the accuracy of these models and enable the generation of models with greater precision to better predict and analyze neuron activity and dynamics (Ravenscroft, 2020).

    Some studies have indicated that an AIS-like region may be present in some Drosophila neurons, based on GAL4-mediated overexpression of Drosophila homologs of vertebrate AIS proteins, such as Ank1 and Shal in MB neurons. The overexpressed proteins were shown to cluster in axons downstream of the dendritic tree, in the MB of the third instar larval and the adult CNS. Using a similar approach, it has been shown that proximal axons of ddaE neurons in the PNS also may contain AIS-like regions. However, the overexpression of proteins may lead to their restriction in cellular compartments as it has been shown that the mobility of transmembrane molecules is restricted to areas before and after dendritic branches, suggesting that there are barriers in axons that restrict protein localization. The DAS reported in this study is of comparable size and location to the AIS-like region previously reported in adult MB neurons. AnkG is key for NaV channel clustering at the vertebrate AIS. The similarity in the reported AIS-like localization of Ank1 localization and Nav enrichment at the DAS as defined in this study indicate that the role of ankyrins in NaV clustering may be conserved in invertebrates, despite differences in ankyrin structure across species. Interestingly, this study found that the distance between the soma and the length of the DAS is correlated, with a longer DAS present in neurons where the DAS is more distal. This is in contrast to what is observed in vertebrates where the more distal the AIS, the shorter the length of the AIS (Ravenscroft, 2020).

    Low levels of Para distribution were observed along long axons, suggesting that AP propagation is maintained by Para across the length of the axon. APs are all-or-nothing signals that rapidly propagate along axons; however, if no NaV channel is present, the signal should decay rapidly. It is argued that the low levels of Para distributed along the length of long axons enable the APs to reach the synapse without signal depletion. This is seen clearly in neurons, such as motor neurons in larval and adult CNS, and neurons in the adult brain that cross from one hemisphere to the other. Neurons with smaller axons may also have Para continually distributed along axons at undetectable levels or may not require Para at all. However, given the expression data discussed below, the latter is unlikely (Ravenscroft, 2020).

    The sparse number of CNS neurons in the third instar larvae that express para is an unanticipated observation. Only 23 ± 1% of elav-positive neurons in the larval CNS express para, and historical gene expression tracing shows that those neurons that are para-negative never express para. These data are also in agreement with the single-cell sequencing data, which document a highly restricted expression pattern for para in the third instar larval CNS. Neurons expressing para also express genes that produce neurotransmitters as well as proteins that have been shown to be upregulated in many Drosophila models of enhanced neuronal activity, both in the third instar larval and adult brain single-cell RNA sequencing datasets. This provides evidence that this restricted cell population is likely capable of firing APs, that para expression is a potential marker for active AP firing neurons, and that only 23 ± 1% of cells in the third instar larvae fit this criterion. Whether the para-negative cells still have electrical activity in lieu of NaV channels, either through passive signaling or using CaV, is unknown. para expression has been demonstrated to positively correlate with neural progenitor proliferation in third instar larvae. The single-cell transcriptomic atlas of the whole third instar larval CNS generated in this study identifies neuronal progenitors at immature, intermediate, and mature maturation steps, and para expression was not detected in any neuroblasts lineage at any stage of maturation (Ravenscroft, 2020).

    This study has identified the DAS, a compartment of Drosophila axons analogous to the AIS in vertebrates. The characterization of NaV channels throughout development in D. melanogaster is reported, uncovering the presence of the DAS in most axons after the dendritic tree, where AP is likely initiated, and continual low-level Para distribution in long axons to maintain AP. Additionally, comprehensive single-cell RNA sequencing of the third instar larval CNS was performed. This single-cell atlas in conjunction with para-T2A-GAL4 revealed that only 23 ± 1% of the cells in the third instar larval brain express para and other genes expressed in AP firing neurons. These data are consistent with the notion that most neurons are developing in third instar larvae, especially in the brain lobes. In contrast, para expression is broad in the adult CNS. The models generated herein should allow the mapping of DAS in any neuron and provide a GAL4 driver to target differentiated, AP firing neurons (Ravenscroft, 2020).

    Control the temporal sequence of neuroblast specification

    Stage-specific inductive signals in the Drosophila neuroectoderm control the temporal sequence of neuroblast specification. To test when identity specification of the various neuroblasts takes place and whether extrinsic signals are involved, heterochronic transplantation experiments were performed. Single neuroectodermal cells from stage 10 donor embryos (after S2) were transplanted into the neuroectoderm of host embryos at stage 7 (before S1) and vice versa. The fate of these cells was examined by determining the makeup of their lineages at stage 16/17. Transplanted cells adjust their fate to the new temporal situation. Late neuroectodermal cells are able to take over the fate of early (S1/S2) neuroblasts. The early neuroectodermal cells preferentially generated late (S4/S5) neuroblasts, despite their reduced time of exposure to the neuroectoderm. Furthermore, neuroblast fates are independent from divisions of neuroectodermal progenitor cells. It is concluded from these experiments that neuroblast specification occurs sequentially under the control of non-cell-autonomous and stage-specific inductive signals that act in the neuroectoderm (Berger, 2001).

    The segmented CNS (ventral nerve cord) of the Drosophila embryo is relatively simple, consisting of approximately 400 cells per hemineuromere. These originate after gastrulation from the ventral neurogenic region of the ectoderm. About 25% of the neuroectodermal cells delaminate into the embryo as CNS progenitor cells, called neuroblasts (NBs). The singling out of the NBs from among neuroectodermal cells is achieved by the activity of proneural and neurogenic genes. In each hemisegment approximately 30 NBs delaminate from the neuroectoderm according to a stereotyped spatiotemporal pattern. Each NB delaminates from a specific region of the neuroectoderm to occupy a particular place within the subectodermal NB layer. The process of delamination has been divided into five successive waves (S1-S5) with particular subpopulations of identified NBs delaminating during each wave. Thus, each NB is characterized by a typical position and time of delamination. Furthermore, each NB expresses a specific set of molecular markers. Finally, the unique identity of each NB is revealed by the production of a characteristic cell lineage (Berger, 2001 and references therein).

    Crucial steps in the specification of the various NB identities appear to take place before delamination by the interpretation of positional information in the neuroectoderm encoded by segmentation genes and dorsoventral patterning genes. Heterotopic transplantation experiments have shown that neuroectodermal cells become committed by these spatial cues to different degrees. For example, whereas dorsal neuroectodermal cells are able to adjust their fate when transplanted to more ventral positions, ventral neuroectodermal cells exhibit firm commitment and produce lineages consistent with their origin. These experiments refer to a given developmental stage (early gastrula, stage 7). However, the time of delamination differs between NBs, and the identity of a given NB correlates with a certain time of delamination. This implies that NB specification requires temporal cues in addition to positional information (Berger, 2001 and references therein).

    The mechanisms behind the temporal sequence of NB specification are unknown. Different modes of regulation could be envisaged. For example, all NB identities, including the respective times of delamination, might become firmly determined at an early stage and are cell-autonomously expressed during further development. Alternatively, progenitor cells might acquire NB-identities sequentially under the influence of extrinsic signals. To test whether the developmental potencies of neuroectodermal progenitor cells change over time and whether inductive signals are involved, the temporal axis was manipulated independently from spatial cues by performing heterochronic transplantations of neuroectodermal cells. Neuroectodermal cells were transplanted from stage 7 donors (early gastrula, before S1) into stage 10 hosts (after S2), and vice versa. The identities assumed by these cells were determined by analyzing their lineages in the host embryos at stage 16/17. In both experimental situations, neuroectodermal cells are able to adjust their fate to the new environment. Late neuroectodermal cells can generate early (S1, S2) NBs. Early neuroectodermal cells preferentially produced late (S3-S5) NB lineages, despite having been exposed to the neuroectoderm for a significantly reduced period of time. Late NB fates are independent of previous divisions of neuroectodermal progenitor cells. These data suggest that extrinsic inductive signals exist in the neuroectoderm that change over time to control the specification of temporal subsets of neuroblasts (Berger, 2001).

    In one set of experiments, neuroectodermal cells from stage 10 embryos were heterochronically transplanted into the neuroectoderm of 2 hours younger, early gastrula (stage 7) hosts. The transplanted cells gave rise to CNS clones, or to epidermal clones, or to mixed CNS/epidermal clones. This shows that despite their more advanced age, the implanted cells participate in the cell interaction process that leads to the decision of neurectodermal cells between an epidermogenic and a neurogenic fate. Remarkably, however, among the cells that follow the neural pathway, about 50% produced lineages typical for early NBs (S1, S2), as for example, NB1-1, MP2, NB2-2 or NB4-2. This indicates that neuroectodermal cells at stage 10, which normally only give rise to late NB lineages, have not lost the potency to assume identities of early NBs. Taken together these data indicate that late ectodermal cells (stage 10) are not irreversibly specified, and that signals exist in the early neuroectoderm (stage7) that are sufficient to induce early NB fates. Thus, instead of being merely based on cell-autonomous properties, the temporal regulation of early NB determination appears to be mediated by extrinsic inductive signals that are active in the early neuroectoderm (Berger, 2001).

    Reduced time of exposure to the neuroectoderm does not prevent formation of late NBs. Having shown that the determination of early NB fates depends on stage specific inductive signals, whether inductive signals are also involved in the generation of late NB fates was tested. Cells from the early neuroectoderm (stage 7) were heterochronically transplanted into the neuroectoderm of stage 10 host embryos. Among 132 identifiable clones obtained from these cells, 24 (19%) were CNS clones and 108 (81%) epidermal clones. Closer analysis of the 24 CNS clones revealed that about 80% (n=19) of them corresponded to lineages typical for late NBs, like 2-1, 5-4, 6-4 or 7-3, and only 20% (n=5) to early NB lineages. Therefore, the transplanted cells tend to adopt to the new temporal environment regarding the identities of NBs to be formed. Although having skipped two hours of exposure to the neuroectoderm, a significant proportion of them can compensate for this lack of time. Thus, the cells are not bound to an intrinsic timer to become specified as late NBs, but are able to react to inductive signals in the late neuroectoderm. The 20% of cells that developed an early NB fate might point to differences in the degrees of commitment of neuroectodermal cells at a given stage or to an insufficient exposure to signaling in the late neuroectoderm under the experimental conditions (Berger, 2001).

    Determination of late NBs does not depend on previous division in the neuroectoderm. As opposed to early NBs the lineages of S4 and S5 NBs, and some of the S3 NBs have an epidermal sister clone. This is due to the postblastodermal division pattern of neuroectodermal progenitors. Progenitors developing as S1 and S2 NBs do not divide before delamination from the neuroectoderm: some of those giving rise to S3 NBs divide, and those giving rise to S4 and S5 NBs (NBs 1-3, 2-1, 2-4, 3-3, 4-3, 4-4, 5-1, 5-4, 5-5 and 7-3) always divide in the neuroectoderm. Only one of the daughter cells that results from this division subsequently delaminates as a late NB, the other remains in the periphery to develop as an epidermoblast. Is this neuroectodermal division required for late NBs to form and become properly specified? When neuroectodermal cells are heterochronously transplanted from stage 7 donors into stage 10 hosts, they are deprived from the phase in which the first wave of divisions normally runs through the neuroectoderm. Most of the CNS clones obtained from these cells corresponded to lineages of late NBs. However, whereas S4 and S5 NBs normally have an obligatory epidermal sister clone, the situation is variable under the experimental conditions. Some of these clones have a sister clone consisting of epidermal cells, whereas the other clones lack an epidermal sister clone (Berger, 2001).

    These data show that: (1) proliferation of individual neuroectodermal progenitors can be influenced by surrounding tissue; (2) late NBs can segregate from the neuroectoderm without having previously divided; and (3) late NBs do not depend on a previous division to acquire an individual identity and to produce their specific and complete CNS lineage. These observations lend further support to the idea that the temporal pattern of NB determination depends on inductive signals in the neuroectoderm instead of following a stereotype cell autonomous clock (Berger, 2001).

    There is ample evidence that the specification of NBs crucially depends on positional information in the neuroectoderm provided by the products of segmentation genes and dorsoventral patterning genes. Part of this information becomes integrated into the cell-autonomous program of the cells before neurogenesis. Another part, however, is subsequently provided by extrinsic signals. For example, the segment polarity gene wingless (wg) is segmentally expressed in a single row of neuroectodermal cells and the secreted Wg protein is non-autonomously required in adjacent anterior and posterior neuroectodermal cells for the formation and specification of NBs. Along the dorsoventral axis, the secreted Spitz and Vein proteins are involved in conferring NB identities. These heterochronic transplantation experiments show that extrinsic signals are also involved in NB specification along the temporal axis. Although neuroectodermal cells of stage 10 embryos normally never produce NBs belonging to the group of S1 and S2 NBs, they do so after being transplanted into stage 7 neuroectoderm. The possibility that the cells follow this fate autonomously after being released from signals that normally inhibit these fates in the late neuroectoderm is incompatible with the following evidence. Cells from the non-neurogenic dorsal ectoderm of stage 10 donors are able to adopt a CNS fate upon heterotopic transplantation, and to become specified as early NBs. However, they are unable to autonomously develop as a NB in cell culture. Thus, the transplanted late cells do react to signals in the early neuroectoderm and adjust their development accordingly. This also seems to be possible in the other direction. Upon transplantation of stage 7 neuroectodermal cells into the neuroectoderm of hosts at stage 10, most of the CNS lineages obtained are typical for NBs that normally delaminate late. Similar to the situation in Drosophila, heterochronic transplantations using the developing ferret brain have revealed an interaction scenario of extrinsic cues and intrinsically changing properties for the sequential birth of neuronal cell types from ventricular zone progenitor cells. Progenitor cells from very young embryos can adjust their fate to older host tissues. By contrast, cells from older tissue transplanted into younger host brains adopt only fates typical of their origin. The latter experiment reveals an irreversible intrinsic change of the developmental properties of older cells. Intrinsic changes over time are likely to occur also in the Drosophila neuroectodermal cells; however, they are reversible under the influence of external signals. It remains to be tested as to how far this is also the case for NBs once they have delaminated from the neuroectoderm (Berger, 2001 and references therein).

    These experiments suggest that the entire temporal sequence of delamination of specific subsets of NBs is not readily determined in the early neuroectoderm but is controlled by the dynamic expression of stage specific signals. Segment-polarity genes play an important role in the formation and identity specification of NBs. They are segmentally expressed in particular rows of neuroectodermal cells. As the expression domains of some of these genes evolve dynamically and, hence, differ at different stages, they are also good candidates for being involved in the temporal control of NB formation/specification. The differential commitment of the late neuroblasts NB 6-4 (S3) and NB 7-3 (S5) has been shown to be mainly controlled by the interplay of the segment polarity genes naked (nkd) and gooseberry (gsb). Mutation of either nkd or gsb leads to the transformation of one NB fate to the other. Interestingly, however, the temporal sequence of their delamination is maintained, i.e. independent from these genes. This suggests that formation and specification of these two NBs is under independent control. Further work will have to test whether this is also the case for other NBs and to uncover the signals that regulate the temporal pattern of NB fate determination (Berger, 2001 and references therein).

    The role of Dichaete in transcriptional regulation during Drosophila embryonic development

    Group B Sox domain transcription factors play conserved roles in the specification and development of the nervous system in higher metazoans. However, comparatively little is known about how these transcription factors regulate gene expression, and the analysis of Sox gene function in vertebrates is confounded by functional compensation between three closely related family members. In Drosophila, only two group B Sox genes, Dichaete and SoxN, have been shown to function during embryonic CNS development, providing a simpler system for understanding the functions of this important class of regulators. Using a combination of transcriptional profiling and genome-wide binding analysis this study conservatively identified over 1000 high confidence direct Dichaete target genes in the Drosophila genome. Dichaete is shown to play key roles in CNS development, regulating aspects of the temporal transcription factor sequence that confer neuroblast identity. Dichaete also shows a complex interaction with Prospero in the pathway controlling the switch from stem cell self-renewal to neural differentiation. Dichaete potentially regulates many more genes in the Drosophila genome and was found to be associated with over 2000 mapped regulatory elements. This analysis suggests that Dichaete acts as a transcriptional hub, controlling multiple regulatory pathways during CNS development. These include a set of core CNS expressed genes that are also bound by the related Sox2 gene during mammalian CNS development. Furthermore, Dichaete was identified as one of the transcription factors involved in the neural stem cell transcriptional network, with evidence supporting the view that Dichaete is involved in controlling the temporal series of divisions regulating neuroblast identity (Aleksic, 2013).

    The core Dichaete binding intervals identified in this study are enriched for Sox binding motifs but significant overrepresentation was also found of binding motifs for Vfl (Zelda), the GAGA-binding factor Trl, and the JAK-STAT pathway transcription factor Stat92E. All three of these factors have been identified as key elements in the regulatory programme that drives the onset of zygotic gene expression in the blastoderm embryo. Dichaete also plays a key role in early zygotic gene expression, regulating the correct expression of pair rule genes, and this study found overlapping Vfl/Dichaete binding at eve, h, and run stripe enhancers. While most of the work on Vfl has focused on understanding its function during the maternal to zygotic transition, the gene is expressed more widely after cellularisation, particularly in the CNS. Indeed recent work has shown a specific role for Vfl in the CNS midline, a tissue where Dichaete is known to be active, and this study found overlapping Vfl/Dichaete binding associated with slit and comm, known Dichaete midline targets. Post cellularisation functions for Trl and Stat92E are well established (Aleksic, 2013).

    These three factors, particularly Vfl and Trl, have been strongly associated with enhancer activity driven by Highly Occupied Target (HOT) regions. HOT regions have been identified in large scale studies of the Drosophila, C. elegans and human genomes, and represent genomic sites where many functionally unrelated transcription factors bind, frequently in the absence of specific binding motifs. The finding that Dichaete binding locations are marked by overrepresentation of binding motifs for factors defining HOT regions, coupled with the widespread gene expression effects of Dichaete mutations, suggests that Dichaete may also play a role in regulatory interactions at HOT enhancers. It is notable that Dichaete, in common with all other characterised Sox proteins, is known to bend DNA upon binding. It is possible that Dichaete activity at HOT regions is mediated by this bending activity, helping to bring together complexes of other regulators. In this view, Dichaete would assist binding of factors at non-canonical target sites by favouring protein-protein interactions. In one of the bona fide Dichaete regulatory elements that have been studied in detail, the slit midline enhancer, Dichaete helps coordinate interactions between the POU factor Vvl and a Sim/Tango heterodimer (Aleksic, 2013).

    Aside from a proposed role at HOT regions, this analysis indicated Dichaete binds to and is active at many characterised regulatory elements. Almost half the enhancers catalogued by RedFly and a substantial fraction of neural enhancers identified by the FlyLight project show evidence of Dichaete regulation. Along with this, an association between Dichaete binding and transcriptional start sites was observed, suggesting one of two possibilities. Either Dichaete directly engages with core promoter elements or looping interactions between Dichaete bound enhancers and the transcriptional machinery results in ChIP or DamID assays capturing these interactions. In this respect it is noted that Dichaete binds in the minor groove of DNA, perhaps making it more likely to capture indirect interactions (Aleksic, 2013).

    Whether Dichaete acts at defined tissue-specific enhancers, HOT regions, core promoters, or all three, this analysis uncovered widespread involvement in specific developmental processes in the embryo. For example, previous studies highlighted a role for Dichaete in hindgut morphogenesis and identified dpp as a likely target gene, since targeted dpp expression in the hindgut of Dichaete mutants was able to partially rescue the phenotype. This new analysis implicates Dichaete in the regulation of many of the key factors responsible for hindgut specification and morphogenesis, with most of the characterised transcription factors or signalling pathway components known to be important for hindgut development bound and regulated by Dichaete. This further emphasises the view that Dichaete plays a hub-like role in controlling regulatory networks. It is noted that hindgut phenotypes and gene expression are unlikely to be functionally compensated by other Sox factors. While the group E gene Sox100B is also expressed in the embryonic hindgut, these is no evidence for synergistic interactions between Dichaete and Sox100B mutants and thus functional compensation by Sox100B is less likely. In contrast, the related group B gene Sox21b is expressed in the hindgut and partially overlaps with Dichaete (McKimmie, 2005). Although deletions encompassing Sox21b show no obvious phenotype, assessing possible functional compensation of Dichaete functions is difficult due to the close proximity of the two genes (~40 kb). It has recently been reported that human SOX2 is involved in gut development where it interacts antagonistically with CDX2. Caudal is a Drosophila orthologue of CDX2 and this study found Dichaete binding and associated repression of cad, hinting at further levels of regulatory network conservation across metazoa (Aleksic, 2013).

    In common with vertebrate group B genes, Dichaete plays a prominent role in the CNS. Many previous studies focused on single genes have shown that Dichaete is involved in neural specification via the regulation of proneural genes in the Achaete-scute complex and the current analysis provides a genomic perspective on this, identifying extensive Dichaete binding across the complex. Importantly, much of this binding coincides with mapped regulatory elements and this study found changes in the expression of complex genes in Dichaete mutants. Dichaete is involved in the temporal cascade that confers specific identities to neuroblasts and their progeny and this analysis provides considerable insights into this role. Dichaete binding was found associated with all of the characterised genes in the temporal cascade, as well as considerable overlapping binding between Dichaete, Hb and Kr, strongly supporting the idea that cross-regulatory interactions between these genes is important for correct neural specification. For example, maintenance of Hb or loss of Cas, the first and last genes in the cascade, lead to prolonged expression of Dichaete and cells remain in a neuroblast state (Maurange, 2008). This analysis suggests that Dichaete may help maintain the temporal cascade expression in the neuroblast (Aleksic, 2013).

    Finally, this analysis uncovered a striking relationship between Dichaete and Pros, with Dichaete negatively regulating pros expression early in neural development. In addition, both proteins show an extensive and highly significant overlap in their binding profiles. The gene expression data indicate that Dichaete and Pros may have antagonistic interactions since genes encoding neuroblast functions (e.g. ase, insc, mira and dpn) were found to be downregulated in Dichaete mutants but upregulated in pros mutants. However, this study also found that genes involved in aspects of neuronal differentiation (e.g. esc, zfh1 and Lim1) are positively regulated by both factors. Taken together it is tempting to speculate that in neuroblasts, when Pros is cytoplasmic, Dichaete positively regulates genes required to maintain the self-renewal state and keeps pros levels down. In the GMC, Dichaete function must be downregulated to allow cells to exit the cell cycle and differentiate, consequently pros expression would be upregulated and the protein translocated to the nucleus by the well-established asymmetric division mechanism, repressing neuroblast genes and promoting differentiation. While Dichaete appears to be uniformly expressed in the neuroectoderm, its expression in neuroblasts is dynamic with many neuroblasts expressing Dichaete transiently. In addition, and related to the subcellular partitioning of Pros, Dichaete is reported to shuttle between cytoplasm and nucleus, at least early in CNS development. Furthermore, Dichaete is dynamically expressed in GMCs and their progeny, consistent with the proposed interaction with Pros (Sanchez-Soriano, 2000). These observations are consistent with the view that control of Dichaete is important for first determining self-renewal versus differentiation, followed by a role in aspects of neuronal differentiation (Aleksic, 2013).

    The emerging view from these studies and previous work with Dichaete is of a transcriptional regulator with multifaceted roles in development. Previous studies have shown that mammalian Sox2 can provide Dichaete function, rescuing Dichaete mutant phenotypes. However, the designation of Dichaete as a group B1 protein based on functional arguments is considered by some to be inconsistent with phylogenetic arguments that firmly place Dichaete in the B2 group. In vertebrates, group B2 proteins act as transcriptional repressors, antagonising group B1 functions. Since very few genes are seen to be upregulated in Dichaete mutants, this analysis suggests that Dichaete may be acting primarily as a transcriptional activator. However, this type of mutant expression study is prone to pleiotropic effects, so further investigation of specific targets and tissues is needed. In vertebrates the group B1 proteins play critical roles in the specification and maintenance of neural stem cells, exactly the functions described for Dichaete. The observed correspondence between Dichaete and Sox2 target genes show that these proteins are not only conserved at the functional level when assayed in mutant rescue experiments but also, remarkably, at the level of the gene regulatory networks they control in the fly and mouse nervous system (Aleksic, 2013).

    One possible explanation for these disparate findings regarding the classification of Dichaete as a group B1 or B2 protein may be provided by the role of Dichaete in the regulation of proneural genes and its early activity on pros. In these specific cases, Dichaete acts to repress these genes in the neuroectoderm while SoxN acts as an activator. It is therefore possible that, in the last common ancestor of the vertebrates and invertebrates when the B1-B2 split occurred, the ancestral Dichaete gene had an limited B2-like repressor role as well as more prominent B1-like activator role in the CNS. As the lineages diverged the vertebrate B2 genes evolved specialised repressor functions while, in the invertebrates, they maintained more basal activator function. Support for the idea that insect Sox genes represent conserved basal functions of more diverged vertebrate family members comes from experiments replacing the mouse group E gene, Sox10, with the fly Sox100B coding sequence. In these studies the fly gene is able to provide substantial Sox10 function in the developing embryo, more so than the Sox8 gene, which is far closer to Sox10 at the sequence level (Aleksic, 2013).

    In summary, this study presents a rigorous analysis of the genomics of the Drosophila group B transcription factor Dichaete, highlighting regulatory input into several key developmental pathways. These studies provide a baseline for more detailed analysis of highly conserved aspects of group B Sox function in neural stem cells and in neuronal differentiation (Aleksic, 2013).

    Global programmed switch in neural daughter cell proliferation mode triggered by a temporal gene cascade

    During central nervous system (CNS) development, progenitors typically divide asymmetrically, renewing themselves while budding off daughter cells with more limited proliferative potential. Variation in daughter cell proliferation has a profound impact on CNS development and evolution, but the underlying mechanisms remain poorly understood. This study found that Drosophila embryonic neural progenitors (neuroblasts) undergo a programmed daughter proliferation mode switch, from generating daughters that divide once (type I) to generating neurons directly (type 0). This typeI> 0 switch is triggered by activation of Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2) expression in neuroblasts. In the thoracic region, Dacapo expression is activated by the temporal cascade (castor) and the Hox gene Antennapedia. In addition, castor, Antennapedia, and the late temporal gene grainyhead act combinatorially to control the precise timing of neuroblast cell-cycle exit by repressing Cyclin E and E2f. This reveals a logical principle underlying progenitor and daughter cell proliferation control in the Drosophila CNS (Baumgardt, 2014).

    Proliferation analysis of the developing Drosophila VNC reveals that most, if not all, lateral NBs initially divide in the type I proliferation mode, generating daughters that divide once. Three specific lineages, as well as many other NBs, subsequently switch to generating daughters that do not divide (type 0 mode). The full extent of the typeI>0 switch is currently difficult to precisely assess for several reasons. One such complicating issue pertains to possible developmental changes in daughter cell-cycle length over time. On this note, however, no obvious change was found in NB5-6T daughter divisions prior to the switch. In addition, if the accelerated decline in daughter division was indeed caused by a lengthening of the cell cycle rather than a typeI>0 switch, a long 'tail' of daughter divisions would be expected, perduring into St16-17. This is not the case; rather, daughter proliferation drops down to almost zero by St16-17. Similarly, no evidence was found for changes in NB cell-cycle length over time in the three specific NB lineages. Another complicating issue pertains to the fact that NBs differ in their time point of delamination, number of division rounds, and time point of switching, so that even if all NBs switched, only a fraction of the NBs would be in their type 0 window at the same time. However, these complications are more likely to lead to under- rather than overappreciation of the extent of the typeI>0 switch, and it is tempting to speculate that it may indeed involve the vast majority of NBs (Baumgardt, 2014).

    The typeI>0 switch is triggered by the onset of Dap expression in NBs at precise stages of lineage progression. The mammalian Dap orthologs, p21CIP1/p27KIP1/p57Kip2, can act as inhibitors of the CycE/Cdk2 complex. By analogy, the mechanism behind the typeI>0 switch is, presumably, that type 0 daughters are prevented from entering the cell cycle by the presence of Dap at the G1/S checkpoint. The onset of Dap expression already in the NB suggests that Dap needs to be present at an early stage in newborn daughters to block their entry into S phase. These findings are also in line with the emerging role for the Cip/KIP family and cell-cycle exit in the mammalian CNS, although there has been no report of a connection to changes in daughter proliferation mode (Baumgardt, 2014).

    No evidence was found for a role of pros in the type 0 mode, and, conversely, no evidence was found for a role of dap in the type I mode. The distinct roles of pros and dap in control of the type I versus 0 modes is further underscored by the expression of E2f, CycE, and Dap. In type I daughters (GMCs), E2f and CycE are rapidly repressed, by pros, and Dap is only weakly expressed at a later stage, around the time point of mitosis. The short window of E2f and CycE expression is still sufficient for the GMC to enter another cell cycle, since Dap expression is absent. As each GMC divides, the postmitotic cells (neurons/glia) are prevented from entering the cell cycle by the lack of E2f and CycE. In type 0 daughters, on the other hand, E2f and CycE expression is robust, but daughters still fail to enter the cell cycle due to the presence of high levels of Dap. These findings point to strikingly different strategies in daughter proliferation control: pros repression of E2f/CycE in type I, and Dap overriding E2f/CycE/Cdk2 in type 0 daughters (Baumgardt, 2014).

    Changes in daughter cell proliferation could perhaps have been envisioned to merely result from a gradual loss of the proliferative potential of each progenitor, as a result of its undergoing many rapid cell cycles. If so, typeI>0 switches could have been predicted to occur somewhat stochastically toward the end stage of each lineage, perhaps loosely linked to the last NB division. In contrast to such simplified models, this study found that the typeI>0 switch can occur many divisions prior to NB exit and that it is programmed to occur at a precise stage during each lineage development. In the thorax, it was found that the precise timing of typeI>0 switches is controlled by the temporal gene cas and the Hox gene Antp, which are expressed at a late stage within NBs. Remarkably, in cas mutants, most, if not all, thoracic typeI>0 lineages fail to enter the type 0 mode. The primary mechanism by which cas and Antp control the switch appears to be by activating the expression of Dap, evident by the reduction of Dap in cas and Antp mutants; by the finding that cas-Antp co-misexpression triggers ectopic Dap expression; and by the finding that cas can be cross-rescued by elav>dap (Baumgardt, 2014).

    The finding that the timing of the typeI>0 switch is scheduled by a temporal gene cascade points to an intriguing regulatory model where daughter cell proliferation mode switches are executed at stereotyped positions within the lineage tree by the activity of specific temporal genes. Since temporal genes also control the progression of NB competence, evident by their roles in cell fate specification, the temporal cascade can act to simultaneously control both cell fate and cell number, thereby ensuring that precise number of each neural cell subtype is produced (Baumgardt, 2014).

    After a stereotyped number of divisions, each NB subtype stops proliferating. This study found that, for many NBs, this is a G1/S decision influenced by the activities of E2f, CycE, and dap. The nuclear localization of Pros was previously identified to be associated with cell-cycle exit in postembryonic NBs. However, previous studies of NB5-6T, and the current study on NB7-3A, do not indicate a general role for pros in NB cell-cycle exit in the embryonic CNS. Instead, in the thorax, the expression levels of E2f, CycE, and Dap are gradually modulated during lineage progression, by the temporal genes cas and grh as well as Antp. Because Cas, Grh, and Antp are progressively activated in thoracic NBs, this brings into view a logical model for timely NB cell-cycle exit where sequential activation of temporal and Hox genes act combinatorially to push E2f, CycE, and/or Dap to limiting levels after a determined number of divisions (Baumgardt, 2014).

    For the majority of NBs in the thorax, cell-cycle exit is followed by quiescence until larval stages. In contrast, for the majority of abdominal NBs, cell-cycle exit is followed by apoptosis. However, for some NBs, such as NB7-3A, apoptosis is the functional exit mechanism. Thus, three general strategies for lineage stop are emerging: (1) cell-cycle exit > quiescence (most thoracic NBs), (2) cell-cycle exit > apoptosis (NB5-6T), and (3) lineage stop by apoptosis (NB7-3A). The balance of E2f, CycE, and Dap is involved in the first two strategies, while the balance of apoptosis gene expression presumably is at the core of the latter strategy (Baumgardt, 2014).

    In addition to the type I and type 0 daughter proliferation modes described here in the embryo, recent studies of Drosophila larval CNS development have identified a third, more prolific, proliferation mode: the type II mode, identified in a small number of larval brain. Type II NBs divide asymmetrically, renewing themselves while budding of daughters that, in turn, undergo multiple rounds of proliferation before finally differentiating. This allows for the generation of very large lineages (some 500 cells) from each individual type II NB (Baumgardt, 2014).

    In mammals, the most obvious equivalent of Drosophila NBs is the radial glia cell (RG), which divides asymmetrically to generate neurons and. During these RG asymmetric divisions, studies have identified several different division modes; RGs dividing asymmetrically to bud off a neuron, to bud off a daughter cell that divides once to generate two neurons, or to bud off daughter cells that themselves divide multiple times before generating neurons. Although mammalian CNS development likely will involve more complex and more elaborate lineage variations, there is, nevertheless, a striking similarity between these alternate mammalian daughter proliferation modes and the type 0, I and II modes now identified in Drosophila. Intriguingly, in line with these analogies between Drosophila and mammals, recent time-lapse studies on the developing primate cortex have revealed a global temporal switch in the proliferation profiles of daughter cells (Betizeau, 2013). It will be interesting to learn if such temporal proliferation changes are intrinsically controlled and if they are stereotypically linked to changes in neural subtype specification also in mammals (Baumgardt, 2014).

    Drosophila embryonic type II neuroblasts: origin, temporal patterning, and contribution to the adult central complex

    Drosophila neuroblasts are an excellent model for investigating how neuronal diversity is generated. Most brain neuroblasts generate a series of ganglion mother cells (GMCs) that each make two neurons (type I lineage), but sixteen brain neuroblasts generate a series of intermediate neural progenitors (INPs) that each produce 4-6 GMCs and 8-12 neurons (type II lineage). Thus, type II lineages are similar to primate cortical lineages, and may serve as models for understanding cortical expansion. Yet the origin of type II neuroblasts remains mysterious: do they form in the embryo or larva? If they form in the embryo, do their progeny populate the adult central complex, as do the larval type II neuroblast progeny? This study presents molecular and clonal data showing that all type II neuroblasts form in the embryo, produce INPs, and express known temporal transcription factors. Embryonic type II neuroblasts and INPs undergo quiescence, and produce embryonic-born progeny that contribute to the adult central complex. These results provide a foundation for investigating the development of the central complex, and tools for characterizing early-born neurons in central complex function (Walsh, 2017).

    It has been difficult to link embryonic neuroblasts to their larval counterparts in the brain and thoracic segments owing to the period of quiescence at the embryo-larval transition, and owing to dramatic morphological changes of the CNS that occur at late embryogenesis. Recent work has revealed the embryonic origin of some larval neuroblasts: the four mushroom body neuroblasts in the central brain and about 20 neuroblasts in thoracic segments. This study used molecular markers and clonal analysis to identify all eight known type II neuroblasts in each brain lobe and show they all form during embryogenesis, perhaps the last-born central brain neuroblasts. It was not possible to identify each neuroblast individually, however, owing to their tight clustering, movements of the brain lobes, and the lack of markers for specific type II neuroblasts (Walsh, 2017).

    The single previously reported embryonic type II neuroblast formed from PntP1+ neuroectodermal cells with apical constrictions called a placode. This study did not investigate this neuroectodermal origin of type II neuroblasts in much detail, but multiple type II neuroblasts were seen developing from PntP1+ neuroectoderm. In the future, it would be interesting to determine whether all type II neuroblasts arise from PntP1+ neuroectoderm or from neuroectodermal placodes. Interestingly, one distinguishing molecular attribute of type II neuroblasts is PntP1, which is not detected in type I neuroblasts. Thus, a candidate for distinguishing type I/type II neuroblast identity is EGF signaling, which can be detected in the three head placodes and is required for PntP1 expression. Clearly, there are more PntP1+ neuroectodermal cells than there are type II neuroblasts, and expression of an EGF negative regulator such as Argos might be necessary to divert some of these neuroectodermal cells away from type II neuroblast specification. The earliest steps of type II neuroblast formation represent an interesting spatial patterning question for future studies (Walsh, 2017).

    Now that the embryonic type II neuroblasts have been identified, it is worth considering whether there are differences between embryonic and larval type II neuroblasts or their INP progeny. To date, molecular markers do not reveal any differences between embryonic and larval type II neuroblasts, with the exception that embryonic neuroblasts transiently express the temporal transcription factor Pdm. Interestingly, type I embryonic neuroblasts require Cas to close the Pdm expression window, whereas this study found that cas mutants do not exhibit extension of the Pdm expression window in the earliest-born type II neuroblast or de novo expression of Pdm in the later-forming neuroblasts. Are there differences between embryonic and larval INPs? Larval INPs mature over a period of 6 h and then divide four to six times with a cell cycle of about 1 h. In contrast, embryonic INPs might have a more rapid maturation because Elav+ neurons were seen within 9D11+ INP lineages by stage 14, just 3 h after the first type II neuroblast forms. This study found that INPs undergo quiescence at the embryo-larval transition, as shown by the pools of INPs at stage 16 that do not stain for the mitotic marker pH3. The fate of these quiescent INPs -- whether they resume proliferation, differentiate or die -- remains to be determined (Walsh, 2017).

    Neuroblasts in the embryonic ventral nerve cord use the temporal transcription factor cascade Hb>Krüppel>Pdm>Cas>Grh to generate neural diversity. This study shows that the type II neuroblasts are among the last neuroblasts to form in the embryonic brain, and that they sequentially express only the late temporal transcription factors Pdm (in the earliest-forming neuroblast) followed by Cas and grh (in all eight type II neuroblasts). It is unknown why most type II neuroblasts skip the early Hb>Krüppel>Pdm temporal transcription factors; perhaps it is due to their late time of formation, although several earlier-forming thoracic neuroblasts also skip Hb (NB3-3), Hb>Krüppel (NB5-5), or Hb>Krüppel>Pdm. This is another interesting spatial patterning question for the future. Furthermore, misexpression of the early factors (Hb and Krüppel) would be unlikely to affect the progeny produced by type II NBs during embryogenesis, as the competence window for Hb (i.e., the stage at which neuroblasts are responsive to Hb expression) closes with the loss of Dan/Danr expression in all neuroblasts at stage 12. Thus, most embryonic type II neuroblasts form after closing of the Hb competence window and would probably be unresponsive (Walsh, 2017).

    Type I neuroblasts show persistent expression of the temporal transcription factors within neurons born during each window of expression (i.e. a Hb+ neuroblast divides to produce a Hb+ GMC which makes Hb+ neurons). In contrast, this study found that type II lineages do not show persistent Cas or grh expression in INPs born during each expression window, but do contain some Cas+ neurons. Both Cas and grh transcription factors can be seen in INPs immediately adjacent to the parental neuroblast, consistent with transient perdurance from the parental neuroblast, but they are typically lacking in INPs more distant. The function of Pdm, Cas and grh in embryonic type II neuroblasts awaits identification of specific markers for neural progeny born during each expression window (Walsh, 2017).

    During larval neurogenesis, virtually all INPs sequentially express the temporal transcription factors Dichaete>Grh>Ey. In contrast, embryonic INPs express only Dichaete. These data, together with the short time frame of embryogenesis, suggest that INP quiescence occurs during the Dichaete window, preventing expression of the later Grh>Ey cascade. Interestingly, INPs in the posterior cluster (presumptive DL1 and DL2 type II neuroblast progeny) completely lack Dichaete; this is similar to the DL1 and DL2 larval lineages, which also do not express Dichaete. It is possible that DL1/DL2 neuroblasts make INPs that generate identical progeny (and thus do not require an INP temporal cascade), or perhaps these two neuroblasts use a novel temporal cascade in both embryonic and larval stages (Walsh, 2017).

    Larval type II neuroblasts produce many intrinsic neurons of the adult central complex. This study shows that embryonic INPs also produce neurons that contribute to the adult central complex. The data show ~54 neurons (64 minus 10 due to 'leaky' expression) born from embryonic-born INPs survive to adulthood and innervate the central complex. It is likely that this is an underestimate, however, because (1) 9D11-gal4 expression is lacking from a few INPs in the embryonic brain and (2) the time to achieve sufficient FLP protein levels to achieve immortalization could miss the earliest born neurons. The variation in immortalization of the widefield ellipsoid body neuron might represent a neuron born early in the type II lineages, thus unlabeled in a subset of embryos. Additionally, some embryonic-born neurons might perform important functions in the larval/pupal stages but die prior to eclosion (Walsh, 2017).

    Further studies will be required to understand the function of neurons born from embryonic type II lineages. It remains to be experimentally determined whether some or all embryonic progeny of type II neuroblasts (1) remain functionally immature in both the larval and adult brain, but serve as pioneer neurons to guide larval-born neurons to establish the central complex, (2) remain functionally immature in the larval brain, but differentiate and function in the adult central complex, or (3) differentiate and perform a function in both the larval and adult CNS. It will be informative to ablate embryonic-born neurons selectively and determine the effect on the assembly of the larval or adult central complex, and their role in generating larval and adult behavior (Walsh, 2017).

    Chromatin remodeling during in vivo neural stem cells differentiating to neurons in early Drosophila embryos

    Neurons are a key component of the nervous system and differentiate from multipotent neural stem cells (NSCs). Chromatin remodeling has a critical role in the differentiation process. However, its in vivo epigenetic regulatory role remains unknown. This study shows that nucleosome depletion regions (NDRs) form in both proximal promoters and distal enhancers during NSCs differentiating into neurons in the early Drosophila embryonic development. NDR formation in the regulatory regions involves nucleosome shift and eviction. Nucleosome occupancy in promoter NDRs is inversely proportional to the gene activity. Genes with promoter NDR formation during differentiation are enriched for functions related to neuron development and maturation. Active histone-modification signals (H3K4me3 and H3K9ac) in promoters are gained in neurons in two modes: de novo establishment to high levels or increase from the existing levels in NSCs. The gene sets corresponding to the two modes have different neuron-related functions. Dynamic changes of H3K27ac and H3K9ac signals in enhancers and promoters synergistically repress genes associated with neural stem or progenitor cell-related pluripotency and upregulate genes associated with neuron projection morphogenesis, neuron differentiation, and so on. These results offer new insights into chromatin remodeling during in vivo neuron development and lay a foundation for its epigenetic regulatory mechanism study of other lineage specification (Ye, 2016).

    Specification of individual adult motor neuron morphologies by combinatorial transcription factor code

    How the highly stereotyped morphologies of individual neurons are genetically specified is not well understood. This study identified six transcription factors (TFs; Ems, Zfh1, Pb, Zfh2, Pros and Toy) expressed in a combinatorial manner in seven post-mitotic adult leg motor neurons (MNs) that are derived from a single neuroblast in Drosophila. Unlike TFs expressed in mitotically active neuroblasts, these TFs do not regulate each other's expression. Removing the activity of a single TF resulted in specific morphological defects, including muscle targeting and dendritic arborization, and in a highly specific walking defect in adult flies. In contrast, when the expression of multiple TFs was modified, nearly complete transformations in MN morphologies were generated. These results show that the morphological characteristics of a single neuron are dictated by a combinatorial code of morphology TFs (mTFs). mTFs function at a previously unidentified regulatory tier downstream of factors acting in the NB but independently of factors that act in terminally differentiated neurons (Enriquez, 2015).

    Neurons are the most morphologically diverse cell types in the animal kingdom, providing animals with the means to sense their environment and move in response. In Drosophila, neurons are generated by neuroblasts (NBs), specialized stem cells dedicated to the generation of neurons and glia. As they divide, NBs express a temporal sequence of transcription factors (TFs) that contribute to the generation of neuronal diversity. For example, in the embryonic ventral nerve cord (VNC), most NBs express a sequence of five TFs (Hunchback, Krüppel, Pdm1/Pdm2, Castor, and Grainyhead), while in medulla NBs and intermediate neural progenitors of the Drosophila larval brain a different series of TFs have been described. In vertebrates, analogous strategies are probably used by neural stem cells, e.g., in the cerebral cortex and retina, suggesting that this regulatory logic is evolutionarily conserved. Nevertheless, although temporally expressed NB TFs play an important role in generating diversity, this strategy cannot be sufficient to explain the vast array of morphologically distinct neurons present in nervous systems. For example, in the Drosophila optic lobe there is estimated to be ~40,000 neurons, classified into ~70 morphologically distinct types, each making unique connections within the fly's visual circuitry neurons (Enriquez, 2015).

    A second class of TFs has been proposed to specify subtypes of neurons. For example, in the vertebrate spinal cord, all motor neurons (MNs) express a common set of TFs at the progenitor stage (Olig2, Nkx6.1/6.2, and Pax6) and a different set of TFs after they become post-mitotic (Hb9, Islet1/2, and Lhx3). Hox6 at brachial and Hox10 at lumbar levels further distinguish MNs that target muscles in the limbs instead of body wall muscles. Subsequently, limb-targeting MNs are further refined into pools, where all MNs in a single pool target the same muscle. Each pool is molecularly defined by the expression of pool-specific TFs, including a unique combination of Hox TFs. In Drosophila embryos, subclasses of MNs are also specified by unique combinations of TFs: evenskipped (eve) and grain are expressed in six MNs that target dorsal body wall, and Hb9, Nkx6, Islet, Lim3, and Olig2 are required for ventral-targeting MNs. However, each neuronal subtype defined by these TFs includes multiple morphologically distinct neurons, leaving open the question of how individual neuronal morphologies are specified neurons (Enriquez, 2015).

    A third class of TFs suggested to be important for neuronal identity is encoded by terminal selector genes. Initially defined in C. elegans, these factors maintain a neuron's terminally differentiated characteristics by, for example, regulating genes required for the production of a particular neurotransmitter or neuropeptide. Consequently, these TFs must be expressed throughout the lifetime of a terminally differentiated neuron. Notably, as with neurons that are from the same subtype, neurons that share terminal characteristics, and are therefore likely to share the same terminal selector TFs, can have distinct morphological identities. For example, in C. elegans two terminal selector TFs, Mec-3 and Unc-86, function together to maintain the expression of genes required for a mechanosensory fate in six morphologically distinct touch sensitive neurons neurons (Enriquez, 2015).

    In contrast to the logic revealed by these three classes of TFs, very little is known about how individual neurons, each with their own stereotyped dendritic arbors and synaptic targets, obtain their specific morphological characteristics. This paper addresses this question by focusing on how individual MNs that target the adult legs of Drosophila obtain their morphological identities. The adult leg MNs of Drosophila offer several advantages for understanding the genetic specification of neuronal morphology. For one, all 11 NB lineages that generate the ~50 leg-targeting MNs in each hemisegment have been defined. More than two-thirds of these MNs are derived from only two lineages, Lin A (also called Lin 15) and Lin B (also called Lin 24), which produce 28 and 7 MNs, respectively, during the second and third larval stages. Second, each leg-targeting MN has been morphologically characterized-both dendrites and axons-at the single-cell level. In the adult VNC, the leg MN cell bodies in each thoracic hemisegment (T1, T2, and T3) are clustered together. Each MN extends a highly stereotyped array of dendrites into a dense neuropil within the VNC and a single axon into the ipsilateral leg, where it forms synapses onto one of 14 muscles in one of four leg segments: coxa (Co), trochanter (Tr), femur (Fe), and tibia (Ti). Not only does each MN target a specific region of a muscle, the pattern of dendritic arbors of each MN is also stereotyped and correlates with axon targeting. The tight correlation between axon targeting and dendritic morphology has been referred to as a myotopic map. The stereotyped morphology exhibited by each MN suggests that it is under precise genetic control that is essential to its function neurons (Enriquez, 2015).

    This study demonstrates that individual post-mitotic MNs express a unique combination of TFs that endows them with their specific morphological properties. Focus was placed on Lin B, which generates seven MNs, and six TFs were identified that can account for most of the morphological diversity within this lineage. Interestingly, these TFs do not cross-regulate each other and are not required for other attributes of MN identity, such as their choice of neurotransmitter (glutamine) or whether their axons target muscles in the periphery, i.e., they remain terminally differentiated leg motor neurons. Consistent with the existence of a combinatorial code, when two or three, but not individual, TFs were simultaneously manipulated nearly complete transformations in morphology were observed. However, removing the function of a single TF, which is expressed in only three Lin B MNs, resulted in a highly specific walking defect that suggests a dedicated role for these neurons in fast walking. Together, these findings reveal the existence of a regulatory step downstream of temporal NB factors in which combinations of morphology TFs (mTFs) control individual neuron morphologies, while leaving other terminal characteristics of neuronal identity unaffected neurons (Enriquez, 2015).

    Inherent in the concept of a combinatorial TF code is the idea that removing or ectopically expressing a single TF will only generate a transformation of fate when a different wild-type code is generated. Consistent with this notion, only when the expression of two or three mTFs were simultaneously manipulated was it possible to partially mimic a distinct mTF code and, as a result, transform the identity of one Lin B MN into another. In contrast, manipulating single TFs typically resulted in aberrant or neo-codes that are not observed in wild-type flies. For example, removing pb function from Lin B resulted in two MNs with a code (Ems+Zfh1) and MN morphology that are not observed in wild-type Lin A and Lin B lineages. Analogously, ectopic Pb expression in Lin A, which normally does not express this TF, generated aberrant codes and MN morphologies. This latter experiment was particularly informative because although Pb redirected a subset of Lin A dendrites to grow in an anterior region of the neuropil, it did not alter the ability of these dendrites to cross the midline. Thus, the dendrites of these MNs had characteristics of both Pb-expressing Lin B MNs (occupying an antero-ventral region) and Pb-non-expressing Lin A MNs (competence to cross the midline). Axon targeting of these MNs was also aberrant: although they still targeted leg muscles, Pb-expressing Lin A MNs frequently terminated in the coxa, which is not a normal characteristic of Pb-expressing Lin B MNs or of any Lin A MN. These observations suggest that the final morphological identity of a neuron is a consequence of multiple TFs executing functions that comprise a complete morphological signature. Some functions, such as the ability to occupy the antero-ventral region of the neuropil, can be directed by a single TF (e.g., Pb), while other functions, such as the ability to accurately target the distal femur, require multiple TFs (e.g., Pb+Ems). Further, because it was possible to generate MNs that have both Lin B and Lin A morphological characteristics, hte results argue against the idea that there are lineage-specific mTFs shared by all progeny derived from the same lineage. Instead, the data are more consistent with the idea that the final morphological identity of an MN depends on its mTF code neurons (Enriquez, 2015).

    Drosophila NBs, and perhaps vertebrate neural stem cells, express a series of TFs that change over time and have therefore been referred to as temporal TFs. For Lin B, the sequence of these factors is unknown, in part because the Lin B NB is not easily identified in the second-instar larval VNC, the time at which it is generating MNs. Nevertheless, each MN derived from Lin B and Lin A has a stereotyped birth order, consistent with the idea that temporal TFs play an important role in directing the identities of MNs derived from these lineages and, therefore, the mTFs they express. For Lin B, this birth order is Co1->Tr1->Fe1->Tr2->Co2->Co3->Co4. Interestingly, according to the mTF code proposed in this study, each of these MNs differs by at most two mTFs in any successive step. For example, Tr1 has the code [Zfh1, Ems, Pb, Zfh2] while Fe1, the next MN to be born, has the code [Zfh1, Ems, Pb]. Thus, it is posited that the sequence of temporal TFs acting in the NB is responsible for directing each successive change in mTF expression in postmitotic MNs (e.g., in the Tr1->Fe1 step, repression of zfh2). Although a link between temporal TFs and TFs expressed in postmitotic neurons has been proposed in Drosophila, the role of these TFs in conferring neuron morphologies is not known. Further, there may be additional diversity-generating mechanisms in lineages that produce many more neurons than the seven MNs generated by Lin B. One additional source of diversity may come from NB identity TFs, which distinguish lineages based on their position. Such spatial information could in principle allow the same temporal TFs to regulate different sets of mTFs in different NB lineages. It is also likely that differences in the levels of some mTFs may contribute to neuronal identities. Consistent with this idea, the levels of Zfh2 and Pros differ in the Lin B MNs expressing these TFs, differences that are consistent in all three thoracic segments and between animals. Further, Zfh1 levels vary between Lin B MNs and its levels control the amount of terminal axon branching. Previous studies also demonstrated that TF levels are important for neuron morphology, including Antp in adult leg MNs derived from Lin A and Cut in the control of dendritic arborization complexity in multidendritic neurons. If the levels of mTFs are important, it may provide a partial explanation for why the transformations of morphological identity generated in this study with the MARCM technique, which cannot control levels, are typically only partially penetrant neurons (Enriquez, 2015).

    Another distinction between temporal TFs and mTFs is that no evidence has been observed of cross-regulation between mTFs. In situations when mTFs were either removed (e.g., pb-/-; emsRNAi) or ectopically expressed (e.g., UAS-pb + UAS-ems) in postmitotic Lin B MARCM clones, the expression of the remaining mTFs was unchanged. In contrast, when an NB lineage is mutant for a temporal TF, the prior TF in the series typically continues to be expressed. These observations suggest that the choice of mTF expression is made in the NB and that once the postmitotic code is established, it is not further influenced by coexpressed mTFs neurons (Enriquez, 2015).

    The data further suggest that mTFs are distinct from terminal selector TFs. In mutants for the mTFs studied here, the resulting neurons remain glutamatergic leg motor neurons: they continue to express VGlut, which encodes a vesicular glutamate transporter, expressed by all Drosophila MNs, and they still exit the VNC to target and synapse onto muscles in the adult legs. Thus, whereas terminal selector TFs maintain the terminal characteristics of fully differentiated neurons, mTFs are required transiently to execute functions required for each neuron's specific morphological characteristics. Together, it is suggested that the combined activities of terminal selector TFs and mTFs specify and maintain the complete identity of each post-mitotic neuron neurons (Enriquez, 2015).

    Although the mTFs defined in this study, e.g., Ems, Pb, and Toy, do not fit the criteria for a terminal selector TF, it is plausible that some TFs function both as mTFs and terminal selector TFs. One example may be Apterous, a TF that is expressed in six interneurons in the thoracic embryonic segments and that functions with other TFs to control the terminal differentiation state of these neuropeptide-expressing neurons. In addition to the loss of neuropeptide expression, these neurons display axon pathfinding defects in the absence of apterous. Despite the potential for overlapping functions, it is conceptually valuable to consider the specification of neuronal morphologies as distinct from other terminal characteristics, as some mTFs regulate morphology without impacting these other attributes. It is also plausible that some of the TFs that have been previously designated as determinants of subtype identity may also be part of mTF codes. For example, eve is required for the identity of dorsally directed MNs inDrosophila embryogenesis, but the TFs required for distinguishing the individual morphologies of these neurons are not known. It may be that Eve is one component of the mTF code and that it functions together with other mTFs to dictate the specific morphologies of these neurons neurons (Enriquez, 2015).

    Flies containing a single pb mutant Lin B clone exhibited a highly specific walking defect: when walking at high speed, these flies were significantly more unsteady compared to control flies. The restriction of this defect to high speeds suggests that the Pb-dependent characteristics of these MNs may be specifically required when the walking cycle is maximally engaged, raising the possibility that Tr1, Tr2, and Fe1 are analogous to so-called fast MNs described in other systems. Further, these data support the idea that the highly stereotyped morphology of these MNs is critical to the wild-type function of the motor circuit used for walking. In particular, the precise dendritic arborization pattern exhibited by these MNs, which is disrupted in the pb mutant, is likely to be essential for their function. Although it cannot be excluded that other pb-dependent functions contribute to this walking defect, these observations provide strong evidence that the myotopic map, in which MNs that target similar muscle types have similar dendritic arborization patterns, is important for the fly to execute specific adult behaviors neurons (Enriquez, 2015).

    Neuronal cell fate diversification controlled by sub-temporal action of Kruppel

    During Drosophila CNS development, neuroblasts express a programmed cascade of five temporal transcription factors that govern the identity of cells generated at different time-points. However, these five temporal genes fall short of accounting for the many distinct cell types generated in large lineages. This study finds that the late temporal gene castor sub-divides its large window in neuroblast 5-6 by simultaneously activating two cell fate determination cascades and a sub-temporal regulatory program. The sub-temporal program acts both upon itself and upon the determination cascades to diversify the castor window. Surprisingly, the early temporal gene Kruppel acts as one of the sub-temporal genes within the late castor window. Intriguingly, while the temporal gene castor activates the two determination cascades and the sub-temporal program, spatial cues controlling cell fate in the latter part of the 5-6 lineage exclusively act upon the determination cascades (Stratmann, 2016).

    This study, along with previous work, found that the temporal gene cascade results in the expression of Cas in the latter part of NB5-6T. cas acts together with spatial input, provided by Antp, hth, exd and lbe to activate col in the NB. col in turn activates ap and eya in the early postmitotic cells, which represents a transient and generic Ap cluster cell fate. col subsequently acts in a feedforward loop of col>ap/eya>dimm>Nplp1 to determine Tv1 cell fate. However, in addition to col, cas activates five other genes, including the last temporal gene grh, and the sub-temporal genes sqz, nab, svp and, as shown in this study, Kr. These five genes engage in a postmitotic cross-regulatory interplay, unique to each of the three cell types, which results in the propagation of the col>ap/eya>dimm>Nplp1 terminal selector cascade exclusively in Tv1, and the ap/eya/dac>dimm/BMP>FMRFa cascade in Tv4, while the Tv2/3 cells acquire a non-peptidergic interneuron identity. The role of Kr is to suppress the sub-temporal gene svp, in order to safeguard the expression of col and dimm, and thereby ensures the propagation of the col>ap/eya>dimm>Nplp1 terminal selector cascade, crucial for specification of the Tv1 cells. The other four genes (grh, sqz, nab, svp) each have unique roles, and act as sub-temporal micromanagers to ensure high fidelity and precision in the sub-division of the cas temporal window (Stratmann, 2016).

    The temporal gene cas plays a pivotal role in the specification process of the different Ap cluster cells due to its activator role on a number of downstream regulators; col, a terminal selector in Tv1 specification, the sub-temporal genes sqz, nab, svp and Kr, as well as the temporal gene grh. Strikingly, cas thus activates both of the two terminal selector feedforward loops (FFLs), and the genes required to refine both FFLs (Stratmann, 2016).

    cas activates Kr and svp, but how is Kr expression then restricted to only Tv1 and svp expression to Tv2/3? For Kr, restricted expression of sqz, nab and svp in Tv2-Tv4, all of which suppress Kr, can explain the confined expression pattern of Kr to Tv1. The gradually restricted expression of svp in Tv2-3 is in turn explained by Kr repressing svp in Tv1, and by grh repressing svp in Tv4. However, because grh misexpression is not sufficient to repress svp, it is tempting to speculate that there exists a similar factor to Kr, being exclusively expressed in the Tv4 cell, acting to suppress svp expression in a highly confined manner to ensure FMRFa/Tv4 specification (Stratmann, 2016).

    Besides its activation by cas, col activation requires additional spatial information, provided by lbe, Antp, hth and exd, which subsequently initializes the generic Ap cluster program, by activating ap and eya. In contrast, cas alone activates grh and the sub-temporal factors, which are then important for the cell diversification, whether by activating or repressing each other's actions, or the FFLs, or partake in the FFL (grh), in order to allocate the correct cell fate to the four Ap cluster neurons. Remarkably, the four spatial inputs (lbe, Antp, hth and exd) act only on col, while the temporal input (cas) acts both on col, as well as the temporal and sub-temporal factors (sqz, nab, svp, Kr and grh). It is tempting to speculate that this may point to a general role for spatial versus temporal cues, and may be logically explained by the fact that spatial cues generally do not display the highly selective temporal expression profile necessary for sub-temporal cell diversification (Stratmann, 2016).

    An unexpected finding in this study pertains to the dual role of Kr, first acting early in the canonical temporal cascade and subsequently late in the sub-temporal cascade, to ensure the specification of the Tv1 cell. The main role of Kr in Tv1 cells is to suppress svp, hence allowing for the maintenance of Col, which itself is critical for the propagation of the terminal FFL, fundamental for Tv1 cell fate. Interestingly, dual expression of Kr, first in the neuroblast and subsequently in neurons, was previously observed in NB3-3, but the functional role of the second Kr expression pulse was not addressed. svp itself also displays a dual expression and function, being expressed early in many NB lineages to suppress hb, then being re-expressed in several lineages, and in NB5-6T it acts to suppress col and dimm. With regards to postmitotic activity, another example of a temporal gene acting postmitotically applies to the role of the last temporal gene, grh, which is necessary and sufficient for FMRFa expression in Tv4 cells, and can trigger ectopic FMRFa in Ap neurons when misexpressed postmitotically. Yet in contrast to Kr, grh does not experience a dual expression profile. Hence, with several examples of dual (Kr and svp) and progenitor versus postmitotic roles of temporal genes (Kr and grh), it is tempting to speculate that this type of temporal multi-tasking may indeed be a common feature for many temporal genes, both in Drosophila and in higher organisms (Stratmann, 2016).

    Neuronal cell fate specification by the convergence of different spatiotemporal cues on a common terminal selector cascade
    Specification of the myriad of unique neuronal subtypes found in the nervous system depends upon spatiotemporal cues and terminal selector gene cascades, often acting in sequential combinatorial codes to determine final cell fate. However, a specific neuronal cell subtype can often be generated in different parts of the nervous system and at different stages, indicating that different spatiotemporal cues can converge on the same terminal selectors to thereby generate a similar cell fate. However, the regulatory mechanisms underlying such convergence are poorly understood. The Nplp1 neuropeptide neurons in the Drosophila ventral nerve cord can be subdivided into the thoracic-ventral Tv1 neurons and the dorsal-medial dAp neurons. The activation of Nplp1 in Tv1 and dAp neurons depends upon the same terminal selector cascade: col->ap/eya->dimm->Nplp1. However, Tv1 and dAp neurons are generated by different neural progenitors (neuroblasts) with different spatiotemporal appearance. It was found that the same terminal selector cascade is triggered by Kr/pdm->grn in dAp neurons, but by Antp/hth/exd/lbe/cas in Tv1 neurons. Hence, two different spatiotemporal combinations can funnel into a common downstream terminal selector cascade to determine a highly related cell fate (Gabilondo, 2016).

    Gene regulatory networks in Drosophila early embryonic development as a model for the study of the temporal identity of neuroblasts

    Genes belonging to the "gap" and "gap-like" family constitute the best-studied gene regulatory networks (GRNs) in Drosophila embryogenesis. Gap genes are a core of two subnetworks controlling embryonic segmentation: (hunchback, hb; Krüppel, Kr; giant, gt; and knirps, kni) and (hb; Kr; pou-domain, pdm; and, probably, castor, cas). Of particular interest is that (hb, Kr, pdm, cas) also specifies the temporal identity of stem cells, neuroblasts, in Drosophila neurogenesis. This GRN controls the sequential differentiation of neuroblasts during the asymmetric cell division. In the last decades, modeling of the patterning of gene ensemble (hb, Kr, gt, kni) in segmentation was in the center of attention. This study now shows that the previously published and extensively studied model at a certain level of external factors is able to reproduce temporal patterns of (hb, Kr, pdm, cas) in neurogenesis with minor evolutionary explicable modifications. This result testifies in favor of a hypothesis that the similarity of two gene ensembles active in segmentation and neurogenesis is a result of co-option of the network architecture in evolution from the common ancestral form. By means of the model dynamical analysis, it is shown that the establishment of the robust patterns in both systems could be explained in terms of the action of attractors in the gap gene dynamical system. This study formulates the common principles underlying the robustness of both GRNs in segmentation and neurogenesis due to the similar functional organization of the gene ensembles as having the same evolutionary origin (Myasnikova, 2020).

    A novel temporal identity window generates alternating Eve(+)/Nkx6(+) motor neuron subtypes in a single progenitor lineage

    Spatial patterning specifies neural progenitor identity, with further diversity generated by temporal patterning within individual progenitor lineages. In vertebrates, these mechanisms generate 'cardinal classes' of neurons that share a transcription factor identity and common morphology. In Drosophila, two cardinal classes are Even-skipped (Eve)(+) motor neurons projecting to dorsal longitudinal muscles, and Nkx6(+) motor neurons projecting to ventral oblique muscles. Cross-repressive interactions prevent stable double-positive motor neurons. The Drosophila neuroblast 7-1 (NB7-1) lineage uses a temporal transcription factor cascade to generate five distinct Eve(+) motor neurons; the origin and development of Nkx6(+) motor neurons remains unclear. This study used a neuroblast specific Gal4 line, sparse labelling and molecular markers to identify an Nkx6(+) VO motor neuron produced by the NB7-1 lineage. Lineage analysis to birth-date the VO motor neuron to the Kr(+) Pdm(+) neuroblast temporal identity window. Gain- and loss-of-function strategies to test the role of Kr(+) Pdm(+) temporal identity and the Nkx6 transcription factor in specifying VO neuron identity. Lineage analysis identifies an Nkx6(+) neuron born from the Kr(+) Pdm(+) temporal identity window in the NB7-1 lineage, resulting in alternation of cardinal motor neuron subtypes within this lineage (Eve>Nkx6>Eve). Co-overexpression of Kr/Pdm generates ectopic VO motor neurons within the NB7-1 lineage - the first evidence that this TTF combination specifies neuronal identity. Moreover, the Kr/Pdm combination promotes Nkx6 expression, which itself is necessary and sufficient for motor neuron targeting to ventral oblique muscles, thereby revealing a molecular specification pathway from temporal patterning to cardinal transcription factor expression to motor neuron target selection. Thus this study shows that one neuroblast lineage generates interleaved cardinal motor neurons fates; that the Kr/Pdm TTFs form a novel temporal identity window that promotes expression of Nkx6; and that the Kr/Pdm > Nkx6 pathway is necessary and sufficient to promote VO motor neuron targeting to the correct ventral muscle group (Seroka, 2020).

    Neural diversity from flies to mice arises from two major developmental mechanisms. First, neural progenitors acquire a unique and heritable spatial identity based on their position along the rostrocaudal or dorsoventral body axes. Second, temporal patterning based on neuronal birth-order results in individual progenitors producing a diverse array of neurons and glia. Temporal patterning is best characterized in Drosophila; neural progenitors (neuroblasts) located in the ventral nerve cord, central brain, and optic lobes all undergo temporal patterning, in which the neuroblast sequentially expresses a cascade of TTFs that specify distinct neuronal identities. Although all neuroblasts undergo temporal patterning, the TTFs are different in each region of the brain. Similar mechanisms are used in the mammalian cortex, retina, and spinal cord, although many TTFs remain to be identified (Seroka, 2020).

    A major open question is how transient expression of TTFs like Kr and Pdm lead to long-lasting specification of molecular and morphological neuronal diversity. Good candidates for integrating spatial and temporal cues to consolidate motor neuron identity are homeodomain transcription factors expressed in post-mitotic motor neurons. In vertebrates, dorsoventral domains of the spinal cord are partitioned into 12 distinct cardinal classes of neurons - each characterized by development from a common progenitor domain, expression of unique homeodomain transcription factors with cross-repressive interactions to stabilize boundaries, and generating neurons with common morphology. This nomenclature is adapted to define Eve+ and Nkx6+ (Flybase: HGTX) motor neurons as two 'cardinal classes' of motor neurons: each class expresses a homeodomain transcription factor (Eve or Nkx6) with cross-repressive interactions, and each class consists of motor neurons with related neuronal morphology (Eve+ motor neurons project to dorsal and lateral longitudinal muscles; Nkx6+ motor neurons project to ventral muscle groups) (Seroka, 2020).

    The Drosophila neuroblast 7-1 (NB7-1) is arguably the best characterized system for understanding TTF expression and function. Similar to most other ventral nerve cord neuroblasts, NB7-1 expresses the canonical TTF cascade Hb-Kr-Pdm-Cas with each TTF inherited by the GMCs born during an expression window, and transiently maintained in the two post-mitotic neurons produced by each GMC. The TTF cascade generates diversity among the five Eve+ U1-U5 motor neuron progeny of NB7-1: Hb specifies U1 and U2, Kr specifies U3, Pdm specifies U4, and Pdm/Cas together specify U5. Identifying TTF target genes, including transcription factors and cell surface molecules, will provide a comprehensive view of how developmental determinants direct neuronal morphology and synaptic partner choices (Seroka, 2020).

    It has long been thought that the cardinal classes of motor neurons derive from distinct progenitors; Eve+ motor neurons derive from NB7-1, NB1-1, and NB4-2 whereas Hb9+ or Nkx6+ motor neurons derive from NB3-1 and others. However, DiI labeling of NB7-1 identified a potentially unknown motor neuron innervating ventral muscles, which is distinct from dorsal and lateral longitudinal muscles targeted by the Eve+ motor neurons. The observed ventral projection in this lineage could reflect transient exuberant outgrowths that are lost during larval life, or they could be due to an uncharacterized motor neuron that forms stable synapses with ventral muscles (Seroka, 2020).

    This study shows that a newly discovered Kr/Pdm TTF window generates an Nkx6+ Eve-motor neuron, born between U3 and U4 in the NB7-1 lineage, that projects to ventral oblique (VO) muscles. It was also shown that overexpression of Kr/Pdm together, or Nkx6 alone, generates ectopic VO motor neurons based on molecular marker expression. Finally, this study demonstrates that Nkx6 is required for proper motor neuron axon targeting to ventral oblique muscles. These results establish a genetic pathway from TTFs (Kr/Pdm), to a cardinal motor neuron transcription factor (Nkx6) to motor axonal targeting. Also the unexpected discovery was made that a single progenitor can alternate production of different cardinal motor neuron classes (Seroka, 2020).

    Kr/Pdm co-expression has been detected in several neuroblast lineages, but until now there has not been evidence that this TTF combination could specify neuronal identity. Previous work showed that the Kr/Pdm window generates a Kr/Pdm GMC, and this study shows that this GMC generates an Nkx6+ ventral-projecting motor neuron. It is unknown whether Kr/Pdm directly or indirectly activate nkx6 expression. The nkx6 gene lies in a 45kb region devoid of genes, and there are only a few, sparse predicted Kr or Pdm binding sites in this genomic expanse. How do Kr and Pdm together specify one fate (Nkx6+ VO motor neuron) whereas Kr or Pdm alone specify completely different fates (Eve+ dorsal motor neurons)? It is likely that Kr/Pdm together activate a different suite of target genes than either alone. For example, Kr/Pdm together may directly activate nkx6 expression, whereas neither alone has that potential. The emergence of single cell transcriptome and ChIP studies will help to reveal how the combination of Kr/Pdm TTFs generates different cell fate output compared to Kr or Pdm alone (Seroka, 2020).

    The production of an Nkx6+ VO motor neuron in Kr/Pdm window interrupts the sequential production of Eve+ dorsal motor neurons in the NB7-1 lineage, resulting in an Eve > Nkx6 > Eve alternation of cardinal motor neuron production within the lineage. This is unusual, as in most cases neurons with similar morphology or function are produced together in a lineage. In mammals, progenitors generate neurons first, followed by glia; no examples are known of neuron>glia>neuron production from a single lineage. Similarly, Drosophila central brain neuroblast lineages produce the mushroom body γ neurons, then α'/ β' neurons, and lastly α/β neurons, with no evidence for alternating or interspersed fates. In the abdominal NB3-3 lineage, the early-born cells are in a mechanosensitive circuit, whereas the late-born cells are in a proprioceptive circuit. The only possible example of interleaved production of two morphological classes of neurons is in the Drosophila lateral antennal lobe neuroblast lineage, which alternate between uniglomerular and multiglomerular (AMMC) projection neurons. The use of clonal and temporal labeling tools will be needed to examine additional lineages to determine the prevalence of lineages producing temporally interleaved neuronal subtypes as in the NB7-1 lineage (Seroka, 2020).

    Overexpression of Kr/Pdm or Nkx6 can induce only 2-3 ectopic VO motor neurons within the NB7-1 lineage. Clearly not all neurons in the lineage are competent to respond to these transcription factors. Early-born temporal identities specified by Hb and Kr (U1-U3) are unaffected by Kr/Pdm or Nkx6 overexpression, which is similar to previous data showing that early temporal fates are not affected by overexpression of later TTFs in multiple lineages. It remains a puzzle why the Kr+ U3 neuron does not switch to a VO fate upon overexpression of Kr/Pdm. There may need to be an equal level of Kr and Pdm to specify VO fate, although this would not explain why Kr/Pdm overexpression converts the Pdm+ U4 motor neuron to a VO fate. Alternatively, there may be an early chromatin landscape that blocks access to relevant Pdm target loci (Seroka, 2020).

    It is noted that the assay of VO neuronal identity was done in newly-hatched larvae. Although motor circuits are functional at this time, larvae grow for five more days. These is no data on whether the ectopic VO motor neurons are functional or are maintained through the life of the larvae. This would be an important question for the future (Seroka, 2020).

    Nkx6 and Eve have cross-repressive interactions (Broihier, 2004), but with some limitations: early-born Eve+ motor neurons are not affected by Nkx6 overexpression. Wild type animals even show sporadic expression of Nkx6 in the Eve+ U2 motor neuron, but in these neurons it has no effect on Eve expression, nor does it promote targeting to ventral oblique muscles. There appears to be a mechanism to block endogenous or overexpressed Nkx6 function in the early lineage of neuroblasts producing Eve+ motor neurons. The mechanism 'protecting' early-born Eve+ neurons from Nkx6 repression of Eve is unknown. Early lineages may lack an Nkx6 cofactor; Nkx6 could act indirectly via an intermediate transcription factor missing in early lineages; the early TTFs Hb or Kr may block Nkx6 function; or the eve locus could be in a subnuclear domain inaccessible to Nkx6 (Seroka, 2020).

    Nkx6 promotes motor neuron specification in both Drosophila and vertebrates. In Drosophila, loss of Nkx6 reduces ventral projecting motor neuron numbers and increases the number of Eve+ neurons, while overexpression increases ventral projecting motor neuron numbers at the expense of Eve+ neurons. In vertebrates the Nkx6 family members Nkx6.1/Nkx6.2 appear to play a broader role in motor neuron specification. Nkx6.1/Nkx6.2 show early expression throughout the pMN domain; mice mutant for both Nkx6 family members lack most somatic motor neurons; and Nkx6.1 overexpression in chick or zebrafish can induce ectopic motor neurons. It would be interesting to investigate whether vertebrate Nkx6.1/Nkx6.2 are required to suppress a specific motor neuron identity, similar to the antagonistic relationship between Nkx6 and Eve in Drosophila (Seroka, 2020).

    Neuroblasts in all regions of the Drosophila CNS (brain, ventral nerve cord, optic lobe) use TTF cascades to generate neuronal diversity, yet less is known about TTF target genes. It is likely that TTFs induce expression of suites of transcription factors that persist in neurons and confer their identity. Examples may include the 'morphology transcription factors' that specify adult leg motor neuron dendrite projections, but in this case it remains unknown whether these transcription factors control all other aspects of adult motor neuron identity. It is possible that 'morphology transcription factors' are one module downstream of a broader regulatory tier similar to the terminal selector genes in C. elegans (Seroka, 2020).

    This study has identified a linear pathway from Kr/Pdm to Nkx6 which specifies VO motor neuron identity. TTFs could act by two non-mutually exclusive mechanisms: inducing a stable combinatorial codes of transcription factors that consolidate neuronal identity, or by altering the chromatin landscape to have a heritable, long lasting effect on motor neuron gene expression. The observation that Nkx6 is maintained in the VO neuron after fading of Kr/Pdm expression supports the former mechanism. Identification of Kr/Pdm or Nkx6 target genes would give a more comprehensive understanding of TTF specification of neuronal identity (Seroka, 2020).

    The results presented in this work lead to several interesting directions. Other embryonic VNC lineages exhibit a Kr/Pdm window; does this window generate neurons in these lineages? Are there common features to neurons born in the Kr/Pdm window? Furthermore, do ectopic VO neurons make functional presynapses with the ventral oblique muscles, and do they have the normal inputs to their dendritic postsynapses? In only a few cases has it been shown the TTF-induced neurons are functionally integrated into the appropriate circuits [30]. Kr and Pdm orthologs have been identified in vertebrates. Looking for dual expression of Kr and Pdm orthologs in vertebrates may reveal a role in specifying temporal identity, similar to evidence for Hb and Cas TTFs having vertebrate orthologs that specify temporal identity (Seroka, 2020).

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