Transcription factors abrupt absent MD neurons and olfactory sensilla absent, small, or homeotic discs 2 Antennapedia AP-2 aristaless Arrowhead Beadex brahma bric à brac broad (common alternative name: Broad Complex) brother of odd with entrails limited buttonhead C15 combgap cousin of atonal cramped crooked legs cryptocephal cut daughterless Dichaete disconnected and disco-related distal-less doublesex Ecdysone-induced protein 74EF ecdysone receptor elbow escargot extradenticle extra machrochaete grain Grunge hairy homothorax klumpfuss Lim1 lozenge moira paired-box neuro pangolin Pcaf pdm-1 period (expressed in adult legs) polyhomeotic pleiohomeotic ptip reversed polarity Rfx rotund SANT domain protein scratch scribbler Sex combs on midleg snail sparkling spineless Sp1 target of Pox-n teashirt twist Ultrabithorax ultraspiracle

Gene systems Epidermal growth factor pathway neurogenic genes proneural genes segment polarity genes Ligands and secreted dawdle decapentaplegic hedgehog shifted spitz Thrombospondin Transforming growth factor beta at 60A vein wingless Other Abl oncogene approximated arc arrow Axin Cad99C Cdk8 costa crossveinless 2 dachs dachsous dachshund death executioner Bcl-2 homologue discs large 1 discs lost dispatched division abnormally delayed Ecdysone-induced protein 63E Enhancer of split m4, Bearded family member Epidermal growth factor receptor expanded fat four-jointed fringe frizzled furrowed Gustatory receptor 5a (common alternative name:Trehalose-sensitivity) Gustatory receptor 66a Gustatory receptor 68a (expressed in male-specific gustatory bristles in the forelegs) headcase heartless hemipterous hibris hippo Inositol 1,4,5,-tris-phosphate receptor legless lethal (2) giant discs 1 liquid facets mastermind mind-bomb Neurotactin - chordotonal neurons Nitric oxide synthase no receptor potential A O-fucosyltransferase 1 Paxillin pickpocket 25 Presenilin prickle Protein tyrosine phosphatase 69D puckered Rab-protein 4 Rab23 rhomboid roughest sarah scarface skittles snoN split ends strabismus strawberry notch string stumble supernumerary limbs tarsal-less tartan terribly reduced optic lobes TNF-receptor-associated factor 1 Trehalose-sensitivity warts Wnt oncogene analog 2 zipper - Myosin II or non-muscle myosin

Axes, boundaries and coordinates in leg development

Dualistic thinking presents a pitfall in any attempt to explain a real world vastly more complex than an either/or perspective. These difficulties are exemplified when formulating models to describe the basis of polarity in Drosophila leg morphogenesis.

A polar coordinate model is appealing because of the circular symmetry of the leg. In this hypothesis cells receive positional information from the disc center. The presumed coordinates are given by distance from the center (radial coordinate) and circumferential location (angular coordinate). Such a model could work in the real world if decapentaplegic or wingless transcription were limited to a quadrant of the disc. The diffusion of their protein products in this case would set the angular coordinates necessary for cell fate specification. The advantage of this system is that it fits in nicely with the radial symmetry of the leg disc (Held, 1995).

A Cartesian model is equally appealing. A wingless-decapentaplegic zone could function as an X axis to specify one coordinate of cellular position while another unknown gene could perform a similar function for a Y axis. In this model positional information is determined by the lateral diffusion of multiple morphogens. The advantage inherent here is that it fits nicely with understanding of determination of positional information during segmentation, when gradients of multiple morphogens like Bicoid and Decapentaplegic establish positional identity along anterior-posterior and dorsal-ventral axes respectively (Held, 1995).

Reality encroches upon these arguments when observations are made of the effects of mutation and of the expression patterns of genes involved in leg morphogenesis. The leg has a clear anterior-posterior boundary, suggesting a Cartesian model, yet expression patterns of Distal-less and aristaless show radial symmetry, suggesting a polar coordinate model. In addition, the sectored expression of wingless is also compatable with a polar model (Held, 1995).

Current thinking inclines toward the Boundary model, a combination of the Cartesian and polar models. The Boundary model assumes that three or more compartments will be specified. This is thought to be the case in leg polarity, and known to be true for segmentation. These compartments would cooperate to cause the production of a specific morphogen at their point of intersection (the center of the disc). The conical concentration gradient formed by the diffusion of this morphogen would then specify a radial coordinate for all cells in the disc (Held, 1995).

In the Boundary model, cell positional identity in the leg disc is defined by both Cartesian and polar coordinates. In Drosophila leg morphogenesis both models have to be called upon to explain all the facts (Held, 1995).

Cell rearrangement and cell division during the tissue level morphogenesis of evaginating Drosophila imaginal discs

The evagination of Drosophila imaginal discs is a classic system for studying tissue level morphogenesis. Evagination involves a dramatic change in morphology and published data argue that this is mediated by cell shape changes. The evagination of both the leg and wing discs has been reexamined and it has been found that the process involves cell rearrangement and that cell divisions take place during the process. The number of cells across the width of the ptc domain in the wing and the omb domain in the leg decreases as the tissue extends during evagination and cell rearrangement was observed to be common during this period. In addition, almost half of the cells in the region of the leg examined divided between 4 and 8 h after white prepupae formation. Interestingly, these divisions were not typically oriented parallel to the axis of elongation. These observations show that disc evagination involves multiple cellular behaviors, as is the case for many other morphogenetic processes (Taylor, 2008).

This study established that cell rearrangement takes place during leg and wing evagination and contributes to the thinning and extension of the appendages. These observations are consistent with the pioneering results of Fristrom (1976) on evagination. The current data also established that cell rearrangement takes place throughout the appendage and is not restricted to a particular region along the proximal/distal axis. However, the observations are also consistent with cell rearrangement being non-uniform as some regions appeared to 'thin' more than others. For example, in the wing the width of the ptc domain at position M5 thinned more than at position M4 (refering to neuronal landmarks). The evaginating leg and wing cells retain their epithelial morphology with extensive apical junctional complexes. Rearrangement requires that cells change neighbors and hence must remove old junctions and generate new ones while maintaining tissue integrity. This problem is not restricted to evaginating discs but is a general one for epithelial tissues and is an issue that has concerned developmental/cell biologists for many years. Important insights into how this could be accomplished come from recent observations on germ band elongation in the Drosophila embryo. Several groups have provided evidence that junctional remodeling plays a key role in cell rearrangement in this epithelial tissue. This mechanism also appears to function in the repacking of pupal wing cells. It is suggested that it also plays a role in leg and wing evagination. No clear evidence is seen for the multicellular rosettes that have been implicated in germ band extension. Perhaps this is due to disc evagination being substantially slower than germ band extension (Taylor, 2008).

No evidence was seen of dramatic coordinated changes in cell shape. There was a small but significant increase in the length along the proximal/distal axis of evaginating omb domain tibia cells that should contribute to elongation. However, the change was not large enough to account for leg morphogenesis. No significant change was seen in cell shape in evaginating ptc domain wing cells although there was a hint of a possible small effect. It is worth noting that in these measurements cells from all positions along the relevant part of the proximal/distal axis were included. Casual observation suggested that there might be small regions with consistent changes but these would likely be counterbalanced by changes in shape elsewhere in the domain (Taylor, 2008).

It was not possible to image the earliest stages of leg disc evagination or the disc cells that form ventral thorax. Thus, these observations were not able to distinguish between the two proposed mechanisms of eversion (i.e., spreading vs. invasion hypotheses). Patterned cell death could in principle play an important role in disc evagination. Previous studies have not seen evidence for patterned cell death during wing blade evagination and the current observations support this conclusion. Cell death has been detected in evaginating legs but this is restricted to the regions of the tarsal segments where the leg joints form and hence is unlikely to contribute to the overall thinning of the omb domain of leg segments (Taylor, 2008).

Based on the literature, it was not expected that cell division takes place during evagination, but the current observations showed that it occurred. The most definitive experiments involved generating clones of cells marked by GFP expression and following these in vivo. These experiments provided compelling evidence for cell division. This was only done for the leg but other experiments provided strong evidence for cell division in evaginating wings. The size of wing clones was larger when they were induced at white prepupae than at the formation of the definitive pupae. Cell division was not rare in evaginating legs, and on average about 40% of the cells divided. Indeed, a majority of the cells divided in about 1/3 of clones examined. This amount of cell division is sufficient to account for the thickening of the omb domain that was observed from 6 to 8 h in developing legs. Observations on the size of wing clones suggested a similar fraction of wing cells divided during evagination. A limitation is that the in vivo imaging technique only allowed effective imaging of clones on the leg surface juxtaposed to the pupal case in the basitarsus and tibia (and occasionally tarsal) segments. Thus, data could not be obtained for much of the leg disc derivatives, and hence the overall proportion of evaginating leg cells that divide cannot be confidently estimated. The spindle in these dividing cells was not imaged but it was inferred that the spindle was not oriented parallel to the elongating axis, based on the position of the resulting daughter cells shortly after division. The two daughter cells usually filled up the area taken up by the parental cell prior to division, which helped in assigning a lineage. The leg epidermis is continuous without free 'space'. Hence, that daughter cells would occupy the space of the parental cell is not surprising. A parallel orientation for the spindle might be expected if the cell division plane was tightly linked to the mechanism of elongation. The inferred orientation of the cell divisions was most often between 46^o and 60^o. Thus, they would increase the number of cells both along the proximal/distal and anterior/posterior (and dorsal/ventral) axes. In the second day pupal leg, the width of the omb domain was narrower than it was in the evaginating leg. This could be a reflection of a later stage of convergent extension. However, legs were not followed throughout this period, other possibilities cannot be ruled out. It is interesting to note that cells in the pupal tibia and basitarsus have a spiral arrangement, and this appears to arise from 6 to 8 h after white prepupae. Thus, this arrangement could be at least in part a consequence of the orientation of the cell divisions (Taylor, 2008).

The fraction of dividing cells varied widely from one clone to another. This was not correlated with particular pupae or legs as both clones where a majority of the cells divided and clones where no cells divided were found in the same pupae and on the same leg. One possibility is that the variation is due to region specific differences. For example, cells in one region of the leg might never divide during evagination while a majority of cells in another region might always divide. No evidence is seen for this but the experiments were not compelling on this point. The observations on the omb domain did not examine a majority of leg cells and in the experiments where MARCM clones were followed, it could not be routinely said exactly where on the leg a clone was located. A second possibility is that the variation is due to the clustered distribution of S phase and mitotic cells in wing and leg discs. Any small clone could comprise a cluster (or not contain a cluster) and this could lead to a great deal of variation in observed cell division. The basis for the clustering is uncertain but could simply represent a pseudo-synchronization due to neighboring sister cells having been born at the same time (Taylor, 2008).

The observations suggest that several different factors play a role in evagination. At the start of evagination, the leg and wing discs are folded and some of the initial elongation is due to an unfolding of the tissue that presumably results from changes in the shape of cells along the apical/basal axis. During the period when leg discs evert and present the apical surface of their epithelial cells to the outside, elongation is also taking place and there is active pulsatile movement. This appears to be related to the movement of hemolymph in the prepupae and blood cells can often be seen to move in step with the pulses. This suggests that hydraulic pressure could be playing a role in eversion and elongation. The leg resembles a cylinder closed on one side (distal tip) and open to the body on the other (proximal). Thus, it is expected that hemolymph is pumped by the heart to produce a mechanical force that could help evert and/or elongate the leg. The pulsatile movement starts to decrease at about 4-4.5 h after white prepupae and largely ends by about 5 h. This is around the time of eversion, but the slowing clearly precedes eversion. It is suggested that the hydraulic pressure of the hemolymph helps drive the early stages of evagination, when the leg is short and unfolding of the tissue plays a major role. It is possible that after this time the increased leg length or increased leg stiffness limits the effectiveness of hemolymph hydraulic pressure. Alternatively, it is possible that there is a decline in the hydraulic pressure due to changes in heart pumping or other prepupal events. The lack of hydraulic pressure may be one reason for the less than optimal evagination of discs seen during in vitro culture (Taylor, 2008).

Mutations in many Drosophila genes result in changes in appendage morphology. It is expected that some of these produce their phenotype by interfering with the observed cell rearrangement. A particularly interesting candidate for such a gene is dachsous (ds), which encodes a large protein with many cadherin domains. Mutations in this gene result in shorter fatter wings and legs with an altered distribution of cells (e.g. an increase in the number of cells along the anterior posterior axis of the wing and a decrease in the number of cells along the proximal/distal axis). However, mutations in this gene are known to alter disc patterning and growth and this may be the cause of the altered shape (Taylor, 2008).

Another group of interesting candidate genes for altering cell rearrangement in evaginating legs is the cellular myosin encoded by zipper and the interacting Sqh (myosin regulatory light chain) and RhoA proteins. Mutations in these genes give rise to a crooked leg phenotype that has been interpreted as being due to the mutations altering cell shape. However, myosin has been implicated in the junctional remodeling associated with cell rearrangements in the extending germ band and it is possible that the leg phenotype is also due to an effect on junctional remodeling required for cell rearrangement. One of the interesting properties of extending germ band cells is the planar polarization of membranes so that the anterior/posterior edges of cells are distinct from the dorsal/ventral edges of cells in their content of proteins such as myosin. No evidence was seen for this in prepupal legs and wings but this point deserves further study as it is possible the experimental conditions were not favorable for seeing this (Taylor, 2008).

Dynamic shape changes of ECM-producing cells drive morphogenesis of ball-and-socket joints in the fly leg

Animal body shape is framed by the skeleton, which is composed of extracellular matrix (ECM). Although how the body plan manifests in skeletal morphology has been studied intensively, cellular mechanisms that directly control skeletal ECM morphology remain elusive. In particular, how dynamic behaviors of ECM-secreting cells, such as shape changes and movements, contribute to ECM morphogenesis is unclear. Strict control of ECM morphology is crucial in the joints, where opposing sides of the skeleton must have precisely reciprocal shapes to fit each other. This study found that, in the development of ball-and-socket joints in the Drosophila leg, the two sides of chitin-based ECM form sequentially. Distinct cell populations produce the 'ball' and the 'socket', and these cells undergo extensive shape changes while depositing ECM. It is proposed that shape changes of ECM-producing cells enable the sequential ECM formation to allow the morphological coupling of adjacent components. These results highlight the importance of dynamic cell behaviors in precise shaping of skeletal ECM architecture (Tajiri, 2010).

This study revealed that the ball and the socket cuticles develop sequentially. The ball-producing activity and the socket-producing activity are allocated to distinct cell populations, and have found that shape changes of these cells that occur simultaneously with their cuticle-secreting activities result in the interlocking ball-and-socket structure. As the ball cuticle builds up, concurrent cell shape changes drive the apical domains of ball-producing cells out of the cavity and bring in the apical domains of the socket-producing cells, resulting in close enwrapment of the ball by the latter cell population. Accordingly, the shape of the resulting socket cuticle conforms to that of the ball. Synchronization between ECM formation and dynamic relocation of the cell surfaces that mediate it thus underlies the building of the complex ECM structure (Tajiri, 2010).

A map of ball-producing and socket-producing cells best summarizes the results of krotzkopf verkehrt (kkv - encoding Chitin Synthase 1) RNAi, and is consistent with the result indicating their continuous association with respective parts of the cuticle during their formation. The ball morphology was severely disrupted by bib>kkv RNAi but not by neur>kkv RNAi, indicating that the ball-producing activity is restricted to the distal subset of bib-expressing cells that do not significantly express neur. Consistently, these cells are in constant contact with the ball cuticle throughout its formation. The cuticle phenotype of neur>kkv RNAi shows that neur-expressing cells are responsible for forming the ventral part of the socket cuticle, with which they continue to associate. Likewise, fng-expressing cells contribute to the formation of the remainder of the socket. Partial disruption of the socket by bib>kkv RNAi should be, to some extent, due to direct blocking of socket production in cells co-expressing bib and neur. Additionally, the impairment of ball formation might somehow interfere with socket formation. Occasional deformation of the ball by neur>kkv RNAi might be caused by marginal expression of neur in the presumptive ball-producing cells (Tajiri, 2010).

Patterns of ECM-producing tissues do play a major role in the regulation of ECM morphology. Previous studies have unraveled how global positional information affects skeletal patterns through the regulation of specification, differentiation and proliferation of ECM-producing cells. There, the morphology of ECM was assumed to be synonymous with that of the cells or tissues that secrete it. The present study illustrates that the skeletal morphology reflects not only the pattern of those cells at one point in time, but also the history of their dynamic behaviors during ECM formation. Secreted apically by the epidermis, the cuticle is monolayered in most parts. In the joints, however, relocation of the secretory surfaces enables formation of a cuticle beneath a pre-formed layer. Cell motility thus allows a tissue of simple configuration to build a complex and essential three-dimensional ECM structure. It is envisioned that movements of ECM-secreting cells probably play important roles in ECM morphogenesis in other systems, especially in formation or adjustment of intricate skeletal structures (Tajiri, 2010).

The morphology of the cuticle, as well as how it develops, correlated well with cell shape changes. This suggested either that the cell-shape changes govern the morphology of the cuticle, and/or vice versa. This study found that the movement of the apical surfaces of the cells was correctly oriented even when the shape of the cuticle was disrupted, indicating that the morphogenesis of the ball-and-socket cuticle is primarily controlled by the way the cells change their shapes as they deposit the cuticle. How do the cells know which way to move? In other words, what is the molecular mechanism that mediates global proximodistal polarity of the leg to direct cell movement? In mutants of well-known planar cell polarity genes, such as frizzled, dishevelled and prickled, extra joints of reverse proximodistal polarity are formed. Nonetheless, the ball-and-socket structure of individual joints remains largely intact, indicating that cell shape changes are correctly guided by a mechanism other than this pathway. Analysis and local disruption of cytoskeletal architecture in the joint region could help answer this question (Tajiri, 2010).

These results do not rule out the possibility that the cuticle plays a permissive role in cell movements. The ECM generally affects cell shape and motility, and chitin-based ECM has been shown to regulate epithelial morphogenesis in some Drosophila tissues. Whether the cuticle provides a permissive environment for cell shape changes in the joint is an important issue to address in future work (Tajiri, 2010).

The formation of reciprocally shaped interfaces is vital for the sake of joint function. The serial progression of ball-and-socket morphogenesis shown here can be compared to ‘mold casting’: (1) the ball enlarges rapidly through stratification, and the cavity expands to accommodate it; and (2) the enlarged cavity then serves as the 'mold' along which the socket cuticle is formed. Hence, the shape of the ball is transmitted to the socket (the 'cast'). Whether or not this model also applies to vertebrate synovial joints is an intriguing question. It has been speculated that, in the chick digit joints, chondrogenic cell differentiation on the distal side might promote its expansion to become convex; at the same time, proliferation of peripheral cells on the proximal side might permit them to grow and wrap themselves around the distal side, thereby becoming concave. If this were the case, that model can be regarded as a modified version of ball-and-socket morphogenesis, one side fitting to the other through cell proliferation instead of cell shape changes. It will then become important to study how cells and ECM collectively undergo morphogenesis in other types of joints and in other species. Unraveling similarities and differences in the modes of joint development would be crucial in a medical sense as well, for understanding various joint pathologies and designing therapies to treat them (Tajiri, 2010).

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).

A common set of DNA regulatory elements shapes Drosophila appendages

Animals have body parts made of similar cell types located at different axial positions, such as limbs. The identity and distinct morphology of each structure is often specified by the activity of different 'master regulator' transcription factors. Although similarities in gene expression have been observed between body parts made of similar cell types, how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development is not known. This study used genome-wide open chromatin profiling to show that among the Drosophila appendages, the same DNA regulatory modules are accessible throughout the genome at a given stage of development, except at the loci encoding the master regulators themselves. In addition, open chromatin profiles change over developmental time, and these changes are coordinated between different appendages. It is proposed that master regulators create morphologically distinct structures by differentially influencing the function of the same set of DNA regulatory modules (McKay, 2013).

This paper addresses a long-standing question in developmental biology: how does a single genome give rise to a diversity of structures? The results indicate that the combination of transcription factors expressed in each thoracic appendage acts upon a shared set of enhancers to create different morphological outputs, rather than operating on a set of enhancers that is specific to each tissue. This conclusion is based upon the surprising observation that the open chromatin profiles of the developing appendages are nearly identical at a given developmental stage. Therefore, rather than each master regulator operating on a set of enhancers that is specific to each tissue, the master regulators instead have access to the same set of enhancers in different tissues, which they differentially regulate. It was also found that tissues composed of similar combinations of cell types have very similar open chromatin profiles, suggesting that a limited number of distinct open chromatin profiles may exist at a given stage of development, dependent on cell-type identity (McKay, 2013).

Different tissues were dissected from developing flies to compare their open chromatin profiles. These tissues are composed of different cell types, each with its own gene expression profile. Formaldehyde-assisted isolation of regulatory elements (FAIRE) data thus represent the average signal across all cells present in a sample. However, data from embryos and imaginal discs indicate that FAIRE is a very sensitive detector of functional DNA regulatory elements. For example, the Dll⁰¹ enhancer is active in 2–4 neurons of the leg imaginal disc; yet, the FAIRE signal at Dll⁰¹ is as strong as the Dll⁰⁴ enhancer, which is active in hundreds of cells of the wing pouch. Thus, FAIRE may detect nearly all of the DNA regulatory elements that are in use among the cells of an imaginal disc. This study does not rule out the existence of DNA regulatory elements that are not marked by open chromatin or are otherwise not detected by FAIRE (McKay, 2013).

Despite this sensitivity, the approach of this study does not identify which cells within the tissue have a particular open chromatin profile. For a given locus, it is possible that all cells in the tissue share a single open chromatin profile or that the FAIRE signal originates from only a subset of cells in which a given enhancer is active. Comparisons between eye-antennal discs, larval CNS, and thoracic discs suggest that the latter scenario is most likely, with open chromatin profiles among cells within a tissue shared by cells with similar identities at a given developmental stage (McKay, 2013).

The observation that halteres and wings share open chromatin profiles demonstrates that Hox proteins like Ubx can differentially interpret the DNA sequence within the same subset of enhancers to modify one structure into another. This is consistent with the idea that morphological differences are largely dependent on the precise location, duration, and magnitude of expression of similar genes, and it is further supported by the similarity in gene expression profiles observed between Drosophila appendages and observed between vertebrate limbs. However, that such dramatic differences in morphology could be achieved by using the same subset of DNA regulatory modules in different tissues genome-wide was not known. The current findings provide a molecular framework to support the hypothesis that Hox factors function as 'versatile generalists,' rather than stable binary switches. The similarity in open chromatin profiles between wings and legs suggests that this framework also extends to other classes of master regulators beyond the Hox genes. It is also noted that, like the Drosophila appendages, vertebrate limbs are composed of similar combinations of cell types that differ in their pattern of organization. Moreover, the Drosophila appendage master regulators share a common evolutionary origin with the master regulators of vertebrate limb development, suggesting that the concept of shared open chromatin profiles may also apply to human development (McKay, 2013).

The data suggest that open chromatin profiles vary both over time for a given lineage and between cell types at a given stage of development. Given the dramatic differences in the FAIRE landscape observed during embryogenesis and between the CNS and the appendage imaginal discs during larval stages, it appears as though the alteration of the chromatin landscape is especially important for specifying different cell types from a single genome. After cell-type specification, open chromatin profiles in the appendages continued to change as they proceeded toward terminal differentiation, suggesting that stage-specific functions require significant opening of new sites or the closing of existing sites. These findings contrast with those investigating hormone-induced changes in chromatin accessibility, in which the majority of open chromatin sites did not change after hormone treatment, including sites of de novo hormone-receptor binding. Thus, it may be that genome-wide remodeling of chromatin accessibility is reserved for the longer timescales and eventual permanence of developmental processes rather than the shorter timescales and transience of environmental responses (McKay, 2013).

Different combinations of 'master regulator' transcription factors, often termed selector genes, are expressed in the developing appendages. Selectors are thought to specify the identity of distinct regions of developing animals by regulating the expression of transcription factors, signaling pathway components, and other genes that act as effectors of identity. One property attributed to selectors to explain their unique power to specify identity during development is the ability to act as pioneer transcription factors. In such models, selectors are the first factors to bind target genes; once bound, selectors then create a permissive chromatin environment for other transcription factors to bind. The finding that the same set of enhancers are accessible for use in all three appendages, with the exception of the enhancers that control expression of the selector genes themselves and other primary determinants of appendage identity, suggests that the selectors expressed in each appendage do not absolutely control the chromatin accessibility profile; otherwise, the haltere chromatin profile (for example) would differ from that of the wing because of the expression of Ubx (McKay, 2013).

What then determines the appendage open chromatin profiles? Because open chromatin is likely a consequence of transcription factor binding, two nonexclusive models are possible. First, different combinations of transcription factors could specify the same open chromatin profiles. In this scenario, each appendage's selectors would bind to the same enhancers across the genome. For example, the wing selector Vg, with its DNA binding partner Sd, would bind the same enhancers in the wing as Dll and Sp1 bind in the leg. In the second model, transcription factors other than the selectors could specify the appendage open chromatin profiles. Selector genes are a small fraction of the total number of transcription factors expressed in the appendages. Many of the non-selector transcription factors are expressed at similar levels in each appendage, and thermodynamic models would predict them to bind the same enhancers. This model could also help to explain how the appendage open chromatin profiles coordinately change over developmental time despite the steady expression of the appendage selector genes during this same period. It is possible that stage-specific transcription factors determine which enhancers are accessible at a given stage of development. This would help to explain the temporal specificity of target genes observed for selectors such as Ubx. Recent work supports the role of hormone-dependent transcription factors in specifying the temporal identity of target genes in the developing appendages (Mou, 2012). Further experiments, including ChIP of the selectors from each of the appendages, will be required to determine the extent to which either of these models is correct (McKay, 2013).

Binding of Ubx results in differential activity of enhancers in the haltere imaginal disc relative to the wing, despite equivalent accessibility of the enhancers in both discs, indicating that master regulators control morphogenesis by differentially regulating the activity of the same set of enhancers. It is likely that functional specificity of enhancers is achieved through multiple mechanisms. These include differential recruitment of coactivators and corepressors, modulation of binding specificity through interactions with cofactors, differential utilization of binding sites within a single enhancer, or regulation of binding dynamics through an altered chromatin context. This last mechanism would allow for epigenetic modifications early in development to affect subsequent gene regulatory events. For example, the activity of Ubx enhancers in the early embryo may control recruitment of Trithorax or Polycomb complexes to the PREs within the Ubx locus, which then maintain Ubx in the ON or OFF state at subsequent stages of development. Consistent with this model, Ubx enhancers active in the early embryo are only accessible in the 2-4 hr time point, whereas the accessibility of Ubx PREs varies little across developmental time or between tissues at a given developmental stage (McKay, 2013).

The current results also have implications for the evolution of morphological diversity. Halteres and wings are considered to have a common evolutionary origin, but the relationship between insect wings and legs is unresolved. The observation that wings and legs share open chromatin profiles supports the hypothesis that wings and legs also share a common evolutionary origin in flies. Because legs appear in the fossil record before wings, the similarity in their open chromatin profiles suggests that the existing leg cis-regulatory network was co-opted for use in creation of dorsal appendages during insect evolution (McKay, 2013).

cis-regulatory architecture of a short-range EGFR organizing center in the Drosophila melanogaster

This study characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE (but not individually) show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional 'ring' enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE many additional leg enhancers were identified, suggesting that this logic is broadly used by many genes during Drosophila limb development (Newcomb, 2018).

The EGFR signaling pathway is widely used in animal development, and is frequently a target in human disease and developmental abnormalities. Yet despite its importance in animal biology, many questions remain about how this pathway functions. Among these questions is whether secreted ligands that activate this pathway can induce distinct cell fates in a concentration-dependent manner. This study tests this idea by specifically eliminating a single source of EGFR ligands from the center of the Drosophila leg imaginal disc, which fate maps to the distal-most region of the adult leg. One plausible scenario is that this single source of secreted EGFR ligands, which is referred to as the EOC, activates distinct gene expression responses at different distances from this source. Alternatively, eliminating ligands secreted from the EOC might only affect gene expression locally, close to or within the EOC. Taken together, the current data are most consistent with the second scenario. This conclusion is largely supported by the observations that CRM deletions that eliminate vn and rho expression from the EOC have mild developmental consequences, both in the L3 leg imaginal discs and adult legs. These phenotypes are significantly weaker than those generated when the entire EGFR pathway is compromised using a temperature sensitive allele of the EGFR receptor. The difference between these two phenotypes is most likely explained by removing only a single source of EGFR ligands in the enhancer deletion experiments versus affecting EGFR signaling throughout the leg disc in the Egfr^tsla experiments. This explanation is further supported by the observation that there are indeed additional CRMs, some of which were defined in this study, that drive EGFR ligand production in more medial ring-like patterns during the L3 stage (Newcomb, 2018).

One possible caveat to these conclusions is that there are a total of seven rho-like protease genes in the Drosophila genome that could, in principle, play a role in distal leg development. This study focused on rho and roughoid ru, based on previous results showing that triple rho ru vn clones generate severe leg truncations that phenocopy strong Egfr^tsla truncations. In addition, it is noted that if other rho family proteases were active in the EOC, leg truncations and patterning defects would not be expected in the leg discs of the rho^rhoE-Df vn^vnE-Df double mutant, because those proteases should be able to produce active Spi. These observations suggest that the remaining five rho-like protease genes play a minor (or no) role in leg development. However, this conclusion will ultimately benefit from further genetic and expression analysis of these additional rho-like genes (Newcomb, 2018).

An additional previous observation that contrasts with the suggestion that EOC activity has only a limited role in specifying distal leg fates is the partial rescue of the PD axis when only a small number of distal leg cells were wild type in legs containing large rho ru vn clones. However, it is noted that even in these 'rescued' legs, medial defects in PD patterning were apparent. It is also noteworthy that in these earlier experiments, only adult legs were examined. When the same experiment was repeated, but L3 discs were analyzed, it was found that rho ru vn clones generated phenotypes that were very similar to those produced by the double vnE rhoE enhancer deletions. Taken together, these observations suggest that timing must be considered in the interpretation of these experiments. When assayed at the late L3 stage, both enhancer deletion and rho ru vn clone experiments argue that EOC activity is limited to specifying only the most distal fates, marked by the expression of al and C15. Starting in mid L3, and perhaps continuing into pupal development, there are additional sources of EGFR ligands that, when compromised, can affect adult leg morphology. Nevertheless, at least at the L3 stage, these data suggest that EGFR ligands produced from the EOC have a limited and local role in specifying distal leg fates (Newcomb, 2018).

Integration of inputs from signaling pathways and organ selector genes at CRMs in order to execute distinct developmental programs is a recurrent theme during animal development. This study identified two leg EGFR ligand CRMs that integrate the inputs from the Wg and Dpp signaling pathways and the leg selector genes Dll and/or Sp1 in a manner that is very similar to a previously characterized leg enhancer DllLT. In addition, when the same regulatory logic was applied to the whole genome, a battery of leg enhancer elements was identified. Interestingly, each of these enhancers drives expression in a specific manner with slightly different timing despite the fact that many of the inputs are shared. It is conceivable that the different expression patterns directed by these enhancers are in part a consequence of additional inputs and/or the difference in the TF binding site grammar. In support of this idea, vnE and rhoE differ in the number of binding sites for many inputs and vnE requires Sp1 while rhoE does not. Both of these differences may contribute to the earlier onset of vnE expression compared to rhoE. The remaining enhancer elements identified in this study direct a plethora of PD-biased leg expression patterns -- ranging from ubiquitous, to central and 'ring' patterns (see Genome-wide analysis of combinatorial inputs of Dll, Sp1, Wg, and Dpp in leg discs), which likely integrate inputs in addition to the ones described here. Future studies of these CRMs would help reveal the complex network of regulation that orchestrates leg development in the fruit fly. Such detailed understanding of the cis-regulatory architecture of fly leg development would likely give insights into organogenesis and evolution in other animals as well (Newcomb, 2018).

The EGFR signaling pathway has tremendous oncogenic potential and understanding the various mechanisms regulating its activation is not only interesting from the point of view of animal development but also has important practical implications. While the core components of the EGFR pathway have been thoroughly studied because of their potent tumorigenic capability in humans, little is known about the transcriptional regulation of EGFR ligands that bind the receptor and activate the pathway. The reiterative use of EGFR signaling in many developmental processes implies that different cis-regulatory elements are likely utilized by each EGFR ligand in different organs and tissues in order to correctly read the diverse cues in any specific developmental context. It is conceivable that genomic variation in EGFR pathway CRMs might lead to a predisposition to different types of EGFR-dependent tumors in humans, since such CRMs may respond to potent growth-promoting signaling pathways, such as Wnt and BMP (Newcomb, 2018).

This study has characterized in detail two Drosophila EGFR CRMs, vnE and rhoE, and showed how they integrate the cues from two transcription factors, Dll and Sp1, and two signaling pathways, Wg and Dpp, in order to execute a leg patterning developmental program. Analogous EGFR CRMs are likely to exist in mammals, especially because complex interactions between BMP, Wnt, Shh, multiple Dlx paralogs and other factors, are implicated in the induction of FGF signaling in mammalian limb development. Consistent with this idea, specific single nucleotide polymorphisms (SNPs) in humans in non-coding loci of genes encoding EGFR ligands have been shown to be associated with different types of cancer. Such loci may be enhancer elements analogous to vnE and rhoE. It is also noted that the regulatory logic uncovered in this study is likely to be relevant to many CRMs and genes that share spatial and temporal expression programs. Exploiting this regulatory logic in other systems might streamline the identification of enhancer elements that will aid in the discovery of mechanisms that are relevant to EGFR-related human disease and developmental birth defects (Newcomb, 2018).

The evolutionary conserved transcription factor Sp1 controls appendage growth through Notch signaling

The appendages of arthropods and vertebrates are not homologous structures, although the underlying genetic mechanisms that pattern them are highly conserved. Members of the Sp family of transcription factors are expressed in the developing limbs and their function is required for limb growth in both insects and chordates. Despite the fundamental and conserved role that these transcription factors play during appendage development, their target genes and the mechanisms in which they participate to control limb growth are mostly unknown. This study analyzed the individual contributions of two Drosophila Sp members, buttonhead (btd) and Sp1, during leg development. Sp1 plays a more prominent role controlling leg growth than btd. A regulatory function of Sp1 in Notch signaling was identified, and a genome wide transcriptome analysis was performed to identify other potential Sp1 target genes contributing to leg growth. The data suggest a mechanism by which the Sp factors control appendage growth through the Notch signaling (Cordoba, 2016).

Understanding the molecular mechanisms that control the specification and acquisition of the characteristic size and shape of organs is a fundamental question in biology. Of particular interest is the development of the appendages of vertebrates and arthropods, i.e., non-homologous structures that share a similar underlying genetic program to build them, a similarity that has been referred to as 'deep homology.' Some of the conserved genes include the Dll/Dlx genes, Hth/Meis and the family of Sp transcription factors. The Sp family is characterized by the presence of three highly conserved Cys2-His2-type zinc fingers and the presence of the Buttonhead (BTD) box just N-terminal of the zinc fingers (Cordoba, 2016).

Members of the Sp family have important functions during limb outgrowth in a range of species from beetles to mice. In vertebrates, Sp6, Sp8 and Sp9 are expressed in the limb bud and are necessary for Fgf8 expression and, therefore, for apical ectodermal ridge (AER) maintenance. Moreover, Sp6/Sp8 phenotypes have been related to the split-hand/foot malformation phenotype (SHFM) and, in the most severe cases, to amelia (the complete loss of the limb) (Cordoba, 2016).

In Drosophila, two members of this family, buttonhead (btd) and Sp1, are located next to each other on the chromosome and share similar expression patterns throughout development. Recently, another member of the family, Spps (Sp1-like factor for pairing sensitive-silencing) has been identified with no apparent specific function in appendage development. The phenotypic analysis of a btd loss-of-function allele and of a deletion that removes both btd and Sp1 led to the proposal that these genes have partially redundant roles during appendage development. However, the lack of a mutant for Sp1 has prevented the analysis of the specific contribution of this gene during development (Cordoba, 2016).

In Drosophila, leg development is initiated in the early embryo by the expression of the homeobox gene Distal-less (Dll) in a group of cells in each thoracic segment. Later on, Dll expression depends on the activity of the Decapentaplegic (Dpp) and Wingless (Wg) signaling pathways, which, together with btd and Sp1, restrict Dll expression to the presumptive leg territory. Therefore, the early elimination of btd and Sp1 completely abolishes leg formation and, in some cases, causes a leg-to-wing homeotic transformation (Estella, 2010). As the leg imaginal disc grows, a proximo-distal (PD) axis is formed by the differential expression of three leg gap genes, Dll, dachshund (dac) and homothorax (hth), which divides the leg into distal, medial and proximal domains, respectively. Once these genes have been activated, their expression is maintained, in part through an autoregulatory mechanism, and no longer relies on Wg and Dpp. Meanwhile, the distal domain of the leg is further subdivided along the PD axis by the activity of the epidermal growth factor receptor (EGFR) signaling pathway through the activation of secondary PD targets such as aristaless (al), BarH1 (B-H1) or bric-a-brac (bab). During these stages, btd and Sp1 control the growth of the leg but are no longer required for Dll expression (Estella, 2010). How btd and Sp1 contribute to the shape and size of the leg and the identity of their downstream effector targets is unknown (Cordoba, 2016).

One important consequence of the PD territorial specification is the generation of developmental borders that activate organizing molecules to control the growth and pattern of the appendage. In the leg, PD subdivision is necessary to localize the expression of the Notch ligands Delta (Dl) and Serrate (Ser), which in turn activate the Notch pathway in concentric rings at the borders between presumptive leg segments. However, it is still unknown how Notch controls leg growth and how the localization of its ligands is regulated. The present study generated a specific Sp1 null mutant, which, in combination with the btd mutant and a deletion that removes both btd and Sp1, allow analysis of the individual contributions of these genes to leg development. This study finds that Sp1 plays a fundamental role during patterning and growth of the leg disc, and that this function is not compensated by btd. The growth-promoting function of Sp1 depends in part on the regulation of the expression of Ser and, therefore, on Notch activity. In addition, other candidate targets of Sp1 affecting leg growth and morphogenesis were identified. Intriguingly, some of these Sp1 potential downstream targets are ecdysone-responding genes. These results highlight a mechanism by which btd and Sp1 control the size and shape of the leg, in part through regulation of the Notch pathway (Cordoba, 2016).

The two Sp family members in Drosophila, Sp1 and btd, display a similar spatial and temporal expression pattern during embryonic and imaginal development. Previous work suggested that btd and Sp1 have partially redundant functions during development. However, the lack of an Sp1 mutant has prevented the detailed analysis of the individual contributions of each gene. This study has generated an Sp1 null mutant that allowed elucidation unambiguously of the individual contributions of each of these genes to leg development (Cordoba, 2016).

Appendage formation starts in early embryos by the activation of Dll (through its early enhancer, Dll-304), btd and Sp1 by Wg, and their expression is repressed posteriorly by the abdominal Hox genes. Some hours later, there is a molecular switch from the early Dll enhancer (Dll-304) to the late enhancer (Dll-LT) to keep Dll expression throughout the embryo-larvae transition restricted to the cells that will form the leg. At this developmental stage, Sp1 and btd play redundant roles in Dll activation, as only the elimination of both genes suppresses Dll expression and Dll-LT activity in the leg primordia. Once Dll expression is activated in the leg disc by the combined action of Wg, Dpp and Btd/Sp1, its expression is maintained in part through an autoregulatory mechanism. At this time point, during second instar, btd and Sp1 are co-opted to control the growth of the leg. The leg phenotype of Sp1 and btd single mutants demonstrates the divergent contributions of each gene to leg growth. Removing btd from the entire leg only slightly affects the growth of proximo-medial segments, whereas loss of Sp1 causes dramatic growth defects along the entire leg. The different phenotypes of Sp1 and btd mutant legs could be a consequence of their distinct expression pattern along the leg PD axis, with btd being expressed more proximally than Sp1 (Cordoba, 2016).

The growth defects observed in Sp1 mutant legs are not due to gross defects in the localization of the different transcription factors that subdivide the leg along the PD axis, nor to defects in the expression of the EGFR ligand vn. By contrast, the results suggest a role for Sp1 in the regulation of the Notch ligand Ser. Notch pathway activation is necessary for the formation of the joints and the growth of the leg, and defects in these two processes were observed in Sp1 mutant legs. Moreover, the results demonstrate that Sp1 is necessary and sufficient for Ser expression at least in the distal domain of the leg and is therefore required for the correct activation of the Notch pathway. These results are consistent with the proposed role of Sp8 in allometric growth of the limbs in the beetle where the number of Ser-expressing rings is reduced in Sp8 knockdown animals (Cordoba, 2016).

The regulation of Ser expression is controlled by multiple CREs that direct its transcription in different developmental territories. Interestingly, although the wing and leg are morphologically different appendages and express a diverse combination of master regulators (e.g. Sp1 selects for leg identity whereas Vg determines wing fate), the same set of enhancers are accessible in both appendages, with the exception of the ones that control the expression of the master regulators themselves. These results imply that appendage-specific master regulators differentially interact with the same enhancers to generate a specific expression pattern in each appendage. The current analysis of Ser CREs identified a specific sequence that is active in the wing and in the leg. In the leg, this CRE reproduced Ser expression in the fourth tarsal segment and require the combined inputs of Sp1 and Ap. It is proposed that Sp1, in coordination with the other leg PD transcription factors, interacts with different Ser CREs to activate Ser expression in concentric rings in the leg. Meanwhile, given the same set of Ser CREs in the wing, the presence of a different combination of transcription factors regulate Ser expression in the characteristic 'wing pattern' (Cordoba, 2016).

Transcriptome analysis identified additional candidate Sp1 target genes that contribute to control the size and shape of the leg. Appendage elongation depends on the steroid hormone ecdysone through several of its effectors, such as Sb. Sb, as well as other genes related to the ecdysone pathway, were misregulated in Sp1 mutant discs. The characteristic change in cell shape that normally occurs during leg eversion does not happen correctly in these mutants. Other genes identified in this study are the Notch pathway targets dys and Poxn, which are both required for the correct development of the tarsal joints. dys and Poxn downregulation is consistent with Sp1 regulation of the Notch ligand Ser. Interestingly, the upregulation of the antenna-specific gene danr in Sp1 mutants might explain the partial transformation of the distal leg to antennal-like structures observed when two copies of Sp1 and one of btd are mutated. Interestingly, btd and Sp1 are only expressed in the antenna disc in a single ring corresponding to the second antennal segment whereas in the leg both genes are more broadly expressed. Consistent with this, misexpression of Sp1 in the antenna transforms the distal domain to leg-like structures, suggesting that different levels or expression domains of Sp1 helps distinguish between these two homologous appendages (Cordoba, 2016).

A considerable group of Hsp-related genes were downregulated in Sp1 mutant legs. Although their contribution to Drosophila leg development is unknown, downregulation of DnaJ-1, the Drosophila ortholog of the human HSP40, affects joint development and leg size, suggesting a potential role of these genes during leg morphogenesis (Cordoba, 2016).

An ancient common mechanism for the formation of outgrowths from the body wall has been suggested. Members of the Sp family are expressed and required for appendage growth in a range of species from Tribolium to mice. Consistent with the current results, knockdown of Sp8/Sp9 in the milkweed bug or the beetle generated dwarfed legs with fused segments that maintain the correct PD positional values. As is the case for Drosophila Sp1 mutants, mouse Sp8-deficient embryos develop with truncated limbs. By contrast, loss of function of Sp6 results in milder phenotypes of limb syndactyly. A progressive reduction of the dose of Sp6 and Sp8 lead to increased severity of limb phenotypes from syndactyly to amelia, suggesting that these genes play partially redundant roles. This phenotypic analysis of Sp1 and btd are consistent with this model, in which Sp1 plays the predominant role in appendage growth and the complete elimination of btd and Sp1 together abolish leg formation. Therefore, Drosophila Sp1 mutants are phenotypically equivalent to vertebrate Sp8 mutants. In vertebrate Sp8 mutant limbs, Fgf8 expression is not maintained and a functional AER fails to form. In Drosophila, FGF signaling does not seem to be involved in appendage development. Nevertheless, another receptor tyrosine kinase, EGFR, is activated at the tip of the leg and act as an organizer to regulate the PD patterning of the tarsus. The current results suggest that Sp1 acts in parallel with the EGFR pathway, as the ligand vn and EGFR target genes maintain their PD positional information in Sp1 mutant legs. However, a potential relationship between Sp1 and the EGFR pathway in later stages of leg development cannot be ruled out (Cordoba, 2016).

The results suggest that the Notch ligand Ser is a target of Sp1, and mediates in part the growth-promoting function of Sp1. Interestingly, members of the Notch pathway in vertebrates, including the Ser ortholog jagged 2 and notch 1 are expressed in the AER and regulate the size of the limb. It would be interesting to investigate further the possible relationship between Sp transcription factors and the Notch pathway in vertebrates, and test whether the functional relationship described in this work is also maintained throughout evolution (Cordoba, 2016).

Rotation of sex combs in Drosophila melanogaster requires precise and coordinated spatio-temporal dynamics from forces generated by epithelial cells

The morphogenesis of sex combs (SCs), a male trait in many species of fruit flies, is an excellent system in which to study the cell biology, genetics and evolution of a trait. In Drosophila melanogaster, where the incipient SC rotates from horizontal to a vertical position, three signal comb properties have been documented: length, final angle and shape (linearity). During SC rotation, in which many cellular processes are occurring both spatially and temporally, it is difficult to distinguish which processes are crucial for which attributes of the comb. This study used a novel approach combining simulations and experiments to uncover the spatio-temporal dynamics underlying SC rotation. The results indicate that 1) the final SC shape is primarily controlled by the inhomogeneity of initial cell size in cells close to the immature comb, 2) the final angle is primarily controlled by later cell expansion and 3) a temporal sequence of cell expansion mitigates the malformations generally associated with longer rotated SCs. Overall, this work has linked together the morphological diversity of SCs and the cellular dynamics behind such diversity, thus providing important insights on how evolution may affect SC development via the behaviours of surrounding epithelial cells (Ho, 2018).

Dynamics of changes in apical cell area during sex comb rotation in Drosophila melanogaster

Epithelia are highly dynamic tissues displaying various types of tissue rearrangements. This study describes the dynamics of changes in apical cell area (ACA) in an epithelial system displaying tissue rearrangement resulting in sex comb rotation on the forelegs of male Drosophila melanogaster. The sex comb is a row of leg bristles which rotates during morphogenesis. The ACA was quantified in the region proximal to the developing sex comb by tracing apical cell boundaries using ImageJ in pupal first leg imaginal discs. Cells display intricate irregular oscillations in size as the comb rotates. However, the net changes in ACA within most of the cells studied are subtle, only 0 to +/-15%. The current working hypothesis suggests these irregular oscillations confer flexibility during tissue rearrangement and can be an important mechanism for tissue homeostasis (Doerksen, 2023).

Asymmetrically deployed actomyosin-based contractility generates a boundary between developing leg segments in Drosophila

Classically, it has been assumed that adhesive differences are a primary means of sorting cells to their respective territories. Yet it is becoming clear that no single, simple mechanism is at play. In the few instances studied, an emergent theme along developmental boundaries is the generation of asymmetry in cell mechanical properties. The repertoire of ways in which cells might establish and then put mechanical asymmetry to work is not fully appreciated since only a few boundaries have been molecularly studied. This study characterize one such boundary in the develop leg epithelium of Drosophila. The region of the pretarsus / tarsus is a known gene expression boundary that also exhibits a lineage restriction. This study show that the interface comprising this boundary is strikingly aligned compared to other cell interfaces across the disk. The boundary also exhibits an asymmetry for both Myosin II accumulation as well as one of its activators, Rho Kinase. Furthermore, the enrichment correlates with increased mechanical tension across that interface, and that tension is Rho Kinase-dependent. Lastly, interfering with actomyosin contractility, either by depletion of myosin heavy chain or expression of a phosphomimetic variant of regulatory light chain causes defects in alignment of the interfaces. These data suggest strongly that mechanical asymmetries are key in establishing and maintaining this developmental boundary (Ly, 2017).

A key component comprising a developmental boundary is the special mechanical properties imposed to its interfaces. Insights into these properties have been gained from the few tissues that have been studied, such as rhombomere boundaries in the vertebrate, but especially the study of several boundaries in Drosophila. The latter studies in Drosophila have afforded much higher resolution so far than study of rhombomeres. Still, relatively few boundaries overall have been studied, and that makes it difficult to draw any generalizations for how the underlying mechanics makes the boundary. This paper reports initial studies on the late-arising developmental boundary necessary for leg segmentation. The pretarsal / tarsal boundary was more aligned than the canonical AP compartment boundary. The rail exhibits an asymmetry in actomyosin accumulation as well as one of its activators, Rho Kinase. This is shown to result in increased tension along the boundary, which is important in aligning its interfaces (Ly, 2017).

Polarized actomyosin enrichment leads to increased cell bond tension along the pretarsal / tarsal interfaces. The fold increase of tension compared with the orthogonal rung interfaces is in line with differences observed in several other tissues, such as the Antero-posterior and dorso-ventral compartment boundaries. Here, along the pretarsal / tarsal interface, actomyosin contractility generates a very smooth, arcing boundary. The alignment is significant, as it is even more aligned than the well-studied AP compartment boundary. In itself, this fact strongly suggests that study of the pretarsal / tarsal boundary will complement the information obtained though study of other developmental boundaries (Ly, 2017).

The data revealing enrichment of the Myosin II regulatory light chain as well as Rho Kinase along rail interfaces strongly implicates contractility in alignment, and the degree of mis-alignment observed in zip mutants supports this contention. Furthermore, treatment with a Rho Kinase inhibitor reduced actomyosin enrichment and released tension along the rail, rapidly generating a less aligned state. In addition, since removal of the Rho Kinase inhibitor led to the rapid re-establishment of alignment, the data collectively argue that asymmetric contractility can drive this alignment event. Still, Rho Kinase inhibitors can affect other protein kinases, such as Atypical Protein Kinase (aPKC). Thus, even though a quite selective Rho Kinase inhibitor was used, it is still possible that another kinase also contributes to alignment, perhaps targeting a factor in addition to the myosin regulatory light chain (Ly, 2017).

The expression of a phosphomimetic form of the Myosin II regulatory light chain generated defects along the rail. The precise mechanism involved awaits live-imaging the formation of the aligned interface. Without that capability in this epithelium, it cannot be determined whether the phosphomimetic form of the Myosin II generated defects due to decreased cycling between on and off states along interfaces normally enriched for myosin, or to increased activity along the normally depleted (rung) interfaces. Nevertheless, regulated contractility is certainly important to alignment (Ly, 2017).

Actomyosin enrichment and the resultant increased tension is a theme observed repeatedly along cell interfaces. Interestingly, the outcome of that increase in tension can be quite different in different circumstances. In some cases, tension stabilizes cell interfaces, as has been observed along the parasegment boundary of the embryonic epithelium, as well as the AP and DV compartment boundaries in developing imaginal disk epithelia. While actomyosin enrichment leads to stabilization in those cases, in other instances, enrichment and the associated increased tension drives interface shrinkage. Those shrinkage outcomes are crucial to the directed junctional remodeling events necessary for convergence extension. Similar shrinkage events are also observed in tissues at steady-state. For example, across the epithelial field in the developing wing, junctional shrinkage events maintain the proper geometry of cell packing. Just how actomoysin enrichment and junctional tension can be directed toward two quite diametrically opposed outcomes, shrinkage or stabilization, is unclear at present. This issue will only be resolved by examining more boundaries of each class, and by identifying more components that act along those interfaces (Ly, 2017).

In fact the pretarsal / tarsal boundary described in this study has several features in common with another interface described previously. In the late embryonic epidermis, well-after convergence and extension, a select set of cells within each parasegment organizes into aligned columns. Those aligning cell columns exhibit enrichments similar to those described here along the smooth, arcing pretarsal / tarsal rail. In addition, in both cases the cells that constitute the boundary assume elongate, rectilinear shapes. A comparison of the mechanics underlying these two alignment events could potentially reveal how actomoysin enrichment and junctional tension can be directed toward stabilization (Ly, 2017).

Besides exhibiting alignment, some boundary interfaces, such as the AP and DV compartment boundaries, are resilient to challenges from neighboring cells, whether from cell division or intercalation. The mechanical basis for this is becoming more clear. The pretarsal / tarsal boundary develops a late-acting lineage-restriction, so it is interesting to consider the degree to which increased tension contributes to the restriction. Interestingly, in depleting or manipulating Myosin II activity the pretarsal/tarsal boundary became very irregular. Yet, no evidence was found for 'invasions' from one territory to the other, at least not in these fixed preparations. This suggests that tension is not sufficient for this restriction in the leg. Perhaps like the DV compartment in the wing a combination of mechanical tension, as seen here, plus oriented divisions and cell elongation contribute to boundary integrity. Alternatively, the affinity properties of the pretarsal versus tarsal cells may well contribute to the lineage restriction (Ly, 2017).

Finally, it is noted that the interfaces flanking the rail are also aligned to a significant degree. This differs from the situation observed along the AP compartment boundary where the adjacent interfaces were used as examples of relatively unaligned interfaces. That raises the interesting question of whether the interfaces flanking the rail are actively aligned. For instance, machinery similar to that deployed along the rail might align the -1 and + interfaces. Alternatively, the flanking interfaces might be aligned only passively, as a consequence of the geometry enforced by the rail interface on the other cell interfaces. If there is an active process aligning the flanking interfaces, MyoII would appear to be minimally involved. No significant enrichment of MyoII was observed along the -1 interface compared to the adjacent rung, and although these interfaces retained some tension, the level was much reduced along the -1 and + interfaces compared to the rails (Ly, 2017).

It is not yet known how the polarized enrichments are first established along the pretarsal / tarsal boundary. There is a fairly well-understood gene regulatory hierarchy that establishes the pretarsal and tarsal territories during the mid third instar period of development. The initially rough borders between the two territories are subsequently refined by further cross-regulatory interactions. Thus, it is no surprise that interfering with the transcriptional regulator, C15, can cause defects along the boundary. In addition, among the factors that are genetically regulated by this transcriptional circuitry are Fasciclin II and the leucine rich proteins, Capricious and Tartan (Caps; Trn). However, it is not known how direct that regulation might be. Moreover, neither removing Fasciclin II, nor both Caps and Trn, generated phenotypes that seemed clarifying. This suggests that key factors remain to be defined. A similar limitation extends to the parasegmental, AP and DV boundaries. While the Wingless, Hedgehog and Notch pathways, respectively, have been implicated at those boundaries, the analyses still leave open the possibility that control by each of those pathways is indirect. Unraveling the direct links from cell signaling to the mechanics of tissue boundaries remains an important goal in studying morphogenesis (Ly, 2017).

The selector genes midline and H15 control ventral leg pattern by both inhibiting Dpp signaling and specifying ventral fate

mid and H15 encode Tbx20 transcription factors that specify ventral pattern in the Drosophila leg. There are at least two pathways for mid and H15 specification of ventral fate. In the first pathway, mid and H15 negatively regulate Dpp, the dorsal signal in leg development. mid and H15 block the dorsalizing effects of Dpp signaling in the ventral leg. In loss- and gain-of-function experiments in imaginal discs, this study shows that mid and H15 block the accumulation of phospho-Mad, the activated form of the Drosophila pSmad1/5 homolog. In a second pathway, mid and H15 must also directly promote ventral fate because simultaneously blocking Dpp signaling in mid H15 mutants does not rescue the ventral to dorsal transformation in most ventral leg structures. mid and H15 act as transcriptional repressors in ventral leg development. The two genes repress the Dpp target gene Dad, the laterally expressed gene Upd, and the mid VLE enhancer. This repression depends on the eh1 domain, a binding site for the Groucho co-repressor, and is likely direct because Mid localizes to target gene enhancers in PCR-ChIP assays. A mid allele mutant for the repressing domain (eh1), mid(eh1), was found to be compromised in gain-of-function assays and in rescue of mid H15 loss-of-function. It is proposed that mid and H15 specify ventral fate through inhibition of Dpp signaling and through coordinating the repression of genes in the ventral leg (Svendsen, 2019).

midline represses Dpp signaling and target gene expression in Drosophila ventral leg development

Ventral leg patterning in Drosophila is controlled by the expression of the redundant T-box Transcription factors midline (mid) and H15. This study shows that mid represses the Dpp-activated gene Daughters against decapentaplegic (Dad) through a consensus T-box binding element (TBE) site in the minimal enhancer, Dad13. Mutating the Dad13 DNA sequence results in an increased and broadening of Dad expression. It was also demonstrated that the engrailed-homology-1 domain of Mid is critical for regulating the levels of phospho-Mad, a transducer of Dpp-signaling. However, mid does not affect all Dpp-target genes as it was demonstrated that brinker (brk) expression is unresponsive to mid. This study further illuminates the interplay between mechanisms involved in determination of cellular fate and the varied roles of mid (Phillips, 2022).

FoxB, a new and highly conserved key factor in arthropod dorsal-ventral (DV) limb patterning

Forkhead box (Fox) transcription factors evolved early in animal evolution and represent important components of conserved gene regulatory networks (GRNs) during animal development. Most of the researches concerning Fox genes, however, are on vertebrates and only a relatively low number of studies investigate Fox gene function in invertebrates. In addition to this shortcoming, the focus of attention is often restricted to a few well-characterized Fox genes such as FoxA (forkhead), FoxC (crocodile) and FoxQ2. Although arthropods represent the largest and most diverse animal group, most other Fox genes have not been investigated in detail, not even in the arthropod model species Drosophila melanogaster. In a general gene expression pattern screen for panarthropod Fox genes including the red flour beetle Tribolium castaneum, the pill millipede Glomeris marginata, the common house spider Parasteatoda tepidariorum, and the velvet worm Euperipatoides kanangrensis, a Fox gene was identified with a highly conserved expression pattern along the ventral ectoderm of arthropod and onychophoran limbs. Functional investigation of FoxB in Parasteatoda reveals a hitherto unrecognized important function of FoxB upstream of wingless (wg) and decapentaplegic (dpp) in the GRN orchestrating dorsal-ventral limb patterning (Heingard, 2019).

This study shows that FoxB genes are expressed along the ventral side of all ventral appendages and that this expression is conserved in species of diverse panarthropod groups, namely the fly Drosophila (Drosophila paralogs Dmfd4/Dmfd5 aka fd96Ca/fd96C), the beetle Tribolium, the millipede Glomeris, the spider Parasteatoda, and the onychophoran Euperipatoides. This suggests a conserved role for FoxB in DV appendage patterning in the entire clade Panarthropoda. Dorsal appendages, like the wings and halteres in Drosophila, in contrast, do not express FoxB, indicating that its function is restricted to ventral appendages. In all ventral appendage types including the highly modified spider opisthosomal appendages (i.e., the book lungs, the tracheal system, and the spinnerets), FoxB is expressed along the ventral ectoderm. This pattern is very similar to that of wg and H15, two other highly conserved ventral limb marker genes (Heingard, 2019).

Exceptions from this rule are the conserved FoxB expression domains in the dorsal tissue of the labrum in Tribolium, Glomeris and Parasteatoda (note that expression of FoxB in the labral discs of Drosophila was not investigated). This apparent discrepancy, however, can be explained by the hypothesis that the labrum is the result of rotation and fusion of a pair of limbs. As a consequence, ventral and dorsal tissue is reversed in the labrum. The second exception concerns the frontal appendages of the onychophoran which do not express FoxB. These appendages are innervated from the protocerebrum and likely are homologous with the arthropod labrum, although expression of FoxB does not support this notion (Heingard, 2019).

The highly conserved expression of FoxB in the limbs of arthropods and the onychophoran strongly suggests an important and evolutionarily conserved function in panarthropod DV limb development. The functional analysis of FoxB in the spider Parasteatoda tepidariorum indeed revealed that FoxB is required for proper DV patterning during limb axis formation. The Class-I phenotype shows an abnormally crooked distal region of pedipalps and legs, most probably explained by a reduction of ventral tissue. Class-I appendages are also broader and softer than wild-type appendages, indicating that the overall integrity of the limbs is disturbed. This becomes even more evident in later stage Class-I appendages which are characterized by the occurrence of abnormal constrictions that finally lead to the complete budding off of limb parts, especially in the distal region (Heingard, 2019).

A very similar phenotype has been reported for wg/Wnt1 and its receptor-encoding gene frizzled-1 (fz1) in the beetle Tribolium. In this study the phenotypes are called 'candy cane' and 'nonpareille'/'pearls on a chain' referring to the bending of the limbs ('candy cane') and the budding off and fusion of distal limb segments ('nonpareille' and 'pearls on a chain'). Tribolium fz1 is expressed ubiquitously, but it fulfils a specific function in limb development as revealed by fz1 knockdown [61]. Although the function of fz1 is not yet studied in other arthropods, it is also ubiquitously expressed in Parasteatoda, allowing for a conserved interaction of Wg and fz1 in spider limb development (Heingard, 2019).

The effect of knockdown of Pt-FoxB and Tc-wg, both of which are expressed in conserved patterns along the ventral side of appendages in all hitherto investigated arthropods, is strikingly similar ('Bandyklubba' phenotype and 'candy cane' phenotype, respectively) suggesting that they might work in the same conserved gene regulatory network (GRN) in DV limb patterning (Heingard, 2019).

In the model system Drosophila melanogaster, the DV limb axis is determined by the action of the dorsal and ventral morphogens Dpp and Wg, respectively. While dpp is specifically expressed in the dorsal sector of the limb imaginal discs, wg is specifically expressed in ventral tissue. The expression of the dorsal morphogen encoding gene dpp is different in the outgrowing appendages of arthropods with direct development, i.e., the vast majority of all arthropods. Instead of being expressed along the dorsal surface of the limbs, its expression is restricted to the tip. Despite these significant differences in gene expression, the so-called topology model has been proposed, that argues for a conserved function of Dpp as dorsal morphogen in a three-dimensional system as represented by directly developing limbs compared to the rather two-dimensional system as represented by the imaginal discs of Drosophila. The T-Box transcription factor optomotor-blind (omb) acts downstream of dpp in Drosophila and is expressed in the dorsal region of the leg imaginal discs. This dorsal omb expression along the developing limbs was previously shown to be conserved in Panarthropoda. The ventral morphogen encoding gene wg is expressed in the ventral sector of the Drosophila leg imaginal disc, and its expression in other arthropods is highly conserved as well. Downstream of wg functions another T-Box transcription factor-encoding gene, H15 (aka midline). Like wg, at least one of the H15 paralogs in each arthropod species is expressed along the ventral side of the outgrowing appendages. In summary, the available data are compatible with the notion that the role of dpp and omb in specifying the dorsal side, as well as the role of wg and H15 in specifying the ventral side are evolutionarily conserved in panarthropods (Heingard, 2019).

After FoxB knockdown in the spider, expression of both ventral marker genes, wg and H15.2, is missing. This indicates that FoxB acts upstream of wg in the GRN required for DV patterning. Since wg is acting upstream of H15 in Drosophila, the lack of H15.2 in FoxB knockdown appendages could be the result of the lack of wg, and thus a secondary effect of FoxB, or it could (as assumed for wg) be under direct control of FoxB. The experimental setup of this study cannot distinguish between these two possibilities, but it would be interesting to study in future experiments (Heingard, 2019).

The expansion of dpp expression along the ventral region in limbs after FoxB knockdown indicates that FoxB normally acts as a repressor of dpp in ventral tissue, either directly, or via wg and/or H15.2. It is noted, however, that no aspect of the topology model predicts the observation that the expression of Pt-dpp is progressively removed from the distal tip in Pt-FoxB knockdown embryos , and therefore this effect of Pt-FoxB RNAi cannot be explained by the model (Heingard, 2019).

Also, the dorsal factor omb is intruding ventral and distal areas of appendages in FoxB knockdown embryos, which suggests that FoxB acts directly (or indirectly via Wg and H15.2) as a repressor of omb. The assumption that Dpp could act as a direct activator of omb is not supported by the data, because the expansion of dpp along the ventral side of the limbs apparently does not cause ectopic expression of omb in this tissue. However, it is also possible that ventral tissue is not competent for omb expression, even in the presence of dpp (Heingard, 2019).

In Drosophila, Hedgehog (Hh) activates dpp and wg in the leg disc due to an early asymmetry that allows ventral and dorsal cells to respond differently to Hh signalling (in dorsal tissue, dpp is activated, and in ventral tissue, wg is activated). Such asymmetry is provided by the relative earlier expression of Wg in ventral tissue. Consequently, in the absence of Wg, Hh would activate dpp in ventral tissue, instead of wg (Heingard, 2019).

This scenario is in line with the current data. Since wg is absent from ventral tissue in FoxB knockdown embryos as a result of the missing function of FoxB, now dpp is dominantly expressed in this tissue. Once the asymmetry between wg and dpp expressing tissue is established, Dpp and Wg act as mutual antagonists in the Drosophila imaginal discs. If this mutual antagonistic function is conserved, or at least the repressive function of Wg on Dpp, this might explain why dpp expands into the now wg-free ventral limb tissue after FoxB knockdown. Again, it was not possible to distinguish between a possible direct or indirect repression of dpp via FoxB or/and Wg. Either way, the data suggest that FoxB is acting at a high level in the GRN orchestrating DV limb patterning (Heingard, 2019).

It has been shown that different regions along the AP axis of the Drosophila leg are under control of different GRNs, or that given GRNs act differently in different regions of the leg. For example, the most proximal region of the Drosophila leg, the coxa, never expresses Distal-less (Dll), a gene that is otherwise involved in the formation of all other podomeres (leg segments). It has also been shown that wg plays a specific role in the development of the coxa. Similarly, it also appears that the proximal region (including the coxa) is patterned differently in the beetle Tribolium. Interestingly, in this study wg appears to have the opposite effect. While distal regions of the legs are affected in wg knockdown and fz1 knockdown embryos, this is not the case for the coxal region. Therefore, it is possible that the proximal region and the distal region (defined as distal to the coxa) are generally regulated differently in arthropods (Heingard, 2019).

The results on Parasteatoda FoxB function support this hypothesis and suggest that the differences between proximal and distal leg development may indeed date back to the last common ancestor of insects and spiders, i.e., the arthropod ancestor. Although Pt-FoxB is expressed all along the ventral side of pedipalps and legs, its knockdown affects only the distal and medial, but not the proximal Pt-wg expression. Similarly, Pt-FoxB knockdown leads to the misexpression of Pt-omb in the distal portions of pedipalps and legs, while medial and proximal portions are not affected. The most intriguing result, however, is the complete change of the Pt-dpp expression pattern after Pt-FoxB RNAi, especially in the distal tip. In this case, the loss of Pt-FoxB influences Pt-dpp expression even in portions of the limbs that never express Pt-FoxB. The reason for this is currently not clear (Heingard, 2019).

In Drosophila, the correct formation of joints depends on the PD patterning system and the so-called leg gap genes. In a combinatorial mode, they activate Delta/Notch signalling and downstream of Delta/Notch signalling act, e.g., the odd-skipped family genes, including odd-skipped (odd) itself. It has been shown that the involvement of Delta/Notch signalling and its downstream factors such as odd in arthropod joint formation is conserved in arthropods beyond Drosophila. In the spider Cupiennius salei, the odd ortholog odd-related-1 (one of three identified odd-related genes in this spider) is expressed in concentric rings in the limbs downstream of Delta/Notch signalling and its function is clearly correlated with that of joint formation. The same expression pattern is seen for odd in the limbs of Parasteatoda (Heingard, 2019).

Remarkably, this study found that odd expression in concentric rings is disturbed after knocking down FoxB, but only in the ventral sector, while expression along the dorsal side of the limbs is not affected (except for the distal region where expression of odd is completely lost). Double in situ revealed that odd is indeed co-expressed with the patches of enhanced expression of FoxB. Together, this implies that odd expression in the limbs is likely under control of the DV patterning system downstream of FoxB function, at least ventrally. Since odd is one of the genes that is involved in joint formation in spiders, this finding is the first potential evidence that joint formation and DV patterning may be connected (Heingard, 2019).

Knockout of crustacean leg patterning genes suggests that insect wings and body walls evolved from ancient leg segments

The origin of insect wings has long been debated. Central to this debate is whether wings are a novel structure on the body wall resulting from gene co-option, or evolved from an exite (outgrowth; for example, a gill) on the leg of an ancestral crustacean. This study reports the phenotypes for the knockout of five leg patterning genes in the crustacean Parhyale hawaiensis and compares these with their previously published phenotypes in Drosophila and other insects. This leads to an alignment of insect and crustacean legs that suggests that two leg segments that were present in the common ancestor of insects and crustaceans were incorporated into the insect body wall, moving the proximal exite of the leg dorsally, up onto the back, to later form insect wings. These results suggest that insect wings are not novel structures, but instead evolved from existing, ancestral structures (Bruce, 2020).

Arp2/3-dependent mechanical control of morphogenetic robustness in an inherently challenging environment

Epithelial sheets undergo highly reproducible remodeling to shape organs. This stereotyped morphogenesis depends on a well-defined sequence of events leading to the regionalized expression of developmental patterning genes that finally triggers downstream mechanical forces to drive tissue remodeling at a pre-defined position. However, how tissue mechanics controls morphogenetic robustness when challenged by intrinsic perturbations in close proximity has never been addressed. Using Drosophila developing leg, this study shows that a bias in force propagation ensures stereotyped morphogenesis despite the presence of mechanical noise in the environment. Knockdown of the Arp2/3 complex member Arpc5 specifically affects fold directionality while altering neither the developmental nor the force generation patterns. By combining in silico modeling, biophysical tools, and ad hoc genetic tools, these data reveal that junctional myosin II planar polarity favors long-range force channeling and ensures folding robustness, avoiding force scattering and thus isolating the fold domain from surrounding mechanical perturbations (Martin, 2021).

Transcriptomic and Genetic Analyses Identify the Kruppel-Like Factor Dar1 as a New Regulator of Tube-Shaped Long Tendon Development

To ensure locomotion and body stability, the active role of muscle contractions relies on a stereotyped muscle pattern set in place during development. This muscle patterning requires a precise assembly of the muscle fibers with the skeleton via a specialized connective tissue, the tendon. Like in vertebrate limbs, Drosophila leg muscles make connections with specific long tendons that extend through different segments. During the leg disc development, cell precursors of long tendons rearrange and collectively migrate to form a tube-shaped structure. A specific developmental program underlies this unique feature of tendon-like cells in the Drosophila model. This study provides for the first time a transcriptomic profile of leg tendon precursors through fluorescence-based cell sorting. From promising candidates, the Kruppel-like factor Dar1 was identified as a critical actor of leg tendon development. Specifically expressed in the leg tendon precursors, loss of dar1 disrupts actin-rich filopodia formation and tendon elongation. These findings show that Dar1 acts downstream of Stripe and is required to set up the correct number of tendon progenitors (Laurichesse, 2021).

The T-box gene optomotor-blind organizes proximodistal leg patterning in the beetle Tribolium castaneum by repressing dorsal Dpp pathway activity

Leg axis formation in Drosophila is organized by Wingless (Wg) and Decapentaplegic (Dpp) that control a number of downstream factors to pattern the dorsoventral (DV) and proximodistal (PD) axis. The T-box genes are important downstream factors mainly involved in dorsoventral leg axis formation. The ventral side is specified by H15 and midline, whereas optomotor-blind (omb) and Dorsocross (Doc1) are factors to specify dorsal cell fates. This study shows that omb also organizes PD leg axis patterning in the beetle Tribolium castaneum. In the legs, Tc-omb is expressed along the dorsal side and represses ventral factors like wg and H15. Intriguingly, removing Tc-omb function leads to the activation of the Dpp pathway along the dorsal side of the legs, thus mimicking normal dpp expression in Drosophila. Dpp activity along the dorsal side leads to altered expression of proximal-distal patterning genes such as Distal-less (Dll) and dachshund (dac). These results indicate a cell-autonomous activation of Dll and repression of dac by dpp. These findings are compatible with the cross-regulatory "cascade model" of proximal-distal leg imaginal disc patterning of Drosophila.

Neural coding of leg proprioception in Drosophila

Animals rely on an internal sense of body position and movement to effectively control motor behavior. This sense of proprioception is mediated by diverse populations of mechanosensory neurons distributed throughout the body. This study investigated neural coding of leg proprioception in Drosophila, using in vivo two-photon calcium imaging of proprioceptive sensory neurons during controlled movements of the fly tibia. The axons of leg proprioceptors are organized into distinct functional projections that contain topographic representations of specific kinematic features. Using subclass-specific genetic driver lines, this study shows that one group of axons encodes tibia position (flexion/extension), another encodes movement direction, and a third encodes bidirectional movement and vibration frequency. Overall, these findings reveal how proprioceptive stimuli from a single leg joint are encoded by a diverse population of sensory neurons, and provide a framework for understanding how proprioceptive feedback signals are used by motor circuits to coordinate the body (Mamiya, 2018).

This study used in vivo calcium imaging to investigate the population coding of leg proprioception in the femoral chordotonal organ (FeCO) of Drosophila. The results reveal a basic logic for proprioceptive sensory coding: genetically distinct proprioceptor subclasses detect and encode distinct kinematic features, including tibia position, directional movement, and vibration. The cell bodies of each proprioceptor subclass reside in separate parts of the FeCO in the leg, and their axons project to distinct regions of the fly VNC. This organization suggests that different kinematic features may be processed by separate downstream circuits, and function as parallel feedback channels for the neural control of leg movement and behavior (Mamiya, 2018).

Claw neurons encode the position of the tibia relative to the femur, club neurons encode bidirectional tibia movement, and hook neurons encode birectional tibia movement. Specifically, each branch of a claw neuron consists of two sub-branches, whose calcium signals increase when the tibia is flexed or extended. Imaging from single claw neurons revealed that individual cells can be narrowly tuned to even more specific tibia angles. These data are consistent with previous reports of angular range fractionation in the locust FeCO. Interestingly, minimal activity in claw axons was observed when the tibia was close to 90°, and no single claw neuron was found tuned to this range in a limited sample. Similar tuning distributions have been observed in multiunit recordings from the FeCO of locusts and stick insects. However, single-unit recordings from these species also revealed the existence of a small number of position-tuned cells with peak activity in this middle range. It is possible that the driver lines that were used did not label the FeCO neurons tuned to this range. It is also possible that this represents a real difference between Drosophila and other insects. The fly FeCO has about half as many neurons as that of the stick insect and locust, and the biomechanics of the organ may also differ between species (Mamiya, 2018).

How does the position tuning of claw neurons relate to natural leg kinematics? When a fly is standing still, the tibia of the front leg rests ~90° relative to the femur; during straight walking, the tibia flexes to ~40° and extends to 120°. Thus, it is predicted that claw neurons are largely silent in a stationary fly, while extension- and flexion-tuned neurons are rhythmically active during walking. Encoding deviations from the natural resting position may reflect an adaptive strategy to minimize metabolic cost (Mamiya, 2018).

Position-encoding claw neurons exhibit response hysteresis (a lag between input and output): both flexion- and extension-tuned sub-branches of the claw showed larger steady-state activity when the tibia is moved in a direction that increases their activity. This response asymmetry is notable because it presents a problem for downstream circuits and computations that rely on a stable readout of tibia angle. Proprioceptive hysteresis has also been described in vertebrate muscle spindles and FeCO neurons of other insects. One possible solution for solving the ambiguities created by hysteresis would be to combine the tonic activity of claw neurons with signals from directionally selective hook neurons. This could allow a neuron to decode tibia position based on past history of tibia movement. However, it is also possible that tibia angle hysteresis is a useful feature of the proprioceptive system, rather than a bug. For example, it has been proposed that hysteresis could compensate for the nonlinear properties of muscle activation in short sensorimotor loops (Mamiya, 2018).

This study identified two functional subclasses of FeCO neurons that respond phasically to tibia movement. Club neurons respond to both flexion and the extension of the tibia, while hook neurons respond only to flexion. In both population and single neuron imaging experiments, directionally selective responses to tibia extension were observe, although it was not possible to identify a specific Gal4 line for this response subclass. The movement sensitivity of the club and hook neurons resembles that of other phasic proprioceptors, including primary muscle spindle afferents, and movement-tuned FeCO neurons recorded in the locust and stick insect. Although the slow temporal dynamics of GCaMP6f did not permit a detailed analysis of velocity tuning, the results indicate that FeCO neurons respond to the natural range of leg speeds encountered during walking. In the future, it will be interesting to investigate how FeCO neurons encode leg movements during walking, and how active movements may be encoded differently from passive movements, for example through presynaptic inhibition of FeCO axon terminals (Mamiya, 2018).

In addition to their directional tuning, this sudy found that club and hook neurons differ in their sensitivity to fast (100-2,000 Hz), low-amplitude (0.9-0.054 μm) tibia vibration. Club neurons are strongly activated by vibration stimuli, but hook neurons are not. This difference in vibration sensitivity is not likely to be caused by a difference in velocity tuning because these differences are relatively small at the range of the speeds experienced during tibia vibration. Rather, it seems that the club neurons have a lower mechanical threshold and/or may be more sensitive to the constant acceleration produced by vibration (Mamiya, 2018).

The functional role of vibration-sensitive FeCO neurons is not entirely clear. Previous studies in stick insects and locusts have found that vibration-tuned FeCO neurons do not contribute to postural reflexes in the same manner as FeCO neurons tuned to joint position and directional movement. This raises the possibility that vibration-tuned chordotonal neurons sense external mechanosensory stimuli. For example, club neurons could monitor substrate vibrations in the environment, which serve as an important communication signal for many insect species. Abdominal vibrations produced during courtship by male Drosophila coincide with pausing behavior in females, and hence increased receptivity to copulation. These vibrations occur at frequencies that match the sensitivity of club neurons (200-2,000 Hz). Therefore, club neurons are well-positioned to mediate intraspecific vibratory communication during courtship or other behaviors (Mamiya, 2018).

Using genetic driver lines for specific FeCO neuron subclasses, this study provides the first detailed anatomical characterization of Drosophila leg proprioceptors. The anatomy and imaging experiments revealed a systematic relationship between the functional tuning of proprioceptor subclasses and their anatomical structure. The cell bodies of the three proprioceptor subclasses are clustered in different regions of the femur, an organization that may reflect biomechanical specialization for detecting position, movement, and vibration. Proprioceptor axons then converge within the leg nerve, before branching within the VNC to form subclass-specific projections that are called the club, claw, and hook. This organization was found to be highly stereotyped across flies (Mamiya, 2018).

The axons of claw neurons split into three symmetric branches, resembling a claw. This unique arborization pattern is suggestive of a Cartesian coordinate system; for example, each branch could represent a different spatial axis. However, this study found that each claw neuron innervates all three branches, and that the X, Y, and Z branches all encode the same stimuli. Specifically, calcium imaging experiments revealed that each claw branch is divided into two sub-branches that are specialized for encoding flexion or extension of the tibia. If each claw branch is functionally similar, what is the purpose of this tri-partite structure? Each branch may target different downstream neurons, or could be independently modulated by presynaptic inhibition. Interestingly, the axons of directionally tuned hook neurons arborized alongside the claw but did not innervate all three of the claw branches. Thus, the X, Y, and Z branches may facilitate integration of positional information with directionally tuned movement signals (Mamiya, 2018).

It was surprising to discover a topographic map of leg vibration frequency within the axon terminals of club neurons. This structure has not previously been described in flies, but resembles the tonotopic map of sensory afferents in the cricket auditory system or the cochlear nucleus in vertebrates. Interestingly, the spatial layout of the frequency map in club axons was consistent across different vibration amplitudes, despite a shift in the peak frequency tuning curve. Recordings from single club neurons suggest that this frequency map is comprised of individual axons that are each tuned to a narrow frequency band. An orderly map of vibration frequency could facilitate feature identification in downstream circuits, for example through lateral inhibition between neighboring axons with shared tuning (Mamiya, 2018).

Neurons in the FeCO population can be generally classified as either tonic (position-encoding) or phasic (movement-encoding). This division has been observed among proprioceptors of many animals, including other insects, crustaceans, and mammals. For example, mammalian muscle spindles are innervated by both phasic (Group 1a) and tonic (Group II) afferents. The same has been found in other primary mechanosensory neurons, including touch, hearing, and vestibular afferents. The ubiquity of tonic and phasic neurons suggests that these two parallel information channels are essential building blocks of sensory circuits. Now that this study has identified genetic tools that delineate tonic and phasic neurons in the proprioceptive system of Drosophila, these circuits have the potential to provide general insights into the utility of this sensory coding strategy (Mamiya, 2018).

Flies possess other chordotonal organs: the most well-studied is the Johnston's organ (JO), which detects antennal movements produced by near-field sound, wind, gravity, and touch. Unlike the FeCO, the JO monitors rotation of a body segment that is not actively controlled by muscles or coupled to the substrate. The JO is also much larger (~500 versus ~135 neurons). Despite these differences, the coding schemes of the two mechanosensory organs share some key similarities. JO neurons can be classified into tonic and phasic classes, some exhibit direction selectivity, and their axon terminals form a rough tonotopic map of frequency. The FeCO and JO share genetic and developmental homology, which suggests that mechanosensory specialization in these organs could arise through similar molecular or biomechanical mechanisms (Mamiya, 2018).

With the advent of new methods for simultaneously monitoring the activity of hundreds or thousands of neurons, a critical challenge has been to link the activity of large neuronal populations to the underlying diversity of specific cell types. Previous efforts have used statistical methods to compare the responses of single neurons to simultaneous optical or electrophysiological population recordings. This study took a different approach, which took advantage of the fact that neurons in the fly can be reliably identified across individuals. Two-photon imaging was first used to monitor activity across a population of proprioceptive sensory neurons during controlled leg movements. From this population data, spatially distinct axon branches were identified that encode specific proprioceptive stimulus features.Genetic driver lines were sought that specifically labeled each axon branch and further characterized their functional tuning with targeted calcium imaging. With this approach, it was possible to identify and characterize the major neuronal subclasses in a key proprioceptive organ (Mamiya, 2018).

With a genetic handle on position, movement, and direction pathways, it should now be possible to trace the flow of proprioceptive signals into downstream circuits and to identify the functional role of specific proprioceptor subclasses within the broader context of motor control and behavior. It is anticipated that Drosophila will provide a useful complement to other model organisms in dissecting fundamental mechanisms of proprioception and deepening understanding of this mysterious 'sixth sense' (Mamiya, 2018).

Parallel transformation of tactile signals in central circuits of Drosophila

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Central processing of leg proprioception in Drosophila

Proprioception, the sense of self-movement and position, is mediated by mechanosensory neurons that detect diverse features of body kinematics. Although proprioceptive feedback is crucial for accurate motor control, little is known about how downstream circuits transform limb sensory information to guide motor output. This study investigated neural circuits in Drosophila that process proprioceptive information from the fly leg. Three cell types from distinct developmental lineages were identified that are positioned to receive input from proprioceptor subtypes encoding tibia position, movement, and vibration. 13Bα neurons encode femur-tibia joint angle and mediate postural changes in tibia position. 9Aα neurons also drive changes in leg posture, but encode a combination of directional movement, high frequency vibration, and joint angle. Activating 10Bα neurons, which encode tibia vibration at specific joint angles, elicits pausing in walking flies. Altogether, these results reveal that central circuits integrate information across proprioceptor subtypes to construct complex sensorimotor representations that mediate diverse behaviors, including reflexive control of limb posture and detection of leg vibration (Agrawal, 2020).

Functional architecture of neural circuits for leg proprioception in Drosophila

To effectively control their bodies, animals rely on feedback from proprioceptive mechanosensory neurons. In the Drosophila leg, different proprioceptor subtypes monitor joint position, movement direction, and vibration. This study investigate how these diverse sensory signals are integrated by central proprioceptive circuits. Signals for leg joint position and directional movement converge in second-order neurons, revealing pathways for local feedback control of leg posture. Distinct populations of second-order neurons integrate tibia vibration signals across pairs of legs, suggesting a role in detecting external substrate vibration. In each pathway, the flow of sensory information is dynamically gated and sculpted by inhibition. Overall, these results reveal parallel pathways for processing of internal and external mechanosensory signals, which are proposed to mediate feedback control of leg movement and vibration sensing, respectively. The existence of a functional connectivity map also provides a resource for interpreting connectomic reconstruction of neural circuits for leg proprioception (Chen, 2021).

This study reports the anatomical structure and functional organization of second-order circuits for leg proprioception in Drosophila. Due to the lack of clear hierarchical structure within the VNC leg neuropil, it has been challenging to infer the flow of proprioceptive sensory signals with existing tools. Therefore genetic driver lines were generated that label specific subtypes of leg proprioceptors and classified candidate second-order neurons based on hemilineage identity. Optogenetics and calcium imaging were used to map the functional connectivity between leg proprioceptors and second-order neurons, followed by EM reconstruction to validate synaptic connectivity and in vivo calcium imaging to understand the function of second-order neurons during leg movement. Spatially targeted and subtype-specific optogenetic stimulation were used to analyze integration of FeCO signals within a subset of second-order neuron classes (Chen, 2021).

Overall, this work reveals the logic of sensory integration in second-order proprioceptive circuits: some populations of second-order neurons integrate tibia vibration signals across pairs of legs, suggesting a role for detection of external substrate vibration. Signals for leg joint position and directional movement converge in other second-order neurons, revealing pathways for local feedback control of leg posture. It is anticipated that this functional wiring diagram (see Summary diagram of circuits processing leg proprioceptive signals from the Drosophila FeCO, based on experiments in this study) will also help guide the interpretation of anatomical wiring diagrams determined through EM reconstruction of VNC circuits (Chen, 2021).

Proprioceptors in the Drosophila FeCO can be classified into three subtypes: club neurons encode bidirectional tibia movement and vibration frequency; claw neurons encode tibia position (flexion or extension); and hook neurons encode the direction of tibia movement.19 Our results show the existence of two distinct central pathways for processing signals from club vs. claw and hook neurons (see Summary diagram of circuits processing leg proprioceptive signals from the Drosophila FeCO, based on experiments in this study). It is proposed that neurons downstream of the club mediate sensing of small mechanical vibrations in the external environment, whereas neurons downstream of the claw and hook provide proprioceptive feedback to motor circuits for controlling the posture and movement of the legs. This division of central pathways for external and internal sensing may be a common motif across limbed animals. Work in a variety of species, including a recent study in mice, has found that many animals can detect low-amplitude, high-frequency, substrate-borne vibrations (Chen, 2021).

Flies may use vibration sensing to monitor acoustic signals in the environment, such as during courtship behavior, or to detect approaching threats. The distinct anatomical organization of neurons downstream of the club vs claw and hook also supports a segregation of vibration sensing and motor control feedback pathways. 9Ba and 10Ba neurons arborize in the mVAC, a common target of descending neuron axons. In contrast, 13Bb arborize in the IntNp, which contains the dendritic branches of the leg motor neurons and premotor interneurons. Based on these differences, it was hypothesized that vibration-sensing neurons interact with ascending and descending signals to and from the brain, whereas neurons downstream of hook and claw axons contribute to local motor control through direct or indirect connections to motor neurons. Leg motor neurons receive position- and movement-tuned proprioceptive input, consistent with feedback from claw and hook neurons (Chen, 2021).

Additional connectomic reconstruction is needed to determine which second-order neurons mediate these feedback connections, but 13Bb neurons are promising pre-motor candidates. VNC neurons postsynaptic to claw and hook axons receive only local input, from individual legs. In contrast, second-order neurons postsynaptic to club axons integrate signals across multiple legs. For example, GABAergic 9Bb neurons pool information from left and right legs in a single VNC segment, whereas cholinergic 10Ba neurons receive convergent input from left and right legs across different segments. Integrating club input across legs may improve detection of external vibration signals, while proprioceptive signals from the claw and hook may be initially processed in parallel to support postural control of individual legs. Bilateral integration also occurs in second-order auditory circuits downstream of the Drosophila Johnston's organ: mechanosensory signals from the two antennae are processed in parallel by second-order neurons in the AMMC but then converge in third-order neurons in the wedge (Chen, 2021).

Although second-order neurons in the vibration pathway integrate club signals across legs, multiple classes of second-order neurons in the motor pathway (13Bb and 8Aa) integrate signals across different FeCO subtypes from the same leg. Using new genetic driver lines that subdivide claw neurons into extension- and flexion-tuned subtypes, it was found that extension-tuned claw and hook neurons converge on 13Bb neurons. These cells are hypothesized to mediate resistance reflexes that stabilize tibia position in response to external perturbations. Prior work in the stick insect has shown that tibia resistance reflexes rely on position and directional movement signals from the FeCO (Chen, 2021).

In support of this hypothesis, another class of neurons in the 13B hemilineage, 13Ba, also encode tibia extension and drive tibia flexion when optogenetically activated. This functional connectivity map reveals interesting parallels with sensorimotor circuitry in the larval Drosophila VNC. Although fly larvae do not have legs, they do possess body wall proprioceptors (class I sensory neurons) and use chordotonal neurons to sense external vibrations in a manner analogous to club neurons in the adult FeCO. As in the adult, larval neurons belonging to lineage 9 (basin neurons) and lineage 8 (eve lateral interneurons) integrate signals from chordotonal sensory neurons and proprioceptors. These examples suggest that some lineage connectivity motifs are likely conserved across the larval and adult nervous systems, which are already known to possess molecular and general anatomical similarities (Chen, 2021).

The results identify a prominent role for inhibition in central processing of proprioceptive information from the FeCO. Of the eight identified second-order cell classes, six are putative inhibitory neurons (i.e., release GABA or glutamate). In other sensory circuits, local inhibitory processing contributes to sharpening spatial and temporal dynamics as well as reducing sensory noise through crossover inhibition (Chen, 2021).

By pharmacologically blocking GABAa and GluCl receptors, we identified a role for inhibition in controlling adaptation within second-order neurons (e.g., 10Ba and 13Bb neurons; Figure 5C). In other cases (20/22Ab or 9Ba; Figure 5A), inhibition was strong enough to completely mask proprioceptive inputs from FeCO axons. We hypothesize that this inhibition may be tuned in certain behavioral contexts, for example, during active movements, to gate the flow of proprioceptive feedback signals in a context-dependent manner. Synaptic transmission in Drosophila can be mediated by chemical synapses, which can be visualized with EM, or electrical gap junctions, which are not typically identifiable at the resolution of current EM volumes. FeCO neurons release acetylcholine but also express gap junction proteins (shakB; data not shown). We therefore used pharmacology to test for the presence of gap junctions between sensory and central neurons. MLA, an effective antagonist of nicotinic acetylcholine receptors in Drosophila eliminated functional connectivity between club and 9Bb neurons but only reduced functional connectivity between club and 10Ba neurons. We observed similar results downstream of the claw: MLA blocked functional connectivity between claw and 13Bb neurons but only reduced functional connectivity between claw and 13Ba neurons (data not shown). These results suggest that second-order proprioceptive circuits receive mixed chemical and electrical input from FeCO neurons. More work is needed to confirm these observations and to investigate the functional significance of why pathways might use one means of signal transmission over the other. One hypothesis is that chemical synapses exhibit adaptation (e.g., synaptic depression), whereas electrical synapses may be more advantageous for sustained synaptic transmission (Chen, 2021).

Each may provide different advantages for pathways that control behavior on a variety of timescales, from slow postural reflexes to rapid escape behaviors (Chen, 2021).

Central processing of sensory signals from the FeCO has been previously studied in other insects, especially the locust and stick insect. In both species, second-order interneurons encode combinations of tibia movement and position and also integrate multimodal signals from different proprioceptive organs (Chen, 2021).

Vibration signals detected by the FeCO appear to be processed by largely segregated populations of VNC interneurons. However, these conclusions were based on mapping of sensory receptive fields, and it was not previously possible to identify specific sources of sensory input, as is done in this study. Overall, comparison of our functional connectivity results in Drosophila with the prior work in other insect species suggests general evolutionary conservation of VNC circuits for leg proprioception and motor control. Although it is currently difficult to identify homologous cell types across insect species, future efforts could leverage conserved developmental programs: the organization of neuroblasts that give rise to the VNC is similar across insect species separated by 350 Ma of evolution (Chen, 2021).

This is an important advantage of using developmental lineages to define VNC cell classes-locusts and stick insects also possess 9A, 10B, and 13B neurons, which could someday be identified based on molecular markers of lineage identity (Chen, 2021).

The functional connectivity approach that was employed in this study has both benefits and drawbacks. On the positive side, it allowed screening a large connectivity matrix of genetically identified sensory and central neurons. Compared to other methods for anatomical mapping (e.g., EM), the use of optogenetics and calcium imaging allowed measuring of connection strength and dynamics across multiple individuals. It was found that second-order VNC neurons varied significantly in their functional connectivity strength and temporal dynamics. 5-fold differences were observed in peak calcium signals in response to optogenetic stimulation with the same light intensity. This range could be due to differences in GCaMP expression or intracellular calcium buffering, but could also reflect differences in synaptic strength between pre- and postsynaptic partners (Chen, 2021).

One limitation of functional connectivity is that it is not possible to measure all possible combinations of pre- and postsynaptic partners. For example, a previous study provided evidence that 9Aa neurons receive input from hook and club neurons, which was not observed in the current screen. This discrepancy could be due to the fact that the driver lines that were used do not label the specific subset of hook and club cells presynaptic to 9Aa neurons. Alternatively, it may be due to differences in signal transmission driven by optogenetic stimulation versus natural tibia movements, as was the case for 9Bb neurons (Chen, 2021).

Functional connectivity mapping also cannot resolve whether inputs are direct, due to the slow kinetics of GCaMP6. This study therefore used sparse, targeted EM tracing to validate some of the functional connections that were identified between FeCO and VNC neurons. A more detailed comparison of functional and anatomical connectivity will require dense, comprehensive reconstruction of the VNC neuropil. Automated reconstruction and manual proofreading have recently led to draft wiring diagrams of neural circuits in the adult Drosophila central brain (Chen, 2021).

NeuroMechFly, a neuromechanical model of adult Drosophila melanogaster

Animal behavior emerges from an interaction between neural network dynamics, musculoskeletal properties and the physical environment. Accessing and understanding the interplay between these elements requires the development of integrative and morphologically realistic neuromechanical simulations. This study presents NeuroMechFly, a data-driven model of the widely studied organism, Drosophila melanogaster. NeuroMechFly combines four independent computational modules: a physics-based simulation environment, a biomechanical exoskeleton, muscle models and neural network controllers. To enable use cases, the minimum degrees of freedom of the leg was defined from real three-dimensional kinematic measurements during walking and grooming. Then, it was shown how, by replaying these behaviors in the simulator, one can predict otherwise unmeasured torques and contact forces. Finally, NeuroMechFly's full neuromechanical capacity was leveraged to discover neural networks and muscle parameters that drive locomotor gaits optimized for speed and stability. Thus, NeuroMechFly can increase understanding of how behaviors emerge from interactions between complex neuromechanical systems and their physical surroundings (Lobato-Rios, 2022).

Single-Cell Atlas of the Drosophila Leg Disc Identifies a Long Non-Coding RNA in Late Development

The Drosophila imaginal disc has been an excellent model for the study of developmental gene regulation. In particular, long non-coding RNAs (lncRNAs) have gained widespread attention in recent years due to their important role in gene regulation. Their specific spatiotemporal expressions further support their role in developmental processes and diseases. This study explored the role of a novel lncRNA in Drosophila leg development by dissecting and dissociating w(1118) third-instar larval third leg (L3) discs into single cells and single nuclei, and performing single-cell RNA-sequencing (scRNA-seq) and single-cell assays for transposase-accessible chromatin (scATAC-seq). Single-cell transcriptomics analysis of the L3 discs across three developmental timepoints revealed different cell types and identified lncRNA:CR33938 as a distal specific gene with high expression in late development. This was further validated by fluorescence in-situ hybridization (FISH). The scATAC-seq results reproduced the single-cell transcriptomics landscape and elucidated the distal cell functions at different timepoints. Furthermore, overexpression of lncRNA:CR33938 in the S2 cell line increased the expression of leg development genes, further elucidating its potential role in development (Tse, 2022).

Sensing of a spore surface protein by a Drosophila chemosensory protein induces behavioral defense against fungal parasitic infections

In addition to innate immunity in a physiological context, insects have evolved behavioral defenses against parasite attacks. This study reports that Drosophila can sense the CFEM (common in fungal extracellular membrane) protein Mcdc9, which acts as a negative virulence factor of the entomopathogenic fungus Metarhizium robertsii. The individual deletions of 18 CFEM genes in Metarhizium followed by fly infection identified three null mutants that could kill the flies more quickly than the wild-type strain, among which Mcdc9 can coat fungal spores and interact with the fly chemosensory protein CheA75a. The deletion of Mcdc9 in the fungus or the knockdown of CheA75a in flies had a similar effect, in which a greater number of fungal spores were left on flies than on the respective controls after topical infection. Thus, similar to the accelerated death of the wild-type flies treated with ΔMcdc9, the CheA75a(RNAi) flies succumbed more quickly than the control insects topically challenged with the wild-type strain. The CheA75a gene is highly transcribed in fly legs and wings, and positive electrophysiological responses were evidenced in tarsal sensilla after stimulation with the Mcdc9 protein. The results imply that this CFEM protein could be sensed as a contact elicitor inducing the hygienic behavior of flies against fungal parasitic infection, which reveals a previously unsuspected mechanism of fungus-insect interactions (Shang, 2023).

Transcription factor paralogs orchestrate alternative gene regulatory networks by context-dependent cooperation with multiple cofactors

In eukaryotes, members of transcription factor families often exhibit similar DNA binding properties in vitro, yet orchestrate paralog-specific gene regulatory networks in vivo. The serially homologous first (T1) and third (T3) thoracic legs of Drosophila, which are specified by the Hox proteins Scr and Ubx, respectively, offer a unique opportunity to address this paradox in vivo. Genome-wide analyses using epitope-tagged alleles of both Hox loci in the T1 and T3 leg imaginal discs, the precursors to the adult legs and ventral body regions, show that ~8% of Hox binding is paralog-specific. Binding specificity is mediated by interactions with distinct cofactors in different domains: the Hox cofactor Exd acts in the proximal domain and is necessary for Scr to bind many of its paralog-specific targets, while in the distal leg domain, the homeodomain protein Distal-less (Dll) enhances Scr binding to a different subset of loci. These findings reveal how Hox paralogs, and perhaps paralogs of other transcription factor families, orchestrate alternative downstream gene regulatory networks with the help of multiple, context-specific cofactors (Feng, 2022).

This study used a combination of whole-genome and mechanistic approaches to understand how serially homologous appendages, such as the fly T1 and T3 legs, obtain their unique morphologies due to the activities of parallel Hox gene networks. The very similar transcriptomes in the three pairs of leg discs suggest that the different morphologies are largely a consequence of changing the expression patterns of the same sets of genes. By comparing the genome-wide DNA-binding profiles of the two relevant Hox paralogs, Scr and Ubx, in their native physiological contexts, this study found hundreds of paralog-specific Hox targets, accounting for ~8% of all binding events for these two Hox proteins. Next, differences in chromatin accessibility and Hox monomer binding preferences were shown to be unlikely to account for paralog-specific binding. Instead, it was demonstrated that interaction with the Hox cofactor Exd explains a large fraction of Scr's paralog-specific binding events. Finally, this study identified Dll as a Hox cofactor in the complementary distal domain of the leg disc. Results from RNA-seq, CBP ChIP, and reporter assays suggest that about 1/3 of the paralog-specific Scr-binding events are functional and lead to tissue-specific gene regulation. Thus, paralog-specific Hox-DNA binding, which is mediated by multiple cofactors including Exd and Dll, contribute significantly to paralog-specific Hox gene networks (Feng, 2022).

Previous in vitro studies provided compelling evidence that the DNA-binding specificities of different Hox-Exd dimers are more divergent from each other than those of Hox monomers, a phenomenon termed latent specificity. There have also been several in vivo examples in which paralog-specific Hox-DNA binding and target regulation was shown to depend on an interaction with Exd. This study shows that, on a genome-wide scale, the interaction with Exd explains a significant fraction of paralog-specific Hox binding, which often leads to paralog-specific gene regulation (Feng, 2022).

Earlier work also suggested that there is a tradeoff between specificity and affinity for Hox-Exd-binding motifs, where high affinity binding motifs are more likely to have low specificity for different Hox-Exd heterodimers. The paralog-specific, Exd-dependent CRMs characterized in this study (ac^-1, h^-1, and fj<^-1), have higher affinity Hox-Exd-binding motifs than those previously described in the shavenbaby (svb) gene: the major Scr-Exd motif in the fj^-1 CRM has an affinity of about 0.06 relative to the optimal motif in the genome, while the motifs in ac^-1-1 and h^-1 have even higher relative affinities of nearly 0.15 and 0.2, respectively. In contrast, the Ubx-Exd-binding motifs in CRMs from svb have a relative affinity of <0.0118. One possible explanation for this difference is that the svb CRMs are active in embryos, which have many different cell types, while the CRMs characterized in this study are active in leg discs, which have significantly less cell-type complexity. Embryonic CRMs may require especially low-affinity binding motifs to distinguish their activities in a context with many cell types. Consistent with this idea, the fkh250 CRM, which is also active in embryos, uses an Scr-Exd-binding motif with a low relative affinity of 0.01718,36. Notably, the relative affinities for the Scr-Exd-binding motifs in ac^-1, h^-1, and fj^-1 are at least eightfold higher than for Ubx-Exd. Manual inspection of other intergenic and intronic loci with Scr_T1 > Ubx_T3 binding suggests that there are many other CRMs that follow this same rule. Thus, for specificity to occur, the most relevant feature may be that the affinity for the 'correct' TFs, in this case Scr-Exd, must be significantly greater compared to the affinity for other 'incorrect' TFs that are co-expressed in the same or homologous cells (Feng, 2022).

Because Exd is only nuclear in a subset of cells during Drosophila development, such as the proximal domain of the leg disc, it was unlikely that Exd was the only Hox cofactor. In fact, CRMs that are directly regulated by Ubx have been described in cells where Exd is not available to be a cofactor. However, it has remained an unresolved question whether non-Exd cofactors are used in these examples. More generally for the leg imaginal disc, the entire distal domain, extending from the trochanter to the tarsus, is without nuclear Exd, yet has Hox-dependent segment-specific morphological characteristics, such as the sex combs on the male T1 leg. Although several candidate TFs have been proposed to be Hox cofactors, none have been confirmed. This study provides evidence that Dll is a distally acting Hox cofactor in leg discs (Feng, 2022).

There are many differences between how Exd and Dll interact with Hox proteins when bound to DNA. The Scr-Exd-binding motif is comprised of two partially overlapping half-sites, while the Scr-Dll motif consists of two HD-binding motifs separated by a spacer of several base pairs. Another difference is that the amount of cooperativity observed for Hox-Exd is far greater than that observed for Hox-Dll. The overlapping nature of the Hox- and Exd-binding motifs may be important for latent specificity, which for Scr requires an Exd-induced conformational change of the homeodomain. In contrast, there is no evidence that latent specificity occurs as a consequence of Hox-Dll binding. Instead, the modest cooperativity observed for the Scr-Dll heterodimer is likely a consequence of increasing Scr-DNA-binding affinity via a protein-protein interaction and closely spaced Dll and Scr-binding motifs (Feng, 2022).

More generally, it is suggested that mode of DNA binding exhibited by Hox-Exd, which is highly cooperative and reveals latent specificity, may be the exception rather than the rule for TF-TF interactions within CRMs, and that the Scr-Dll example, with weak cooperativity between TFs stemming from a protein-protein interaction, may be the more common mode of interaction to distinguish the binding of paralogous TFs. In support of this notion, a systematic in vitro study identified 315 TF-TF interactions, only five of which exhibited latent specificity (Feng, 2022).

The TALE homeodomain proteins, which include Exd and Hth, are very ancient TFs that were present before the split of plants and animals, and TALE-mediated nuclear localization analogous to the Hth-Exd example in flies has been described in plants. In contrast, the Hox gene family is only present in metazoans, and Dll is specific to bilaterians. Moreover, it has been proposed that Dll initially functioned in the CNS, and was later co-opted to pattern the distal appendage. Based on these observations, it is plausible that the Hox-Dll interaction evolved more recently than the Hox-Exd interaction, accounting for why Exd interacts with all Hox paralogs, while Dll may be a more limited Hox cofactor. This is supported by the results from a small-scale bimolecular fluorescence complementation (BiFC) screen that revealed Dll interacts with some Hox proteins, but not others (Feng, 2022).

Notably, the combined activities of Exd and Dll still do not account for all ScrT1 > UbxT3-binding events genome-wide and reporter analysis suggests the presence of additional, yet to be identified Hox cofactors that have the capacity to promote Scr-specific binding. It is suggested that the Hox-Dll mode of binding uncovered in this study may be representative of additional TFs that also have the ability to promote paralog-specific Hox binding and activity at specific CRMs. Further, it is noted that the differentiation of the T1 and T3 leg fates is a continuous developmental process and that the observations described in this study are limited to the late 3rd instar stage. Nevertheless, it is expected that the principles governing Hox paralog specificity uncovered in this study will likely extend to other developmental stages and tissues. Finally, although this study focused on the role of paralog-specific TF-DNA binding, there may be additional mechanisms that do not depend on differences in DNA binding between paralogous TFs that also contribute to their specific functions (Feng, 2022).

Characterisation of the role and regulation of Ultrabithorax in sculpting fine-scale leg morphology

Hox genes are expressed during embryogenesis and determine the regional identity of animal bodies along the antero-posterior axis. However, they also function post-embryonically to sculpt fine-scale morphology. To better understand how Hox genes are integrated into post-embryonic gene regulatory networks, this study further analysed the role and regulation of Ultrabithorax (Ubx) during leg development in Drosophila melanogaster. Ubx regulates several aspects of bristle and trichome patterning on the femurs of the second (T2) and third (T3) leg pairs. Repression of trichomes in the proximal posterior region of the T2 femur by Ubx is likely mediated by activation of the expression of microRNA-92a and microRNA-92b by this Hox protein. Furthermore, this study identified a novel enhancer of Ubx that recapitulates the temporal and regional activity of this gene in T2 and T3 legs. Transcription factor (TF) binding motif analysis was used in regions of accessible chromatin in T2 leg cells to predict and functionally test TFs that may regulate the Ubx leg enhancer.The role of the Ubx co-factors Homothorax (Hth) and Extradenticle (Exd) in T2 and T3 femurs was also tested. Several TFs were found that may act upstream or in concert with Ubx to modulate trichome patterning along the proximo-distal axis of developing femurs and that the repression of trichomes also requires Hth and Exd. Taken together these results provide insights into how Ubx is integrated into a post-embryonic gene regulatory network to determine fine-scale leg morphology (Buffry, 2023).

References

Agrawal, S., Dickinson, E. S., Sustar, A., Gurung, P., Shepherd, D., Truman, J. W. and Tuthill, J. C. (2020). Central processing of leg proprioception in Drosophila. Elife 9. PubMed ID: 33263281

Azevedo, A. W., Dickinson, E. S., Gurung, P., Venkatasubramanian, L., Mann, R. S. and Tuthill, J. C. (2020). A size principle for recruitment of Drosophila leg motor neurons. Elife 9. PubMed ID: 32490810

Baek, M. and Mann, R. S. (2009). Lineage and birth date specify motor neuron targeting and dendritic architecture in adult Drosophila. J Neurosci 29: 6904-6916. Pubmed: 19474317

Brierley, D. J., Rathore, K., VijayRaghavan, K. and Williams, D. W. (2012). Developmental origins and architecture of Drosophila leg motoneurons. J Comp Neurol 520: 1629-1649. Pubmed: 22120935

Bruce, H. S. and Patel, N. H. (2020). Knockout of crustacean leg patterning genes suggests that insect wings and body walls evolved from ancient leg segments. Nat Ecol Evol 4(12): 1703-1712. PubMed ID: 33262517

Buffry, A. D., Kittelmann, S. and McGregor, A. P. (2023). Characterisation of the role and regulation of Ultrabithorax in sculpting fine-scale leg morphology. Front Cell Dev Biol 11: 1119221. PubMed ID: 36861038

Chen, C., Agrawal, S., Mark, B., Mamiya, A., Sustar, A., Phelps, J. S., Lee, W. A., Dickson, B. J., Card, G. M. and Tuthill, J. C. (2021). Functional architecture of neural circuits for leg proprioception in Drosophila. Curr Biol. PubMed ID: 34637749
Summary:

Cordoba, S., Requena, D., Jory, A., Saiz, A. and Estella, C. (2016). The evolutionary conserved transcription factor Sp1 controls appendage growth through Notch signaling. Development 143(19):3623-3631. PubMed ID: 27578786

Doerksen, A., Mulder, M., Ingram, S., Nelson, D., Defehr, J., Reimer, E., Atallah, J. and Malagon, J. N. (2022). Dynamics of changes in apical cell area during sex comb rotation in Drosophila melanogaster. MicroPubl Biol 2022. PubMed ID: 36606080

Estella, C. and Mann, R. S. (2010). Non-redundant selector and growth-promoting functions of two sister genes, buttonhead and Sp1, in Drosophila leg development. PLoS Genet 6: e1001001. PubMed ID: 20585625

Feng, S., Rastogi, C., Loker, R., Glassford, W. J., Tomas Rube, H., Bussemaker, H. J. and Mann, R. S. (2022). Transcription factor paralogs orchestrate alternative gene regulatory networks by context-dependent cooperation with multiple cofactors. Nat Commun 13(1): 3808. PubMed ID: 35778382

Fristrom, D. (1976). The mechanism of evagination of imaginal discs of Drosophila melanogaster: III. Evidence for cell rearrangement. Dev. Biol. 54: 163-171. PubMed ID: 825402

Heingard, M., Turetzek, N., Prpic, N. M. and Janssen, R. (2019). FoxB, a new and highly conserved key factor in arthropod dorsal-ventral (DV) limb patterning. Evodevo 10: 28. PubMed ID: 31728178

Held, L. I. (1995). Axes, boundaries and coordinates: the ABCs of fly leg development. Bioessays 17: 721-732. PubMed ID: 7661853

Ho, E. C. Y., Malagon, J. N., Ahuja, A., Singh, R. and Larsen, E. (2018). Rotation of sex combs in Drosophila melanogaster requires precise and coordinated spatio-temporal dynamics from forces generated by epithelial cells. PLoS Comput Biol 14(10): e1006455. PubMed ID: 30303951

Lacin, H., Zhu, Y., Wilson, B. A. and Skeath, J. B. (2014). Transcription factor expression uniquely identifies most postembryonic neuronal lineages in the Drosophila thoracic central nervous system. Development 141(5): 1011-1021. PubMed ID: 24550109

Lacin, H. and Truman, J. W. (2016). Lineage mapping identifies molecular and architectural similarities between the larval and adult Drosophila central nervous system. Elife 5: e13399. PubMed ID: 26975248

Lacin, H., Chen, H. M., Long, X., Singer, R. H., Lee, T. and Truman, J. W. (2019). Neurotransmitter identity is acquired in a lineage-restricted manner in the Drosophila CNS. Elife 8. PubMed ID: 30912745

Lacin, H., Williamson, W. R., Card, G. M., Skeath, J. B. and Truman, J. W. (2020). Unc-4 acts to promote neuronal identity and development of the take-off circuit in the Drosophila CNS. Elife 9. PubMed ID: 32216875

Landgraf, M., Jeffrey, V., Fujioka, M., Jaynes, J. B. and Bate, M. (2003). Embryonic origins of a motor system: motor dendrites form a myotopic map in Drosophila. PLoS Biol 1: E41. Pubmed: 14624243

Laurichesse, Q., Moucaud, B., Laddada, L., Renaud, Y., Jagla, K. and Soler, C. (2021). Transcriptomic and Genetic Analyses Identify the Kruppel-Like Factor Dar1 as a New Regulator of Tube-Shaped Long Tendon Development. Front Cell. Dev Biol 9: 747563. PubMed ID: 34977007

Lobato-Rios, V., Ramalingasetty, S. T., Ozdil, P. G., Arreguit, J., Ijspeert, A. J. and Ramdya, P. (2022). NeuroMechFly, a neuromechanical model of adult Drosophila melanogaster. Nat Methods 19(5): 620-627. PubMed ID: 35545713

Ly, D., Resch, E., Ordiway, G. and DiNardo, S. (2017). Asymmetrically deployed actomyosin-based contractility generates a boundary between developing leg segments in Drosophila. Dev Biol 429(1): 165-176. PubMed ID: 28689737

Martin, E., Theis, S., Gay, G., Monier, B., Rouviere, C. and Suzanne, M. (2021). Arp2/3-dependent mechanical control of morphogenetic robustness in an inherently challenging environment. Dev Cell 56(5): 687-701.e687. PubMed ID: 33535069

McKay, D. J. and Lieb, J. D. (2013). A common set of DNA regulatory elements shapes Drosophila appendages. Dev Cell 27: 306-318. PubMed ID: 24229644

Mou, X., Duncan, D. M., Baehrecke, E. H. and Duncan, I. (2012). Control of target gene specificity during metamorphosis by the steroid response gene E93. Proc Natl Acad Sci U S A 109: 2949-2954. PubMed ID: 22308414

Mamiya, A., Gurung, P. and Tuthill, J. C. (2018). Neural coding of leg proprioception in Drosophila. Neuron 100(3):636-650. PubMed ID: 30293823

Newcomb, S., Voutev, R., Jory, A., Delker, R. K., Slattery, M. and Mann, R. S. (2018). cis-regulatory architecture of a short-range EGFR organizing center in the Drosophila melanogaster leg. PLoS Genet 14(8): e1007568. PubMed ID: 30142157

Pechmann, M. and Prpic, N. M. (2022). The T-box gene optomotor-blind organizes proximodistal leg patterning in the beetle Tribolium castaneum by repressing dorsal Dpp pathway activity. Dev Biol 482: 124-134. PubMed ID: 34942194

Phillips, L. A., Atienza, M. L., Ryu, J. R., Svendsen, P. C., Kelemen, L. K. and Brook, W. J. (2022). midline represses Dpp signaling and target gene expression in Drosophila ventral leg development. Biol Open 11(5). PubMed ID: 35608103

Shang, J., Tang, G., Yang, J., Lu, M., Wang, C. Z. and Wang, C. (2023). Sensing of a spore surface protein by a Drosophila chemosensory protein induces behavioral defense against fungal parasitic infections. Curr Biol 33(2): 276-286. PubMed ID: 36423638

Shepherd, D., Sahota, V., Court, R., Williams, D. W. and Truman, J. W. (2019). Developmental organization of central neurons in the adult Drosophila ventral nervous system. J Comp Neurol 527(15): 2573-2598. PubMed ID: 30919956

Svendsen, P. C., Phillips, L. A., Deshwar, A. R., Ryu, J. R., Najand, N. and Brook, W. J. (2019). The selector genes midline and H15 control ventral leg pattern by both inhibiting Dpp signaling and specifying ventral fate. Dev Biol. PubMed ID: 31299230

Tajiri, R., Misaki, K., Yonemura, S. and Hayashi, S. (2010). Dynamic shape changes of ECM-producing cells drive morphogenesis of ball-and-socket joints in the fly leg. Development 137(12): 2055-63. PubMed ID: 20501594

Taylor, J. and Adler, P. N. (2008). Cell rearrangement and cell division during the tissue level morphogenesis of evaginating Drosophila imaginal discs. Dev. Biol. 313(2): 739-51. PubMed ID: 18082159

Tse, J., Li, T. H., Zhang, J., Lee, A. C. K., Lee, I., Qu, Z., Lin, X., Hui, J. and Chan, T. F. (2022). Single-Cell Atlas of the Drosophila Leg Disc Identifies a Long Non-Coding RNA in Late Development. Int J Mol Sci 23(12). PubMed ID: 35743238

Tuthill, J. C. and Wilson, R. I. (2016). Parallel transformation of tactile signals in central circuits of Drosophila. Cell 164: 1046-1059. PubMed ID: 26919434

Genes involved in organ development

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.