The Interactive Fly

Genes involved in tissue and organ development

The Drosophila Leg

Axes, boundaries and coordinates in leg development

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

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

Developmental origins and architecture of Drosophila leg motoneurons

A common set of DNA regulatory elements shapes Drosophila appendages

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

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

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

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

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

Genes involved in leg morphogenesis

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 46o and 60o. 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 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 Egfrtsla 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 Egfrtsla 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 rhorhoE-Df vnvnE-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).

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).


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Genes involved in leg morphogenesis

Genes involved in organ development

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