The Interactive Fly
Genes involved in tissue and organ development
Developmental origins and architecture of Drosophila leg motoneurons
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).
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).
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).
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).
Baek, M. and Mann, R. S. (2009). Lineage and birth date specify motor neuron targeting and dendritic architecture in adult Drosophila. J Neurosci 29: 6904-6916. Pubmed: 19474317
Brierley, D. J., Rathore, K., VijayRaghavan, K. and Williams, D. W. (2012). Developmental origins and architecture of Drosophila leg motoneurons. J Comp Neurol 520: 1629-1649. Pubmed: 22120935
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Landgraf, M., Jeffrey, V., Fujioka, M., Jaynes, J. B. and Bate, M. (2003). Embryonic origins of a motor system: motor dendrites form a myotopic map in Drosophila. PLoS Biol 1: E41. Pubmed: 14624243
Tajiri, R., Misaki, K., Yonemura, S. and Hayashi, S. (2010). Dynamic shape changes of ECM-producing cells drive morphogenesis of ball-and-socket joints in the fly leg. Development 137(12): 2055-63. PubMed ID: 20501594
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Genes involved in organ development
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