The Interactive Fly
Genes involved in tissue and organ 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).
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).
Fristrom, D. (1976). The mechanism of evagination of imaginal discs of Drosophila melanogaster: III. Evidence for cell rearrangement. Dev. Biol. 54: 163-171. PubMed Citation: 825402
Held, L. I. (1995). Axes, boundaries and coordinates: the ABCs of fly leg development. Bioessays 17: 721-732. PubMed Citation: 7661853
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 Citation: 20501594
Taylor, J. and Adler, P. N. (2008). Cell rearrangement and cell division during the tissue level morphogenesis of evaginating Drosophila imaginal discs. Dev. Biol. 313(2): 739-51. PubMed Citation: 18082159
Genes involved in organ development
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