Crossveinless: Biological Overview | References
Gene name - crossveinless
Cytological map position - 5A13-5A13
Function - extracellular transport
Symbol - cv
FlyBase ID: FBgn0000394
Genetic map position - X:5,584,029..5,587,117 [+]
Classification - Twisted gastrulation (Tsg) protein conserved region
Cellular location - secreted
In the early Drosophila embryo, Bone morphogenetic protein (BMP) activity is positively and negatively regulated by the BMP-binding proteins Short gastrulation (Sog) and Twisted gastrulation (Tsg). A similar mechanism operates during crossvein formation, utilizing Sog and a new member of the tsg gene family, encoded by the crossveinless (cv) locus. The initial specification of crossvein fate in the Drosophila wing requires signaling mediated by Dpp and Gbb, two members of the BMP family. cv is required for the promotion of BMP signaling in the crossveins. Large sog clones disrupt posterior crossvein formation, suggesting that Sog and Cv act together in this context. sog and cv can have both positive and negative effects on BMP signaling in the wing. Moreover, Cv is functionally equivalent to Tsg, since Tsg and Cv can substitute for each other's activity. It is also confirmed that Tsg and Cv have similar biochemical activities: Sog/Cv complex binds a Dpp/Gbb heterodimer with high affinity. Taken together, these studies suggest that Sog and Cv promote BMP signaling by transporting a BMP heterodimer from the longitudinal veins into the crossvein regions (Shimmi, 2005b).
One interesting aspect of BMP signaling in many developmental contexts is that its activity can be regulated at the extracellular level by a number of secreted factors. In Drosophila, these include the products of the short gastrulation (sog), twisted gastrulation (tsg), and tolloid (tld) genes. All three genes were identified as modulators of BMP signaling in the early embryo, and their developmental functions have been well characterized. At the blastoderm stage, BMP signals provided by the dorsally expressed Decapentaplegic (Dpp), and by the generally expressed Screw (Scw), a second ligand that forms a heterodimer with Dpp (Shimmi, 2005a), instruct cells to adopt either amnioserosa or dorsal ectoderm fate. Proper subdivision into these two cell types requires the action of Sog, Tsg, and Tld. Sog and Tsg are BMP-binding proteins that make a high-affinity complex with the Dpp/Scw heterodimer. This complex reduces BMP signaling in the dorsal-lateral regions by blocking the ability of the heterodimer to bind to receptors. Thus, a major role of the Sog/Tsg complex is to antagonize signaling, and similar activity has been found for the vertebrate homologs Chordin and Tsg (Shimmi, 2005b and references therein).
However, the Sog/Tsg complex also stimulates BMP signaling in the dorsal-most cells of Drosophila embryo; it is thought to do so by protecting the ligand from degradation and enabling it to diffuse over long distances. Since sog is expressed in ventral-lateral cells adjacent to the dorsal cells that express dpp, scw, and tsg, the net flux of Sog towards the dorsal side provides a driving force that concentrates BMP heterodimer in the dorsal-most region of the embryo. The ligand is then released for signaling by Tld, an extracellular metalloprotease that cleaves Sog in a BMP-dependent manner. Concentration of the heterodimer to the dorsal-most cells by this facilitated transport mechanism provides a high level signal that specifies amnioserosa cell fate, while dorsal-lateral cells receive less BMP signal and become dorsal ectoderm (Shimmi, 2005b and references therein).
The ability of Tsg to stimulate BMP signaling is apparently not limited to the early Drosophila embryo. Vertebrate Tsg can stimulate BMP signaling in some circumstances, and is required to stimulate high levels of BMP signaling during axis formation in the zebrafish embryo. However, in these cases, Tsg may act, not via a transport mechanism, but by antagonizing Chordin's ability to inhibit BMP signaling. Tsg increases the rate at which Chordin and Sog are cleaved and thus inactivated by Tolloid-like protease. Nonetheless, zebrafish Chordin can also apparently stimulate BMP signaling in some circumstances (Shimmi, 2005b and references therein).
This paper reports another context in which both Sog and a novel Tsg family member stimulate BMP signaling at the developing crossveins in the Drosophila pupal wing. The Drosophila wing has proven to be an attractive model system for elucidating molecular mechanisms that regulate growth and patterning. A major attribute of this system is the stereotypical array of veins that develop along the wing surfaces. These thickenings of the ectodermal cuticle serve both structural support roles for flight and act as channels for the supply of nutrients to the wing cells. For the geneticist, they provide a key set of morphological landmarks for identification of genes that affect the patterning process. Analysis of many classical mutations that alter vein cell fate and patterning have revealed the fundamental roles played by three highly conserved growth factor signaling pathways. For the five longitudinal veins (L1-L5) that form along the proximal-distal axis, a key initiating event is the localized expression of Epidermal growth factor (EGF) signaling components in the vein primordial cells during late imaginal disc development. In response to EGF receptor signaling, Delta is expressed along the veins and induces Notch to inhibit vein formation in neighboring cells. Subsequently, during pupal stages, EGF receptor signaling induces expression of dpp within the developing longitudinal veins. Expression of dpp in the longitudinal veins is required for maintenance of EGF receptor signaling and final vein differentiation, especially at the distal tips (Shimmi, 2005b and references therein).
In addition to the five longitudinal veins, two other shorter veins form perpendicular to the longitudinal veins; these are the anterior crossvein (ACV), which forms between L3 and L4, and the posterior crossvein (PCV), which forms between L4 and L5. Unlike the longitudinal veins, the crossveins do not rely on early EGF signaling for their initial specification. Instead, their formation is initiated during pupal stage by localized BMP signaling, which requires Dpp. However, in the case of the PCV, Dpp is not initially produced in the crossvein, but instead diffuses into the PCV region from the longitudinal veins. Dpp does not act alone during this process since mutations in glass bottom boat (gbb), a member of the BMP5/6/7 subfamily, also eliminate the PCV. Gbb is widely expressed during pupal wing development, but an analysis of gbb mutant clones has suggested that the active BMP component for PCV specification might be a heterodimer of Dpp and Gbb formed in the longitudinal veins, since the PCV is lost only when the clone includes cells of the longitudinal veins where Dpp is produced (Shimmi, 2005b and references therein).
Like the embryo, modulation of BMP signaling in the PCV also appears to involve several additional secreted proteins. For example, mutations in tolloid-related (tlr; also known as tolkin) and crossveinless 2 (cv-2) eliminate PCV formation by preventing BMP signals in the primordial PCV cells. The tlr gene encodes a Tolloid-like metalloprotease that is able to cleave Sog, while cv-2 encodes a protein containing 5 cysteine-rich (CR) domains, similar to the BMP-binding modules found in Sog (Shimmi, 2005b).
The similarity of these proteins to those involved in patterning the early Drosophila embryo suggests that correct specification of the PCV likely involves establishing a spatially regulated distribution of BMP ligand(s) through the activity of extracellular modulatory factors. This report provides additional evidence supporting this hypothesis by cloning the crossveinless (cv) gene and analyzing its function. cv encodes a new member of the tsg gene family. Like mutations in cv-2 and tlr, loss of cv prevents accumulation of phosphorylated Mad (pMad), the active form of the major transcription effector of BMP signaling, in the crossvein cells. In addition, ectopic expression studies were used to show that Cv and Tsg have functionally related activities, since each can substitute for the other in vivo, and ectopic expression of Cv and Sog phenocopies co-expression of Tsg and Sog. These observations led to a re-examination of the role of Sog during wing vein development. Previous clonal analyses suggested that Sog acts as a dedicated antagonist of BMP signaling and helps maintain longitudinal vein integrity. However, this study shows that Sog is in fact required for BMP signaling in the PCV, since large sog clones inhibit PCV formation. These data suggest that, as in the early embryo, Sog plays a dual role in promoting and inhibiting BMP signaling. In light of these results, the biochemical properties of Cv were examined, and it was found that, like Tsg, it can form a high-affinity oligomeric complex with Sog and BMP heterodimers (Shimmi, 2005b).
Taken together, these results suggest that similar mechanisms govern PCV development and early embryonic development. In the embryo, a Dpp/Scw heterodimer specifies amnioserosa fate following ligand transport from lateral to dorsal-most regions through the action of Sog and Tsg. Processing of the complex by Tld then enables signaling in a restricted spatial domain. It is speculated that formation of the PCV likely requires selective transport of a Dpp/Gbb heterodimer from the longitudinal veins to the PCV competent zone through the action of Sog and the Tsg-like protein Cv. As in the embryo, the Tolloid-related enzyme may release the ligand through processing of the Sog/Cv/BMP complex to generate a spatially restricted pattern of signaling in the PCV. This example illustrates how, in different developmental contexts, related molecules and common mechanistic processes can achieve new patterning outcomes (Shimmi, 2005b).
The formation of Drosophila wing veins is a very sensitive system for examining the activity of BMP signaling within the context of a developmental patterning process. Two distinct aspects of the BMP signaling process in veins have been recognized. (1) BMP signals are produced by the developing longitudinal veins where they act locally to help maintain the vein fate earlier specified by EGF signaling. (2) BMP signals produced in the longitudinal veins act at longer range to initiate BMP signaling in the crossveins (Ralston, 2005). This study shows evidence that this long-range signaling requires the activity of both Sog and a Tsg-like molecule encoded by the cv gene. Thus, within the context of the crossveins, Cv and Sog play positive roles in BMP signaling. Based on analogy to the embryonic patterning system, these results suggest that Cv and Sog aid in the transport of BMP ligands from producing cells to receiving cells in the posterior crossvein competent zone (Shimmi, 2005b).
Since Tsg and Cv showed a similar domain structure, attempts were made to determine if they were functionally equivalent by expressing one in place of the other during either embryonic or pupal development. These experiments showed that these two products are, to some extent, genetically interchangeable. However, Tsg and Cv may have been optimized for a particular developmental function that likely represents interactions with a particular ligand, i.e., Dpp/Scw heterodimers in the case of Tsg and Dpp/Gbb heterodimers in the case of Cv. A recent phylogenetic comparison of the Cv and Tsg proteins from different insect species suggests that these two proteins fall into distinct families, one Cv-like and one Tsg-like (Vilmos, 2005). In addition, under conditions of overexpression, cv and tsg exhibit enhanced genetic interactions with different BMP ligands. For instance, cv interacts better with gbb than with dpp (Vilmos, 2005). While no difference was seen in the ability of Sog and Tsg versus Sog and Cv to bind to Dpp/Gbb heterodimers, these data are qualitative. Thus, it is possible that these two protein complexes could have different affinities for different ligands that are optimal for their particular developmental function (Shimmi, 2005b).
A similar observation has recently been made for Tld and Tlr proteins. These two metalloproteases show very similar overall structure and both cleave Sog in the same positions, but with different kinetics and site preferences (Serpe, 2005). In this case, the two proteins cannot substitute for the other and it has been proposed that this represents optimization of catalytic activity for a fast (Tld in the embryo) or slow (Tlr in pupal wing vein) developmental function (Shimmi, 2005b).
In the early embryo, Tsg and Sog function together to help redistribute BMP signals from their broad initial distribution profiles throughout the dorsal half of the embryo into a narrow stripe of cells centered on the dorsal midline. In this model, Tsg and Sog play both positive and negative roles. The positive role comes via transport and the resulting increase in BMP concentration at the dorsal midline. The negative role comes from blocking access of the ligand to receptors in the lateral regions during BMP transport (Shimmi, 2005b).
The process of PCV formation appears remarkably similar, at least in terms of the BMP signaling components employed. Both Sog and the Tsg-like molecule Cv are required for BMP signaling, as indicated by the accumulation of pMad, in the developing crossveins. Thus, both Cv and Sog play positive roles in augmenting BMP signaling during crossvein formation. This positive role may also come from facilitating transport of BMPs, since co-expression of Cv and Sog in the posterior compartment resulted in ectopic vein formation and BMP signaling gains in the anterior compartment. Similar, although less penetrant, effects on anterior venation have been observed when Sog alone is misexpressed (Shimmi, 2005b).
In addition, Cv and Sog may also inhibit BMP signaling around the longitudinal veins. A sog mutant clonal analysis has demonstrated a requirement for Sog in keeping longitudinal veins straight and narrow. In the absence of Sog, the veins meandered. A similar effect on the longitudinal veins is seen when cv is lost, with an expansion in pMad accumulation around the longitudinal veins. Thus, Cv and Sog may function together to restrict the range of Dpp signaling along the longitudinal veins (Shimmi, 2005b).
Two other similarities between embryonic patterning and PCV formation are worth noting. In the embryo, the Tld metalloprotease is required to release ligand from the inhibitor complex of Sog and Tsg. Likewise, the Tolloid-related protease Tlr is required for PCV formation. Tlr is expressed in the pupal wings, when it is required for crossvein pMad, and has recently been shown to process Sog at the same three sites as does Tld (Serpe, 2005). Thus, it seems likely that Tlr is needed to release a BMP ligand for signaling in the PCV competent zone (Shimmi, 2005b).
There may also be strong similarities between the embryo and crossvein patterning in their use of ligands. In the embryo, both Dpp and Scw are needed to specify the amnioserosa, while for PCV specification, both Dpp and Gbb are required. Sog and Tsg show the highest affinity for the heterodimer of Dpp/Scw in the embryo, suggesting that this is the primary transported ligand. Similarly, the ligand with highest affinity for Cv and Sog is a heterodimer of Dpp and Gbb. Interestingly, gbb mutant clonal analysis has shown that the PCV is lost only when a gbb clone encompasses adjacent longitudinal vein material. Since the longitudinal veins serve as the source of Dpp during PCV specification (Ralston, 2005), these observations are consistent with the notion that a heterodimer of Dpp and Gbb is the primary ligand that specifies PCV formation (Shimmi, 2005b).
Although similarities between embryonic dorsoventral patterning and PCV formation are striking, there are clear differences. The most notable is the geometry of the system. Why is the long-range signaling from the longitudinal veins limited to the crossvein regions? Examination of the expression patterns of several components may provide some clues. Tkv expression is reduced in the crossveins, and since binding to receptor is a major impediment to diffusion in wing discs, this might enhance net flux of ligand into the area of reduced Tkv expression. However, down-regulation of tkv in the PCV actually depends on high levels of BMP signal (Ralston, 2005). Therefore, it is not likely that reduced Tkv expression provides a channel for ligand flow; rather, it may reinforce a flux direction that is initiated by other means (Shimmi, 2005b).
In this regard, it is notable that sog expression is also reduced in the crossvein regions and this is independent of BMP signaling (Ralston, 2005). As in the embryo, Sog flux from areas of high expression, i.e., intervein regions in the wing, into areas of low expression, the crossvein zones, might provide the proper positional information. Consistent with this view is the observation that uniform expression of Sog eliminates the initial stages of crossvein development. However, there are inconsistencies in this simple model. While misexpression of Sog can lead to loss of the crossvein, normally positioned crossveins appear when Sog misexpression is coupled with ubiquitous expression of Cv-2 (Ralston, 2005), suggesting that crossvein positioning can be independent of the sog expression pattern. Similarly, loss of sog from clones does not induce ectopic crossveins (this study) (Shimmi, 2005b).
Another possibility is that the cleavage of Sog is spatially regulated. In the embryo, Tld is expressed in the dorsal domain, and since its ability to process ligand is dependent on the Dpp concentration, the processing rate will be highest at the dorsal midline. However, in the pupal wing, tlr is not expressed at higher levels in the crossvein zones; instead, it is high in the entire intervein (Serpe, 2005). Therefore, it is not clear how the ligand would be released from a complex of Cv and Sog specifically in the crossveins. Moreover, uniform expression of tlr causes only mild expansion of the crossveins (Shimmi, 2005b).
Perhaps the key to understanding the differences between the embryonic patterning process and that of the crossveins will be determining the mechanism of action of other gene products that are required for crossvein formation. These include cv-c, cv-d, detached, and the cv-2 gene products. Among these genes, only the cv-2 product has been identified; it is a large secreted factor that contains CR domains, similar to those found in Sog, and is expressed in the developing crossveins. The major distinction between Sog and Cv-2 is that Cv-2 contains a Von Willebrand type D domain found on many blood-clotting proteins that is not present in Sog. In Chordin, the CR modules are responsible for BMP binding and vertebrate Cv-2 homologs have also been shown to bind BMPs. Depending on the assay used, vertebrate Cv-2 homologs can either inhibit or promote signaling. Cv-2 does not seem to be required in the early embryo, yet it is essential for crossvein formation. Drosophila Cv-2, like its vertebrate counterparts, can also bind BMPs and, although it is a secreted protein, Cv-2 can associate with the cell surface. One possibility is that it captures BMPs, perhaps from a Sog/Cv complex, and keeps them close to the cell surface and in this way promotes BMP signaling by keeping the local BMP concentration high. It may also play a more direct role as a coreceptor (Shimmi, 2005b).
Nonetheless, while cv-2 is expressed in the crossveins, and is required for BMP signaling there, ubiquitous expression of cv-2 does not disrupt the positioning of the crossveins, even when coupled with ubiquitous expression of sog. Thus, other genes must act in conjunction with or upstream of these BMP modulators to help establish the crossvein competent zone. It is interesting to note in this regard that mutations in CDC42 induce ectopic crossveins, suggesting that it might be involved in the process that selects the site of crossvein formation (Shimmi, 2005b).
Normally, tsg is expressed only in the early blastoderm embryo. However, sog is expressed at several other developmental stages. In fact, this was one of the motivations to look for additional Tsg homologs, so that it might be determined if Sog always utilizes a Tsg-like partner or whether in some developmental processes it might act alone. One late embryonic process in which Sog has been implicated is to regulate tracheal morphogenesis. As in vein formation, tracheal patterning requires input from the EGFR and Dpp pathways. However, in this case, each pathway is antagonistic to the other. Normally, sog is expressed as a dorsal stripe abutting the tracheal pits, and in sog mutant embryos, hyper-activation of Dpp leads to a loss of dorsal trunk and a reduction in visceral branches. It was therefore interesting that cv is also expressed in and around the tracheal pits, but tsg is not expressed at this stage. However, no alteration was observed in tracheal development in cv mutants. Indeed, these embryos appear fully viable and the resulting adults are fertile. These results suggest that Cv has no other essential role in development. The pattern of cv expression around the tracheal pits may reflect a prior evolutionary involvement in tracheal development that is now provided by Sog alone or perhaps by Sog in conjunction with some other unknown BMP modulatory factor (Shimmi, 2005b).
The Twisted gastrulation (Tsg) proteins are modulators of bone morphogenetic protein (BMP) activity in both vertebrates and insects. The crossveinless (cv) gene of Drosophila encodes a new tsg-like gene. Genetic experiments show that cv, similarly to tsg, interacts with short gastrulation (sog) to modulate BMP signalling. Despite this common property, Cv shows a different BMP ligand specificity as compared with Tsg, and its expression is limited to the developing wing. These findings and the presence of two types of Tsg-like protein in several insects suggest that Cv represents a subgroup of the Tsg-like BMP-modulating proteins (Vilmos, 2005).
A tsg-like gene (CG12410) was found on the first chromosome between CG3160 and CG3149. It encodes a protein with about 50% homology to the Tsg protein and the same molecular topology: two cysteine-rich (CR) domains connected by a variable hinge domain. A comparison of tsg-like genes in insects suggests two subgroups in the tsg-like family typified by cv and tsg. Of the five insects for which complete genomes are available, Drosophila melanogaster, Drosophila pseudoobscura and Drosophila simulans have both a tsg-like and a cv-like gene, whereas the mosquito and bee seem to have only a cv-like gene (Vilmos, 2005).
To determine the function of CG12410, the element EP1349 that shows no mutant phenotype was excised; it is located about 700 base pairs (bp) 5' of the exon containing the predicted start codon. Four strains were recovered that delete portions of CG12410 and all showed a recessive visible crossveinless phenotype with loss of the anterior crossveins (ACV) and the posterior crossveins. Two of the four mutants were strict recessive visibles (cv18, cv43), whereas the other two (cv34, cv51) showed semilethality (22% and 53%) not linked to the cv locus (Vilmos, 2005).
As flies heterozygous for cv18 and cv1 have the same phenotype as cv1 homozygotes, CG12410 is allelic to cv. Further evidence for allelism was obtained by rescuing both cv1 and cv18 hemizygotes with CG12410 using UAS>EP1349 under the control of the ptc>Gal4 driver (Vilmos, 2005).
Although the most obvious phenotype is the absence of crossveins and a delta at the tips of the L3 and L4 veins as originally described (Bridges, 1920; Waddington, 1940), it was also found that the longitudinal veins in cv mutants show poorly defined edges and trajectories often broadening and meandering along their length in a manner similar to that seen in sog- wing tissue, suggesting that cv has a role in refining the domains where veins and crossveins form (Vilmos, 2005).
The nature of the cv mutations was determined by PCR. As cv18, cv34, cv43 and cv51 alleles delete a region that extends from the P-element insertion site past the ATG start codon to the second intron of cv, these alleles are considered as physically verified nulls. The cv1 mutation is due to a 412 retrotransposon inserted in the second intron of cv that introduces two poly(A) addition signals that should terminate the cv transcript prematurely (Vilmos, 2005).
The cv52 and cv12 mutations show no phenotype; however, they delete all the DNA from the insertion to either the adjacent gene CG3160. Thus, regulatory sequences necessary for cv function do not extend past 475 bp upstream of the cv12 breakpoint (Vilmos, 2005).
Endogenous cv messenger RNA was detected only in the developing pupal wing, with no evidence of earlier expression. Expression of cv first appears as diffuse staining in the regions of the vein primordia 24-28 h after pupariation (APF) and later refines to stripes of 2-3 cells localized at the vein-intervein boundaries and disappears by 40 h APF. By comparison, dpp and the sog-like cv-2 are expressed in the vein domain at these times, whereas sog and gbb are expressed in the intervein regions with concentrations at the boundaries that are coincident with cv (Vilmos, 2005).
Expression of UAS>cv along the anterior-posterior (A/P) border rescues both the ACV and the PCV in cv mutants, whereas tsg does not rescue either crossvein. Thus, anterior expression of cv can restore function in posterior cells. Similarly, cv expressed in the embryo does not rescue tsg mutants. Thus, Tsg and Cv are not functionally interchangeable proteins (Vilmos, 2005).
It has been well documented that the loss of BMP signalling in wings produces two phenotypes, one being reduction of wing size and the other loss of veins. The first phenotype involves an early abrogation of long-range BMP signalling, whereas the second results from a late local loss of signalling in veins (Vilmos, 2005).
It has also been shown that Tsg can inhibit BMP-like ligands by synergizing with Sog, or in other environments can promote BMP activity by displacing an inhibitory fragment of Sog generated by proteolytic cleavage. To compare the activities of Cv and Tsg, transgene combinations were expressed under the control of the wing driver A9>Gal4. Excess cv alone can induce small fragments of extra veins and a delta phenotype, consistent with a mild pro-BMP activity. In contrast, coexpression of cv and sog produces a phenotype resembling early loss of BMP signalling in the organizer that runs along the A/P boundary (i.e. reduction in size and loss of intervein regions). Interestingly, coexpression of cv and sog along the A/P border affects structures throughout the wing, whereas expression of these proteins in the posterior compartment affects only posterior structures. The asymmetric activity of cv+sog suggests a restricted mobility due either to local inhibition of a long-range signal such as Dpp or Gbb or to the existence of an asymmetric inhibitor of diffusion of Cv/Sog-containing complexes (Vilmos, 2005).
It has been postulated that Cv might synergize with Cv-2, a second Sog-like protein; however, when Cv-2 is coexpressed with either Cv or Tsg, no evidence is seen of the strong inhibition of BMP signalling observed when Cv or Tsg is coexpressed with Sog. On further underscoring a difference between Sog and Cv-2, both Cv and Sog mutants show similar effects on wing veins (loss of crossveins and expanded vein tips), whereas Cv-2 mutants show loss of vein tips as well as crossveins. Thus, Cv-2 is not interchangeable with Sog (Vilmos, 2005).
To evaluate ligand specificity, the ability was tested of Cv and Sog to suppress the wing disruptions caused by overexpressing Dpp and Gbb. Expression of cv, tsg or sog alone has no detectable effect on the overexpression of dpp. However, when expressed together with sog, cv and tsg behave fairly differently when challenged with excess Dpp. Although tsg with sog can rescue the effect of excess dpp, cv with sog does not suppress the dpp overexpression phenotype at all. Cv and Tsg also differ in their effect on Gbb overexpression. Although Cv or Sog alone has no effect on Gbb overexpression, Cv together with Sog re-establishes the intervein tissue and the longitudinal veins with a series of expanded crossveins between L2 and L3. In contrast, Tsg alone suppresses fairly effectively the excess Gbb effect, whereas Tsg together with Sog leads to an intermediate level of rescue. These observations indicate that Cv and Tsg have distinct activities with respect to the Dpp and Gbb ligands and also different requirements for Sog to inhibit BMP (Vilmos, 2005).
Skeletal muscles are readily characterized by their location within the body and by the number and composition of their constituent muscle fibers. This study characterized a mutation that causes a severe reduction in the number of fibers comprising the tergal depressor of the trochanter muscle (TDT, or jump muscle), which functions in the escape response of the Drosophila adult. The wild-type TDT comprises over 20 large muscle fibers and four small fibers. In crossveinless (cv) mutants, the number of large fibers is reduced by 50%, and the number of small fibers is also occasionally reduced. This reduction in fiber number arises from a reduction in the number of founder cells contributing to the TDT at the early pupal stage. Given the role of cv in TGFβ signaling, whether this pathway directly impacts TDT development was determined. Indeed, gain- and loss-of-function manipulations in the TGFβ pathway resulted in dramatic increases and decreases, respectively, in TDT fiber number. By identifying the origins of the TDT muscle, from founder cells specified in the mesothoracic leg imaginal disc, it was also demonstrated that the TGFβ pathway directly impacts the specification of founder cells for the jump muscle. These studies define a new role for the TGFbeta pathway in the control of specific skeletal muscle characteristics (Jaramillo, 2009).
The specification and action of founder cells is crucial to normal muscle development in Drosophila, where founder cells impart unique phenotypes for each muscle. This study showns that the founder cell model also holds true for the adult tubular muscles such as the TDT, or jump, muscle. More importantly, the specification of TDT founder cells arises from activation of the TGFβ pathway, a pathway that has not been previously implicated in controlling founder cell number at the adult stage of development. These data demonstrate that it is most probably the activation of the TGFβ pathway within myoblasts that impacts founder cell specification, since in the overexpression experiments a Gal4 driver was used that is active in the adult myoblasts (Jaramillo, 2009).
The TGFβ pathway has roles in a range of developmental processes, and the addition of adult muscle development here extends a detailed list. In mammals, TGFβ can affect muscle development in several ways, including inhibition of differentiation and inhibition of muscle regeneration in vivo. Interestingly, the TGFβ molecule myostatin acts in mammalian muscle development to modulate the number of muscle fibers: in myostatin mutants in a variety of mammalian models, there is a profound increase in both muscle fiber number and muscle mass. Despite these similarities, there is not yet sufficient evidence to suggest that Cv-mediated modulation of the Dpp pathway plays a similar role in Drosophila to that played by myostatin in mammals. This is at least partly because the effects of cv mutants that were observed are restricted to a small subset of the adult musculature. A Drosophila gene named Myoglianin and encoding a molecule with strong sequence similarity to myostatin has been described, although no mutant alleles have been characterized (Jaramillo, 2009).
Genetic interaction data suggest roles for both Dpp and Gbb in TDT fiber specification. Each of these ligands function in wing vein development; thus, a combinatorial role for them in founder cell specification would not be unprecedented. It is also noted that the Drosophila genome encodes a number of additional TGFβ-related molecules, and such molecules, in addition to Dpp and Gbb, might contribute to TDT founder cell specification (Jaramillo, 2009).
Although manipulation of the TGFβ pathway showed clear effects upon the numbers of TDT founder cells, it is also noted that inhibition of the pathway, via UAS-Dad, caused a decrease in the number of total myoblasts as visualized by MEF2 staining. This observation suggests that, in addition to founder cell specification, the TGFβ pathway in adult myoblasts impacts either myoblast proliferation or myoblast survival. This observation is consistent with the finding that, in cv mutants, the number of founder cells reduces slightly as pupal development proceeds (Jaramillo, 2009).
Specification of TDT founder cell number appears to be subject to significant variability in Drosophila. This observation contrasts sharply with many of the other muscles of the animal, which show relatively invariant fiber numbers. These include the skeletal body wall muscles of the larva, and the indirect flight muscles of the adult. Perhaps the variability in TDT fiber number is a reflection of the multi-step TGFβ pathway responsible for its specification, where variation in one or a few of the signaling components required for founder cell specification will ultimately impact the number of founder cells specified. Similar to the studies presented in this paper, a genetic approach should be able to identify additional genes whose products function in this founder specification pathway and are responsible for the strain-specific differences in TDT fiber number that have been characterized. The identified genes might encode novel new members of the TGFβ signaling pathway active in myoblasts, or might identify mild alleles of known pathway members (Jaramillo, 2009).
Search PubMed for articles about Drosophila Crossveinless
Bridges, C. B. (1920). The mutant crossveinless in Drosophila melanogaster. Proc. Natl. Acad. Sci. 6: 660-663. PubMed ID: 16576553
Jaramillo, M. S., Lovato, C. V., Baca, E. M. and Cripps, R. M. (2009). Crossveinless and the TGFβ pathway regulate fiber number in the Drosophila adult jump muscle. Development 136(7): 1105-13. PubMed ID: 19244280
Ralston, A. and Blair, S. S. (2005). Long-range Dpp signaling is regulated to restrict BMP signaling to a crossvein competent zone. Dev. Biol. 280: 187-200. PubMed ID: 15766758
Shimmi, O., Umulis, D., Othmer, H. and O'Connor, M. B. (2005a). Facilitated transport of a Dpp/Scw heterodimer by Sog/Tsg leads to robust patterning of the Drosophila blastoderm embryo. Cell 120(6): 873-86. PubMed ID: 15797386
Shimmi, O., Ralston, A., Blair, S. S. and O'Connor, M. B. (2005b). The crossveinless gene encodes a new member of the Twisted gastrulation family of BMP-binding proteins which, with Short gastrulation, promotes BMP signaling in the crossveins of the Drosophila wing. Dev. Biol. 282(1): 70-83. PubMed ID: 15936330
Serpe, M., Ralston, A., Blair, S. S. and O'Connor, M. B. (2005). Matching catalytic activity to developmental function: tolloid-related processes Sog in order to help specify the posterior crossvein in the Drosophila wing. Development 132(11): 2645-56. PubMed ID: 15872004
Vilmos, P., et al. (2005). Crossveinless defines a new family of Twisted-gastrulation-like modulators of bone morphogenetic protein signalling. EMBO Rep. 6: 262-267. PubMed ID: 15711536
Waddington, C. H. (1940). The genetic control of wing development in Drosophila. J. Genet. 41: 75-139
date revised: 18 July 2009
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