BIOLOGICAL OVERVIEW

Formation of the longitudinal veins (LVs) of the Drosophila wing involves the interplay among Dpp, Egf and Notch pathways. Formation of crossveins (CVs: see Derivatives of the wing disc) present a paradoxical problem. As shown both morphologically and using molecular markers, the definitive CVs are not formed until long after the initial specification of the LVs. The CVs therefore must form within territory that has already been specified as intervein. The CVs must also interconnect with existing LVs at a time when the Delta expressed by the LVs is thought to inhibit vein formation in adjacent cells. Mechanisms must exist that override both intervein specification and the lateral inhibition of veins, allowing the formation of continuous, interconnected vein tissue. BMP-like signaling plays a special role in the formation of the CVs from within intervein territory. BMP-like signals also help maintain the connections between the LVs and the margin of the wing. crossveinless 2 (cv-2) is a critical factor in these processes, as it is expressed more highly in the CVs and the ends of the LVs and is required for the high levels of BMP-like signaling observed in these regions (Conley, 2000). The cv-2 mutation was first identified by Benedetto Nicoletti in 1962 (FlyBase). The structure of the Cv-2 protein strongly suggests that these effects are direct, and that Cv-2 is a novel player in the BMP-like signaling pathway (Conley, 2000).

Formation of the longitudinal veins (LVs) of the Drosophila wing involves the interplay among Dpp, Egf and Notch pathways. Formation of crossveins (CVs: see Derivatives of the wing disc) present a paradoxical problem. As shown both morphologically and using molecular markers, the definitive CVs are not formed until long after the initial specification of the LVs. The CVs therefore must form within territory that has already been specified as intervein. The CVs must also interconnect with existing LVs at a time when the Delta expressed by the LVs is thought to inhibit vein formation in adjacent cells. Mechanisms must exist that override both intervein specification and the lateral inhibition of veins, allowing the formation of continuous, interconnected vein tissue. BMP-like signaling plays a special role in the formation of the CVs from within intervein territory. BMP-like signals also help maintain the connections between the LVs and the margin of the wing. crossveinless 2 (cv-2) is a critical factor in these processes, as it is expressed more highly in the CVs and the ends of the LVs and is required for the high levels of BMP-like signaling observed in these regions (Conley, 2000). The cv-2 mutation was first identified by Benedetto Nicoletti in 1962 (FlyBase). The structure of the CV-2 protein strongly suggests that these effects are direct, and that Cv-2 is a novel player in the BMP-like signaling pathway (Conley, 2000).

During development, the dorsal and ventral epithelia of the Drosophila wing form a precisely patterned array of veins. The specification and maintenance of vein cells requires the cooperation and interaction between several different signaling pathways. The roles played by two such pathways have been relatively well characterized: the MAPK signaling mediated by the Drosophila Egf Receptor (Egfr) stimulates vein formation, while Notch signaling inhibits and refines vein formation. Both these pathways are active from the earliest stages of vein formation, at mid-late third instar, and are required to maintain and refine vein fates until at least 30 hours after pupariation. The mechanisms that localize Egfr signaling to the veins are not completely understood, but involve regulating ligand expression, ligand activation and the sensitivity of cells to active ligand. The Notch ligand Delta is expressed along the veins, apparently in response to high Egfr signaling; Delta induces Notch target gene expression and inhibits vein formation in neighboring cells. A third pathway, the BMP-like signaling mediated by the ligands Dpp and Gbb, also plays a role in vein specification. In wing discs, dpp is expressed just anterior to the anteroposterior compartment boundary, between L3 and L4, and plays a role setting up the axes of the wing. It has not yet been determined whether the reception of BMP-like signals initiates vein specification, although high BMP activity has been detected near the fourth longitudinal vein (LV) at late third instar. However, at later pupal stages, dpp expression is lost from the compartment boundary and rises along the veins; this expression is required for maintaining the fate of most veins and ectopic signaling can induce ectopic veins (Conley, 2000 and references therein).

Although Gbb expression is not higher in pupal veins, it also helps maintain vein formation, either by raising general levels of signaling or by interacting with localized modulators of signaling. Both Dpp and Gbb vein signals may be mediated largely by the type I receptor Thickveins (Tkv), rather than the alternate type I receptor Saxophone (Sax). Cells lacking Tkv do not form veins, but removal of Sax does not reliably remove veins. However, not all veins are equally sensitive to reductions in Dpp and Gbb signaling. The hypomorphic gbb⁴ mutation shows complete loss of the cross veins (CVs), but only slight loss of the ends of the LVs. Sog encodes a Chordin-like molecule that inhibits BMP-like signaling; both Sog and Chordin are thought to bind to and sequester ligands, preventing the activation of receptors. Overexpressing Sog in the wing specifically blocks formation of the CVs and the ends of the LVs. The secreted Tolloid proteases, similar to vertebrate BMP1s, can increase BMP signaling by cleaving and inactivating Chordin or Sog. Loss of tolkin (also known as tolloid-related) blocks formation of the CVs and the tips of the LVs. Overexpressing a dominant negative form of Sax again induces a similar phenotype (Conley, 2000 and references therein).

Such phenotypes are very reminiscent of the crossveinless class of mutations in Drosophila (reviewed in Garcia-Bellido, 1992). Strong reductions in crossveinless 2 (cv-2) function have been shown to remove the posterior CV (PCV), the anterior CV (ACV), and the ends of the LVs. However, despite the possibility that the crossveinless genes encode novel players in BMP-like signaling, none have been characterized and the sensitivity of CVs to BMP-like signaling has not been explained. Evidence is presented that cv-2 encodes a novel member of the BMP-like signaling pathway, expressed in and required for high levels of BMP-like signaling in the developing cross veins. The Cv-2 protein contains five cysteine-rich domains similar to those known to bind BMP-like ligands, strongly suggesting that Cv-2 directly modulates Dpp or Gbb activity (Conley, 2000 and references therein).

While MAPK signaling appears to play the primary role in the initial specification of most or all of the LV proveins in third instar discs, BMP-like signaling helps maintain the LVs during later stages. After the first day of pupal development, dpp is lost from the L3-L4 intervein and is expressed along the LVs and, later, along the CVs; this expression is required to reinforce vein formation. Mad is indeed activated in all of the veins during pupal stages. However, Mad activity is higher and appears earlier in the CVs and the ends of the LVs, the regions that are especially sensitive to reductions in BMP-like signaling. Mad activation may play the initial role during CV formation, preceding the activation of MAPK. Higher Mad activity is detected in the CVs before the loss of DSRF expression which is thought to be mediated by MAPK activity. Ectopic dpp signaling is capable of inducing vein formation in regions lacking high MAPK activity (Conley, 2000).

CV formation is sensitive to reductions in Gbb and Dpp expression, and the genetic interactions of both dpp and gbb with cv-2 mutations indicate that both Gbb and dpp play a role in the local activation of Mad in the CVs. However, localized expression of gbb or dpp cannot apparently account for the initial activation of Mad near the CVs. At the stage when Mad activity is first detected in the CVs, dpp is expressed along the LVs but is not detectable in the CVs, and anti-Gbb staining did not obviously emphasize the CVs. While it is possible that other BMP-like ligands locally activate Mad, the only other BMP-like ligand known in Drosophila, Screw, has not been detected in pupae. It is therefore likely that other localized factors potentiate Mad activity in the CVs. These results suggest that Cv-2 is such a factor (Conley, 2000).

cv-2 is expressed at higher levels in the developing CVs and the ends of the LVs, and is required for Mad activation in these regions. The structure of the Cv-2 protein is consistent with a direct role in Dpp or Gbb signaling. Cv-2 contains a putative signal or transmembrane region, and CR and VWFd domains typical of secreted proteins, suggesting that it acts either extracellularly or in the secretory pathway. Moreover, the strong similarity between the five closely apposed CR domains in Cv-2 and the CR domains in Chordin, Sog and Procollagen suggest that Cv-2 may directly bind Dpp or Gbb (Conley, 2000).

Chordin and Procollagen CRs bind BMPs and can mediate Chordin-like inhibition of BMP signaling. Xenopus Tsg contains a partial CR that can also bind BMPs (Oelgeschlager, 2000). Since Sog overexpression induces cv-2-like phenotypes (Yu, 1996), Sog and Cv-2 have opposing effects on vein formation. Sog is expressed in the interveins during CV formation and is likely to diffuse to non-expressing cells (Yu, 1996). To raise BMP-like activity, localized factors may have to overcome the Sog-mediated inhibition of signaling (Conley, 2000 and references therein).

Thus, one possible role for Cv-2 is to protect or release Gbb or Dpp from Sog. This is similar to the role proposed for Xenopus Tsg (Oelgeschlager, 2000). While clones lacking sog do not induce ectopic CVs (Yu, 1996), such clones might be rescued by the diffusion of Sog from outside the clone. Sog can act over long distances; overexpression of Sog in the anterior compartment of the wing can block PCV formation in the posterior (Conley, 2000 and references therein).

Another possible role for Cv-2 is in the activation or stability of the ligands themselves. BMPs are initially expressed as inactive precursors that must be cleaved and stabilized during secretion. Interestingly, Thrombospondin, which also contains a Chordin-like CR region, is required for the activation of a cleaved but latent form of TGFbeta1 in vivo (Crawford, 1998). Cv-2 also contains a partial VWFd domain. VWFd domains are found in a number of secreted proteins, including Von Willebrand Factors (VWFs) and Mucins, and both directly bridge protein multimers and regulate the formation of intermolecular and intramolecular disulfide bonds. Cv-2 lacks the portion of the VWFd domain that has been proven to directly bridge VWFs or Mucins. Cv-2 does, however, contain a CGLCG motif, which in VWFs and Mucins is required for the formation of protein multimers; this region is similar to a motif found in disulfide isomerases, and thus may regulate the formation of disulfide bonds (Conley, 2000 and references therein).

The presence of multiple CR domains and a VWFd domain makes Cv-2 different from Chordin, Sog, Tsg or CRIM1, but similar to the recently identified Xenopus protein Kielin which, like Cv-2, can regulate BMP-dependent patterning (Matsui, 2000). Kielin and Cv-2 are not identical, however. The much longer 2,327 amino acid Kielin has 28 CR domains instead of the five found in Cv-2, followed by a full rather than a partial VWFd domain, and has an amino-terminal region, similar to the amino-terminal domain of Thrombospondin, which is lacking in Cv-2. Thus, while it is possible that these proteins are homologs, one or both would have to have been severely modified from its original form. Cv-2 and Kielin also differ functionally. While Cv-2 is required for high levels of BMP-like signaling in the CVs, ectopic Kielin inhibits some (but not all) forms of BMP-like signaling during axis formation in Xenopus. This functional difference may be caused by the structural differences between these proteins. Alternatively, other factors may regulate the ability of these two proteins to either inhibit or potentiate signaling. This would not be surprising in a situation where multiple ligand-binding proteins compete for available ligand. The cleavage of Chordin or Sog by Tolloid apparently lowers their affinity for ligand. In the Drosophila embryo, this is thought to convert Sog from an inhibitor to an activator of signaling, since Sog is required for the highest levels of BMP-like activity in a process that requires Tolloid. Xenopus Tsg potentiates BMP signaling, possibly by protecting ligand that would otherwise be sequestered by the cleaved form of Chordin; however, Tsg may also sequester ligand by forming a complex with full-length Chordin (Conley, 2000 and references therein).

The activity of Cv-2 thus may be highly context dependent and, indeed, the evidence suggests that other locally expressed factors are required for Cv-2ís activity. While cv-2 is necessary for Mad activity surrounding the CVs, misexpression of cv-2 message at levels at or above normal levels has little effect on vein formation or other types of Mad-dependent development. Flies that have misexpressed cv-2 throughout embryogenesis, larval and pupal life are viable and appear normal. Only the very high levels of wing expression driven by ap-Gal4 induce ectopic vein phenotypes, and these are limited to the regions near where Mad activity is normally high. It is therefore unlikely that Cv-2 alone is responsible for localizing Mad activation. Other extracellular regulators of Mad activity are similar in this regard: for instance, ectopic Tsg expression has little effect on the Drosophila embryo, and overexpression in the wing causes a mild phenotype similar to that observed after misexpression of Cv-2. The remaining crossveinless loci provide obvious candidates for the missing factors (Conley, 2000 and references therein).

GENE STRUCTURE

cDNA clone length - 2498 bases

Exons - 6

PROTEIN STRUCTURE

Amino Acids - 751

Structural Domains

Intron prediction programs predict that the putative cv-2 sequence contains six exons; a strong TATA box is found 550 bp upstream and a predicted poly(A) site 1.2 kb downstream of the coding sequence. The amino half of the putative Cv-2 protein contains five adjacent cysteine-rich domains, similar to those found in a number of secreted and transmembrane proteins, including vertebrate Chordin, Sog and Procollagens, the mammalian and C. elegans CRIM1 proteins, and Xenopus Kielin. The CRs in Chordin and Procollagen IIA bind BMPs and can mediate Chordin-like biological activity during axis formation in Xenopus embryos. The spacing of cysteines is especially well conserved in the CxxCxC and CCxxC motifs. The second Cv-2 CR is the most divergent in terms of cysteines, lacking two cysteine residues. The Chordin and Sog CRs also contain a conserved tryptophan that is shared by the second and fifth Cv-2 CRs. The CRs are similar to Von Willebrand Factor Type C domains (VWFc), but most regions in these CRs are shorter than the equivalent regions in the canonical VWFc, and the first and second Cv-2 CRs only weakly match VWFc domains (Conley, 2000).

The portion of Cv-2 carboxy terminal to the CRs contains a region highly similar to the amino two thirds of the Von Willebrand Factor Type D domain (VWFd). VWFd domains are found in a number of secreted proteins, including Von Willebrand Factors and Mucins, and are involved in the regulation of disulfide bonds and the formation of protein multimers. The similarity ends at Cys646; the remaining portion of Cv-2 shares no significant similarity with any known protein, but does contain a potential Furin cleavage site (Conley, 2000).

The amino terminus of Cv-2 contains a hydrophobic region predicted to act as either a signal peptide, with a likely cleavage site between amino acids 53 and 54, or as an uncleaved transmembrane anchor. Cv-2 does not contain any obvious secretory retention signals, such as KDEL, suggesting that this protein acts extracellularly, being either secreted or membrane bound. This, and the presence of domains typical of secreted proteins, makes it likely that Cv-2 acts either extracellularly or in the secretory pathway. Despite sharing similar CR domains, Cv-2 is not an obvious ortholog of Chordin, Sog or CRIM1. The number and arrangement of CRs differs, and the portion of Cv-2 outside the CRs is not similar to Chordin, Sog or CRIM1, as these lack a VWFd domain. Cv-2 is most similar to the Xenopus Kielin protein (Matsui, 2000) in that Kielin contains a region of repeated CR domains, followed by a VWFd domain. Kielin is much longer than Cv-2, however, having 28 CR domains, a full rather than a partial VWFd domain, and an amino-terminal region, similar to the amino-terminal domain of Thrombospondin, that is lacking in Cv-2. Nonetheless, BLAST analyses indicate that Cv-2ís VWFd domain is more similar to the VWFd domain of Kielin than to other VWFd domains in the database, suggesting that the two might share a common ancestor (Conley, 2000).

The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The mouse and human homologs of the Drosophila Cv-2 protein have been identified. The mouse gene is located on chromosome 9A3 while the human locus maps on chromosome 7p14. CV-2 is expressed dynamically during mouse development, in particular in regions of high BMP signaling such as the posterior primitive streak, ventral tail bud and prevertebral cartilages. It is concluded that CV-2 is an evolutionarily conserved extracellular regulator of the Dpp/BMP signaling pathway (Coffinier, 2002).

Proteins that bind to bone morphogenetic proteins (BMPs) and inhibit their signalling have a crucial role in the spatial and temporal regulation of cell differentiation and cell migration by BMPs. A chick homologue of crossveinless 2, a Drosophila gene that was identified in genetic studies as a promoter of BMP-like signalling, has been identified. Chick Cv-2 has a conserved structure of five cysteine-rich repeats similar to those found in several BMP antagonists, and a C-terminal Von Willebrand type D domain. Cv-2 is expressed in the chick embryo in a number of tissues at sites at which elevated BMP signalling is required. One such site of expression is premigratory neural crest, in which at trunk levels threshold levels of BMP activity are required to initiate cell migration. When overexpressed, Cv-2 can weakly antagonise BMP4 activity in Xenopus embryos, but in other in vitro assays Cv-2 can increase the activity of co-expressed BMP4. Furthermore, increased expression of Cv-2 causes premature onset of trunk neural crest cell migration in the chick embryo, indicative of Cv-2 acting to promote BMP activity at an endogenous site of expression. It is therefore proposed that BMP signalling is modulated both by antagonists and by Cv-2 that acts to elevate BMP activity (Coles, 2004).

Drosophila Crossveinless-2 (dCV-2) is required for local activation of Mad phosphorylation in the fruit fly wing and has been postulated to be a positive regulator of BMP-mediated signaling. In contrast, the presence of 5 Chordin-like cysteine-rich domains in the CV-2 protein suggests that CV-2 belongs to a family of well-established inhibitors of BMP function that includes Chordin and Sog. A human homolog of Drosophila CV-2 (hCV-2) has been identified. Purified recombinant hCV-2 protein inhibits BMP-2 and BMP-4 dependent osteogenic differentiation of W-20-17 cells, as well as BMP dependent chondrogenic differentiation of ATDC5 cells. Interestingly, hCV-2 messenger RNA is expressed at high levels in human primary chondrocytes, whereas expression in primary human osteoblasts is low. These results suggest that hCV-2 may regulate BMP responsiveness of osteoblasts and chondrocytes in vivo. Taken together this study shows that contrary to the function predicted from the fruit fly, Crossveinless-2 is a novel inhibitor of BMP function (Binnerts, 2004).

One extracellular regulatory molecule is the Chordin/Short gastrulation protein (Chordin/Sog), a secreted protein that acts as an antagonist to BMP/Dpp. Chordin/Sog contains four cysteine-rich (CR) domains that bind to and inactivate BMP/Dpp. In contrast, a positive regulator has been identified in Drosophila. Named crossveinless 2 (cv-2), this molecule contains five CR domains at the N-terminal half and a von Willebrand factor D domain at the C-terminal part. Genetic data suggest that Cv-2 potentiates Dpp signaling. Chick and mouse CV-2 genes have been isolated and found to be secreted and enhance BMP signaling. Expression patterns were closely related to those of BMPs, supporting the likelihood of a tight link. These data show that CV-2 is a conserved, positive regulator of BMP signaling and that CR domain proteins act as both positive and negative modulators of BMP signaling (Kamimura, 2004).

date revised: 20 January 2005

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