Transforming growth factor beta at 60A

Targets of Activity

Animals lacking Tgfbeta-60A function died at late larval/early pupal stages. One of the striking phenotypes of Tgfbeta-60A mutant larvae is a transparent appearance due to the lack of fat body. In roughly 50% of the Tgfbeta-60A larvae, the gastric caecae are reduced in length, consistent with the expression of Tgfbeta-60A in the gastric caecae (Doctor, 1992). These phenotypes are similar to those of the Mad mutant larvae. During embryogenesis, Tgfbeta-60A is expressed throughout the visceral mesoderm of the developing midgut (Doctor, 1992), suggesting a function for Tgfbeta-60A in gut development. Indeed, embryos lacking Tgfbeta-60A fail to form the first constriction. The homeotic gene Antennapedia (Antp) is expressed in the visceral mesoderm around the first constriction and is required for its formation. Antp expression was examined in Tgfbeta-60A mutant embryos. Consistent with the lack of the first constriction, Antp expression is greatly reduced. Thus, Tgfbeta-60A is required for the formation of the first constriction of the midgut, likely through positively regulating the expression of Antp in the visceral mesoderm. This function of Tgfbeta-60A is independent of dpp signaling, since mutations in dpp or its receptors are known to disrupt the formation of the second but not the first constriction (Chen, 1998).

The identification of mutations in Tgfbeta-60A as dominant enhancers of tkv 6 in the imaginal discs raises the possibility that Tgfbeta-60A is required for optimal signaling by the dpp pathway. To determine if there is a general requirement for Tgfbeta-60A in dpp signaling, the effects of Tgfbeta-60A mutations were examined on dpp signaling in the visceral mesoderm where both dpp and Tgfbeta-60A are expressed. dpp is expressed in two discrete domains in the visceral mesoderm. The anterior domain of dpp coincides with the gastric caecae primordia, which are immediately anterior to the expression domain of Sex combs reduced (Scr) in parasegment (ps) 4. The failure to initiate dpp expression in ps3 in dpp shv mutants results in anterior expansion of Scr expression and arrested outgrowth of the gastric caecae, indicating a role for dpp in repressing Scr in ps3. tkv 6 homozygotes are homozygous viable, so it is not surprising that all the midgut gene expression patterns examined are essentially normal. Scr expression in tkv 6 and Tgfbeta-60A mutants is normal. However, in tkv 6 and Tgfbeta-60A double mutants, the Scr expression extends anteriorly into ps3 as it does in dpp shv mutants, suggesting that Tgfbeta-60A activity is required in ps3 for optimal dpp signaling (Chen, 1998).

To test whether 60A also acts synergistically with dpp elsewhere in the midgut, the gene expression of dpp and Ultrabithorax (Ubx) in ps7 and wingless (wg) in the adjacent ps8 were examined. Parasegment 7 expression of dpp is activated by the homeotic gene Ubx and maintained by an autostimulatory circuit involving dpp, Ubx and wg. The proper expression of all three genes is interdependent and critical for maintaining a stable cellular differentiation commitment. The expression of dpp and wg in the visceral mesoderm is required for the induction of the homeotic gene labial (lab) in the underlying endoderm. The absence of dpp function in ps7 disrupts the autoregulatory loop and reduces the expression of Ubx, wg, dpp and lab in ps7, leading to the absence of the second constriction in dpp mutant embryos (Chen, 1998 and references).

Flies mutant for either tkv 6 or Tgfbeta-60A have normal expression of dpp, wg and Ubx. However, in tkv 6 Tgfbeta-60A double mutants, dpp expression in ps3 and ps7 is greatly reduced. The initiation of dpp expression at earlier stages is not affected in the double mutants, suggesting that the reduction of dpp expression results from failure to maintain its expression at later stages. Similarly, in Mad mutants, the initiation of dpp expression in ps3 and ps7 is not affected but the maintenance of dpp expression does not occur. This is because dpp expression is activated directly by Ubx and only its maintenance requires positive feedback involving dpp signaling. In the double mutants, Ubx expression in ps7 and wg expression in ps8 are greatly reduced, suggesting the disruption of the positive regulatory loop. The reduction of dpp in ps3 in the double mutants may explain the observed derepression of Scr (Chen, 1998).

Ubx is required for repressing Antp in ps6. In Ubx mutants, the Antp domain is extended posteriorly into ps7, indicating a homeotic transformation of ps7 into ps6. A similar phenotype is observed for tkv null embryos. Consistent with the argument that a lack of Tgfbeta-60A compromises dpp signaling, there is also a posterior expansion of Antp in tkv 6 Tgfbeta-60A double mutants. Interestingly, due to the Tgfbeta-60A mutation, the endogenous Antp expression is absent: there is only ectopic Antp in ps7, where Ubx would normally be expressed (Chen, 1998 and references).

The expression of labial in the endoderm was also examined. Consistent with the gene expression changes in the visceral mesoderm, lab expression is not affected by tkv 6 or Tgfbeta-60A mutations. However, it is greatly reduced in tkv 6 Tgfbeta-60A double mutants. The gut of the double mutants only forms two chambers instead of the normal four chambers. This phenotype likely results from the failure to form the first constriction due to lacking Tgfbeta-60A function and the failure to form the second constriction due to lacking dpp signaling. It is unclear why the position of the only constriction observed in the double mutants is somewhat more anterior than a normal third constriction. The gene expression changes in the midgut of the tkv 6 Tgfbeta-60A double mutants are consistent with Tgfbeta-60A playing a role in augmenting Tgfbeta-60A signaling (Chen, 1998).

Bmp signals directly repress bag of marbles in germline stem cells

The Drosophila ovary is an attractive system to study how niches control stem cell self-renewal and differentiation. The niche for germline stem cells (GSCs) provides a Dpp/Bmp signal, which is essential for GSC maintenance. bam is both necessary and sufficient for the differentiation of immediate GSC daughters (cystoblasts). Bmp signals directly repress bam transcription in GSCs in the Drosophila ovary. Similar to dpp, gbb encodes another Bmp niche signal that is essential for maintaining GSCs. The expression of phosphorylated Mad (pMad), a Bmp signaling indicator, is restricted to GSCs and some cystoblasts, which have repressed bam expression. Both Dpp and Gbb signals contribute to pMad production. bam transcription is upregulated in GSCs mutant for dpp and gbb. In marked GSCs mutant for two essential Bmp signal transducers (Med and punt) bam transcription is also elevated. Finally, Med and Mad are shown to directly bind to the bam silencer in vitro. This study demonstrates that Bmp signals maintain the undifferentiated or self-renewal state of GSCs, and directly repress bam expression in GSCs by functioning as short-range signals. Thus, niche signals directly repress differentiation-promoting genes in stem cells in order to maintain stem cell self-renewal (Song, 2004).

This study reveals a new function for gbb in the regulation of GSCs in the Drosophila ovary. Loss of gbb function leads to GSC differentiation and stem cell loss, similar to dpp mutants. gbb is expressed in somatic cells but not in germ cells, suggesting that gbb is another niche signal that controls GSC maintenance. Like dpp, gbb contributes to the production of pMad in GSCs and also functions to repress bam expression in GSCs. As in the wing imaginal disc, gbb also probably functions to augment the dpp signal in the regulation of GSCs through common receptors in the Drosophila ovary. In both dpp and gbb mutants, pMad accumulation in GSCs is severely reduced but not completely diminished. Since the dpp or gbb mutants used in this study do not carry complete loss-of-function mutations, it remains possible that complete elimination of either dpp or gbb function is sufficient for eradicating pMad accumulation in GSCs. Alternatively, both dpp and gbb signaling are required independently for full pMad accumulation in GSCs, and thus disrupting either one of them only partially diminishes pMad accumulation in GSCs. The lethality of null dpp and gbb mutants, and the difficulty in completely removing their function in the adult ovary, prevent these possibilities from being tested directly (Song, 2004).

Interestingly, dpp overexpression results in complete suppression of cystoblast differentiation and complete repression of bam transcription in the germ cells, whereas gbb overexpression does not have obvious effects on cystoblast differentiation or bam transcription. Even though the UAS-gbb transgene and the c587 driver for gbb overexpression have been demonstrated to function properly, it is possible that active Gbb proteins are not produced in inner sheath cells and somatic follicle cells because of a lack of proper factors that are required for Gbb translation and processing in those cells, which could explain why the assumed gbb overexpression does not have any effect on cystoblast differentiation. However, since active Dpp proteins can be successfully achieved using the same expression method, and Dpp and Gbb are closely related Bmps, it is unlikely that active Gbb proteins are not produced in inner sheath cells and follicle cells. Alternatively, dpp and gbb signals could have distinct signaling properties, and dpp may play a greater role in regulating GSCs and cystoblasts. Recent studies have indicated that Dpp and Gbb have context-dependent relationships in wing development. In the wing disc, duplications of dpp are able to rescue many but not all of the phenotypes associated with gbb mutants, suggesting that dpp and gbb have not only partly redundant functions but also distinct signaling properties. In the wing and ovary, gbb and dpp function through two Bmp type I receptors, sax and tkv. The puzzling difference between gbb and dpp could be explained by context-dependent modifications of Bmp proteins, which render different signaling properties in different cell types. It will be of great future interest to better understand what causes Bmps to have distinct signaling properties (Song, 2004).

All the defined niches share a commonality, structural asymmetry, which ensures stem cells and their differentiated daughters receive different levels of niche signals. In order for a niche signal to function differently in a stem cell and its immediately differentiating daughter cell that is just one cell away, it has to be short-ranged and localized. This study shows that Bmp signaling mediated by Dpp and Gbb results in preferential expression of pMad and Dad in GSCs. Bmp signaling appears to elicit different levels of responses in GSCs and cystoblasts, suggesting that the cap cells are likely to be a source for active short-ranged Bmp signals. These observations support the idea that Bmp signals are active only around cap cells. Consistently, when GSCs lose contact with the cap cells following the removal of adherens junctions they move away from the niche and then are lost. As gbb and dpp mRNAs are broadly expressed in the other somatic cells of the germarium besides cap cells, localized active Bmp proteins around cap cells could be generated by localized translation and/or activation of Bmp proteins. As they can function as long-range signals, it remains unclear how Dpp and Gbb act as short-range signals in the GSC niche (Song, 2004).

Bmp signaling and bam expression are in direct opposition in Drosophila ovarian GSCs. bam is actively repressed in GSCs through a defined transcriptional silencer. These observations lead to a model in which Bmp signals from the niche maintain adjacent germ cells as GSCs by actively suppressing bam transcription and thus preventing differentiation into cystoblasts. The levels of pMad are correlated with the amount of bam transcriptional repression in GSCs and cystoblasts. In the wild-type germarium, pMad is highly expressed in GSCs and some cystoblasts where bam is repressed. In other cystoblasts and differentiated germline cysts, pMad is reduced to very low levels, and thus bam transcriptional repression is relieved. In the GSCs mutant for dpp, gbb or punt, pMad levels are severely reduced, and bam begins to be expressed. The repression of bam transcription as a result of dpp overexpression seems to be a rapid process; bam mRNA is reduced to below detectable levels two hours after dpp is overexpressed. This suggests that repression of bam transcription by Bmp signaling could be direct. Furthermore, Med and Mad can bind to the defined bam silencer in vitro, which also supports the idea that Bmp signaling acts directly to repress bam transcription. Dpp signaling has also been shown to repress brinker (brk) expression in the wing imaginal disc and in the embryo. The repression of brk expression by Dpp signaling is mediated by the direct binding of Mad and Med to a silencer element in the brk promoter. Since the brk silencer is very similar to the bam silencer, the results suggest that bam repression in GSCs is also mediated directly by Dpp and Gbb in a similar manner (Song, 2004).

It remains unclear how the binding of Med and Mad to the bam silencer results in bam transcriptional repression in GSCs. For the brk silencer, Dpp signaling and Shn are both required to repress brk expression in the Drosophila wing disc and embryo. Mad and Med belong to the Smad protein family, which are known to function as transcriptional activators by recruiting co-activators with histone acetyltransferase activity. In the wing disc, Shn is proposed to function as a switch factor that converts the activating property of Mad and Med proteins into a transcriptional repressor property. Possibly, the Mad-Med complex could also recruit Shn to the bam repressor element. Consistent with the possible role of Shn in repressing bam expression in GSCs is the observation that GSCs that lose shn function differentiate, and thus are lost. Also, it remains possible that Mad and Med could recruit a repressor other than Shn when binding to the bam repressor element. In the future, it will be very important to determine whether Shn itself is a co-repressor for Mad/Med proteins or whether it directly recruits a co-repressor to repress bam transcription in GSCs (Song, 2004).

Gbb/Bmp signaling is essential for maintaining germline stem cells and for repressing bam transcription in the Drosophila testis.

Stem cells are responsible for replacing damaged or dying cells in various adult tissues throughout a lifetime. They possess great potential for future regenerative medicine and gene therapy. However, the mechanisms governing stem cell regulation are poorly understood. Germline stem cells (GSCs) in the Drosophila testis have been shown to reside in niches, and thus these represent an excellent system for studying relationships between niches and stem cells. Bmp signals from somatic cells are essential for maintaining GSCs in the Drosophila testis. Somatic cyst cells and hub cells express two Bmp molecules, Gbb and Dpp. Genetic analysis indicates that gbb functions cooperatively with dpp to maintain male GSCs, although gbb alone is essential for GSC maintenance. Furthermore, mutant clonal analysis shows that Bmp signals directly act on GSCs and control their maintenance. In GSCs defective in Bmp signaling, expression of bam is upregulated, whereas forced bam expression in GSCs causes the GSCs to be lost. This study demonstrates that Bmp signals from the somatic cells maintain GSCs, at least in part, by repressing bam expression in the Drosophila testis. dpp signaling is known to be essential for maintaining GSCs in the Drosophila ovary. This study further suggests that both Drosophila male and female GSCs use Bmp signals to maintain GSCs (Kawase, 2004).

To determine the sources for Gbb and Dpp in the testis, RT-PCR was used to study the presence of gbb and dpp mRNAs in the purified hub cells, somatic cyst cells and germ cells using fluorescent-activated cell sorting (FACS). The hub cells were marked by the upd-gal4 driven UAS-GFP expression. The somatic cyst cells and somatic stem cells were marked by the c587-gal4-driven UAS-GFP. vasa is a germline-specific gene. The germ cells were marked by a vasa-GFP transgene. The tips of the testes were isolated and dissociated, and the GFP-positive cells were purified from the dissociated testicular cells by FACS. As a control, vasa mRNAs were present in the whole testis and isolated germ cells but were absent in the somatic cyst cells and hub cells. Interestingly, gbb and dpp mRNAs were present in the hub cells and the somatic cysts/somatic stem cells but were absent in the germ cells. In addition, dpp mRNAs appeared to be less abundant than gbb mRNAs in the testis. These results indicate that both Dpp and Gbb are probably somatic cell-derived Bmp signals that directly regulate GSC maintenance in the testis (Kawase, 2004).

Protein Interactions

Expression of a Tgfbeta-60A cDNA in Drosophila S2 cells was used to determine that Tgfbeta-60A encodes a preproprotein precursor that is proteolytically processed to yield secreted amino- and carboxy-terminal polypeptides. The carboxy-terminal peptides are recovered as disulfide-linked homodimers (Doctor, 1992).

Structurally unrelated neural inducers in vertebrate and invertebrate embryos have been proposed to function by binding to BMP4 or Dpp, respectively, and preventing these homologous signals from activating their receptor(s). The functions of various forms of the Drosophila Sog protein were examined using the discriminating assay of Drosophila wing development. Misexpression of Drosophila Sog, or its vertebrate counterpart Chordin, generates a very limited vein-loss phenotype. This sog misexpression phenotype is very similar to that of viable mutants of glass-bottom boat (gbb), which encodes a BMP family member. Consistent with Sog selectively interfering with Gbb signaling, Sog can block the effect of misexpressing Gbb, but not Dpp in the wing. In contrast to the limited BMP inhibitory activity of Sog, carboxy-truncated forms of Sog, referred to as Supersog, have been identified which when misexpressed cause a broad range of dpp minus mutant phenotypes. Evidence is provided that Twisted gastrulation (Tsg) functions in the embryo to generate a Supersog-like activity, perhaps by modifying the enzymic activity of Tolloid, the enzyme that processes Sog (Yu, 2000).

The predicted Sog protein is 1038 amino acids in length and contains four cysteine-rich (CR) domains in the extracellular domain. The metalloprotease Tld cleaves Sog at three major sites. Supersog1 is an N-terminal fragment of Sog including CR1 plus another 114 amino acids, and contains an additional 33 amino acids derived from vector sequences at its C terminus. Supersog2, which contains the same amino acids as Supersog1 but terminates abruptly at the end of Sog sequences, also generates Supersog phenotypes, albeit slightly weaker than those observed with Supersog1. Supersog4 is an N-terminal fragment of Sog ending 80 amino acids before CR2 and includes 130 sog 3' UTR derived amino acids (Yu, 2000).

In line with its phenotypic effects, Supersog can block the effects of both misexpressing Dpp and Gbb in the wing. Vertebrate Noggin, in contrast, acts as a general inhibitor of Dpp signaling, which can interfere with the effect of overexpressing Dpp, but not Gbb. Evidence suggests that Sog processing occurs in vivo and is biologically relevant. Overexpression of intact Sog in embryos and adult wing primordia leads to the developmentally regulated processing of Sog. This in vivo processing of Sog can be duplicated in vitro by treating Sog with a combination of the metalloprotease Tolloid (Tld) plus Twisted Gastrulation (Tsg), another extracellular factor involved in Dpp signaling. In accord with this result, coexpression of intact Sog and Tsg in developing wings generates a phenotype very similar to that of Supersog. Evidence is provided that tsg functions in the embryo to generate a Supersog-like activity, since Supersog can partially rescue tsg minus mutants. Consistent with this finding, sog minus and tsg minus mutants exhibit similar dorsal patterning defects during early gastrulation. These results indicate that differential processing of Sog generates a novel BMP inhibitory activity during development and, more generally, that BMP antagonists play distinct roles in regulating the quality as well as the magnitude of BMP signaling (Yu, 2000).

Cysteine repeat domains and adjacent sequences determine distinct Bone morphogenetic protein modulatory activities of the Drosophila Sog protein

The Drosophila short gastrulation gene encodes a large extracellular protein (Sog) that inhibits signaling by BMP-related ligands. Sog and its vertebrate counterpart Chordin contain four copies of a cysteine repeat (CR) motif defined by 10 cysteine residues spaced in a fixed pattern and a tryptophan residue situated between the first two cysteines. This study presents a structure-function analysis of the CR repeats in Sog, using a series of deletion and point mutation constructs, as well as constructs in which CR domains have been swapped. This analysis indicates that the CR domains are individually dispensable for Sog function but that they are not interchangeable. These studies reveal three different types of Sog activity: intact Sog, which inhibits signaling mediated by the ligand Glass bottom boat (Gbb), a more broadly active class of BMP antagonist referred to as Supersog, and a newly identified activity, which may promote rather than inhibit BMP signaling. Analysis of the activities of CR swap constructs indicates that the CR domains are required for full activity of the various forms of Sog but that the type of Sog activity is determined primarily by surrounding protein sequences. Cumulatively, this analysis suggests that CR domains interact physically with adjacent protein sequences to create forms of Sog with distinct BMP modulatory activities (Yu, 2004).

The Sog CR domains are defined by a set of 10 cysteine residues with a conserved spacing and a single tryptophan residue located between the first two cysteines. The function of the tryptophan residues was examined by mutating them individually or all to alanine. The finding that all four single W --> A mutants have wild-type Sog function as assayed by misexpression in the wing, either alone or when coexpressed with Tsg, indicates that none of these residues is individually essential for either Sog or Tsg + Sog (Supersog) activities. This finding is also consistent with the results of deleting the individual CR domains. When all four tryptophans were mutated to alanine, however, the Sog-like activity remained relatively unaffected but this mutant was greatly compromised in its ability to interact with Tsg to generate a Supersog-like activity. These results suggest that the tryptophan residues in two or more CRs can mediate functional interaction with Tsg and that Sog residues outside of the four conserved tryptophans are not sufficient on their own to mediate this interaction. However, deletion of the stem region also eliminates the functional Tsg interaction since a mutant lacking the stem and CR1 fails to interact with Tsg while a mutant lacking just CR1 interacts fully. The requirement for the stem region in interacting with Tsg is consistent with both CR and stem sequences being essential for Supersog activity (Yu, 2004).

Truncated forms of Sog consisting of CR1, the stem, and CR2 behave differently from either Sog or Supersog constructs when misexpressed in the wing or embryo. The strongest form of this novel Sog activity is observed when both CR3 and CR4 are deleted (e.g., SogCR1,2). Several lines of evidence suggest that Sog CR1,2 functions by promoting BMP signaling. (1) The effect of misexpressing SogCR1,2 on gene expression in wing discs is most similar to that of misexpressing an activated Sax receptor or the putative Sax ligand Gbb. This profile of gene response is quite distinct from that resulting from misexpression of activated or dominant-negative forms of the BMP receptor or EGF-receptor pathway, which is the other major signaling system regulating early vein development. (2) Misexpression of SogCR1,2 in pupal wings by heat shock results in significant ectopic expression of the rho gene, which is a good measure of BMP vs. EGF-R pathway activation during this stage. (3) Expression of SogCR1,2 in the early embryo broadens the dorsal expression domain of the BMP target gene zen (Yu, 2004).

One attractive model for a positive function of Sog such as that potentially mediated by SogCR1,2 is that in addition to binding to BMPs and preventing them from gaining access to the receptors, Sog might also act as a carrier of BMPs to either protect them from degradation or possibly transport them dorsally. Since the CR3 and CR4 domains appear to be those most critical to inhibition of Gbb/Scw activity, it is possible that the apparent positive activity of SogCR1,2 is the result of removing the Scw/Gbb inhibitory activity, while leaving a carrier function intact. The difference between the activity of SogCR1,2 and Supersog molecules, which lack the CR2 domain, might be explained if CR1 and CR2 can interact in SogCR1,2 to prevent CR1 and/or adjacent sequences from interacting with Dpp, thus neutralizing this remaining potential BMP inhibitory activity. It is currently unclear what relationship, if any, the SogCR1,2 activity has to the positive function of Sog required to sustain dorsal expression of race in the early embryo. SogCR1,2, unlike intact Sog, is unable to rescue race at a distance, but can broaden dorsal zen expression, which intact Sog does not do. The apparent differences between these activities may be the result of threshold-dependent effects in the early embryo or may reflect a fundamentally different mode of action (Yu, 2004).

An important question is whether truncated forms of Sog similar to SogCR1,2 or SogDeltaCR4 are generated and function in vivo. It is known that Tld can cleave Sog in vitro to generate products of approximately the same size as these constructs, and Sog-reactive bands of approximately the same size are observed in early embryos and pupal wings. In addition, forms of Chordin similar in structure to SogCR1,2 and Supersog are produced in humans as the result of alternative RNA splicing . Further analysis of the production and activity of SogCR1,2-like molecules will be required to determine the relevance of such forms in vivo and to determine the mechanism by which they may act on the BMP pathway (Yu, 2004).

Analysis of Sog mutants in which individual CRs are deleted or single conserved tryptophan residues are mutated to alanine suggests that the CR domains perform partially overlapping functions since none of them is absolutely essential for intact Sog activity (e.g., inhibition of Gbb/Scw) or for interaction with Tsg to create a Dpp inhibitory activity. Nonetheless, these experiments also suggest that the CR domains are not equivalent. For example, deletion of CR3 or CR4 had much greater effects in reducing the SOG-like activity than did deletion of CR1 or CR2. Moreover, the fact that Supersog, which contains only CR1, can inhibit Dpp while SogCR4 apparently interferes selectively with Gbb, suggested that CR1 might bind Dpp while CR4 bound Gbb. The results of the CR swap experiments performed in the contexts of SogCR4, Supersog1, and SogCR1,2 are quite informative in resolving this question and provide a surprising answer, namely that sequences adjacent to CR domains are the primary determinants of BMP specificity (Yu, 2004).

While replacing a CR domain in swap constructs typically resulted in greatly reduced activity or inactivity of UAS-transgenes tested as single- or multicopy insertions, those that had activity generated phenotypes similar to those of the parent constructs. These data suggest that the CRs are required in a context-specific fashion to boost the activity levels of the various forms of Sog. These findings, although limited in scope, suggest that the primary determinant of the quality of Sog activity lies within the sequences surrounding the CRs rather than within the CR domains themselves. These non-CR sequences may correspond to identified repetitive motifs such as the SR repeats or the circularly permuted SOG/CHRD domains. The simplest interpretation of this result is that CRs contribute to defining the strength and surrounding sequences determine the specificity of Sog activities. This model of Sog function is consistent with the finding that deletion of any single CR does not eliminate Sog-like activity (e.g., inhibition of Scw/Gbb) or the ability to interact with Tsg (e.g., inhibition of Dpp) and that both the CR1 domain and adjacent stem sequences are required for Supersog activity. The fact that the conserved tryptophans in the CR domains are required in aggregate, but not individually, for interaction with Tsg and that stem sequences are also required for this interaction lends additional support to the view that the CRs function in a partially redundant fashion in conjunction with non-CR sequences. One potential explanation for the results reported here is that the CR domains alone bind to BMPs, but do so with little selectivity. The surrounding sequences may provide specificity by interacting with only a subset of BMPs, thereby increasing the affinity of adjacent CRs for particular BMPs. Alternatively, surrounding sequences may alter the conformation of CR domains or sterically limit their interactions with BMPs, rendering them more selective. Further biochemical analysis will be required to determine the degree to which CRs affect affinity of the various Sog forms for particular BMPs and the mechanism by which surrounding sequences may confer specificity for binding particular BMPs (Yu, 2004).

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

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. 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). 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. 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. 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, 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. 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).

Crossveinless defines a new family of Twisted-gastrulation-like modulators of bone morphogenetic protein signalling

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).

Dpp and Gbb exhibit different effective ranges in the establishment of the BMP activity gradient critical for Drosophila wing patterning

Morphogen gradients ensure the specification of different cell fates by dividing initially unpatterned cellular fields into distinct domains of gene expression. It is becoming clear that such gradients are not always simple concentration gradients of a single morphogen; however, the underlying mechanism of generating an activity gradient is poorly understood. This study indicates that the relative contributions of two BMP ligands, Gbb and Dpp, to patterning the wing imaginal disc along its A/P axis, change as a function of distance from the ligand source. Gbb acts over a long distance to establish BMP target gene boundaries and a variety of cell fates throughout the wing disc, while Dpp functions at a shorter range. On its own, Dpp is not sufficient to mediate the low-threshold responses at the end points of the activity gradient, a function that Gbb fulfills. Given that both ligands signal through the Tkv type I receptor to activate the same downstream effector, Mad, the difference in their effective ranges must reflect an inherent difference in the ligands themselves, influencing how they interact with other molecules. The existence of related ligands with different functional ranges may represent a conserved mechanism used in different species to generate robust long range activity gradients (Bangi, 2006a).

Dual function of the Drosophila Alk1/Alk2 ortholog Saxophone shapes the Bmp activity gradient in the wing imaginal disc

Wing patterning in Drosophila requires a Bmp activity gradient created by two Bmp ligands, Gbb and Dpp, and two Bmp type I receptors, Sax and Tkv. Gbb provides long-range signaling, while Dpp signals preferentially to cells near its source along the anteroposterior (AP) boundary of the wing disc. How each receptor contributes to the signaling activity of each ligand is not well understood. This study shows that while Tkv mediates signals from both Dpp and Gbb, Sax exhibits a novel function for a Bmp type I receptor: the ability to both promote and antagonize signaling. Given its high affinity for Gbb, this dual function of Sax impacts the function of Gbb in the Bmp activity gradient more profoundly than does Dpp. It is proposed that this dual function of Sax is dependent on its receptor partner. When complexed with Tkv, Sax facilitates Bmp signaling, but when alone, Sax fails to signal effectively and sequesters Gbb. Overall, this model proposes that the balance between antagonizing and promoting Bmp signaling varies across the wing pouch, modulating the level and effective range, and, thus, shaping the Bmp activity gradient. This previously unknown mechanism for modulating ligand availability and range raises important questions regarding the function of vertebrate Sax orthologs (Bangi, 2006b).

These data clarify the respective roles of Sax and Tkv in mediating Bmp signaling during wing patterning. This analysis shows that Tkv is responsible for mediating both Dpp and Gbb signals, and that Sax has a much more complex role in wing patterning than previously appreciated; Sax not only promotes signaling but also antagonizes signaling by limiting the availability of primarily the Gbb ligand. Both the antagonistic and signal promoting functions of Sax were revealed not only by gain-of-function studies but importantly, also by loss-of-function analyses. Loss of the antagonistic function of endogenous sax is evident: (1) as a broadening the pMad profile when the wing disc completely lacks sax function; and (2) as a non-autonomous increase in pMad levels in wild-type cells abutting the boundary of sax null clones. Loss of Sax-mediated signaling itself is evident: (1) in sax mutant discs as a reduction in the peak pMad levels along the AP boundary; and (2) in sax clones as a cell-autonomous reduction in pMad accumulation. Gain-of-function or overexpression studies indicate that the balance of Sax and Tkv levels in wing disc cells is crucial for proper signaling and, thus, wing patterning. Altogether, these results indicate that Sax is important in modulating Bmp signaling across the wing disc by both mediating and blocking Bmp signals, and, thus, shaping the Bmp activity gradient. How can the novel function of Sax as an antagonist be reconciled at the molecular level with the ability of Sax to promote signaling (Bangi, 2006b)?

Given that Tkv is required for all Bmp signaling in the wing disc, the simplest explanation for the fact that Sax signaling appears to depend on the presence of Tkv is that Sax can only promote signaling in a receptor complex also containing Tkv. Three different forms of Bmp receptor complexes can potentially form in wing disc cells, those composed of two type II receptor molecules and either two Tkv, two Sax or one molecule of each: Tkv-Tkv, Sax-Sax and Tkv-Sax. Overexpressing Tkv or Sax in wing disc cells enabled shifting of the balance between the relative levels of these two molecules, artificially enriching for the formation of receptor complexes homomeric for type I molecules Tkv-Tkv or Sax-Sax. Disrupting the balance of endogenous Tkv to Sax levels by overexpressing Sax immediately reveals the antagonistic function of Sax, consistent with the idea that excess Sax could be sequestering ligand in Sax-Sax receptor complexes which signal either very poorly or not at all. However, overexpression of Tkv, enriching for Tkv-Tkv complexes with high affinity for Dpp and lower affinity for Gbb, leads to increased signaling given sufficient ligand. The third receptor complex, Tkv-Sax, probably accounts for the contribution of Sax to the promotion of Bmp signaling and probably signals in vivo more efficiently than Tkv-Tkv, based on the fact that pMad levels are lower inside clones devoid of Sax than the pMad levels seen in cells at an equivalent position along the AP axis elsewhere on the disc. Loss of Tkv, by definition, eliminates signaling by both Tkv-Tkv and Tkv-Sax, leaving only Sax-Sax containing receptor complexes, which are clearly unable to elicit a pMad-mediated signal on their own. Thus, the model predicts that removing Sax function results in two opposing consequences: (1) a reduction in total Bmp signaling caused by loss of Tkv-Sax complexes, and (2) an increased availability of Bmp ligand and potential signaling caused by loss of Sax-Sax complexes. Several biochemical studies support the putative existence of functional Sax-Tkv receptor complexes. Heteromeric complexes involving different vertebrate type I receptors have been shown to contribute to a single signaling receptor complex and in Drosophila S2 cells both Sax and Tkv appear to be necessary to produce a synergistic signal (Bangi, 2006b).

It is important to note that increasing wild-type Tkv levels in the presence versus absence of excess ligand results in very different phenotypic outcomes. In contrast to Sax, increasing Tkv in the presence of excess ligand leads to a larger increase in Bmp signaling. However, at endogenous ligand levels, as Tkv levels are experimentally increased, a loss of Bmp signaling is seen that is indicative of the preference of Tkv for binding Dpp over Gbb. Clearly, both Gbb and Dpp become limiting in the presence of excess Tkv, with low level Tkv overexpression preferentially limiting Dpp-dependent signaling, while higher levels of overexpression limit both. Clearly, although overexpression of ligand and receptor together reveals a significant difference in the signaling ability of Tkv and Sax, overexpression of receptor alone in the absence of increased ligand appears to reflect only receptor ligand-binding preference (Bangi, 2006b).

Such experimental manipulations of Tkv levels can lead to the loss of Bmp signaling by limiting the range of Bmp signaling, but unlike sax, loss of endogenous tkv function never leads to an increase in Bmp signaling. Furthermore, there is no indication that Tkv is required for or involved in the antagonistic function of Sax. At endogenous levels, Sax-Sax complexes, unlike Tkv-Tkv or Tkv-Sax complexes, appear to modulate the range of Bmp signaling by sequestering ligand without any associated signaling, and, thus, Sax identifies a new previously unrecognized Bmp modulator whose signaling ability appears to depend on which receptor it partners (Bangi, 2006b).

The fact that both Dpp and Gbb are dependent on Tkv for signaling has significant implications regarding the Bmp activity gradient, given that removal of Tkv at any point along the gradient results in the loss of both Gbb and Dpp signaling, not just Dpp signaling. When both ligands are present at similar levels, the higher affinity of Dpp for Tkv means the contribution of Dpp to total Bmp signaling will be more significant than that of Gbb, and movement of Dpp across the wing disc will be affected more strongly by Tkv than that of Gbb. Thus, Gbb should and does contribute more significantly to the low points of the Bmp activity gradient, especially since competition with Dpp for binding to Tkv will also be lower in these regions (Bangi, 2006b).

These findings from receptor and ligand overexpresion experiments suggest that both the antagonistic and signal promoting functions of Sax impact Gbb signaling most significantly because of their preferential interaction. For example, although localized loss of Sax from the peripheral cells of the wing pouch leads to ectopic induction of brk, loss in more central cells does not, suggesting that the relative contribution of Sax to overall Bmp signaling is less in the central cells where Tkv must contribute more significantly given the higher level of Dpp near the AP boundary. The greater contribution of Sax to total signaling in the more peripheral cells of the wing pouch is consistent with its higher affinity for Gbb and the long-range nature of Gbb versus Dpp (Bangi, 2006b). Similarly, removal of Sax from just anterior compartment cells results in brk repression in both the anterior and posterior compartments suggesting that in the absence of Sax, anteriorly expressed Gbb can signal to the posterior-most cells of the wing pouch to effectively repress brk expression beyond its normal domain. This result indicates that endogenous Sax normally functions to not only restrict the level of Gbb signaling but also the range of Gbb. The role that Sax plays in promoting Gbb function, in particular, is detected only when sax function is completely eliminated and gbb function is also significantly compromised (Bangi, 2006b).

Given that Tkv is also required for mediating Gbb signals, of the two proposed receptor complexes that could mediate Gbb signaling (Tkv-Tkv and Tkv-Sax), which is preferentially used by Gbb in wild-type cells? It is clear that Tkv-Sax complexes are not obligatory for Gbb signaling since Gbb signaling is not abolished in sax mutants. The fact that removing Sax does not cause a gbb loss-of-function phenotype indicates that enough Gbb is made available by the loss of Sax antagonism and can signal to compensate for losing that region of total signaling that Sax normally promotes. The fact that pMad levels within a sax clone are lower then endogenous levels indicates that signaling in the clone cells containing only Tkv-Tkv is less efficient than the neighboring cells that have wild-type levels of both Sax and Tkv (Bangi, 2006b).

A synergy has been observed between co-expressed constitutively active (CA) Tkv and Sax in the early embryo and between Tkv and Sax in S2 cells in response to Dpp-Scw heterodimers, since only Dpp homodimers are able to signal efficiently in the absence of Sax. A likely, albeit minimal, contribution of Dpp-Gbb heterodimers to long-range wing patterning has been detected (Bangi, 2006a) making it is possible that Tkv-Sax complexes could respond to Dpp-Gbb heterodimers and such complexes could be particularly efficient at signaling. Given the dual function of Sax, the relative levels of Sax to Tkv are likely to be crucial for establishing a synergistic interaction. The ability of Tkv-Sax containing complexes to mediate ligand homodimers has not yet been determined in vivo and it is also not yet completely clear if the antagonism by Sax can affect heterodimers as well as homodimers. The current data indicate that the ability of Sax to promote signaling must reside with Tkv-Sax-containing complexes and the strong contribution of Gbb to the low points of the gradient with a minimal contribution by Dpp leaves open the possibility that Dpp-Gbb can signal, in addition to Gbb-Gbb, to cells far from the AP boundary (Bangi, 2006b).

Overexpression studies in the follicle cells of the Drosophila ovary produce the same results as those in the wing, indicating that the ability of Sax to block Gbb signaling is not limited to the developing wing. However, in contrast to studies in the wing disc, loss of sax from the follicle cells, as well as the embryonic midgut and neuromuscular synapse produces mutant phenotypes indicative of a loss of ligand function. It is possible that the contribution of Sax to signal promotion in these tissues may be stronger than its antagonistic function. The phenotypic outcome of sax loss of function in a particular process probably depends on the relative numbers of Sax-Sax and Sax-Tkv complexes on the cell surface and the relative binding affinity of a given Bmp ligand for these two complexes. What regulates the composition of type I receptors in a signaling complex is not yet known (Bangi, 2006b).

The ability of the Sax to block Bmp signaling may reflect its requirement to have input from another molecule to activate its kinase domain. When activated by in vitro mutagenesis, Sax and its vertebrate orthologs Alk1/Alk2 (Acvrl1 and Acvr1 - Mouse Genome Informatics) are able to phosphorylate Bmp specific R-Smads, but ligand-induced activation of Sax or Alk1/2 kinase has not been reported. Interestingly, a ligand-induced Bmp receptor complex containing Alk2 and ActRII is unable to phosphorylate Smad1. Furthermore, Alk1 has been shown to require a different type I receptor (Alk5) to activate its kinase domain. Although it has been suggest that the Alk2/ActRII complex might be unstable in vitro, it is also possible that activation of Alk2 (and of its Drosophila ortholog Sax) may depend on its partner type I receptor and/or which ligand is bound, or some other protein. Although Gbb fails to activate Sax-Sax, perhaps another Bmp ligand (i.e. Scw) can. Similarly, endoglin, related to the co-receptor betaglycan, could be important in modulating Alk1-dependent signaling given that mutations in either gene give rise to hereditary hemorrhagic telangiectasia. Sax may require a different type I receptor partner, i.e. Tkv, to activate its kinase or transduce a signal, and such a requirement may be a universal feature of the Alk1/Alk2/Sax subgroup of Bmp type I receptors (Bangi, 2006b).

The robustness of morphogen gradients may depend on negative-feedback mechanisms to buffer against environmental and genetic fluctuations. Clearly, Sax plays a crucial role in modulating the range of the Bmp activity gradient from analysis at both the level of Bmp-dependent target gene expression and the final pattern of the adult wing. The identification of the antagonistic nature of a Bmp type I receptor to modulate signaling activity by sequestering ligand without transducing a signal provides a new mechanism that contributes to the robustness of the Bmp activity gradient. It is proposed that the dual function of Sax is crucial for buffering the wing disc Bmp activity gradient against local fluctuations in ligand levels (environmental, genetic or experimentally induced). Whether this mechanism of signal modulation is evolutionarily conserved remains to be determined, but the fact that the vertebrate Sax orthologs Alk1 and Alk2 have been shown biochemically to exhibit antagonistic behaviors in vitro is interesting. Detailed analysis of these orthologs in developmental contexts will be crucial to determine whether the robustness of vertebrate Bmp activity gradients also depends on the modulation of ligand availability by specific receptors (Bangi, 2006b).

Crimpy inhibits the BMP homolog Gbb in motoneurons to enable proper growth control at the Drosophila neuromuscular junction

The BMP pathway is essential for scaling of the presynaptic motoneuron arbor to the postsynaptic muscle cell at the Drosophila neuromuscular junction (NMJ). Genetic analyses indicate that the muscle is the BMP-sending cell and the motoneuron is the BMP-receiving cell. Nevertheless, it is unclear how this directionality is established as Glass bottom boat (Gbb), the known BMP ligand, is active in motoneurons. This study demonstrates that crimpy (cmpy) limits neuronal Gbb activity to permit appropriate regulation of NMJ growth. cmpy was identified in a screen for motoneuron-expressed genes and encodes a single-pass transmembrane protein with sequence homology to vertebrate Cysteine-rich transmembrane BMP regulator 1 (Crim1). Cysteine-rich repeat (CRR)-containing single-pass transmembrane protein are present in a large number of BMP-interacting proteins in vertebrates and invertebrates. This structurally related family includes extracellular antagonists, such as Drosophila Short gastrulation (Sog) and vertebrate Chordin (Chrd), which are believed to interfere with receptor-ligand interactions. It also includes proteins such as gremlin and sclerostin that can interact with BMPs intracellularly and are thought to interfere with BMP activity, at least in part, by altering ligand activation or secretion. A targeted deletion of the cmpy locus was generated; loss-of-function mutants exhibit excessive NMJ growth. In accordance with its expression profile, tissue-specific rescue experiments indicate that cmpy functions neuronally. The overgrowth in cmpy mutants depends on the activity of the BMP type II receptor Wishful thinking, arguing that Cmpy acts in the BMP pathway upstream of receptor activation and raising the possibility that it inhibits Gbb activity in motoneurons. Indeed, the cmpy mutant phenotype is strongly suppressed by RNAi-mediated knockdown of Gbb in motoneurons. Furthermore, Cmpy physically interacts with the Gbb precursor protein, arguing that Cmpy binds Gbb prior to the secretion of mature ligand. These studies demonstrate that Cmpy restrains Gbb activity in motoneurons. A model is presented whereby this inhibition permits the muscle-derived Gbb pool to predominate at the NMJ, thus establishing the retrograde directionality of the pro-growth BMP pathway (James, 2011).

Gbb has been proposed to cue presynaptic motoneurons to the size of their postsynaptic muscle partners. However, muscles have not been established as the primary source of Gbb at the NMJ. In fact, motoneuron-derived Gbb has a crucial retrograde activity at the motoneuron-interneuron synapse, demonstrating that motoneuronal Gbb is active. The present work demonstrates that motoneurons express Cmpy, a Gbb antagonist. It is proposed that Cmpy restrains motoneuronal activity of Gbb at the NMJ, thus establishing the muscle as the predominant source of the pro-growth BMP signal. Potential mechanisms for Cmpy function at the NMJ and the relationship of Cmpy with intracellular and extracellular BMP antagonists are discussed (James, 2011).

Interest in CG13253/Crimpy was sparked by its restricted expression in the VNC and was reinforced by the presence of a predicted transmembrane domain and CRR. The presence of these two sequence elements renders Cmpy similar to vertebrate Crim1. In mice, Crim1 hypomorphs have been described and display pleiotropic defects in multiple organ systems (Pennisi, 2007). Notably, Crim1 is expressed in developing motoneuron and interneuron populations in the developing mouse and chick spinal cord, although LOF studies have not addressed a neuronal function. A Crim1 homolog has also been described in zebrafish, where it is linked to vascular and somitic development, and in C. elegans, where RNAi-mediated knockdown of crm-1 (cysteine-rich motor neuron protein 1) suggests a pro-BMP function in the control of body size (Fung, 2007). Cell culture studies provide evidence that Crim1 binds Bmp4/7 and antagonizes the production and processing of the preprotein in the Golgi (Wilkinson, 2003). Interestingly, Crim1 interacts with Bmp4/7 at the cell surface and inhibits BMP secretion into the medium (Wilkinson, 2003), raising the possibility that Crim1 antagonizes BMP signaling by multiple cellular mechanisms (James, 2011).

CRR-containing proteins are established modulators of BMP signaling in vertebrates and invertebrates. In Drosophila, posterior wing crossvein specification requires local activation of the BMP pathway, and loss of BMP signaling yields a crossveinless phenotype. BMP ligands are produced in neighboring longitudinal wing veins and are transported to the posterior crossvein. Ligand activity is differentially regulated by the secreted CRR-containing proteins Sog and Crossveinless 2 (Cv-2). Sog and Cv-2 both have pro- and anti-BMP activity, although their mode and range of action differ. Sog is proposed to act at long range, and its anti-BMP activity is thought to derive from sequestering BMPs from their receptors, whereas its pro-BMP activity is likely to arise from transporting BMP ligands through tissues. By contrast, Cv-2 is proposed to act at short range and binds heparan sulfate proteoglycans and the type I receptor Tkv (James, 2011).

The biphasic activities of Sog and Cv-2 serve to emphasize the complex modes of extracellular regulation of BMPs by CRR-containing proteins, as well as to draw attention to possible differences between BMP regulation in the wing and Cmpy-dependent BMP regulation at the NMJ. Although overexpression of Cmpy suppresses Gbb overexpression phenotypes in the wing, cmpy LOF mutants do not display wing vein phenotypes. Cmpy does not function during early embryogenesis, when the BMP homolog Decapentaplegic acts as a classical morphogen in dorsoventral patterning. In both the early embryo and the wing, BMP activity is shaped over many cell diameters by extracellular CRR-containing proteins. Sog and Cv-2 play essential extracellular roles in establishing the magnitude and directionality of BMP signaling. By contrast, Gbb is proposed to act locally at the NMJ to couple pre- and postsynaptic growth (James, 2011).

The close apposition of the cells that send and receive BMP at the NMJ might relieve a requirement for long-range extracellular regulation of the ligand. Instead, it is proposed that a primary challenge at the NMJ is to establish the cellular source of the BMP signal, as Gbb is present both in motoneurons and muscle. In this case, cell-autonomous regulation of the ligand could provide a mechanism for the motoneuron to discriminate between motoneuron- and muscle-derived pools. Consistent with this model, evidence is presented that Cmpy binds Gbb prior to processing and inhibits its growth-promoting activity in motoneurons. In this manner, the Cmpy-Gbb interaction might provide motoneurons with an effective mechanism for distinguishing autocrine and paracrine Gbb signals within the NMJ microenvironment (James, 2011).

CRR-containing BMP antagonists were initially identified from their extracellular roles in the establishment of BMP morphogenetic gradients. It will be interesting to determine whether additional CRR-containing proteins function intracellularly as more short-range BMP-dependent signaling interactions are thoroughly described. Consistent with this idea, several mammalian CRR-containing proteins bind precursor forms of BMP and inhibit BMP activity or secretion in a cell-autonomous manner. Gremlin is a BMP antagonist that is expressed in differentiated cells, including neurons. When co-expressed with Bmp4, gremlin binds to the precursor form of Bmp4 and inhibits secretion. sclerostin, another BMP antagonist, inhibits Bmp7 secretion when the proteins are co-expressed in osteocytes. These studies argue that intracellular modulation of ligand production contributes to BMP signaling directionality in vertebrates (James, 2011).

The work presented in this study suggests that Cmpy antagonizes Gbb activity in motoneurons prior to ligand secretion. To further delineate the Cmpy-Gbb relationship, it will be important to map their localization patterns in motoneurons using compartment-specific markers. Although attempts to generate anti-Cmpy antibodies have been unsuccessful, generation of transgenic flies carrying epitope-tagged Cmpy might enable an analysis of Cmpy subcellular localization. Cmpy-mediated inhibition of Gbb at the NMJ might rely upon restricted localization of Cmpy to this subcellular locale; however, the possibility that Cmpy regulates Gbb activity at the central synapse remains open. Investigation of the localization pattern of Cmpy in motoneurons will begin to address the issue of Cmpy function at these distinct synapses (James, 2011).

An analysis of Gbb distribution, trafficking and secretion in motoneurons in cmpy mutants will indicate the stage of Gbb processing at which Cmpy is likely to act. Studies on mammalian sclerostin provide precedent for an intracellular mechanism for BMP inhibition, as sclerostin sequesters Bmp7 preprotein, leading to its intracellular retention and proteasomal degradation. Interestingly, Cmpy contains only a single, low-threshold CRR. These motifs modulate interactions with mature secreted ligand, suggesting that sequences outside of the CRR mediate interactions with the precursor form of Gbb. Indeed, interaction of Cmpy with Gbb is dependent on C-terminal sequences, including an arginine/lysine-rich domain at the extreme C-terminus. Likewise, the intracellular interaction of gremlin with the precursor form of Bmp4 is not modulated by its cysteine-rich region, but rather by an arginine/lysine-rich domain. The sequence similarities between the BMP interaction domains in gremlin and Crimpy raise the possibility that these proteins antagonize BMP activity by a conserved mechanism (James, 2011).

This study has focused on Cmpy regulation of Gbb in the anatomical development of the NMJ. In addition, Gbb regulates baseline neurotransmission and synaptic homeostasis at the NMJ. Motoneurons precisely compensate for impaired postsynaptic neurotransmitter receptor sensitivity by increasing presynaptic neurotransmitter release. This homeostatic response requires Gbb, which is not itself the acute retrograde homeostatic signal but rather establishes the competence of motoneurons to receive the homeostatic signal. A number of genetic manipulations indicate that the roles of Gbb in regulating synaptic homeostasis, basal neurotransmission and NMJ morphology are separable. Perhaps surprisingly, neuron-specific Gbb rescues both synaptic homeostasis and baseline neurotransmitter release in gbb null animals. By contrast, whereas muscle-derived Gbb rescues synaptic homeostasis in gbb null animals, it does not significantly rescue baseline synaptic function, arguing that neuronal- and muscle-derived pools of Gbb serve distinct functions. Although the data indicate that Cmpy antagonizes autocrine Gbb signaling in motoneurons to restrain morphological expansion at the NMJ, it is likely that motoneuronal Gbb has an independent role in regulating functional development of the NMJ. If so, the Cmpy-Gbb complex might be active and could elicit a signaling outcome distinct from that of the muscle-derived pool of Gbb. Physiological analyses of cmpy mutants, as well as an investigation of Gbb trafficking and secretion at the NMJ in cmpy mutants, should provide crucial insight into this important question (James, 2011).

More broadly, this study is of relevance to the regulation of signal release in neurons. By definition, neurotransmitter is released from the presynaptic compartment and received by neurotransmitter receptors on the postsynaptic side. However, signaling pathway activity is not circumscribed in this way and may occur at short or long range at multiple subcellular positions. Hence, neurons are likely to possess fine-regulatory mechanisms controlling the release of, and response to, extracellular cues. The present work provides insight into the regulation of signaling molecules in neurons and suggests that the mechanisms that control signaling specificity in the developing nervous system are only beginning to be uncovered (James, 2011).

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.