dSno facilitates baboon signaling in the Drosophila brain by switching the affinity of Medea away from Mad and toward dSmad2

A screen for modifiers of Dpp adult phenotypes led to the identification of the Drosophila homolog of the Sno oncogene (dSno; termed snoN in FlyBase). The SnoN locus is large, transcriptionally complex and contains a recent retrotransposon insertion that may be essential for SnoN function. This is an intriguing possibility from the perspective of developmental evolution. SnoN is highly transcribed in the embryonic central nervous system and transcripts are most abundant in third instar larvae. SnoN mutant larvae have proliferation defects in the optic lobe of the brain very similar to those seen in baboon (Activin type I receptor) and Smad2 mutants. This suggests that SnoN is a mediator of Baboon signaling. SnoN binds to Medea and Medea/SnoN complexes have enhanced affinity for Smad2. Alternatively, Medea/SnoN complexes have reduced affinity for Mad such that, in the presence of SnoN, Dpp signaling is antagonized. It is proposed that SnoN functions as a switch in optic lobe development, shunting Medea from the Dpp pathway to the Activin pathway to ensure proper proliferation. Pathway switching in target cells is a previously unreported mechanism for regulating TGFß signaling and a novel function for Sno/Ski family proteins (Takaesu, 2006).

Studies in mammalian cells showed that, when overexpressed, Sno is an antagonist of TGFß/Activin signaling. Overexpression of dSno with A9.Gal4 (throughout the presumptive wing blade) resulted in small wings with multiple vein truncations at 100% penetrance. A9.Gal4:UAS.dSno pupal wing discs were examined for Drosophila serum response factor (dSRF) expression, an intervein marker repressed by Dpp signaling. In A9.Gal4:UAS.dSno pupal discs, dSRF expression is highly irregular with no obviously downregulated regions corresponding to vein primordia. These wing and disc phenotypes are strongly reminiscent of those expressing the dominant-negative allele Mad1 (DNA binding defective but competent to bind Medea) with a variety of drivers, including A9.Gal4 (100% penetrant) and 69B.Gal4. Mad1 dominant-negative effects are due to the titration of Medea into nonfunctional complexes. The similarity of dSno and Mad1 phenotypes suggests that overexpression of dSno antagonizes BMP signaling (Takaesu, 2006).

This was further tested this by coexpressing dSno with Medea or Mad or dSmad2. Coexpression of dSno with Medea or Mad rescues the dSno phenotype to nearly wild type in size and vein pattern. In dSno and Medea coexpressed wings, reduced size was completely eliminated and multiple vein defects were reduced to 28% penetrance. In dSno and Mad coexpressed wings, reduced size was completely eliminated and multiple vein defects were reduced to 19% penetrance. Alternatively, coexpression of dSno with dSmad2 has little effect on the dSno phenotype. In dSno and dSmad2 coexpressed wings, reduced size and multiple vein defects remained 100% penetrant. Coexpression of Mad1 and dSno significantly enhanced the dSno phenotype. One hundred percent of Mad1 and dSno coexpressing the wings are more abnormal than those expressing either dSno or Mad1. The coexpressing wing is very small and veinless and resembles wings expressing UAS.Dad (Dpp antagonist) or dpp class II disc mutants (e.g., dppd5). The enhanced phenotype suggests that dSno and Mad1 antagonize BMP signaling in distinct ways that have additive effects (Takaesu, 2006).

Experiments with a constitutively activated form of the Dpp type I receptor Thickveins (CA-Tkv) are also consistent with this hypothesis. One hundred percent of A9.Gal4:UAS.CA-Tkv wings are overgrown and have numerous ectopic veins as well as vein truncations. This phenotype is suppressed in 98% of the individuals when UAS.dSno is coexpressed with UAS.CA-Tkv. In fact, the coexpression phenotype is not much different from A9.Gal4:UAS.dSno alone, indicating that dSno antagonism of Dpp signaling is fully epistatic to activated Tkv. Finally, ubiquitous overexpression of dSno in the embryonic ectoderm with 32B.Gal4 resulted in discless larvae—a phenotype seen in Mad and Medea null genotypes and in dpp class V disc mutants ( e.g., dppd12. It is concluded that overexpression of dSno antagonizes BMP signaling (Takaesu, 2006).

In Drosophila, as in vertebrates, two TGFß subfamilies are present. The bone morphogenetic protein (BMP) subfamily member Dpp signals through its type I receptor Thickveins to its dedicated transducer Mad (Smad1 homolog) and the Co-Smad Medea (Smad4 homolog). The TGFß/Activin subfamily member activin signals through its type I receptor Baboon to its dedicated transducer dSmad2 and Medea. This study shows that dSno binds Medea and then functions as a mediator of Activin signaling by enhancing the affinity of Medea for dSmad2. Antagonism for BMP signaling likely arises as a secondary consequence of dSno overexpression. Examination of dSno loss-of-function mutants shows that dSno is required in cells of the optic lobe of the brain to maintain proper rates of cell proliferation. Given that Dpp signaling is essential for neuronal differentiation in the optic lobe, these data suggest that dSno functions as a switch that shunts Medea from the Dpp pathway to the Activin pathway to ensure a proper balance between differentiation and proliferation in the brain (Takaesu, 2006).

Protein Interactions

When expressed alone in test cells, Baboon is unable to bind TGF-beta, activin, or bone morphogenetic protein 2. However, Baboon binds activin efficiently when coexpressed with the distantly related Drosophila activin receptor Atr-II (Punt), with which it forms a heteromeric complex. Baboon can also bind activin in concert with mammalian activin type II receptors (ActR-II and ActR-IIB). Maternal Baboon transcripts are abundant in the oocyte and widespread during embryo development and in the imaginal discs of the larva. The structural properties, binding specificity, and dependence on type II receptors define Atr-I as an activin type I receptor from D. melanogaster. These results indicate that the heteromeric kinase structure is a general feature of this receptor family (Brummel, 1999).

In Drosophila, Mad and Medea, both of which mediate Dpp signaling, are the only activating Smads that have been identified so far. Because Babo only appears to induce Smad2/3 responsive promoters in mammalian cell culture, the Drosophila ESTs (Berkeley Genome Project) database was searched for new Smad-like genes. One clone with significant homology to vertebrate Smad2/3 was identified. Using this clone as a probe, a Drosophila ovarian cDNA library was screened to obtain the full-length cDNA, which was sequenced and named dSmad2 (accepted FlyBase name: Smad on X). This cDNA encodes a protein of 486 amino acids and contains a carboxy-terminal SSXS motif, which is found in other receptor-regulated SMADs. dSmad2 sequence alignment with other known Smads reveals ~50% overall identity to Mad or human Smad1 and ~70% identity to human Smad2 or Smad3. Furthermore, the MH2 domain represents the region of highest homology with >90% identity between dSmad2 and either Smad2 or Smad3. In the MH1 domain, dSmad2 lacks the two inserts that are found in the MH1 domain of Smad2. In the linker, dSmad2 contains a PY motif that is present in other Smads, but in addition has a glycine, serine, and glutamine-rich insert (amino acid residues 177-251) that is absent in either hSmad2 or hSmad3 (Brummel, 1999).

The expression pattern of dSmad2 in embryonic and larval tissue was determined by in situ hybridization. High expression of dSmad2 is observed in preblastoderm stage embryos, indicating that dSmad2 is a maternally supplied product. The maternal message is rapidly turned over during the blastoderm stage, and the first zygotic expression of dSmad2 is detected during early gastrulation in the ventrally invaginating mesoderm. Enriched mesodermal expression remains throughout embryogenesis, particularly in the visceral mesoderm surrounding the midgut. In third instar larvae, expression is seen in all the imaginal discs. Most notable, however, is the enriched expression in the brain lobes, specifically in the optic proliferation centers, in which the most pronounced effects on cell proliferation are observed in babo mutants (Brummel, 1999).

The specificity of Smad interactions with receptors and nuclear targets is dictated by the MH2 domain. This suggests that dSmad2 might function in a Drosophila TGF/Activin-like signaling pathway that involves Babo. To investigate this possibility, the ability of Babo to mediate the phosphorylation of dSmad2 was tested. COS-1 cells were transfected with an epitope-tagged dSmad2 either alone or together with wild-type or constitutively active versions of Babo. Analysis of dSmad2 in the absence of signaling shows some basal phosphorylation of the protein. However, on coexpression of activated Babo, a strong increase in phosphorylation of the protein is observed. Previous work has shown that receptor-dependent phosphorylation of Smads occurs on the last two serines of the protein. To confirm that Babo induces phosphorylation of dSmad2 on these residues, the last two serines were mutated to alanines (dSmad2-2SA). Unlike wild-type dSmad2, this mutant is not phosphorylated by activated Babo. These data suggest that dSmad2 is a downstream target of Babo and is phosphorylated on the last two serine residues in the carboxyl terminus (Brummel, 1999).

One functional consequence of phosphorylating receptor-regulated Smads is the induction of heteromeric complex formation with the common partner Smad4. In Drosophila, the Smad4 homolog Medea similarly associates with Mad and is required for a subset of Dpp signaling. Thus, an investigation was carried to see whether Babo-dependent phosphorylation of dSmad2 might induce association with Medea. In the absence of signaling, dSmad2 and Medea form some heteromeric complexes, however, the level of complex formation is substantially increased on cotransfection with a constitutively active form of Babo. Furthermore, use of dSmad2-2SA abolishes this receptor-dependent increase in heteromeric complex formation. These data indicate that phosphorylation of dSmad2 on the last two serines is necessary for receptor-dependent induction of heteromeric complexes of dSmad2 and Medea (Brummel, 1999).

Because recognition of type I receptors by Smads requires activation by the type II receptor, a test was carried out to see whether Punt, the Drosophila type II receptor can function to activate Babo. In cultured cells, transient transfection of either Punt or Babo alone has no effect on the 3TP promoter. However, overexpression of Punt together with Babo leads to a strong induction of the promoter. This is consistent with previous observations that type II and type I ser/thr kinase receptors have intrinsic affinity for each other and on overexpression can associate and signal in the absence of ligand. To test whether dSmad2 interacts with Punt/Babo complexes, a kinase-deficient Babo, which functions to stabilize Smad-receptor interactions, was used. When dSmad2 is immunoprecipitated in the presence of wild-type receptors, no complexes can be detected. However, when the kinase-deficient form of Babo is utilized, receptor complexes coprecipitating with dSmad2 are readily detected. In addition, when the phosphorylation site mutant of dSmad2 (2SA) is tested, stable complex formation is detected between the mutant protein and wild-type receptor complexes. The association of Mad with Punt-Babo receptor complexes was also tested. Thus, dSmad2 interacts transiently and specifically with Punt-Babo receptor complexes. Taken together, these functional and biochemical analyses strongly suggest that dSmad2 is a Drosophila homolog of Smad2/Smad3 and functions as a downstream signaling component that directly interacts with Babo (Brummel, 1999).

Interactions between the various components of the putative activin pathway of Drosophila were characterized. In vertebrates, R-Smads have been shown to associate with, and be phosphorylated by, specific type I receptors. Baboon, along with thick veins (tkv) and saxophone (sax) have been cloned in searches for Drosophila TGFbeta-like receptors. Since Sax and Tkv participate in the Dpp pathway, and biochemical studies have shown that Tkv activates Mad, it was possible that Baboon could be the receptor responsible for activating Smox. In addition, a previous study (Wrana, 1994) had shown that Baboon could bind human Activin, supporting the view that Smox, along with Baboon, may comprise part of a Drosophila activin pathway (Das, 1999).

The activation of Smox was studied by two methods. First, the ability of Baboon to phosphorylate Smox was examined. An antiphosphoserine antibody was used that has been shown to recognize the ligand-dependent phosphorylation of R-Smads, including Smads 1, 2, 3 and 5 and Drosophila Mad. N-terminally FLAG-tagged Smox (FLAG-Smox) was transfected into COS cells with baboon and the Drosophila type II receptor punt, and cells were treated with Activin A. Cells were then lysed and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting using antiphosphoserine antibody. Co-expression of baboon and punt induces a dramatic increase in the phosphorylation of Smox. The phosphorylation of Smox in the absence of Activin A may have been induced by the spontaneous association of type I and type II receptors in transfected cells. The expression of punt alone induces a weak phosphorylation of Smox, which may be due to the interaction of Punt with endogenous type I receptors in COS cells. Mutations of Thr-204 in the TGFbeta type I receptor and corresponding threonine or glutamine residues in other type I receptors to acidic amino acids leads to the constitutive activation of these type I receptors. The constitutively active form of Punt (CA-Punt) induces a weak phosphorylation of Smox. As expected, however, a kinase-inactive (dn) form of Punt (Punt-KR) does not phosphorylate Smox (Das, 1999).

Since Mad has been shown to be phosphorylated by Tkv, another Drosophila type I receptor, the specificity of activation of the Drosophila R-Smads, Smox and Mad, by type I receptors, was examined. Punt was used as the type II receptor in these experiments, since it has been shown to bind both Activin and BMP-like ligands, together with Punt and Tkv, respectively. In the presence of Punt, Baboon induces the phosphorylation of Smox but not of Mad, while MAD is phosphorylated by the constitutively active form of Tkv (CA-Tkv), but Smox is not. Thus, Smox acts as a downstream component of Baboon, whereas MAD acts downstream of Tkv (Das, 1999).

R-Smads translocate into the nucleus following phosphorylation and oligomerization. The nuclear translocation of Smox was examined using transfected COS cells. COS cells were transfected with expression plasmids for Smox, Baboon and punt, and the subcellular localization of Smox was determined by immunofluorescent staining. In unstimulated cells, Smox is distributed throughout the cell, but after activation by Baboon and Punt, Smox accumulates in the nucleus (Das, 1999).

A potential interaction between Patched and Baboon

Patched regulates Drosophila head development by promoting cell proliferation in the eye-antennal disc. During head morphogenesis, Patched positively interacts with Smoothened, which leads to the activation of Activin type I receptor Baboon and stimulation of cell proliferation in the eye-antennal disc. Thus, loss of Ptc or Smoothened activity affects cell proliferation in the eye-antennal disc and results in adult head capsule defects. Similarly, reducing the dose of smoothened in a patched background enhances the head defects. Consistent with these results, gain-of-function Hedgehog interferes with the activation of Baboon by Patched and Smoothened, leading to a similar head capsule defect. Expression of an activated form of Baboon in the patched domain in a patched mutant background completely rescues the head defects. These results provide insight into head morphogenesis and reveal an unexpected non-canonical positive signaling pathway in which Patched and Smoothened function to promote cell proliferation as opposed to repressing it (Shyamala, 2002).

Thus, a novel pathway has been uncovered by which Ptc promotes proliferation of cells in the eye-antennal disc to generate the Drosophila head capsule. Ptc, together with the enigmatic transmembrane protein Smo, promotes activation of Babo, the Activin type I receptor, to stimulate cell proliferation. Previous studies have shown that Ptc is a repressor of Smo, and the interaction of Hh and Ptc relieves this repression on Smo, allowing Smo to activate downstream genes. Ptc signaling is also known to be a suppressor of cell proliferation and loss of function for Ptc in vertebrates, for example, leads to nevoid basal carcinomas. The results described here show that Ptc signaling, in concert with Smo, can also promote cell proliferation and that this is via activation of downstream genes. Thus, these results reveal an intriguing and non-canonical mode of action by this pathway during head morphogenesis (Shyamala, 2002).

The loss of the head capsule in ptc mutants is not due to cell death, no inappropriate and massive cell death has been observed in the eye-antennal disc by the TUNEL assays. However, a lack of BrdU incorporation is observed as well as fewer phospho-histone-positive cells in the eye-antennal disc. Lack of differentiation of cells of the eye-antennal discs can also give rise to similar head capsule defects. For example, pharate adults mutant for the headcase gene show severe head capsule defects with resemblance to ptc mutants. However, in headcase mutants, the morphology, the size and the shape of the eye-antennal discs are normal and the head capsule defects appear to be due to a failure in the differentiation of cells of the eye-antennal disc. In ptc mutants, the morphology, organization, and size of the eye-antennal disc are severely affected by late 3rd instar larvae and the primary cause for the head capsule defects is loss of cell proliferation. This conclusion is further supported by the fact that an activated form of Babo completely rescues the head capsule defects in ptc mutants. babo is a known player in promoting cell proliferation and is required only for cell proliferation but not for cell differentiation in the imaginal discs. Moreover, in vitro culture of eye-antennal discs indicate that the differentiation per se is not affected in ptc mutants. Therefore, it is concluded that Ptc promotes cell proliferation in the eye-antennal disc during head development (Shyamala, 2002).

The results indicate that Ptc-Smo signaling leads to the activation of Babo. During Activin signaling, Activin binds to Activin type II receptor, which promotes physical interaction between type II and type I receptors and the phosphorylation of type I receptor. Both type I and type II receptors are transmembrane serine/threonine kinases. Phosphorylation of the type I receptor results in the activation of its kinase activity and the phosphorylation of downstream transcription activators such as the Smad proteins, resulting in their nuclear localization. In Drosophila, analysis of null mutants for the type I receptor babo, as well as analysis of babo germline clones, indicates that babo is not required during embryogenesis but is essential during pupal development and adult viability. The major defect in babo mutants is a reduction of cell proliferation in the imaginal discs and brain tissue. It has also been shown that in tissue culture experiments, a constitutively active form of Babo can signal to vertebrate TGF-ß/Activin, but not to BMP-responsive promoters. The activated Babo then interacts with Drosophila Smad2 to effect the nuclear localization of this transcription factor (Shyamala, 2002).

These results, that expression of an activated form of Babo in the ptc-expression domain in the eye-antennal disc of ptc mutants completely rescues the head capsule defects, indicates that Ptc-Smo signaling ultimately leads to activation of Babo and promotes cell proliferation in the eye-antennal disc. Since babo and ptc show transheterozygous interaction, it is tempting to speculate that the interaction between Ptc and Babo might be direct. A transheterozygous interaction is generally observed in several cases where the two proteins associate with one another, in cases such as the receptor-ligand pairs Notch and Delta. However, it is also possible that Ptc-Smo signaling and Babo signaling represent parallel pathways that converge at the point of cell cycle control. In this scenario, partial reduction in each could have a synergistic negative affect on cell proliferation, while overexpression of one (i.e. activated Babo) could compensate for loss of the other. Yet another possibility would be that the Pt-Smo pathway activates one of the Activin-like ligands. While the results indicate that there is no transheterozygous genetic interaction between ptc and punt (the inferred type II receptor for Activin), the possibility cannot be ruled out that the Ptc-Smo pathway does not interact with Punt. This is due to the fact that a lack of transheterozygous interaction does not mean that the two players do not interact, as it actually depends on what is limiting. Nonetheless, the finding that Ptc, together with Smo stimulates cell proliferation and the interfacing of Ptc-signaling with Babo-signaling in this process provides new insight into the process of head development (Shyamala, 2002).

The Drosophila type II receptor, Wishful thinking, binds BMP and myoglianin to activate multiple TGFβfamily signaling pathways

Wishful thinking (Wit) is a Drosophila transforming growth factor-β (TGFβ) superfamily type II receptor most related to the mammalian bone morphogenetic protein (BMP) type II receptor, BMPRII. To better understand its function, a biochemical approach was undertaken to establish the ligand binding repertoire and downstream signaling pathway. It was observed that BMP4 and BMP7, bound to receptor complexes comprised of Wit and the type I receptor Thickveins and Saxophone to activate a BMP-like signaling pathway. Further it was demonstrated that both Myoglianin and its most closely related mammalian ligand, Myostatin, interacted with a Wit and Baboon (Babo) type II-type I receptor complex to activate TGFβ/activin-like signaling pathways. These results thereby demonstrate that Wit binds multiple ligands to activate both BMP and TGFβ-like signaling pathways. Given that Myoglianin is expressed in muscle and glial-derived cells, these results also suggest that Wit may mediate Myoglianin-dependent signals in the nervous system (Lee-Hoeflich, 2005).

To provide insight into the molecular mechanisms of Wit that contribute to the biological functions of Wit, this study has characterized the Wit interacting ligands, their compatible type I receptor partners, and their downstream signaling pathways. The binding of BMP7, the mammalian ligand most related to Gbb, to Wit is in agreement with and gives biochemical evidence for results obtained from genetic analysis indicating that Wit mediates Gbb-activated BMP signaling in collaboration with the type I receptors, Tkv and Sax. The demonstration of the binding of BMP4, a functional ortholog of Dpp, to the receptor complex also suggests the possibility of Wit mediating Dpp signals. Dpp is not expressed in muscle or motoneurons but Wit is widely expressed in the central nervous system from embryonic stages suggesting that this putative Dpp signaling might regulate early developmental processes other than NMJ formation. The data showing that a receptor complex comprised of Wit and Tkv can activate MAD phosphorylation is also in agreement with the observation of impaired phosphorylation of MAD in Wit deficient flies and provides further support for a role of Wit in mediating BMP signaling (Lee-Hoeflich, 2005).

While the expression of dActivin in the developing nervous system and its proposed function in neuronal remodeling downstream of Wit or Punt have led to a suggestion that dActivin might induce Wit-mediated activin signaling, this study observed that mammalian activin, which is most closely related to dActivin, does not bind to Wit. One possible explanation for this discrepancy is that Wit might bind ligands other than dActivin and that this indirectly compensates for the lack of Punt-dActivin interaction. An attempt to produce dActivin in mammalian cells using a heterologous system was unsuccessful, thus the possibility cannot be eliminated that mammalian and Drosophila activins have different binding specificities. Generation of dActivin null flies or cell clones and testing for functional equivalence in rescue experiments should help resolve this issue (Lee-Hoeflich, 2005).

Alternatively, it is speculated that Wit might mediate activin signaling via Myoglianin since myostatin, the mammalian ligand most closely related to Myoglianin, activates a TGFβ/activin-like pathway. Accordingly, it was found that both myoglianin and myostatin bind to the Wit and Babo receptor complex. Furthermore, it was observed that coexpression of Wit and Babo induces dSmad2 phosphorylation and mediates myostatin-induced transcriptional activation of a TGFβ/activin-responsive reporter. In agreement to these observations, ectopic expression of Wit induces dSmad2 phosphorylation in insect S2 cells. Retrograde signaling between target-derived factors and the presynaptic terminal is crucial for NMJ development. Since Drosophila Myoglianin is abundantly expressed in muscle at late developmental stages and since Wit-mediated retrograde signaling had been identified previously, it is postulated that Myoglianin might activate a novel retrograde Wit signaling pathway. Interestingly, myostatin inhibits the BMP7 signaling response by competitive binding to type II receptor, ActRIIB, thus the binding of Myoglianin to Wit might also affect Gbb-mediated signaling and thus contribute to NMJ formation. Generation of flies harboring myoglianin loss-of-function mutations will shed light on these issues. These observations underscore the diverse mechanisms controlling Wit signaling and add impetus to further experiments in the context of the Wit receptor (Lee-Hoeflich, 2005).

Baboon/dSmad2 TGF-ß signaling is required during late larval stage for development of adult-specific neurons

The intermingling of larval functional neurons with adult-specific neurons during metamorphosis contributes to the development of the adult Drosophila brain. To better understand this process, the development was studied of a dorsal cluster (DC) of Atonal-positive neurons that are born at early larval stages but do not undergo extensive morphogenesis until pupal formation. DCNs are ~40 clustered neurons located in the dorso-lateral central brain. They are part of the Drosophila adult visual system and innervate the optic lobes. Baboon(Babo)/dSmad2-mediated TGF-ß signaling, known to be essential for remodeling of larval functional neurons, is also indispensable for proper morphogenesis of these adult-specific neurons. Mosaic analysis reveals slowed development of mutant DC neurons, as evidenced by delays in both neuronal morphogenesis and atonal expression. Similar phenomena were observed in other adult-specific neurons. Babo/dSmad2 operates autonomously in individual neurons and specifically during the late larval stage. These results suggest that Babo/dSmad2 signaling prior to metamorphosis may be widely required to prepare neurons for the dynamic environment present during metamorphosis (Zheng, 2006).

The evolutionarily conserved TGF-ßs and their signaling molecules are involved in diverse biological processes. Interestingly, similar signaling cues often elicit different responses in different cells. Consistent with this notion, Babo/dSmad2 is implicated in mediating distinct morphogenetic processes in different neurons, and the involvement of widespread TGF-ß signaling is further suggested in post-embryonic fly brain development before the prepupal ecdysone peak. Given that many vertebrate TGF-ß signaling molecules are dynamically present in the postnatal brain, it is tempting to speculate that similar mechanisms may help modulate neural circuitry in higher organisms during periods of extensive morphogenesis (Zheng, 2006).

Spatially and/or temporally controlled genetic manipulations were used to provide several insights into Babo/dSmad2's roles in postmitotic neuronal morphogenesis. First, mutant clones of interest exhibit similar phenotypes in various mosaic organisms, supporting the cell-autonomous involvement of Babo/dSmad2/Punt and arguing against interference from unlabeled background clones. Second, knocking down Punt at different developmental stages indicated a specific requirement of TGF-ß signaling in prewandering larvae, providing an argument against the direct involvement of Babo/dSmad2/Punt in adult-specific neurons' extensive morphogenesis during early metamorphosis. Third, the defects observed in single-cell versus Nb mutant clones are similar, suggesting a requirement of TGF-ß signaling in postmitotic neurons rather than their precursors. Fourth, phenotypic analysis through development reveals a delay in the postmitotic development of mutant neurons, as evidenced by slow morphogenesis as well as late onset of subtype-specific GAL4 drivers. Interestingly mutant neurons acquire stereotyped, although abnormal morphologies. For instance, mutant DC axons often stall and occasionally get repelled from the junction between the central brain and the optic lobe. It may be that the optic lobe becomes impermeable to late-arriving DC neurites or that mutant neurons intrinsically lack the ability to penetrate the protocerebral-optic lobe interface. The subtler dendritic phenotypes may be because dendrites have nearby targets and undergo little morphogenesis before pupal formation (Zheng, 2006).

Due to the presence of extensively overlapping neurites, previous studies utilizing dendritic and axonal markers were unable to identify the dendrites of DC neurons on the ipsilateral optic lobe. To exclude interference from the neurites coming from the opposing side, MARCM clones were created only on one side of the brain and several different dendritic and axonal markers were tested. The selective targeting of GFP-tagged Synaptobrevin (a presynaptic marker) to the contralateral processes was demeonstrated versus an accumulation of Dscam (exon 17.1)-GFP (a dendritic marker) on the ipsilateral side. Thus, DC neurons send their dendrites to the ipsilateral optic lobe and axons to the contralateral one, directly connecting the two optic lobes. It appears that they receive input from the nearby optic lobe and output onto the contralateral lobular complex, chiasm and medulla. Even though dendrites and axons from contralateral DC neurons innervate similar regions of the optic lobes, they may not make direct connections, since they occupy different focal planes in confocal micrographs (Zheng, 2006).

Insights regarding the roles of DC neurons are few and therefore current ideas about their functions are speculative. Ablation of the ato-expressing neurons through ectopic expression of cell-death genes by ato-GAL4 leads to failed or delayed eclosion of the flies. This indicates a potential role for these neurons in eclosion but it is currently impossible to distinguish between the requirements for DC neurons versus other ato-expressing neurons. The insights regarding connectivity provided in this paper are consistent with the model that DC neurons handle simultaneous processing of information from both optic lobes (Zheng, 2006).

Given that Babo/dSmad2 is required around the mid-3rd instar stage for high-level expression of EcR-B1 in the larval functional MB gamma neurons, it was of interest to enquire if stage-specific TGF-ß signaling is also required for timely differentiation of these neurons prior to metamorphosis. The expression of EcR-B1 in dSmad2 mutant MB Nb clones at various late larval stages and observed a 12-h delay in the onset of EcR-B1 expression in dSmad2 mutant MB gamma neurons. As reported previously, 100% of dSmad2 mutant clones of gamma neurons fail to remodel their neurites during early metamorphosis, thus the delayed EcR-B1 expression may block the MB gamma neurons' responses to the prepupal ecdysone peak. These phenomena imply that TGF-ß and ecdysone signals act sequentially in the postembryonic development of the Drosophila brain (Zheng, 2006).

Loss of Babo/dSamd2-mediated TGF-ß/Activin signaling leads to delayed neuronal morphogenesis and delayed expressions of genes. It was of interest to discover if advanced neuronal development or expression of certain genes would occur if the Babo/dSmad2 pathway was activated early. To test this hypothesis, a transgene encoding a constitutively active (CA) form of Babo-a was ectopically expressed in the MB or DC neurons with GAL4-OK107 and ato-GAL4, respectively. No early or increased expression of either EcR-B1 or Atonal was detected, nor were precocious morphological changes in MB or DC neurons observed. Of course, it is possible that the activity of the ato-GAL4/(CA)Babo is too late to affect the morphogenesis of DC neurons, but the onset of GAL4-OK107 is more than 2 days ahead of the requirement of endogenous Babo/dSmad2 signaling. Thus, the Drosophila TGF-ß/Activin pathway may play a permissive role in promoting the timely differentiation of postmitotic neurons or additional temporally controlled signaling may be involved (Zheng, 2006).

Nevertheless, analysis of Babo/dSmad2's functions in various neurons reveals the potential role of global TGF-ß signaling in preparing fly brains for metamorphosis. In the larval functional neurons that remodel during early metamorphosis, Babo/dSmad2 acts to upregulate expression of a certain ecdysone receptor isoform before the prepupal ecdysone peak. At the same developmental stage, TGF-ß signaling promotes morphological differentiation of larval immature neurons. Interestingly, these distinct developmental processes may both be controlled by transcriptional regulation. Apparent involvement of transcriptional controls in Babo/dSmad2-dependent neuronal morphogenesis is evidenced by a significant delay in the expression of the subtype-specific postmitotic markers, ato-GAL4, GAL4-EB1 and EcR-B1. It is speculated that such stage-specific global TGF-ß signaling may temporally coordinate diverse developmental programs and may help to synchronize development of neurons that are born sequentially over a broad window within individual lineages. Nevertheless, better understanding of the physiological significance awaits characterization of the involved TGF-ß(s) and their modes of secretion/action (Zheng, 2006).

Although transcriptional regulation of distinct target genes may underlie different Babo/dSmad2 functions, much remains to be investigated regarding how activation of the same molecules can elicit distinct nuclear responses. TGF-ß signaling leads to translocation of phosphorylated R-Smad proteins, which might complex with co-Smad. Because Smad proteins alone confer little DNA-binding specificity, their induction of specific genes possibly depends on transcription factors that form complexes with nuclear Smads. Some of these factors may be ubiquitous and available in diverse cells, while others may be differentially restricted to activate gene expression in various cell type-specific manners. One also wonders if differential involvement of Punt versus Wit (or of Babo-a versus Babo-b or of Medea and/or dActivin versus the Activin-like protein) results in qualitatively and/or quantitatively distinct patterns of TGF-ß signaling leading to different cellular responses (Zheng, 2006).

Interestingly, Babo is apparently needed for prompt cell differentiation/growth in both neural and non-neural tissues during late larval development. However, loss of Babo does not appear to affect the ultimate cell fates. An alternative model is suggested for TGF-ß's mechanism of action in promoting cell differentiation/growth. It is proposed that independent genetic programs control cell fate determination and the rate of differentiation, and that there is a common molecular network that determines the rate of cell differentiation/growth. It is possible that activation of Babo/dSmad2 may simply intensify a similar genetic program in different cells to facilitate distinct, already ongoing, but otherwise slowly-progressing cell type-specific development. However, it is also possible that cell fate and the rate of differentiation are extensively intertwined. Future identification of Babo/dSmad2 downstream signaling targets in various tissues should provide some fundamental insights into both organism development and brain function (Zheng, 2006).

The metalloprotease Tolloid-related and its TGF-β-like substrate Dawdle regulate Drosophila motoneuron axon guidance

Proper axon pathfinding requires that growth cones execute appropriate turns and branching at particular choice points en route to their synaptic targets. The Drosophila metalloprotease tolloid-related (tlr) is required for proper fasciculation/defasciculation of motor axons in the CNS and for normal guidance of many motor axons enroute to their muscle targets. Tlr belongs to a family of developmentally important proteases that process various extracellular matrix components, as well as several TGF-ß inhibitory proteins and pro-peptides. Tlr is a circulating enzyme that processes the pro-domains of three Drosophila TGF-ß-type ligands, and, in the case of the Activin-like protein Dawdle (Daw), this processing enhances the signaling activity of the ligand in vitro and in vivo. Null mutants of daw, as well as mutations in its receptor babo and its downstream mediator Smad2, all exhibit axon guidance defects that are similar to but less severe than tlr. It is suggestd that by activating Daw and perhaps other TGF-ß ligands, Tlr provides a permissive signal for axon guidance (Serpe, 2006).

Mutants in the metalloprotease tlr cause lethality during larval and pupal stages of development; however, the cause of the lethality has not been determined. Since a small percentage of larvae (about 15%) die as soon as they hatch, the need for Tlr may start during embryogenesis. Beginning at stage 13, Tlr protein expression is found in the muscles, a subset of cells in the central nervous system that include many glia and the corpus allatum portion of the ring gland. The distinct pattern of expression of tlr in the CNS and muscles, together with the observation that rare tlr mutant escapers exhibit impaired movement, prompted an examination of nervous system development in tlr mutants. To look for global defects, all CNS axons in mutant embryos of the strong allelic combination tlrex[2-41]/tlrE1 were stained with the monoclonal antibody mAB BP102. This analysis did not reveal any gross abnormalities in formation of longitudinal or commissural tracts. Next the Fas2 monoclonal antibody mAb 1D4 which, at stages 16 and 17, highlights motor axon tracts in the periphery and six longitudinal bundles within the CNS, was used. In tlrex[2-41]/tlrE1 mutants, the Fas2-positive longitudinal bundles are wavy and irregular and the outer bundle is discontinuous or missing. This phenotype is of variable penetrance because embryos that had mild defects were found as well as embryos with severe, interaxonal adhesion defects (Serpe, 2006).

In abdominal segments A2-A7, motor axons exit the CNS within the intersegmental nerve (ISN) and segmental nerve (SN) roots; these then split into five pathways that innervate 30 muscle fibers. The ISN develops fairly early and reaches the terminus region near muscle 1 at stage 16 of embryogenesis. In tlrex[2-41]/tlrE1 mutants, ISN growth appeared delayed: 85% of ISNs (140 hemisegments examined) reached their final destination by late stage 16, whereas 15% of ISNs were still at the secondary branch point, around muscle 2. By stage 17, the ISN reached its terminal position in most hemisegments of the tlrex[2-41]/tlrE1, but the terminal arbors were thin or bifurcated (Serpe, 2006).

The SNa has a bifurcated morphology. The posterior branch of SNa innervates muscles 5 and 8, and the anterior branch innervates muscles 21-24. To reach muscle 24, the anterior branch makes a characteristic turn at stage 16. In tlrex[2-41]/tlrE1 mutant animals, SNa did not turn, but instead stalled or produced random branches at this point (Serpe, 2006).

The SNb branch innervates the ventral muscles 7, 6, 13 and 12, and contains the axons of RP1, 3, 4 and 5. The development of the SNb involves two key sets of contacts, the first at muscle 28 and 14, where SNb axons leave the common pathway and enter the ventral muscle field, and the second near muscle 30, where stage 16 SNb growth cones shift their trajectory to extend along a more interior muscle layer. At early stage 17, the SNb forms a linear synaptic branch at the muscle 6/7 cleft, a 'blobby' synapse at the proximal edge of muscle 13 (referred as the 13/30 synapse), and a linear synapse at 12/13. In tlrex[2-41]/tlrE1 mutant animals, the appearance of the SNb proximal synapse (6/7) was normal; however, the SNb bundles stalled at the 13/30 'blob' with an occasional thin bundle exiting and extending towards the 12/13 cleft. The thinned SNb appeared to reach the target muscles at random locations and produced very short synapses. The overall appearance was that of stalled growth cones with frail axons perhaps trying to achieve some sort of innervation at the 12/13 synapse. Such unsuccessful attempts to compensate for the lack of proper 12/13 innervation were observed in 46% of the tlrex[2-41]/tlrex[2- 41] hemisegments, in 48% of the tlrex[2-41]/tlrE1 hemisegments, and in 56% of the tldP1/tlrE1 hemisegments. Since tlrP1 is a deletion comprising both tld and tlr genes, and tlrE1 contains a stop codon within the tlr ORF, the slightly lower penetrance of defects in the case of tlrex[2-41]/tlrex[2-41] animals could be due to some residual tlr muscle expression. Such minimal expression might be the result of using an alternative exon, upstream of the breakpoints of the tlrex[2-41] deficiency, that is computationally predicted by Flybase, although it was not recovered in any of the extant tlr cDNAs (Serpe, 2006).

The fact that Tlr was able to rescue mutant animals when supplied from either the muscle or the nerve suggests that its precise spatial expression pattern may not be important for function. Since Tlr is a secreted protein, it is possible that it could gain access to its substrate from the hemolymph. In fact, in addition to expression in muscle and glia, tlr is also heavily expressed in the corpus allatum of the ring gland, a known secretory tissue. To determine if Tlr could rescue mutant animals when expressed exclusively in secretory or circulating cells, a series of ring gland or hemocyte drivers (Cg, hml, phantom) were used and full rescue of tlrex[2-41]/tlrE1 lethality and axon guidance defects was found in all cases. Furthermore, hemolymph samples from wild-type animals, but not from tlr mutants, contained the processed activated Tlr protein. Moreover, significant levels of HA-tagged Tlr were detected in hemolymph samples collected from animals in which a UAS-tlr-HA transgene was overexpressed in various tissues, including glial cells and muscle. These results support the hypothesis that Tlr is secreted and circulates in the hemolymph and need not be supplied locally by either the muscle or glial cells in order to promote proper axon guidance (Serpe, 2006).

The divergent TGF-β ligand Dawdle utilizes an activin pathway to influence axon guidance in Drosophila

Axon guidance is regulated by intrinsic factors and extrinsic cues provided by other neurons, glia and target muscles. Dawdle (Daw), a divergent TGF-β superfamily ligand expressed in glia and mesoderm, is required for embryonic motoneuron pathfinding in Drosophila. In daw mutants, ISNb and SNa axons fail to extend completely and are unable to innervate their targets. Daw initiates an activin signaling pathway via the receptors Punt and Baboon (Babo) and the signal-transducer Smad2. Mutations in these signaling components display similar axon guidance defects. Cell-autonomous disruption of receptor signaling suggests that Babo is required in motoneurons rather than in muscles or glia. Ectopic ligand expression can rescue the daw phenotype, but has no deleterious effects. These results indicate that Daw functions in a permissive manner to modulate or enable the growth cone response to other restricted guidance cues, and support a novel role for activin signaling in axon guidance (Parker, 2006).

Cell signaling assays and phenotypic analyses indicate that Daw affects motoneuron pathfinding by acting through Put, Babo and Smad2. Supporting this idea, the incidence of ISNb pathfinding defects increases when animals with a single copy of the receptors Put and Babo are further depleted of Daw ligand. Mutations in Daw and its receptors result in a similar range and penetrance of phenotypes, arguing that Daw is the primary contributor to activin signaling in motoneuron pathfinding and that the canonical pathway can fully account for the ability of Daw to influence axon guidance. The slightly higher penetrance of ISNb defects in babo as compared with daw maternal/zygotic nulls (59% versus 50%), raises the possibility that an additional ligand could contribute to embryonic motor axon guidance. Both Activin and Myoglianin can bind Babo, and are expressed in neural or muscle cells compatible with such a role. Intriguingly, overexpression of Activin (and to a lesser extent Myg) can partially rescue daw- pathfinding defects. However, an assessment of their roles in axon pathfinding must await the recovery of mutations in these genes. Furthermore, daw may have other functions in addition to embryonic pathfinding. A majority of daw mutants die during pupal stages despite the fact that pathfinding defects are largely corrected by the third larval instar (Parker, 2006).

Daw could act as a paracrine signal from the muscle or glia to influence motoneurons. Alternatively, it could provide an autocrine signal that supports glial or muscle growth/function and affects axon outgrowth indirectly. The data show that cell-autonomous disruption of activin signaling in muscles or glia does not disrupt motoneuron pathfinding, ruling out an autocrine mechanism. By contrast, expression of BaboΔI and PutΔI receptors in motoneurons effectively phenocopies daw-, suggesting that axon guidance defects could arise from the inability of motoneurons to respond to a paracrine Daw signal. Interestingly, the retrograde Gbb/BMP signal transduced by Wit/Tkv and Mad that regulates synapse morphology and function in larval motoneurons, shows minimal crosstalk despite acting in the same tissue. Disruption of BMP signaling, by expression of TkvΔI in motoneurons or mutations in wit, does not affect axon guidance although it affects neuromuscular junction (NMJ) function (Parker, 2006).

The Drosophila Activin-like ligand Dawdle signals preferentially through one isoform of the Type-I receptor Baboon

How TGF-beta-type ligands achieve signaling specificity during development is only partially understood. This study shows that Dawdle, one of four Activin-type ligands in Drosophila, preferentially signals through Baboc, one of three isoforms of the Activin Type-I receptor that are expressed during development. In cell culture, Dawdle signaling is active in the presence of the Type-II receptor Punt but not Wit, demonstrating that the Type-II receptor also contributes to the specificity of the signaling complex. During development, different larval tissues express unique combinations of these receptors, and ectopic expression of Baboc in a tissue where it is not normally expressed at high levels can make that tissue sensitive to Dawdle signaling. These results reveal a mechanism by which distinct cell types can discriminate between different Activin-type signals during development as a result of differential expression of Type-I receptor isoforms (Jensen, 2009).

The data presented in this study demonstrate that the gene for the Drosophila Activin receptor, Baboon, encodes three isoforms that differ only in their extracellular ligand-binding domains and that one of these isoforms, Baboc, is uniquely required for signaling by the Activin-like ligand Dawdle. The ability to express ligand-specific receptor isoforms is likely to have important implications for cells during development. For example, different profiles of receptor expression, combined with different ligand/receptor affinities, would allow neighboring cells to receive different levels of Activin signaling, even if they are exposed to the same suite of Activin ligands. Restricted receptor expression patterns also offer a means to enable systemically delivered ligands such as Daw to still exhibit tissue-specific effects. One example of this regulation may occur at the Drosophila neuromuscular junction, where multiple Activin-like ligands are expressed either pre- or post-synaptically. Differential expression of Baboon isoforms may be an important way for the muscle and neuron to distinguish between different Activin inputs (Jensen, 2009).

Because Daw appears to signal though a specific Type-I receptor isoform that is not necessary for dAct signaling, it is curious that the two ligands appear to function redundantly in one case, the regulation of neuroblast proliferation. This is especially surprising since baboc is not highly expressed in the brain, at least as measured by low-cycle RT-PCR. One possible explanation for this discrepancy is that baboc may be expressed only in a small subset of cell types in the brain, like neuroblasts, and whole-brain RT-PCR using a pan-babo 5' primer was biased towards the more prevalent baboa transcript. However, attempts to examine the tissue distribution of individual isoforms by in situ hybridization using isoform-specific mRNA probes have not been successful. Ultimately, isoform-specific antibodies may be necessary to elucidate higher-resolution spatial expression patterns of the three isoforms. Such studies, together with a more careful analysis of each ligand’s expression pattern and the generation of isoform-specific loss-of-function mutants, will help elucidate the extent of potential functional redundancies between ligands and if specific receptor isoforms regulate unique biological processes (Jensen, 2009).

Baboon is not the only Drosophila Type-I receptor with multiple isoforms: the Drosophila BMP receptors Sax and Tkv also have several isoforms that differ in their extracellular regions. Similarly, recent work has uncovered Type-I receptor isoforms that are divergent in their extracellular domains in many mammalian species. In both of these cases, however, the divergent isoforms do not affect the cysteine box as seen in the Babo isoforms. For this reason it is not clear if, or to what degree, these more subtle changes might affect affinity of ligand-binding or the ability to form signaling complexes. Genes encoding mammalian Type-II receptors also produce multiple isoforms that differ in their extracellular domains, and some of these isoforms can bind the same ligand with different affinities or bind different ligands with different affinities. Some of these isoforms are also expressed tissue-specifically. When coupled with these findings, this study has uncovered an evolutionarily conserved mechanism by which cells can regulate their response to TGF-β ligands via the type of receptor isoforms that they express. The many potential combinations of Type-I and Type-II isoforms likely enable a cell to fine-tune its response when presented with numerous TGF-β family members, especially in mammals, where 33 ligands appear to signal via a limited set of 5 Type-II and 7 Type-I receptors (Jensen, 2009).

In Drosophila, only one isoform of each Type-II receptor has been found, but because flies express so few ligands, many combinations of Type-I–Type-II signaling complexes may not be needed. For example, the Drosophila genome encodes two Type-II receptors and three isoforms of Babo, giving six combinations of homomeric Type-I/Type-II receptor complexes. The fly genome also encodes four Activin-style ligands (e.g., with nine cysteines versus the seven found in BMPs): dActivin, Dawdle, Myoglianin and Maverick. It is possible, therefore, that each ligand could have a specific combination of high affinity receptors for signaling. This possibility was examined using S2 signaling assays, but it was not possible to see reproducible signaling in vitro from dActivin, Myoglianin, and Maverick, even in the presence of every combination of receptors. Perhaps, in addition to expressing different isoforms of Baboon, cells also control other Activin-like signals by regulating expression of a necessary co-receptor not found in S2 cells. Indeed, a co-receptor is required by the ligand Nodal to phosphorylate Smads 2/3 in vertebrates (Jensen, 2009).

In summary, demonstration of Type-I receptor isoform-specific signaling reveals an additional mechanism by which signal specificity can be achieved within the TGF-β pathway. These findings suggest that, by regulating which complement of receptor isoforms they express, together with different ligand/receptor affinities, distinct cell types within a tissue may be able to discriminate between Activin-family signals during development. This specificity may allow a tissue or cell type to maintain a unique level of Activin signaling, different from its neighbor’s, despite similar exposure to several systemically expressed ligands that can all activate a common intracellular Activin signaling cascade (Jensen, 2009).

Glia instruct developmental neuronal remodeling through TGF-β signaling

Glia secrete myoglianin, a TGF-β ligand, to instruct developmental neural remodeling in Drosophila. Glial myoglianin upregulates neuronal expression of an ecdysone nuclear receptor that triggers neurite remodeling following the late-larval ecdysone peak. Thus glia orchestrate developmental neural remodeling not only by engulfment of unwanted neurites but also by enabling neuron remodeling (Awasaki, 2011).

To establish and refine functional neural circuits, neurons alter connections as the organism matures. In Drosophila, larval brain neural circuits are remodeled into adult ones during metamorphosis. Neurons forming functional larval neural circuits prune their neural projections by local degeneration in early metamorphosis and re-extend their neurites to form the adult-specific neural circuits. This phenomenon requires activation of TGF-β signaling in the remodeling neurons. TGF-β signaling upregulates expression of the B1 isoform of the ecdysone receptor (EcR-B1) at the late larval stage. The pruning of larval projections is then triggered by the steroid molting hormone ecdysone (Awasaki, 2011).

The activin-β gene (Actβ), which encodes a Drosophila activin/TGF-β family molecule, is expressed in the developing larval brain. Temporal inhibition of Actβ with its dominant-negative form or double-stranded RNA (dsRNA) partially suppresses the expression of EcR-B1 in the wandering larvae. In a previous study, it was proposed that Actβ is a candidate ligand for TGF-β signaling in neuronal remodeling. However, the recently isolated Actβ null mutant, Actβed80 had no developmental defects in the remodeling of the mushroom body γ neurons. This observation excludes Actβ as a principal ligand for TGF-β–dependent remodeling of mushroom body neurons. In addition, the Actβ null mutant grows normally until the pharate adult stage, contrasting the embryonic lethality that results from the ubiquitous induction of the dominant-negative form or dsRNA of Actβ. These contradictory phenomena suggest that off-target effects occur when Actβ is suppressed with dominant-negative proteins or RNA interference (Awasaki, 2011).

Notably, myoglianin (myo), which encodes another Drosophila TGF-β ligand (Lo, 1999), is temporally expressed in the brain of third instar larvae. Although no myo transcripts could be detected in the brain of early larvae, intense signals for myo transcripts were seen in subsets of glial cells in the cortex and inner regions of the central brain after the mid third instar larval stage. myo is selectively expressed in two subtypes of larval glial cells: the larval cortex and astrocyte-like glial cells. The cortex glia surround the cell body of each mature neuron and the astrocyte-like glia infiltrate into brain neuropile. The glial processes of both types are in the vicinity of, if not directly contacting, the larval mushroom body γ neurons (Awasaki, 2011).

To determine whether myo governs mushroom body remodeling, the glial expression of myo was silenced by targeted RNAi. dsRNA or microRNA (miRNA) against myo was selectively expressed in glia using the pan-glial GAL4 driver repo-GAL4. myo transcripts were no longer detectable after induction of myo dsRNA in pan-glial cells. The pruning and re-extension of mushroom body γ axons were examined by immunostaining with antibody to Fasciclin 2 (Fas2). In wild-type animals, the perpendicular γ axonal branches in the larval mushroom body lobes are completely pruned by 18 h after puparium formation (APF). γ neurons subsequently re-extended axons horizontally to form the midline-projecting γ lobe in adult brains. This remodeling was blocked by pan-glial induction of myo RNAi. The perpendicular axonal branches of γ neurons persisted through early metamorphosis, and the abnormally retained larval neurites coexisted with the α/β lobes in the adult mushroom bodies that failed to remodel. Direct visualization of mushroom body γ neurons validated the above observations with antibody to Fas2. The myo-silenced brains, including their glial network, were otherwise grossly normal. These observations indicate that a loss of myo expression in glia has no detectable effect on glial cells but adversely affects mushroom body remodeling (Awasaki, 2011).

myo was knocked down using glial subtype–specific drivers. Notably, only cortex glia–specific silencing could marginally block mushroom body remodeling and elicit mild mushroom body lobe defects in about 60% of adult mushroom bodies. However, silencing myo in both larval cortex and astrocyte-like glia fully recapitulated the mushroom body remodeling defects caused by the pan-glial induction of myo RNAi. These findings indicate that myo from two glial sources acts redundantly to govern mushroom body remodeling (Awasaki, 2011).

Next, to determine whether glial-derived myo is required for upregulation of EcR-B1 in remodeling mushroom body γ neurons, EcR-B1 expression in late-larval mushroom bodies in wild-type larvae was compared with expression in myo RNAi larvae. Although the characteristic pattern of EcR-B1 enrichment was detected in wild-type larvae, no such enrichment was detected in myo RNAi-expressing larvae. For example, the strong nuclear signal of EcR-B1 in the mushroom body γ neurons was no longer discernible. When EcR-B1 expression was selectively restored in the mushroom body γ neurons of animals expressing myo RNAi in glia, no defect in mushroom body remodeling could be detected. These findings suggest that the neuronal phenotypes resulting from glial myo RNAi can be effectively rescued by neuronal induction of EcR-B1. These results indicate that the glia-derived Myo instructs mushroom body remodeling via upregulation of neuronal EcR-B1 (Awasaki, 2011).

Remodeling of larval olfactory projection neurons is under the control of the same TGF-β and ecdysone signaling as the mushroom body γ neurons. The loss of glial myo blocked EcR-B1 expression and neurite remodeling of projection neurons, and the remodeling defect was substantially rescued by projection neuron–specific induction of transgenic EcR-B1. These results suggest that glia-derived Myo acts broadly to pattern neuronal remodeling via upregulation of EcR-B1 (Awasaki, 2011).

Using an FRT-mediated inter-chromosomal recombination technique, a deletion mutant, myoΔ1 was generated. Organisms homozygous for myoΔ1 showed no developmental delay through the third instar larval stage. However, mutant larvae prepupate on the surface of or in the food and are developmentally arrested before head inversion. In the myo mutant pupae (0 h APF) or prepupae (2–6 h APF), no enhancement of EcR-B1 expression was detected in the clustered cell bodies of mushroom body γ neurons. When myo expression was restored using myo-GAL4 to drive UAS-myo, the myo mutants grew into pharate or eclosed adults. These animals showed enriched EcR-B1 in the larval brain and they underwent normal mushroom body remodeling. In contrast, when myo expression was restored with myo-GAL4, but excluded in glia by using repo-GAL80 to selectively block GAL4 function in all glial cells, no enrichment of EcR-B1 was detected in the late larval or prepupal brains, and no evidence was found of neuronal remodeling in the pharate adults. These results with myo null mutants substantiate the notion that myo expression in glia governs neuronal remodeling via upregulation of neuronal EcR-B1 expression (Awasaki, 2011).

Does Myo activate TGF-β signaling through the Baboon (Babo) receptor that, contrasting with Myo, acts cell-autonomously to enable neuronal remodeling? There are three Babo isoforms with different ligand-binding domains. Babo-A has been implicated in governing neuron remodeling. To determine whether Myo activity requires Babo-A, their relationship was examined by epistasis. Ubiquitous expression of Myo induced larval lethality. If Myo signals through Babo-A, silencing babo-A should suppress the Myo-induced larval lethality. Attempts were made to deplete specific Babo isoforms by miRNAs to isoform-specific exons. Targeted induction of babo-A miRNA, but not babo-B or babo-C miRNA, effectively blocked mushroom body remodeling. When such isoform-specific miRNAs were co-induced with the myo transgene, only miRNA against babo-A potently suppressed the larval lethality that resulted from ectopic Myo expression. These epistasis results provide in vivo evidence that Myo and Babo-A act in a linear pathway to upregulate EcR-B1 and enable neuronal remodeling (Awasaki, 2011).

Remodeling of larval neurons occurs promptly as the larvae cease activity and become pupae. The tight temporal control of this developmental neuronal remodeling requires a timely induction of the EcR-B1 in these neurons. Three pathways, including TGF-β signaling, the cohesin protein complex and the FTZ-F1 nuclear receptor, are essential for the late-larval upregulation of EcR-B1. The nature and source of the TGF-β signaling become increasingly clear with the finding that Myo, in addition to Babo and dSmad2, is indispensable for the upregulation of EcR-B1. Myo can bind with the Babo/Wit receptor complex in culture (Lee-Hoeflich, 2005). Notably, Myo is produced by glia and is required in glia for neuronal expression of EcR-B1. Namely, glial cells directly instructed the neural remodeling through secretion of Myo. Glial cells further participate in the execution of neuronal remodeling through facilitation of neurite pruning by engulfment of the unwanted neuronal processes. Thus, glial cells orchestrate developmental neural remodeling and may have active roles in dynamically modulating mature neuronal connections (Awasaki, 2011).

R-Smad competition controls activin receptor output in Drosophila

Animals use TGF-β superfamily signal transduction pathways during development and tissue maintenance. The superfamily has traditionally been divided into TGF-β/Activin and BMP branches based on relationships between ligands, receptors, and R-Smads. Several previous reports have shown that, in cell culture systems, 'BMP-specific' Smads can be phosphorylated in response to TGF-β/Activin pathway activation. Using Drosophila cell culture as well as in vivo assays, this study found that Baboon, the Drosophila TGF-β/Activin-specific Type I receptor, can phosphorylate Mad, the BMP-specific R-Smad, in addition to its normal substrate, dSmad2. The Baboon-Mad activation appears direct because it occurs in the absence of canonical BMP Type I receptors. Wing phenotypes generated by Baboon gain-of-function require Mad, and are partially suppressed by over-expression of dSmad2. In the larval wing disc, activated Baboon cell-autonomously causes C-terminal Mad phosphorylation, but only when endogenous dSmad2 protein is depleted. The Baboon-Mad relationship is thus controlled by dSmad2 levels. Elevated P-Mad is seen in several tissues of dSmad2 protein-null mutant larvae, and these levels are normalized in dSmad2; baboon double mutants, indicating that the cross-talk reaction and Smad competition occur with endogenous levels of signaling components in vivo. In addition, it was found that high levels of Activin signaling cause substantial turnover in dSmad2 protein, providing a potential cross-pathway signal-switching mechanism. It is proposed that the dual activity of TGF-β/Activin receptors is an ancient feature, and several ways this activity can modulate TGF-β signaling output are discussed (Peterson, 2012).

This report presents experimental results showing that the Baboon receptor can directly phosphorylate Mad in cell culture and in vivo, and that this cross-talk activity is tightly controlled by the availability of dSmad2. These findings extend the initial report describing canonical signaling between Baboon and dSmad2 where dSmad2, but not Mad, was shown to be a substrate of Baboon in mammalian cells. There are several possible reasons why Baboon-Mad activity is observed in Drosophila cells but was missed in the initial report. First, it is possible that Mad binding to Baboon may be too weak or transient to be detected by immunoprecipitation. Alternatively, endogenous Smad2/3 may have blocked the interaction in heterologous cell systems, or species-specific co-factors may facilitate Mad-Baboon binding (Peterson, 2012).

The strong functional conservation of TGF-β superfamily proteins prompts a comparison of the current results in Drosophila with studies describing cross-pathway signaling in mammalian systems. The limited number of pathway members and the efficacy of RNAi in Drosophila enabled distinguishing between competing models presented for mammalian epithelial cells. With regard to the key mechanistic question of whether TGF-β Type I receptors can directly phosphorylate the BMP R-Smads, the observation of Mad phosphorylation in the absence of BMP Type I receptors is inconsistent with the model of heteromeric TGF-β/BMP Type I receptor complexes proposed in a previous study, but is consistent with the model of direct phosphorylation of BMP R-Smads by TGF-β/Activin receptors. It is possible that both types of mechanisms exist, but are differentially utilized depending on the ligands and receptors present in the particular tissue being examined. Even in Drosophila, it is noteed that direct action of Baboon on Mad does not preclude the possibility that mixed receptors form active signaling complexes in some situations. Mixed receptor complexes have been detected in Drosophila cell culture under over-expression conditions, but their functionality is unknown. A similar point can be made regarding mixed Smad oligomers detected in mammalian epithelial cells. In adult wing assay, this study found that the Babo* phenotype depended primarily on Mad and that dSmad2 was not required. If the wing development defect was caused by the activity of a complex containing both P-Mad and P-dSmad2, then removal of either one should have blocked the Babo* phenotype. Again, this observation does not argue against the formation or activity of mixed R-Smads complexes in some contexts, but shows that productive signaling by cross-pathway phosphorylation can take place independently of such complexes (Peterson, 2012).

One key observation in this report concerning the mechanism of cross-pathway signal regulation is that the degree to which it occurs, both in cell culture and in vivo, appears to be regulated by competition between the R-Smads, likely for receptor binding. Further work is required to determine how general this mechanism might be. Epithelial cell culture models showed that cross-talk is important for the TGF-β-induced migratory switch. The results in the larval wing disc and gut represent the first examples of cross-talk in vivo, and it is expected that additional examples will be found in various animals and tissues. With regard to developmental studies, other systems should be evaluated to see if loss-of-function mutations of Smad2/3 orthologs lead to increased signaling through BMP R-Smads. Additionally, several human diseases have been attributed to mutations in TGF-β components. A cautionary implication of this work is that mutations in the TGF-β branch may have unanticipated loss- or gain-of-function influences on the BMP branch (Peterson, 2012).

Curiously, TGF-β/Activin Type I receptors appear to have gained or retained cross-phosphorylation activity throughout evolution, but BMP Type I receptors do not appear to have reciprocal activity. Phosphorylation of dSmad2 by activated Drosophila Type I BMP receptors, or in response to various ligands, has never been seen. Likewise, no phosphorylation of Smad2/3 by BMP receptors has been reported in vertebrate cells. Why is there this distinction? The growing number of complete genome sequences has allowed phylogenetic reconstruction of the evolution of TGF-β signaling. Apparently all metazoans, even the simple placazoan, have a complex TGF-β network containing both TGF-β/Activin and BMP subfamilies. It is tempting to speculate that a single receptor with dual Smad targets could have played a transitional role in the expansion of the signaling network. The all-or-none nature of the TGF-β-superfamily network (both BMPs and TGF-β/Activins present, or none) in extant organisms, however, does not provide support for this idea. What it does support is the possibility that relationships between core pathway proteins stabilized hundreds of millions of years ago. If the dual-kinase activity of Baboon orthologs has been available to all metazoans during the radiation of animal forms, this activity would be expected to be deployed by different animals and different cell types in diverse ways. Several phenotypes are currently being investigated that differ between baboon and dSmad2 mutants to determine which depend on Babo-Mad cross-talk and which might depend on other forms of Smad-independent signaling (Peterson, 2012).

The different responses of adult wings and larval imaginal tissue upon Baboon activation illustrate that TGF-β pathway wiring and output can vary with developmental context. Given the relationship between Babo and dSmad2, the cross-talk activity can be viewed two ways. From one perspective, the response to loss of dSmad2 depends on the level of Baboon signaling: only cells with Baboon activity can produce P-Mad by cross-talk. From the other perspective, the response of wildtype cells to Baboon stimulation depends on dSmad2 levels: efficient cross-talk will only occur in the absence of dSmad2 (Peterson, 2012).

Given the dominant control that dSmad2 exerts on the ability of Baboon to phosphorylate Mad, the simplest model is that output of TGF-β ligand stimulus in a given cell depends on the expression level of dSmad2. Although dSmad2 is widely expressed, as are Smad2/3 proteins in other animals, different subsets of cells might express different ratios of R-Smads that could influence the signaling output (Peterson, 2012).

The observation that Baboon activity can lower the overall level of dSmad2 protein offers an additional regulatory possibility, where the TGF-β/Activin signal itself can influence its response. In the substrate-switch model, a cell exposed to a prolonged Activin signal would eventually degrade enough of its dSmad2 pool to allow Baboon signaling through Mad. It is not known which tissues, if any, require Baboon-to-Mad signaling in normal developmental contexts. In some cases, dSmad2 over-expression leads to mad loss-of-function phenotypes, which could represent disruption of a sensor incorporating dSmad2 concentration as an input and Mad activity as an output. Proteosomal degradation of activated Smad proteins is a well documented mechanism of signal attenuation, but the current observation of bulk degradation adds a new dimension because it can redirect receptor signaling. The mechanism of signal-dependent bulk dSmad2 degradation is unknown, and preliminary experiments do not support a simple ubiquitylation-proteosomal pathway. Regardless of the molecular details, the observed reduction of total dSmad2 available for receptor competition is the key parameter in the competition model. Since activated R-Smad proteins can also be dephosphorylated to rejoin the pool of would-be substrates, the relative rates of recycling and bulk degradation would be predicted to influence the substrate switch to Mad (Peterson, 2012).

What could be gained by the substrate switch? A general answer is that multiple interactions permit diverse regulatory schemes. For example, cross-talk would permit Mad signaling in a cell that is not exposed to BMP ligands or is otherwise not competent to transduce such signals. Other modes of signal integration are intriguing, such as the conditional formation of mixed R-Smad complexes. Another type of regulatory link that could contribute to the evolutionary maintenance of an integrated dSmad2-Babo-Mad triad considered. If dSmad2 transcription were a target for P-Mad, this would affect the substrate switch. In once case, Baboon signaling to Mad would be triggered by dSmad2 depletion and serve to up-regulate Smad2 to return balance to the system. In contrast, if dSmad2 were down-regulated by P-Mad, this would stabilize the switch to the Baboon-Mad interaction. Additional studies are required to explore these intriguing possibilities (Peterson, 2012).

baboon: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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