baboon

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., dpp^d5). 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., dpp^d12. 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).

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 tlr^ex[2-41]/tlr^E1 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 tlr^ex[2-41]/tlr^E1 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 tlr^ex[2-41]/tlr^E1 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 tlr^ex[2-41]/tlr^E1, 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 tlr^ex[2-41]/tlr^E1 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 tlr^ex[2-41]/tlr^E1 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 tlr^ex[2-41]/tlr^{ex[2- 41]} hemisegments, in 48% of the tlr^ex[2-41]/tlr^E1 hemisegments, and in 56% of the tld^P1/tlr^E1 hemisegments. Since tlr^P1 is a deletion comprising both tld and tlr genes, and tlr^E1 contains a stop codon within the tlr ORF, the slightly lower penetrance of defects in the case of tlr^ex[2-41]/tlr^ex[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 tlr^ex[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 tlr^ex[2-41]/tlr^E1 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).

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