Smad on X


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).

Protein Interactions

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).

It has been shown that R-Smads transiently associate with the appropriate type I receptors, an association that is rendered more stable by the use of kinase-defective versions of the type I receptors. To ascertain whether the phosphorylation of Smox by Baboon might be direct, the physical interaction of Smox with Baboon was investigated. COS cells were transfected with expression plasmids for Smox, Baboon and punt, affinity-labelled with 125I-activin A, and subjected to immunoprecipitation with anti-FLAG antibody for FLAG-Smox. Smox interacts with the kinase-defective form of Baboon, but not with the wild-type Baboon. This finding suggests that Smox physically interacts with Baboon. Consistent with previous findings that Smads are rapidly released from the receptor following phosphorylation, the interaction could not be seen when the wild-type Baboon was used (Das, 1999).

Co-Smads have been identified in C. elegans, Drosophila and vertebrates. They differ from the R-Smads in that they are not phosphorylated on the C terminal serine residues in the'SSXS' motif. However, they form complexes with R-Smads and translocate to the nucleus, where they have been shown to bind DNA and stimulate transcription. In vertebrates, the Co-Smad Smad4, has been shown to participate as a partner in the TGFbeta, activin and BMP signaling pathways. Medea is the only Co-Smad known in Drosophila, and hence a possible association with Smox was tested. FLAG-Smox and myc-Medea were transfected into COS cells in the presence or absence of Baboon and Punt. Cells were lysed and immunoprecipitated with anti-FLAG antibodies. The association of Smox and Medea could only be observed in the presence of the receptors. These data show that Smox and Medea form a complex in the presence of the receptors and indicate that the ability of Co-Smads to function via multiple pathways is an ancient and conserved property (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).

The ability of Smox to activate transcription was studied using a heterologous promoter reporter construct (CAGA)-MLP-Luc. In vertebrates, Smad3 and Smad4, but not Smad1 or Smad2, bind directly to the sequence 'AG(C/A)CAGACA' (CAGA boxes), both in vitro and in vivo; furthermore, stimulation by TGFbeta and Activin, but not by BMPs, induces the transcriptional activation of this reporter construct. Failure of Smad2 to bind the CAGA box is due to the presence of a 30-amino acid insert next to the DNA binding domain in the MH1 domain of Smad2, which is not found in Smad3 nor, significantly, in Smox. Smox does however, have a putatively inactive DNA binding domain, and in addition, preliminary data reveal that Smox does not bind to the CAGA DNA sequence, suggesting that it may behave much like the vertebrate Smad2 in its DNA binding characteristics. Smox or Medea alone weakly induces transcriptional activation, but the co-transfection of Smox and Medea cause a dramatic increase in the luciferase activity. This observation suggests that Smox regulates the expression of target genes activated by activin/TGFbeta-like ligand(s) in Drosophila (Das, 1999).

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).

To resolve the apparent contradiction between the effect on signaling of dSno overexpression (antagonism) and the role identified in dSno mutants (mediation), dSno was examined biochemically. Expression constructs encoding Flag or T7 epitope-tagged Medea, Mad, and dSmad2 were used and various combinations were co-expressed in COS1 cells. It was possible to clearly detect interaction of Medea with both dSmad2 and Mad by co-immunoprecipitation. A T7-tagged dSno expression construct was generated and coexpressed with Flag-dSmad2, Mad, or Medea or with a control vector. Complexes were isolated on Flag agarose and analyzed for the presence of coprecipitating dSno by T7 Western blot. T7-dSno was readily detectable in complexes isolated from cells expressing Medea, but not Mad or dSmad2 (Takaesu, 2006).

Since Medea is a shared partner for both Mad and dSmad2, whether co-complexes containing Medea and dSno together with either Mad or dSmad2 was tested. COS1 cells were transfected with T7-dSno and Flag-Mad or dSmad2 with or without T7-Medea. T7-dSno was present in a complex with Flag-dSmad2 only when T7-Medea was also present. Interestingly, approximately equal amounts of Medea and dSno appeared to coprecipitate with dSmad2, suggesting that much of the Medea that interacts with dSmad2 in this assay is also bound to dSno. In contrast, dSno was not detected in complex with Mad, even when Medea was present, even though Medea clearly interacted with Mad in this assay. These results suggest that dSno interacts specifically with Medea and that the dSno-Medea complex can interact with dSmad2 but not with Mad (Takaesu, 2006).

To test whether incorporation of dSno affected formation of the Medea-dSmad2 complex, Flag-Medea and T7-dSmad2 were coexpressed with or without dSno. The amount of dSmad2 that coprecipitated with Flag-Medea was clearly increased in the presence of dSno. In the reverse of this experiment, it was also observed that an increase in T7-tagged Medea present in Flag-dSmad2 precipitates when dSno was coexpressed. These results suggest that dSno may promote the formation of Medea-dSmad2 complexes (Takaesu, 2006).

To test whether dSno has any effect on the formation of Mad-Medea complexes, similar experiments were performed in which Flag-Medea and Western blotted was isolated for coprecipitating T7-Mad or dSmad2 in the presence or absence of coexpressed dSno. The interaction of Medea with Mad was more readily detectable than with dSmad2. However, inclusion of dSno again increased the interaction between Medea and dSmad2. In contrast, no increase was seen in the Medea–Mad interaction when dSno was coexpressed and it appeared that increasing dSno expression decreased the amount of Mad that coprecipitated with Flag-Medea. Thus it appears that dSno not only may promote interaction of Medea with dSmad2, but also may compete with Mad for Medea interaction, suggesting that dSno may play a role in determining the pathway specificity of Medea (Takaesu, 2006).

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).

Van Bortle, K., Peterson, A. J., Takenaka, N., O'Connor, M. B. and Corces, V. G. (2015). CTCF-dependent co-localization of canonical Smad signaling factors at architectural protein binding sites in D. melanogaster. Cell Cycle 14(16):2677-87. PubMed ID: 26125535

CTCF-dependent co-localization of canonical Smad signaling factors at architectural protein binding sites in D. melanogaster

The transforming growth factor beta (TGF-beta) and bone morphogenic protein (BMP) pathways transduce extracellular signals into tissue-specific transcriptional responses. During this process, signaling effector Smad proteins translocate into the nucleus to direct changes in transcription, but how and where they localize to DNA remain important questions. This study has mapped Drosophila TGF-beta signaling factors Mad, dSmad2, Medea and Schnurri genome-wide in Kc cells and find that numerous sites for these factors overlap with the architectural protein CTCF Depletion of CTCF by RNAi results in the disappearance of a subset of Smad sites, suggesting Smad proteins localize to CTCF binding sites in a CTCF-dependent manner. Sensitive Smad binding sites are enriched at low occupancy CTCF peaks within topological domains, rather than at the physical domain boundaries where CTCF may function as an insulator. In response to Decapentaplegic, CTCF binding is not significantly altered, whereas Mad, Medea, and Schnurri are redirected from CTCF to non-CTCF binding sites. These results suggest that CTCF participates in the recruitment of Smad proteins to a subset of genomic sites and in the redistribution of these proteins in response to BMP signaling (Van Bortle, 2015).

TGF-β effector proteins have been shown to co-localize with mammalian CTCF in a CTCF-dependent manner at just 2 individual loci. This observation has been extended to Drosophila using a genome-wide approach, providing evidence that architectural protein CTCF and canonical Smad signaling proteins, both highly conserved from fly to humans, co-localize on a global scale. Context-specific features were uncovered in which Smad localization is dependent or independent of CTCF binding. Interestingly, genome-wide analysis identifies Mad, dSmad2, Medea, and Schnurri binding to previously characterized response elements even in the absence of DPP ligand, in which levels of phosphorylated Mad are undetectable. This signal-independent clustering of signaling proteins suggests that the genomic TGF-β signaling response is not as simple as regulating binary 'off vs. on' states, dependent on phosphorylated Mad. However, attempts to map the genomic landscape of phosphorylated-Mad before and after DPP stimulation were unsuccessful, likely due to issues with currently available p-Mad antibodies. Though it was not possible to determine the role of phosphorylation as a determinant in Mad localization, it is conceivable that phosphorylation of Mad might play a role in regulating the resident time of DNA-binding, the recruitment of additional regulatory partners, or the ability to establish functional long-range interactions (Van Bortle, 2015).

Smad co-binding at dCTCF sites is sensitive to dCTCF depletion at low occupancy dCTCF target sequences for which Smad consensus sequences are depleted, whereas high occupancy dCTCF binding sites co-bound by additional architectural proteins remain unaffected. The dCTCF-independent recruitment of Smads to high occupancy APBSs suggests that additional architectural proteins may redundantly recruit Smads, or simply provide an accessible chromatin landscape to which Mad, Medea, and dSmad2 can associate. Nevertheless, dCTCF-dependent localization of Smad proteins to specific low occupancy elements is consistent with the CTCF-dependent nature of Smad binding at both the APP and H19 promoters in humans. It is speculated that dCTCF-dependent Smad localization to low occupancy APBSs within topological domains may represent regulatory elements involved in enhancer-promoter interactions, whereas dCTCF-independent high occupancy APBSs are involved in establishing higher-order chromosome organization. What role Smads might play in establishing or maintaining such long-range interactions relevant to chromosome architecture, or whether Smads and other transcription factors simply localize to high occupancy APBSs due to chromatin accessibility, remains difficult to address. However, it has been recently shown that high occupancy APBSs are distinct from analogous transcription factor hotspots, suggesting some level of specificity, most likely governed by protein-protein interactions, decides which factors can associate and where. Alternatively, the enrichment of ChIP-seq signal at high occupancy APBSs may, to some degree, reflect indirect association via long-range interactions with regulatory elements directly bound by Smad proteins. This possibility raises a potential explanation for why Smad ChIP signal is independent of dCTCF binding at high occupancy APBSs (Van Bortle, 2015).

Surprisingly, DPP-activated phosphorylation of Mad does not lead to significant changes in dCTCF binding, whereas Mad, Medea, and Schnurri levels increase at regulatory elements away from dCTCF. These results suggest that TGF-β signaling in Kc167 cells redirects Smad binding to genomic loci independent of architectural proteins, and that architectural proteins may facilitate binding of nuclear Smad proteins in the absence of signaling. The complete loss of Smad ChIP signal at numerous dCTCF binding sites enriched for the core dCTCF consensus sequence nevertheless provides compelling evidence that recruitment of Smad proteins is directly governed by Drosophila CTCF at a subset of binding sites. These results establish CTCF as an important determinant of Smad localization and, depending on the cell-type specific binding patterns of CTCF, suggest that CTCF might also influence the tissue-specific localization of Smad proteins analogous to master regulatory transcription factors in multi-potent stem cells (Van Bortle, 2015).

Smad on X : Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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