Smad on X



The expression pattern of Smox in embryonic and larval tissue was determined by in situ hybridization. High expression of Smox is observed in preblastoderm stage embryos, indicating that Smox is a maternally supplied product. The maternal message is rapidly turned over during the blastoderm stage, and the first zygotic expression of Smox 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).

To gain insights into the functions of Smox in the absence of mutations, the expression patterns of Smox were examined by whole mount in situ hybridizations using digoxigenin-labelled RNA. The expression of Smox is observed in the imaginal discs of third-instar larvae, where cells are undergoing proliferation and differentiation. Specific patterns of Smox expression are observed in the laminar furrow (LF) of the outer proliferation center (OPC) of the brain, immediately adjacent to where the lamina precursor cell (LPC) mitoses give rise to the lamina neurons, and in the morphogenetic furrow (MF) of the eye disc. The MF is a dorso-ventral indentation that traverses the eye disc from posterior to anterior and brings about a wave of cell cycle changes and cell fate determinations in its wake, to effect the proper patterning of the ommatidia of the eye. Strikingly similar expression patterns are observed for Baboon, with widespread expression in the discs and specific patterns in the LPCs of the brain and in the MF of the eye disc. Smox is therefore expressed in the same tissues as Baboon, and could act to transduce its signal. These data further strengthen the idea that Smox and Babo function together in vivo, and relay the same activin-like signal (Das, 1999).

Interestingly, dpp is expressed in two spots at the termini of the target region of the growing retinal axons, which corresponds to the edges of the OPC of the brain. dpp has important patterning functions in the brain, and dpp pathway mutants (e.g. Mad mutants) have significantly smaller brains, suggesting that the dpp and activin-like pathways may act in concert with each other to regulate proliferation and patterning during brain development. dpp is also expressed in the MF of the eye disc, and its roles there include both patterning and proliferative functions. It is possible that one or both of these roles are effected in conjunction with the activin-like pathway.


Metamorphosis of the Drosophila brain involves pruning of many larval-specific dendrites and axons followed by outgrowth of adult-specific processes. From a genetic mosaic screen, two independent mutations were recovered that block neuronal remodeling in the mushroom bodies (MBs). These phenotypically indistinguishable mutations affect Baboon function, a Drosophila TGF-ß/activin type I receptor, and Smad on X (Smox, or dSmad2), its downstream transcriptional effector. Punt and Wit, two type II receptors, act redundantly in this process. In addition, knocking out Activin-beta (dActivin) around the mid-third instar stage interferes with remodeling. Binding of the insect steroid hormone ecdysone to distinct Ecdysone receptor isoforms induces different metamorphic responses in various larval tissues. Interestingly, expression of the Ecdysone receptor B1 isoform (EcR-B1) is reduced in activin pathway mutants, and restoring EcR-B1 expression significantly rescues remodeling defects. It is concluded that the Drosophila Activin signaling pathway mediates neuronal remodeling in part by regulating EcR-B1 expression (Zheng, 2003).

It was of interest to identify possible ligands that participate in the remodeling process. Seven TGF-β type ligands are present in the Drosophila genome. Three of these, dpp, scw, and gbb, are clearly of the BMP family. The remaining, maverick (mav), myoglianin (myo), dActivin (dAct), and activin-like-protein (alp), have not been assigned either genetically or biochemically to a particular family or signaling pathway. Phylogenetic considerations place dAct clearly within the Activin subfamily, while Myo is most similar to BMP-11 and GDF-8, and Mav and Alp are equidistant from both the BMP and TGF-β/Activin subgroups. Therefore, possible involvement of dAct in the Babo signaling was examined (Zheng, 2003).

First, in situ hybridization revealed that dAct is widely expressed in larval brains. Next, when conditioned media from cells expressing dAct was added to S2 cells transfected with Smox, it was found that this ligand is able to stimulate phosphorylation of Smox, while the prototypical BMP ligand Dpp is not. Finally, attempts were made to knock out dAct activity using two independent approaches and dAct, like Babo, was found to be essential for both optic lobe development and EcR-B1 expression in larval brains. Since dAct mutations are currently unavailable, attempts were made to produce a partial loss-of-function condition by overexpression of a dominant-negative form of the protein or RNAi. All TGF-β type ligands that have been examined dimerize and are processed prior to secretion. Previous studies have shown that overexpression of a cleavage-defective form of a particular ligand can interfere with processing and secretion of endogenous ligand. Therefore, a cleavage defective form of dAct (CMdAct) was expressed using either a general GAL4 driver (tubP-GAL4) or an MB-specific driver (GAL4-OK107). CNS development was observed to be retarded only when the CMdAct is ubiquitously expressed and not when it is expressed in MBs. This suggests that dAct does not function within MBs in an autocrine-like fashion. Poor development of the optic lobes is apparent in the tubP-GAL4>CMdAct larval brains, similar to that observed in babo and punt mutant larvae. More importantly, EcR-B1 expression is largely absent in γ neurons of animals that ubiquitously express CMdAct, similar to what is observed in babo mutants. Hs-GAL4-mediated transient expression of CMdAct around the mid-third instar stage also blocks both optic lobe development and EcR-B1 expression (53%). Consistent results are obtained after induction of RNAi using a hairpin-loop dAct construct (UAS-HLdAct). For instance, no EcR-B1 expression was detected in 65% of the late third instar larval brains that were heat shocked to express UAS-HLdAct transiently around the mid-third instar stage. Again, absence of EcR-B1 expression is tightly associated with poor optic lobe development. Similar treatments yield no detectable phenotypes when UAS-CMdAct/UAS-HLdAct is absent or replaced with other UAS-transgenes, such as UAS-mCD8-GFP and UAS-antisense dActivin. In addition, punt mutants, despite having small brains, continue to show EcR-B1 expression. Taken together, these results suggest that dAct, like Babo and dSmox, is indispensable for EcR-B1 expression in the CNS of wandering larvae (Zheng, 2003).

Therefore, from forward genetic mosaic screens, it was found that the Babo TGF-β/Activin type I receptor and a well-known TGF-β/Activin receptor downstream effector, Smox, are both cell autonomously required for remodeling of MB neurons during metamorphosis, providing definitive evidence for involvement of TGF-β/Activin signaling in neuronal plasticity. No evidence exists for any cell fate change in babo mutant MB neurons. For instance, expression of multiple cell type-specific markers remains normal, and mutant γ neurons, unlike wild-type α'/β' neurons, consistently acquire mature dendritic morphological features before metamorphosis. In addition, MB γ neurons that are born at various stages all commit to expressing EcR-B1 in response to TGF-β signaling at the same time and after they all develop into morphologically mature neurons. Therefore, TGF-β signaling probably plays a direct role in programming neuronal plasticity and is not required for cell specification (Zheng, 2003).

TGF-β signaling is implicated in regulating neuronal plasticity in diverse organisms. For instance, environmental cues regulate synthesis of a TGF-β-related ligand (DAF-7) in a pair of chemosensory neurons in C. elegans to direct entry into and exit from an alternative third larval stage called the dauer larva. Dauer formation involves arrest of all postembryonic cell divisions and remodeling of various tissues throughout the body. Given that the DAF-7 TGF-β ligand is primarily sensed by neurons expressing appropriate TGF-β receptors and Smads, it is likely that changes in TGF-β activities directly mediate neuronal remodeling and in turn orchestrate diverse dauer entry/exit responses outside the nervous system. However, it remains to be elucidated whether and how TGF-β signaling regulates neuronal projections and connections in individual remodeling neurons during the entry into and exit from the dauer stage (Zheng, 2003).

In Drosophila, recent data suggest that a BMP signaling pathway controls synaptic growth and function at the neuromuscular junction (NMJ). Whether this pathway also contributes to activity-dependent remodeling at the NMJ remains to be determined. It is interesting to note, however, that in this pathway Wit acts as a BMP receptor, and it can not be substituted for by Punt. In contrast, the activin pathway described here appears to be able to utilize either Punt or Wit for signaling. This may reflect selectivity in the binding of some ligands to one receptor, but not the other. Additional studies will be required to resolve this issue. Since many components of several different TGF-β signaling pathways show pronounced expression in different parts of the developing and postnatal rodent brain, the demonstration that TGF-β/Activin signaling cell-autonomously controls plasticity of MB neurons may provide novel insights into how neuroplasticity is dynamically regulated in higher organisms (Zheng, 2003).

Changes in gene expression are believed to mediate most TGF-β-dependent biological processes. The observation that restoration of EcR-B1 expression significantly rescues remodeling defects in babo mutant neurons supports the model that the Babo/dSmox-mediated TGF-β signaling mediates neuronal remodeling via upregulation of the EcR-B1 expression. Interestingly, ecdysone has also been implicated in regulating synaptic efficacy at the Drosophila NMJ, as has BMP signaling. However, as yet no connection between TGF-β signaling and the ecdysone pathway has been established in this system. In C. elegans, the DAF-7 TGF-β ligand as well as the DAF-12 nuclear hormone receptor are involved in dauer formation. In response to hormonal signals, DAF-12 and EcR coordinate changes in diverse tissues during dauer formation and metamorphosis, respectively. Therefore it might be a common theme that TGF-β signaling patterns tissue-specific responses to steroid hormones in diverse organisms by regulating expression and/or activities of specific steroid hormone receptors (Zheng, 2003).

Several lines of evidence support the model that patterned EcR-B1 expression in the late third instar larval CNS is likely established as a consequence of stage-regulated, cell type-specific responses to TGF-β signaling: (1) dActivin is broadly expressed in the CNS, while expression of EcR-B1 is selectively restricted; (2) despite the persistent presence of dActivin expression during all developmental stages and the fact that γ neurons are born at different times, programming of EcR-B1 expression in γ neurons does not occur until the mid-third instar stage; (3) ubiquitous expression of activated Babo fails to activate EcR-B1 expression ectopically. Determining how TGF-β signaling induces such stage-specific, cell type-dependent responses will provide mechanistic cues for how EcR-B1 is differentially expressed to pattern metamorphosis of the CNS. Possible models might include differential expression of a Smox cofactor or the requirement for a second signal that cooperates with the dActivin signal. It will be important to determine how dActivin reaches its target MB neurons. As has been recently suggested for BMP signaling at the NMJ, this might involve retrograde signaling from the MB synapse or it may occur via a juxtacrine mechanism from nearby cells (Zheng, 2003).

No direct connection has been shown between TGF-β signaling and regulation of cytoskeleton dynamics. Consistent with vital roles of TGF-β pathways in regulating gene expression, the results suggest that Babo/Smox signaling might simply lead to activation of EcR-B1 expression to capacitate MB neuronal remodeling during metamorphosis. Although this study provides novel insights into how differential expression of EcR isoforms is achieved, the challenge now is how ecdysone-induced transcriptional hierarchies mediate complex cytoskeletal changes in remodeling neurons. Identifying mutations that block various aspects of the MB neuronal remodeling in mosaic organisms will continue to shed new light on the molecular mechanisms underlying neuronal plasticity (Zheng, 2003).

Transgenic analysis of the Smad family of TGF-ß signal transducers

Smad signal transducers are required for transforming growth factor-ß-mediated developmental events in many organisms including humans. However, the roles of individual human Smad genes (hSmads) in development are largely unknown. It was hypothesized that an hSmad performs developmental roles analogous to those of the most similar Drosophila Smad gene (dSmad). Six hSmad and four dSmad transgenes were expressed in Drosophila using the Gal4/UAS system and their phenotypes were compared. Phylogenetically related human and Drosophila Smads induce similar phenotypes supporting the hypothesis. In contrast, two nearly identical hSmads generate distinct phenotypes. When expressed in wing imaginal discs, hSmad2 induces oversize wings while hSmad3 induces cell death. This observation suggests that a very small number of amino acid differences, between Smads in the same species, confer distinct developmental roles. These observations also suggest new roles for the dSmads, Medea and Dad, in Drosophila Activin signaling (see Drosophila Activins Activin-ß and Activin Like Protein at 23B; the Drosophila Activin receptor is Baboon) and in potential interactions between these family members. Overall, the study demonstrates that transgenic methods in Drosophila can provide new information about non-Drosophila members of developmentally important multigene families (Marquez, 2001).

dSmad2, hSmad2, and hSmad3 can transduce TGF-ß/Activin signals. hSmad4 and possibly Med can transduce signals for both TGF-ß subfamilies. hSmad4 forms complexes with hSmad2 and with hSmad3. These relationships suggest that these Smads will produce similar phenotypes (Marquez, 2001).

UAS.dSmad2, UAS.hSmad2, UAS.Med, and UAS.hSmad4 induce similar wing phenotypes. When expressed with A9.Gal4, each produces moderately large wings. A9.Gal4 is expressed throughout the wing disc. A comparison of wing surface areas reveals that UAS.dSmad2, UAS.hSmad2, and UAS.Med wings are ~22% larger than wild type. UAS.hSmad4 wings are 16% larger than wild type. It has been shown that dSmad2 transduces dActivin signals that modestly stimulate cell proliferation in wing development. The influence of dActivin signals on wing cell proliferation is much smaller than that of Dpp signals. Ectopic dActivin signaling results in an ~30% increase in wing size. The moderately large size of the common wing phenotype suggests that UAS.hSmad2, UAS.Med, and UAS.hSmad4 simulate dActivin-, rather than Dpp-, mediated cell proliferation (Marquez, 2001).

Consistent with their ptc.Gal4 phenotypes, UAS.Med/A9.Gal4 and UAS.hSmad4/A9.Gal4 wings also show ectopic veins. These wings have a distal crossvein between L2 and L3 and duplications of L2, L3, and L4 at the margin. The presence of wing size and vein phenotypes provides the first evidence that Med, like its counterpart hSmad4, can signal for both TGF-ß subfamilies. UAS.Med and UAS.hSmad4 influences vein formation like Dpp/BMP signaling Smads (UAS.Mad and UAS.hSmad1) and moderately increase wing size like TGF-ß/Activin signaling Smads (UAS.dSmad2 and UAS.hSmad2) (Marquez, 2001).

In contrast, UAS.hSmad3/A9.Gal4 wings are smaller than wild type (~32%). These wings have essentially wildtype venation although loops are occasionally present. The dramatic difference in wing size suggests that UAS.hSmad3 cannot participate in a dActivin pathway that stimulates cell proliferation. The UAS.hSmad3 wing phenotype suggests that UAS.hSmad3 can inhibit cell proliferation or stimulate apoptosis during wing development (Marquez, 2001).

UAS.hSmad3 consistently generates distinct phenotypes from the phylogenetically related Smads UAS.dSmad2 and UAS.hSmad2. UAS.dSmad2 and UAS.hSmad2 are viable with all Gal4 lines while UAS.hSmad3 is lethal with any Gal4 line with significant embryonic expression. UAS.hSmad3 lethal phenotypes often include small size, brown patches of necrotic tissue, and in pharate adults the absence of entire limbs. In cell culture, ectopic expression of hSmad3 induces apoptosis in lung epithelial cells. The UAS.hSmad3 phenotypes observed suggest the hypothesis that UAS.hSmad3 expressed in flies also stimulates apoptosis (Marquez, 2001).

To test this hypothesis, UAS.hSmad3/ptc.Gal4 wing discs were stained with acridine orange, an indicator of cell death. UAS.hSmad3/ptc.Gal4 discs show significantly larger numbers of dead cells than control discs containing only ptc.Gal4 or only UAS.hSmad3. The UAS.hSmad3/ptc.Gal4 discs show clusters of dead cells in the anterior and posterior compartments. Surprisingly, the distribution of dead cells does not correspond to the expression pattern of ptc.Gal4. However, wing discs are known to compensate for cell death in one compartment by inducing cell death in the other compartment. This compensation mechanism appears to be operating in UAS.hSmad3/ptc.Gal4 discs, leading to the observed distribution of dead cells (Marquez, 2001).

To determine if UAS.hSmad3 induces cell death only with ptc.Gal4, UAS.hSmad3/en.Gal4 wing discs were stained. There are many more dead cells in UAS.hSmad3/en.Gal4 wing discs than in UAS.hSmad3/ptc.Gal4 wing discs. This is likely due to the larger domain of expression for en.Gal4 (throughout the posterior compartment) vs. ptc.Gal4 (a central stripe). UAS.hSmad3/en.Gal4 discs are small and dysmorphic, possibly due to the large amount of UAS.hSmad3-induced cell death (Marquez, 2001).

A comparison of the amino acid sequences of hSmad2 and hSmad3 reveals 91% overall identity and 97% identity in the MH2 domain for these Smads. Given this extensive identity, UAS.hSmad2/ptc.Gal4 wing discs were stained with acridine orange. It was reasoned that perhaps the large wings seen in UAS.hSmad2/ptc.Gal4 flies are the result of overproliferation in response to UAS.hSmad2-induced cell death. However, the level of cell death in UAS.hSmad2/ptc.Gal4 and UAS.hSmad2/en.Gal4 wing discs is similar to the ptc.Gal4 control and the UAS.hSmad2 control discs. These results suggest that hSmad2 and hSmad3 can have distinct developmental roles (Marquez, 2001).

Thus phylogenetically related Smad family members (Mad/hSmad1, dSmad2/hSmad2, Med/hSmad4, and Dad/hSmad6/hSmad7) induce similar phenotypes. This result supports the hypothesis that an hSmad performs roles in human development analogous to the ones their dSmad counterpart plays in Drosophila development. It is suggested that the developmental roles of hSmads can now be more profitably investigated using clues from dSmads. For example, tinman is a Mad/Med target gene for Dpp signals during the subdivision of the embryonic mesoderm. On the basis of these results, the highly conserved human homologs of tinman are candidate targets of hSmad1 and hSmad4 in human mesodermal cells (Marquez, 2001).

Many of the phenotypes observed reinforce known roles for dSmads. For example, the moderately large wing phenotype seen with UAS.dSmad2 is consistent with a role in a dActivin pathway that stimulates cell proliferation in wing development. However, other phenotypes suggest new roles for dSmads. For example, moderately large wings generated with several Gal4 lines suggest that Media participates in dActivin signaling. The tiny wings generated with MS1096.Gal4 suggest that Dad may have the ability to antagonize both Dpp and dActivin signaling. In addition, the common truncated leg phenotype generated by Medea, hSmad6, and hSmad7 suggests that Med may interact with antagonist Smads such as Dad. These potential roles for Med and Dad are consistent with activities already shown for their human counterparts. For example, hSmad4, hSmad6, and hSmad7 can influence signals from both TGF-ß subfamilies in cell culture and hSmad4 can interact with hSmad6 in Xenopus injection assays (Marquez, 2001).

The demonstration that UAS.hSmad3 can induce cell death in wing discs is the first instance where cell death is associated with TGF-ß/Activin signaling in Drosophila. Previous studies connected cell death in imaginal discs with the loss of Dpp signals. Increased cell death is seen in eye and wing discs in certain dpp mutants. The ability of hSmad3 to induce cell death is consistent with the colon tumor phenotype of Smad3 knockout mice. Tumors may result from the overgrowth of colon cells not properly targeted for apoptosis in the absence of Smad3 (Marquez, 2001).

The distinct phenotypes generated by UAS.hSmad2 and UAS.hSmad3 suggest that Smads with similar sequences induce similar phenotypes only when comparing Smads from different species. Expression of the nearly identical Smads, hSmad2 and hSmad3, always generates distinct phenotypes. Consistent with these results, functional differences between these Smads have been reported in the regulation of Activin-inducible genes in cell culture (Marquez, 2001).

What then are the critical amino acid differences between these nearly identical hSmads? Since hSmad2 and dSmad2 are more similar in the MH1 domain it is believed that amino acid differences between hSmad2 and hSmad3 in the MH1 domain are likely responsible for their distinct phenotypes. One obvious sequence difference between hSmad2 and hSmad3 is a 30-amino-acid insertion in the MH1 domain of hSmad2. Two cell culture studies suggest that this insertion is the source of functional differences between hSmad2 and hSmad3. However, this is unlikely to be the source of the observed different ptc.Gal4 phenotypes of UAS.hSmad2 and UAS.hSmad3. Neither hSmad3 nor dSmad2 have the insertion, yet it is dSmad2 and hSmad2 that display similar phenotypes. Amino acid differences between hSmad2 and hSmad3 in the MH1 domain outside the insertion are likely to be responsible for their distinct phenotypes. It has been claimed that the amino-terminal region of the MH1 domain, not the insertion, is responsible for observed functional differences between hSmad2 and hSmad3 (Marquez, 2001 and references therein).

In summary, this analysis of hSmad and dSmad transgenes supports the hypothesis that phylogenetically related Smads fulfill developmental roles that are conserved between humans and Drosophila. The results also suggest a number of new hypotheses regarding roles for human and Drosophila Smads in pattern formation, cell proliferation, and cell death. The data suggest that a small number of amino acid differences between two very similar Smads in the same species can confer distinct activities. Overall, this study demonstrates that transgenic methods in Drosophila can provide new information about mammalian members of developmentally important multigene families (Marquez, 2001).

The metalloprotease Tolloid-related and its TGF-ß-like substrate Dawdle, signaling through Babo and Smad2 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 suggested that by activating Daw and perhaps other TGF-ß ligands, Tlr provides a permissive signal for axon guidance (Serpe, 2006).

Drosophila Activin-β and the Activin-like product Dawdle function redundantly to regulate proliferation in the larval brain

The Drosophila Activin-like ligands Activin-β and Dawdle control several aspects of neuronal morphogenesis, including mushroom body remodeling, dorsal neuron morphogenesis and motoneuron axon guidance. This study shows that the same two ligands act redundantly through the Activin receptor Babo and its transcriptional mediator Smad2 (Smox), to regulate neuroblast numbers and proliferation rates in the developing larval brain. Blocking this pathway results in the development of larvae with small brains and aberrant photoreceptor axon targeting, and restoring babo function in neuroblasts rescued these mutant phenotypes. These results suggest that the Activin signaling pathway is required for producing the proper number of neurons to enable normal connection of incoming photoreceptor axons to their targets. Furthermore, as the Activin pathway plays a key role in regulating propagation of mouse and human embryonic stem cells, the observation that it also regulates neuroblast numbers and proliferation in Drosophila suggests that involvement of Activins in controlling stem cell propagation may be a common regulatory feature of this family of TGF-β-type ligands (Zhu, 2008).

It is not entirely clear how Activin signaling in neuroblast lineages maintains the wild-type number of brain neuroblasts. Approximately 100 neuroblasts per brain lobe are formed during embryogenesis, and most go quiescent at the embryonic/larval transition. From L1-L3 they progressively re-enter the cell cycle to resume their cell lineages. Perhaps the slower cell cycle of Activin pathway mutants inhibits exit from quiescence and promotes premature differentiation. Mutations in trol, which encodes a heparin sulfate proteoglycan Perlecan, prevent neuroblast reactivation and lead to a severe reduction in neuroblast numbers and brain size. Many TGF-β ligands bind to heparin sulfate proteoglycans, and thus part of the effect of Activin signaling on brain size might be mediated by Perlecan or other proteoglycans such as the glypican Dally (Zhu, 2008). The small brain size is not just caused by a reduced number of neuroblasts, however; babo mutant clones that contained a single neuroblast produced fewer daughter cells in a given time window than did wild-type neuroblasts, presumably due to the increased expression of Cyclin A and the delay in metaphase exit. One additional possibility is that the Activin signal may affect neuroblast temporal identity progression in larval neuroblast lineages, similar to the effect of temporal identity mutations on embryonic neuroblast lineages, leading to the failure to produce early, mid or late subsets of larval lineages. Testing this hypothesis awaits the development of markers for different neurons within optic lobe neuroblast lineages (Zhu, 2008).

The importance of Activin in regulating neuroblast proliferation is reminiscent of the positive role that Activin/Nodal signaling plays in regulating the cell cycle of mouse and human ES cells. In those cells, as in Drosophila neuroblasts, Activin/Nodal signaling enhances, but is not absolutely required for, cell proliferation. Another point of potential similarity is that the mES cells endogenously produce an Activin/Nodal signal leading to an autocrine/paracrine regulation of proliferation. While in situ data are not of sufficient resolution to unambiguously assign expression of actβ and daw to particular cell types, both are expressed in the optic proliferation zones where neuroblasts are highly concentrated. It is also possible that some ligand may be supplied by the innervating photoreceptors. Activin is strongly expressed in R7 and 8 and like hh and spitz may provide a tropic signal that simulates proliferation in the target tissue (Zhu, 2008).

Lastly, it is interesting to note that Activins are not the only TGF-β-like factors required for proliferation of Drosophila neuroblasts. The BMP family member Dpp is expressed in four regions in each brain lobe. Two lie in the dorsal and ventral margins of the posterior optic zone neuroepithelium near what has been termed the lamina glial precursor region, whereas the other two smaller zones are more interior at the base of the inner proliferation zone. In the brain, the dpp loss-of-function phenotype is remarkably similar to that seen in babo mutants. A potential trivial explanation for the similarity in phenotypes might be that Activin signaling is required for dpp expression, or vice versa. However, dpp is still expressed in babo mutants, and daw and actβ are both still expressed in dpp mutants, although it is difficult to know in each case whether the levels are equivalent. Thus, both Dpp and Activin signaling appear to be required to stimulate brain neuroblast proliferation (Zhu, 2008).

In addition to regulating proliferation in the brain, Dpp signaling plays a major role in regulating proliferation in other tissues, including the imaginal discs. Once again, Activins may collaborate with BMPs in regulating proliferation in this tissue. In particular, it is noted that babo mutants show ectopic P-H3 staining within the morphogenetic furrow of the eye disc, which is also observed in loss-of-function mutants in the Dpp receptor Tkv. Furthermore, babo mutant wing disc clones can grow large in contrast to clones mutant in dpp signaling components, although the overall sizes of babo mutant discs are not affected proportionally as much as is the brain. Therefore, the way in which BMP and Activin inputs regulate the cell cycle might be different in discs versus the brain, or the two tissues might exhibit different sensitivities to common inputs (Zhu, 2008).

How Activins and BMPs affect the cell cycle is not entirely clear. In the wing disc, Dpp signaling through Tkv/Mad has been shown to promote the G1-S transition. In the brain, babo mutants exhibit a decrease in the M/S ratio, which could be due to a decrease in cells at the G2/M phase of the cell cycle. Consistent with this view, it was found that Cyclin A levels are enhanced in babo mutants and that heterozygosity for a Cyclin A mutation suppresses the babo phenotype. This is very similar to that seen in dally mutants, which also affect brain development by causing a delay in the G2-M transition within the outer proliferation centers. Just as was found for babo mutants, heterozygosity for Cyclin A suppresses the dally cell cycle defect (Zhu, 2008).

One attractive model for how both BMPs and Activins contribute to cell cycle progression is that they regulate the cycle at different points: Activins at G2-M and BMPs at G1-S. Alternatively, since previous work has suggested that Cyclin A probably has roles in regulating both G2-M and G1-S transitions in Drosophila and since Smads can form heterotrimers, it may be that a composite signal composed of a Smad2/Mad/Medea heterotrimer acts at several points in the cell cycle. Interestingly, several potential target genes that are regulated by both Activin and BMP signals in the larval brain have been identified by microarray studies using activated receptors, but no obvious candidates for genes that might influence proliferation are evident within the list (Zhu, 2008).

Lastly, it is noted that daw plays a role in motoneuron axon guidance in the embryo, while actβ has been implicated in mushroom body remodeling and in regulating the terminal steps in photoreceptor R8 targeting during pupal stages. Since both ligands are expressed in each of these tissues, it is possible that there may be functional redundancy that limits the severity of the previously observed phenotypes. Consistent with this view, both actβ and daw modulate neurotransmission at the neuromuscular junction, and the double mutant phenotypes are more severe than those seen in the single mutants, similar to their redundant function in regulating neuroblast proliferation. In conclusion, all available data suggest that Activin signaling plays at least two important roles in Drosophila nervous system development. First, it ensures that the proper numbers of cells are produced in the CNS; and second, it helps establish correct functional connections between neurons and their synaptic partners (Zhu, 2008).

Haemocytes control stem cell activity in the Drosophila intestine

Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes were recruited to the intestine and secreted the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switched their response to DPP by inducing expression of Thickveins, a second type I receptor that had previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promoted infection resistance, but also contributed to the development of intestinal dysplasia in ageing flies. The study proposes that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes are recruited to the intestine and secrete the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switch their response to DPP by inducing expression of Thickveins, a second type I receptor that has previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in ageing flies. It is proposed that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

The results extend the current model for the control of epithelial regeneration in the wake of acute infections in the Drosophila intestine. It is proposed that the control of ISC proliferation by haemocyte-derived DPP integrates with the previously described regulation of ISC proliferation by local signals from the epithelium and the visceral muscle, allowing precise temporal control of ISC proliferation in response to tissue damage, inflammation and infection (Ayyaz, 2015).

The association of haemocytes with the intestine is extensive, and can be dynamically increased on infection or damage. In this respect, the current observations parallel the invasion of subepithelial layers of the vertebrate intestine by blood cells that induce proliferative responses of crypt stem cells during infection. A role for macrophages and myeloid cells in promoting tissue repair and regeneration has been described in adult salamanders and in mammals, where TGFβ ligands secreted by these immune cells can inhibit ISC proliferation, but can also contribute to tumour progression. The results provide a conceptual framework for immune cell/stem cell interactions in these contexts (Ayyaz, 2015).

The observation that DPP/SAX/SMOX signalling is required for UPD-induced proliferation of ISCs suggests that SAX/SMOX signalling cooperates with JAK/STAT and EGFR signalling in the induction of ISC proliferation. Accordingly, while constitutive activation of EGFR/RAS or JAK/STAT signalling in ISCs is sufficient to promote ISC proliferation cell autonomously, this study found that this partially depends on Smox. Even in these gain-of-function conditions, ISC proliferation can thus be fully induced only in the presence of basal SMOX activity. As short-term overexpression of DPP in haemocytes does not induce ISC proliferation, it is further proposed that DPP/SAX/SMOX signalling can activate ISCs only when JAK/STAT and/or EGFR signalling are activated in parallel. However, long-term overexpression of DPP in haemocytes results in increased ISC proliferation, suggesting that chronic activation of immune cells disrupts normal signalling mechanisms and results in ISC activation even in the absence of tissue damage (Ayyaz, 2015).

BMP TGFβ signalling pathways are critical for metazoan growth and development and have been well characterized in flies. Multiple ligands, receptors and transcription factors with highly context-dependent interactions and function have been described. This complexity is reflected by the sometimes conflicting studies exploring DPP/TKV/SAX signalling in the adult intestine. These studies consistently highlight two important aspects of BMP signalling in the adult Drosophila gut: ISCs can undergo opposite proliferative responses to BMP signals; and there are various sources of DPP that differentially influence ISC function in specific conditions. By characterizing the temporal regulation of BMP signalling activity in ISCs, the results resolve some of these conflicts: it is proposed that early in the regenerative response, haemocyte-derived DPP triggers ISC proliferation by activating SAX/SMOX signalling, and ISC quiescence is re-established by muscle-derived DPP as soon as TKV becomes expressed. Of note, some of the conflicting conclusions described in the literature may have originated from problems with the genetic tools used in some studies. This study have used two independent RNAi lines (BL25782 and BL33618) that effectively decrease dpp mRNA levels in haemocytes when expressed using HmlΔ::Gal4 (Ayyaz, 2015).

The close association of haemocytes with the type IV collagen Viking suggests that the stimulation of ISC proliferation by haemocyte-derived DPP may also be controlled at the level of ligand availability, as suggested previously for DPP from other sources. The regulation of SAX/SMOX signalling by DPP observed in this study is surprising, but consistent with earlier reports showing that SAX can respond to DPP in certain contexts. Biochemical studies have suggested that heterotetrameric complexes between the type II receptor PUNT and the type I receptors SAX and TKV can bind DPP, and complexes with TKV/TKV homodimers preferentially bind DPP, and complexes with SAX/SAX homodimers preferentially bind GBB. In the absence of TKV, SAX has been proposed to sequester GBB, shaping the GBB activity gradient, but to fail to signal effectively. Expression of GBB in the midgut epithelium has recently been described, and ligand heterodimers between GBB and DPP are well established. Consistent with earlier reports, this study found that GBB knockdown in ECs significantly reduces ISC proliferation in response to infection. Complex interactions between haemocyte-derived DPP, epithelial GBB, and ISC-expressed SAX, PUNT and TKV thus probably shape the response of ISCs to damage, and will be an interesting area of further study (Ayyaz, 2015).

Similar complexities exist in the regulation of transcription factors by SAX and TKV. Canonically, SMOX is regulated by Activin ligands (Activin, Dawdle, Myoglianin and maybe more), and the type I receptor Baboon. This study has tested the role of Activin and Dawdle in ISC regulation, and, in contrast to DPP, this study could not detect a requirement for these factors in the induction of ISC proliferation after Ecc15 infection. Furthermore, the data establish a requirement for haemocyte-derived DPP as well as for SAX expression in ISCs in the nuclear translocation of SMOX after a challenge. This study thus indicates that in this context, SAX responds to DPP and regulates SMOX. Regulation of SMOX by SAX has been described before, yet SAX is also known to promote MAD phosphorylation, but only in the presence of TKV. Consistent with such observations, this study has detected MAD phosphorylation in ISCs only in the late recovery phase on bacterial infection, when TKV is simultaneously induced in ISCs. During this recovery phase, ISCs maintain high SAX expression, but SMOX nuclear localization is not detected anymore, suggesting that SAX cannot activate SMOX in the presence of TKV, and might actually divert signals towards MAD instead. The data also suggest that Medea (the Drosophila SMAD4 homologue) is not required for SMOX activity. Although surprising, this observation is consistent with recent reports that SMAD proteins in mammals can translocate into the nucleus and activate target genes in a SMAD4-independent manner. The specific signalling readouts in ISCs when these cells are exposed to various BMP ligands and are expressing different combinations of receptors are thus likely to be complex (Ayyaz, 2015).

The current findings demonstrate that the control of ISC proliferation by haemocyte-derived DPP is critical for tolerance against enteropathogens, but contributes to ageing-associated epithelial dysfunction, highlighting the importance of tightly controlled interactions between blood cells and stem cells in this tissue. Nevertheless, where haemocytes themselves are required for normal lifespan, loss of haemocyte-derived DPP does not impact lifespan. One interpretation of this finding is that beneficial (improved gut homeostasis) and deleterious (for example, reduced immune competence of the gut epithelium) consequences of reduced haemocyte-derived DPP cancel each other out over the lifespan of the animal. It will be interesting to test this hypothesis in future studies. Ageing is associated with systemic inflammation, and a role for immune cells in promoting inflammation in ageing vertebrates has been proposed. In humans, recruitment of immune cells to the gut is required for proper stem cell proliferation in response to luminal microbes, and prolonged inflammatory bowel disease further contributes to cancer development. It is thus anticipated that conserved macrophage/stem cell interactions influence the aetiology and progression of such diseases. The data confirm a role for haemocytes in age-related intestinal dysplasia in the fly intestine, and provide mechanistic insight into the causes for this deregulation. It can be anticipated that similar interactions between macrophages and intestinal stem cells may contribute to the development of IBDs, intestinal cancers, and general loss of homeostasis in the ageing human intestine (Ayyaz, 2015).

Myostatin-like proteins regulate synaptic function and neuronal morphology

Growth factors of the TGF-beta superfamily play key roles in regulating neuronal and muscle function. Myostatin (or GDF8) and GDF11 are potent negative regulators of skeletal muscle mass. However, expression of both Myostatin and its cognate receptors in other tissues, including brain and peripheral nerves, suggests a potential wider biological role. This study shows that Myoglianin (MYO), the Drosophila homolog of Myostatin and GDF11, regulates not only body weight and muscle size, but also inhibits neuromuscular synapse strength and composition in a Smad2-dependent manner. Both Myostatin and GDF11 affected synapse formation in isolated rat cortical neuron cultures, suggesting an effect on synaptogenesis beyond neuromuscular junctions. This study also shows that Myoglianin acts in vivo to inhibit synaptic transmission between neurons in the escape response neural circuit of adult flies. Thus, these anti-myogenic proteins act as important inhibitors of synapse function and neuronal growth (Augustin, 2017).

Growth factors regulate many aspects of tissue development, growth and metabolism. Myostatin and GDF11 are highly homologous members of the TGF-β superfamily of growth factors. While GDF11 plays a role in a variety of systems, the role of Myostatin appears to be confined to skeletal and cardiac muscles (Augustin, 2017).

Despite the previously described roles of MYO in neural remodelling and synapse refinement (Awasaki, 2011; Yu, 2013) very little is known about the impact of Myoglianin on synaptic physiology. This study first established muscle-derived MYO as a negative regulator of both spontaneous and evoked response at the NMJ, demonstrating its role as a broad regulator of synaptic transmission. The highly coordinated apposition of active zones and glutamate receptors underlies their ability to regulate synaptic strength and plasticity of the larval NMJ. This study has shown that muscle expression of myo inversely affects the NMJ quantity of Brp and GluRIIA, critical pre-and post-synaptic proteins, and determinants of evoked neurotransmitter release and quantal size (i.e., postsynaptic sensitivity to presynaptically released transmitter), respectively. While it is possible that MYO exerts its influence on synaptic strength through other mediators, GluRIIA and Brp are their likely downstream effectors. The electrophysiological results, obtained using the GAL4-UAS system for targeted manipulation of myo, differ from the ones obtained recently using a genetic null myo mutant showing slightly reduced miniature amplitudes (Kim, 2014). The likely explanation is that compensatory effects happen in other tissues in the tissue-specific knockdown animals that cannot occur in genetic nulls, especially for systemic type factors. The other possible explanation is differential cross regulation between different (MYO-like) ligands in genetic null vs tissue knockdown animals. These results thus indicate the relevance of tissue specificity of MYO action, and of myo expression levels, in regulating synaptic function, and emphasize the need for caution when interpreting results from different types of gene manipulations (Augustin, 2017).

myo expression was detected in the glial cells of the larval neuromuscular junction. While Drosophila NMJ contains at least 2 subtypes of glia, myo expression appears confined to the 'repo-positive' subtype both in the central (Awasaki, 2011) and peripheral nervous system. The dual muscle and glial presence makes MYO ideally positioned for regulating NMJ function. Due to the small size of the compartment, however, glia-derived MYO likely has a modulatory role at the neuromuscular junction (Augustin, 2017).

This study also found that muscle suppression of Myoglianin, a Drosophila homolog of Myostatin and GDF11, promotes increased larval weight and body-wall muscle size in developing larvae, resembling the effect of Myostatin knockdown in mammals. Interestingly, pan-glial expression of myo negatively affected larval wet weight, but not the size of somatic myofibers, suggesting previously unsuspected systemic roles for glial cells (Augustin, 2017).

Smad2 is a mediator of MYO action on both evoked response and postsynaptic sensitivity, with MAD having a minor effect on the latter. While MAD primarily functions as a cytoplasmic transducer of BMP signalling, it has been demonstrated that, under certain conditions, MAD can be phosphorylated in response to Activin pathway activation (Peterson, 2012; Augustin, 2017 and references therein).

This study detected elevated levels of phosphorylated Akt and GSK-3/Shaggy in larval somatic muscles of animals with reduced myo expression in this tissue. In flies and mammals, the Akt- mTOR axis promotes skeletal muscle growth, and phosphorylation-induced inhibition of GSK-3/Shaggy induces hypertrophy in skeletal myotube. The effects of attenuated myo expression on larval tissue size, however, do not appear to be mediated by Smad2 (or MAD) activation as their overexpression does not reverse the weight phenotype in 'low myo' background. Indeed, 'non-Smad' signalling pathways have been demonstrated for various TGF-β ligands in vertebrates and Drosophila. In addition to its role as an inhibitor of the NMJ growth and active zone formation in developing Drosophila larvae, GSK-3β is also a critical promoter of synaptic plasticity, possibly through regulation of glutamate receptor function or trafficking. This work has revealed Shaggy as a mediator of reduced MYO action, and as a negative regulator of synaptic strength at the larval NMJ. While MYO likely affects both sides of the synapse directly, an unlikely but possible scenario is that presynaptic motoneuron responds to a retrograde signal released from muscle/glial cells at the NMJ in response to an induction by MYO. An attractive hypothesis is that MYO negatively regulates presynaptic release directly, in conjunction with muscle-secreted Gbb, a positive regulator of neuromuscular synapse development and growth. The effects of MYO could also be mediated through the transmembrane protein Plum, previously proposed to regulate connectivity at the larval NMJ by sequestrating Myoglianin (Yu, 2013; Augustin, 2017 and references therein).

Myostatin negatively regulates synaptic function and neuronal morphology This study found that injections of Myostatin into rapidly growing larvae abolish the positive effect of myo down-regulation on NMJ strength and composition, and reverse the elevated muscle p-Akt levels. Furthermore, both Myostatin and GDF11 surpressed the growth of neuronal processes and perturbed the formation of synapses in cultured brain neurons, suggesting a direct action on neurons and regulation of synaptogenesis beyond neuromuscular junctions. Recently, Myostatin transcript and protein were detected in the mouse hippocampus and olfactory system neurons, respectively, and Myostatin type I (Alk4/5) and type II (ActIIB) receptors were found to be expressed in the mammalian nervous system. The current results therefore expand on these findings, suggesting functional relevance for Myostatin in both peripheral and central nervous system, and beyond its action as a canonical regulator of skeletal muscle growth. These novel roles remain to be further explored (Augustin, 2017).

This study expanded analysis of the functional relevance of MYO in the nervous system by demonstrating its importance in a non-NMJ synapse. Specifically, Myoglianin plays a role in the development of a mixed electro-chemical synapse in the Drosophila escape response pathway, likely by regulating the density of shakB-encoded gap junctions at the GF-TTMn synapse (Blagburn, 1999). These findings implicate MYO as a broad negative regulator of neuronal function across the nervous system and developmental stages (Augustin, 2017).

This work thus reveals broad and novel roles for anti-myogenic TGF-β superfamily of proteins in the nervous system and suggests new targets for interventions into synaptic function across species (Augustin, 2017).

Effects of Mutation

Tiling of R7 axons in the Drosophila visual system is mediated both by transduction of an activin signal to the nucleus and by mutual repulsion

The organization of neuronal wiring into layers and columns is a common feature of both vertebrate and invertebrate brains. In the Drosophila visual system, each R7 photoreceptor axon projects within a single column to a specific layer of the optic lobe. The restriction of terminals to single columns is refered as tiling. In a genetic screen based on an R7-dependent behavior, the Activin receptor Baboon and the nuclear import adaptor Importin-α3 were identified as being required to prevent R7 axon terminals from overlapping with the terminals of R7s in neighboring columns. This tiling function requires the Baboon ligand, dActivin, the transcription factor, dSmad2, and retrograde transport from the growth cone to the R7 nucleus. It is proposed that dActivin is an autocrine signal that restricts R7 growth cone motility, and it was demonstrated that dActivin acts in parallel with a paracrine signal that mediates repulsion between R7 terminals (Ting, 2007).

Previous anatomical studies have highlighted two prominent features of neuropil organization in the fly visual system: (1) the axons of most neuron classes arborize in characteristic layers of the brain and (2) they then remain restricted either to one column or a small number of adjacent columns. To gain insight into the developmental mechanisms that regulate these aspects of axon targeting, focus was placed on the R7 photoreceptor neuron. Previous studies have characterized mechanisms controlling the precise layer termination of R7 growth cones. This paper analyzed the mechanisms that restrict R7 terminals to the correct columns. The latter process is regulated by two partially redundant pathways: a paracrine signal that mediates repulsion between adjacent R7 axons and an autocrine Activin signal that is transduced by retrograde transport and import of the transcription factor Smad2 into the nucleus by a component of the classical nuclear import pathway, Importin-α3 (Ting, 2007).

A prominent organizing feature of the medulla is the restriction of axons and their terminals, including those of R7, R8, and the lamina monopolar neurons (except L4), to single columns. This phenomenon is similar to the tiling of processes observed in both the peripheral and central nervous systems. Ablation experiments in both fly and zebrafish support the view that repulsive interactions between processes of different cells of the same class prevent overlap of dendritic and axonal receptive fields. Consistent with this model, wild-type R7 terminals only invade adjacent columns from which R7s have been removed. However, an R7 axon does not invade even empty columns unless it is under 'competitive pressure' from additional R7 axons within its own column, suggesting that a second, intrinsic mechanism also restricts R7 terminals (Ting, 2007).

Activin signaling is required for tiling of R7 terminals: loss of babo, dSmad2, or dActivin causes R7 axons to invade adjacent occupied targets. Because these mutant axons invade even when they are not under competitive pressure, it is hypothesized that Activin affects an intrinsic property of R7 terminals such as their motility or ability to initiate synaptogenesis. In support of this model, it was found that virtually all R7 axons lacking babo, dSmad2, or dActivin initially extend filopodia beyond the R7-temporary layer at 17 hr APF, although these retract by 40 hr APF, and expression of a constitutively active Baboon in R7s affects growth cone morphology. Although models in which Activin signaling also mediates repulsion among R7 terminals cannot be ruled out, R7s unable to respond to Activin are still partly repelled by their neighbors, indicating the existence of repulsive mechanisms that are redundant with Activin. Recent studies have demonstrated that Dscam2 mediates repulsion between L1 growth cones in a layer immediately distal to the R7 terminals (Millard, 2007). Because Dscam2 is not expressed in R7, other cell-surface proteins must mediate the repulsive interactions between adjacent R7 terminals. The identification of Activin's involvement in R7 tiling paves the way to identifying such molecules by allowing the removal of a pathway that is functionally redundant with them (Ting, 2007).

Members of the TGF-β superfamily have been widely implicated in regulating axon guidance and synaptogenesis by both transcription-dependent and -independent mechanisms. This study found that loss of dSmad2 from R7s resembles loss of Activin, suggesting that the tiling of R7 terminals requires changes in transcription. In support of this model, it was found that restriction of R7 terminals also requires imp-α3, which is required for the accumulation of dSmad2 in R7 nuclei. Whereas some previous vertebrate studies have suggested that individual Smads are imported by Importin-α, others argue instead that active Smad complexes can enter the nucleus by an importin-independent mechanism. The results provide the first genetic evidence that Smad function can require Importin-α-mediated nuclear import and may help reconcile previous results by demonstrating that different cell types import dSmad2 by different mechanisms (although R7s require imp-α3, pigment cells do not). It is noted that imp-α3 mutant R7s have more frequent defects in tiling than babo or dSmad2 mutant R7s, suggesting that imp-α3 may transport additional nuclear proteins that, redundantly with the Activin pathway, restrict R7 terminals (Ting, 2007).

In addition to their classical nuclear import function, Importins have been implicated in mediating retrograde transport of signals from growth cones to the nucleus. Both Imp-α3 and dSmad2 are found throughout the length of R7 axons, and like loss of Activin signaling, disrupting retrograde axonal transport affects the intrinsic mechanism that restricts R7 terminals. These results are consistent with a model in which the Activin signal is received by R7 growth cones, and dSmad2 bound to Imp-α3 is transported through the axon and ultimately into the nucleus (Ting, 2007).

Surprisingly, Activin appears to be required in the R neurons and likely in R7s themselves: disrupting Activin in all R neurons causes R7 terminals to invade adjacent targets, and among the photoreceptors, only R7 and R8 express Activin. An attempt to test whether Activin is specifically required in R7s met with only partial success: sevenless-Gal4 (sev-Gal4) was used to express UAS-ActivinDN in R7s but not R8s and it was found that the resulting R7s temporarily overshoot their initial target layer, a phenotype also caused by loss of Babo or dSmad2. Thus, Activin can function as an autocrine effector. Unfortunately, the sev-Gal4 driver is no longer expressed by 40–50 hr APF, the time at which Activin prevents R7s from invading adjacent columns, and sev-Gal4/ActivinDN R7s appear normal at this time point (Ting, 2007).

Nonetheless, the finding that R7s and/or R8s are the source of Activin raises two mechanistic questions. First, if the R7 or R8 neurons themselves are providing the signal and the signal is simply transduced into the R7 nucleus, why might Activin, as has been argued, be secreted in the target region and received by the R7 growth cone (i.e., rather than being secreted and received by the cell body)? One possibility is that R7s use Activin to coordinate their developmental program with that of other cells within the medulla. For example, one could imagine that both R7 growth cones and their postsynaptic targets would encounter the Activin signal in the medulla at the same time, allowing them to coordinate their preparations for mutual synaptogenesis. A second question is, therefore, how might Activin signaling be coordinated with the R7 growth cones' arrival in the medulla? It was found that the Activin-processing enzyme Tolloid-related (Tlr) is located both at the R7-temporary target layer (at 17 hr APF) and at the final R7 target layer (at 50 hr APF) and that Tlr mutants exhibit severe R7 retinotopic map defects. One possibility is therefore that the medulla localization of Tlr might confer spatial and temporal specificity on Activin expressed by R7 and/or R8 (Ting, 2007).

Although Activin is also expressed in R8s, no evidence was found that Activin affects R8 axons, as shown by the fact that neither babo mutant R8s nor R8s expressing GMR-Gal4/UAS-ActivinDN exhibited connectivity defects. However, the possibility cannot be ruled out that redundancy obscures such a role (for example, there is no straightforward method of removing adjacent R8s) (Ting, 2007).

In the mushroom body, dActivin signaling results in upregulation of the ecdysone receptor gene, EcR-B1. Although EcR-B1 is expressed in essentially all photoreceptor neurons, three lines of evidence indicate that EcR-B1 is not the target gene of Activin signaling in R7s: the expression level of EcR-B1, as judged by anti-EcR-B1 staining, was not altered in babo mutant R7 clones; forced expression of EcR-B1 did not rescue babo mutant R7 defects; and USP R7 mutants did not phenocopy babo. In the dorsal cluster of Atonal-positive neurons, Babo-mediated signaling, via an EcR-independent pathway, mediates morphogenesis and axonal extension. The versatility of Activin signaling likely reflects its ability to regulate the expression of different genes in a context-dependent manner. It is speculated that dActivin signaling activates a transcriptional program that not only restricts growth cone motility once R7s are within their target layer but also promotes synaptogenesis. Such a model could explain the observed strong defects in R7-mediated behavior despite the infrequent specific defects in R7 tiling. Identifying the transcriptional targets of Activin signaling in R7s will likely provide insight into these processes (Ting, 2007).

Neuroendocrine regulation of Drosophila metamorphosis requires TGFβ/Activin signaling

In insects, initiation of metamorphosis requires a surge in the production of the steroid hormone 20-hydroxyecdysone from the prothoracic gland, the primary endocrine organ of juvenile larvae. This study shows that blocking TGFβ/Activin signaling, specifically in the Drosophila prothoracic gland (PG), results in developmental arrest prior to metamorphosis. The terminal, giant third instar larval phenotype results from a failure to induce the large rise in ecdysteroid titer that triggers metamorphosis. It was further demonstrated that activin signaling regulates competence of the prothoracic gland to receive prothoracicotropic hormone (PTTH) and insulin signals, and that these two pathways act at the mRNA and post-transcriptional levels, respectively, to control ecdysone biosynthetic enzyme expression. This dual regulatory circuitry may provide a cross-check mechanism to ensure that both developmental and nutritional inputs are synchronized before initiating the final genetic program leading to reproductive adult development. As steroid hormone production in C. elegans and mammals is also influenced by TGFβ/Activin signaling, this family of secreted factors may play a general role in regulating developmental transitions across phyla (Gibbons, 2011).

Previous work in a number of holometabolous insects, including Drosophila, has highlighted the importance of the PTTH and insulin signaling pathways in stimulating 20E production in the PGs to trigger metamorphosis. This study demonstrates that PG competence to respond to these two essential metamorphic stimuli in Drosophila is crucially dependant on TGFβ/Activin signaling, which controls production of the primary receptors for these two pathways. The ability of activins to act as either direct or indirect permissive signals in the PG is interesting as competence factors have long been thought to play a central role in modulating cellular responses to hormonal signals and one generally recognized mechanism through which they function is by regulating the expression of receptors for a variety of signals. In the mammalian ovary, Activin has been shown to induce the expression of the receptor for follicle stimulating hormone (FSH) in rat granulosa cells, contributing to the stage-specific response of the developing follicles to FSH stimulation. It is also interesting to note in this regard that in Bombyx, the prothoracic gland, has been shown to be refractory to PTTH signals at certain stages and that Torso (the PTTH receptor) levels fluctuate dramatically during the 5th instar stage, potentially accounting for the lack of PTTH response at certain times. At present it is not known whether Torso or InR levels in the PG fluctuate during normal Drosophila larval development, nor is it know whether activins play a role in regulating Torso and InR levels in response to some specific timing or nutritional cue. It is possible that activins simply impart constitutive expression of these receptors in the PG as part of a general developmental program responsible for endowing the PG cells with their steroidogenic capacity. As the general morphological features of the PG cells are not perturbed by Activin signal knockdown, even when using the phantom-Gal4 driver, which is activated in the early embryo, it is not suspected that activins are required for specification of the gland itself. However, this conclusion must be tempered by the fact that RNAi knockdown is probably not complete and the phantom-Gal4 driver may not turn on until after gland formation is largely finished. To fully rule out a role of activins in gland specification, null germline clones for babo or dSmad2, encoding respectively for a receptor and signaling protein of the activin pathway, need to be analyzed, and at this time it is not clear that any such alleles exist for either gene (Gibbons, 2011).

The lack of true null mutations in both babo and dSmad2 may also account for the stronger phenotype produced by PG-specific knockdown of babo or dSmad2 compared with the reported babo genetic loss-of-function phenotype. Previous studies examining the phenotype of babo zygotic mutants revealed a 2-3 day developmental delay in puparium formation, but up to 30% of the mutant larvae do initiate metamorphosis. By contrast, elimination of activin signal reception only in the PG produces a stronger phenotype where virtually 100% of the knockdown larvae arrest development without puparium formation. Although residual Babo function is a possible explanation for this difference, it is pointed out that another explanation exists that highlights the difficulties associated with interpreting tissue-specific knockdown experiments. It is theoretically possible to produce a stronger whole animal phenotype by tissue-specific knockdown than by a genetic null mutation, owing to potential compensatory changes in other mutant tissues in the genetically null animal. This is especially true when the factor potentially regulates multiple systemic signals in different tissues. For example, the ligand Actβ is produced in insulin-producing cells (IPCs) of the brain. If Actβ negatively regulates insulin production or secretion, then, in a babo genetic null background, upregulation of insulin signal upon loss of Actβ signaling in IPCs may compensate for reduced insulin reception capacity in the PGs. This compensatory mechanism may enable a percentage of the babo mutant larvae to undergo puparium formation. By contrast, knockdown of activin signal reception in the PG alone, as in the case of phantom>babo RNAi animals, would probably not lead to this compensatory response and thus results in a stronger developmental arrest phenotype. Consistent with this view, it was found that using a ubiquitous driver, such as daughterless-Gal4, to knockdown dSmad2 in all tissues, including the PG, does not lead to developmental arrest, whereas using a PG specific driver does. These potential complications in evaluating phenotypic differences obtained using genetic mutations and tissue-specific knockdown methods should be borne in mind as they are likely to be observed more frequently with the increasing use of tissue-specific knockdown analyses in numerous model organisms (Gibbons, 2011).

The issue of which activin-like ligand(s) are responsible for providing the competence signal and whether they are regulated by particular developmental or nutritional cues is also important to answer but is problematic because of redundancy concerns. It is suspected that Actβ probably plays a role as it is expressed in numerous neurosecretory cells including the insulin-producing cells (IPCs), which innervate the heart tube and thereby probably provide systemic delivery of this ligand to many tissues. In addition, overexpression of this ligand in the PG causes stage precocious pupation, similar to that produced by expression of activated the activin receptor Babboon (Babo). However, the one available Actβ loss-of-function mutation does not produce substantial developmental delay, and the majority of larvae (>90%) instead undergo slightly precocious puparation consistent with potential upregulation of insulin signaling, as suggested above. Likewise, mutations in daw, a second Activin-like ligand that is produced in the PG, do not elicit major developmental delay when fed a yeast-enriched diet. However, when strong daw alleles are combined with the one available Actβ mutation, then only 25-35% of the larvae are able to initiate pupariation on rich food. This observation suggests a functional redundancy between these ligands for regulating developmental timing, similar to their previously noted redundant roles in regulating neuroblast proliferation in the larval brain (Zhu, 2008). The residual pupation ability of the daw-Actβ double mutants may be accounted for by the compensation mechanism described above or further functional redundancy provided by the two other Activin-like ligands, Myo and Mav. At present, no mutations are available in these genes (Gibbons, 2011).

Although these observations clearly show that loss of ecdysone (E) biosynthetic enzyme expression underlies the dSmad2 developmental arrest phenotype, it cannot be said with certainty that this downturn is due solely to the loss of insulin and PTTH signaling or whether dSmad2 might also participate directly in regulating biosynthetic enzyme gene expression. In addition, whether dSmad2 binds directly to target sequences within Torso and InR regulatory sequences also remains uncertain as no dSmad2 responsive elements have been identified for any gene in Drosophila. It is interesting to note, however, that one other molecular process, SUMOlyation, has recently been implicated in regulating E biosynthetic enzyme expression and localization in the PG (Talamillo, 2008). Knockdown of smt3, the sole SUMO-encoding gene in Drosophila, in the PG results in third instar larval arrest phenotype that is rescuable by feeding the larvae 20E. In addition, these larvae show low levels of Disembodied (Dib) protein accumulation in the PG (Talamillo, 2008). These phenotypes are strikingly similar to those seen in dSmad2 RNAi larvae. Interestingly, previous studies have demonstrated that Medea can be SUMOlyated in vitro, providing a potential link between SUMOlyation and Activin signaling. However, no SUMOylated forms of Medea or dSmad2 have been detected under various signaling conditions in vivo or in S2 cells. Additional studies will be required to determine whether SUMOlyation and Activin signaling are linked in a common pathway or whether they act through independent means to control steroid production in the PG (Gibbons, 2011).

Despite the uncertainty in determining whether one or more ligands act as a PG competence factor, the observation that TGFβ/Activin signaling regulates metamorphosis may highlight an ancient and conserved role for these factors in regulating developmental transitions in many organisms. For example, the nematode worm C. elegans employs the TGFβ pathway to make a nutritionally dependent decision on whether to continue the normal development program into a mature adult or to enter a developmentally arrested stage known as dauer. The activation of the TGFβ pathway promotes normal development whereas its inactivation results in dauer formation (for a review, see Fielenbach, 2008). Interestingly, a key target of the TGFβ pathway is daf-9, which encodes a cytochrome P450 protein. DAF-9 is involved in the synthesis of dafachronic acid, an ecdysone-like steroid hormone that prevents the developmental arrest as a dauer. Likewise, in mammals, Activin signaling probably affects pubertal timing by controlling sex steroid synthesis. Activin signaling can stimulate the production of the sex steroid hormone estradiol by enhancing the activity of CYP 450 aromatase. These observations from C. elegans and mammalian studies, together with the finding that Drosophila Activin signaling also regulates cytochrome P450 expression, although probably by indirect means, strongly indicate that TGFβ/Activin signaling is a common means by which developmental transitions are regulated across species (Gibbons, 2011).

Previously, it was found that complete loss of PTTH signaling delayed metamorphosis but did not completely block it (McBrayer, 2007). This observation led to a speculation that a second metamorphic signal, perhaps supplied by insulin, eventually facilitated pupation in the PTTH negative larvae. The finding in this study that simultaneous knockdown of both pathways by eliminating dSmad2 in the PG leads to third instar developmental arrest and that resupplying activity in either pathway promotes metamorphosis is consistent with this idea. Even more intriguing is this finding that the two metamorphic signals provided by PTTH and insulin appear to regulate the steroid biosynthetic capacity of the gland in two distinct ways: PTTH at the level of biosynthetic enzyme mRNA accumulation and insulin at the biosynthetic enzyme protein level (see Model for TGFβActivin regulation of metamorphosis). The observation that loss of PTTH signal reception in dSmad2 PG knockdown larvae reduces the steady-state mRNA levels of the E biosynthetic enzymes is consistent with earlier PTTH-neuron ablation results with one exception. In the present studies, downregulation of only dib, spok and nvd was obaserved in response to reduction in Torso expression, whereas in PTTH-neuron ablated larva, transcription of phantom and sad are also reduced (McBrayer, 2007). One possible explanation for this discrepancy is that the five biosynthetic enzymes exhibit different sensitivities to the strength of the PTTH signal. In PTTH-neuron ablation experiments, all signaling is lost, whereas in the dSmad2 knockdown, it is likely that some Torso expression remains and provides enough signal to activate phantom and sad. Consistent with this view, it was found that in the rescued larvae, where PTTH signaling was resupplied in the PG using activated Ras, the mRNA levels of the phantom and sad genes are dramatically upregulated compared with the other three E biosynthetic enzymes, indicating they are very sensitive to the PTTH signal (Gibbons, 2011).

The effects of insulin signaling on protein levels is intriguing and has not been examined previously, although there are two reports to indicate that insulin signaling also affects transcription of at least two biosynthetic enzymes, dib and phantom. However, the reported effects were modest (between 30% to 2 fold) and it was not clear whether the determinations were normalized for differences in body size and/or ring gland size, which are crucial when considering the small reported differences. In the current measurements, ring gland-brain complexes were used that were of similar size and staging, and no differences were detected in transcription of biosynthetic enzymes between the dSmad2 knockdown animals and those in which InR expression was restored to the PG. Instead, significant differences were observed in biosynthetic enzyme protein levels. The known role of insulin in modulating translational capacity of cells is consistent with the idea that these changes in biosynthetic enzyme levels are the result of translational differences. Whether this effect is through Tor, which has been shown to be an important mediator of developmental timing in the PG, and its modulation of S6 kinase, remains to be determined. In addition, effects on protein stability cannot be excluded, and further studies examining protein turnover will also be required to address this issue fully. Overall, this two-tiered regulatory control of biosynthetic enzyme expression may better enable the larva to fine-tune its ecdysone level depending on conditions, or perhaps may serve as a coincidence detector to ensure that both developmental and nutritional conditions are appropriate before triggering a terminal developmental program such as metamorphosis (Gibbons, 2011).

Anterograde Activin signaling regulates postsynaptic membrane potential and GluRIIA/B abundance at the Drosophila neuromuscular junction

Members of the TGF-beta superfamily play numerous roles in nervous system development and function. In Drosophila, retrograde BMP signaling at the neuromuscular junction (NMJ) is required presynaptically for proper synapse growth and neurotransmitter release. This study analyzed whether the Activin branch of the TGF-beta superfamily also contributes to NMJ development and function. Elimination of the Activin/TGF-beta type I receptor babo, or its downstream signal transducer smox, does not affect presynaptic NMJ growth or evoked excitatory junctional potentials (EJPs), but instead results in a number of postsynaptic defects including depolarized membrane potential, small size and frequency of miniature excitatory junction potentials (mEJPs), and decreased synaptic densities of the glutamate receptors GluRIIA and B. The majority of the defective smox synaptic phenotypes were rescued by muscle-specific expression of a smox transgene. Furthermore, a mutation in actβ, an Activin-like ligand that is strongly expressed in motor neurons, phenocopies babo and smox loss-of-function alleles. These results demonstrate that anterograde Activin/TGF-beta signaling at the Drosophila NMJ is crucial for achieving normal abundance and localization of several important postsynaptic signaling molecules and for regulating postsynaptic membrane physiology. Together with the well-established presynaptic role of the retrograde BMP signaling via Glass bottom boat and Wishful thinking, these findings indicate that the two branches of the TGF-beta superfamily are differentially deployed on each side of the Drosophila NMJ synapse to regulate distinct aspects of its development and function (Kim, 2014).

Numerous reports have now implicated the Activin/TGF-β and BMP branches of the TGF-β superfamily in regulating neuronal development, synaptic plasticity and cognitive behavior. Accordingly, members from both subfamilies are widely expressed in the nervous system and are co-expressed in multiple regions of vertebrate and invertebrate brains. It is therefore quite likely that ligands of both subfamilies co-exist within the extracellular space and in some cases, act on the same neurons. Lending support to this idea, pyramidal neurons in the CA3 region of the rat hippocampus are known to accumulate both phosphorylated Smad2 and Smad1/5/8, transcriptional transducers of the canonical Activin/TGF-β and BMP-type signaling, respectively. The activation of these two closely-related signaling pathways in common sets of neurons, or different cells of a common neuronal circuit raises the intriguing question of whether the two pathways play different or redundant roles during neuronal development and function (Kim, 2014).

This study utilized the Drosophila neuromuscular junction to address this issue since ligands of both Activin/TGF-β and BMP families are expressed in both muscle and motor neurons. The data, together with previous studies on the role of BMP signaling at the NMJ, clearly demonstrate that the two pathways influence NMJ synaptogenesis in different ways. The Activin/TGF-β pathway is necessary for achieving the proper densities of GluRIIA, GluRIIB and Dlg in postsynaptic muscle membrane, while the BMP pathway has a smaller effect on the distribution of these postsynaptic proteins. In addition, the Activin/TGF-β pathway was dispensable for maintaining overall synaptic growth and homeostasis, both of which are strongly affected by mutations in the BMP pathway. In addition, tissue-specific rescue experiments indicate that the postsynaptic reception of Activin/TGF-β signaling is important in regulating synaptic GluR abundance, whereas BMP signal reception is known to act in the presynaptic motor neurons to promote synaptic growth. These observations suggest that each pathway influences NMJ synapse development and function by acting mainly in either the pre- or postsynaptic cell (Kim, 2014).

Interestingly, the BMP and Activin/TGF-β pathways have also been recently found to control different aspects of the Drosophila innate immune response (Clark, 2011). In this case BMP signaling suppresses the expression of multiple antimicrobial peptide genes following wounding, whereas the Activin/TGF-β pathway limits melanization after bacterial infection in adult flies. Therefore, it appears that the division of labor between these subpathways is not limited to just the nervous system, rather it may be the norm when these related signaling pathways act in concert to regulate a common biological process (Kim, 2014).

The fact that the pathways actually differ in how they affect a complex biological process is not surprising given that the different R-Smads are likely to have different selectivity in gene activation. Within motor neurons, BMP signaling promotes microtubule formation in axons and directly regulates expression of trio, a Rac GEF, that acts as a major regulator of actin cytoskeleton in many types of cells. Thus, it is likely that BMP signaling modulates synaptic growth, in part, by changing the structure and dynamics of the actin and microtubule cytoskeleton within motor neurons. BMP signaling also regulates the transcription of twit, a gene encoding a L-6 neurotoxin-like molecule that controls the frequency of presynaptic spontaneous vesicle release (Kim, 2012; Kim, 2014 and references therein).

Targets of Drosophila Activin/TGF-β signaling in any tissue are less well characterized. Within the central brain, glial-derived Myo signals through Smox to control expression of the Ecdysone B1 receptors in remodeling mushroom body neurons. However, it is not clear if EcR-B1 is a direct or indirect target of smox transcriptional regulation. It is also unclear if Ecdysone signaling plays a role in regulating synaptogenesis at the NMJ, although it may play a role during metamorphic remodeling of the NMJ as it does for the mushroom body neurons. The only other known targets of Smox are InR, Pi3K and Akt, all of which are Insulin signaling components and are reduced in the Drosophila prothoracic gland in the absence of Activin/TGF-β signaling. Once again the effect may be indirect, but this finding is interesting since Insulin signaling components have been shown to control synaptic clustering of GluRs (Kim, 2014).

The clustering of GluRs and Dlg at the NMJ have been shown to be regulated by both transcriptional and post-transcriptional mechanisms. For example, a recent genetic screen identified longitudinals lacking (lola), a BTN-Zn finger transcription factor, as an essential regulator of GluR and dPak expression in muscles. In contrast, the current studies on Activin/TGF-β signaling suggest, at least for GluRIIA, that this pathway functions at the post-transcriptional level since this study found that overexpression of glurIIA-gfp using an exogenous promotor and transcriptional activator does not lead to an enrichment of GluRIIAGFP at synaptic sites of Activin/TGF-β pathway mutants. This phenotype is reminiscent of that found for certain mutants in the NF-κB signaling system. Loss of Dorsal (an NF-κB homolog), Cactus (an IκB related factor), or Pelle (an IRAK kinase) leads to a substantial reduction of GluRIIA and a slight reduction of Dlg postsynaptic localization at the NMJ and a concomitant reduction in mEJP size. In addition, as was found for loss of Activin/TGF-β signaling, exogenously-expressed GluRIIA-myc did not reach the synaptic surface in NF-κB signaling mutants consistent with a possible role of Activin/TGF-β signaling in regulating NF-κB signaling. However, even if future studies show that the relationship is true, the Activin/TGF-β pathway likely regulates additional factors since its loss also affects GluRIIB levels and muscle resting potential, neither of which is altered in NF-κB pathway mutants. Interestingly, the regulation of GluRIIB levels by Activin/TGF-β signaling does appear to be at the level of transcription, indicating that this signaling pathway likely affects GluR clustering at the NMJ via both transcriptional and post-transcriptional mechanisms (Kim, 2014).

Analysis of Activin/TGF-β signaling at the NMJ, coupled with previous studies on BMP signaling and the novel ligand Maverick, indicates that TGF-β ligands are produced in, and act upon, all three cell types that contribute to NMJ function, specifically the motor neuron, wrapping glia, and muscle (see Model of controlling NMJ development and function by Activin/TGF-β and BMP pathways). This leads to the important issue of how directionality of TGF-β signaling at the NMJ is regulated. One possibility is that ligands are sequestered, either inside the secreting cells or on their surfaces, so that they have limited access to receptors on the opposing pre or postsynaptic membrane. For example, Gbb is produced both in muscle and motor neurons, leading to the issue of how directional signaling from muscle to motor neurons is achieved. On the postsynaptic muscle, Gbb release is potentiated by dRich (Rho GTPase activating protein at 92B), a Cdc42 selective Gap while in the presynaptic neuron Crimpy, a Drosophila homolog of the vertebrate Crim1 gene, has been shown to bind to a precursor form of Gbb. The Gbb/Crimpy complex is thought to either interfere with secretion or activation of motor neuron-derived Gbb thus ensuring that only muscle-derived Gbb activates the retrograde BMP signal at the NMJ. Since there are a large number of characterized TGF-β superfamily binding proteins, Drosophila homologs of some of these factors such as the BMP binding proteins Cv-2, Sog, Tsg and Dally, or the Activin-binding protein Follistatin, may sequester and regulate levels of active ligands within the NMJ. Sequestering mechanisms may also provide direction control by facilitating autocrine as opposed to juxtacrine signaling. If ligand-binding proteins are associated with the membrane surface of the ligand-producing cell, they may facilitate delivery of the ligand to receptors on the producing cell, thus enhancing autocrine signaling. It is interesting in this regard that in the developing Drosophila retina, Actβ appears to signal in an autocrine fashion to control photoreceptor connectivity in the brain (Kim, 2014).

Activin-type ligands are secreted from glia, motor neuron and muscle. The Activin-type ligands induce Babo-mediated phosphorylation of Smox that facilitates association with Med. In the muscle, the phospho-Smox/Med complexes activate the transcription of glurIIB and an unknown factor controlling post-transcriptional process or stability of glurIIA mRNA. In the motor neuron, the phospo-Smox/Med complex controls spontaneous release of synaptic vesicles via unknown mechanism(s). On the other hand, glia-secreted Mav stimulates Mad phosphorylation in the muscle resulting in increased gbb transcription. Gbb protein is released from the muscle and binds Tkv/Sax and Wit complex on the motor neuron leading to an accumulation of phospho-Mad in the nuclei by an unknown mechanism. The resultant phospho-Mad/Med complex activates the transcription of trio whose product promotes synaptic bouton formation (Kim, 2014).

Another important mechanism to control signal direction is likely to be tissue-specific receptor expression. For example, Wit is highly enriched in motor neurons compared to muscle, and this may help ensure that Gbb released from the postsynaptic muscle signals to the presynaptic motor neuron. Type I receptor diversity may be even more important in controlling directionality since at least 2 isoforms of Tkv and three isoforms of Babo have been identified. In the case of Babo, Activin-like ligands have a clear preference for signaling through different receptor isoforms, and these isoforms show differential tissue expression (Kim, 2014).

An additional factor to be considered in understanding TGF-β superfamily signal integration within different NMJ cell types is the possibility of canonical versus non-canonical and/or cross-pathway signaling. For example, in mushroom body neurons Babo can signal in a non-Smad dependent manner through Rho1, Rac and LIM kinase1 (LIMK1) to regulate axon growth and target recognition. Whether this mechanism, or another non-canonical pathway is operative at the NMJ is unclear. Cross-pathway signaling has also recently been identified in Drosophila. In this example, loss of Smox protein in the wing disc has been shown to up-regulate Mad activity in a Babo-dependent manner. Double mutants of babo and smox suppress the cross-pathway signal. As is described in this study, smox protein null mutations lead to significantly more severe GluR and mEJP defects than strong babo mutations alone, and this phenotype is suppressed in double mutants. Thus, as in wing discs, loss of Smox protein likely leads to ectopic Mad activity in muscles that further decrease GluR expression and/or localization at the NMJ. Consistent with this view, this study found that loss of Mad actually increases GluRIIB localization, suggesting that Mad acts negatively to regulate GluRIIB in muscle. One possible model to explain the Smox/Mad data is that normally the Babo/Smox signal inhibits Mad signaling which is itself a repressive signal for GluR accumulation. Thus, in babo mutants, total GluR levels decrease due to the loss of smox and therefore an increase in the repressive Mad signal. In the smox protein null mutant even more repressive Mad signal is generated by Babo further hyperactivating Mad activity leading to even lower levels of GluR accumulation. In medea mutants the activity of both pathways is reduced thereby returning the level of GluR levels close to normal. Additional experiments employing various single and double mutants, together with tissue-specific expression of various ligands, receptor isoforms and ligand-binding proteins will be needed to fully elucidate how vectorial TGF-β signaling is accomplished at the NMJ. Likewise, the identifcation of directly responding target genes and how they are influenced by both Smox and Mad signals is needed to fully appreciate how these two TGF-β signaling branches regulates NMJ functional activity (Kim, 2014).


Abdollah, S., et al. (1997). TbetaRI phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling. J. Biol. Chem. 272: 27678-27685. PubMed Citation: 9346908

Abe, T., et al. (2005). Notch signaling modulates the nuclear localization of carboxy-terminal-phosphorylated smad2 and controls the competence of ectodermal cells for activin A. Mech. Dev. 122: 671-680. 15817224

Augustin, H., McGourty, K., Steinert, J. R., Cocheme, H. M., Adcott, J., Cabecinha, M., Vincent, A., Halff, E. F., Kittler, J. T., Boucrot, E. and Partridge, L. (2017). Myostatin-like proteins regulate synaptic function and neuronal morphology. Development 144(13):2445-2455. PubMed ID: 28533206

Awasaki, T., Huang, Y., O'Connor, M. B. and Lee, T. (2011). Glia instruct developmental neuronal remodeling through TGF-beta signaling. Nat Neurosci 14(7): 821-823. PubMed ID: 21685919

Bai, S. and Cao, X. (2002). A nuclear antagonistic mechanism of inhibitory Smads in transforming growth factor-beta signaling. J. Biol. Chem. 277: 4176-4182. 11711531

Batut, J., Howell, M. and Hill, C. S. (2007). Kinesin-mediated transport of Smad2 is required for signaling in response to TGF-beta ligands. Dev. Cell 12(2): 261-74. Medline abstract: 17276343

Blagburn, J. M., Alexopoulos, H., Davies, J. A. and Bacon, J. P. (1999). Null mutation in shaking-B eliminates electrical, but not chemical, synapses in the Drosophila giant fiber system: a structural study. J Comp Neurol 404(4): 449-458. PubMed ID: 9987990

Blokzijl, A., ten Dijke, P. and Ibanez, C. F. (2002). Physical and functional interaction between GATA-3 and Smad3 allows TGF-ß regulation of GATA target genes. Curr. Biol. 12: 35-45. 11790301

Brown, J. D., et al. (1999). MEKK-1, a component of the stress (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, can selectively activate Smad2-mediated transcriptional activation in endothelial cells. J. Biol. Chem. 274(13): 8797-805. PubMed Citation: 10085121

Brummel, T., et al. (1999). The Drosophila activin receptor baboon signals through dSmad2 and controls cell proliferation but not patterning during larval development. Genes Dev. 13: 98-111. PubMed Citation: 9887103

Callery, E. M., Smith, J. C. and Thomsen, G. H. (2005). The ARID domain protein dril1 is necessary for TGF(beta) signaling in Xenopus embryos. Dev. Biol. 278(2): 542-59. 15680369

Candia, A. F., et al. (1997). Cellular interpretation of multiple TGF-beta signals: intracellular antagonism between activin/BVg1 and BMP-2/4 signaling mediated by Smads. Development 124(22): 4467-4480. PubMed Citation: 9409665

Chen, C.-R., et al. (2002). E2F4/5 and p107 as smad cofactors linking the TGFß receptor to c-myc repression. Cell 110: 19-32. 12150994

Chen, X., et al. (1997). Smad4 and FAST-1 in the assembly of activin-responsive factor. Nature 389: 85-89. PubMed Citation: 9288972

Clark, R. I., Woodcock, K. J., Geissmann, F., Trouillet, C. and Dionne, M. S. (2011). Multiple TGF-beta superfamily signals modulate the adult Drosophila immune response. Curr Biol 21: 1672-1677. PubMed ID: 21962711

Collart, C., et al. (2005). The novel Smad-interacting protein Smicl regulates Chordin expression in the Xenopus embryo. Development 132: 4575-4586. 16192311

Cordenonsi, M., et al. (2003). Links between tumor suppressors: p53 is required for TGF-ß gene responses by cooperating with smads. Cell 113: 301-314. 12732139

Crease, D. J., Dyson, S. and Gurdon, J. B. (1998). Cooperation between the activin and wnt pathways in the spatial control of organizer gene expression. Proc. Natl. Acad. Sci. 95(8): 4398-4403. PubMed Citation: 9539748

Cui, X. M., et al. (2005). Overexpression of Smad2 in Tgf-beta3-null mutant mice rescues cleft palate. Dev. Biol. 278(1): 193-202. 15649471

Dai, F., Lin, X., Chang, C. and Feng, X. H. (2009). Nuclear export of Smad2 and Smad3 by RanBP3 facilitates termination of TGF-beta signaling. Dev. Cell 16(3): 345-57. PubMed Citation: 19289081

Daniels, M., et al. (2004). Negative regulation of Smad2 by PIASy is required for proper Xenopus mesoderm formation. Development. 131: 5613-5626. 15496439

Das, P., et al. (1999). Drosophila dSmad2 and Atr-I transmit activin/TGFbeta signals. Genes to Cells 4: 123-134. PubMed Citation: 10320478

Datta, P. K., Blake, M. C. and Moses, H. L. (2000). Regulation of plasminogen activator inhibitor-1 expression by transforming growth factor-beta -induced physical and functional interactions between smads and Sp1. J. Biol. Chem. 275(51): 40014-9. 11054406

de Caestecker, M. P., et al. (1998). Smad2 transduces common signals from receptor serine-threonine and tyrosine kinases. Genes Dev. 12(11): 1587-92. 9620846

Dunn, N. R., et al. (2004). Combinatorial activities of Smad2 and Smad3 regulate mesoderm formation and patterning in the mouse embryo. Development 131: 1717-1728. 15084457

Dunn, N. R., et al. (2005). Mice exclusively expressing the short isoform of Smad2 develop normally and are viable and fertile. Genes Dev. 19(1): 152-63. 15630024

Dutta, D. J., Zameer, A., Mariani, J. N., Zhang, J., Asp, L., Huynh, J., Mahase, S., Laitman, B. M., Argaw, A. T., Mitiku, N., Urbanski, M., Melendez-Vasquez, C. V., Casaccia, P., Hayot, F., Bottinger, E. P., Brown, C. W. and John, G. R. (2014). Combinatorial actions of Tgfβ and Activin ligands promote oligodendrocyte development and CNS myelination. Development 141: 2414-2428. PubMed ID: 24917498

Feng, X. H., et al. (1998). The tumor suppressor Smad4/DPC4 and transcriptional adaptor CBP/p300 are coactivators for smad3 in TGF-beta-induced transcriptional activation. Genes Dev. 12(14): 2153-2163. PubMed Citation: 9679060

Feng, X.-H., Lin, X. and Derynck, R. (2000). Smad2, Smad3 and Smad4 cooperate with Sp1 to induce p15Ink4B transcription in response to TGF-beta. EMBO J. 19: 5178-5193. PubMed Citation: 11013220

Feng, X. H., et al. (2002). Direct interaction of c-Myc with Smad2 and Smad3 to inhibit TGF-ß-mediated induction of the CDK inhibitor p15Ink4B. Mol. Cell 9: 133-143. 11804592

Ferguson, C. A., et al. (2001). The role of effectors of the activin signalling pathway, activin receptors IIA and IIB, and Smad2, in patterning of tooth development. Development 128: 4605-4613. 11714685

Fielenbach, N. and Antebi, A. (2008). C. elegans dauer formation and the molecular basis of plasticity. Genes Dev. 22: 2149-2165. PubMed Citation: 18708575

Fukuchi, M., et al. (2001). Ligand-dependent degradation of Smad3 by a ubiquitin ligase complex of ROC1 and associated proteins. Mol. Biol. Cell 12: 1431-1443. 11359933

Garcia-Campmany, L. and Marti, E. (2007). The TGFbeta intracellular effector Smad3 regulates neuronal differentiation and cell fate specification in the developing spinal cord. Development 134(1): 65-75. Medline abstract: 17138664

Guo, X., et al. (2008). Axin and GSK3-β control Smad3 protein stability and modulate TGF-β signaling. Genes Dev. 22: 106-120. PubMed citation: 18172167

Gao, S., et al. (2009). Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-β signaling. Mol. Cell 36: 457-468. PubMed Citation: 19917253

Gibbens, Y. Y., Warren, J. T., Gilbert, L. I. and O'Connor, M. B. (2011). Neuroendocrine regulation of Drosophila metamorphosis requires TGFβ/Activin signaling. Development 138(13): 2693-703. PubMed Citation: 21613324

Hata, A., et al. (1997). Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4. Nature 388(6637): 82-87

Henderson, K. D. and Andrew, D. J. (1998). Identification of a novel Drosophila SMAD on the X chromosome. Biochem. Biophys. Res. Commun. 252(1): 195-201

Heyer, J., et al. (1999). Postgastrulation Smad2-deficient embryos show defects in embryo turning and anterior morphogenesis. Proc. Natl. Acad. Sci. 96(22): 12595-600.

Hocevar, B. A., et al. (2001). The adaptor molecule Disabled-2 links the transforming growth factorß receptors to the Smad pathway. EMBO J. 20: 2789-2801. 11387212

Hoodless, P. A., et al. (1999), Dominant-negative Smad2 mutants inhibit Activin/Vg1 signaling and disrupt axis formation in Xenopus. Dev. Biol. 207(2): 364-79

Howell, M. and Hill, C. S. (1997). XSmad2 directly activates the activin-inducible, dorsal mesoderm gene XFKH1 in Xenopus embryos. EMBO J. 16(24): 7411-7421

Howell, M., Inman, G. J. and Hill, C. S. (2002). A novel Xenopus Smad-interacting forkhead transcription factor (XFast-3) cooperates with XFast-1 in regulating gastrulation movements. Development 129: 2823-2834. 12050132

Inman, G. J., Nicolas, F. J. and Hill, C. S. (2002). Nucleocytoplasmic shuttling of Smads 2, 3, and 4 permits sensing of TGF-ß receptor activity. Molec. Cell 10: 283-294. 12191474

Ito, Y., et al. (2001). Overexpression of Smad2 reveals its concerted action with Smad4 in regulating TGF-ß-mediated epidermal homeostasis. Dev. Bio. 236: 181-194. 11456453

James, D., Levine, A. J., Besser, D. and Hemmati-Brivanlou, A. (2005). TGFß/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132: 1273-1282. 15703277

Kim, M. J. and O'Connor, M. B. (2014). Anterograde Activin signaling regulates postsynaptic membrane potential and GluRIIA/B abundance at the Drosophila neuromuscular junction. PLoS One 9: e107443. PubMed ID: 25255438

Kim, N. C. and Marques, G. (2012). The Ly6 neurotoxin-like molecule Target of wit regulates spontaneous neurotransmitter release at the developing neuromuscular junction in Drosophila. Dev Neurobiol 72: 1541-1558. PubMed ID: 22467519

Kim, R. H., et al. (2000). A novel Smad nuclear interacting protein, SNIP1, suppresses p300-dependent TGF-ß signal transduction. Genes Dev. 14: 1605-1616. PubMed ID: 10887155

Ku, M., et al. (2005). Positive and negative regulation of the transforming growth factor beta/activin target gene goosecoid by the TFII-I family of transcription factors. Mol. Cell. Biol. 25(16): 7144-57. 16055724

Kurisaki, A., et al. (2001). Transforming growth factor-ß induces nuclear import of Smad3 in an importin-ß1 and Ran-dependent manner. Mol. Biol. Cell 12: 1079-1091. 11294908

Lo, R. S., Wotton, D. and Massague, J. (2001). Epidermal growth factor signaling via Ras controls the Smad transcriptional co-repressor TGIF. EMBO J. 20: 128-136

Lu, J., et al. (2005). SMAD pathway mediation of BDNF and TGFß2 regulation of proliferation and differentiation of hippocampal granule neurons. Development 132: 3231-3242. 15958511

Marquez, R. M., et al. (2001). Transgenic analysis of the Smad family of TGF-ß signal transducers in Drosophila melanogaster suggests new roles and new interactions between family members. Genetics 157: 1639-1648. 11290719

Millard, S. S., et al. (2007). Dscam2 mediates axonal tiling in the Drosophila visual system, Nature 447: 720-724. PubMed citation: 17554308

McBrayer, Z., et al. (2007). Prothoracicotropic hormone regulates developmental timing and body size in Drosophila. Dev. Cell 13: 857-871. PubMed Citation: 18061567

Nakao, A., et al. (1997). TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J. 16(17): 5353-5362

Nishihara, A., et al. (1998). Role of p300, a transcriptional coactivator, in signalling of TGF-beta. Genes Cells 3(9): 613-23

Oh, S. P., et al. (2002). Activin type IIA and IIB receptors mediate Gdf11 signaling in axial vertebral patterning. Genes Dev. 16: 2749-2754. 12414726

Peterson, A. J., Jensen, P. A., Shimell, M., Stefancsik, R., Wijayatonge, R., Herder, R., Raftery, L. A. and O'Connor, M. B. (2012). R-Smad competition controls activin receptor output in Drosophila. PLoS One 7: e36548. PubMed ID: 22563507

Podos, S. D., Hanson, K. K., Wang, Y. C. and Ferguson, E. L. (2001). The DSmurf ubiquitin-protein ligase restricts BMP signaling spatially and temporally during Drosophila embryogenesis. Dev. Cell 1: 567-578. 11703946

Prunier, C., et al. (1999). Evidence that Smad2 is a tumor suppressor implicated in the control of cellular invasion. J. Biol. Chem. 274: 22919-22922. PubMed ID: 10438456

Qin, B. Y., et al. (2002). Smad3 allostery links TGF-ß receptor kinase activation to transcriptional control. Genes Dev. 16: 1950-1963. 12154125

Randall, R. A., et al. (2002). Different Smad2 partners bind a common hydrophobic pocket in Smad2 via a defined proline-rich motif. EMBO J. 21: 145-156. 11782434

Ring, C., et al. (2002). The role of a Williams-Beuren syndrome-associated helix-loop-helix domain-containing transcription factor in activin/nodal signaling. Genes Dev. 16: 820-835. 11937490

Schohl, S. and Fagotto, F. (2002). ß-catenin, MAPK and Smad signaling during early Xenopus development. Development 129: 37-52. 11782399

Serpe, M. and O'Connor, M. B. (2006). The metalloprotease Tolloid-related and its TGF-ß-like substrate Dawdle regulate Drosophila motoneuron axon guidance. Development 133: 4969-4979. Medline abstract: 17119021

Shimizu, A., et al. (1998). Identification of receptors and Smad proteins involved in activin signalling in a human epidermal keratinocyte cell line. Genes Cells 3(2): 125-34.

Souchelnytskyi, S., et al. (1997). Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factor-beta signaling. J. Biol. Chem. 272: 28107-28115

Stroschein, S. L., et al. (2001). Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN. Genes Dev. 15: 2822-2836. 11691834

Takaesu, N. T., et al. (2006). dSno facilitates baboon signaling in the Drosophila brain by switching the affinity of Medea away from Mad and toward dSmad2. Genetics 174(3): 1299-313. Medline abstract: 16951053

Talamillo A., et al. (2008). Smt3 is required for Drosophila melanogaster metamorphosis. Development 135: 1659-1668. PubMed Citation: 18367553

Ting, C.-Y., et al. (2007). Tiling of R7 axons in the Drosophila visual system is mediated both by transduction of an activin signal to the nucleus and by mutual repulsion. Neuron 56(5): 793-806. PubMed citation: 18054857

Topper, J. N., et al. (1998). CREB binding protein is a required coactivator for Smad-dependent, transforming growth factor beta transcriptional responses in endothelial cells. Proc. Natl. Acad. Sci. 95(16): 9506-11

Tsukazaki, T., et al. (1998). SARA, a FYVE domain protein that recruits Smad2 to the TGFbeta receptor. Cell 95(6): 779-91

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

von Gersdorff, G., et al. (2000). Smad3 and Smad4 mediate transcriptional activation of the human Smad7 promoter by transforming growth factor beta. J. Biol. Chem. 275(15): 11320-6.

Wang, J., Tokarz, R. and Savage-Dunn, C. (2002). The expression of TGFß signal transducers in the hypodermis regulates body size in C. elegans. Development 129: 4989-4998. 12397107

Wang, J. Mohler, W. A. and Savage-Dunn, C. (2005). C-terminal mutants of C. elegans Smads reveal tissue-specific requirements for protein activation by TGF-ß signaling. Development 132: 3505-3513. 16000380

Wang, Y. and Levy, D. E. (2006). C. elegans STAT cooperates with DAF-7/TGF-β signaling to repress dauer formation. Curr. Biol. 16(1): 89-94. 16401427

Weinstein, M., et al. (1998). Failure of egg cylinder elongation and mesoderm induction in mouse embryos lacking the tumor suppressor smad2. Proc. Natl. Acad. Sci. 95(16): 9378-83

Wrana, J. L., et al. (1994). Mechanism of activation of the TGF-beta receptor. Nature 370: 341-347

Wu, G., et al. (2000). Structural basis of Smad2 recognition by the Smad anchor for receptor activation. Science 287: 92-97

Wu, J. W., et al. (2002). Crystal structure of a phosphorylated Smad2: recognition of phosphoserine by the MH2 domain and insights on smad function in TGF-ß signaling. Mol. Cell 8: 1277-1289. 11779503

Xiao, Z., et al. (2000). A distinct nuclear localization signal in the N terminus of Smad 3 determines its ligand-induced nuclear translocation. Proc. Natl. Acad. Sci. 97: 7853-7858. PubMed Citation: 10884415

Zhu C. C., et al. (2008). Drosophila activin- and the activin-like product dawdle function redundantly to regulate proliferation in the larval brain. Development 135: 513-521. PubMed Citation: 18171686

Xu, J. and Attisano, L. (2000). Mutations in the tumor suppressors Smad2 and Smad4 inactivate transforming growth factor beta signaling by targeting Smads to the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. 97(9): 4820-5. PubMed Citation: 10781087

Xu, L., et al. (2002). Smad2 nucleocytoplasmic shuttling by nucleoporins CAN/Nup214 and Nup153 feeds TGFß signaling complexes in the cytoplasm and nucleus. Molec. Cell 10: 271-282. 12191473

Xu, W., et al. (2000). Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type beta transforming growth factor. Proc. Natl. Acad. Sci. 97(11): 5924-9. PubMed Citation: 10811875

Yamakawa, N., Tsuchida, K. and Sugino, H. (2002). The rasGAP-binding protein, Dok-1, mediates activin signaling via serine/threonine kinase receptors. EMBO J. 21: 1684-1694. 11927552

Yanagisawa, J., et al. (2000). Convergence of transforming growth factor-beta and vitamin D signaling pathways on SMAD transcriptional coactivators. Science 283(5406): 1317-21. PubMed Citation: 10037600

Yoon, S. J., et al. (2011). HEB and E2A function as SMAD/FOXH1 cofactors. Genes Dev. 25(15): 1654-61. PubMed Citation: 21828274

Yoshida, Y., et al. (2003). Tob proteins enhance inhibitory Smad-receptor interactions to repress BMP signaling. Mech. Dev. 120: 629-637. 12782279

Yu, X. M., Gutman, I., Mosca, T. J., Iram, T., Ozkan, E., Garcia, K. C., Luo, L. and Schuldiner, O. (2013). Plum, an immunoglobulin superfamily protein, regulates axon pruning by facilitating TGF-beta signaling. Neuron 78(3): 456-468. PubMed ID: 23664613

Yun, C.-H., et al. (2007). Negative regulation of Activin/Nodal signaling by SRF during Xenopus gastrulation. Development 134: 769-777. Medline abstract: 17259304

Zhang, Y., Feng, X. H. and Derynck, R. (1998). Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-beta-induced transcription. Nature 394: 909-913. PubMed Citation: 9732876

Zheng. X., et al. (2006). Baboon/dSmad2 TGF-ß signaling is required during late larval stage for development of adult-specific neurons. EMBO J. 25: 615-627. 16437159

Zhu, C. C., et al. (2008). Drosophila Activin-β and the Activin-like product Dawdle function redundantly to regulate proliferation in the larval brain. Development 135(3): 513-21. PubMed Citation: 18171686

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

date revised: 3 November 2015

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