InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

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).

Several lines of evidence argue against an instructive role Dawdle. First, axon pathfinding does not require restricted expression of daw. Guidance defects associated with daw mutants can be rescued by daw expression in sites of endogenous transcription, and ectopically in motoneurons. Second, in daw mutants, axons do not extend into inappropriate areas or show ectopic branching, phenotypes typical of mutations in Sema-2a, Netrin-A and Netrin-B that provide spatial guidance cues or target recognition. Finally, misexpression of Daw did not cause mistargeting of axons, indicating no apparent spatial sensitivity to Daw. These data are therefore consistent with a permissive role in which Daw enables and/or modulates the response of the growth cone to other restricted cues (Parker, 2006).

Adhesive forces as well as chemo-attractant and repellent cues are likely to be modulated by secreted factors. There is increasing evidence to suggest that matrix metalloproteases (MMPs) as well members of the ADAMs family of disintegrin-containing metalloproteases are likely candidates for modulating the activity of guidance cues. Mutations in the Drosophila ADAM10 homolog kuzbanian (kuz), for example, exhibit both axon extension and guidance defects where many Fas2-positive axons inappropriately cross the CNS midline. In Xenopus, application of general metalloprotease inhibitors to exposed brain preparations yields severe disruption of the retinal ganglion cell axon projections as they extend through the brain to their targets in the optic tectum. More recent experiments employing selective MMP-inhibitors suggest that axon behavior at specific guidance choice points in this system is likely to involve MMPs (Serpe, 2006).

How these metalloproteases affect guidance choices is not clear, although one simple model is that they might digest components in the extracellular matrix (ECM) to clear a path for extending axons. However, recent observations suggest that they may play more direct roles by processing components of different guidance pathways. For example, kuz mutations genetically interact with slit and robo mutations suggesting that Kuz might proteolytically modify one of these two components (Serpe, 2006).

This report demonstrates that mutations in the Drosophila metalloprotease tolloid-related (tlr), also known as tolkin, result in fasciculation defects both within the CNS and at choice points in the periphery as motor axons traverse to their target muscles. The choice point defects are similar to those seen in sidestep (side), beat and lar (leukocyte-antigen-related-like) mutants, suggesting that Tlr functions in conjunction with, or parallel to, these pathways. Tlr is a member of the BMP-1/Tolloid family of Astacin-like metalloproteases. In vertebrates, these proteases process a number of different ECM components; however, their best-characterized developmental role is to regulate the activity of TGF-ß-type ligands. This is accomplished in two conceptually similar ways, either by processing extracellular inhibitors such as Sog or Chordin that normally bind to the ligand and prevent it from binding receptor, or by processing the N-terminal pro-domains of ligands such as Myostatin and GDF-11 that would otherwise remain bound to the C-terminal ligand domain and prevent it from binding receptor. This study tested if Tlr processes the pro-domains of various Drosophila TGF-ß-type ligands and found that in vitro it cleaves Myoglianin (Myo), which is the Drosophila homolog of Myostatin, Activin (Act), and the Activin-like protein Dawdle (Daw). In the case of Daw, this processing is shown to enhance Daw signaling activity in vitro; daw-null mutants exhibit axon guidance defects similar to, but less severe than, tlr. Daw is likely to signal through the canonical Activin pathway to mediate axon guidance because germline mutant clones of babo, the type I receptor for Daw, as well as Smad2 (Smox - Flybase), the main downstream transcriptional mediator of activin-like signaling, also produce axon guidance defects similar to daw mutants. Since Tlr supplied either in motoneurons, muscles or the hemolymph is able to rescue tlr mutants, it is suggested that Tlr is likely to provide a permissive signal rather than a spatially instructive cue for guidance, perhaps by activating several TGF-ß ligands (Serpe, 2006).

Mutant Tlr embryos exhibit numerous defects in motoneuron axon guidance beginning at embryonic stage 16-17 and persisting into larval stages. One set of substrates for Tlr are the pro-domains of several TGF-ß-type ligands. In the case of Daw, processing of its pro-domain by Tlr leads to enhanced signaling abilities in a cell culture assay. Since daw mutants also exhibit axon guidance errors, albeit less severe and persistent than those of tlr mutants, these observations suggest (but do not prove) that Tlr processing of inhibitory pro-domains enhances TGF-ß signaling to regulate nerve branching and innervation, perhaps by altering adhesiveness at particular choice points and target cues on muscles (Serpe, 2006).

Previous work characterizing the attraction/repression and adhesive/non-adhesion forces acting during axon guidance revealed remarkable plasticity in the growth cone responses to modulation by both intrinsic and extrinsic factors. Specifying an axon's trajectory is therefore not just a simple matter of selecting the appropriate set of guidance receptors and delivering them to the growth cone. The growth cone must also be able to modulate its responsiveness en route. Tlr and Daw appear to be required for a process that allows proper modulation of this en route responsiveness of the growing axons and perhaps also muscle site target selection (Serpe, 2006).

How might Tlr and Daw regulate axon guidance and target site selection? An attractive model is that Tlr processing of Daw and other TGF-ßs in muscles might provide a chemoattractant signal that collaborates with other muscle-derived attractants such as Side to guide motor axons to their appropriate innervation sites, similar to the way in which the morphogen sonic hedgehog has been shown to collaborate with netrin 1 in mediating midline axon guidance to the floor plate in mice. Although Tlr and Daw both show localized expression in muscle, the observation that tlr mutants can be rescued by expression of a tlr transgene in a wide variety of tissues, and that the activated form of the protein is a normal constituent of the hemolymph, suggests that it may not provide a directional cue for axon guidance. Likewise, daw mutants can be rescued by transgene expression from multiple tissues (Parker, 2006). These results are more consistent with Tlr and Daw providing a permissive signal rather then providing a directional cue(s) (Serpe, 2006). At least one response to a permissive cue is likely to be mediated through a canonical TGF-ß pathway because maternal loss of both babo, the Drosophila type I receptor for Daw, and Smad2, the primary transcriptional transducer of Daw signaling, both produce axon guidance defects similar to daw mutants. Daw signaling is likely to control the transcription of other gene products that directly regulate axon fasciculation/guidance, perhaps by modulating production of an attractant on muscle or a repellent on motor nerves, and thereby to integrate this new pathway with previously identified mediators of attractive and repulsive forces. It is interesting to note in this regard that double mutants of side and tlr show strongly enhanced phenotypes as compared with each single mutant, suggesting that these two products act in parallel, as opposed to in a linear pathway (Serpe, 2006).

One last point with respect to Daw function is that it is likely to regulate other aspects of Drosophila development in addition to motor nerve axon guidance. This follows from the fact that mutations in daw lead to lethality in the pupal stage, yet by this time motor axon guidance defects are largely corrected. Therefore, the lethality is likely to result from defects in some other process. This might reflect a more general role of daw in guiding axons other than those from motoneurons to their targets, or maybe Daw and Tlr regulate other functional aspects of motoneurons or glial cells that remain to be identified (Serpe, 2006).

Although the daw and tlr loss-of-function axon guidance phenotypes are similar in both penetrance and the spectrum of defects that they exhibit at embryonic stage 17, there are significant differences between the two. In particular, many tlr defects persist into the larval third instar stage, whereas daw embryonic mutant phenotypes are corrected by this time. In addition, tlr mutant embryos have significant fasciculation defects in the Fas2-positive longitudinal bundles within the ventral ganglia that are not seen in daw mutants (Serpe, 2006).

One possible explanation for these differences is that other TGF-ß molecules might act redundantly with Daw. Among the uncharacterized ligands, Myo in particular is intriguing in this regard as its expression overlaps daw in muscle and glia cells. mav expression also overlaps that of both daw and myo by virtue of being broadly expressed in most embryonic tissues. BMP ligands might also be involved because microarray analysis has shown that the Activin and BMP pathways share several transcriptional targets in brain tissue. In one simple scenario, daw mutants might be corrected because of redundancy with mav, myo or one of the BMP ligands, whereas tlr mutants exhibit more severe defects because lack of Tlr might affect the activation of multiple ligands. At present, mutants that disrupt mav and myo are not available to assess possible functional overlap of their products with Daw and each other (Serpe, 2006).

Although it is believed that Tlr regulates motor axon guidance in part by processing latent complexes of Daw and other TGF-ß ligands, it is quite possible that TGF-ß-type molecules are not the only Tlr substrates relevant to this process. Metalloproteases may interact directly with the guidance signaling pathway via either ligand modification or processing of their receptors. For example, metalloproteases regulate cell-surface expression of DCC or robo receptors, and ADAM family members have been implicated in terminating the interaction between the ephrins and their receptors. Alternatively, metalloprotease may cleave components of the ECM thereby clearing a path for axon extension through the extracellular environment (Serpe, 2006).

The activation of latent TGF-ß-type ligands for regulating axon guidance might be a conserved and ancient mechanism. In C. elegans, the unc-129 locus codes for a TGF-ß ligand that is 33% identical to human BMP7 and mediates motor axon attraction to the dorsal midline. In vertebrates, BMPs are roof-plate secreted chemorepellents for commissural axons. Whether either of these examples also utilizes a Tld-like enzyme in the processing of a latent complex of the BMP ligands attached to their pro-domains remains to be determined (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).

REGULATION

Identification of new Tlr substrates

Enzymes of the BMP-1/Tolloid family have been shown to process a number of substrates including the BMP inhibitors Sog and Chordin, components of the ECM, and the pro-domains of several TGF-ß-type factors including Myostatin, which is a key regulator of muscle mass, and GDF-11, which is a negative feedback inhibitory signal in neurogenesis. Myostatin and GDF-11, the mature ligand remains associated with the pro-domain after secretion in a non-covalent latent complex. Additional processing of the pro-domain by Tld-like enzymes releases the ligand from the complex enabling it to signal (Serpe, 2006).

Since TGF-ß-type ligands have been implicated in some aspects of axon guidance in both C. elegans and the mouse, it was asked whether the role of Tlr in Drosophila motoneuron axon guidance might involve processing of a latent TGF-ß-type ligand. Drosophila has four non-BMP TGF-ß-type ligands comprising Act, Activin-like protein (now called Dawdle), Myo and Mav that are, as yet, poorly characterized. A scan of the pro-domain sequences of these ligands revealed at least one putative BMP-1/Tolloid cleavage site in each pro-peptide that resembled the cleavage sites characterized in Myostatin and GDF11 (Serpe, 2006).

To test directly if any of these pro-peptides is a substrate for Drosophila Tolloid proteins, a V5/His tag was introduced at the N-terminus of the pro-domains and the tagged proteins were produced in Drosophila S2 cells. Act, Myo and Daw are secreted and processed at the predicted multibasic, subtilisin-like maturation site just prior to the ligand domain, yielding a pro-peptide of the appropriate size. This processing is independent of Tlr and occurs for all TGF-ß-type ligands. For example, in the case of Myo, a pro-domain of `50 kD (subtilisin generated) was generated in the conditioned media that corresponds to the calculated 48 kD myoglianin pro-domain. When the ligand was produced in cells co-transfected with a Tlr construct, the pro-domain was further processed at an additional site, producing a ~30 kD tagged fragment. The identity of the cleavage site within the pro-domain of each ligand was confirmed by alanine substitution mutagenesis of four residues on each side of the putative cleavage site. The Tld enzyme also cleaves these sites, consistent with the observation that Tld can rescue Tlr axon guidance defects when expressed at a low level (Serpe, 2006).

Processing of the Daw pro-domain by Tlr/Tld activates Daw in a cell-based signaling assay

The stability of the Daw, Myo and Act pro-domains in the absence of Tlr and their rapid disappearance upon induction of protease expression is similar to the behavior of the vertebrate Myostatin and GDF11 pro-domains in the presence of Tld proteases. In these cases, cleavage of the pro-domains results in activation of latent ligand complexes leading to enhanced signaling. It was asked if cleavage of the Act, Daw or Myo pro-domains by Drosophila Tolloids had any effect on the signaling ability of the ligands. This hypothesis was tested using a tissue culture-based signaling assay. Both the BMP pathway that signals through Mad, and the Activin pathway that signals through Smad2 were examined, by transfecting S2 cells with either tagged Mad or Smad2 constructs and presenting them with various ligands. For example, the control ligand Dpp signals through the BMP pathway: cells treated with 10 ng Dpp had a 5-fold increase in the level of phosphorylated Mad (Mad-P) as compared with mock treated cells. Under the experimental conditions, none of the other ligands tested appeared to elicit a strong BMP-like signal. Act signaled weakly through Smad2 with or without Tld. Cells treated with Daw, in the absence of Tlr or Tld, increased their level of Smad2-P 2-fold compared with the control. More importantly, cells treated with Daw+Tlr or Tld exhibited a much stronger increase in the relative level of Smad2-P, suggesting that Tld proteins enhanced Daw signaling properties. Neither Myo nor Mav produced any significant signal under the experimental conditions, either with or without Tlr. The signaling advantage of adding Tld or Tlr was completely lost for DawTm, a mutant form of Daw in which the Tld/Tlr cleavage site was destroyed (Serpe, 2006).

To determine if Tlr could enhance Daw signaling in vivo, gain-of-function phenotypes of daw were examined with or without additional tlr in various tissues. Indeed, when daw was overexpressed in hemocytes using the Cg-Gal4 driver less than 10% pupal lethality (in more than 200 progeny) was observed; Cg-Gal4>UAS-tlr1 animals had no detectable phenotype. By contrast, when both tlr and daw were overexpressed, the pupal lethality of Cg-Gal4>UAS-tlr1,UAS-daw animals exceeded 50%. In addition, transheterozygous combinations of tlr and daw showed enhanced axon guidance defects as compared with the single heterozyous animals, consistent with a role for Tlr in activating Daw in vivo. Taken together, these data show that Drosophila Tolloids cleave various TGF-ß-ligand pro-domains and, at least in the case of Daw, this cleavage activates the ligand, most likely by releasing it from a latent complex with its inhibitory pro-domain (Serpe, 2006).

daw-null mutants exhibit axon guidance defects similar to tlr mutants

The observed pattern of daw expression in the muscles where tlr is abundant, is consistent with the possibility that Daw could be cleaved and activated in vivo by Tlr. If Daw is a physiologically relevant substrate for Tlr, then one might expect that the daw mutant phenotype should exhibit some overlap with the tlr mutant phenotype. To examine this, daw-null mutants were generated by excising a P-element located just upstream of the daw gene. Two deletions, daw^ex11 and daw^ex32, were obtained in which parts of the Daw coding sequence, including the transcriptional and the translational start sites, were deleted. Both mutations produce lethality in the larval-pupal stages and the lethality was rescued by ubiquitous expression of a daw transgene, indicating that no other essential gene was removed by these excisions (Serpe, 2006).

Examination of the nervous system of stage-17 daw-null mutant embryos revealed very mild fasciculation defects in the CNS with thinning and breaks of the exterior (third) bundle of pioneer axon fascicles. Stage-17 daw mutant embryos exhibited various motoneuron misprojections including ISN delays, stalling and misrouting of the SNa, particularly no turning towards muscle 24, and stalling of SNb at the 13/30 synapse accompanied by defective 12/13 innervation. Overall, the axon guidance defects of daw mutants were similar in nature, but weaker in intensity and penetrance than the tlr mutant defects. In addition, unlike tlr mutants in which defects persist into the third instar stage, daw mutant third-instar larvae exhibited relatively normal innervation at the 12/13 muscle cleft. The delayed nature of the innervation defects lead to the renaming of this locus from alp to dawdle. These results suggest that the inability to properly activate the daw ligand may contribute to the tlr phenotype (Serpe, 2006).

Other TGF-ß pathway components exhibit axon guidance defects

To determine if the daw axon guidance defects arise because of a loss of signaling through a canonical TGF-ß pathway, several additional pathway components were examined for axon guidance defects. Since a cell culture signaling assay revealed that Daw signals through Smad2, which is phosphorylated by the type I receptor encoded by the babo locus, babo and Smad2 mutant embryos were examined for axon guidance defects. In both cases, zygotic loss-of-function mutants showed no increase in defects. However, when the maternal contribution of each was also removed by making germline clones, a very similar distribution and penetration of defects was observed in stage 17 embryos as was found for daw mutants (Serpe, 2006).

In Drosophila, two different type II receptors, Punt (Put) and Wishful thinking (Wit), mediate signaling by TGF-ß ligands. Since wit is not expressed in S2 insect cells, but these cells are able to transduce a signal to Smad2, and since wit mutants have a normal pattern of muscle innervation at the end of embryogenesis, the analysis focused on put mutants. Like babo and Smad2, put is maternally supplied to the embryo; its maternal loss results in severely ventralized embryos with a highly disorganized CNS. However, certain combinations of put alleles are temperature-sensitive, allowing elimination of put activity by temperature shift. At permissive temperatures of 18-20°C no significant embryonic axon guidance defects were observed in put^135-22/put⁶² animals. At temperatures above 25°C, put^135-22/put⁶² exhibited gross head involution and dorsal closure defects making it difficult to score for axon guidance defects. However, analysis of put^135-22/put⁶² embryos that were developed at 23-25°C revealed axon guidance defects similar to those seen in tlr, daw, babo and Smad2 mutant embryos. Also, no increase was found in the penetrance or severity of defects in put, wit double-mutant embryos, suggesting that Wit is not redundant with Put for this activity. Together these data suggest that Daw signals through a canonical TGF-ß pathway involving Babo, Put and Smad2 to elicit a transcriptional output crucial for fulfilling proper axon guidance in late Drosophila embryos (Serpe, 2006).

Drosophila Follistatin exhibits unique structural modifications and interacts with several TGF-beta family members

Follistatin (FS) is one of several secreted proteins that modulate the activity of TGF-β family members during development. The structural and functional analysis of Drosophila Follistatin (dFS) reveals important differences between dFS and its vertebrate orthologues: it is larger, more positively charged, and proteolytically processed. dFS primarily inhibits signaling of Drosophila Activin (dACT) but can also inhibit other ligands like Decapentaplegic (DPP). In contrast, the presence of dFS enhances signaling of the Activin-like protein Dawdle (DAW), indicating that dFS exhibits a dual function in promoting and inhibiting signaling of TGF-β ligands. In addition, FS proteins may also function in facilitating ligand diffusion. Mutants of daw are rescued in significant numbers by expression of vertebrate FS proteins. Since two PiggyBac insertions in dfs are not lethal, it appears that the function of dFS is non-essential or functionally redundant (Bickel, 2008).

Polypeptide cytokines of the transforming growth factor β (TGF-β) family control a wide range of developmental and physiological functions in higher eukaryotes. This diverse group of signaling molecules provides positional information required for axis formation and tissue specification, controls various processes such as tissue growth, cell death, and pathfinding of axons in the nervous system, and prevents differentiation of embryonic stem cells. Many components of this pathway have been linked to tumor formation in humans. The highest degree of sequence conservation between various family members is found within the C-terminal domains, which are released as dimers by proteolytic processing. Similarities in sequence and biological activities allow these factors to be divided into at least two distinct subgroups: Bone Morphogenetic Proteins (BMPs) and Activins/Inhibins/TGF-βs. The latter group exhibits an additional intramolecular disulfide bond at the N-terminus after processing. In Drosophila, there are four Activins/TGF-βs, Drosophila Activin (dACT), Dawdle (DAW, also known as Activin-like protein at 23, ALP23, and Anti-Activin, AACT), Myoglianin (MYO), and Maverick (MAV), and three BMP-type ligands, Decapentaplegic (DPP), Screw (SCW), and Glass Bottom Boat (GBB). Each ligand dimer forms a complex with two type II and two type I receptor serine/threonine kinases that phosphorylate SMAD transcription factors. BMP-type ligands signal primarily through the type I receptors thick veins (TKV) and Saxophone (SAX) and activate Mothers against DPP (MAD). Activins/TGF-β-type ligands are believed to signal through the type I receptor Baboon (BABO), which in turn activates primarily dSMAD2 but to a minor extent also MAD (Bickel, 2008)

TGF-β signaling is regulated by various extracellular proteins. Antagonists like Follistatin (FS), Noggin, Chordin/Short Gastrulation, and DAN/Cerberus bind ligands and prevent interactions with receptors and signaling. In some species, they exhibit overlapping and redundant functions. Recently, it was shown that the simultaneous depletion of FS, Noggin, and Chordin in Xenopus tropicalis results in transformation of ventral into dorsal tissue during embryogenesis (Bickel, 2008).

Follistatin was first identified as an inhibitor of Activin in vertebrates. Subsequent studies showed that it also binds other ligands with lower affinities including BMP 2, 4, 6, 7, and Myostatin. Knockout mice of fs die shortly after birth. They are smaller and exhibit defects in skeletal and muscle development. Recently, the crystal structure of the human FS-Activin complex was resolved. It provides valuable insight into the function of the different FS domains and a basis to explain the mechanism of ligand inhibition (Bickel, 2008 and references therein)

This study has analyzed the function of Drosophila Follistatin (dFS). Like vertebrate FS proteins, dFS is subdivided into a N-terminal domain (N) and three FS domains (FS1-3). However, dFS is substantially larger than its vertebrate homologues due to a large basic insertion into FS1. Interestingly, dFS is proteolytically processed, and small processed forms of dFS are able to bind to ligands like dACT. This result suggests a possible different inhibitory mechanism: ligands bound to processed dFS can bind to type II receptors but cannot recruit type I receptors. Consequently, processed dFS might not only sequester ligands but also prevent unbound ligands from interacting with receptor complexes. Among the seven Drosophila TGF-β ligands, dFS primarily inhibits dACT but can also inhibit signaling of other ligands like DPP. In contrast, dFS can augment signaling of the TGF-β member DAW. These results suggest that dFS might exhibit dual functions in facilitating and inhibiting TGF-β signaling. Analysis of two PiggyBac insertions in dFS reveals that they affect dfs expression. Since homozygous animals of these lines are viable and phenotypically wild type, it is assumed that the function of dFS is non-essential or functionally redundant. Taken together, this study reveals interesting differences between the mechanisms of modulating TGF-β signaling by dFS and its vertebrate orthologues (Bickel, 2008)

The primary structure of dFS shows both similarities and differences compared to its vertebrate orthologues. dFS is divided into a N-terminal domain (N) and three FS domains (FS1-3) that can be further subdivided into EGF-like and Kazal protease inhibitor-like domains. Compared to its vertebrate orthologues, dFS is substantially larger due to an insertion of 260 amino acids. The insertion contains 55 positively charged amino acids (pI > 10) and is located after the heparin binding site in FS1. In contrast to Drosophila, vertebrates produce a second, long isoform of FS by alternative splicing (FS-315). This form contains a C-terminal extension with many negatively charged amino acids that reduce adhesion to sulfates of proteoglycans at the cell surface. FS-315 is the major mammalian circulating form (Bickel, 2008)

To analyze the function of dFS, dfs cDNA was cloned into expression vectors for tissue culture cells and transgenic flies. From comparison to vertebrate orthologues, the start site was assumed to be at nucleotide 577. At this position, the SignalP server website predicts a signal sequence (MALLIGLLLLNFRLTAA-GTCW) that is cleaved equivalently to the vertebrate FS proteins. However, expression of a construct that contains this predicted signal sequence but lacks the first 279 nucleotides does not result in a translated protein in culture cells and growth inhibition in transgenic flies. In contrast, the complete cDNA is translated in tissue culture cells and inhibits growth in vivo. There are three potential start codons upstream of the predicted signal peptide sequence. Based on the consensus for Drosophila start sites, the most likely start codon is at nucleotide 181-183 (CAACA-ATG). It is noted that there is a full-length dfs cDNA that is 671 nucleotides longer, GH04473 (NM_144119). This cDNA extends the coding sequence by 62 additional amino acids. Although conserved between different Drosophila species, the absence of this additional sequence in the shorter cDNA does not prevent translation and secretion of a growth inhibitory protein. Interestingly, the full-length cDNA does not encode an N-terminal signal peptide sequence either. Instead, several protein analysis programs predict three potential transmembrane domains within the 211 amino acid leader. Thus, in contrast to vertebrate FS proteins, the secretion of dFS might involve an initial membrane-anchoring step (Bickel, 2008)

There are several differences in the structure of dFS and its vertebrate orthologues. One unique characteristic of dFS is the unusual signal trailer that contains three potential transmembrane domains. The results suggest that cleavage likely occurs after the third transmembrane domain at the equivalent position of vertebrate FS proteins. No evidence was found that this feature affects secretion or alters the function of dFS (Bickel, 2008)

A major difference between dFS and its vertebrate orthologues is the large basic insertion. This modification is likely consequential and may alter the activity of dFS. Based on protein folding prediction and the structure of the human FS-Activin complex, the insertion is not well structured and is located after the heparin binding site within the EGF sub-domain of FS1. Since this area projects away from Activin, the positively charged amino acids likely increase the affinity of dFS to heparin sulfate proteoglycans on the cell surface and do not contribute to Activin binding. It is conceivable that this feature leads to a reduction of diffusion and an increase in local concentration of dFS. In addition, it may also reduce diffusion of ligands and enhance the stability of ligand-receptor complexes. Vertebrates have adopted an opposite strategy. They generate a long form of FS by alternative splicing, which contains a negatively charged C-terminal tail that reduces interactions with heparin sulfate. The long form is the major endocrine form in humans. Since it does not efficiently interact with the cell surface, it is not clear whether this form primarily inhibits Activin signaling or actually enhances Activin circulation by preventing unwanted interactions with neighboring cells (Bickel, 2008)

The FS-Activin complex shows that the N domain binds to the wrist region of Activin blocking interaction with the type I receptor. The FS2 domain binds to the type II receptor binding site of Activin. In humans, two FS proteins can encircle an Activin dimer preventing interactions with both types of receptors. It was found experimentally that Activin bound to FS still can interact with the type II receptor. This result is explained if only one molecule of FS is bound to an Activin dimer leaving the second Activin monomer free to bind to receptors. Unlike vertebrate FS proteins, this study found that dFS is proteolytically processed at the C-terminus into two major forms. Based on the migration of epitope tagged forms of dFS on Western blots, the small form contains at least the N and FS1 domains and lacks FS3 and probably part of FS2. Since the small form is immunoprecipitated by dACT, it appears that it can bind to dACT. What is the possible role of this small processed form? The following model suggests that the small form could potentially be a stronger inhibitor: if two small processed dFS proteins without FS2 bind to dACT monomers, they prevent interactions with type I but not type II receptors. A dFS-dACT-PUT complex is inactive, since dACT cannot recruit the type I receptor BABO. In this scenario, the small dFS forms would not only inactivate bound dACT but also reduce interactions of free dACT with type II receptors. If type II receptors were limiting, the small dFS forms would be able to reduce dACT signaling more efficiently than the full-length protein. This hypothesis can be tested by expressing transgenes that encode the small form of dFS. To perform these experiments, the structures of the small and large forms of dFS are currently being determined (Bickel, 2008)

In most experiments, dFS was seen to function as an inhibitor of TGF-β signaling. As a major exception, dFS enhances rather than inhibits DAW signaling in tissue culture assays. Interestingly, unlike other ligands, DAW does not increase but reduces the viability of animals that over-express dFS. This observation suggests that over-expression of dFS may not only be lethal due to reducing the activities of various TGF-β family members but also due to increased signaling of ligands like DAW. If DAW and dFS physically interact, the question arises how dFS can increase ligand signaling. In the early embryo, it has been shown that Short Gastrulation (SOG), the Drosophila orthologues of the vertebrate Chordin protein, not only inhibits signaling of DPP but also facilitates diffusion, which is necessary for the formation of the peak levels of the DPP gradient. The formation of the DPP gradient depends on the equilibrium between bound and unbound DPP by SOG. Similarly, it is conceivable that ligand binding by the positively charged dFS can reduce ligand diffusion. If the concentration or the affinity of dFS for a ligand is high, the ligand is not released or bound again, and signaling is inhibited. Alternatively, if the concentration of dFS or its affinity for a ligand is low, dFS binding can locally increase ligand concentration, while subsequent ligand release will enhance signal transduction. Consistent with such a model, in tissue culture assays dFS rather increases than decreases the level of MAD activation by DPP, while presumably higher levels of dFS inhibit DPP signaling in the wing. Such a dual function could probably be at work during dorsal-ventral axis formation. While dact is not expressed in the early embryo, dfs is present in a dorsal stripe. This pattern corresponds with the highest levels of DPP signaling. Expression of dfs is rather late in the formation of the DPP gradient and is potentially regulated by DPP. It is conceivable that initial low levels of dFS could, like SOG, function to increase the local concentration of DPP and contribute to the formation and maintenance of the dorsal peak activity of DPP signaling. In contrast, higher levels of dFS protein that accumulate by the end of the dorsal-ventral axis formation process may inhibit DPP and contribute to terminating the dorsal DPP signal. Recently, a computational model that described ligand distribution and signaling in the presence of a cell surface BMP-binding protein was developed that supports this idea. A dual function of FS proteins in facilitating and inhibiting TGF-β signaling is also supported by the finding that fs mutants in mice exhibit overlapping phenotypes with activin knock out animals. Consequently, FS was originally described as enhancer of Activin signaling. Finally, facilitating diffusion and redistribution of dACT and potentially other ligands is probably the best mechanism to explain why dFS and vertebrate FS proteins from frogs and humans can partially compensate for the lack of DAW. It is speculated that the vertebrate FS proteins exhibit lower affinities for Drosophila ligands like dACT and diffuse better since they lack the basic insertion of dFS. Most likely, these differences could explain why expression of the vertebrate but not dFS can rescue small but significant numbers of daw mutant animals. Taken together, it is possible that distinct affinities for dFS account for the different interactions seen with various ligands. Further studies are necessary to investigate such a possible dual function of dfs in early embryos and during development (Bickel, 2008).

Two PiggyBac insertions affect dfs transcription. Interestingly, both lines are homozygous viable and do not show any obvious pattern defects. These results suggest that the function of dFS not essential for viability. In mice, a mutation in fs results in various defects with lethal consequences. In contrast, FS, Chordin, and Noggin exhibit overlapping functions in X. tropicalis. It is necessary to reduce all three inhibitors to transform ventral into dorsal tissue during embryogenesis of this species. Although there is no Noggin-like protein in Drosophila, it is possible that dFS also shares overlapping functions with SOG. Expression of sog overlaps with dfs in many tissues, and it is possible that SOG can substitute for dFS in its absence (Bickel, 2008)

The analysis of dfs RNA levels in homozygous f00897 flies shows that they are substantially reduced. Thus, this insertion can be regarded as a hypomorphic allele. It is conceivable that the amount of protein synthesized in this mutant is sufficient for normal development. However, further reduction of dFS by combining f00897 with the deficiency Df(2R)Exel7135 that entirely removes dfs does not affect viability either. In contrast to f00897, the PiggyBac insertion in e03941 clearly disrupts transcription of a full-length mRNA. Based on the location of the insertion, the truncated mRNA likely encodes an altered protein that still contains the N, FS1, and FS2 domains. It lacks the entire FS3 domain and likely contains additional C-terminal amino acids due to altered or absence of splicing. Since the small form is still able to bind dACT, a partially functional dFS protein could still be present in e03941 flies, if proper processing would occur. However, it is unlikely that such an altered protein is processed correctly into the small form of dFS. Since no homozygous lethal lines were obtained in the FRT-mediated deletion screen, it appears that dfs is not lethal. Taken together, the lack of any obvious phenotypes in homozygous f00897 and e03941 lines suggests that dFS is not essential for normal development or is redundant like in some vertebrate species (Bickel, 2008)

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

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

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

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

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

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

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

Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila

Reduced insulin/IGF signaling increases lifespan in many animals. To understand how insulin/IGF mediates lifespan in Drosophila, chromatin immunoprecipitation-sequencing analysis was performed with the insulin/IGF regulated transcription factor dFOXO in long-lived insulin/IGF signaling genotypes. Dawdle, an Activin ligand, is bound and repressed by dFOXO when reduced insulin/IGF extends lifespan. Reduced Activin signaling improves performance and protein homeostasis in muscles of aged flies. Activin signaling through the Smad binding element inhibits the transcription of Autophagy-specific gene 8a (Atg8a) within muscle, a factor controlling the rate of autophagy. Expression of Atg8a within muscle is sufficient to increase lifespan. These data reveal how insulin signaling can regulate aging through control of Activin signaling that in turn controls autophagy, representing a potentially conserved molecular basis for longevity assurance. While reduced Activin within muscle autonomously retards functional aging of this tissue, these effects in muscle also reduce secretion of insulin-like peptides at a distance from the brain. Reduced insulin secretion from the brain may subsequently reinforce longevity assurance through decreased systemic insulin/IGF signaling (Bai, 2013).

Insulin/IGF-1 signaling modulates longevity in many animals. Genetic analysis in C. elegans and Drosophila shows that insulin/IGF-1 signaling requires the DAF-16/FOXO transcription factor to extend lifespan, while in humans several polymorphisms of FoxO3A are associated with exceptional longevity. Although many downstream effectors of FOXO have been identified through genome-wide studies, the targets of FOXO responsible for longevity assurance upon reduced insulin signaling are largely unknown. This study found 273 genes targeted by Drosophila FOXO using ChIP-Seq with two long-lived insulin mutant genotypes. Focused was placed on daw, an Activin ligand, which is transcriptionally repressed by FOXO upon reduced insulin/IGF signaling. Inactivation of daw and of its downstream signaling partners babo and Smox extend lifespan. These results are reminiscent of observations from C. elegans where reduced TGF-β/dauer signaling extends longevity. Notably, the lifespan extension of TGF-β/dauer mutants (e.g. daf-7 (e1372) mutants) can be suppressed by daf-16 mutants, suggesting that TGF-β signaling intersects with the insulin/IGF-1 pathway for longevity in C. elegans. In phylogenetic analysis, DAF-7, Daw and mammalian Activin-like proteins share common ancestry. Activin signaling, in response to insulin/IGF-1, may thus represent a taxonomically conserved longevity assurance pathway (Bai, 2013).

Longevity benefits of reduced Activin (TGF-β/dauer) in C. elegans were resolved only when the matricide or 'bagging' (due to progeny hatching within the mother) was prevented by treating daf-7(e1372) mutants with 5-fluorodeoxyuridine (FUdR) to block progeny development. This approach made it possible to distinguish the role of Activin in somatic aging from the previously recognized influence of BMP (Sma/Mab signaling) upon reproductive aging in C. elegans. Activin, of course, is a somatically expressed regulatory hormone of mammalian menstrual cycles that induces follicle-stimulating hormone (FSH) in the pituitary gland. In young females, FSH is suppressed within a cycle when maturing follicles secrete the related TGF-β hormone Inhibin. In mammalian reproductive aging, the effect of Activin in the pituitary becomes unopposed as the stock of primary follicles declines, thus inducing elevated production of FSH. This study now found that reduced Activin but not BMP signaling favors somatic persistence in Drosophila. These parallels between reproductive and somatic aging among invertebrate models and humans suggest that unopposed Activin signaling is pro-aging while favoring reproduction (Bai, 2013).

Reduced insulin/IGF signaling extends lifespan through interacting autonomous and non-autonomous actions. Reducing IIS in some distal tissues has been shown to slow aging because this reduces insulin secretion from a few neurons: reducing IIS by increasing dFOXO in fat body or muscle extends Drosophila fly lifespan while decreasing IPC production of systemically secreted DILP2. This study has identified Activin as a direct, downstream target of insulin/dFOXO signaling within muscles that has the capacity to non-autonomously regulate lifespan. Knockdown of Activin in muscle but not in fat body is sufficient to prolong lifespan. RNAi for muscle Activin signaling led to decreased circulating DILP2 and increased peripheral insulin signaling. Muscle is thus proposed to produce a signaling factor, a myokine, which impacts organism-wide aging and metabolism (Bai, 2013).

Aging muscle may produce different myokine-like signals in response to their physiological state. Aged muscles degenerate in many ways including changes in composition, mitochondria, regenerative potential and within-cell protein homeostasis. Protein homeostasis is normally maintained, at least in part, by autophagy. Loss of macroautophagy and chaperone-mediated autophagy with age will accelerate the accumulation of damaged proteins. Expression of Atg8a in Drosophila CNS is reported to extend lifespan by 56% (Simonsen, 2008), while recent studies find elevated autophagy in long-lived mutants including those of the insulin/IGF-1 signaling pathway. The current results show that insulin/IGF signaling can regulate autophagy through its control of Activin via dFOXO. Poly-ubiquitinated proteins accumulate in aging Drosophila while lysosome activity and macroautophagy decline. Muscle performance with age (flight, climbing) was preserved by inactivating Activin within this tissue. This genetic treatment also reduced the accumulation of protein aggregates. These effects are mediated by blocking the transcription factor Smox, which otherwise represses Atg8a. Smox directly regulates Atg8a through its conserved Smad binding motif (AGAC AGAC). These results, however, contrast with an observation where TGF-β1 promotes autophagy in mouse mesangial cells (Bai, 2013).

Insulin/IGF-1 signaling is a widely conserved longevity assurance pathway. The data indicate that reduced insulin/IGF-1 retards aging at least in part through its FOXO-mediated control of Activin. Furthermore, affecting Activin only in muscle is sufficient to slow its functional decline as well as to extend lifespan. Autophagy within aging muscle controls these outcomes, and it is now found that Activin directly regulates autophagy through Smox-mediated repression of Atg8a. If extrapolated to mammals, pharmaceutical manipulations of Activin may reduce age-dependent muscle pathology associated with impaired autophagy, and potentially increase healthy and total lifespan through beneficial signaling derived from such preserved tissue (Bai, 2013).

Transforming growth factor beta/Activin signaling functions as a sugar-sensing feedback loop to regulate digestive enzyme expression

Organisms need to assess their nutritional state and adapt their digestive capacity to the demands for various nutrients. Modulation of digestive enzyme production represents a rational step to regulate nutriment uptake. However, the role of digestion in nutrient homeostasis has been largely neglected. This study analyzed the mechanism underlying glucose repression of digestive enzymes in the adult Drosophila midgut. Glucose represses the expression of many carbohydrases and lipases. The data reveal that the consumption of nutritious sugars stimulates the secretion of the transforming growth factor β (TGF-β) ligand, Dawdle, from the fat body. Dawdle then acts via circulation to activate TGF-β/Activin signaling in the midgut, culminating in the repression of digestive enzymes that are highly expressed during starvation. Thus, this study not only identifies a mechanism that couples sugar sensing with digestive enzyme expression but points to an important role of TGF-β/Activin signaling in sugar metabolism (Chng, 2004).

Digestive enzymes expression is subjected to complex regulation. However, apart from the regulation of magro (lipase) by the nutrient-sensitive DHR96 and dFOXO (Karpac, 2013). It is noteworthy that an arbitrary threshold for RNA-seq analysis has rejected several genes whose repression was more subtle. For this, it has been have independently verified Amy-p, Amy-d, CG9466, CG9468, and CG6283 to be repressed by glucose through qRT-PCR. Thus, the actual repertoire of carbohydrases and lipases affected by glucose could be potentially larger (Karpac, 2013).

To date, little is known about the contribution of digestion on sugar homeostasis. It seems likely that glucose repression of carbohydrases and lipases is aimed at reducing the amount of sugars and lipids that are available for absorption. Consistent with this view, glucose transmembrane transporters were also found among genes that were downregulated by dietary glucose. A high-sugar diet in Drosophila is associated with dire consequences such as hyperglycemia, insulin resistance, and increased fat accumulation. Thus, reducing both carbohydrases and lipases expression may restrict the nutritional load available for absorption into the circulation when carbohydrate stores in the organism are sufficient and fats are accumulating. In accordance with this, early postprandial glucose level was elevated in the hemolymph when TGF-β/Activin pathway function was compromised in the midgut, a condition associated with elevated digestive enzymes expression. However, when the levels of TAG, glycogen, glucose, and trehalose were monitored after 2 weeks on a high-sugar diet, no significant differences were observed between flies whereby Smad2 or Babo were knocked down in the midgut and control. Sugar homeostasis is a tightly regulated process involving multiple tissues. One possibility would be that the postprandial increase in glucose was counteracted by early acting satiety response when hemolymph glucose level passed a certain threshold, thus limiting the net amount of glucose entering the circulation. Clearly, the role of glucose repression in sugar homeostasis and metabolism warrants additional research. An understanding of how the repertoire of digestive enzymes respond to other nutriments in the diet will provide insights into how an organism may rebalance its diet after ingestion and improve understanding of nutrients homeostasis (Karpac, 2013).

In this study, it was also shown that digestive enzyme repression is induced only by nutritious carbohydrates in the diet. Arabinose, a sweet-tasting sugar with no nutritional value, and L-glucose, another nonutilizable sugar did not suppress amylase and maltase expression. Hence, postprandial activation of gustatory receptors in the gut are considered to be an unlikely mechanism for glucose repression of digestive enzymes. Instead, all these are suggestive of an underlying sugar-sensing mechanism to ensure that carbohydrate digestive capacity toward utilizable carbohydrate sources are not comprised until nutritional sufficiency is attained (Karpac, 2013).

In Drosophila, sugar homeostasis is often associated with the AKH and insulin signaling, whereas insulin signaling is also modulated by proteins and amino acids in the diet. Recently, it has been shown that Daw expression is modulated by insulin signaling, and Daw was identified as a target of dFOXO (Bai, 2013), raising the possibility that glucose repression may be similarly affected by insulin signaling. Surprisingly, disrupting both AKH and insulin signaling did not compromise glucose repression. Instead, this study identified a key role for TGF-β/Activin signaling in this process. Whereas Daw expression may be modulated by insulin signaling, the results clearly showed that glucose repression is mediated through an insulin-independent mechanism. More recently, Ghosh (2014) has demonstrated that Daw is required for insulin secretion, suggesting that the TGF-β/Activin pathway may function upstream of the insulin signaling. It is also noteworthy that, whereas compromising insulin signaling is known to raise circulating sugar levels, this did not affect the ability of flies to repress digestive enzymes in response to dietary glucose. One possible explanation is that Daw expression in response to glucose is dependent on the nutritional state perceived cell autonomously by the fat body cells. Thus, if nutrient sensing in these cells is not compromised, Daw induction and glucose repression can be achieved. Future research should clarify the mechanism underlying Daw induction by nutritious sugar and define the possible interactions between TGF-β/Activin and other sugar-sensing mechanisms (Karpac, 2013).

The TGF-β/Activin pathway in Drosophila has been previously studied in the context of larval brain development, neuronal remodeling, wing disc development, and, more recently, aging and pH homeostasis. This study addresses the physiological function of the TGF-β/Activin pathway in the adult midgut. When the TGF-β/Activin signaling was disrupted in the adult midgut, glucose repression was abolished. Conversely, increasing TGF-β/Activin signaling in the midgut, through the overexpression of the constitutive active form of Babo or Smad2, was sufficient to repress both amylase and maltase expression. Furthermore, glucose repression is mediated by the TGF-β ligand Daw, produced and secreted from the fat body, a metabolic tissue functionally analogous to the mammalian liver and adipose tissue. Thus, this study uncovers a physiological role for the TGF-β/Activin pathway in adapting carbohydrate and lipase digestion in response to the nutritional state of the organism. Because many features of digestion and absorption are conserved between flies and mammals, it will be of interest to investigate the role of TGF-β/Activin pathway in mammalian digestion (Karpac, 2013).

Recent studies have attributed a role for Daw in aging and pH homeostasis, two processes tightly linked to metabolism. Thus, it is likely that Daw induced from the fat body in response to carbohydrate in the diet will induce a more global response instead of a local response, affecting only digestive enzyme expression. As such, Daw may act as a central mediator for glucose homeostasis by regulating sugar level in the circulation. When there are sufficient carbohydrates in the diet, Daw expression restricts the expression of carbohydrase and glucose transporters. Concurrently, at the postabsorption level, Daw in the circulation may act directly or indirectly (via insulin signaling) to maintain circulating sugar level. A broader role for Daw in sugar homeostasis is reinforced by the findings that Daw mutant larvae were more sensitive to a high-sugar diet. Similarly, this study found overexpression of Daw, but not Myo, Mav, or Actβ, renders flies sensitive to sugar starvation. Along this line, in C. elegans, the TGF-β signaling is reported to be elevated and required in neurons for satiety. There were also several observations that hyperglycemia is linked to increased TGF-β activity in mammals. Hence, the role of TGF-β/Activin signaling in sugar homeostasis requires further investigation in Drosophila and other organisms (Karpac, 2013).

In conclusion, this study revealed a remarkable resilience in the regulation of carbohydrate and lipid-acting enzymes expression to ensure that digestive capacity in the midgut is not compromised before certain metabolic criteria in the fat body is attained. The study also unraveled a role of the TGF-β/Activin-signaling pathway in the adult Drosophila midgut, which has not been appreciated. It reinforced the notion that the gut is not a passive tube for nutriment flow. Rather, it dynamically modulates digestive enzyme expression in response to the organism’s nutritional state through endocrine signals derived from other metabolic tissues (Karpac, 2013).

DEVELOPMENTAL BIOLOGY

Embryonic

Analysis of daw expression revealed maternally-provided mRNA in presyncitial stage embryos. Zygotic daw transcripts were seen in the mesoderm from stage 6 to 8, and at higher levels after stage 9. At stage 13, expression was also detected in the visceral mesoderm and oenocytes. Somatic muscle expression is significantly reduced by stage 15. Instead, at stage 16, prominent expression was seen in the ventral nerve cord (VNC), in median, intermediate and lateral groups of cells in a segmental pattern. Absence of daw expression in the VNC of glial cells missing (gcm^N7-4) mutants that lack glia, as well as double staining with anti-Repo, indicated that the daw-positive cells in the VNC correspond to glia. Additional sites of transcription were the fat body, the ring gland, cells in the maxillary segment, hindgut and the posterior spiracles. In larvae, daw was expressed in the outer proliferative center of the optic lobe and in the central brain, the wing and leg imaginal discs, and in larval bodywall muscles. Northern blots detected a 2.7 kb transcript at high levels in late embryonic and larval stages and in adult males and females (Parker, 2006).

To determine if daw is a biologically relevant substrate for the secreted Tolloid-related (Tlr), the daw expression pattern was analyzed. daw is expressed ubiquitously in the early embryo, but in the later stages of embryogenesis the expression is enriched in mesoderm, muscle, and in a subset of cells in the CNS that are, based on position, likely to be glial. In the third instar larval stage, daw expression was seen in many glia within the brain lobes and ventral ganglion. The muscle expression is very strong at later developmental stages, as seen in the ventral muscle field of a third-instar filet. daw expression was also observed in all larval imaginal discs, and in the cells of the tracheal system (Serpe, 2006).

EFFECTS OF MUTATION

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

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

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

An important question is whether Daw functions as an instructive cue that provides directional information, or as a permissive factor that promotes axon outgrowth. Several lines of evidence argue against an instructive role. (1) Axon pathfinding does not require restricted expression of daw. Guidance defects associated with daw mutants can be rescued by daw expression in sites of endogenous transcription, and ectopically in motoneurons. (2) In daw mutants, axons do not extend into inappropriate areas or show ectopic branching, phenotypes typical of mutations in Sema-2a, Netrin-A and Netrin-B that provide spatial guidance cues or target recognition. (3) Misexpression of Daw did not cause mistargeting of axons, indicating no apparent spatial sensitivity to Daw. These data are therefore consistent with a permissive role in which Daw enables and/or modulates the response of the growth cone to other restricted cues (Parker, 2006).

Both daw and Smad2 mutants display similar errors in pathfinding, suggesting that daw acts at the transcriptional level by altering the expression of one or more molecules that regulate growth cone response or motility. No evidence was found for cell fate changes in the embryonic nervous system of daw mutants; and pathfinding defects can be rescued using OK6-Gal4 that initiates expression in motoneurons at stage 15, well after neuronal cell fates are specified. Furthermore, no guidance errors were found in daw third-instar larvae. These results argue that motoneurons are correctly specified and that the embryonic pathfinding errors are compensated by larval stages (Parker, 2006).

Daw signaling could act on a wide range of transcriptional targets in the neuron. Mutations in several genes that function as guidance cues, such as plexin A, Sema-1a and side, show ISNb and SNa phenotypes similar to daw mutants. Thus daw could alter the activity of or the response to these guidance cues. Plexin A and Sema-1a are expressed in neurons and mediate local repulsion. Sidestep, a muscle-derived attractant is unlikely to be directly regulated, however components involved in the response to side could be downstream of daw. Interestingly, overexpression of the IgCAM Fas2 that promotes axon fasciculation also results in stalling defects reminiscent of the daw phenotype. Thus, Daw could act on Fas2 or Beat-Ia, which potentially downregulates Fas2 in motoneurons, to decrease adhesiveness at specific choice points in response to cues directing defasciculation. Other possible targets are the RPTPs that affect fasciculation and outgrowth. Whereas the Lar-null phenotype is significantly stronger than that of daw, the combinatorial loss of RPTP10D, RPTP69D and RPTP99A mimics the loss of Daw activity in ISNb and SNa pathways. Finally, daw mutants show phenotypic overlap with mutations in the actin-microtubule crosslinking proteins Pod1 and Shot, and in the actin-binding protein Profilin, which are required for axon outgrowth. This raises the possibility that Daw signaling could control the expression or activity of genes involved in regulating cytoskeletal dynamics. Future epistasis studies will resolve whether Daw acts in conjunction with, or parallel to, known pathways that regulate axon fasciculation and extension (Parker, 2006).

The consequences of mutations in daw are often less severe and limited to a subset of axon pathways affected by the genes discussed above, suggesting that Daw could in part act redundantly with other proteins. Alternatively, daw activity could be spatially restricted to select axon pathways by localized expression of receptors, coreceptors or other pathway components. For example, in the Drosophila CNS, only axons expressing the Derailed receptor are sensitive to the Wnt5 repulsive cue, and are hence directed away from the posterior into the anterior commissure. Alternatively, receptor expression could be dynamically modulated, as seen in the downregulation of Robo in commissural neurons by transient expression of Commissureless, which results in local insensitivity to Slit at the midline. Although mRNA for the Daw-receptors Babo and Put can be detected throughout the VNC, it remains to be seen whether the proteins are enriched in a subset of growth cones, restricting the response to Daw. A further possibility is that the activity of the ligand itself could be spatially regulated. A recent study has shown that mutations in tolloid-related (tlr; tolkin - Flybase) that encodes a metalloprotease of the BMP1/Tolloid family, display persistent pathfinding defects (Meyer, 2006). tlr is required for activation of a latent Daw complex (Serpe, 2006). Intriguingly, this study found that daw/+; tlr/+ embryos show pathfinding defects consistent with a functional link between the two genes. However, localized activation of Daw by Tlr appears unlikely because Tlr is present in the hemolymph and circulates throughout the embryo. Finally, Daw interaction with the extracellular matrix or HSPGs could result in localized presentation of the ligand to the extending growth cone. HSPGs are known to modulate BMP activity by affecting ligand stability and receptor interaction. An intriguing possibility is that in addition to functioning as ligands for Lar, HSPGs could also function in axon guidance by enhancing Daw signaling (Parker, 2006).

This study provides the first evidence that an activin pathway, acting through its transcription factor Smad2, can direct axon pathfinding. The mechanism by which Daw functions stands in contrast to previous studies implicating BMP/TGF-ß ligands in direct regulation of growth cone motility independent of a nuclear response. In vertebrates, BMP7/GDF7 heterodimers secreted by the spinal cord roof plate mediate repulsion of commissural axons away from the dorsal midline. Exposure to BMP7 for as little as 30 minutes resulted in growth cone collapse in cultured neurons. BMP7 can also stimulate formation of dendritic arbors by directly regulating the cytoskeleton. This Smad-independent effect requires interaction of the BMP type-II receptor with LIMK1 that regulates the actin-depolymerising factor cofilin, and suggests a potential mechanism by which BMPs may influence growth cone motility as well. In Drosophila, a BMP/Wit pathway acting through LIMK1 promotes synapse stabilization at the NMJ although this is independent of ADF/Cofilin, suggesting that at least part of this mechanism is conserved (Parker, 2006).

In C. elegans, the TGF-ß family member UNC-129 functions as a target-derived chemoattractant for dorsally projecting motor axons. UNC-129 is also likely to exploit an unconventional mechanism to direct motoneuron guidance, as mutations in the single type-II receptor DAF-4 or the Smad signal transducer do not cause pathfinding defects. Thus TGF-ß family members may utilize both canonical (as seen for Daw) and noncanonical strategies to regulate neuronal guidance, depending on context. It remains to be determined if an activin signaling pathway, comparable to Daw, plays a role in vertebrate axon guidance. The finding that axons from ventrally-derived retinal ganglion cells fail to enter the optic nerve head in Bmpr1b-deficient mice implicates a conventional BMP signal-transduction pathway in vertebrate growth cone guidance. Conversely, the recent finding that LIMK1 and cofilin have been implicated in axon outgrowth in mushroom body neurons, raises the possibility that an activin/BMP ligand acting through Wit could initiate this process in the larval brain (Parker, 2006).

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).

EVOLUTIONARY HOMOLOGS

Long-lasting modifications in synaptic transmission depend on de novo gene expression in neurons. The expression of activin, a member of the transforming growth factor (TGF-β) superfamily, is upregulated during hippocampal long-term potentiation (LTP). Activin increased the average number of presynaptic contacts on dendritic spines by increasing the population of spines that were contacted by multiple presynaptic terminals in cultured neurons. Activin also induced spine lengthening, primarily by elongating the neck, resulting in longer mushroom-shaped spines. The number of spines and spine head size were not significantly affected by activin treatment. The effects of activin on spinal filamentous actin (F-actin) morphology were independent of protein and RNA synthesis. Inhibition of cytoskeletal actin dynamics or of the mitogen-activated protein (MAP) kinase pathway blocked not only the activin-induced increase in the number of terminals contacting a spine but also the activin-induced lengthening of spines. These results strongly suggest that activin increases the number of synaptic contacts by modulating actin dynamics in spines, a process that might contribute to the establishment of late-phase LTP (Shoji-Kasai, 2007).

Regeneration of the retina in amphibians is initiated by the transdifferentiation of the retinal pigmented epithelium (RPE) into neural progenitors. A similar process occurs in the early embryonic chick, but the RPE soon loses this ability. The factors that limit the competence of RPE cells to regenerate neural retina are not understood; however, factors normally involved in the development of the eye (i.e., FGF and Pax6) have also been implicated in transdifferentiation. Therefore, whether activin, a TGFβ family signaling protein shown to be important in RPE development, contributes to the loss in competence of the RPE to regenerate retina was tested. It was found that addition of activin blocks regeneration from the RPE, even during stages when the cells are competent. Conversely, a small molecule inhibitor of the activin/TGFβ/nodal receptors can delay, and even reverse, the developmental restriction in FGF-stimulated neural retinal regeneration (Sakami, 2008).

Studies of the olfactory epithelium model system have demonstrated that production of neurons is regulated by negative feedback. A locally produced signal, the TGFβ superfamily ligand GDF11, regulates the genesis of olfactory receptor neurons by inhibiting proliferation of the immediate neuronal precursors (INPs) that give rise to them. GDF11 is antagonized by follistatin (FST), which is also produced locally. This study shows that Fst^-/- mice exhibit dramatically decreased neurogenesis, a phenotype that can only be partially explained by increased GDF11 activity. Instead, a second FST-binding factor, activin βB (ACTβB), inhibits neurogenesis by a distinct mechanism: whereas GDF11 inhibits expansion of INPs, ACTβB inhibits expansion of stem and early progenitor cells. Data is presented supporting the concept that these latter cells, previously considered two distinct types, constitute a dynamic stem/progenitor population in which individual cells alternate expression of Sox2 and/or Ascl1. In addition, it was demonstrated that interplay between ACTβB and GDF11 determines whether stem/progenitor cells adopt a glial versus neuronal fate. Altogether, the data indicate that the transition between stem cells and committed progenitors is neither sharp nor irreversible and that GDF11, ACTβB and FST are crucial components of a circuit that controls both total cell number and the ratio of neuronal versus glial cells in this system. Thus, these findings demonstrate a close connection between the signals involved in the control of tissue size and those that regulate the proportions of different cell types (Gokoffski, 2011).

Age-dependent neuroendocrine signaling from sensory neurons modulates the effect of dietary restriction on rongevity of Caenorhabditis elegans

Dietary restriction extends lifespan in evolutionarily diverse animals. Little is known about how neuroendocrine signals influence the effects of dietary restriction on longevity. This study shows that DAF-7/TGFbeta (see Drosophila Daw and Activin-β), which is secreted from the C. elegans amphid, promotes lifespan extension in response to dietary restriction in C. elegans. DAF-7 produced by the ASI pair of sensory neurons acts on DAF-1/TGFbeta receptors (see Drosophila Thickveins) expressed on interneurons to inhibit the co-SMAD DAF-3 (see Drosophila Medea). Increased activity of DAF-3 in the presence of diminished or deleted DAF-7 activity abrogates lifespan extension conferred by dietary restriction. It was also observe that DAF-7 expression is dynamic during the lifespan of C. elegans, with a marked decrease in DAF-7 levels as animals age during adulthood. This age-dependent diminished expression contributes to the reduced sensitivity of aging animals to the effects of dietary restriction. These studies establish a molecular link between the neuroendocrine physiology of C. elegans and the process by which dietary restriction can extend lifespan (Fletcher, 2017).

Genetic control of encoding strategy in a food-sensing neural circuit

Neuroendocrine circuits encode environmental information via changes in gene expression and other biochemical activities to regulate physiological responses. Previous work has shown that daf-7 TGFbeta and tph-1 tryptophan hydroxylase expression in specific neurons encode food abundance to modulate lifespan in Caenorhabditis elegans, and uncovered cross- and self-regulation among these genes. This study extends these findings by showing that these interactions between daf-7 and tph-1 regulate redundancy and synergy among neurons in food encoding through coordinated control of circuit-level signal and noise properties. This analysis further shows that daf-7 and tph-1 contribute to most of the food-responsiveness in the modulation of lifespan. A computational model was applied to capture the general coding features of this system. This model agrees with a previous genetic analysis and highlights the consequences of redundancy and synergy during information transmission, suggesting a rationale for the regulation of these information processing features.

REFERENCES

Search PubMed for articles about Drosophila dawdle

Alic, N., Andrews, T. D., Giannakou, M. E., Papatheodorou, I., Slack, C., Hoddinott, M. P., Cocheme, H. M., Schuster, E. F., Thornton, J. M. and Partridge, L. (2011). Genome-wide dFOXO targets and topology of the transcriptomic response to stress and insulin signalling. Mol Syst Biol 7: 502. PubMed ID: 21694719

Bai, H., Kang, P., Hernandez, A. M., Tatar, M. (2013). Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila. PLoS Genet 9: e1003941. PubMed ID: 24244197

Bickel, D., Shah, R., Gesualdi, S. C. and Haerry, T. E. (2008). Drosophila Follistatin exhibits unique structural modifications and interacts with several TGF-beta family members. Mech. Dev. 125(1-2): 117-29. PubMed citation: 18077144

Chng, W. B., Sleiman, M. S., Schupfer, F. and Lemaitre, B. (2014). Transforming growth factor beta/Activin signaling functions as a sugar-sensing feedback loop to regulate digestive enzyme expression. Cell Rep 9: 336-348. PubMed ID: 25284780

Diana, G., Patel, D. S., Entchev, E. V., Zhan, M., Lu, H. and Ch'ng, Q. (2017). Genetic control of encoding strategy in a food-sensing neural circuit. Elife 6. PubMed ID: 28166866

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

Fletcher, M. and Kim, D. H. (2017). Age-dependent neuroendocrine signaling from sensory neurons modulates the effect of dietary restriction on longevity of Caenorhabditis elegans. PLoS Genet 13(1): e1006544. PubMed ID: 28107363

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

Ghosh, A. C. and O'Connor, M. B. (2014). Systemic Activin signaling independently regulates sugar homeostasis, cellular metabolism, and pH balance in Drosophila melanogaster. Proc Natl Acad Sci U S A 111: 5729-5734. PubMed ID: 24706779

Gokoffski, K. K., et al. (2011). Activin and GDF11 collaborate in feedback control of neuroepithelial stem cell proliferation and fate. Development 138(19): 4131-42. PubMed Citation: 21852401

Jensen, P. A., Zheng, X., Lee, T. and O'Connor, M. B. (2009). The Drosophila Activin-like ligand Dawdle signals preferentially through one isoform of the Type-I receptor Baboon. Mech. Dev. 126(11-12): 950-7. PubMed Citation: 19766717

Karpac, J., Biteau, B. and Jasper, H. (2013). Misregulation of an adaptive metabolic response contributes to the age-related disruption of lipid homeostasis in Drosophila. Cell Rep 4: 1250-1261. PubMed ID: 24035390

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

Meyer, F. and Aberle, H. (2006). At the next stop sign turn right: the metalloprotease Tolloid-related 1 controls defasciculation of motor axons in Drosophila. Development 133: 4035-4044. Medline abstract: 16971470

Parker, L., Ellis, J. E., Nguyen, M. Q. and Arora, K. (2006). The divergent TGF-β ligand Dawdle utilizes an activin pathway to influence axon guidance in Drosophila. Development 133(24): 4981-91. Medline abstract: 17119022

Sakami, S., Etter, P. and Reh, T. A. (2008). Activin signaling limits the competence for retinal regeneration from the pigmented epithelium. Mech. Dev. 125(1-2): 106-16. PubMed citation: 18042353

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

Shoji-Kasai, Y., et al. (2007). Activin increases the number of synaptic contacts and the length of dendritic spine necks by modulating spinal actin dynamics. J. Cell Sci. 120(Pt 21): 3830-7. Medline abstract: 17940062

Simonsen, A., Cumming, R. C., Brech, A., Isakson, P., Schubert, D. R. and Finley, K. D. (2008). Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila. Autophagy 4: 176-184. PubMed ID: 18059160

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

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