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).
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).
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, dawex11 and dawex32, 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).
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 put135-22/put62 animals. At temperatures above 25°C, put135-22/put62 exhibited gross head involution and dorsal closure defects making it difficult to score for axon guidance defects. However, analysis of put135-22/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).
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)
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 (gcmN7-4) 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).
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 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).
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).
Search PubMed for articles about Drosophila Dawdle
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
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-þ-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
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
date revised: 20 August 2008
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