Gene name - dawdle
Synonyms - Activin Like Protein at 23B (Alp23B)
Cytological map position- 23B1-23B2
Function - ligand
Symbol - daw
FlyBase ID: FBgn0031461
Genetic map position - 23B1-23B2
Classification - TGF-ß superfamily ligand
Cellular location - secreted
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).
A Blast search of the BDGP database for sequences with homology to human activin identified a genomic clone DS07149, and two cDNA clones GH14433 and RE17443. GH14433 contains a 1762 bp ORF flanked by 543 bp of 5' sequence and 405 bp of 3' untranslated region (UTR) (daw-A. RE17443 represents an alternate isoform with a unique first exon derived from a distinct promoter, resulting in a transcript that is shorter by 146 bp (daw-B). Both transcripts contain common coding exons and encode an identical predicted protein of 586 amino acids. This gene was initially called Activin Like Protein at 23B (Alp23B, CG16987). To avoid confusion with Abnormal leg pattern (Alp) and to better reflect the mutant phenotype, the locus is referred to as daw. The daw ORF shows several features characteristic of TGF-ß superfamily ligands. The first in-frame methionine is followed by a signal sequence indicating that the protein is likely to be secreted. Two consensus cleavage sites for furin-like proteases, RRRK (464-467) and RQKR (469-472), would generate a mature ligand of 114 (or 119) amino acids. Within the ligand domain, Daw shares 35% identity with human activinC, TGF-ß3 and BMP3. However, the presence of nine rather than seven cysteines at stereotypic positions characteristic of activin and TGF-ß subfamilies, suggests that Daw represents a divergent activin/TGF-ß ligand in Drosophila (Parker, 2006).
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).
date revised: 28 May 2008
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