In the Drosophila ovary, germline stem cells (GSCs) are maintained primarily by bone morphogenetic protein (BMP) ligands produced by the stromal cells of the niche. This signaling represses GSC differentiation by blocking the transcription of the differentiation factor Bam. Remarkably, bam transcription begins only one cell diameter away from the GSC in the daughter cystoblasts (CBs). How this steep gradient of response to BMP signaling is formed has been unclear. This study shows that Fused (Fu), a serine/threonine kinase that regulates Hedgehog, functions in concert with the E3 ligase Smurf to regulate ubiquitination and proteolysis of the BMP receptor Thickveins in CBs. This regulation generates a steep gradient of BMP activity between GSCs and CBs, allowing for bam expression on CBs and concomitant differentiation. Similar roles for Fu were observed during embryonic development in zebrafish and in human cell culture, implying broad conservation of this mechanism (Xia, 2010).
Previous studies have demonstrated that BMP/Dpp signals from the niche play primary roles in the self-renewal of GSCs by silencing bam transcription. However, the mechanism by which the differentiating CBs avoid the control of BMP/Dpp and activate bam remains poorly understood. This study has provided direct evidence that the differentiating daughter cells of GSCs, known as CBs, become resistant to BMP signaling through degradation of Tkv in CBs. Fu functions as an antagonistic factor in BMP/Dpp signaling by regulating Tkv degradation during the differentiation of CBs. Moreover, both genetic and biochemical evidence is provided that Fu acts in concert with Smurf, a HECT domain-containing ubiquitin E3 ligase, to regulate the ubiquitination of Tkv in the CB, thereby generating a steep gradient of response to BMP signaling between GSCs and CBs for their fate determination. Finally, a conserved role is shown for fu in antagonizing BMP/ TGFβ signals in zebrafish embryonic development as well as in human cell cultures. These findings not only reveal a conserved function of fu in controlling BMP/TGFβ signal-mediated developmental processes, but also provide a comprehensive view of mechanisms that produce both self-renewal and asymmetry in the division of stem cells (Xia, 2010).
Observations of the existence of a BMP resistance mechanism that controls the proper division of GSCs through the regulation of Tkv prompted an exploration of how Tkv was regulated. Using immunoprecipitation followed by mass spectrometry analysis, it was identified that Fu associates with the Tkv protein. Given that previous studies demonstrated that a loss of fu leads to early germ cell proliferation and a tumorous germarium phenotype and that biochemical evidence showed that Fu forms a complex with Tkv and affects its stability, it was subsequently identified that Fu as a component negatively regulates BMP/Dpp signaling by interacting with the BMP/Dpp type I receptor, Tkv (Xia, 2010).
BMP/TGFβ signals play pivotal roles in controlling diverse normal developmental and cellular processes. In the canonical BMP/TGFβ pathway, the receptors and Smad proteins are the essential components for BMP/TGFβ signal transduction. However, this pathway is known to be modulated by additional factors to reach physiological levels in a cellular context-dependent manner. Smurfs and HECT domain-containing proteins have been shown to antagonize BMP/TGFβ signals through the regulation of the stability of either receptors or Smads in vertebrates. In Drosophila, Smurf has previously been implicated in regulating proteolysis of phosphorylated Smad proteins in somatic cells. In the ovary, Smurf was also proposed to downregulate the level of BMP to promote CB differentiation. The mechanism underlying the action of Smurf in Drosophila early germline cells remains elusive. This study has shown that Fu, Smurf, and Tkv could form a trimeric complex in S2 cells. Importantly, both Fu and Smurf are required for ubiquitination of Tkv in S2 cells and for turnover of Tkv in germ cells. Combined with genetic evidence, it is proposed that Fu and Smurf likely function in a common biochemical process by controlling Tkv degradation. The present study reveals a mechanism by which Fu serves as an essential component in the Smurf-mediated degradation of the BMP/TGFβ receptor, thereby terminating BMP/TGFβ signaling and negatively regulating the downstream target genes of BMP/TGFβ (Xia, 2010).
Because Fu is a putative serine/threonine protein kinase, the question becomes how Fu acts on Tkv regulation in concert with Smurf. Given that knockdown of fu does not significantly change the pattern of autoubiquitination of Smurf itself, it is therefore likely that Tkv is a strong candidate substrate for Fu kinase. Although there is no assay system for analyzing the kinase activity of Fu presently, in this study, mutagenesis assays were perfomred and it was identified that the S238 in Tkv is important for Tkvca to respond to Fu and is critical for Tkvca ubiquitination and degradation. Of note, it was found that the ubiquitin- resistant form of Tkvca [TkvcaS238A] blocks CB differentiation. A previous study has shown that the S189 site in TGF-β type-I receptor, the corresponding site of S238 in Tkv, was phosphorylated in the cell culture system. The current results suggest that Fu likely acts on Tkv through targeting and phosphorylating the S238 site and subsequently leads to Tkv ubiquitination and degradation by Smurf. Nevertheless, it would be advantageous to develop a kinase assay system for Fu to determine whether the S238 site in Tkv is an authentic phosphorylation site for Fu kinase in the future (Xia, 2010).
Previous genetic analyses revealed that Fu plays an evolutionarily conserved role in the proper activation of the Hh pathway and functions downstream of the Hh receptor. Increasing evidence has shown that the kinase Fu regulates the Hh-signaling complex by targeting Cos2. However, the function of Fu as a component in the Hh pathway is not consistent with its spatiotemporal expression pattern during development. For example, Hh signaling only plays a role in zebrafish embryonic development at late stages, but Fu is expressed ubiquitously at both the early and the late stages of zebrafish embryonic development. These findings suggest that Fu may have Hh-independent functions in different physiological conditions. In this study, by using several different systems, including Drosophila germline, zebrafish embryo, and human tissue cultures, it was demonstrated that Fu is indeed required for balancing proper BMP/TGFβ signals in different developmental processes. Given that both Fu and Smurf are evolutionarily conserved proteins, it would be interesting to determine whether the Fu/Smurf complex also plays roles in other signaling pathways (Xia, 2010).
The Hippo pathway has been implicated in controlling organ size and tumorigenesis and the underlying molecular mechanisms have attracted intensive attentions. This work identified dSmurf as a new regulator of Wts, a core component of the Hippo pathway, in Drosophila. The data revealed that Wts and dSmurf colocalize to cytoplasm and physically form an immunoprecipitated complex in S2 cells. Sufficient knock-down of dSmurf increases the protein abundance of Wts and thus increases phosphorylation level at S168 of Yki, the key downstream target of Wts in the Hippo pathway. Genetic epistasis assays showed that halving dosage of dSmurf dominantly enhances the phenotype caused by overexpression of Wts and restrains Yki activity in Drosophila eyes. This works defines a novel role of dSmurf in animal development through modulating Wts turnover and thereby Hippo signal transduction, implying that targeting dSmurf may be a promising therapeutic strategy to manipulate the Hippo pathway in pathological conditions (Cao, 2014).
Smurf1 is expressed uniformly at the blastoderm stage and broadly but weakly at later stages of embryogenesis (Podos, 2001).
The phenotypes caused by the two Smurf1 mutations were examined. Both Smurf1 mutants are viable and fertile as homozygotes and exhibit no significant defects in adult structures derived from imaginal discs. However, mutation of both the maternal and zygotic components of Smurf1 activity resulted in embryonic lethality. Therefore, Smurf1 encodes an essential gene that is required for normal embryogenesis (Podos, 2001).
The penetrance of the observed lethality was significantly greater for Smurf115C (99%) than for Smurf111R (26%), even though both alleles result from Hobo insertions within the Smurf1 coding region. The specific phenotypes of Smurf111R are similar to those of Smurf115C, but with lower penetrance and expressivity in every aspect examined. These differences suggest either that Smurf111R retains residual activity or that Smurf115C encodes a truncated protein with antimorphic properties. Because no deficiencies are available for this region of the Drosophila genome, it is not possible to distinguish between these alternatives (Podos, 2001).
A function of Smurf1 in Dpp signaling is revealed by the novel posterior cuticular hole of Smurf1 mutant embryos. The hindgut, which expresses brachyenteron (byn) throughout development, is defective in Smurf1 mutants. In wild-type embryos, by stage 14 the posterior segments have aligned and fused dorsally over the hindgut. In contrast, the hindgut in stage 14 Smurf1 mutant embryos is a foreshortened, widened structure that bulges to the dorsal surface of the embryo where it likely interferes with epidermal closure. Moreover, the apical epithelial protein Crumbs, which marks the lumen of the hindgut, is located on the exposed hindgut surface. It is concluded that the dorsal cells of the hindgut have lost their integrity by this stage, splaying the tube open and exposing its interior surface (Podos, 2001).
To determine the stage of embryogenesis at which a hindgut defect is first visible, the developmental progression of byn transcription was examined in Smurf1 mutant embryos. Although the initial hindgut specification appears normal, by stage 10 the dorsal hindgut cells cease to express byn in at least some Smurf115C mutant embryos. As mutations in byn cause reaper expression in the hindgut primordium at stage 10 and significant apoptosis by stage 14, this local downregulation of byn is likely sufficient to cause the ensuing loss of dorsal hindgut integrity (Podos, 2001).
It is propose that temporal deregulation of Dpp signaling, coupled with an increased level of signaling, directly causes the hindgut defect. In wild-type embryos, the descendants of the cells that had high levels of P-Mad staining at stage 6 gradually downregulate P-Mad levels during stages 8 and 9. By stage 10, P-Mad staining is not readily observable in any of these cells, except those that form the amnioserosa. Conversely, in Smurf1 embryos, P-Mad staining is maintained at high levels in all of these cells through stage 10, but is downregulated by stage 12. The prolonged presence of P-Mad in Smurf1 mutant embryos reflects a generalized requirement for Smurf1 in the downregulation of Dpp signals in multiple tissues during embryogenesis (Podos, 2001).
The perdurance of P-Mad correlates with, and is the likely cause of, the ectopic expression of Dpp target genes within the developing hindgut. At the onset of gastrulation in wild-type embryos, Dpp-dependent zen transcription is present in the amnioserosa primordium in the trunk region of the embryo, but is excluded by an unknown mechanism from the dorsal hindgut primordium. zen expression is then rapidly downregulated and completely disappears by stage 8. In contrast, zen expression in Smurf1 mutant embryos persists at stage 8, and has been observed as late as stage 10. Prior to gastrulation, zen expression in Smurf1 embryos is excluded from the hindgut primordium. Strikingly, however, at stage 8, zen expression extends into the adjacent dorsal hindgut territory. This ectopic zen expression might follow directly from its temporal deregulation, if the mechanism that normally restricts its expression from the presumptive hindgut is not active past stage 7 (Podos, 2001).
Three genetic criteria indicate that defects in Dpp signaling directly cause the hindgut phenotype in Smurf1 mutant embryos: (1) the hindgut defects are not observed in Smurf1 mutant embryos that lack one copy of dpp; (2) a complete loss of zen function substantially suppresses the Smurf115C phenotype, restoring the embryonic hindgut to a tubular morphology and an interior location. It is noted that the hindgut of Smurf115C; zen double mutant embryos often fails to adopt the normal hook-shaped trajectory, suggesting that the deregulation of other target genes also contributes to the Smurf1 phenotype. (3) The hindgut defect was also suppressed in sog; Smurf115C double mutant embryos. While Sog antagonizes BMP signaling in the ventrolateral ectoderm, a positive activity of Sog is also required at the onset of gastrulation to promote the Dpp-dependent specification of amnioserosa at the dorsal midline. It is proposed that the blastoderm-specific Dpp signaling in the dorsal-most region of sog;Smurf1 embryos is reduced to a level that, even in the absence of temporal downregulation of P-Mad, does not elicit the observed Smurf1 hindgut defect (Podos, 2001).
The temporal deregulation of Dpp signaling in Smurf1 mutant embryos may cause additional defects in organogenesis. In the dorsal stomodeum of wild-type stage 12 embryos, each of three proneural cells acts as a tip cell to cause stereotypical epithelial invaginations that ultimately reorganize to give rise to the stomatogastric nervous system. In Smurf1 mutant embryos, which have prolonged P-Mad staining in the dorsal stomodeum, there is a variability in the number and shape of these invaginations. However, it has not yet been ascertained whether elevated Dpp signaling in the stomodeum is the direct cause of this defect. In conclusion, these results demonstrate that Smurf1 is essential for the temporal downregulation of Dpp signals necessary at the onset of gastrulation and that, for at least the hindgut, failure of this downregulation causes specific defects in organogenesis much later during embryogenesis (Podos, 2001).
The available experimental data support the hypothesis that the cap cells (CpCs) at the anterior tip of the germarium form an environmental niche for germline stem cells (GSCs) of the Drosophila ovary. Each GSC undergoes an asymmetric self-renewal division that gives rise to both a GSC, which remains associated with the CpCs, and a more posterior located cystoblast (CB). The CB upregulates expression of the novel gene, bag of marbles (bam), which is necessary for germline differentiation. Decapentaplegic (Dpp), a BMP2/4 homolog, has been postulated to act as a highly localized niche signal that maintains a GSC fate solely by repressing bam transcription. The role of Dpp in GSC maintenance has been examined in more detail. In contrast to the above model, it is found that an enhancer trap inserted near the Dpp target gene, Daughters against Dpp (Dad), is expressed in additional somatic cells within the germarium, suggesting that Dpp protein may be distributed throughout the anterior germarium. However, Dad-lacZ expression within the germline is present only in GSCs and to a lower level in CBs, suggesting there are mechanisms that actively restrict Dpp signaling in germ cells. One function of Bam is to block Dpp signaling downstream of Dpp receptor activation, thus establishing the existence of a negative feedback loop between the action of the two genes. Moreover, in females doubly mutant for bam and the ubiquitin protein ligase Smurf, the number of germ cells responsive to Dpp is greatly increased relative to the number observed in either single mutant. These data indicate that there are multiple, genetically redundant mechanisms that act within the germline to downregulate Dpp signaling in the Cb and its descendants, and raise the possibility that a Cb and its descendants must become refractory to Dpp signaling in order for germline differentiation to occur (Casanueva, 2004).
The prevalent model for Dpp action within the ovary is that it is a local niche signal whose activity is permissive for GSC maintenance. In this model, only GSCs within the niche are exposed to Dpp protein and removal of the CB from the niche lessens or eliminates exposure to the ligand. Moreover, the only postulated function of Dpp is to repress the transcription of bam within the GSCs. The data presented in this paper reveal additional aspects of Dpp function in GSC maintenance. The results strongly suggest that Dpp ligand is not restricted to the niche but rather is present throughout the anterior germarium. Data is presented that the observed specificity of Dpp signaling to the GSCs and CBs is due to functionally redundant mechanisms that operate in the germline to actively downregulate Dpp signaling during GSC differentiation. One of these mechanisms is Bam itself, thus establishing a negative feedback loop between the actions of the two genes. These findings indicate GSC differentiation is correlated with downregulation of Dpp signaling, raising the possibility that Dpp signaling plays an active role in GSC maintenance, and that GSC differentiation requires both the presence of Bam and the absence of Dpp signaling (Casanueva, 2004).
If GSCs and CBs are exposed to equivalent amounts of Dpp protein, as is suggested by both the transcription pattern of the Dpp gene and the expression of Dad-lacZ in the CpCs of the niche and the ISCs posterior to the niche, then it is likely that the observed reduction in Dad-lacZ expression between the GSC and the CB results from intracellular modulation of the strength of the Dpp signal. One hallmark of the GSC is its invariant plane of division. It is proposed that the differential Dpp signaling between the GSC and CB sign results from an intracellular modulation of Dpp signal strength between the two daughter cells, either by the asymmetric segregation of one or more cellular components that modulate Dpp signaling, or by loss of a contact-based niche signal that elevates Dpp signaling preferentially within the GSCs. Removal of the CB cell from the niche thus results in partial downregulation of Dpp signaling. A lower level of Dpp signaling in the CB cell results in the transcription of Bam, which plays multiple roles in CB differentiation, one of which is to cause the daughters of the CB cell to become refractory to further Dpp signaling. Thus, sequential regulatory mechanisms cooperate to ensure an irreversible change in the fate of the GSC cell within two generations (Casanueva, 2004).
Loss-of-function mutations in Smurf and gain-of-function mutations in sax increase the number of GSCs, suggesting these genes may perturb the proposed intracellular modulation of Dpp signaling that occurs between the GSC and CB. However, these data are not sufficient to determine whether this proposed modulatory pathway acts through direct regulation of the functions of one or both of these gene products, or whether the proposed pathway acts in parallel to these genes. In the embryo, loss of Smurf activity results in a ligand-dependent elevation of Dpp signaling that has greater, but not indefinite, perdurance (Podos, 2001), suggesting that Dpp signaling in Smurf mutants, and by inference sax mutants, is still responsive both to the amount of ligand and to the presence of other negative regulatory mechanisms. In the ovary, the Dad-lacZ-expressing germ cells in the Smurf and sax mutants fill the region of the anterior germarium that roughly corresponds to the spatial extent of Dad-lacZ expression in the somatic cells of region 1 and 2A of a wild-type germarium, suggesting that potentially all germ cells in region 1 and 2A of the Smurf and sax germaria are equally and fully responsive to the Dpp ligand. It is proposed that GSCs in the Smurf and sax germaria ultimately undergo normal differentiation because in the more posterior regions of the germaria the amount of Dpp ligand may be reduced to a level that allows bam transcription, which further reduces Dpp signaling and causes cyst differentiation (Casanueva, 2004).
The reduction in Dpp signaling between the GSC and the CB releases Bam from Dpp-dependent transcriptional repression, and one, but not the only, function of Bam is to downregulate Dpp signaling downstream of receptor activation prior to overt GSC differentiation. This is the first molecular action ascribed to Bam, and these data could provide an entry point to elucidate the biochemical basis of the function of Bam in CB differentiation. Further work will be necessary to determine whether the action of Bam on the Dpp pathway is direct or indirect, whether Bam action results in the reduction or complete elimination of Dpp signaling in the developing cysts, and which step in the intracellular Dpp signal transduction pathway or expression of Dpp target genes is affected by Bam action. However, it is possible that initial insights into Bam function can be made by comparing the thresholds for Dpp signaling readouts in the developing wing disc of the larva to the data obtained in the germarium. In the wing disc, Dpp diffuses from a limited source to form a gradient throughout the disc that displays different thresholds for multiple signaling readouts. Specifically, Dad-lacZ is transcribed in response to high and intermediate levels of Dpp, but does not respond to the lowest levels of ligand. An antibody exists that recognizes the active phosphorylated form of Mad, pMad. In the wing disc, high level staining with the pMad antibody is present in only a subset of cells that express high levels of Dad-lacZ, suggesting that in this tissue the pMad antibody is less sensitive to Dpp signaling than is Dad-lacZ expression. Intriguingly, in the ovariole pMad staining is visible in the GSCs, CBs and the developing cysts. Because Dad-lacZ expression was never observed in the developing cysts, these results could suggest that the relative sensitivities of these two reagents are reversed within the germline. Alternatively, if the reagents have the same relative sensitivities in the two tissues, the data suggest that Bam could act, probably at a post-transcriptional level, to downregulate Dpp signaling downstream of Mad activation (Casanueva, 2004).
The pattern of Dad-lacZ expression observed in the Smurf; bam and sax; bam double mutant ovarioles is qualitatively different from that observed in any of the single mutant ovarioles. Although Dad-lacZ expression is observed only at the anterior tip of the germarium of each single mutant, many, but not all, of the double mutant ovarioles contain germ cells throughout the ovariole that express high levels of Dad-lacZ. From these data, it is concluded that two redundant pathways downregulate Dpp signaling in the germline, and that in the single mutants, the action of the remaining active pathway is sufficient to constrain Dpp responsiveness to the anterior tip of the germarium. However, not all doubly mutant ovarioles display a spatial expansion of Dpp signaling, and this variability can even be observed in ovarioles from a single female. It is proposed that the observed variability results because the Smurf and sax mutations have modulatory effects on Dpp signaling that are both dependent on the presence of ligand and are sensitive to additional mechanisms that downregulate Dpp signaling. In both the Smurf; bam and sax; bam ovarioles, the germ cells that express Dad-lacZ are observed throughout the ovariole, but are more likely to be near somatic cells. It is possible that the variability in Dad-lacZ expression occurs because of a non-uniform distribution of the Dpp ligand. Nevertheless, there is not a consistent correlation between the domains of Dad-lacZ expression in the somatic and germ cells, suggesting that there may be additional germline intrinsic factors that affect Dpp signaling (Casanueva, 2004).
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date revised: 25 March 2015
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