Daughters against dpp
The TGF-β family member Decapentaplegic (Dpp) is a key regulator of patterning and growth in Drosophila development. Previous studies have identified a short DNA motif called the silencer element (SE), which recruits a trimeric Smad complex and the repressor Schnurri to downregulate target enhancers upon Dpp signaling. The minimal enhancer of the dad gene was isolated, and a short motif was discovered that was termed the activating element (AE). The AE is similar to the SE and recruits the Smad proteins via a conserved mechanism. However, the AE and SE differ at important nucleotide positions. As a consequence, the AE does not recruit Schnurri but rather integrates repressive input by the default repressor Brinker and activating input by the Smad signal transducers Mothers against Dpp (Mad) and Medea via competitive DNA binding. The AE allows the identification of hitherto unknown direct Dpp targets and is functionally conserved in vertebrates (Weiss, 2010).
A 520-bp fragment was discovered within the second intron of the dad gene. This fragment induces an expression pattern very similar to that of the endogenous dad gene in embryonic, larval and adult tissues and contains evolutionarily conserved and largely overlapping binding sites for Smad and Brk proteins within a short sequence element that was called activating element (AE). The Smad and Brk proteins bind in a competitive manner to the AE, a mechanism similarly proposed for zen and Ubx enhancer elements. By precise targeted mutations, Brk binding was selectively abolished, and it was possible to unlink Smad and Brk input. Notably, the AE assembles a high-affinity trimeric complex of full-length Mad and Medea proteins. In Drosophila, such complexes have so far only been demonstrated for a so-called silencer element (SE) (Pyrowolakis, 2004). Therefore, this study presents the first example of such complex formation on a short-sequence element in the context of a gene activated by Dpp (Weiss, 2010).
The AE very closely resembles the SE, but despite their analogy, AE and SE differ in several key aspects. Because of the arrangement of the Smad binding sites, they are both able to recruit a complex of Mad and Medea. However, only the SE includes the second thymidine, which is essential for the recruitment of the repressor Shn (Pyrowolakis, 2004). Furthermore, the AE identified in the dad enhancer is able to interact with the Brk repressor. Brk competes with Mad for binding to the AE, which fulfills the consensus sequence derived from analysis of the SE with regard to Mad binding (GRCGNC) as well as the sequence for Brk binding (TGGCGYY). In contrast, Brk does not bind to the SEs described (Pyrowolakis, 2004). Thus, the AE and SE use a very similar sequence to exert opposite effects. These results provide a striking molecular scenario for Dpp signaling readout, based on the assembly of a trimeric Smad complex and its recruitment of a corepressor (Shn) or its competition with a dedicated repressor of the pathway (Brk) (Weiss, 2010).
The role of the zinc finger transcription factor Schnurri (Shn) in mediating the nuclear response to Dpp during adult patterning has been investigated. Using clonal analysis, it has been shown that wing imaginal disc cells mutant for shn fail to transcribe the genes spalt, optomotor blind, vestigial, and Dad, that are known to be induced by dpp signaling. shn clones also ectopically express brinker, a gene that is downregulated in response to dpp, thus implicating Shn in both activation and repression of Dpp target genes. Loss of shn activity affects anterior-posterior patterning and cell proliferation in the wing blade, in a manner that reflects the graded requirement for Dpp in these processes. Furthermore, shn is expressed in the pupal wing and plays a distinct role in mediating dpp-dependent vein differentiation at this stage. The absence of shn activity results in defects that are similar in nature and severity to those caused by elimination of Mad, suggesting that Shn has an essential role in dpp signal transduction in the developing wing. These data are consistent with a model in which Shn acts as a cofactor for Mad (Torres-Vazquez, 2000).
Decapentaplegic (Dpp), a homolog of vertebrate bone morphogenic protein 2/4, is crucial for embryonic patterning and cell fate specification in Drosophila. Dpp signaling triggers nuclear accumulation of the Smads Mad and Medea, which affect gene expression through two distinct mechanisms: direct activation of target genes and relief of repression by the nuclear protein Brinker (Brk). The zinc-finger transcription factor Schnurri (Shn) has been implicated as a co-factor for Mad, based on its DNA-binding ability and evidence of signaling dependent interactions between the two proteins. A key question is whether Shn contributes to both repression of brk as well as to activation of target genes. During embryogenesis, brk expression is derepressed in shn mutants. However, while Mad is essential for Dpp-mediated repression of brk, the requirement for shn is stage specific. Analysis of brk;shn double mutants reveals that upregulation of brk does not account for all aspects of the shn mutant phenotype. Several Dpp target genes are also expressed at intermediate levels in double mutant embryos, demonstrating that shn also provides a brk-independent positive input to gene activation. Shn-mediated relief of brk repression establishes broad domains of gene activation, while the brk-independent input from Shn is crucial for defining the precise limits and levels of Dpp target gene expression in the embryo (Torres-Vazquez, 2001).
Genetic evidence implicates both Shn and Mad in dpp-dependent repression of brk. In the wing disc, cells that lack Mad or shn ectopically express brk and fail to activate the Dpp-responsive genes optomotor-blind, vestigial, spalt and Dad. Abolition of shn or Mad activity results in upregulation of brk in the embryo and in the absence of shn ectopic Dpp cannot suppress brk expression. Since Shn and Mad interact directly, an attractive hypothesis is that a Shn/Mad complex is involved in the Dpp-dependent repression of brk. It has recently been suggested that Dpp signaling bifurcates downstream of Mad/Med into a Shn-dependent pathway, leading to brk repression and a Shn-independent pathway that triggers gene activation. According to this model, Shn acts primarily as a dedicated repressor that switches Mad from a transcriptional activator to a transcriptional repressor on the brk promoter. However several lines of evidence from this study are incompatible with such an interpretation (Torres-Vazquez, 2001).
Analysis of Dpp-responsive gene expression in brk; shn double mutants has allowed an assessment the brk-independent input from shn to gene activation at different developmental stages in a range of tissues. Although it has not been demonstrated that each of these markers is a direct target of Dpp signaling, three categories of responses can be distinguished based on these studies. In the first group (class A), exemplified by dpp in the leading edge of the dorsal ectoderm, expression in the double mutant is indistinguishable from that in brk- embryos. Thus, shn contributes to class A gene expression primarily by relief of brk repression. Promoters belonging to class B include Dad and pnr in the dorsal ectoderm during germband extension. Expression of class B genes is downregulated in the double mutant compared with brk- embryos, but is equivalent to wild-type levels. It is inferred from this result that in the absence of Brk and Shn, Mad-mediated activation may be sufficient for expression within the normal domain, but cannot sustain the lateral expansion encountered in brk mutants. A third category of responses (class C) includes dpp and Ubx in the midgut, and sna in the primordia of the wing/haltere imaginal discs. Genes in this class show significantly reduced levels of expression in the double mutant, not only relative to brk- but also compared with wild-type animals. Class C promoters incorporate a brk-independent positive input from shn that is necessary for wild-type levels of expression. The inability of ectopic Dpp to induce sna expression in shn mutants demonstrates the essential nature of the requirement for Shn in activation of class C genes (Torres-Vazquez, 2001).
It is evident that repression of brk is crucial for expression of all three classes of genes described, and as such accounts for a significant part of the positive input from shn to gene activation. In addition, the data suggest that Mad and Shn contribute equally to repression of brk and regulation of class A genes. However, the fact that brk activity is only partially epistatic with respect to class B and C promoters, indicates that the majority of genes examined in this study integrate positive inputs from shn, as well as negative inputs from brk. The near wild-type expression of class B genes in double mutant embryos suggests that the brk-independent input from shn may be crucial at the margins of the expression domains and may be less significant in regions of the embryo that receive moderate to high levels of Dpp signaling. In contrast, the positive input from shn to class C targets appears to be important throughout the domain of expression. The observation that genes such as dCreb-A and Scr, which are repressed by dpp signaling, and which are also sensitive to loss of brk, raises the possibility that Dpp regulates their expression indirectly. In this event, the dpp target genes that mediate repression of dCreb-A and Scr would belong to classes A and C, respectively (Torres-Vazquez, 2001).
The partial restoration of dpp target gene expression in the double mutants relative to shn- embryos provides a basis for interpreting the cuticle phenotype. Homozygous brk;shn animals as well as brk;tkv mutants have an intermediate phenotype in that they show rescue of the dorsal closure defect observed in shn and tkv mutants, but they also display a reduced dorsal epidermis compared with brk-null embryos. Both dpp and pnr have been implicated in dorsal closure, which results from movement of the epidermal cells over the amnioserosa and their suturing at the midline. In light of this, the recovery of their expression in the dorsalmost ectodermal cells in the double mutants correlates well with the restoration of dorsal closure. Likewise, the compromised expression of dorsal ectodermal markers such as Dad and pnr in brk;shn embryos relative to brk null animals, provides molecular correlates for the ventralization observed in the double mutants (Torres-Vazquez, 2001).
Transforming growth factor ß signaling mediated by Decapentaplegic and Screw is known to be involved in defining the border of the ventral neurogenic region in the fruitfly. A second phase of Decapentaplegic signaling occurs in a broad dorsal ectodermal region. The dorsolateral peripheral nervous system forms within the region where this second phase of signaling occurs. Decapentaplegic activity is required for development of many of the dorsal and lateral peripheral nervous system neurons. Double mutant analysis of the Decapentaplegic signaling mediator Schnurri and the inhibitor Brinker indicates that formation of these neurons requires Decapentaplegic signaling, and their absence in the mutant is mediated by a counteracting repression by Brinker. Interestingly, the ventral peripheral neurons that form outside the Decapentaplegic signaling domain depend on Brinker to develop. The role of Decapentaplegic signaling on dorsal and lateral peripheral neurons is strikingly similar to the known role of Transforming growth factor ß signaling in specifying dorsal cell fates of the lateral (later dorsal) nervous system in chordates (Halocythia, zebrafish, Xenopus, chicken and mouse). It points to an evolutionarily conserved mechanism specifying dorsal cell fates in the nervous system of both protostomes and deuterostomes (Rusten, 2002).
The embryonic abdominal (A) PNS of Drosophila consists of three bilateral clusters of neurons (ventral, lateral and dorsal) per segment, which can be most especially appreciated in the serially homologous segments A1-A7. In order to investigate whether the second phase of Dpp signaling is necessary for patterning the PNS, mutant alleles for a gene involved in the Dpp signaling pathway, schnurri (shn), were examined. This gene encodes a zinc-finger transcription factor that is necessary for the transcription of some Dpp target genes and binds directly to the main Dpp mediator Mothers against Dpp (Mad). Unlike the zygotic mutants of dpp, scw, tolloid (tld) or mad, shn mutants have no effect on the initial dpp/scw governed dorsoventral patterning of the blastoderm. They express normally the early Dpp target genes, such as pannier (pnr, stage 7), dpp itself in the dorsal ectoderm (stage 9) and Krüppel (Kr) (which is a marker for the amnioserosa), showing that the dorsal ectoderm is correctly specified. By contrast, several Dpp target genes that are expressed following the second phase of Dpp signaling are affected in shn zygotic mutants: at stage 11, the expression of genes responsive to Dpp signaling, such as dad, pnr, spalt or dpp itself is reduced or lost. Thus, any failures in PNS formation, which are observed in shn mutant embryos, must originate from the second rather than the first phase of Dpp signaling and are likely to be mediated by Shn. PNS malformations were sought in strong shn zygotic mutant embryos using the ubiquitous PNS neuronal marker 22C10. Homozygous shn1 and shnk00401 fail to undergo dorsal closure and show severe defects of PNS development. A strong reduction in number of neurons is observed, especially in the dorsal and lateral PNS clusters, although it is difficult to determine exactly which neurons are affected because of the dorsal closure failure. Therefore, another allele, shnk04412, which does undergo dorsal closure, was also examined. In these embryos, position and identity of PNS neurons could be more clearly assigned. In homozygosity, as well as in transheterozygosity over shn1, this mutant shows a reduction in the number of dorsal and lateral neurons, similar to the other mutants analyzed. These results are consistent with a role for Shn-mediated Dpp signaling in the formation of the dorsal and lateral PNS (Rusten, 2002).
In the Drosophila ovary, germline stem cell (GSC) self-renewal is controlled by both extrinsic and intrinsic factors. The Bmp signal from niche cells controls GSC self-renewal by directly repressing a Bam-dependent differentiation pathway in GSCs. pelota (pelo), which has been previously shown to be required for Drosophila male meiosis, was identified in a genetic screen as a dominant suppressor of the dpp overexpression-induced GSC tumor phenotype. Pelo acts in controlling GSC self-renewal by repressing a Bam-independent differentiation pathway. In pelo mutant ovaries, GSCs are lost rapidly owing to differentiation. Results from genetic mosaic analysis and germ cell-specific rescue show that it functions as an intrinsic factor to control GSC self-renewal. In pelo mutant GSCs, Bmp signaling activity detected by Dad-lacZ expression is downregulated, but bam expression is still repressed. Furthermore, bam mutant germ cells are still able to differentiate into cystocytes without pelo function, indicating that Pelo is involved in repressing a Bam-independent differentiation pathway. Consistent with its homology to the eukaryotic translation release factor 1alpha, Pelo is shown to be localized to the cytoplasm of the GSC. Therefore, Pelo controls GSC self-renewal by repressing a Bam-independent differentiation pathway possibly through regulating translation. Since Pelo is highly conserved from Drosophila to mammals, it may also be involved in the regulation of adult stem cell self-renewal in mammals, including humans (Xi, 2005).
pelo was identified in a genetic screen looking for genes that can suppress dpp overexpression-induced GSC-like tumors, suggesting that pelo must somehow genetically interact with the dpp/Bmp pathway. To further reveal the relationship between pelo and Bmp signaling, the dose effect of pelo on dpp-induced GSC-like tumor formation was carefully examined. Ovarioles overexpressing dpp by the c587 gal4 driver contain only single germ cells resembling GSCs. Among the dpp-overexpressing ovarioles also carrying one copy of the pelo1 mutation, 36% of them showed the same tumor phenotype, but the rest of the ovarioles contained differentiated germline cysts, developing egg chambers and even mature eggs, which could explain why pelo was identified in the suppressor screen. Among the dpp-overexpressing ovarioles also carrying two copies of the pelo1 mutations, only 13.8% of them contained only GSC-like single germ cells, while 49.8% of them had a mixture of single germ cells and developing cysts. Interestingly, the rest (36.4%) were reminiscent of the pelo GSC loss phenotype only. These results suggest that pelo functions as one of the Bmp downstream components or in a pathway parallel to the Bmp signaling pathway to control GSC self-renewal (Xi, 2005).
To further understand how pelo modulates Bmp signaling activity, the expression of a Bmp direct target gene, Dad, was examined in the pelo mutant GSCs. Dad-lacZ is a lacZ enhancer trap line for Dad. Its expression is the strongest in the GSCs, and is quickly downregulated in the differentiating cystoblasts. The pelo1 mutant GSCs marked by loss of ubi-GFP expression were generated by the FLP-mediated FRT recombination, and then were analyzed for Dad-lacZ expression 2 weeks after clone induction. Consistent with the idea that pelo is involved in modulating Bmp signaling, 69% of the marked mutant pelo GSCs (GFP negative) showed the downregulation of Dad-lacZ expression in comparison with their neighboring wild-type GSCs (GFP-positive). It was further asked whether pelo is also involved in Bmp-mediated bam repression in GSCs, since Bmp signaling has been shown to directly represses bam transcription in GSCs. A bam-GFP transgene (a GFP reporter driven by a bam promoter) is repressed in GSCs, while its expression is upregulated in the differentiating cystoblasts. The marked pelo mutant GSCs (lacZ negative) were generated by the FLP-mediated FRT recombination and were examined for bam expression. Only about 5% of the marked pelo mutant GSCs (lacZ negative) showed slight upregulation of bam-GFP in comparison with their neighboring unmarked wild-type GSCs (lacZ positive), while the rest of the marked pelo1 mutant GSCs did not upregulate bam-GFP expression. These findings indicate that Pelo is involved in modulating Bmp signaling in GSCs but plays little or no role in regulating Bmp-mediated bam repression, and further suggest that it functions in one branch of the responses of the Bmp signaling pathway to regulate GSC self-renewal (Xi, 2005).
Before this work, pelo had not been shown to be involved in regulating any signaling pathways in any organisms. The Bmp pathway is a major signaling pathway that is essential for controlling GSC self-renewal and division in the Drosophila ovary. The Bmp signaling activities can be reliably monitored by expression of Dad in GSCs. It is anticipated that pelo must somehow interact with the Bmp pathway in controlling GSC self-renewal, since pelo was also identified as a dominant suppressor of Dpp overexpression-induced GSC-like tumors. In this study, GSCs mutant for pelo are shown to downregulate Dad. These findings indicate that Pelo participates in Bmp signaling to control expression of dpp target genes in GSCs such as Dad (Xi, 2005).
Intracellular signaling of the TGF-beta superfamily is mediated by Smad proteins, which are now grouped into three classes. Two Smads have been identified in Drosophila. Mothers against dpp (Mad) is a pathway-specific Smad, whereas Daughters against dpp (Dad) is an inhibitory Smad genetically shown to antagonize Dpp signaling. The identification of a common mediator Smad, that is closely related to human Smad4, is reported. Mad forms a heteromeric complex with Drosophila Smad4 (Medea) upon phosphorylation by Thick veins (Tkv), a type I receptor for Dpp. Dad stably associates with Tkv and thereby inhibits Tkv-induced Mad phosphorylation. Dad also blocks hetero-oligomerization and nuclear translocation of Mad. Mad exists as a monomer in the absence of Tkv stimulation. Tkv induces homo-oligomerization of Mad, and Dad inhibits this step. A model for Dpp signaling by Drosophila Smad proteins is reported (Inoue, 1998).
Dad has been genetically shown to inhibit Dpp signaling in vivo (Tsuneizumi, 1997). The molecular basis of this inhibitory effect has been examined by studying the effect of Dad on Mad phosphorylation by Tkv. Various combinations of Mad, Dad, and Tkv-QD (a constitutively active form of Tkv) were introduced into COS cells. In the first experiment, cells were labeled with [32P]orthophosphate in vivo, and incorporation of radioactivity into Mad was detected. Dad inhibits phosphorylation of Mad by Tkv-QD. Next, anti-phosphoserine antibody was used. As in the orthophosphate labeling, phosphorylation of Mad diminishes in the presence of Dad (Inoue, 1998).
In vertebrates, inhibitory Smads such as Smad6 and Smad7 have been shown to stably associate with type I receptors (Hayashi, 1997; Imamura, 1997; Nakao, 1997; Hata, 1998). The interaction of Mad or Dad with Tkv was examined. Cells were transfected with an appropriate combination of expression plasmids, affinity labeled with iodinated BMP-2, and subjected to immunoprecipitation with antibodies against Mad or Dad. Pathway-specific Smads associate with type I receptors upon ligand stimulation. The interaction can be observed when the type I kinases are rendered inactive or when the C-terminal phosphorylation sites of the Smads are modified to be resistant to phosphorylation. Mad interacts with the kinase-defective form of Tkv, whereas the interaction of Mad with wild type Tkv is still detectable. The interaction of Mad with Tkv might thus be more stable than that of mammalian Smads with receptors. Dad interacts with wild-type Tkv as efficiently as with the kinase-defective form of Tkv. Notably, almost the same amount of Tkv is immunoprecipitated with Mad and Dad, although the expression level of Mad is much higher than that of Dad. Thus the affinity of Dad with Tkv seems to be higher than that of Mad. Stable interaction is also observed with immunoprecipitation followed by Western blotting. Finally, the interaction of Mad with Tkv is hampered by expression of Dad. Dad thus inhibits phosphorylation of Mad by Tkv by competing with Mad in association with the receptor (Inoue, 1998).
Oligomerization of the Smad proteins is a critical step in their activation. Most of the cancer-derived mutations of Smad4 or Smad2, as well as mutations of the Mad and sma genes causing developmental defects, are mapped to their MH2 domains. Based on the crystal structure of the Smad4 MH2 domain, these mutations can be sorted into three groups: those that are located in the hydrophobic core and destabilize the overall structure, those that disrupt hetero-oligomeric interaction, and those that disrupt homo-oligomeric complex formation. Tkv-QD causes hetero-oligomerization of Mad with Medea. The effect of Dad on the hetero-oligomerization was examined. Whether Dad can inhibit Tkv-QD-induced complex formation of Mad and human Smad4 was tested. The hetero-oligomerization of Mad with Smad4 is efficiently blocked. Dad thus blocks a critical step in the activation of Mad (Inoue, 1998).
Smad2 and Smad3 associate with each other in a TGF-beta-dependent manner. This finding suggested that pathway-specific Smads may exist as monomers in the absence of ligand stimulation and form oligomeric complexes upon phosphorylation by type I receptors. This hypothesis was tested for Mad. Mads exist as monomers in the absence of Tkv-QD, and Tkv-QD induces homo-oligomerization of Mad. The activation of Mad by Tkv-QD thus appears to consist of a sequential linkage of phosphorylation, homo-oligomerization, hetero-oligomerization, and nuclear translocation. When Dad is coexpressed, the homo-oligomer formation of Mad induced by Tkv-QD is inhibited (Inoue, 1998).
Smad proteins translocate into the nucleus after phosphorylation and oligomerization. The effect of Dad on this step has been examined. COS cells were transfected with various combinations of Mad, Dad, and Tkv-QD, and the subcellular localization of Mad was determined by immunofluorescence microscopy. Mad is localized throughout the cell in unstimulated cells, and Tkv-QD induces nuclear accumulation of the Mad proteins. When Dad is coexpressed, nuclear translocation of Mad is blocked. The percentage of cells displaying predominant nuclear staining increases from 12% to 96% upon Tkv stimulation and decreases to 11% in the presence of Dad. This finding again demonstrates that Dad inhibits Mad activation by Tkv (Inoue, 1998).
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