BIOLOGICAL OVERVIEW

The pattern-organizing mechanism governed by Decapentaplegic (Dpp) involves a negative-feedback circuit in which Dpp induces expression of its own antagonist, Daughters against dpp (Dad). Dad shares weak homology with Drosophila Mad (Mothers against dpp), a protein required for transduction of Dpp signals. In contrast to Mad or the activated Dpp receptor, whose overexpression hyperactivates the Dpp signaling pathway, overexpression of Dad blocks Dpp activity. Expression of Dad together with either Mad or the activated receptor rescues phenotypic defects induced by each protein alone. Dad can also antagonize the activity of a vertebrate homolog of Dpp, bone morphogenetic protein (BMP-4), as evidenced by induction of dorsal or neural fate following overexpression in Xenopus embryos. Thus this feedback loop appears to be conserved in vertebrate development (Tsuneizumi, 1997).

The Drosophila wing is divided into two compartments along its anteroposterior (A/P) axis. The compartment boundary between these regions serves as the source of an organizing activity that patterns both anterior and posterior compartments. This activity is mediated, at least in part, by the long-range action of Dpp, which is expressed by cells along the A/P compartment boundary. Dpp is thought to act as a morphogen to inform target cells of their position along the A/P axis, but as yet, little is known about how cells interpret the distribution of Dpp protein. An enhancer trap screen was conducted to identify genes whose transcription is controlled by Dpp. Two enhancer trap lines in the same locus (89E/F), P1883 and 1(3)1E4, were identified whose expression patterns are similar to those of Dpp during embryonic and imaginal development. The gene whose expression is reflected in these enhancer traps has been named Daughters against dpp (Dad). In these enhancer trap lines, beta-galactosidase is expressed in a wide stripe that straddles the A/P compartment boundary of the imaginal discs, in contrast to Dpp, whose expression is confined to the anterior side. This pattern of expression suggests that Dad expression is positively regulated by the secreted Dpp molecule. To test whether Dad responds to Dpp signaling, its expression has been examined in P1883 wing discs in which a UAS-dpp transgene was transcribed in a ring around a wing pouch under the control of a Gal4 driver. Ectopic Dpp expression results in abnormally large discs and in ectopic expression of Dad in a broad ring around a wing pouch. Identical results were obtained when another transgene was used -- UAS-tkv ^Q253D -- which encodes a constitutively active form of the major type-I Dpp receptor, Thick veins (Tkv). In addition, expression of Dad is not detected in cells that lack a functional Tkv Dpp receptor. These results indicate that Dpp signaling is necessary and sufficient for Dad expression in the developing wing (Tsuneizumi, 1997).

To analyse Dad function, the gene was ectopically expressed using the Gal4-UAS system and patterning defects were examined in the wing. When Dad is misexpressed along the wing margin, the wing loses its margin and, in extreme cases, only a tiny winglet forms. Because dpp is required not only for pattern formation, but also for cell proliferation, clones of cells that have lost Dpp responsiveness as a result of mutation of genes encoding tkv or punt (a type II Dpp receptor) do not survive in the wing blade. The similarities in wing phenotypes caused by loss of Dpp responsiveness and those caused by ectopic expression of Dad suggest that Dad antagonizes the Dpp signaling pathway. In contrast, conditions that mimic the effects of hyperactivating the Dpp signaling pathway, such as overexpressing Tkv^Q253Dor Mad, cause outgrowth of wing tissue. When Dad is expressed together with either Tkv^Q253D or Mad, phenotypic defects caused by overexpression of each protein alone are nullified and nearly wild-type wings form (Tsuneizumi, 1997).

Effects were examined of ectopic expression of Dad and Mad on a Dpp target gene, optomotor-blind (omb), which is positively regulated by dpp in wing discs. Omb is expressed in a broad stripe straddling Dpp expressing cells, as is Dad, but unlike Dad, it is not expressed along the entire A/P boundary. Clones of cells that express Dad or Mad, or both, have effects that are cell autonomous. Omb expression is absent in Dad-overexpressing cells. When Mad-overexpressing clones fall (respectively) either inside, or near the normal Omb-expression domain, higher level, or ectopic expression of Omb is observed. In contrast, when Mad-overexpressing cells are situated distal to the endogenous Omb-expression domain, ectopic expression of Omb is not detected. Thus, overexpressed Mad may be dependent on Dpp signaling for activation, whereas elevated levels of Mad may lower the threshold concentration of Dpp required to induce Omb expression. When both Dad and Mad are overexpressed in the same cells, Omb expression is barely affected. The observation that overexpression of Dad does not reduce expression of endogenous Dpp, together with the fact that patterning defects induced by overexpression of Dad are rescued by overexpression of Mad or Tkv^Q253D, suggests that antagonism of the Dpp signal by Dad occurs after reception of the Dpp signal at the cell surface and before control of transcription of target genes (Tsuneizumi, 1997).

To confirm that Dad represses Omb expression, somatic Dad mutant clones were analysed. Dad mutants were induced by excising the P element of the 1(3)1E4 enhancer trap line; one of these, Dad ^271-68, is associated with a deletion of the entire C-terminal domain after amino acid 391. Omb is derepressed autonomously in Dad ^271-68clones in wing discs, indicating that Dad normally represses Omb expression. It is inferred that Dad negatively modulates the level of Dpp signaling, at least during wing development (Tsuneizumi, 1997).

Components of the Dpp signaling pathway are highly conserved between arthropods and chordates, so an investigation was carried out to see whether Dad can antagonize signaling by BMP-4, a vertebrate homolog of Dpp. In embryos of Xenopus laevis, BMP-4 functions to specify ventral mesodermal and epidermal fates. Blockage of BMP signaling during gastrulation induces the formation of secondary dorsal axes in intact embryos and neuralizes the fate of explanted ectodermal cells. Similar patterning defects are observed following ectopic expression of Dad in Xenopus embryos. Microinjection of DAD mRNA into dorsal blastomeres of 4-cell embryos produces no detectable patterning defects, whereas injection into ventral cells induces formation of a secondary axis in 90% of embryos. The induced axes contain muscle and neural tissue, but not notochord. In some cases, a cyclopic eye differentiates in the secondary axis, although the frequency of eye induction varies, ranging from 3%-38% in different experiments. Ectodermal explants (animal caps) from Dad-injected embryos elongate and form darkly pigmented cement glands, whereas explants from control embryos retain a rounded epidermal appearance. Dad-injected explants express cement gland- ( XAG) and neural-specific (N-CAM, OtxA) genes but not a dorsal mesodermal gene, alpha-actin or a panmesodermal gene, Xbra, indicating that Dad can directly mediate neural induction in the absence of mesoderm. To test whether Dad can antagonize BMP function, BMP-4 and DAD mRNAs were co-injected into dorsal blastomeres of 4 cell embryos. Injection of BMP-4 mRNA alone causes a loss of anterior and dorsal structures, yielding an average dorsoanterior index, whereas co-injection of DAD mRNA almost completely rescues the ventralized phenotype. These results suggest that Dad can antagonize BMP signaling in Xenopus embryos. Given that components of the Dpp/BMP signaling pathway are highly conserved between insects and vertebrates, it is likely that a homolog of Dad exists that modulates the amplitude or duration of BMP signaling during vertebrate embryogenesis (Tsuneizumi, 1997).

Although Dad is a distantly related member of the SMAD family, it is unique among SMADs in antagonizing, rather than transducing, TGF-beta-like signals. Oddly enough, Dad appears to participate in a direct negative feedback loop in that it antagonizes the very signaling pathway (that is, Dpp) that is required for induction of its own expression. This relationship between Dpp, which positively regulates the level of expression of Dad, and Dad, which negatively regulates the level of Dpp activity, suggests that the final outcome of Dpp signaling may not be directly proportional to the graded concentration of Dpp protein but may depend on the balance between transduction of Dpp signals by activated Mad, and antagonism of Dpp signals by Dad. Mechanistically, Dad may interact directly with Mad to modulate Dpp signaling because co-expression of Dad and Mad rescue phenotypic defects induced by either protein alone. Precedence for direct interaction between different members of the SMAD family exists in that the SMADs form multimeric complexes. Alternatively, Dad may antagonize signaling by interacting directly with the receptors. A vertebrate SMAD protein, Smad2, stably associates with receptors and blocks TGFbeta-dependent transcriptional responses when its conserved three C-terminal serine residues are substituted with alanine residues. In contrast, wild-type Smad2 transiently associates with receptors and transduces the signal. Dad does not have these C-terminal serines and may stably associate with receptors. A new member of SMADs, Smad7, which does not have these C-terminal serines, has been reported to inhibit TGF-beta signaling by interacting stably with the receptor. The data suggest the existence of a Dad negative feedback circuit that might stabilize the gradient of positional information emanating from Dpp expressing cells (Tsuneizumi, 1997).

Germline stem cell number in the ovary is regulated by mechanisms that control Dpp signaling: Dad as an indicator of the strength of Dpp signaling

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).

GSCs mutant for pelota downregulate Dad

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).

GENE STRUCTURE

cDNA clone length - 3.8 kb

PROTEIN STRUCTURE

Amino Acids - 568

Structural Domains

The deduced amino-acid sequence of Dad shows limited homology to Drosophila Mad, a protein that is required for intracellular transduction of the Dpp signal . A family of structurally related proteins, termed SMAD proteins, has been identified in Caenorhabditis elegans and in vertebrates. It has been postulated that these proteins are phosphorylated in response to receptor activation, after which they translocate to the nucleus and function as transcription factors. SMAD proteins share conserved amino- and carboxy-terminal domains separated by a variable proline-rich region. Although Dad shares significant homology with other SMAD family members within the carboxy-terminal domain, the amino-terminal domain is less well conserved. The carboxy-terminal domain of Dad shares the highest homology with human Smad6, which lacks an amino-terminal homology domain (Tsuneizumi, 1997).

date revised: 22 November 2022

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