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).

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 September 2000

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