Interplay of signal mediators of decapentaplegic (Dpp): molecular characterization of mothers against dpp, Medea, and daughters against dpp.

Decapentaplegic (Dpp) plays an essential role in Drosophila development, and analyses of the Dpp signaling pathway have contributed greatly to understanding of the actions of the TGF-beta superfamily. 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. Here we report the identification of a common mediator Smad in Drosophila, which is closely related to human Smad4. 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. We also show that Mad exists as a monomer in the absence of Tkv stimulation. Tkv induces homo-oligomerization of Mad, and Dad inhibits this step. Finally, we propose a model for Dpp signaling by Drosophila Smad proteins.

Xenopus Smad8 acts downstream of BMP-4 to modulate its activity during vertebrate embryonic patterning.

Bone morphogenetic proteins (BMPs) participate in the development of nearly all organs and tissues. BMP signaling is mediated by specific Smad proteins, Smad1 and/or Smad5, which undergo serine phosphorylation in response to BMP-receptor activation and are then translocated to the nucleus where they modulate transcription of target genes. We have identified a distantly related member of the Xenopus Smad family, Smad8, which lacks the C-terminal SSXS phosphorylation motif present in other Smads, and which appears to function in the BMP signaling pathway. During embryonic development, the spatial pattern of expression of Smad8 mirrors that of BMP-4. We show that an intact BMP signaling pathway is required for its expression. Overexpression of Smad8 in Xenopus embryos phenocopies the effect of blocking BMP-4 signaling, leading to induction of a secondary axis on the ventral side of intact embryos and to direct neural induction in ectodermal explants. Furthermore, Smad8 can block BMP-4-mediated induction of ventral mesoderm-specific gene expression in ectodermal explants. Overexpression of Smad8 within dorsal cells, however, causes patterning defects that are distinct from those reported in BMP-4-deficient embryos, suggesting that Smad8 may interact with additional signaling pathways. Indeed, overexpression of Smad8 blocks expression of Xbra in whole animals, and partially blocks activin signaling in animal caps. In addition, Smad8 inhibits involution of mesodermal cells during gastrulation, a phenotype that is not observed following blockade of activin or BMPs in Xenopus. Together, these results are consistent with the hypothesis that Smad8 participates in a negative feedback loop in which BMP signaling induces the expression of Smad8, which then functions to negatively modulate the amplitude or duration of signaling downstream of BMPs and, possibly, downstream of other transforming growth factor-beta (TGF-beta) family ligands.

Daughters against dpp modulates dpp organizing activity in Drosophila wing development.

The family of TGF-beta signalling molecules play inductive roles in various developmental contexts. One member of this family, Drosophila Decapentaplegic (Dpp) serves as a morphogen that patterns both the embryo and adult. We have now isolated a gene, Daughters against dpp (Dad), whose transcription is induced by Dpp. 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 signalling 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 homologue of Dpp, bone morphogenetic protein, as evidenced by induction of dorsal or neural fate following overexpression in Xenopus embryos. We conclude that the pattern-organizing mechanism governed by Dpp involves a negative-feedback circuit in which Dpp induces expression of its own antagonist, Dad. This feedback loop appears to be conserved in vertebrate development.

Physiological and biochemical analysis of the effects of alkaline phosphatase overproduction in Escherichia coli.

Overexpression of the Escherichia coli phoA gene, coding for alkaline phosphatase (PhoA), on multicopy plasmids caused a severe defect in the precursor processing (secretion) of PhoA, beta-lactamase, and the outer membrane protein OmpA. This secretion defect continued even after the repression of phoA expression, indicating that protein secretion was irreversibly impaired in cells. Among the secretory proteins, only OmpA gradually secreted posttranslationally. The inverted inner membrane vesicles prepared from cells with the secretion defect showed appreciably reduced translocation activity in vitro. But the membrane vesicles retained the ability to generate a proton motive force which, together with ATP, is essential as an energy source for the efficient secretion of proteins in E. coli. An appreciable amount of incompletely translocated PhoA molecules was detected in the inner membranes of cells with the secretion defect.

Induction of specific protein tyrosine phosphatase transcripts during differentiation of mouse embryonal carcinoma (F9) cells.

We have investigated the pattern of PTPase transcript expression during in vitro differentiation of mouse embryonal carcinoma (F9) cells. While the transcripts of most PTPases were unchanged or undetected during embryonal differentiation induced by retinoic acid, several PTPase transcripts exhibited distinct patterns of induction. Mutant cells defective in differentiation did not display the induction of some of these PTPase transcripts. Interestingly, three out of the four PTPase transcripts induced were the same PTPase transcripts induced during in vitro erythroid differentiation of mouse erythroleukemia (MEL) cells [(1994) J. Biol. Chem. 269, 4709-4712] [corrected]. The possible role played by specific PTPases in cell differentiation is discussed.

Induction of specific protein tyrosine phosphatase transcripts during differentiation of mouse erythroleukemia cells.

We reported previously that most of the phosphotyrosine-containing cellular proteins were quickly dephosphorylated at the very early stage of erythroid differentiation of mouse erythroleukemia (MEL) cells. These and other experimental results implicated a specific protein tyrosine phosphatase(s) (PTPase(s)) involved in the commitment of the erythroid differentiation. We have investigated the pattern of transcripts of PTPases during MEL cell differentiation and found that while the transcripts of most PTPases were unchanged or undetected in the cells, transcripts for two PTPases (PTP beta 2 and RIP) exhibited distinct patterns of induction at a very early stage of differentiation. Some of the mutant cells defective in differentiation did not show the induction of these PTPase transcripts. We discuss the possible role played by the PTPases in the commitment of MEL cell differentiation.

Synergistic induction of erythroid differentiation of mouse erythroleukemia (MEL) cells by inhibitors of topoisomerases and protein tyrosine kinases.

In vitro erythroid differentiation of mouse erythroleukemia (MEL) cells was induced by combinations of topoisomerase and protein kinase inhibitors. Neither inhibitor alone exhibited inducing activity. Although inhibitors of topoisomerases I and II were equally effective in the synergistic induction of erythroid differentiation, only inhibitors of tyrosine kinases, not of serine/threonine kinases, exhibited synergistic activity. The erythroid differentiation induced by the combination of topoisomerase and protein tyrosine kinase inhibitors was distinguished from that induced by typical erythroid inducing agents such as DMSO or HMBA by (1) earlier hemoglobin accumulation in the cells and (2) insensitivity to specific inhibitors (dexamethasone and sodium orthovanadate) of MEL cell differentiation.

Induction of in vitro differentiation of mouse embryonal carcinoma (F9) cells by inhibitors of topoisomerases.

To investigate the possible involvement of topoisomerases in embryonal differentiation, we examined the effect of topoisomerase inhibitors on the in vitro differentiation of mouse embryonal carcinoma F9 cells. We found that camptothecin, teniposide (VM-26), or genistein, specific inhibitors of topoisomerases, induced morphological as well as biochemical changes (production of tissue plasminogen activator, synthesis of laminin, and disappearance of stage-specific embryonic antigen 1) specific to F9 cell differentiation. Since these changes were indistinguishable from those observed in F9 differentiation induced by retinoic acid (plus dibutyryl cyclic AMP), it was suggested that inhibition of cellular topoisomerase activities triggered F9 cell differentiation into parietal endoderm-like cells in the same manner as retinoic acid (plus dibutyryl cyclic AMP). Experiments using differentiation-resistant mutant F9 cell lines, however, indicated that the molecular cascade involved in topoisomerase inhibitor-induced differentiation involves different steps from those functioning in the retinoic acid-induced differentiation cascade.