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Ventralizing transcriptional repressors in the Vox/Vent family have been proposed to be important regulators of dorsoventral patterning in the early embryo. While the zebrafish genes vox (vega1) and vent (vega2) both have ventralizing activity in overexpression assays, loss-of-function studies are needed to determine whether these genes have distinct or redundant functions in dorsoventral patterning and to provide critical tests of the proposed regulatory interactions among vox, vent and other genes that act to establish the dorsoventral axis. We show that vox and vent are redundant repressors of dorsal fates in zebrafish. Mutants that lack vox function have little or no dorsoventral patterning defect, and inactivation of either vox or vent by injection of antisense morpholino oligonucleotides has little or no effect on the embryo. In contrast, embryos that lack both vox and vent function have a dorsalized phenotype. Expression of dorsal mesodermal genes, including chordin, goosecoid and bozozok, is strongly expanded in embryos that lack vox and vent function, indicating that the redundant action of vox and vent is required to restrict dorsal genes to their appropriate territories. Our genetic analysis indicates that the dorsalizing transcription factor Bozozok promotes dorsal fates indirectly, by antagonizing the expression of vox and vent. In turn, vox and vent repress chordin expression, restricting its function as an antagonist of ventral fates to the dorsal side of the embryo. Our results support a model in which BMP signaling induces the expression of ventral genes, while vox and vent act redundantly to prevent the expression of chordin, goosecoid and other dorsal genes in the lateral and ventral mesendoderm.

T-box transcription factors are critical regulators of early embryonic development. We have characterized a novel zebrafish T-box transcription factor, hrT(H15-related T box) that is a close relative of Drosophila H15and a recently identified human gene. We show that Drosophila H15and zebrafish hrTare both expressed early during heart formation, in strong support of previous work postulating that vertebrate and arthropod hearts are homologous structures with conserved regulatory mechanisms. The timing and regulation of zebrafish hrTexpression in anterior lateral plate mesoderm suggest a very early role for hrTin the differentiation of the cardiac precursors. hrTis coexpressed with gata4and nkx2.5not only in anterior lateral plate mesoderm but also in noncardiac mesoderm adjacent to the tail bud, suggesting that a conserved regulatory pathway links expression of these three genes in cardiac and noncardiac tissues. Finally, we analyzed hrTexpression in pandoramutant embryos, since these have defects in many of the tissues that express hrT,including the heart. hrTexpression is much reduced in the early heart fields of pandoramutants, whereas it is ectopically expressed subsequently. Using hrTexpression as a marker, we describe a midline patterning defect in pandoraaffecting the anterior hindbrain and associated midline mesendodermal derivatives. We discuss the possibility that the cardiac ventricular defect previously described in pandoraand the midline defects described here are related.

We applied the method of Guarente et al. [Guarente, L., Lauer, G., Roberts, T.M. & Ptashne, M. (1980) Cell 20, 543-553] to construct plasmids that direct expression in Escherichia coli of the human fibroblast interferon (F-IF) gene. Two plasmids were recovered. One directs efficient synthesis of a protein whose primary sequence is that of pre-F-IF and the other, that of mature F-IF. Extracts of bacteria synthesizing mature F-IF display antiviral activity characteristic of human F-IF. This activity is lower than that expected from the differential rate of synthesis of the protein. We have detected no such activity in extracts of bacteria synthesizing pre-F-IF.

A new approach to the isolation of mutations in mammalian genes was developed which permits both the selection of infrequently occurring mutants that alter the cellular morphology of recipient cells and the rapid reisolation of the mutant gene. The adenovirus type 5 13S early region 1a (E1a) gene was mutagenized in vitro with sodium bisulfite and then efficiently transferred into cells with a retrovirus shuttle vector. Three classes of mutants of the 13S E1a gene product were isolated, each of which induced a distinct morphological alteration. The mutant E1a gene was reisolated from each cell line, and the precise nucleotide changes were determined. The E1a-induced morphological alterations were further examined by the construction of single and double point mutations within different regions of the polypeptides by utilizing the amino acid substitutions obtained from the original mutants. The results suggest that each of the three regions of highly conserved amino acids within the E1a 13S polypeptide has a distinct role in the alteration of cellular morphology and the activation of gene expression.

The early patterning of mesoderm in the Xenopus embryo requires signals from several intercellular factors, including mesoderm-inducing agents that belong to the fibroblast growth factor (FGF) and TGF-beta families. In animal hemisphere explants (animal caps), basic FGF and the TGF-beta family member activin are capable of converting pre-ectodermal cells to a mesodermal fate, although activin is much more effective at inducing dorsal and anterior mesoderm than is basic FGF. Using a dominant-negative form of the Xenopus type 1 FGF receptor, we show that an FGF signal is required for the full induction of mesoderm by activin. Animal caps isolated from embryos that have been injected with the truncated FGF receptor and cultured with activin do not extend and the induction of some genes, including cardiac actin and Xbra, is greatly diminished, while the induction of other genes, including the head organizer-specific genes gsc and Xlim-1, is less sensitive. These results are consistent with the phenotype of the truncated FGF receptor-injected embryo and imply that the activin induction of mesoderm depends on FGF, with some genes requiring a higher level of FGF signaling than others.

Antisera were prepared against the amino acid sequences encoded within the N-terminal half of the adenovirus 12 (Ad12) early region 1A (E1A) gene. This was accomplished by construction of a plasmid vector which encoded the N-terminal 131 amino acids of Ad12 E1A joined in frame to the coding sequence of beta-galactosidase. After induced synthesis in Escherichia coli, the Ad12 E1A-beta-galactosidase fusion protein (12-1A-FP) was extracted with urea and used to raise antibodies in rabbits. The 12-1A-FP antisera immunoprecipitated major phosphoproteins of 39,000 and 37,000 apparent molecular weights from Ad12-transformed and infected cells. The 12-1A-FP antisera also immunoprecipitated E1A phosphoproteins from Ad5-transformed and infected cells. Immunospecificity of the 12-1A-FP antisera was demonstrated by the ability of 12-1A-FP antigen to block immunoprecipitation of E1A proteins. Furthermore, E1A proteins immunoprecipitated from in vivo-labeled cells comigrated with those translated in vitro by RNA that had been hybridization selected to E1A DNA.

The Wnt pathway regulates the early dorsal-ventral axis in Xenopus through a complex of beta-catenin and HMG box transcription factors of the Lef/Tcf family. We show that the promoter of the dorsalizing homeo box gene siamois is a direct target for the beta-catenin/XTcf-3 complex, establishing a link between the Wnt pathway and the activation of genes involved in specifying the dorsal axis. By injecting siamois reporter constructs into the animal pole of Xenopus embryos, we show that a 0.8-kb fragment of the siamois promoter is strongly activated by beta-catenin. The proximal 0.5 kb, which is also activated by beta-catenin, contains three Lef/Tcf-binding sites. Mutations in these sites eliminate the beta-catenin-mediated activation of siamois and show that siamois is regulated by the beta-catenin/XTcf-3 complex, in combination with additional transcriptional activators. When expressed at the equator of the embryo, the siamois promoter is activated to much higher levels on the dorsal side than the ventral side. Ectopic ventral expression of beta-catenin raises the ventral expression of the siamois promoter to the dorsal levels. Conversely, ectopic dorsal expression of dominant-negative XTcf-3 abolishes the dorsal activation of the siamois promoter. Furthermore, elimination of the Lef/Tcf sites elevates the ventral expression of siamois, revealing a repressive role for XTcf-3 in the absence of beta-catenin. Finally, we find that the endogenous siamois activator, although present throughout the dorsal side of the embryo, is most potent in the dorsal vegetal region. We propose that the dorsal activation of siamois by the beta-catenin/XTcf-3 complex combined with the ventral repression of siamois by XTcf-3 results in the restriction of endogenous siamois expression to the dorsal side of Xenopus embryos.

Isolated explants from the animal hemisphere of Xenopus embryos were incubated with Xenopus basic fibroblast growth factor (XbFGF) or human activin A. XbFGF incubation resulted in the rapid activation of mitogen-activated protein kinase (MAPK) and ribosomal S6 protein kinase (pp90rsk) in a dose-dependent manner with the highest levels of activation occurring at 50 ng/ml. Maximal activation occurred within 6-10 min after the addition of growth factor, and the activity of both kinases declined to unstimulated levels after 30 min. Activin was unable to activate either MAPK or pp90rsk in the Xenopus explants to a substantial level, although it induced dorsal mesoderm better than XbFGF under the same experimental conditions. The regulatory protein Xwnt-8 did not activate MAPK, nor did it enhance the activation of MAPK by XbFGF. XbFGF was able to activate MAPK through at least the midgastrula stage, suggesting that this family of growth factors may have a role in gastrula-stage events.

Adenovirus early region 1A (E1A), which gives rise to three overlapping transcripts, was inserted into a murine leukemia virus-derived vector, and recombinant viruses were used to prepare permanent cell lines of NIH 3T3 cells containing DNA copies of the individual 13S, 12S, and 9S mRNAs. Integrated proviral copies of the recombinant genomes were rescued as bacterial plasmids from each of the cell lines, and the DNA sequence of E1A was demonstrated to be a precise copy of the individual transcripts. The DNA copies were shown to be expressed as part of the full-length retroviral transcript by S1 nuclease analysis, and the synthesis of their encoded polypeptides was demonstrated by immunoprecipitation. Those cell lines expressing the polypeptide encoded by the 13S transcript were shown to contain that function required for regulating the accumulation of mRNAs from adenovirus early genes by their ability to complement the adenovirus type 5 E1A deletion mutant dl312. Cell lines expressing polypeptides encoded by the 13S, 12S, and 9S transcripts showed characteristic alterations in morphology. Two-dimensional gel electrophoresis of total cellular protein derived from the three cell lines demonstrated that each E1A gene product elicits specific alterations in the patterns of proteins expressed. Studies of the expression of two specific genes, those encoding fibronectin and collagen type 1, indicated that the observed alteration in levels of the two proteins results from a reduction in RNA levels induced by E1A functions.

Human adenovirus (Ad)-encoded early region 1A (E1A) tumor (T) antigens have been implicated in the positive regulation of viral early genes, the positive and negative regulation of some cellular genes, and cell immortalization and transformation. To further study the Ad E1A T antigens and to facilitate their purification, we have cloned cDNA copies of the Ad12 E1A 13S mRNA and 12S mRNA downstream of a hybrid Escherichia coli trp-lac (tac) promoter. Up to 8% of the protein synthesized in E. coli cells transformed by each of the two different Ad12 E1A cDNA constructs were immunoprecipitated as a Mr 47,000 protein by antibody to a synthetic peptide encoded in the Ad12 E1A DNA sequence. Both proteins produced in E. coli appear to be authentic and complete Ad12 E1A T antigens because they possess (i) the Ad12 E1A NH2-terminal amino acid sequence predicted from the DNA sequence; (ii) the Ad12 E1A COOH-terminal sequence, as shown by immunoprecipitation with anti-peptide antibody; and (iii) a molecular weight and an acidic isoelectric point similar to that of the E1A T antigens synthesized in Ad12-infected and transformed mammalian cells. The T antigens were purified to near homogeneity in yields of 100-200 micrograms per g wet weight of transformed E. coli cells.

Simian adenovirus 7 (SA7) is a highly oncogenic virus, capable of causing tumors in hamsters upon the direct injection of viral DNA. We determined the transcriptional organization of the transforming region and compared it with that of the human adenoviruses. This analysis demonstrated that there are two independently promoted transcription units similar to the E1a and E1b regions of the human adenoviruses. The nucleotide sequence of the SA7 E1a region demonstrated considerable homology with the human adenoviruses, both in the sequences that regulate E1a expression and in the encoded polypeptides. The amino acid homology was reflected in the ability of SA7 to complement the growth of human adenoviruses mutant in the E1a region. Furthermore, we found two regions of amino acid homology unique to SA7 and the highly oncogenic human adenovirus 12.

A 16-amino acid peptide, H2N-Arg-Glu-Gln-Thr-Val-Pro-Val-Asp-Leu-Ser-Val-Lys-Arg-Pro-Arg-Cys-COOH (peptide 204), targeted to the common C-terminus of human adenovirus 12 (Ad12) tumor antigens encoded by the E1A 13S mRNA and 12S mRNA, has been synthesized. Antibody prepared in rabbits against peptide 204 immunoprecipitated two proteins of apparent Mr 47,000 and 45,000 from extracts of [35S]methionine-labeled Ad12-early infected KB cells and a 47,000 protein from extracts of the Ad12-transformed hamster cell line, HE C19. Immunoprecipitation analysis of infected and transformed cells labeled with 32Pi showed that both major Ad12 E1A T antigens are phosphoproteins. Immunofluorescence microscopy of Ad12-early infected KB cells with antipeptide antibody showed the site of E1A protein concentration to be predominantly nuclear. E1A proteins were detected by immunofluorescence at 4 to 6 h postinfection and continued to increase until at least 18 h postinfection. Antipeptide 204 antibody was used to analyze the proteins synthesized in Escherichia coli cells transformed by plasmids containing cDNA copies of the Ad12 E1A 13S mRNA or 12S mRNA under the control of the tac promoter (D. Kimelman, L. A. Lucher, M. Green, K. H. Brackmann, J. S. Symington, and M. Ptashne, Proc. Natl. Acad. Sci. U.S.A., in press). A major protein of ca. 47,000 was immunoprecipitated from extracts of each transformed E. coli cell clone. Two-dimensional gel electrophoretic analysis of immunoprecipitates revealed that the T antigens synthesized in infected KB cells, transformed hamster cells, and transformed E. coli cells possess very similar molecular weights and acidic isoelectric points of 5.2 to 5.4.