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Fig. 7. Overexpression of hyperventralizing xTsg constructs in zebrafish embryos inhibits dorsoanterior development. (A) Uninjected sibling. Wild-type zebrafish embryos were injected at the one-cell stage with 400 pg wild-type xTsg mRNA (B-D) or with 330 pg xTsgW67G mRNA (E-G). Embryos injected with wild-type xTsg mRNA in two independent experiments (n=144) displayed a range of dorsalization (as classified by Mullins et al., 1996): 13% Class 1 (not shown), 44% Class 2 (B), 17% Class 3 (C) and 24% Class 4 (D). Of the embryos displaying Class 1 or 2 dorsalizations, 51% also exhibited duplication of the terminal ventral fin (inset in B, indicated by arrowheads), suggestive of a tail ventralization in zebrafish. Of 199 embryos injected with 330 pg xTsgW67G mRNA in two independent experiments, 73% were moderately ventralized to levels comparable with chordino (E). 14% showed a phenotype more severe than chordino (F) and 3% displayed an even more ventralized phenotype (G). At higher doses (800 pg), xTsgW67G mRNA caused phenotypes stronger than chordino in 86.8% of embryos (n=91), with 18.7% of the type shown in G and 67% of the type shown in F. Note the almost complete absence of brain and trunk somites in G. Injection of 200 pg xTsgC59A mRNA into 60 embryos (not shown) also caused ventralization, with 82% appearing similar to E, and 5% resembling F.





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