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Fig. 6. Long-range patterning of neuroectoderm by XWnt3A is direct. (A) Titration of Xwnt3A and Xβcat* mRNAs. For every mRNA concentration (in pg/nl), 15 embryos were injected at four- to eight-cell stages vegetally into two opposite blastomeres (2.5 nl/blastomere), cultured until tailbud stage and scored for the induction of secondary body axes. Comparable results were obtained in four independent experiments. (B) RT-PCR analysis of expression of the indicated marker genes at (a) early gastrula stage (stage 10+) and (b) neural plate stage (stage 15) in whole embryos (we) and animal caps cut from embryos injected at the eight-cell stage into the four animal blastomeres with no (co), 0.25 ng/blastomere Xwnt3A or 2.5 ng/blastomere Xβcat* mRNAs. H4, histone4 for normalization; –RT, negative control without reverse transcriptase. (C) Pigmented donors uninjected (co) or injected with 0.25 ng/blastomere Xwnt3A or 2.5 ng/ blastomere Xβcat* mRNA were grafted as depicted in Fig. 5A into the presumptive host anterior neural plate. Embryos were analysed at neural plate stage (stage 15) for expression of Bf1, En2 and Krox20. Frontal views are shown (dorsofrontal in b′′,c′′). Between 21 and 63 embryos were analysed for every type of transplant in six independent experiments. Note that Xβcat*-expressing grafts do not induce changes in host marker gene expression unlike Xwnt3A (arrows), even at 5 ng/blastomere (not shown). |
