Figure 2. Dorsal genes are inaccessible at the onset of Gastrulation.

A. Embryos were cultured in 0.1xMMR and Wnt signaling was activated by exposing embryos to a 10-12 minute pulse of 0.3M LiCl at the indicated stages. Activation of Wnt signaling in cleavage stage embryos leads to radial dorsalization (center). Activation of Wnt signaling at the late blastula stage leads to posteriorization (right). Controls are untreated stage 33 tadpoles. For this and following experiments, ≥ 80% of embryos treated with LiCl at the cleavage stage showed dorsalization at tadpole stages, with complete lack of trunk structures, no obvious somites, expanded and frequently circumferential eyes, and frequently expanded cement gland (DAI ≥ 8; (Kao and Elinson, 1988; Karimi et al., 2018)). Similarly, ≥80% of embryos exposed to LiCI at the late blastula stage demonstrated anterior truncation/posteriorization, with reduced or absent forebrain and small or absent cement gland, frequently with small or absent eyes and microcephaly. B. Wnt signaling was activated by exposure to LiCl at cleavage stage (stage 6) or the blastula stage (stage 9) and expression of dorsal Wnt target genes sia1 and nodal3.1 and non-dorsal Wnt target genes (hoxa1, hoxd1, and cdx2) was measured by RT-qPCR in whole embryos at the midgastrula stage (stage 10.5). Gene expression in all samples was normalized to ODC and then expression in embryos exposed to LiCl at stage 6 (blue bars) and stage 9 (orange bars) was normalized to untreated (grey) and are presented as mean values for fold change in expression for 5 biological replicates except for nodal3.1 which had 3 replicates. Error bars represent standard error of the mean (SEM). C. Representative sequencing tracks for the dorsal Wnt target genes sia1 and nodal3.1, the organizer marker Gsc, and non-dorsal Wnt target genes hoxa1, hoxd1, and cdx2. Y-axis represents normalized reads scaled by 1,000,000/total alignments. Scale bar represents 1kb.