DEV01901 Supplementary Material

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• Supplemental Figure 1 - Fig. S1. The posteriorzed anus phenotype is an indicator of low BMP signaling in Xenopus. (A) Control tadpole. White arrowheads point to the proctodeum or anus. (B) Embryos injected with low doses of Bmp4 MO (4 ng total) have a posteriorzed anus and diminished ventral fin tissues. (C) Posteriorized anus phenotype and loss of ventral fin arising from injection of 120 pg DBmp7 mRNA. This dominant-negative BMP7 construct was generated for this study by deleting the mature growth factor domain from human BMP7 by PCR.

• Supplemental Figure 2 - Fig. S2. BMP4 depletion leads to a reduction of ventral markers and expansion of dorsal markers. Embryos were injected radially at the 4-cell stage with Bmp4 MO (12 ng total), fixed at late gastrula (stage 12) and analyzed for the designated markers (n=10 per experimental set). Blastopore views are shown, dorsal to the top. (A,B) Bmp4 expression is reduced in Bmp4 MO-injected embryos, demonstrating that BMP4 positively regulates its own expression. (C,D) Sizzled expression in the ventral gastrula center at stage 12 is abolished in BMP4-depleted embryos. (E,F) Expression of Mix1, a ventral endo-mesodermal marker (Mead et al., 1996), is downregulated in the absence of BMP4 activity. (G,H) Expression of Vent1, a direct target of Bmp signaling, in the ventral side of the closing blastopore is significantly reduced by Bmp4 MO in ventral mesoderm, while its more diffuse lateral expression is unaffected. (I,J) Expanded expression of the pan-neural marker Sox2 in Bmp4 MO-injected embryos. (K,L) The future forebrain/midbrain territory is increased in embryos depleted for BMP4, as shown by the expression of Otx2. At later developmental stages, this expansion regulates and is not as apparent. (M,N) Narrower and longer expression domain of Xnot in the nascent notochord, suggesting that convergence and extension movements in the BMP4-depleted gastrula are enhanced. (O,P) Formation of mesoderm is unaffected in BMP4-depleted embryos, as shown by the expression of the pan-mesodermal marker Xbra.

• Supplemental Figure 3 - Fig. S3. UV treatment induces radial expression of Bmp4 and its target gene Sizzled, without affecting mesoderm formation. Fertilized eggs were subjected to UV irradiation for 1 minute and tested for expression of the designated markers at late gastrula stage. Blastopore views are shown, dorsal to the top (n=8 per experimental set). (A) In the wild-type control gastrula, the dorsal side, or future neural plate, is devoid of Bmp4 expression. (B) UV-irradiated embryos display radial Bmp4 expression. (C) Sizzled expression is confined in the ventral center of the embryo at stage 12. (D) Ubiquitous Sizzled expression is seen in UV-treated embryos, indicating high BMP signaling. (E,F) Expression of the pan-mesodermal marker Xbra is unaffected in UV-treated embryos.