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Fig. S4. Upregulation of canonical Wnt signaling during gastrulation leads to a loss of blood. Embryos were incubated in the absence (control) or presence of LiCl for 20 minutes at the onset of gastrulation (st. 10). Expression of scl was analyzed by WMISH at stage 32. |
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Fig. 1. Signals from the ectoderm are required to induce blood formation in the mesoderm during gastrulation. (A) Illustration of ectoderm removal assay. The ventral half of embryos was isolated and ectoderm was either retained or removed from explants at stages 11, 12 or 13. Explants were allowed to develop until intact siblings reached the tailbud stage (stage 34) and assayed for expression of globin by WMISH. (B) Photographs of representative globin stained explants mounted in PBS (top two rows) or in Murrays clear (bottom row). (C, D) Quantitation of percent of explants expressing globin when cultured in the presence (C) or absence (D) of ectoderm. Results are pooled from 2–3 independent experiments for each group. |
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Fig. 7. Non-canonical Wnt signaling is required in ectodermal and mesodermal cells for primitive erythropoiesis. (A) RNA encoding Dsh∆DEP (500 pg) was injected into each blastomere of 4-cell embryos as illustrated and expression of scl and globin was analyzed by qPCR at stage 17 and stage 35, respectively. (B–D) RNA encoding Dsh∆DEP (750 pg) was injected into each ventral animal pole or ventral vegetal pole blastomere of 8-cell embryos as illustrated. (C) Expression of globin was analyzed by WMISH at stage 34 and globin staining in the posterior VBI (pVBI) was scored as barely or not detectable (−/+), moderately decreased (++) or strong (+++) using the scale illustrated on the right in panel C. (D) Expression of globin was determined by Northern blotting at stage 34. Levels of globin transcripts are normalized to and reported as a percentage of globin levels in control embryos below each lane. In this blot, the control lane contains RNA from CS2+ injected embryos and is the same control shown for Fig. 6 since all samples were run on the same gel. Results were replicated in two additional experiments. |
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Fig. S5. Inhibition of non-canonical Wnt signaling in dorsal cells does not interfere with globin expression in the posterior VBI. RNA encoding Dsh∆DEP was injected near the dorsal midline of 4-cell embryos and expression of globin was analyzed by WMISH at stage 34. |
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Fig. 6. Upregulation of zygotic canonical Wnt signaling in either ectodermal or mesodermal cells disrupts primitive erythropoiesis. (A and B) Empty CS2+vector DNA or CS2+ßcatenin DNA (100 pg per blastomere) was injected into both ventral animal pole or vegetal pole cells of 8-cell embryos as illustrated and expression of globin was analyzed by WMISH at stage 34. Globin staining in the posterior VBI (pVBI) of experimental embryos was scored as barely or not detectable (−/+), moderately decreased (++) or strong (+++) using the scale illustrated in panel A. (C) Embryos were injected as illustrated in panel A and expression of globin was determined by Northern blotting at stage 34. Levels of globin transcripts are normalized to and reported as a percentage of globin levels in CS2+ embryos below each lane. Results shown were replicated in one additional experiment. |
