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Fig. S2. Inhibition of BMP signaling by use of HS:Noggin transgenic line and chemical inhibitor DMH1 blocks embryonic myelopoiesis, erythropoiesis, and vasculogenesis. (A) Diagram showing the transgene in HS:Noggin transgenic line. A 15-min heat shock induces myc-noggin protein expression within 30 min. Noggin levels remain steady for at least 24 h after treatment. (B) In situ hybridization on sections of stage 33 embryos shows that induced noggin is expressed across the section in HS:Noggin transgenic siblings. Embryos were heat-shocked for 15 min at 35 °C at stage 32 and fixed 2 h after the heat shock treatment. (C) Inhibition of BMP signaling in heat-shocked noggin (HS:N) and DMH1-treated embryos compared to heat-shocked wild type siblings (wt) or DMSO solvent-treated controls (C) is verified by the loss of P-Smad1/5/8 signal in Western blotting. Embryos were treated from stage14, and DLPs were explanted at stage 25, about 24 h after heat shock, further confirming that noggin protein is active a day after heat shock. Smad1 staining and actin staining were used as loading controls. (D) Timing of BMP requirement in ventral blood island. Inhibition of BMP signaling by DMH1 from stage 12 leads to a strong reduction in primitive myeloid cells originating from both first (mpo, spib) and second (ventral spib staining, red arrow) waves of myelopoiesis. DMH1 treatment blocks hematopoiesis as well as endothelial development when administered from stage 12, at the end of gastrulation. Expression of erythropoiesis markers hba3 and tal1 is absent, and expression of myelopoiesis markers spib and mpo is severely reduced. Endothelial gene expression of aplnr and tek in vitelline vessels, neighboring the ventral blood island, is also absent (red arrows). Aplnr and tek are expressed in the head and the cardinal vein precursors but at a lower level. DMH1 treatment from stage14, early neurulation, eliminates erythroid gene expression, as in treatment starting from stage 12. Spib expression in the periphery of ventral blood island are also absent (red arrow) whereas patrolling myeloid cells, marked by spib and mpo, are unaffected. DMH1 treatment from stage 14 does not block aplnr expression. In 12 of 20 embryos, aplnr expression was expanded to the most ventral tissues in the territory of blood island (blue arrow). DMH1 treatment from stage 14 reduces tek expression (23 of 23 embryos), but in 7 of 23 embryos tek expression was also observed in ventral blood island (blue arrow). (E) DMH1 treatment from stage 12 blocks myeloid gene expression of mpo, spib, and runx1 in anterior hemangioblast/aVBI at stage 16. (F) qRT-PCR analysis of dissected stage 16 aVBI tissue treated with DMH1 from stage 12 shows expression of blood genes gata2, tal1/scl, runx1, and mpo are reduced in stage 16 aVBI tissue, whereas fli1 expression is normal. |
