| |
Fig. S3. BMP is required before stage 28 for the specification hemogenic endothelium. (A) Runx1 WISH of heat-shock time course. Embryos were heat-shocked for 30 min at 35 °C at the stage indicated and were heat-shocked every 24 h until stage 39, when the embryos were collected for analysis. Runx1-expressing cells were then stained by WISH. Embryos are shown from the lateral view, with anterior to the left and dorsal to the top. Green arrows mark the position of the DA, stained for runx1 in control siblings as well as HS:Noggin embryos treated from stage 30. Red arrow shows that runx1 staining in the DA is missing when embryos are heat-shocked starting from stage 20 and 26. HS:Noggin embryos were sorted by visual inspection of shuffled fin and protruding proctodaeum phenotype (blue arrow). Proportion of embryos for which that image represents is shown in bottom right corner. (B) BMP signaling is not required for dll4 expression. Dll4 WISH of heat-shock time course. Embryos were treated as in A and stained for Dll4. The arterial marker dll4 is expressed at all stages analyzed albeit disorganized. (C) Runx1 WISH of DMH1 treatment time course. Embryos were cultured in 0.1× MBS containing 100 μM DMH1 from the stage indicated and were cultured to stage 39, when the embryos were collected for analysis. Runx1-expressing cells were then stained by WISH. Embryos are shown from the lateral view, zoomed into the trunk region, with anterior to the left and dorsal to the top. Green arrows mark the position of the DA, stained for runx1 in control siblings as well as stage 28 DMH1-treated embryos. The red arrow shows that runx1 staining in the DA is missing when embryos are treated starting from stage 24 and decreased when treated starting from stage 26. The blue box highlights stage 28; after that time point, there is no effect on runx1 expression. (D and E) BMP is not required for runx1 maintenance in Xenopus DA. BMP was inhibited from stage 28 by either 100 μM DMH1 treatment or by noggin heat-shock induction. Embryos were collected at stage 43. D, Top shows runx1-expressing cells stained by WISH on wax sections. Sections are 10-μm transversal slices taken through the trunk of the embryo. D, Middle shows embryos, visualized from the ventral aspect, with anterior to the left. The green arrow indicates normal intestinal morphogenesis, which is absent in Bmp-inhibited embryos. D, Bottom shows stage 43 embryo tails, shown from the lateral view, with anterior to the left and dorsal at the top. Bmp-inhibited embryos have an irregular tail fin, whereas control embryos have a smooth edge to the tail fin. Proportion of embryos for which that image represents is shown in bottom right corner in all figures. Images and numbers are from one experiment and are representative of at least three biological replicates except in sectioned embryos. (F) DMH1 inhibits Smad1/5/8 phosphorylation from stage 32 to, and from stage 32–39 without affecting Smad2 phosphorylation level. Treatment with TGFB inhibitors SB505124 and SB431542 from stage 32–39 reduces Smad2 phosphorylation (WB with Millipore anti-Psmad2 Ab no. 04-953) without affecting phospho Smad1/5/8 levels. Smad1 is expressed relatively uniformly between matched DMSO and chemical inhibitor treatments. |
