Fig. 6.

BMP2a together with BMP5 functions in PAA progenitor specification. (A,A′) The expression of etv2 in embryos injected with indicated MO was analyzed by in situ hybridization (A). Injection doses: bmp2a MO, 2 ng; bmp2b MO, 0.3 ng; bmp4 MO, 2 ng; bmp5 MO, 4 ng. The average numbers of etv2⁺ PAA angioblast clusters were quantified from three independent experiments, and the group values are expressed as mean±s.d. (A′). Student's t-test (**P<0.01, ***P<0.001; NS, no significant difference). (B) Knockdown of bmp2a and bmp5 disrupted the specification of PAA progenitors. Wild-type embryos were injected with bmp2a and bmp5 MOs at the one-cell stage. The resulting embryos were harvested for in situ hybridization. (C) Wild-type and indicated mutant embryos were harvested at 36 hpf for immunofluorescence assay with anti-p-Smas1/5/8 antibody. Nuclei were counterstained with DAPI. There is a distinct decrease of p-Smad1/5/8 in the bmp2a^−/−;bmp5^−/− double mutants. Scale bar: 50 μm. (D-E′) Expression analysis of nkx2.5 (D) and etv2 (E) in bmp2a^−/− or bmp5^−/− embryos and bmp2a^−/−;bmp5^−/− double mutants by in situ hybridization. The average numbers of etv2⁺ angioblast clusters were quantified from three independent experiments and the group values were expressed as mean±s.d. (E′). Student's t-test was used to determine the significance of differences between wild-type animals and each mutant, and one-way ANOVA test was performed to analyze the statistical differences between bmp2a^−/−;bmp5^−/− double mutants and bmp2a^−/− or bmp5^−/− embryos. **P<0.01, ***P<0.001.