Fig. 7

Mynn promotes BMP signaling through competing with Ppm1a for Smad binding

(A) Presence of Mynn attenuates the interaction of Ppm1a and Smad1. HEK293T cells were co-transfected with Myc-Smad1 and HA-Ppm1a together with or without Flag-Mynn, and then harvested for immunoprecipitation.

(B) Mynn weakens the Ppm1a-mediated dephosphorylation of p-Smad1/5/9. HEK293T cells were transfected with the indicated constructs. CaALK1 is a constitutively active form of BMP type 1 receptor ALK1 (Q233D). The expression levels of p-Smad1/5/9 and total Smad1 were detected by Western blots with appropriate antibodies.

(C) Overexpression of mynn can restore the decrease of BMP signal activity caused by excessive Ppm1a. Tg(BRE:dGFP) embryos were injected with indicated mRNAs at the one-cell stage and subjected to immunostaining for GFP (green) at 48 hpf. Scale bars, 50 μm. Injection dosages: mCherry mRNA, 400 pg; ppm1a mRNA, 400 pg; mynn mRNA, 300 pg.

(D and E) Ppm1a-induced pharyngeal cartilage defects were compromised by overexpression of mynn. Wild-type embryos were injected with 400 pg ppm1a mRNA together with or without 300 pg mynn mRNA at the one-cell stage, and stained at 96 hpf for cartilage with Alcian blue. Representative pictures of different classes of defects in pharyngeal cartilages were shown in (D). The percentage of embryos with different degrees of cartilage defects was calculated (E). Scale bars, 200 μm.

(F and G) Knockdown of ppm1a could restore the CNCC proliferation defects in mynn^−/− mutants. mynn^−/− mutant embryos were injected with 5 ng ppm1a MO at the one-cell stage and incorporated with BrdU at 34 hpf, and then harvested at 36 hpf for immunostaining with anti-BrdU (red) and anti-GFP (green) antibodies (F). Scale bars, 50 μm. The number of BrdU and GFP double positive cells in the second pharyngeal arch was calculated (G). Error bars indicate ±S.D. Student’s t-test, ∗∗∗, p < 0.001.

(H) The differentiation defects of CNCCs in mynn mutant embryos were partially recovered by ppm1a knockdown. mynn^−/− embryos were injected with 5 ng ppm1a MO at the one-cell stage and harvested at 60 hpf for immunostaining with anti-Col2 (green) antibody. pq, palatoquadrate; ep, ethmoid plate; ch, ceratohyal. Scale bars, 50 μm.