Figure 2.

Temporal alteration of Nkx2-5 expression in U1/U2 double-deletion mice during heart developmental progression

(A) Schematic of the strategy for the double-knockout (DKO) mice generation.

(B) Gross morphological examination of E9.5 hearts from DKO mice. DKO mice showed severe SHF defects. The arrow indicated the underdeveloped outflow tract. The asterisk indicated the open atrioventricular chamber. The arrowhead indicated the single ventricle. Scale bar, 200 μm.

(C) Survival analysis of DKO strains determined by heterozygote intercrosses at three indicated developmental stages. p-value was calculated by chi-squared test.

(D) RT-qPCR for Nkx2-5 in WT, Het (Heterozygotes), and DKO embryonic hearts at E8.25, E8.75, and E9.5. RNA expression levels were normalized to 18s rRNA. Data were mean ± s.e.m. p-value was calculated by Student’s t-test.

(E, G, I, and K) Whole-mount in situ hybridization of Nkx2-5 in WT and DKO embryos at E7.5 (E), E8.25 (G), E8.75 (I), and E9.5 (K). Scale bar, 500 μm in (K) and 200 μm in (E, G, and I).

(F, H, J, and L) Immunofluorescence staining of NKX2-5 (red), TNNI3 (green), and DAPI (blue) in WT and DKO at E7.5 (F), E8.25 (H), E8.75 (J), and E9.5 (L). Insets of heart regions were shown in higher magnification on the right. Scale bar, 100 μm.