Hey2 negatively regulates the expression of CPC-associated genes. (A-F) WISH staining for SHF markers nkx2.5, mef2cb and ltbp3 at 24 hpf in hey2 heterozygous (A-C) and hey2 mutant (D-F) embryos. (G-I) Quantitative RT-PCR at 24 hpf of mef2cb, ltbp3 and nkx2.5 gene expression. (J,K) Confocal image of Mef2 immunostaining in Tg(myl7:EGFP) hey2 heterozygous (J) and mutant (K) embryos. Arrowheads indicate Mef2+ cells in the arterial pole of the heart. (L,M) Total number of Mef2+ cells in the arterial pole of the heart (L) and in the heart proper (M, with chambers counted separately) in control and hey2 mutant embryos at 48 hpf (N=3, n=4). (N-S) RNA in situ hybridization analysis of expression of nkx2.5, mef2cb and hey2 at 16.5 hpf in hey2 heterozygous (N-P) and mutant (Q-S) embryos. (T-V) Quantitative RT-PCR analysis showing relative expression levels of mef2cb, nkx2.5 and hey2 at 16.5 hpf in hey2 heterozygous and mutant embryos (gene expression normalized to β-actin, fold difference relative to control; N=3, n=3).
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