Fig. S4

Effect of hey2 knockdown on CPC gene expression.

(A-F) RNA in situ hybridization analysis of nkx2.5, ltbp3 and mef2cb gene expression at 24 hpf in WT (A-C) and hey2 MO (D-F) embryos. Note the expanded expression of transcript levels at the poles of the heart tube in morphant embryos (D-F, arrowheads) compared to controls (A-C). Asterisks indicate the heart tube. (G-J) Quantitative RTPCR analysis for mef2cb (G), ltbp3 (H), nkx2.5 (I) and hey2 (J) gene expression between control and hey2 MO embryos at 24 hpf. (K-N) RNA in situ hybridization analysis at 16.5 hpf in control (K, L) and hey2 MO (M, N) embryos for nkx2.5 and mef2cb expression. (O-Q) Quantitative RT-PCR analysis comparing relative expression in control and hey2 morphant embryos for mef2cb (O), nkx2.5 (P) and hey2 (Q) at 16.5 hpf (gene expression is normalized to β–actin, fold difference is relative to controls). (R-U) RNA in situ hybridization analysis at 12 hpf in control (R) and hey2 MO (S) embryos and at 11 hpf (T and U) in hey2 heterozygous (T) and mutant (U) embryos. Dotted line indicates whole embryo outline, arrowheads highlight low levels of nkx2.5 expression in the ALPM of hey2 mutants (U).