(A) RT-qPCR analysis of Isl1 (n = 8), Nkx2.5 (n = 8), Flk1 (n = 7), Hand1 (n = 6), Hand2 (n = 6), and Tbx5 (n = 6) expression in E9.5 embryonic hearts and the adjacent mesoderm (20–21 somites) after chemical induction of hypoxia responses. PBS-injected mice were used as control. The m34b4 gene was used as a reference for normalization. **P < 0.01; ***P < 0.001, t test. (B) In situ hybridization of C57BL/6 E9.25 embryos (18 somites) for either Isl1 mRNA (upper panel) or Nkx2.5 mRNA (lower panel) after chemical induction of hypoxia responses (30 mg CoCl₂/kg body weight). Arrows indicate the NKX2.5⁺ or ISL1⁺ cardiogenic region. Representative images from 2 independent experiments are shown. (C) Western blot analysis of ISL1, NKX2.5, and sarcomeric α-actinin levels in cardiac mesoderm containing adjacent outflow tract isolated from E9.5 embryos with or without CoCl₂ treatment. α-Tubulin was used as protein-loading control. Two separate experiments (3 individual litters per experiments) were analyzed, yielding similar results. (D) Analysis of ISL1⁺ cell proliferation in mock (n = 4) or CoCl₂-treated (n = 4) E9.5 embryos by immunostaining for ISL1 and phospho-histone H3 (Ser10) (pH3). The percentages of pH3/ISL1 double-positive cells in cardiac mesoderm are shown. At least 6 sections from each embryo were counted. **P < 0.01, ANOVA with Dunnett’s post hoc correction. (E) TUNEL assay of ISL1⁺ cells after CoCl₂ treatment. The percentages of TUNEL-positive ISL1⁺ CPCs are shown. At least 12 sections from each embryo were counted. ANOVA with Dunnett’s post hoc test was used to calculate significance (n = 3). (F) Immunofluorescence-based quantification of ISL1⁺NKX2.5^–, ISL1⁺NKX2.5⁺, and ISL1^–NKX2.5⁺ cells after FACS-based cell sorting of ISL1⁺ cells from E8.0 ISL1^nGFP/+ embryos and 2-day cultivation of isolated cells under either normoxic or hypoxic conditions. Quantification of different cell populations was achieved by counting all immunostained cells in a multiwell dish. *P < 0.05; ***P < 0.001, t test (n = 3). (G) Number of Isl1-Cre⁺ Rosa26^Nkx2.5 embryos after breeding of heterozygous Isl1-Cre⁺ mice with Rosa26^Nkx2.5 mice at different developmental stages. (H) Whole-mount views of E9.5 (left panels) and E10.5 embryos (right panels). Boxed areas are enlarged. Representative images are shown. Arrows indicate shortened outflow tract in Isl1-Cre⁺ Rosa26^Nkx2.5 embryos. Scale bars: 100 μm. (I) H&E staining of E15.5 hearts isolated from either Isl1-Cre^– Rosa26^WT (upper panel) or Isl1-Cre⁺ Rosa26^Nkx2.5 (lower panel) mice. Thirty embryos from 5 litters including 2 Isl1-Cre⁺ Rosa26^Nkx2.5 embryos were analyzed. Arrows point to individual cardiac defects named in the figure. Scale bars: 200 μm.