(A) E11.5 control and DKO embryo immunofluorescence (IF) for TdTomato showing NCC lineage (red), costained for GLP, G9a, H3K9me1, H3K9me2, or H3K9me3 (green). Scale bars: 5 μm. (B) Masson’s trichrome staining of palatal shelves (boxes) showing cleft palate in P0 DKO embryo. Scale bars: 500 μm. (C) H&E staining of P0 transverse sections, with black arrowheads denoting ligamentum arteriosum (LA) in control or patent ductus arteriosus (PDA) in DKO. Ao, aorta; PA, pulmonary artery. Scale bars: 500 μm. (D) RUNX2 (green) or SP7 (cyan) and TdTomato (red) IF in oral cavity (DAPI, blue). Scale bars: 500 μm. (E) Smooth muscle actin (SMA) (green) and TdTomato (red) IF of branchial arch arteries (DAPI, blue). Scale bars: 500 μm (left), 50 μm (right). (F) RNA-seq from E11.5 branchial arch TdTomato⁺ NCCs shown as heatmaps with 5 of the top 10 genes from lineage-specific gene sets differentially enriched in control or DKO. (G–I) H3K9me2 signal from TdTomato⁺ NCCs. (G) H3K9me2-enriched gene locus. (H) Distributions across all genes and DKO up- or downregulated genes. Wilcoxon’s rank-sum tests, median of all genes (dashed line). ***P < 2.2 × 10^-16. Boxes represent 25th–75th percentile range, solid horizontal lines indicate the median, and whiskers are 10th–90th percentiles. (I) Fold enrichment at DKO differentially expressed genes (DEGs) using Genomic Association Tester.