SUMMARY The abnormal perturbation in gene regulation during palatogenesis may lead to cleft palate, a major congenital birth defect in humans and mice. However, a comprehensive multi-omic map of the developing secondary palate at single-cell resolution is lacking. In this study, we performed single-cell multiome sequencing and profiled chromatin accessibility and gene expression simultaneously within the same cells (n = 36,154) isolated from mouse secondary palate across embryonic days (E) 12.5, E13.5, E14.0, and E14.5. Application of optimal transport reconstructed five trajectories, representing continuous differentiation of multipotent cells into different subpopulations in later stages. By linking open chromatin signals to gene expression changes, we discovered a list of lineage-determining transcription factors, such as Shox2 for the anterior and Dlx1/2 for the posterior palatal mesenchymal trajectories. In conclusion, this study charted epigenetic and transcriptional dynamics during palatogenesis, which provides a valuable resource for the community and facilitate future research in cleft palate. Highlights The first report on building a single-cell multi-omics atlas with joint chromatin accessibility and gene expression measurements from the same cells during the development of mouse secondary palate. Application of optimal transport calculated fate probabilities to different terminal states and recovered continuous landscapes during mouse secondary palate development. By linking cis -regulatory DNA elements to target genes, we characterized a series of transcription factors governing the differentiation of cranial neural crest-derived multipotent cells to the anterior and posterior palatal mesenchymal trajectories, respectively. Transcription factors Shox2 and Dlx1/2 exhibited top regulatory roles for the anterior and posterior palatal mesenchymal trajectories, respectively, showing significant enrichment in both motif accessibility and gene expression.

Single-cell multi-omics decodes regulatory programs during development of mouse secondary palate