Single-cell multi-omics decodes regulatory programs during development of mouse secondary palate [bulk RNA-seq]

Perturbations in gene regulation during palatogenesis can lead to cleft palate, which is among the most common congenital birth defects. However, currently there is no comprehensive multiomics map of the developing secondary palate. Here, we perform single-cell multiome sequencing and profile 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. We construct five trajectories representing continuous differentiation of cranial neural crest-derived multipotent cells into distinct lineages. By linking open chromatin signals to gene expression changes, we characterize the underlying lineage-determining transcription factors. In silico perturbation analysis identifies transcription factors SHOX2 and MEOX2 as important regulators of the development of the anterior and posterior palate, respectively. In conclusion, our study chart epigenetic and transcriptional dynamics in palatogenesis, serving as a valuable resource for further cleft palate research. The anterior (n=3) and posterior (n=3) 1/3 palatal shelves of embryonic day (E) 14.0 C57BL/6J mice were microdissected, isolated, and then subjected to bulk RNA-seq.腭发育过程中的基因调控紊乱可引发腭裂，而腭裂是最常见的先天性出生缺陷之一。然而，目前尚无针对发育中次级腭的完整多组学图谱。本研究采用单细胞多组学测序（single-cell multiome sequencing）技术，对胚胎第12.5天（E12.5）、E13.5、E14.0及E14.5的小鼠次级腭组织分离得到的36154个细胞内的染色质可及性与基因表达进行了同步检测分析。我们构建了五条分化轨迹，用以展现颅神经嵴来源的多能干细胞向不同细胞谱系的连续分化过程。通过将开放染色质信号与基因表达变化进行关联分析，我们解析了调控细胞谱系决定的核心转录因子。经计算机模拟扰动分析，我们分别确定转录因子SHOX2与MEOX2为腭前部与腭后部发育的关键调控因子。综上，本研究阐明了腭发育过程中的表观遗传与转录调控动态变化，可为后续腭裂相关研究提供极具价值的参考资源。本研究还对胚胎第14.0天（E14.0）的C57BL/6J小鼠前1/3与后1/3腭突（各3例）进行显微解剖、分离后开展了批量RNA测序（bulk RNA-seq）。