Sox9 in the Epicardium: Implications for Cell Invasion, Differentiation, and Coronary Vascular Development

The epicardium is the mesothelial lining of the heart which acts as a source of progenitor cells during heart development, giving rise to an invasive population of mesenchymal cells which differentiate into cardiac fibroblasts, mural cells, and other cell types essential for heart structure and function. Previously, we showed that epicardial-specific deletion of the gene encoding SRY-box transcription factor 9 (SOX9) impairs epicardial-derived cell invasion and reduces their contribution to the atrioventricular valve mesenchyme. In this study, we use single-cell RNA-sequencing to investigate broader roles of Sox9 in the epicardium as it relates to epicardial invasion, differentiation, and vascular development. We identified transcriptional changes indicative of decreased epicardial-to-mesenchymal transformation consistent with histological observations. Sox9-deficient epicardial cells exhibited elevated expression of vascular smooth muscle cell genes, suggesting that Sox9 may influence epicardial cell fate decisions. Immunofluorescence analyses revealed defective epicardial attachment and decreased epicardial-derived cell invasion into the ventricular myocardium associated with delayed coronary plexus formation. This study expands our understanding of the role of Sox9 in epicardial biology, demonstrating an important function in regulating epicardial cell invasion, differentiation, and coronary vasculature development. These insights provide a foundation for further investigations into epicardial-mediated mechanisms underlying congenital heart abnormalities. This study focused on single-cell RNA-sequencing to further characterize the role of SOX9 in epicardial development. Transcriptomic analyses of E14.5 epicardial-Sox9 knockout and control hearts indicated dysregulation of genes implicated in epicardial invasion, differentiation, and vascular development.

心外膜（epicardium）是心脏的间皮衬层，在心脏发育过程中作为祖细胞来源，可产生具有侵袭性的间充质细胞群，这些细胞可分化为心脏成纤维细胞、壁细胞以及其他对心脏结构和功能至关重要的细胞类型。既往研究表明，编码SRY框转录因子9（SOX9）的基因在心外膜特异性敲除后，会损伤心外膜来源细胞的侵袭能力，并降低其对房室瓣间充质的贡献。本研究通过单细胞RNA测序（single-cell RNA-sequencing），探究了Sox9在心外膜中与心外膜侵袭、分化及血管发育相关的更广泛作用。我们鉴定到了与组织学观察一致的、提示心外膜-间充质转化减弱的转录组变化。Sox9缺陷的心外膜细胞表现出血管平滑肌细胞基因的表达上调，这表明Sox9可能影响心外膜细胞的命运决定。免疫荧光分析显示，心外膜附着异常，且心外膜来源细胞侵袭进入心室心肌的能力降低，同时伴随冠状丛形成延迟。本研究拓展了我们对Sox9在心外膜生物学中作用的认知，证实其在调控心外膜细胞侵袭、分化及冠状血管发育中发挥重要功能。这些发现为进一步研究心外膜介导的先天性心脏异常相关机制奠定了基础。本研究聚焦单细胞RNA测序，以进一步阐明SOX9在心外膜发育中的作用。对胚胎14.5天（E14.5）心外膜Sox9敲除小鼠及对照小鼠心脏的转录组分析显示，与心外膜侵袭、分化及血管发育相关的基因存在表达失调。