Exploring the role of the RNA-binding protein SRSF3 in the developing heart

Understanding the molecular processes that govern cardiovascular development has the potential to inform regenerative strategies for disease states. Due to their versatile roles, epicardial cells are an attractive therapeutic target. However, using epicardial cells for therapy is hindered by our limited understanding of epicardial biology. The embryonic epicardium is one of the main regulators of cardiovascular development, functioning to secrete essential growth factors and produce epicardium-derived cells (EPDCs) that contribute most coronary vascular smooth muscle cells and cardiac fibroblasts. Due to its complex biology, the molecular mechanisms that control epicardial formation and differentiation have not been fully elucidated. SRSF3 is an RNA-binding protein that has essential roles in RNA processing, and it is highly expressed in the proepicardium and later in the epicardial layer during heart development. Deletion of Srsf3 from the murine proepicardium using the Tg(Gata5-Cre) or embryonic day (E) 8.5 induction of Wt1^CreERT2 led to proliferative arrest and impaired epithelial-to-mesenchymal transition (EMT), which prevented proper formation and function of the epicardial layer. The epicardial requirement for SRSF3 was further assessed in a mouse epicardial cell line using RNA interference, which revealed that SRSF3 is required for production of Cyclin D1 and for EMT in response to TGFβ. Induction of Srsf3 deletion with the Wt1^CreERT2 after the proepicardial stage resulted in disrupted sprouting from the sinus venosus and elevated hypoxia at E13.5. Single-cell RNA-sequencing showed SRSF3-depleted epicardial cells were removed by E15.5. The remaining non-targeted cells became hyperproliferative and compensated for the loss, with normal epicardial contribution being detected at E17.5. Due to the compensation mechanism, the existence of epicardial subpopulations was explored through assessment of published heterogeneous marker genes: Sema3d, Scx, Tcf21, Tbx18 and Wt1, using RNAscope, PrimeFlow and Single-cell RNA-Sequencing, in both wild-type and SRSF3 mutants. Complete overlap of the markers was detected, with no evidence of epicardial subpopulations, raising questions regarding the specificity of previously reported Cre lines. This research supports a role for SRSF3 as a master regulator of RNA metabolism in epicardial cells, however, further studies are required to dissect the exact molecular mechanisms involved.