Abstract Body: During organogenesis, epicardial cells undergo epithelial-to-mesenchymal transition (EMT), contributing essential cell types and paracrine signalling to the growing heart. The epicardium is integral to heart regeneration in lower vertebrates and neonatal mammalian injured hearts. That said, prospects to harness the epicardium for therapeutic applications to effect adult heart repair, heavily depend on improved insight into its intrinsic properties in development. Cell fate decisions underpinning EMT are directed by transcription factors such as Wilms’ tumour 1 (WT1). Whilst a requirement for Wt1 in heart development is established, the mechanisms underpinning its activation remain elusive. We identified two evolutionary conserved regions (ECRs) shared in mouse and human, located within intron 1 of Wt1 locus. We hypothesised these regulatory sequences direct locus activation and EMT to support normal heart development.

Here, we used CRISPR/Cas9 gene-editing technology to generate mice carrying a sequence deletion containing one ECR or a deletion comprising both ECRs. Extensive survival analysis, high-resolution episcopic microscopy (HREM), qPCR, immunostaining, confocal microscopy and epicardial explants were used to characterise heart formation. Mendelian ratios indicated an overall underrepresentation of Wt1^ΔECR/ΔECR mutants in adulthood. HREM revealed smaller hearts, incidence of myocardial non-compaction and spongy interventricular septum with muscular and membranous defects, tricuspid hypoplasia and enlarged aortic valves, as well as abnormal patterning of coronary artery stems in mutant hearts. Expression of Wt1 was markedly reduced in Wt1^ΔECR/ΔECR hearts but not kidneys, suggesting intronic enhancers are cardiac-specific. Whole-mount and tissue immunostaining revealed abnormal coronary vessel and innervation patterning in the subepicardium, as well as reduced EMT and myocardial non-compaction/hyper-trabeculation in Wt1^ΔECR/ΔECR.

Collectively, we demonstrated a requirement for novel Wt1 intronic enhancers regulating locus activity and essential epicardial EMT-associated biological processes in normal heart development. Importantly, observation of septum and semilunar valve defects in ΔECR hearts suggests an hitherto unrecognised role for WT1-driven EMT, opening new avenues of research to improve our understanding of congenital heart disease affecting at least 1:150 live births, with remarkably two thirds of cases having unknown aetiology.