Multi-omic dissection of enhancer, transcription factor and ncRNA regulation of epicardial EMT in cardiac development
J Nunes Vieira, M Valenzuela Sanchez, T Dias, H Scheucher, Q Dang, C Roque Silva, N Owens, K Zvonickova, E Bell, L Bell, J Kalisch Smith, B Davies, D Sparrow, P Riley, A RedpathAbstract
Background
Epicardial epithelial-to-mesenchymal transition (EMT) is essential for myocardial growth and coronary formation. In adult mammals, epicardial EMT becomes markedly restricted, limiting regenerative capacity. Defining the regulatory architecture that drives epicardial EMT during development is therefore critical for identifying mechanisms that could be reactivated for cardiac repair.
Methods
Single-cell RNA-sequencing, ATAC-sequencing and CUT&RUN were integrated to map chromatin accessibility and transcription factor occupancy during epicardial EMT. CRISPR/Cas9 genome editing was used to generate enhancer-deleted mouse models and new lineage-tracing tools. Embryonic phenotypes were assessed using high-resolution episcopic microscopy and confocal imaging. High-throughput microRNA screening and metabolic profiling were performed in an epicardial EMT model to identify additional regulatory pathways with translational potential.
Results
A cardiac-specific enhancer within the Wt1 locus was identified as essential for normal heart development. Enhancer deletion reduced Wt1 expression and caused myocardial non-compaction, septal defects, abnormal valvulogenesis and disrupted coronary patterning, demonstrating a central role in epicardial–myocardial communication. Gene-regulatory network analysis highlighted transcription factors predicted to optimise Wt1 activity. Parallel studies revealed a requirement for Slit-dependent signalling in epicardial EMT, with loss-of-function models showing impaired subepicardial patterning and reduced EMT markers. MicroRNA screening identified ncRNAs capable of modulating EMT, while metabolic assays showed coordinated increases in mitochondrial respiration and glycolysis during epicardial activation.
Conclusions
This multi-omic analysis defines enhancer-driven, transcription factor-mediated and ncRNA-dependent mechanisms controlling epicardial EMT. By identifying regulatory elements, signalling pathways, microRNA modulators and metabolic switches, this work provides a mechanistic foundation for strategies aimed at reactivating epicardial plasticity to support cardiac regeneration.