An epithelial sheet, the epicardium, lines the surface of the heart. In the developing embryo, the epicardium expresses the transcriptional regulator Wilm's Tumor Gene 1 (Wt1). Through incompletely understood mechanisms, Wt1 inactivation derails normal heart development. We investigated mechanisms by which Wt1 regulates heart development and epicardial epithelial to mesenchymal transition (EMT). We used genetic lineage tracing approaches to track and isolate epicardium and epicardium derivatives in hearts lacking Wt1 (Wt1(KO)). Wt1(KO) hearts had diminished proliferation of compact myocardium and impaired coronary plexus formation. Wt1(KO) epicardium failed to undergo EMT. Wt1(KO) epicardium expressed reduced Lef1 and Ctnnb1 (β-catenin), key components of the canonical Wnt/β-catenin signaling pathway. Wt1(KO) epicardium expressed decreased levels of canonical Wnt downstream targets Axin2, Cyclin D1, and Cyclin D2 and exhibited decreased activity of the Batgal Wnt/β-catenin reporter transgene, suggestive of diminished canonical Wnt signaling. Hearts with epicardium-restricted Ctnnb1 loss of function resembled Wt1(KO) hearts and also failed to undergo epicardial EMT. However, Ctnnb1 inactivation did not alter WT1 expression, positioning Wt1 upstream of canonical Wnt/β-catenin signaling. Wnt5a, a prototypic non-canonical Wnt with enriched epicardial expression, and Raldh2, a key regulator of retinoic acid signaling confined to the epicardium, were also markedly downregulated in Wt1(KO) epicardium. Hearts lacking Wnt5a or Raldh2 shared phenotypic features with Wt1(KO). Although Wt1 has been proposed to regulate EMT by repressing E-cadherin, we detected no change in E-cadherin in Wt1(KO) epicardium. Collectively, our study shows that Wt1 regulates epicardial EMT and heart development through canonical Wnt, non-canonical Wnt, and retinoic acid signaling pathways.
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