Epicardial epithelial-mesenchymal transition (EMT) is hypothesized to generate cardiovascular progenitor cells that differentiate into various cell types, including coronary smooth muscle and endothelial cells, perivascular and cardiac interstitial fibroblasts and cardiomyocytes. Here we show that an epicardial-specific knockout of Wt1 leads to a reduction of mesenchymal progenitor cells and their derivatives. We demonstrate that Wt1 is essential for repression of the epithelial phenotype in epicardial cells and during Embryonic Stem (ES) cell differentiation, through direct transcriptional regulation of Snail (Snai1) and E-cadherin (Cdh1), two of the major mediators of EMT. Some mesodermal lineages fail to form in Wt1 null embryoid bodies but this effect is rescued by the expression of Snai1, underlining the importance of EMT in generating these differentiated cells. These new insights into the molecular mechanisms regulating cardiovascular progenitor cells and EMT will shed light on the pathogenesis of heart diseases and may help the development of cell based therapies.
Wt1 regulates the epithelial-mesenchymal transition (EMT) in the epicardium and the reverse process (MET) in kidney mesenchyme. The mechanisms underlying these reciprocal functions are unknown. Here, we show in both embryos and cultured cells that Wt1 regulates Wnt4 expression dichotomously. In kidney cells, Wt1 recruits Cbp and p300 as coactivators; in epicardial cells it enlists Basp1 as a corepressor. Surprisingly, in both tissues, Wt1 loss reciprocally switches the chromatin architecture of the entire Ctcf-bounded Wnt4 locus, but not the flanking regions; we term this mode of action "chromatin flip-flop." Ctcf and cohesin are dispensable for Wt1-mediated chromatin flip-flop but essential for maintaining the insulating boundaries. This work demonstrates that a developmental regulator coordinates chromatin boundaries with the transcriptional competence of the flanked region. These findings also have implications for hierarchical transcriptional regulation in development and disease.
SUMMARYEpicardial-derived signals are key regulators of cardiac embryonic development. An important part of these signals is known to relate to a retinoic acid (RA) receptor-dependent mechanism. RA is a potent morphogen synthesised by Raldh enzymes, Raldh2 being the predominant one in mesodermal tissues. Despite the importance of epicardial retinoid signalling in the heart, the molecular mechanisms controlling cardiac Raldh2 transcription remain unknown. In the current study, we show that Wt1-null epicardial cells display decreased expression of Raldh2 both in vivo and in vitro. Using a RA-responsive reporter, we have confirmed that Wt1-null epicardial cells actually show reduced synthesis of RA. We also demonstrate that Raldh2 is a direct transcriptional target of Wt1 in epicardial cells. A secondary objective of this study was to identify the status of RA-related receptors previously reported to be critical to epicardial biology (PDGFR,; RXR). PDGFR and PDGFR mRNA and protein levels are downregulated in the absence of Wt1, but only Pdgfra expression is rescued by the addition of RA to Wt1-null epicardial cells. RXR mRNA levels are not affected in Wt1-null epicardial cells. Taken together, our results indicate that Wt1 critically regulates epicardial RA signalling via direct activation of the Raldh2 gene, and identify a role for Wt1 in the regulation of morphogen receptors involved in the proliferation, migration, and differentiation of epicardial and epicardially-derived cells (EPDC). DEVELOPMENT 1094as previously described (Pérez-Pomares et al., 2002) using anti-PDGFR, anti-PDGFR and anti-RXR antibodies (eBioscience 14-1401-81; Sc-432; Sc-553, respectively). A non-specific rabbit IgG was used instead of the primary antibodies for negative controls. Immortalised epicardial cell lines (MEEC)Hearts from control (Wt1 GFP/+ ) and Wt1-null mice (Wt1 GFP/-) carrying the 'immorto' transgene (Jat et al., 1991) were isolated at E11.5 and cultured on 24-well gelatinised dishes containing DMEM. After 24 hours, hearts were removed and epicardial cells attached to the surface were cultured and propagated as described previously Martínez-Estrada et al. (Martínez-Estrada et al., 2010). Treatments with retinoic acid (all-trans, Sigma) were performed at 1 M concentration (24 hours). Control for the treatments was incubation with the RA vehicle (ethanol 100°, 1:1000). Western blottingWestern blots were performed as described previously (Martínez-Estrada et al., 2010). Luciferase and RA reporter assaysThe Raldh2 upstream region (830 bp) was amplified by PCR from mouse genomic DNA and cloned into the pGL4.10 vector (Promega). Primer sequences were: forward, 5Ј-AAAGCTAGCTCTCCCACGCT ATCTGG -AGT-3Ј; reverse, 5Ј-AAAAGATCTGATCTCGCTGGAAG TCATGG-3Ј. PCR product was digested with NheI and BglII and cloned into the corresponding sites upstream of the firefly luciferase construct in the pGL4.10 vector.This reporter construct (100 ng) was transfected in immortalised epicardial cells, in the presence of the indicated amounts of ...
The epicardium plays a crucial role in embryonic heart development and adult heart repair; however, the molecular events underlying its maturation remain unknown. Wt1, one of the main markers of the embryonic epicardium, is essential for epicardial development and function. Here, we analyse the transcriptomic profile of epicardialenriched cells at different stages of development and from control and epicardial-specific Wt1 knockout (Wt1KO) mice. Transcriptomic and cell morphology analyses of epicardial cells from epicardial-specific Wt1KO mice revealed a defect in the maturation process of the mutant epicardium, including sustained upregulation of Bmp4 expression and the inability of mutant epicardial cells to transition into a mature squamous phenotype. We identified Bmp4 as a transcriptional target of Wt1, thus providing a molecular basis for the retention of the cuboidal cell shape observed in the Wt1KO epicardium. Accordingly, inhibition of the Bmp4 signalling pathway both ex vivo and in vivo rescued the cuboidal phenotype of the mutant epicardium. Our findings indicate the importance of the cuboidal-to-squamous transition in epicardial maturation, a process regulated by Wt1.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.