image: (A) Immunohistochemistry of Dlc1 in E12.5 mouse embryos. AVC, atrio-ventricular canal; A, atrium; V, ventricle. Scale bar, 500/100 μm. (B) Schematic illustration of the in vitro hanging drop differentiation process. (C–E) Real-time PCR analysis of common Dlc1 and two transcript (NM_015802 and NM_001194940.2) expression during mESC differentiation. n = 3. Data were shown as mean ± standard deviation; **P < 0.01 and ***P < 0.001.
Credit: Wei Wei, Xuechao Jiang, Wenxiang Shi, Junmei Zhou, Rang Xu
A recent study published in Genes & Diseases by researchers from Shanghai Jiao Tong University School of Medicine investigated the role of Dlc1 in cardiac progenitor differentiation by constructing a Dlc1 knockout mESC (mouse embryonic stem cell) monoclonal cell line using CRISPR/Cas9 to mimic cardiomyocyte differentiation.
Initial analysis revealed that Dlc1 was mainly expressed in the myocardium and endocardium, and its expression was down-regulated during the specification of the gastrulating mesoderm, and up-regulated during the differentiation of cardiac progenitors, suggesting that Dlc1 was involved in the differentiation of cardiac progenitor cells.
Using the CRISPR/Cas9 gene-editing technique, the authors constructed three different Dlc1-knockout Nkx2.5-emGFP mESC cell lines that label cardiac progenitors and selected cell line 2 for further investigations. Additional experiments and RNA sequencing revealed that Dlc1 deficiency: i) promotes the differentiation and accumulation of cardiac progenitors, affecting subsequent cardiomyocyte lineage specification, and ii) promotes mesodermal or endodermal cell specification, causing the accumulation of cardiac progenitors but perturbs their further differentiation into cardiomyocytes.
Previous studies have shown that the DLC1 protein regulates multiple development-related signaling pathways, EGFR/Akt/NF-κB, Wnt/β-catenin, and RhoA/ROCK, of which WNT signaling is critical during mesoderm formation and late cardiac progenitor specification — processes essential for myocardial cell differentiation.
Bioinformatics analyses and rescue experiments using the Wnt pathway inhibitor XAV-939 showed that Dlc1 knockdown promotes mesoderm specification by activating canonical Wnt signaling, thereby enhancing cardiac progenitor differentiation. Mechanistically, Dlc1 knockdown activates the canonical Wnt pathway by reducing β-catenin degradation and promoting its nuclear translocation.
Collectively, the results of this study show that Dlc1 expression changes dynamically during mESC differentiation into cardiomyocytes. Furthermore, Dlc1 deficiency promotes mesoderm lineage specification by activating canonical Wnt signaling, leading to the accumulation of cardiac progenitors and blocking their further differentiation into cardiomyocytes, thus identifying a new candidate gene in cardiomyocyte differentiation.
Journal
Genes & Diseases