Dlc1 deletion activates the canonical Wnt pathway at an early stage and affects subsequent cardiac differentiation in mouse embryonic stem cells

Abstract

Cardiomyocyte differentiation is an important process during heart development that involves the specification of a series of cardiac lineage cells differentiated from mesendoderm to cardiac mesoderm, precisely regulated by multiple genes and signaling pathways. Our study aims to explore the role of the deleted in liver cancer 1 (DLC1) gene in cardiomyocyte differentiation and the molecular mechanisms underlying it. We constructed a Dlc1 knockout mouse embryonic stem cell (mESC) line using CRISPR/Cas9 and an mESC-cardiomyocyte differentiation model to investigate the role of the Dlc1 gene in the cardiac progenitor differentiation process. Flow cytometry, real-time PCR, teratoma formation assay, RNA sequencing, and other cellular and molecular biology assays were used to detect the cellular processes and differentiation ability of the knockout cell line at different stages. We found that the cardiac contractility of embryonic bodies was weakened by Dlc1 deletion at the late stage of differentiation. Moreover, Dlc1 deficiency promoted mesoderm specification and the subsequent differentiation and accumulation of cardiac progenitors, which may later impair cardiomyocyte generation. RNA sequencing analysis revealed that Dlc1 deficiency promoted mesoderm specification by activating canonical Wnt signaling, which subsequently enhanced cardiac progenitor differentiation. These results were confirmed by rescue experiments with the Wnt pathway inhibitor XAV-939. Furthermore, we found that the Dlc1 knockout activated canonical Wnt signaling by affecting the degradation and nuclear transportation of β-catenin. In conclusion, Dlc1 is involved in regulating embryonic stem cell cardiac differentiation by affecting canonical Wnt signaling at an early stage.