Associate Professor Rice University, United States
Introduction: Early in embryonic development, the primitive streak (PS) forms during gastrulation and further differentiates into endoderm and mesoderm subtypes. This process involves epithelial-mesenchymal transition (EMT), marked by cadherin switching, where E-Cadherin (E-Cad) is downregulated, and N-Cadherin is upregulated. While the induction routes from human embryonic stem cells (hESCs) to PS and mesoderm subtypes have been specified, the interplay among signaling pathways (ACTIVIN, BMP, and WNT), EMT, and cell fate commitment remains unclear. To address this gap, we investigated cadherin switching dynamics in mesoderm differentiation using hESCs as a model.
Materials and
Methods: hESCs were used to investigate the dynamics of cadherin switching during mesoderm differentiation. WNT, BMP, and Activin/Nodal signaling pathways were modulated during PS and mesodermal induction for observation of the effects on cadherin switching and cell fate outcomes through immunofluorescent staining and confocal microscopy. A dual-reporter hESC line with fluorescently tagged E-Cad and N-Cad at the endogenous loci, in addition to a membrane fluorescent marker, was generated to visualize and quantify cadherin dynamics in real time. Live-cell confocal imaging was employed to monitor E-Cad downregulation and N-Cad upregulation.
Results, Conclusions, and Discussions: Our results demonstrated that signaling pathways regulated cadherin switching independently from the fate outcome. Modulating WNT, BMP, or Activin/Nodal signaling during PS induction on day 1 had little effect on cell fates but significantly impacted cadherin switching. BMP inhibited cadherin switching, while ACTIVIN and WNT promoted it. Protocols generating anterior, mid, or posterior PS progenitors on day 1 efficiently induced paraxial or lateral mesoderm on day 2, but the degree of cadherin switching was influenced by the day 1 protocol. The plasticity of PS progenitors was dependent on E-Cad, as E-Cad knockout cells showed varied fate outcomes based on the day 1 protocol. Real-time analysis using the dual-reporter system revealed that E-Cad downregulation preceded N-Cad upregulation by approximately 4 hours. ACTIVIN was confirmed to promote EMT, with nuanced effects of BMP on EMT dynamics. These findings indicate that cadherin switching and cell fate decisions can be separately modulated, with cadherins preserving cellular plasticity during differentiation. The novel cadherin reporter system established in this study provides a powerful tool for quantitative, real-time analysis of EMT during development and cancer studies.
