Disabled-2 is required for mesoderm differentiation of murine embryonic stem cells.

A variety of signaling networks are implicated in the control of mesoderm differentiation. Previous studies demonstrated that Disabled-2 (DAB2) is a multifunctional protein involved in growth factor signaling and embryonic development. In this study, we investigated DAB2 expression and function during in vitro mesoderm differentiation of murine embryonic stem cells (ESCs). We found that DAB2 was up-regulated when ESCs were co-cultured with OP9 stromal cells for mesoderm differentiation. DAB2 was also up-regulated when ESCs were induced for embryoid body formation. Expression of DAB2 short hairpin small interfering RNA (shDAB2) did not alter the puripotency of ESCs. However, shDAB2 disrupted ESCs cell-cell adhesion and affected embryoid body and colony formation that subsequently impeded mesoderm differentiation of ESCs. Immunofluorescent staining revealed that disorganization of beta-catenin and plakoglobin cellular distribution may account for the aberrant cell-cell adhesion in DAB2-deficient cells. Accordingly, DAB2 was identified as a plakoglobin-binding partner with the interaction mediated by the phosphotyrosine binding domain of DAB2 and the Asn-Pro-Asp-Tyr (NPDY) motif of plakoglobin. Molecular analysis and transcriptome profiling also revealed that DAB2 was involved in the regulation of insulin-like growth factor 2-mediated signaling and in the expression of p53, asparagine synthetase and glutathione peroxidase 2. Expression screening of 52 ESCs-related miRNAs further unveiled the interplay between DAB2 and the signaling networks associated with cell death, differentiation and development. This study thereby defines a role of DAB2 in fate determination of ESCs and suggests the presence of a DAB2-associated regulatory circuit in the control of mesoderm differentiation.