Disruption of the mouse rbp-j gene alters differentiation of cell lineages derived from mesoderm

Notch proteins are transmembrane receptors which influence differentiation, proliferation and apoptosis in many developmental systems. Four different mammalian Notch receptors have been described. After ligand binding and activation, the Notch intracellular domain (NIC) is released from the cytoplasmic membrane and translocates into the nucleus to act as a transcription factor. NIC binds to DNA via the adapter protein RBP-J (also termed CBF-1) which is an essential component for the signalling of all 4 Notch receptors. Binding of NIC converts the transcriptional repressor RBP-J into a transcriptional activator. RBP-J−/− mice die early in development from multiple defects. To investigate the role of RBP-J in the development of mesoderm-derived cell lineages, we analysed the differentiation of RBP-J−/− embryonic stem cells (ES) using the OP9 stromal co-culture system. RBP-J−/− ES differentiated normally into mesodermal Flk-1(VEGF-R2)+ cells but further differentiation of these cells was drastically altered. RBP-J−/− ES generated 60 times more cardiac muscle colonies and nearly twice as many endothelial colonies as RBP-J−/+ or RBP-J+/+ ES. RBP-J−/− cells generated c-Kit+ Sca-1+ AA4.1+ hematopoietic stem sells and mature erythroid, myeloid, B-lymphoid cells in vitro. Furthermore, transplantation experiments revealed that RBP-J−/− ES can also give rise to B cells in vivo. Our data demonstrate that the RBP-J signalling pathway is an important regulator of myogenic and endothelial differentiation and/or proliferation. Moreover, we show that RBP-J is not required for the generation of erythroid, myeloid and B lymphoid cells from embryonic stem cells.