B-1 lymphoid cells develop independently of Notch signaling during mouse embryonic development.

The in vitro generation from pluripotent stem cells (PSCs) of different blood cell types, in particular those that are not replenished by hematopoietic stem cells (HSCs) like fetal-derived tissue-resident macrophages and innate-like lymphocytes, is of a particular interest. In order to succeed in this endeavor, a thorough understanding of the pathway interplay promoting lineage specification for the different blood cell types is needed. Notch signaling is essential for the HSC generation and their derivatives, but its requirement for tissue-resident immune cells is unknown. Using mouse embryonic stem cells (mESCs) to recapitulate murine embryonic development, we have studied the requirement for Notch signaling during the earliest B-lymphopoiesis and found that Rbpj-deficient mESCs are able to generate B-1 cells. Their Notch-independence was confirmed in ex vivo experiments using Rbpj-deficient embryos. In addition, we found that upregulation of Notch signaling was needed for the emergence of B-2 lymphoid cells. Taken together, these findings indicate that control of Notch signaling dosage is critical for the different B-cell lineage specification and provides pivotal information for their in vitro generation from PSCs for therapeutic applications.