1117 Introduction Hematopoietic cells differentiate from mesoderm during embryogenesis, in close association with endothelial cells. Definitive hematopoietic progenitors and stem cells originate in distinct sites in the embryo, including the yolk sac (YS) (Yoder et al., 1997), the umbilical and vitelline arteries (de Bruijn et al., 2000), the para-aortic splanchnopleura (P-Sp) (Cumano et al., 2001) and the aorta/genital ridge/mesonephros (AGM) region (Medvinsky and Dzierzak, 1996). The first hematopoietic cells detected during mouse embryonic development are the primitive erythroid cells of the YS at embryonic day (E) 7. One day later, before circulation between the embryo and YS is established, multipotent hematopoietic stem cells (HSCs) have been isolated from the intra-embryonic P-Sp (Cumano et al., 2001) indicating that intra-embryonic hematopoietic cells can originate independently of the YS. In the mouse, the P-Sp forms from the splanchnic mesoderm (the endoderm-associated mesoderm) and the whole region develops into aorta, gonads and mesonephros and is subsequently called AGM. Around E10-11, the HSC activity is autonomously generated in this region (reviewed by Ling and Dzierzak, 2002). The developmental origin and the genetic program of embryonic HSC emergence in the YS and the P-Sp/AGM in some aspects are divergent. Yolk sac blood cells originate simultaneously with the surrounding endothelial cells, consistent with the idea of developing from a common progenitor or hemangioblast (Palis and Yoder, 2001). By contrast, P-Sp/AGM hematopoietic cells emerge in close association to the presumably differentiated aortic endothelium. The lineage relationships and molecular events leading to their differentiation are not completely understood. Immunohistochemical analyses of the AGM region reveal overlapping expression of hematopoietic and endothelial markers in the clusters of cells that emerge from the ventral wall of the aorta. However, Aml1/Cbfa2 (Runx1 – Mouse Genome Informatics) transcription factor has been shown specifically to be involved in the development of intra- embryonic hematopoiesis without affecting the main vasculature (North et al., 1999). The analysis of recently developed transgenic mice, which enable specific labeling of emerging HSCs, provides supportive evidence that true HSCs originate among the cells residing in the endothelial layer (Ma et al., 2002). Besides Aml1 (North et al., 2002), Gata2 (Tsai et al., 1994; Tsai and Orkin, 1997) and Scl (Tal1 – Mouse Genome Informatics) (Porcher et al., 1996; Robb et al., 1996) are also expressed in hematopoietic clusters and endothelial- like cells lining the ventral wall of the dorsal aorta at E10-11 and there is now strong evidence that all these transcription Definitive hematopoiesis in the mouse embryo originates from the aortic floor in the P-Sp/AGM region in close association with endothelial cells. An important role for Notch1 in the control of hematopoietic ontogeny has been recently established, although its mechanism of action is poorly understood. Here, we show detailed analysis of Notch family gene expression in the aorta endothelium between embryonic day (E) 9.5 and E10.5. Since Notch requires binding to RBPjκ transcription factor to activate transcription, we analyzed the aorta of the para-aortic splanchnopleura/AGM in RBPjκ mutant embryos. We found specific patterns of expression of Notch receptors, ligands and Hes genes that were lost in RBPjκ mutants. Analysis of these mutants revealed the absence of hematopoietic progenitors, accompanied by the lack of expression of the hematopoietic transcription factors Aml1/Runx1, Gata2 and Scl/Tal1. We show that in wild-type embryos, a few cells lining the aorta endothelium at E9.5 simultaneously expressed Notch1 and Gata2, and demonstrate by chromatin immunoprecipitation that Notch1 specifically associated with the Gata2 promoter in E9.5 wild-type embryos and 32D myeloid cells, an interaction lost in RBPjκ mutants. Consistent with a role for Notch1 in regulating Gata2, we observe increased expression of this gene in 32D cells expressing activated Notch1. Taken together, these data strongly suggest that activation of Gata2 expression by Notch1/RBPjκ is a crucial event for the onset of definitive hematopoiesis in the embryo. Key words: Notch, Mouse, Rbpsuh Summary RBPjκ-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells Àlex Robert-Moreno 1 , Lluís Espinosa 1 , José Luis de la Pompa 2 and Anna Bigas 1, * 1 Centre Oncologia Molecular, IDIBELL-Institut de Recerca Oncologica, Hospitalet, Barcelona 08907, Spain 2 Department of Immunology and Oncology, Centro Nacional de Biotecnología, CSIC. Darwin, 3. Campus de Cantoblanco, Madrid 28049, Spain *Author for correspondence (e-mail: abigas@iro.es) Accepted 22 December 2004 Development 132, 1117-1126 Published by The Company of Biologists 2005 doi:10.1242/dev.01660 Research article Development and disease Development