Hematopoietic stem cell (HSC) specification occurs in the embryonic aorta and requires Notch activation; however, most of the Notch-regulated elements controlling de novo HSC generation are still unknown. Here, we identify putative direct Notch targets in the aorta-gonad-mesonephros (AGM) embryonic tissue by chromatin precipitation using antibodies against the Notch partner RBPj. By ChIP-on-chip analysis of the precipitated DNA, we identified 701 promoter regions that were candidates to be regulated by Notch in the AGM. One of the most enriched regions corresponded to the Cdca7 gene, which was subsequently confirmed to recruit the RBPj factor but also Notch1 in AGM cells. We found that during embryonic hematopoietic development, expression of Cdca7 is restricted to the hematopoietic clusters of the aorta, and it is strongly up-regulated in the hemogenic population during human embryonic stem cell hematopoietic differentiation in a Notch-dependent manner. Down-regulation of Cdca7 mRNA in cultured AGM cells significantly induces hematopoietic differentiation and loss of the progenitor population. Finally, using loss-of-function experiments in zebrafish, we demonstrate that CDCA7 contributes to HSC emergence in vivo during embryonic development. Thus, our study identifies Cdca7 as an evolutionary conserved Notch target involved in HSC emergence.

J. Guiu was a recipient of Formación Personal Investigador (FPI) BES-2008-005708. This research was funded by grants from the Ministerio de Economía y Competitividad, FEDER (SAF2007-60080, PLE2009-0111, SAF2010-15450, and SAF2013-40922R), Red Temática de Investigación Cooperativa en Cáncer (RTICC; RD06/0020/0098 and RD12/0036/0054), Association for International Cancer Research (AICR; 13-0064), Agència de Gestió d’Ajuds Universitaris i de Recerca (AGAUR; 2009SGR-23, CONES2010-0006) to A. Bigas, the Spanish Association against Cancer Research (AECC) to A. Bigas and P. Menendez, Fondo de Investigación Sanitaria (FIS; PI10/00449) to P. Menendez, and ZonMW TOP (40-00812-98-11068) to E. Dzierzak and E. De Pater.