The first adult-repopulating hematopoietic stem cells (HSCs) are detected starting at day 10.5 of gestation in the aorta-gonads-mesonephros (AGM) region of the mouse embryo. Despite the importance of the AGM in initiating HSC production, very little is currently known about the regulators that control HSC emergence in this region. We have therefore further defined the location of HSCs in the AGM and incorporated this information into a spatial and temporal comparative gene expression analysis of the AGM. The comparisons included gene expression profiling (1) in the newly identified HSC-containing region compared with the region devoid of HSCs, (2) before and after HSC emergence in the AGM microenvironment, and (3) on populations enriched for HSCs and their putative precursors. Two genes found to be up-regulated at the time and place where HSCs are first detected, the cyclin-dependent kinase inhibitor p57Kip2/Cdkn1c and the insulinlike growth factor 2, were chosen for further analysis. We demonstrate here that they play a novel role in AGM hematopoiesis. Interestingly, many genes involved in the development of the tissues surrounding the dorsal aorta are also upregulated during HSC emergence, suggesting that the regulation of HSC generation occurs in coordination with the development of other organs. (Blood. 2009;114:4645-4653) IntroductionHematopoietic stem cells (HSCs) are at the center of the hematopoietic system. Their proliferative and multilineage differentiation potential endows them with the capacity to regenerate every blood cell type throughout the entire life of a person. For this reason, they are subject to tight regulatory processes to ensure an adequate supply of blood cells without the risk of HSC depletion or the development of blood-related malignancies.Insight into the basic mechanisms of HSC regulation can be gained from the study of how these cells are first generated and expanded during development. The first cells that display adult HSC characteristics in transplantation assays are detected at embryonic day (E) 10.5 in a region of the embryo that includes the developing dorsal aorta, gonads, and mesonephros (AGM region). 1 Subdissections have localized this first HSC activity to the dorsal aorta and the associated vitelline and umbilical vessels. 2 Subsequently, adult HSC-type cells are also found in the yolk sac and the placenta. 1,3,4 It is currently unclear why HSCs are harbored in multiple sites during development and whether they have multiple origins. After E12, HSC numbers start declining in the AGM as the fetal liver (FL) becomes colonized and an adult-type hematopoietic system is further established (reviewed in Dzierzak and Speck 5 ).Little is known about how HSCs are first generated in the AGM. In particular, the identification of the direct precursors of HSCs, the definition of the components of the regulatory microenvironment, and the discovery of cell-intrinsic regulators are issues that have only recently started to be addressed. There is now good evidence to suggest that HSCs...

Key Points• Emerging HSCs require Jak2 and Pi3k signaling for proliferation and survival.• Embryonic HSCs are unaffected by the JAK2V617F mutation.The regulation of hematopoietic stem cell (HSC) emergence during development provides important information about the basic mechanisms of blood stem cell generation, expansion, and migration. We set out to investigate the role that cytokine signaling pathways play in these early processes and show here that the 2 cytokines interleukin 3 and thrombopoietin have the ability to expand hematopoietic stem and progenitor numbers by regulating their survival and proliferation. For this, they differentially use the Janus kinase (Jak2) and phosphatidylinositol 3-kinase (Pi3k) signaling pathways, with Jak2 mainly relaying the proproliferation signaling, whereas Pi3k mediates the survival signal. Furthermore, using Jak2-deficient embryos, we demonstrate that Jak2 is crucially required for the function of the first HSCs, whereas progenitors are less dependent on Jak2. The JAK2V617F mutation, which renders JAK2 constitutively active and has been linked to myeloproliferative neoplasms, was recently shown to compromise adult HSC function, negatively affecting their repopulation and self-renewal ability, partly through the accumulation of JAK2V617F-induced DNA damage. We report here that nascent HSCs are resistant to the JAK2V617F mutation and show no decrease in repopulation or selfrenewal and no increase in DNA damage, even in the presence of 2 mutant copies. More importantly, this unique property of embryonic HSCs is stably maintained through ‡1 round of successive transplantations. In summary, our dissection of cytokine signaling in embryonic HSCs has uncovered unique properties of these cells that are of clinical importance.