Identification of novel regulators of hematopoietic stem cell development through refinement of stem cell localization and expression profiling

Abstract

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...

Concise Review: From Greenhouse to Garden: The Changing Soil of the Hematopoietic Stem Cell Microenvironment During Development

“…Supporting the idea that these populations are also functionally dissimilar is our finding that AGM HSCs respond differently from E14.5 FL and adult BM HSCs to the deletion of the cyclin-dependent kinase inhibitor, p57Kip2 (Cdkn1c). Loss of p57Kip2 in both E14.5 FL and adult BM HSCs leads to reduced self-renewal and maintenance, respectively [22,23], whereas we have observed an expansion of HSCs in p57Kip2 null AGMs [24]. More recently, it has been shown that despite expression of both retinoic acid receptors (RARs) a and c on HSCs, the functionally predominant RAR transitions throughout development.…”

“…The niche is the convergence of metabolic, humoral, paracrine, neural, structural, and physical signals that act to regulate stem cells [42]. For HSCs, it is HSCs emerge/mature from pre-HSCs [3] No de novo generation, but rapid expansion of HSCs [18] Largely quiescent unless activated [90,91] Response to p57kip2 knockout Increased repopulation ability [24] Reduced repopulation in tertiary recipients [22] Conditional knockout results in reduced repopulation capacity [23] …”

Leucine-rich Repeat-containing G-protein-coupled Receptor 5 Marks Short-term Hematopoietic Stem and Progenitor Cells during Mouse Embryonic Development

“…In E11.5 AGMs, Lgr5, and Ki67 double positive cells were observed mainly within the intra-aortic clusters ( Fig. 2A), where HSCs emerged and matured (13,14). In E12.5 embryos, the main HSC expansion sites switched to the fetal liver, where proliferative Lgr5-GFP cells increased from 52% at E11.5 to 65.8% at E12.5 (Fig.…”

“…In mouse embryos, the generation of HSCs initiates in AGM on E10.5, and continues very rapidly through E12 (12)(13)(14)(15). A pool of HSCs is formed predominantly in fetal liver on E12 by recruiting definitive HSCs via the blood circulation from extra-hepatic sources (AGM region and/or placenta) (15,16).…”

Innate immunity as orchestrator of stem cell mobilization

“…1,2 The later appearance of definitive HSPCs in the aorta-gonad-mesonephros region raises the question whether cells identified in this region (i) arrive from the yolk sac at the time when the heart begins to beat and initiates blood flow, 3 (ii) are specified in the aortic wall from a local pool of stem cells as a response to blood-flow shearing forces 4 or (iii) are derived from a subpopulation of primitive mesodermal/germ line cells that migrate through the embryo proper on their way to colonize the genital ridges. 5 Later, at the second trimester of gestation, HSPCs accumulate in the fetal liver (FL), which becomes a major hematopoietic organ in the developing embryo. 6 Finally, at the beginning of the third trimester of gestation, HSPCs leave the FL and begin to colonize in the developing bone marrow (BM).…”