Summary Hematopoietic stem cells (HSCs) are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors are implicated in regulating mouse HSC development, but which cells in the aorta-gonad-mesonephros (AGM) microenvironment produce these factors is unknown. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation were involved in the AGM HSC-generative microenvironment. CyTOF analysis of CD45 + AGM cells revealed predominance of two hematopoietic cell types, mannose-receptor positive macrophages and mannose-receptor negative myeloid cells. We show here that macrophage appearance in the AGM was dependent on the chemokine receptor Cx3cr1. These macrophages expressed a pro-inflammatory signature, localized to the aorta, and dynamically interacted with nascent and emerging intra-aortic hematopoietic cells (IAHCs). Importantly, upon macrophage depletion, no adult-repopulating HSCs were detected, thus implicating a role for pro-inflammatory AGM-associated macrophages in regulating the development of HSCs.

Translational regulation by oncogenic proteins may be a rapid and efficient mechanism to modulate gene expression. We report here the identification of the CEBPB gene as a target of translational regulation in myeloid precursor cells transformed by the BCR/ABL oncogene. Expression of CEBPB was repressed in 32D-BCR/ABL cells and reinduced by imatinib (STI571) via a mechanism that appears to depend on expression of the CUG-repeat RNA-binding protein CUGBP1 and the integrity of the CUG-rich intercistronic region of c/ebp␤ mRNA. Constitutive expression or conditional activation of wildtype CEBPB induced differentiation and inhibited proliferation of 32D-BCR/ABL cells in vitro and in mice, but a DNA binding-deficient CEBPB mutant had no effect. The proliferation-inhibitory effect of CEBPB was, in part, mediated by the CEBPB-induced GADD45A gene. Because IntroductionChronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder arising from neoplastic transformation of the hematopoietic stem cell. 1,2 Its clinical course involves progression from a protracted chronic phase (CML-CP), characterized by accumulation of apparently normal neutrophils, to a rapidly fatal blast crisis (CML-BC), characterized by clonal expansion of differentiationarrested myeloid or lymphoid blasts. CML is consistently associated with a reciprocal translocation of the long arms of chromosomes 9 and 22, 3,4 which generates the BCR/ABL fusion gene, in turn translated in the p210 BCR/ABL oncoprotein in almost all patients. 5 Expression activity of p210 BCR/ABL is necessary and sufficient for hematopoietic cell transformation and disease maintenance as demonstrated by in vitro assays, leukemogenesis in mice, and the antileukemia effects of imatinib, a specific BCR/ABL tyrosine kinase inhibitor. [6][7][8][9] BCR/ABL-dependent transformation of hematopoietic cells involves the assembly of multiprotein complexes and the phosphorylation of various substrates, which is essential to generate proliferative and antiapoptotic signals [10][11][12] and is often accompanied by transcriptional and posttranscriptional changes in gene expression. In regard to the latter mechanism, BCR/ABL can regulate both positively and negatively mRNA translation and protein stability. [13][14][15] We recently identified translation-regulatory mechanisms involving increased expression of RNA-binding proteins that modulate MDM2 and CEBPA levels in BCR/ABL-expressing cells. 16,17 Enhanced expression of MDM2 by increased expression of the RNA-binding protein La reduces the susceptibility of BCR/ABLexpressing cells to apoptosis induced by DNA-damaging agents. 16 Suppression of CEBPA expression by increased expression of the RNA-binding protein hnRNPE2 is important for the differentiation arrest of BCR/ABL-expressing leukemic cells as indicated by the rapid induction of differentiation on reactivation of CEBPA expression or activity. 17,18 Because translational regulation by BCR/ABL might not be limited to MDM2 and C/EBPA, we sought to identify other translation-regula...

Whereas hundreds of cells in the mouse embryonic aorta transdifferentiate to hematopoietic cells, only very few establish hematopoietic stem cell (HSC) identity at a single time point. The Gata2 transcription factor is essential for HSC generation and function. In contrast to surface-marker-based cell isolation, Gata2-based enrichment provides a direct link to the internal HSC regulatory network. Here, we use iterations of indexsorting of Gata2-expressing intra-aortic hematopoietic cluster (IAHC) cells, single-cell transcriptomics, and functional analyses to connect HSC identity to specific gene expression. Gata2-expressing IAHC cells separate into 5 major transcriptomic clusters. Iterative analyses reveal refined CD31, cKit, and CD27 phenotypic parameters that associate specific molecular profiles in one cluster with distinct HSC and multipotent progenitor function. Thus, by iterations of single-cell approaches, we identify the transcriptome of the first functional HSCs as they emerge in the mouse embryo and localize them to aortic clusters containing 1-2 cells.