SUMMARYHematopoietic stem cells (HSCs), which are defined by their capacity to reconstitute adult conventional mice, are first found in the dorsal aorta after 10.5 days post coitus (dpc) and in the fetal liver at 11 dpc. However, lympho-myeloid hematopoietic progenitors are detected in the dorsal aorta from 9 dpc, raising the issue of their role in establishing adult hematopoiesis. Here, we show that these progenitors are endowed with long-term reconstitution capacity, but only engraft natural killer (NK)-deficient Rag2c -/-mice. This novel population, called here immature HSCs, evolves in culture with thrombopoietin and stromal cells, into HSCs, defined by acquisition of CD45 and MHC-1 expression and by the capacity to reconstitute NK-competent mice. This evolution occurs during ontogeny, as early colonization of fetal liver by immature HSCs precedes that of HSCs. Moreover, organ culture experiments show that immature HSCs acquire, in this environment, the features of HSCs.

No abstract

Although the expression of Pitx2, a bicoid family homeodomain transcription factor, is highly regulated during hematopoiesis, its function during this process was not documented; we thus studied hematopoiesis in Pitx2-null mice. We found that Pitx2 ؊/؊ embryos display hypoplastic livers with reduced numbers of hematopoietic cells, but these cells had normal hematopoietic potential, as evidenced by colony-forming assays, immature progenitor cell assays, and long-term repopulation assays. Because the microenvironment is also crucial to the development of normal hematopoiesis, we established Pitx2 ؊/؊ and Pitx2 ؉/؉ stromas from fetal liver and studied their hematopoietic supportive capacity. We showed that the frequency of cobblestone area-forming cells was 4-fold decreased when using Pitx2 ؊/؊ stromal cells compared with Pitx2 ؉/؉ stromal cells, whatever the Pitx2 genotype of hematopoietic cells tested in this assay. This defect was rescued by expression of Pitx2 into Pitx2 ؊/؊ fetal liver stromal cells, demonstrating a major and direct role of Pitx2 in the hematopoietic supportive capacity of fetal liver stroma. Finally, we showed a reduced capacity of MS5 stromal cells expressing Pitx2 RNAi to support human hematopoiesis. Altogether these data showed that Pitx2 has major functions in the hematopoietic supportive capacity of fetal liver and adult bone marrow stromal cells. IntroductionPitx2, a bicoid-type homeobox protein, plays an important role in the development of multiple organs. 1 Pitx2 is expressed in many tissues during development, including derivatives of the first branchial arch, the eyes, the brain, the pituitary gland, the mandible, the heart, the lungs, and the limbs. [1][2][3][4][5][6][7][8][9] The Pitx2 gene is expressed as 3 mRNA isoforms derived by either alternative splicing, for Pitx2a and Pitx2b, or from an internal promoter located upstream exon 4 for Pitx2c ( Figure 1A). Exons 5 and 6, which encode the homeodomain and the COOH terminus of the protein, are common to all isoforms. Among these 3 isoforms, Pitx2c shows an asymmetrical pattern of expression during development on the left side of the embryo at the lateral plate mesoderm stage as well as later during development in the heart, gut, and lung. In contrast, all 3 isoforms are expressed symmetrically in the head mesoderm and the lateral body wall. Therefore, only the c isoform of Pitx2 was found to be involved in the determination of left-right asymmetry during development. [10][11][12][13][14][15][16] Mutations in the homeodomain of Pitx2 are one cause of Rieger syndrome in humans, an autosomal-dominant disorder. 3,17,18 It is characterized by craniofacial and umbilical stump abnormalities.The phenotype of mice heterozygous for a null Pitx2 allele mimics human Rieger syndrome and is characterized by eye and tooth abnormalities. 4 Homozygous null embryos do not survive beyond embryonic day 14.5 after coitus due to severe cardiac and other developmental defects. Organogenesis of the eyes, teeth, and pituitary gland is also severely imp...