Embryonic Sources of Definitive Hemopoietic Stem Cells

A review of one of the key problems of experimental hematology: the origin of hemopoietic stem cells in the development of vertebrates (amphibians, birds, and mammals). The appearance and functioning of two independent sources of hemopoietic stem cells (extra- and intraembryonic) were considered in amphibians, birds, and mammals. The contribution of each source to the formation of definitive hemopoietic tissue was analyzed. It was shown for amphibians and birds that intraembryonic organs such as the dorsolateral plate and the mesenchyme of dorsal aorta are involved in the formation of adult hemopoietic tissue, while the extraembryonic organs such as ventral islets and the yolk sac are devoid of true stem cells and provide only for the primary, transient hemopoiesis. New data have been considered concerning the previously unknown intraembryonic hemopoietic organ in mammals, a region of aorta–gonad–mesonephros arising in embryogenesis simultaneously with the yolk sac. Two extreme views on the involvement of stem cells of all these organs in the formation of definitive hemopoiesis have been considered. The data are provided on the interaction of the embryonic hemopoietic stem cells and the hemopoietic microenvironment of adult recipients.     

The stepwise specification of embryonic stem cells to hematopoietic fate is driven by sequential exposure to Bmp4, activin A, bFGF and VEGF

The differentiation of embryonic stem (ES) cells offers a powerful approach to study mechanisms implicated in cell fate decision. A major hurdle, however, is to promote the directed and efficient differentiation of ES cells toward a specific lineage. Here, we define in serum-free media the minimal factor requirement controlling each step of the differentiation process, resulting in the production of highly enriched hematopoietic progenitors. Four factors - Bmp4, activin A, bFGF (Fgf2) and VEGF (VegfA) - are sufficient to drive the selective and efficient differentiation of mouse ES cells to hematopoiesis. Each of these factors appears to regulate a step of the process: Bmp4 promotes the very efficient formation of mesoderm; bFGF and activin A induce the differentiation of these mesodermal precursors to the hemangioblast fate; and VEGF is required for the production of fully committed hematopoietic progenitors. The stimulation of mesodermal precursors by bFGF and activin A switches on very rapidly the hematopoietic program, allowing us to dissect the molecular events leading to the formation of the hemangioblast. Runx1, Scl (Tal1) and Hhex expression is upregulated within 3 hours of stimulation, whereas upregulation of Lmo2 and Fli1 is observed later. Interestingly, increased expression levels of genes such as cMyb, Pu.1 (Sfpi1), Gata1 and Gata2 are not observed at the onset of hemangioblast commitment. This stepwise control of differentiation is extremely efficient, giving rise to a very high frequency of hematopoietic precursors, and provides an optimal system for understanding the molecular machineries involved in blood progenitor commitment.     

Specification and maintenance of the spinal cord stem zone

Epiblast cells adjacent to the regressing primitive streak behave as a stem zone that progressively generates the entire spinal cord and also contributes to paraxial mesoderm. Despite this fundamental task, this cell population is poorly characterised, and the tissue interactions and signalling pathways that specify this unique region are unknown. Fibroblast growth factor (FGF) is implicated but it is unclear whether it is sufficient and/or directly required for stem zone specification. It is also not understood how establishment of the stem zone relates to the acquisition of spinal cord identity as indicated by expression of caudal Hox genes. Here, we show that many cells in the chick stem zone express both early neural and mesodermal genes; however, stem zone-specific gene expression can be induced by signals from underlying paraxial mesoderm without concomitant induction of an ambivalent neural/mesodermal cell state. The stem zone is a site of FGF/MAPK signalling and we show that although FGF alone does not mimic paraxial mesoderm signals, it is directly required in epiblast cells for stem zone specification and maintenance. We further demonstrate that caudal Hox gene expression in the stem zone also depends on FGF and that neither stem zone specification nor caudal Hox gene onset requires retinoid signalling. These findings thus support a two step model for spinal cord generation - FGF-dependent establishment of the stem zone in which progressively more caudal Hox genes are expressed, followed by the retinoid-dependent assignment of spinal cord identity.     

Definitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrafish embryo

Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed primitive and definitive. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass and a distinct population of macrophages that arises from cephalic mesoderm. Based on previous gene expression studies, definitive hematopoiesis has been suggested to begin with the generation of presumptive hematopoietic stem cells (HSCs) along the dorsal aorta that express c-myb and runx1. Here we show, using a combination of gene expression analyses, prospective isolation approaches, transplantation, and in vivo lineage-tracing experiments, that definitive hematopoiesis initiates through committed erythromyeloid progenitors (EMPs) in the posterior blood island (PBI) that arise independently of HSCs. EMPs isolated by coexpression of fluorescent transgenes driven by the lmo2 and gata1 promoters exhibit an immature, blastic morphology and express only erythroid and myeloid genes. Transplanted EMPs home to the PBI, show limited proliferative potential, and do not seed subsequent hematopoietic sites such as the thymus or pronephros. In vivo fate-mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. Additional fate mapping of mesodermal derivatives in mid-somitogenesis embryos suggests that EMPs are born directly in the PBI. These studies provide phenotypic and functional analyses of the first hematopoietic progenitors in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.     

Null mutation of the Lmo4 gene or a combined null mutation of the Lmo1/Lmo3 genes causes perinatal lethality, and Lmo4 controls neural tube development in mice

The LIM-only family of proteins comprises four members; two of these (LMO1 and LMO2) are involved in human T-cell leukemia via chromosomal translocations, and LMO2 is a master regulator of hematopoiesis. We have carried out gene targeting of the other members of the LIM-only family, viz., genes Lmo1, Lmo3 and Lmo4, to investigate their role in mouse development. None of these genes has an obligatory role in lymphopoiesis. In addition, while null mutations of Lmo1 or Lmo3 have no discernible phenotype, null mutation of Lmo4 alone causes perinatal lethality due to a severe neural tube defect which occurs in the form of anencephaly or exencephaly. Since the Lmo1 and Lmo3 gene sequences are highly related and have partly overlapping expression domains, we assessed the effect of compound Lmo1/Lmo3 null mutations. Although no anatomical defects were apparent in compound null pups, these animals also die within 24 h of birth, suggesting that a compensation between the related Lmo1 and 3 proteins can occur during embryogenesis to negate the individual loss of these genes. Our results complete the gene targeting of the LIM-only family in mice and suggest that all four members of this family are important in regulators of distinct developmental pathways.     

Chemical and immunological characterization of proteoglycans of embryonic chick calvaria

We have previously demonstrated in quail embryos grafted on chick yolk sacs the existence of intraembryonic stem cells responsible for definitive hemopoiesis. In order to determine the origin of these cells, we now examine the diffuse hemopoietic processes within the avian embryo's mesoderm. At 4–5 days of incubation in the two species, basophilic cells were found throughout the dorsal mesentery. At 6–8 days these cells became very numerous and built up dense foci at the level of branching of the anterior and posterior cardinal veins. These cells often infiltrated the wall of lymph spaces and channels and were also present in the lumen of blood vessels. Such locations support the interpretation that these basophilic cells represent early stages of hemopoietic differentiation. At 8–10 days, erythropoiesis or granulopoiesis was seen in the foci, which then regressed rapidly. The foci maximal development coincided with the period of colonization of the intraembryonic organ rudiments. In “yolk sac chimeras,” the foci were always constituted by quail cells, indicating their intraembryonic origin. The primordial origin of the intramesodermal cells remains to be determined. A likely source might be the ventral wall of the aorta which appeared to shed cells into the lumen and into the mesentery in the 3-day embryo.     

The origin of hematopoietic stem cells in the ontogenesis of vertebrates

A review of the origin of stem blood cells in ontogeny of vertebrates is presented. The comparative analysis of the data on laying, determination and migration of the hemopoietic precursor cells during embryogenesis in various taxonomic groups (teleosteans, urodeleans, anurans, avians and mammals) is performed. The change of the hemopoietic site and erythroid cells populations has been described. The data on sources of blood cell precursors and the origin of hemopoietic cells in the primordiums of hemopoietic organs were classified. A conclusion has been reached that in the course of evolution the hemopoietic anlage is gradually divided into two parts: one part migrates to the extraembryonic (ventral) mesoderm and another one remains intraembryonically and gives rice to the predecessors of definitive hemopoietic stem cells.