The role of meis1 in primitive and definitive hematopoiesis during zebrafish development

Background

The Meis1 protein represents an important cofactor for Hox and Pbx1 and is implicated in human and murine leukemias. Though much is known about the role of meis1 in leukemogenesis, its function in normal hematopoiesis remains largely unclear. Here we characterized the role of the proto-oncogene, meis1, during zebrafish primitive and definitive hematopoiesis.

Design and Methods

Zebrafish embryos were stained with o-dianisidine to detect hemoglobin-containing cells and Sudan black to quantify neutrophils. The numbers of other cells (scl-, gata1- and alas2-positive cells) were also quantified by measuring the corresponding stained areas of the embryos. We used anti-Meis1 antibody and whole mount immunohistochemistry to determine the pattern of expression of Meis1 during zebrafish development and then analyzed the functional role of Meis1 by knocking-down the meis1 gene.

Results

Using antisense morpholino oligomers to interrupt meis1 expression we found that, although primitive macrophage development could occur unhampered, posterior erythroid differentiation required meis1, and its absence resulted in a severe decrease in the number of mature erythrocytes. Furthermore a picture emerged that meis1 exerts important effects on later stages of erythrocyte maturation and that these effects are independent of gata1, but under the control of scl. In addition, meis1 morpholino knock-down led to dramatic single arteriovenous tube formation. We also found that knock-down of pbx1 resulted in a phenotype that was strikingly similar to that of meis1 knock-down zebrafish.

Conclusions

These results imply that meis1, jointly with pbx1, regulates primitive hematopoiesis as well as vascular development.     

Crystal structure of HLA-G: a nonclassical MHC class I molecule expressed at the fetal-maternal interface

HLA-G is a nonclassical major histocompatibility complex class I (MHC-I) molecule that is primarily expressed at the fetal-maternal interface, where it is thought to play a role in protecting the fetus from the maternal immune response. HLA-G binds a limited repertoire of peptides and interacts with the inhibitory leukocyte Ig-like receptors LIR-1 and LIR-2 and possibly with certain natural killer cell receptors. To gain further insights into HLA-G function, we determined the 1.9-A structure of a monomeric HLA-G complexed to a natural endogenous peptide ligand from histone H2A (RIIPRHLQL). An extensive network of contacts between the peptide and the antigen-binding cleft reveal a constrained mode of binding reminiscent of the nonclassical HLA-E molecule, thereby providing a structural basis for the limited peptide repertoire of HLA-G. The alpha3 domain of HLA-G, a candidate binding site for the LIR-1 and -2 inhibitory receptors, is structurally distinct from the alpha3 domains of classical MHC-I molecules, providing a rationale for the observed affinity differences for these ligands. The structural data suggest a head-to-tail mode of dimerization, mediated by an intermolecular disulfide bond, that is consistent with the observation of HLA-G dimers on the cell surface.     

The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of definitive hematopoiesis

In the adult, platelets are derived from unipotential megakaryocyte colony-forming cells (Meg-CFCs) that arise from bipotential megakaryocyte/erythroid progenitors (MEPs). To better define the developmental origin of the megakaryocyte lineage, several aspects of megakaryopoiesis, including progenitors, maturing megakaryocytes, and circulating platelets, were examined in the murine embryo. We found that a majority of hemangioblast precursors during early gastrulation contains megakaryocyte potential. Combining progenitor assays with immunohistochemical analysis, we identified 2 waves of MEPs in the yolk sac associated with the primitive and definitive erythroid lineages. Primitive MEPs emerge at E7.25 along with megakaryocyte and primitive erythroid progenitors, indicating that primitive hematopoiesis is bilineage in nature. Subsequently, definitive MEPs expand in the yolk sac with Meg-CFCs and definitive erythroid progenitors. The first GP1bbeta-positive cells in the conceptus were identified in the yolk sac at E9.5, while large, highly reticulated platelets were detected in the embryonic bloodstream beginning at E10.5. At this time, the number of megakaryocyte progenitors begins to decline in the yolk sac and expand in the fetal liver. We conclude that the megakaryocyte lineage initially originates from hemangioblast precursors during early gastrulation and is closely associated both with primitive and with definitive erythroid lineages in the yolk sac prior to the transition of hematopoiesis to intraembryonic sites.     

Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis

A critical issue for clinical utilization of human ES cells (hESCs) is whether they can generate terminally mature progenies with normal function. We recently developed a method for efficient production of hematopoietic progenitors from hESCs by coculture with murine fetal liver-derived stromal cells. Large numbers of hESCs-derived erythroid progenitors generated by the coculture enabled us to analyze the development of erythropoiesis at a clone level and investigate their function. The results showed that the globin expression in the erythroid cells in individual clones changed in a time-dependent manner. In particular, embryonic ε-globin-expressing erythroid cells from individual clones decreased, whereas adult-type β-globin-expressing cells increased to ≈100% in all clones we examined, indicating that the cells undergo definitive hematopoiesis. Enucleated erythrocytes also appeared among the clonal progeny. A comparison analysis showed that hESC-derived erythroid cells took a similar differentiation pathway to human cord blood CD34⁺ progenitor-derived cells when examined for the expression of glycophorin A, CD71 and CD81. Furthermore, these hESC-derived erythroid cells could function as oxygen carriers and had a sufficient glucose-6-phosphate dehydrogenase activity. The present study should provide an experimental model for exploring early development of human erythropoiesis and hemoglobin switching and may help in the discovery of drugs for hereditary diseases in erythrocyte development.     

Beyond the increasing complexity of the immunomodulatory HLA-G molecule

Human leukocyte antigen G (HLA-G) is a nonclassic major histocompatibility complex (MHC) class I molecule that functions as an immunomodulatory molecule capable of protecting fetal tissues from the maternal immune system. The relevance of HLA-G in other contexts was investigated soon afterward. Numerous studies have sought (and some have shown) the relevance of HLA-G in pathologic conditions, such as transplantation, autoimmunity, and cancer and hematologic malignancies. One of the main goals of the current research on HLA-G is now to use it in the clinic, either for diagnosis or as a therapeutic tool/target. For this, precise knowledge on the nature and functions of HLA-G is critical. We highlight here what we consider are recent key basic findings on the immunomodulatory function of HLA-G. These strengthen the case for considering HLA-G as clinically relevant.     

CD98: a type II transmembrane glycoprotein expressed from the beginning of primitive and definitive hematopoiesis may play a critical role in the development of hematopoietic cells

In our search for cell surface markers expressed on hematopoietic stem cells and/or very early progenitor cells we found that the Joro 177 monoclonal antibody (MoAb) bound to most hematopoietic cells in day 8/8.5 yolk sac, day 12 fetal liver, and day 13 fetal thymocytes; it stained hematopoietic stem cells and less immature lymphoid, myeloid, and erythroid-lineage cells, but not most thymocytes and splenic lymphocytes in adult mice. Joro 177 MoAb stimulated tyrosine phosphorylation of an integral of 124-kD protein and induced homotypic aggregation of lymphoid progenitor cells. Importantly, Joro 177 MoAb inhibited cell survival/growth and consequently the generation of lymphoid, myeloid, and erythroid lineage cells in vitro from early Lin- hematopoietic precursors. Joro 177 MoAb induced apoptosis of hematopoietic progenitor cells. Molecular cloning and expression indicated that Joro 177 MoAb recognizes a type II transmembrane protein, which is the mouse homologue of the human CD98 heavy chain gene. We suggest that CD98 is a cell membrane receptor involved in the control of cell survival/death of hematopoietic cells.     

Negative regulation of primitive hematopoiesis by the FGF signaling pathway

Hematopoiesis is controlled by multiple signaling molecules during embryonic and postnatal development. The function of the fibroblast growth factor (FGF) pathway in this process is unclear. Here we show that FGF plays a key role in the regulation of primitive hematopoiesis in chicks. Using hemoglobin mRNA expression as a sensitive marker, we demonstrate that timing of blood differentiation can be separated from that of initial mesoderm patterning and subsequent migration. High FGF activity inhibits primitive blood differentiation and promotes endothelial cell fate. Conversely, inhibition of FGFR activity leads to ectopic blood formation and down-regulation of endothelial markers. Expression and functional analyses indicate that FGFR2 is the key receptor mediating these effects. The FGF pathway regulates primitive hematopoiesis by modulating Gata1 expression level and activity. We propose that the FGF pathway mediates repression of globin gene expression and that its removal is essential before terminal differentiation can occur.     

Both primitive and definitive blood cells are derived from Flk-1+ mesoderm

Emerging evidence suggests that all hematopoietic and endothelial cells originate from Flk-1(+) mesoderm in the mouse. However, this concept has not been completely proven, especially for the origin of blood cells. Using either Flk1(+/Cre);Rosa26R-EYFP or Flk1(+/Cre);Rosa26R-LacZ mice, we permanently marked Flk-1(+) cells and their progenies to determine the relationship between hematopoietic tissues and cells that express Flk-1. In embryos, all blood cells within the yolk sac and aorta were of Flk-1(+) origin. In addition, nearly all CD45(+) cells in bone marrow and circulating blood in adults were of Flk-1(+) origin. These results provide clear evidence that all blood cells, primitive and definitive, in mice are derived from Flk-1(+) mesodermal cells.     

Regulation of primitive hematopoiesis in zebrafish embryos by the death receptor gene

OBJECTIVE: We investigated the regulatory mechanism of primitive hematopoiesis in zebrafish (Danio rerio) embryos with particular reference to the role of a death receptor (zDR) gene, based on a morpholino (MO) knockdown approach. METHODS: MOs targeting the zDR and chordin (Chd) were injected into naturally spawned embryos at one- to four-cell stage. A random sequence (RS) MO was used as a control. Effects on hemoglobin formation (Hb), apoptosis, and lineage-specific gene expression were examined. Embryos injected with zDR, Chd, and RS-MOs were denoted zDR(mo), zChd(mo), and zRS(mo), respectively. Those co-injected with Chd+zDR-MOs and Chd+RS-MOs were abbreviated zChd+DR(mo) and zChd+RS(mo). RESULTS: zDR mRNA expression was restricted to the intermediate cell mass of wild-type (WT) and zChd(mo) embryos. At 48 hours postfertilization, zDR(mo) embryos showed increased Hb compared with WT or zRS(mo) embryos (2.36 x 10(-2) +/- 1.13 x 10(-3) vs 1.85 x 10(-2) +/- 5.60 x 10(-4) vs 1.79 x 10(-2) +/- 1.31 x 10(-3) U, p < 0.05). zChd+DR(mo) embryos also showed increased Hb compared with zChd(mo) or zChd+RS(mo) embryos (4.60 x 10(-2) +/- 2.79 x 10(-3) vs 3.17 x 10(-2) +/- 1.07 x 10(-3) vs 3.05 x 10(-2) +/- 1.25 x 10(-3) U, p < 0.05). zDR-MO reduced apoptosis, as shown by reduced terminal transferase-mediated dUTP nick end-labeling staining in zChd+DR(mo) compared with zChd+RS(mo) embryos and caspase-3 activity in zDR(mo) vs zRS(mo) (0.525 +/- 0.094 vs 0.953 +/- 0.113 U, p < 0.05), and zChd+DR(mo) vs zChd+RS(mo) embryos (0.247 +/- 0.121 vs 1.180 +/- 0.082, p < 0.05). zChd+DR(mo) embryos showed upregulation of erythroid-specific embryonic hemoglobin gene expression but not that of a myeloid-specific myeloperoxidase gene. CONCLUSION: Knockdown of zDR in zebrafish embryos decreased apoptosis and increased Hb, suggesting that zDR may regulate primitive hematopoiesis during development.     

alpha4-Integrin(+) endothelium derived from primate embryonic stem cells generates primitive and definitive hematopoietic cells

The mechanism of commencement of hematopoiesis in blood islands of the yolk sac and the aorta-gonad-mesonephros (AGM) region during primate embryogenesis remains elusive. In this study, we demonstrated that VE-cadherin(+)CD45(-) endothelial cells derived from nonhuman primate embryonic stem cells are able to generate primitive and definitive hematopoietic cells sequentially, as revealed by immunostaining of floating erythrocytes and colony-forming assay in cultures. Single bipotential progenitors for hematopoietic and endothelial lineages are included in this endothelial cell population. Furthermore, hemogenic activity of these endothelial cells is observed exclusively in the alpha4-integrin(+) subpopulation; bipotential progenitors are 4-fold enriched in this subpopulation. The kinetics of this hemogenic subpopulation is similar to that of hemogenic endothelial cells previously reported in the yolk sac and the AGM region in vivo in that they emerge for only a limited time. We suggest that VE-cadherin(+)CD45(-)alpha4-integrin(+) endothelial cells are involved in primitive and definitive hematopoiesis during primate embryogenesis, though VE-cadherin(-)CD45(-)alpha4-integrin(+) cells are the primary sources for primitive hematopoiesis.     

Mofarotene (Ro 40-8757) inhibits hematopoiesis in vitro by preventing maturation from primitive progenitor cells

The effect of the arotinoid mofarotene (Ro 40-8757; 4-[2-[p-[(E)- 2(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)- propenyl]phenoxy]ethyl]morpholine) on stromal cell-mediated hematopoiesis was examined in murine long-term bone marrow cultures. Whether added at week 2 to regenerating cultures or at week 4 to plateau-phase cultures, mofarotene strongly inhibited total cell production in a dose-dependent manner. Progenitor cell production was also inhibited, but to a lesser extent. When added at the initiation of culture, 1 mumol/L mofarotene did not affect formation of the adherent layer, but production of total nucleated cells and progenitors was inhibited over the next 10 weeks by 95% and 96%, respectively. However, after mofarotene treatment ceased, progenitor cell levels began increasing immediately, and cell production reached plateau levels comparable with those of control cultures within 4 weeks. Hematopoiesis was maintained for 14 more weeks, indicating that long-term culture- initiating cells survived the treatment. Assays of spleen colony- forming units (CFU-S) in the adherent layers showed an enrichment of day-13 CFU-S relative to the more mature day-9 CFU-S. Mofarotene did not inhibit colony formation by bone marrow cells stimulated by exogenous growth factors and did not decrease production of growth factors by stromal cells in the cultures, as determined by functional assays and by mRNA levels. These results suggest that mofarotene blocks differentiation of very primitive progenitors, inhibiting production of more mature hematopoietic elements.     

Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells

We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34(+) cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sac-derived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I (zeta(2)epsilon(2)) and the orthochromatic erythroblasts hemoglobin Gower II (alpha(2)epsilon(2)). This suggests that the switch from Hb Gower I to Hb Gower II, the first hemoglobin switch in humans is a maturation switch not a lineage switch. We also show that extending the coculture of the hESCs with immortalized fetal hepatocytes to 35 days yields CD34(+) cells that differentiate into more developmentally mature, fetal liver-like erythroblasts, that are smaller, express mostly fetal hemoglobin, and can enucleate. We conclude that hESC-derived erythropoiesis closely mimics early human development because the first 2 human hemoglobin switches are recapitulated, and because yolk sac-like and fetal liver-like cells are sequentially produced. Development of a method that yields erythroid cells with an adult phenotype remains necessary, because the most mature cells that can be produced with current systems express less than 2% adult beta-globin mRNA.     

Placental expression of the nonclassical MHC class I molecule Mamu-AG at implantation in the rhesus monkey

During human implantation trophoblasts mediate attachment of the embryo to the uterine epithelium and invade and reorganize vessels of the maternal endometrium to initiate blood flow to the intervillous space. Expression of the nonclassical MHC class I molecule HLA-G by invading trophoblasts may play a central role in their protection from recognition by the maternal immune system; however, the ontogeny of trophoblast HLA-G expression during the earliest stages of implantation is difficult to evaluate in human pregnancy. We previously identified a novel nonclassical MHC class I molecule, Mamu-AG, which is expressed in the rhesus monkey placenta and shares many unique characteristics of HLA-G. Immunocytochemical analysis with a Mamu-AG-specific mAb and locus-specific in situ hybridization of rhesus implantation sites 7-12 days after embryo attachment (days 14-19 of pregnancy) demonstrated that Mamu-AG molecules are expressed predominantly in cytotrophoblasts invading the maternal vessels and endometrium, whereas syncytiotrophoblasts covering trophoblastic lacunae or newly formed chorionic villi remained largely Mamu-AG-negative. By day 36 of pregnancy, Mamu-AG glycoprotein also was expressed in villous syncytiotrophoblasts, and accumulation of Mamu-AG glycoprotein was noted at the border between maternal decidua and fetal trophoblasts. The ontogeny of a nonclassical MHC class I molecule at the implantation site supports the hypothesis that its expression is important for the establishment of maternal-fetal immune tolerance.