A 2-kb c-mpl promoter fragment is sufficient to direct expression to the megakaryocytic lineage and sites of embryonic hematopoiesis in transgenic mice

Thrombopoietin receptor c-mpl is expressed on hematopoietic progenitors and cells of the megakaryocytic lineage. The c-mpl promoter may, therefore, be useful for directing the expression of transgenes. We tested whether a 2-kb genomic DNA fragment comprising the putative c-mpl regulatory elements and most of the 5'-untranslated region of mouse c-mpl is able to direct the expression of a reporter gene to hematopoietic cells in transgenic mice. As a reporter gene we used the human placental alkaline phosphatase (PLAP). In adult transgenic mice, PLAP expression was specifically detected in megakaryocytes and platelets. Embryos showed PLAP reporter gene expression already in the yolk sac at embryonic day 6.5 (E6.5) and in blood islands at E7.5. At E9.5, expression was found in blood vessels of the yolk sac and the embryo proper, followed by high levels of expression in the fetal liver at E11.5. Expression in E6.5 yolk sac is compatible with a function of c-mpl and its ligand, thrombopoietin, in the earliest stages of embryonic hematopoiesis.     

Requirement of gp130 signaling for the AGM hematopoiesis

OBJECTIVE: Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region during mouse development and remarkably expands in the liver at a later stage of ontogeny. gp130 is a signal transducing receptor component shared by all the IL-6 family cytokines, whose gene ablation in mouse results in the significant reduction in the fetal liver hematopoiesis. The present study aims to evaluate the role of gp130 signaling in the fetal mouse AGM hematopoiesis. METHODS AND MATERIALS: Mouse AGM regions from the wild-type and gp130-deficient mice on embryonic day 11.5 were dissociated and cultured with a mixture of cytokines, including one which activates gp130. Wild-type human gp130 and its mutant constructs were introduced into cultured gp130-deficient AGM cells using retrovirus system. To further analyze gp130 downstream signaling, a dominant-negative mutant of STAT3 was also introduced. RESULTS: The gp130 deficiency in the culture of fetal mouse AGM cells resulted in the failure of the expansion of the c-kit(+), Sca-1(+), and lineage markers(-) population. Such failure was rescued by introduction of a wild-type gp130 expression construct but not its mutant constructs having no ability to activate STAT3. In the normal AGM cell culture, introduction of a dominant-negative form of STAT3 in which Y(705) was changed to phenylalanine suppressed the expansion of hematopoietic cell colonies. CONCLUSION: gp130 plays an indispensable role in the expansion of hematopoietic precursor cells in the fetal mouse AGM. In particular, the activation of STAT3 by gp130 is found to be important in this process.     

Development of day-8 colony-forming unit-spleen hematopoietic progenitors during early murine embryogenesis: spatial and temporal mapping

The ontogeny of the hematopoietic system in mammalian embryos occurs during the yolk sac (YS) and the fetal liver (FL) stages. Events leading to the establishment of hematopoiesis in the FL remain obscure. The appearance of colony-forming units-spleen (CFU-S) in the FL is preceded by a gradual increase of CFU-S in the YS and a more rapid increase in the AGM region (area comprising dorsal aorta, gonads, and mesonephros) during day 10 of development (Medvinsky et al, Nature 364:64, 1993). By this time, the AGM CFU-S attain a high frequency equivalent to that found in the adult bone marrow. The analogous area gives rise to adult hematopoiesis in amphibians and probably in birds. We present here a more complete picture of CFU-S development during transition from the pre-liver to liver stage of hematopoiesis. (1) Dissectional analysis of the mouse AGM region shows the presence of CFU- S both around the dorsal aorta and in the uro-genital ridges. (2) The embryonic gut also shows low but distinctive CFU-S activity. This initial intrabody pattern of CFU-S distribution in murine embryogenesis parallels that found for primordial germ cells. (3) The beginning of definitive liver hematopoiesis is accompanied by wide dissemination of CFU-S in the embryonic tissues. (4) Comparison of spleen colonies arising from the AGM and YS has shown morphologic differences. In contrast to simple erythroid constitution of the YS colonies, a broader variety of cells are found within the AGM-derived colonies that are similar to those derived from 11-day FL. These data suggest a lineage relationship for hematopoietic progenitors between the AGM region and the FL.     

Expression of blood group A antigens in human bone marrow cells

We have studied the appearance of blood group A-activity during hematopoiesis in human bone marrow cells by the use of the blood group A-specific lectin from Vicia cracca. Cells that bound the lectin were identified using antiserum against the lectin followed by rosetting with protein A-containing Staphylococcus aureus cells. Only cells of the erythroid lineage from blood group A individuals formed staphylococcal rosettes. A-activity occurred in basophilic normoblasts and later stages of erythropoiesis, whereas pronormoblasts were negative. The appearance of blood group A-activity coincided roughly with the onset of hemoglobin synthesis and slightly later than the expression of the major sialoglycoprotein of erythrocytes, glycophorin A. Glycophorin A did not, however, contain blood group A-activity when analyzed by immunoprecipitation and gel electrophoresis.     

Stromal cell lines from the aorta-gonado-mesonephros region are potent supporters of murine and human hematopoiesis

OBJECTIVE: The hematopoietic system is nurtured by a supportive stroma environment allowing maintenance and differentiation of hematopoietic stem cells (HSC). However, only a limited number of these stromal cell clones support hematopoiesis in the absence of cytokine supplementation. So far, only two bone marrow-derived stromal cell lines (OP9 and S17) are capable of inducing hematopoietic differentiation of totipotent murine and human embryonic stem cells (ESC). Here, the potential of more than 100 stromal cell lines developed from the aorta-gonado-mesonephros (AGM) region was investigated in supporting adult and embryonic hematopoiesis. In addition, extensive phenotypic analysis should elucidate possible mechanisms involved in maintenance of hematopoietic stem cell function. METHODS: More than 100 stromal cell clones derived from the AGM region of E10.5 mouse embryos were isolated. Hematopoietic stem cell support was tested for adult murine and human cord blood hematopoietic stem cells and hematopoietic cells derived from murine ESC. Genotypic and phenotypic characterization was performed including gene array analysis. RESULTS: It was demonstrated that multiple clones showed high efficiency in supporting maintenance and expansion of primitive murine and human hematopoietic progenitors. In addition, we demonstrated for the first time that AGM stromal cell lines are also potent inducers of hematopoietic differentiation of murine ESC. Microarray analysis of AGM lines revealed a characteristic genotype with expression of genes involved in regulating hematopoiesis as well as mesodermal and early B cell development. CONCLUSION: These AGM stromal cell lines may be of value in elucidating molecular mechanisms regulating early stem cell development and hematopoietic differentiation from ES-derived mesoderm.     

Embryonic beginnings of adult hematopoietic stem cells

Hematopoietic stem cells (HSC) are at the foundation of the adult hematopoietic system. HSC give rise to all blood cells through a complex series of proliferation and differentiation events that occur throughout the lifespan of the individual. Because of their clinical importance in transplantation protocols, recent research has focused on the developmental origins and potential of embryonic HSC. In both mammalian and non-mammalian vertebrate embryos, two independent anatomical sites have been found to generate hematopoietic cells. The yolk sac (or its equivalent in amphibians, the ventral blood islands) participates in a first transient wave of hematopoiesis by producing primitive erythrocytes. Importantly, adult-type HSCs emerge autonomously in a second wave of hematopoietic generation in an intraembryonic region surrounding the dorsal aorta, the aorta-gonads-mesonephros (AGM) region. In this review, we will discuss research advances in the field of developmental hematopoiesis, with a particular emphasis on the cellular origins of AGM HSC and their regulation by the embryonic hematopoietic microenvironment.     

Extracellular signal-regulated kinase 2 is necessary for mesoderm differentiation

The extracellular signal-regulated kinase (ERK) is a component of the mitogen-activated protein kinase cascade. Exon 2 of erk2 was deleted by homologous recombination and resulted in embryonic lethality at embryonic day 6.5. erk2 mutant embryos did not form mesoderm and showed increased apoptosis but comparable levels of BrdUrd incorporation, indicating a defect in differentiation. erk2 null embryonic stem (ES) cells exhibited reduced total ERK activity upon serum stimulation, augmented ERK1 phosphorylation, and decreased downstream p90Rsk phosphorylation and activity; yet ES cell proliferation was unaffected. Mutant ES cells were capable of forming mesoderm; however, treatment of mutant ES cells with the mitogen-activated protein kinase kinase inhibitor PD184352 decreased total ERK activity and expression of the mesodermal marker brachyury, suggesting that ERK1 can compensate for ERK2 in vitro. Normal embryos at embryonic day 6.5 expressed activated ERK1/2 in the extraembryonic ectoderm, whereas erk2 mutant embryos had no detectable activated ERK1/2 in this region, suggesting that activated ERK1 was not expressed, and therefore cannot compensate for loss of ERK2 in vivo. These data indicate that ERK2 plays an essential role in mesoderm differentiation during embryonic development.     

Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro

In this report we demonstrate a role for Runx1 (AML1) at the hemangioblast stage of hematopoietic and endothelial development in embryonic stem (ES) cell-derived embryoid bodies (EBs). Runx1 is expressed in EBs during the appearance of precursors with hemangioblast properties, the blast colony-forming cells (BL-CFCs). Cell sorting studies revealed that all BL-CFCs within EBs express Runx1. Runx1-deficient EBs consistently generate 10- to 20-fold fewer blast colonies than wild-type controls and display a complete block in definitive hematopoiesis. Despite this defect, Runx1-/- EBs and yolk sacs from mutant embryos generate normal numbers of primitive erythroid precursors. These observations clearly demonstrate that Runx1 functions early in hematopoietic development, and they support the interpretation that the primitive erythroid lineage is established early by a subset of BL-CFCs that develop in a Runx1-independent fashion.     

Induction of hematopoietic commitment and erythromyeloid differentiation in embryonal stem cells constitutively expressing c-myb

To provide insight into the mechanisms by which c-myb regulates hematopoiesis, we analyzed the expression of markers for multiple hematopoietic lineages in differentiating parental embryonic stem (ES) cells and in ES cells transfected with c-myb or with a mutant c-myb deficient in DNA binding and assessed the ability of these cells to undergo hematopoietic commitment and colony formation. Undifferentiated ES cells transfected with intact c-myb, but not cells transfected with mutant c-myb, expressed CD34, c-kit, GATA1, and flt3 mRNA as well as surface CD34, c-kit, and flt3 product. In contrast, the kinetics of GATA-2 mRNA expression was identical in parental and Myb-transfected ES cells. Transient expression assays suggested transactivation of gene expression dependent on interaction with Myb binding sites in the CD34 and GATA1 5' flanking regions. Undifferentiated parental and c-myb mutant-transfected ES cells were not clonogenic, whereas c-myb transfectants formed erythromyeloid colonies in methylcellulose cultures in the absence of added hematopoietic growth factors and, at higher frequency, in the presence of kit and flt-3 ligands. Colony formation was suppressed by treatment with antisense oligodeoxynucleotides specifically downregulating c-kit and flt-3 expression. These findings indicate that c-myb regulates hematopoietic commitment and progenitor cell proliferation and differentiation through the activation of certain genes that define the stem/progenitor cell compartment.     

Core-binding factor beta (CBFbeta), but not CBFbeta-smooth muscle myosin heavy chain, rescues definitive hematopoiesis in CBFbeta-deficient embryonic stem cells

Core-binding factor beta (CBFbeta) is the non-DNA-binding subunit of the heterodimeric CBFs. Genes encoding CBFbeta (CBFB), and one of the DNA-binding CBFalpha subunits, Runx1 (also known as CBFalpha2, AML1, and PEBP2alphaB), are required for normal hematopoiesis and are also frequent targets of chromosomal translocations in acute leukemias in humans. Homozygous disruption of either the Runx1 or Cbfb gene in mice results in embryonic lethality at midgestation due to hemorrhaging in the central nervous system, and severely impairs fetal liver hematopoiesis. Results of this study show that Cbfb-deficient mouse embryonic stem (ES) cells can differentiate into primitive erythroid colonies in vitro, but are impaired in their ability to produce definitive erythroid and myeloid colonies, mimicking the in vivo defect. Definitive hematopoiesis is restored by ectopic expression of full-length Cbfb transgenes, as well as by a transgene encoding only the heterodimerization domain of CBFbeta. In contrast, the CBFbeta-smooth muscle myosin heavy chain (SMMHC) fusion protein generated by the inv(16) associated with acute myeloid leukemias (M4Eo) cannot rescue definitive hematopoiesis by Cbfb-deficient ES cells. Sequences responsible for the inability of CBFbeta-SMMHC to rescue definitive hematopoiesis reside in the SMMHC portion of the fusion protein. Results also show that the CBFbeta-SMMHC fusion protein transdominantly inhibits definitive hematopoiesis, but not to the same extent as homozygous loss of Runx1 or Cbfb. CBFbeta-SMMHC preferentially inhibits the differentiation of myeloid lineage cells, while increasing the number of blastlike cells in culture.     

Conditional requirement for the Flk-1 receptor in the in vitro generation of early hematopoietic cells.

Genetic studies in mice have previously demonstrated an intrinsic requirement for the vascular endothelial growth factor (VEGF) receptor Flk-1 in the early development of both the hematopoietic and endothelial cell lineages. In this study, embryonic stem (ES) cells homozygous for a targeted null mutation in flk-1 (flk-1 (-/-)) were examined for their hematopoietic potential in vitro during embryoid body (EB) formation or when cultured on the stromal cell line OP9. Surprisingly, in EB cultures flk-1 (-/-) ES cells were able to differentiate into all myeloid-erythroid lineages, albeit at half the frequency of heterozygous lines. In contrast, although flk-1 (-/-) ES cells formed mesodermal-like colonies on OP9 monolayers, they failed to generate hematopoietic clusters even in the presence of exogenous cytokines. However, flk-1 (-/-) OP9 cultures did contain myeloid precursors, albeit at greatly reduced percentages. This defect was rescued by first allowing flk-1 (-/-) ES cells to differentiate into EBs and then passaging these cells onto OP9 stroma. Thus, the requirement for Flk-1 in early hematopoietic development can be abrogated by alterations in the microenvironment. This finding is consistent with a role for Flk-1 in regulating the migration of early mesodermally derived precursors into a microenvironment that is permissive for hematopoiesis.     

Caspase-3 has a nonapoptotic function in erythroid maturation

Caspase-3 plays a central role in apoptosis. It is also activated in normal erythropoiesis, with its activity peaking early during development (erythroid colony-forming unit [CFU-E] stage). In the present study, we have reduced the expression and subsequent enzymatic activity of caspase-3 by transfection of small interfering RNA (siRNA) directed to caspase-3 in a differentiating human erythroid culture system. We find that siRNA treatment yields a 50% reduction in cells that undergo enucleation with no change in the fraction of cells that undergo apoptosis, measured throughout the culture. Furthermore, a substantial fraction of treated cells are unable to complete the transition from pronormoblasts to basophilic normoblasts. These results demonstrate that caspase-3 is required for efficient erythropoiesis in this model system.