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Zebrafish are one of the preeminent model systems for the study of blood development (hematopoiesis), hematopoietic stem and progenitor cell (HSPC) biology, and hematopathology. The zebrafish hematopoietic system shares strong similarities in functional populations, genetic regulators, and niche interactions with its mammalian counterparts. These evolutionarily conserved characteristics, together with emerging technologies in live imaging, compound screening, and genetic manipulation, have been employed to successfully identify and interrogate novel regulatory mechanisms and molecular pathways that guide hematopoiesis. Significantly, perturbations in many of the key developmental signals controlling hematopoiesis are associated with hematological disorders and disease, including anemia, bone marrow failure syndromes, and leukemia. Thus, understanding the regulatory pathways controlling HSPC production and function has important clinical implications. In this review, we describe how the blood system forms and is maintained in zebrafish, with particular focus on new insights into vertebrate hematological regulation gained using this model. The interplay of factors controlling development and disease in the hematopoietic system combined with the unique attributes of the zebrafish make this a powerful platform to discover novel targets for the treatment of hematological disease., (© 2017 Elsevier Inc. All rights reserved.)

The developmental relationship between the blood and endothelial cell (EC) lineages remains unclear. In the extra-embryonic blood islands of birds and mammals, ECs and blood cells are closely intermixed, and blood island precursor cells in the primitive streak express many of the same molecular markers, leading to the suggestion that both lineages arise from a common precursor, called the hemangioblast. Cells within the blood island of Xenopus also coexpress predifferentiation markers of the blood and EC lineages. However, using multiple assays, we find that precursor cells in the Xenopus blood island do not normally differentiate into ECs, suggesting that classic hemangioblasts are rare or nonexistent in Xenopus. What prevents these precursor cells from developing into mature ECs? We have found that bone morphogenetic protein (BMP) signaling is essential for erythroid differentiation, and in the absence of BMP signaling, precursor cells adopt an EC fate. Furthermore, inhibition of the erythroid transcription pathway leads to endothelial differentiation. Our results indicate that bipotential endothelial/erythroid precursor cells do indeed exist in the Xenopus blood island, but BMP signaling normally acts to constrain EC fate. More generally, these results provide evidence that commitment to the erythroid lineage limits development of bipotential precursors toward an endothelial fate.

Granulocyte colony-stimulating factor (Gcsf) drives the proliferation and differentiation of granulocytes, monocytes, and macrophages (mφs) from hematopoietic stem and progenitor cells (HSPCs). Analysis of the zebrafish genome indicates the presence of 2 Gcsf ligands, likely resulting from a duplication event in teleost evolution. Although Gcsfa and Gcsfb share low sequence conservation, they share significant similarity in their predicted ligand/receptor interaction sites and structure. Each ligand displays differential temporal expression patterns during embryogenesis and spatial expression patterns in adult animals. To determine the functions of each ligand, we performed loss- and gain-of-function experiments. Both ligands signal through the Gcsf receptor to expand primitive neutrophils and mφs, as well as definitive granulocytes. To further address their functions, we generated recombinant versions and tested them in clonal progenitor assays. These sensitive in vitro techniques indicated similar functional attributes in supporting HSPC growth and differentiation. Finally, in addition to supporting myeloid differentiation, zebrafish Gcsf is required for the specification and proliferation of hematopoietic stem cells, suggesting that Gcsf represents an ancestral cytokine responsible for the broad support of HSPCs. These findings may inform how hematopoietic cytokines evolved following the diversification of teleosts and mammals from a common ancestor.

The first site exhibiting hematopoietic activity in mammalian development is the yolk-sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in blast colony-forming cells to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk-sac hematopoiesis and vascular development in Ldb1(-/-) mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells. Transcriptome analysis and correlation with the genome-wide binding pattern of Ldb1 in hemangioblasts revealed a number of direct-target genes and pathways misregulated in the absence of Ldb1. The regulation of essential developmental factors by Ldb1 defines it as an upstream transcriptional regulator of hematopoietic/endothelial development. We show the complex interplay that exists between transcription factors and signaling pathways during the very early stages of hematopoietic/endothelial development and the specific signaling occurring in hemangioblasts in contrast to more advanced hematopoietic developmental stages. Finally, by revealing novel genes and pathways not previously associated with early development, our study provides novel candidate targets to manipulate the differentiation of hematopoietic and/or endothelial cells.

Erythroid ontogeny is characterized by overlapping waves of primitive and definitive erythroid lineages that share many morphologic features during terminal maturation but have marked differences in cell size and globin expression. In the present study, we compared global gene expression in primitive, fetal definitive, and adult definitive erythroid cells at morphologically equivalent stages of maturation purified from embryonic, fetal, and adult mice. Surprisingly, most transcriptional complexity in erythroid precursors is already present by the proerythroblast stage. Transcript levels are markedly modulated during terminal erythroid maturation, but housekeeping genes are not preferentially lost. Although primitive and definitive erythroid lineages share a large set of nonhousekeeping genes, annotation of lineage-restricted genes shows that alternate gene usage occurs within shared functional categories, as exemplified by the selective expression of aquaporins 3 and 8 in primitive erythroblasts and aquaporins 1 and 9 in adult definitive erythroblasts. Consistent with the known functions of Aqp3 and Aqp8 as H2O2 transporters, primitive, but not definitive, erythroblasts preferentially accumulate reactive oxygen species after exogenous H2O2 exposure. We have created a user-friendly Web site (http://www.cbil.upenn.edu/ErythronDB) to make these global expression data readily accessible and amenable to complex search strategies by the scientific community.

Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Transcription factors have long been recognised as powerful regulators of mammalian development yet it is largely unknown how individual key regulators operate within wider regulatory networks. Here we have used a combination of global gene expression and chromatin-immunoprecipitation approaches during the early stages of haematopoietic development to define the transcriptional programme controlled by Runx1, an essential regulator of blood cell specification. Integrated analysis of these complementary genome-wide datasets allowed us to construct a global regulatory network model, which suggested that key regulators are activated sequentially during blood specification, but will ultimately collaborate to control many haematopoietically expressed genes. Using the CD41/integrin alpha 2b gene as a model, cellular and in vivo studies showed that CD41 is controlled by both Scl/Tal1 and Runx1 in fully specified blood cells, and initiation of CD41 expression in E7.5 embryos is severely compromised in the absence of Runx1. Taken together, this study represents the first global analysis of the transcriptional programme controlled by any key haematopoietic regulator during the process of early blood cell specification. Moreover, the concept of interplay between sequentially deployed core regulators is likely to represent a design principle widely applicable to the transcriptional control of mammalian development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Etv2 (Ets Variant 2) has been shown to be an indispensable gene for the development of hematopoietic cells (HPCs)/endothelial cells (ECs). However, how Etv2 specifies the mesoderm-generating HPCs/ECs remains incompletely understood. In embryonic stem cell (ESC) differentiation culture and Etv2-null embryos, we show that Etv2 is dispensable for generating primitive Flk-1(+)/PDGFRα(+) mesoderm but is required for the progression of Flk-1(+)/PDGFRα(+) cells into vascular/hematopoietic mesoderm. Etv2-null ESCs and embryonic cells were arrested as Flk-1(+)/PDGFRα(+) and failed to generate Flk-1(+)/PDGFRα(-) mesoderm. Flk-1(+)/Etv2(+) early embryonic cells showed significantly higher hemato-endothelial potential than the Flk-1(+)/Etv2(-) population, suggesting that Etv2 specifies a hemato-endothelial subset of Flk-1(+) mesoderm. Critical hemato-endothelial genes were severely down-regulated in Etv2-null Flk-1(+) cells. Among those genes Scl, Fli1, and GATA2 were expressed simultaneously with Etv2 in early embryos and seemed to be critical targets. Etv2 reexpression in Etv2-null cells restored the development of CD41(+), CD45(+), and VE-cadherin(+) cells. Expression of Scl or Fli1 alone could also restore HPCs/ECs in the Etv2-null background, indicating that these 2 genes are critical downstream targets. Furthermore, VEGF induced Etv2 potently and rapidly in Flk-1(+) mesoderm. We propose that Flk-1(+)/PDGFRα(+) primitive mesoderm is committed into Flk-1(+)/PDGFRα(-) vascular mesoderm through Etv2 and that up-regulation of Etv2 by VEGF promotes this commitment.

The primitive hematopoietic stem/progenitor cells (HSPCs) during embryonic hematopoiesis are thought to be short-lived (SL) with limited self-renewal potential. The fate and consequence of these short-lived HSPCs, once reprogrammed into "long-lived" in a living animal body, remain unknown. Here we show that targeted expression of a dominant-negative C/ebpα (C/ebpαDN) in the primitive SL-HSPCs during zebrafish embryogenesis extends their life span, allowing them to survive to later developmental stage to colonize the definitive hematopoietic sites, where they undergo a proliferative expansion followed by erythropoietic dysplasia and embryonic lethality because of circulation congestion. Mechanistically, C/ebpαDN binds to a conserved C/EBP-binding motif in the promoter region of bmi1 gene, associated with a specific induction of bmi1 transcription in the transgenic embryos expressing C/ebpαDN. Targeted expression of Bmi1 in the SL-HSPCs recapitulates nearly all aberrant phenotypes induced by C/ebpαDN, whereas knockdown of bmi1 largely rescues these abnormalities. The results indicate that Bmi1 acts immediately downstream of C/ebpαDN to regulate the survival and self-renewal of HSPCs and contribute to the erythropoietic dysplasia.

STAT1 mediates response to interferons and regulates immunity, cell proliferation, apoptosis, and sensitivity of Fanconi Anemia cells to apoptosis after interferon signaling; the roles of STAT1 in embryos, however, are not understood. To explore embryonic functions of STAT1, we investigated stat1b, an unstudied zebrafish co-ortholog of human STAT1. Zebrafish stat1a encodes all five domains of the human STAT1-alpha splice form but, like the human STAT1-beta splice variant, stat1b lacks a complete transactivation domain; thus, two unlinked zebrafish paralogs encode protein forms translated from two splice variants of a single human gene, as expected by sub-functionalization after genome duplication. Phylogenetic and conserved synteny studies showed that stat1b and stat1a arose as duplicates in the teleost genome duplication (TGD) and clarified the evolutionary origin of STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 by tandem and genome duplication. RT-PCR revealed maternal expression of stat1a and stat1b. In situ hybridization detected stat1b but not stat1a expression in embryonic hematopoietic tissues. Morpholino knockdown of stat1b, but not stat1a, decreased expression of the myeloid and granulocyte markers spi and mpo and increased expression of the hematopoietic progenitor marker scl, the erythrocyte marker gata1, and hemoglobin. These results suggest that zebrafish Stat1b promotes myeloid development at the expense of erythroid development., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Edification of the human hematopoietic system during development is characterized by the production of waves of hematopoietic cells separated in time, formed in distinct embryonic sites (ie, yolk sac, truncal arteries including the aorta, and placenta). The embryonic liver is a major hematopoietic organ wherein hematopoietic stem cells (HSCs) expand, and the future, adult-type, hematopoietic cell hierarchy becomes established. We report herein the identification of a new, transient, and rare cell population in the human embryonic liver, which coexpresses VE-cadherin, an endothelial marker, CD45, a pan-hematopoietic marker, and CD34, a common endothelial and hematopoietic marker. This population displays an outstanding self-renewal, proliferation, and differentiation potential, as detected by in vitro and in vivo hematopoietic assays compared with its VE-cadherin negative counterpart. Based on VE-cadherin expression, our data demonstrate the existence of 2 phenotypically and functionally separable populations of multipotent HSCs in the human embryo, the VE-cadherin(+) one being more primitive than the VE-cadherin(-) one, and shed a new light on the hierarchical organization of the embryonic liver HSC compartment.

The vitelline artery is a temporary structure that undergoes extensive remodeling during midgestation to eventually become the superior mesenteric artery (also called the cranial mesenteric artery, in the mouse). Here we show that, during this remodeling process, large clusters of hematopoietic progenitors emerge via extravascular budding and form structures that resemble previously described mesenteric blood islands. We demonstrate through fate mapping of vascular endothelium that these mesenteric blood islands are derived from the endothelium of the vitelline artery. We further show that the vitelline arterial endothelium and subsequent blood island structures originate from a lateral plate mesodermal population. Lineage tracing of the lateral plate mesoderm demonstrates contribution to all hemogenic vascular beds in the embryo, and eventually, all hematopoietic cells in the adult. The intraembryonic hematopoietic cell clusters contain viable, proliferative cells that exhibit hematopoietic stem cell markers and are able to further differentiate into myeloid and erythroid lineages. Vitelline artery-derived hematopoietic progenitor clusters appear between embryonic day 10 and embryonic day 10.75 in the caudal half of the midgut mesentery, but by embryonic day 11.0 are sporadically found on the cranial side of the midgut, thus suggesting possible extravascular migration aided by midgut rotation.

Children with Down syndrome exhibit 2 related hematopoietic diseases: transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Both exhibit clonal expansion of blasts with biphenotypic erythroid and megakaryocytic features and contain somatic GATA1 mutations. While altered GATA1 inhibits erythro-megakaryocytic development, less is known about how trisomy 21 impacts blood formation, particularly in the human fetus where TMD and AMKL originate. We used in vitro and mouse transplantation assays to study hematopoiesis in trisomy 21 fetal livers with normal GATA1 alleles. Remarkably, trisomy 21 progenitors exhibited enhanced production of erythroid and megakaryocytic cells that proliferated excessively. Our findings indicate that trisomy 21 itself is associated with cell-autonomous expansion of erythro-megakaryocytic progenitors. This may predispose to TMD and AMKL by increasing the pool of cells susceptible to malignant transformation through acquired mutations in GATA1 and other cooperating genes.

Previous studies revealed that mAb BB9 reacts with a subset of CD34(+) human BM cells with hematopoietic stem cell (HSC) characteristics. Here we map BB9 expression throughout hematopoietic development and show that the earliest definitive HSCs that arise at the ventral wall of the aorta and surrounding endothelial cells are BB9(+). Thereafter, BB9 is expressed by primitive hematopoietic cells in fetal liver and in umbilical cord blood (UCB). BB9(+)CD34(+) UCB cells transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice contribute 10-fold higher numbers of multilineage blood cells than their CD34(+)BB9(-) counterparts and contain a significantly higher incidence of SCID-repopulating cells than the unfractionated CD34(+) population. Protein microsequencing of the 160-kDa band corresponding to the BB9 protein established its identity as that of somatic angiotensin-converting enzyme (ACE). Although the role of ACE on human HSCs remains to be determined, these studies designate ACE as a hitherto unrecognized marker of human HSCs throughout hematopoietic ontogeny and adulthood.

Gene expression profiling demonstrated that components of the cholinergic system, including choline acetyltransferase, acetylcholinesterase and nicotinic acetylcholine receptors (nAChRs), are expressed in embryonic stem cells and differentiating embryoid bodies (EBs). Triggering of nAChRs expressed in EBs by nicotine resulted in activation of MAPK and shifts of spontaneous differentiation toward hemangioblast. In vivo, non-neural nAChRs are detected early during development in fetal sites of hematopoiesis. Similarly, in vivo exposure of the developing embryo to nicotine resulted in higher numbers of hematopoietic progenitors in fetal liver. However postpartum, the number of hematopoietic stem/progenitor cells (HSPC) was decreased, suggesting an impaired colonization of the fetal bone marrow with HSPCs. This correlated with increased number of circulating HSPC and decreased expression of CXCR4 that mediates migration of circulating cells into the bone marrow regulatory niche. In addition, protein microarrays demonstrated that nicotine changed the profile of cytokines produced in the niche. While the levels of IL1alpha, IL1beta, IL2, IL9 and IL10 were not changed, the production of hematopoiesis-supportive cytokines including G-CSF, GM-CSF, IL3, IL6 and IGFBP-3 was decreased. This correlated with the decreased repopulating ability of HSPC in vivo and diminished hematopoietic activity in bone marrow cultures treated with nicotine. Interestingly, nicotine stimulated the production of IL4 and IL5, implying a possible role of the cholinergic system in pathogenesis of allergic diseases. Our data provide evidence that the nicotine-induced imbalance of the cholinergic system during gestation interferes with normal development and provides the basis for negative health outcomes postpartum in active and passive smokers.

The TAL-1/SCL and LYL-1 genes encode two closely related basic helix-loop-helix transcription factors involved in child T-acute lymphoblastic leukemia through chromosomal rearrangements and transcriptional deregulation. During ontogeny, Tal-1/SCL is required for hematopoietic cell generation, both in the yolk sac, where erythro-myeloid cells are first produced, then in the intra-embryonic compartment, where hematopoietic stem cells independently arise. We describe here the expression pattern of lyl-1 in mouse embryos from 7 to 14 days post coitus using in situ hybridization, as well as beta-Galactosidase (beta-Gal) expression in lyl-1-lacZ knock-in embryos, which express a C-terminally truncated Lyl-1 protein fused to the beta-Galactosidase (Lyl-1Delta/beta-Gal). In addition, we compare lyl-1 expression pattern with that of tal-1/scl. Similar to Tal-1/SCL, Lyl-1 mRNA expression occurs in the developing cardiovascular and hematopoietic systems. However, contrary to tal-1/scl, lyl-1 is not expressed in the developing nervous system. In lyl-1-lacZ knock-in heterozygous and homozygous embryos, beta-Gal expression completely correlates with Lyl-1 mRNA expression in the intra-embryonic compartment and is present: (1) in the developing hematopoietic system, precisely where hematopoietic stem cells emerge, and thereafter in the fetal liver; (2) in the developing vascular system; and (3) in the endocardium. In contrast, whereas Lyl-1 mRNA is expressed in yolk sac-derived endothelial and hematopoietic cells, Lyl-1Delta/beta-Gal is either absent or poorly expressed in these cell types, thus differing from Tal-1/SCL, which is highly expressed there at both mRNA and protein levels.

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.

The Lmo2 transcription factor, a T-cell oncoprotein, is required for both hematopoiesis and angiogenesis. To investigate the fate of lmo2-expressing cells and the transcriptional regulation of lmo2 in vivo, we generated stable transgenic zebrafish that express green fluorescent protein (EGFP) or DsRed under the control of an lmo2 promoter. A 2.5-kb fragment contains the cis-regulatory elements required to recapitulate endogenous lmo2 expression in embryonic hematopoietic and vascular tissues. We further characterized embryonic Lmo2+ cells through transplantation into vlad tepes (vlt), an erythropoietic mutant. These Lmo2+ primitive wave donor cells differentiated into circulating hematopoietic cells and extended the life span of vlt recipients, but did not demonstrate long-term repopulation of the erythroid lineage. Promoter analysis identified a 174-bp proximal promoter that was sufficient to recapitulate lmo2 expression. This element contains critical ETS-binding sites conserved between zebrafish and pufferfish. Furthermore, we show that ets1 is coexpressed with lmo2, and overexpression experiments indicate that ets1 can activate the lmo2 promoter through this element. Our studies elucidate the transcriptional regulation of this key transcription factor, and provide a transgenic system for the functional analysis of blood and blood vessels in zebrafish.

Though a topic of medical interest for centuries, our understanding of vertebrate hematopoietic or "blood-forming" tissue development has improved greatly only in recent years and given a series of scientific and technical milestones. Key among these observations was the description of procedures that allowed the transplantation of blood-forming activity. Beyond this, other advances include the creation of a variety of knock-out animals (mice and more recently zebrafish), microdissection of embryonic and fetal blood-forming tissues, hematopoietic stem (HSC) and progenitor cell (HPC) colony-forming assays, the discovery of cytokines with defined hematopoietic activities, gene transfer technologies, and the description of lineage-specific surface antigens for the identification and purification of pluripotent and differentiated blood cells. The availability of both murine and human embryonic stem cells (ESC) and the delineation of in vitro systems to direct their differentiation have now been added to this analytical arsenal. Such tools have allowed researchers to interrogate the complex developmental processes behind both primitive (yolk sac or extraembryonic) and definitive (intraembryonic) hematopoietic tissue formation. Using ES cells, we hope to not only gain additional basic insights into hematopoietic development but also to develop platforms for therapeutic use in patients suffering from hematological disease. In this review, we will focus on points of convergence and divergence between murine and human hematopoiesis in vivo and in vitro, and use these observations to evaluate the literature regarding attempts to create hematopoietic tissue from embryonic stem cells, the pitfalls encountered therein, and what challenges remain.

The steps to leukemia following an in utero fusion of MLL (HRX, ALL-1) to a partner gene in humans are not known. Introduction of the Mll-AF9 fusion gene into embryonic stem cells results in leukemia in mice with cell-type specificity similar to humans. In this study we used myeloid colony assays, immunophenotyping, and transplantation to evaluate myelopoiesis in Mll-AF9 mice. Colony assays demonstrated that both prenatal and postnatal Mll-AF9 tissues have significantly increased numbers of CD11b(+)/CD117(+)/Gr-1(+/-) myeloid cells, often in compact clusters. The self-renewal capacity of prenatal myeloid progenitors was found to decrease following serial replating of colony-forming cells. In contrast, early postnatal myeloid progenitors increased following replating; however, the enhanced self-renewal of early postnatal myeloid progenitor cells was limited and did not result in long-term cell lines or leukemia in vivo. Unlimited replating, long-term CD11b/Gr-1(+) myeloid cell lines, and the ability to produce early leukemia in vivo in transplantation experiments, were found only in mice with overt leukemia. Prenatal Mll-AF9 tissues had reduced total (mature and progenitor) CD11b/Gr-1(+) cells compared with wild-type tissues. Colony replating, immunophenotyping, and cytochemistry suggest that any perturbation of cellular differentiation from the prenatal stage onward is partial and largely reversible. We describe a novel informative in vitro and in vivo model system that permits study of the stages in the pathogenesis of Mll fusion gene leukemia, beginning in prenatal myeloid cells, progressing to a second stage in the postnatal period and, finally, resulting in overt leukemia in adult animals.

Hematopoiesis has been considered hierarchical in nature, but recent data suggest that the system is not hierarchical and is, in fact, quite functionally plastic. Existing data indicate that engraftment and progenitor phenotypes vary inversely with cell cycle transit and that gene expression also varies widely. These observations suggest that there is no progenitor/stem cell hierarchy, but rather a reversible continuum. This may, in turn, be dependent on shifting chromatin and gene expression with cell cycle transit. If the phenotype of these primitive marrow cells changes from engraftable stem cell to progenitor and back to engraftable stem cell with cycle transit, then this suggests that the identity of the engraftable stem cell may be partially masked in nonsynchronized marrow cell populations. A general model indicates a marrow cell that can continually change its surface receptor expression and thus responds to external stimuli differently at different points in the cell cycle.

During embryogenesis there is a sequential, temporal appearance of increasingly more-complex hematopoietic cells beginning with unipotential progenitors, proceeding to multipotential (myeloid, erythroid and lymphoid) progenitors and culminating with adult-repopulating hematopoietic stem cells. Current research has established an important role for the aorta-gonads-mesonephros region of the mouse embryo in the generation of multipotential progenitors and hematopoietic stem cells. Comparisons of normal and hematopoietic-cell-mutant mouse embryos have revealed several genes pivotal in hematopoietic stem cell generation/function. Other genes have been implicated in the critical generation of lymphoid lineage potential. Thus, an understanding of the cellular and molecular interactions within the midgestation aorta-gonads-mesonephros region offers insight into the mechanisms of hematopoietic lineage specification during ontogeny and perhaps will lead to a more complete knowledge of the adult hematopoietic system.

Integrin alphaIIb is a cell adhesion molecule expressed in association with beta3 by cells of the megakaryocytic lineage, from committed progenitors to platelets. While it is clear that lymphohemopoietic cells differentiating along other lineages do not express this molecule, it has been questioned whether mammalian hemopoietic stem cells (HSC) and various progenitor cells express it. In this study, we detected alphaIIb expression in midgestation embryo in sites of HSC generation, such as the yolk sac blood islands and the hemopoietic clusters lining the walls of the major arteries, and in sites of HSC migration, such as the fetal liver. Since c-Kit, which plays an essential role in the early stages of hemopoiesis, is expressed by HSC, we studied the expression of the alphaIIb antigen in the c-Kit-positive population from fetal liver and adult bone marrow differentiating in vitro and in vivo into erythromyeloid and lymphocyte lineages. Erythroid and myeloid progenitor activities were found in vitro in the c-Kit(+)alphaIIb(+) cell populations from both origins. On the other hand, a T cell developmental potential has never been considered for c-Kit(+)alphaIIb(+) progenitors, except in the avian model. Using organ cultures of embryonic thymus followed by grafting into athymic nude recipients, we demonstrate herein that populations from murine fetal liver and adult bone marrow contain T lymphocyte progenitors. Migration and maturation of T cells occurred, as shown by the development of both CD4(+)CD8- and CD4-CD8(+) peripheral T cells. Multilineage differentiation, including the B lymphoid lineage, of c-Kit(+)alphaIIb(+) progenitor cells was also shown in vivo in an assay using lethally irradiated congenic recipients. Taken together, these data demonstrate that murine c-Kit(+)alphaIIb(+) progenitor cells have several lineage potentialities since erythroid, myeloid, and lymphoid lineages can be generated., ((C)2002 Elsevier Science (USA).)

This article describes the isolation of a novel cell population (B220(lo)c-kit(+)CD19(-)) in the fetal liver that represents 70% of T-cell precursors in this organ. Interestingly, these precursors showed a bipotent T-cell and natural killer cell (NK)- restricted reconstitution potential but completely lacked B and erythromyeloid differentiation capacity both in vivo and in vitro. Moreover, not only mature T-cell receptor (TCR)alphabeta(+) peripheral T cells but also TCRgammadelta(+) and TCRalphabeta(+)CD8alphaalpha(+) intestinal epithelial cells of extrathymic origin were generated in reconstituted mice. The presence of this population in the fetal liver of athymic embryos indicates its prethymic origin. The comparison of the phenotype and differentiation potential of B220(lo)c-kit(+)CD19(-) fetal liver cells with those of thymic T/NK progenitors indicates that this is the most immature common T/NK cell progenitor so far identified. These fetal liver progenitors may represent the immediate developmental step before thymic immigration.

At different developmental stages, candidate human hematopoietic stem cells (HSCs) are present within the CD34+ CD38- population. By means of xenotransplantation, such CD34+CD38- cells were recently shown to engraft the hematopoietic system of fetal sheep and nonobese diabetic severe combined immunodeficient adult mice. Here it is demonstrated that, after their injection into murine blastocysts, human cord blood (CB)-derived CD34+ and CD34+ CD38- cells repopulate the hematopoietic tissues of nonimmunocompromised murine embryos and that human donor contribution can persist to adulthood. It is further observed that human hematopoietic progenitor cells are present in murine hematopoietic tissues of midgestational chimeric embryos and that progeny of the injected human HSCs activate erythroid-specific gene expression. Thus, the early murine embryo provides a suitable environment for the survival and differentiation of human CB CD34+ CD38- cells.

The human RUNX3/AML2 gene belongs to the 'runt domain' family of transcription factors that act as gene expression regulators in major developmental pathways. Here, we describe the expression pattern of Runx3 during mouse embryogenesis compared to the expression pattern of Runx1. E10.5 and E14.5-E16.5 embryos were analyzed using both immunohistochemistry and beta-galactosidase activity of targeted Runx3 and Runx1 loci. We found that Runx3 expression overlapped with that of Runx1 in the hematopoietic system, whereas in sensory ganglia, epidermal appendages, and developing skeletal elements, their expression was confined to different compartments. These data provide new insights into the function of Runx3 and Runx1 in organogenesis and support the possibility that cross-regulation between them plays a role in embryogenesis.

The Krüppel-like factor (KLF) family of genes encodes transcriptional regulatory proteins that play roles in differentiation of a diverse set of cells in mammals. For instance, the founding member KLF1 (also known as EKLF) is required for normal globin production in mammals. Five new KLF genes have been isolated from the zebrafish, Danio rerio, and the structure of their products, their genetic map positions, and their expression during development of the zebrafish have been characterized. Three genes closely related to mammalian KLF2 and KLF4 were found, as was an ortholog of mammalian KLF12. A fifth gene, apparently missing from the genome of mammals and closely related to KLF1 and KLF2, was also identified. Analysis demonstrated the existence of novel conserved domains in the N-termini of these proteins. Developmental expression patterns suggest potential roles for these zebrafish genes in diverse processes, including hematopoiesis, blood vessel function, and fin and epidermal development. The studies imply a high degree of functional conservation of the zebrafish genes with their mammalian homologs. These findings further the understanding of the KLF genes in vertebrate development and indicate an ancient role in hematopoiesis for the Krüppel-like factor gene family.

In vitro studies have indicated that the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression is regulated at the posttranscriptional level by the AU-rich element (ARE) sequence present in its 3' untranslated region (UTR). This study investigated the importance of the ARE in the control of GM-CSF gene expression in vivo. For this purpose, transgenic mice bearing GM-CSF gene constructs containing or lacking the ARE (GM-CSF AU(+) or GM-CSF AU(-), respectively) were generated. Both transgenes were under the transcriptional control of the immediate early promoter of the cytomegalovirus (CMV) to ensure their early, widespread, and constitutive expression. The regulation imposed by the ARE was revealed by comparing transgene expression at day 14 of embryonic development (E14); only the ARE-deleted but not the ARE-containing construct was expressed. Although GM-CSF AU(+) embryos were phenotypically normal, overexpression of GM-CSF in E14 GM-CSF AU(-) embryos led to severe hematopoietic alterations such as abnormal proliferation of granulocytes and macrophages accompanied by an increased number of peroxidase-expressing cells, their putative progenitor cells. These abnormalities compromise development because no viable GM-CSF AU(-) transgenic pups could be obtained. Surprisingly, by E18, significant accumulation of transgene messenger RNA was also observed in GM-CSF AU(+) embryos leading to similar phenotypic abnormalities. Altogether, these observations reveal that GM-CSF ARE is a developmentally controlled regulatory element and highlight the consequences of GM-CSF overexpression on myeloid cell proliferation and differentiation.

The helix-loop-helix transcription factor SCL (TAL1) is indispensable for blood cell formation in the mouse embryo. We have explored the localization and developmental potential of cells fated to express SCL during murine development using SCL-lacZ mutant mice in which the Escherichia coli lacZ reporter gene was 'knocked in' to the SCL locus. In addition to the hematopoietic defect associated with SCL deficiency, the yolk sac blood vessels in SCL(lacZ/lacZ) embryos formed an abnormal primary vascular plexus, which failed to undergo subsequent remodeling and formation of large branching vessels. Intraembryonic vasculogenesis in precirculation SCL(lacZ/lacZ) embryos appeared normal but, in embryos older than embryonic day (E) 8.5 to E9, absolute anemia leading to severe hypoxia precluded an accurate assessment of further vascular development. In heterozygous SCL(lacZ/w) embryos, lacZ was expressed in the central nervous system, vascular endothelia, and primitive and definitive hematopoietic cells in the blood, aortic wall, and fetal liver. Culture of fetal liver cells sorted for high and low levels of beta galactosidase activity from SCL(lacZ/w) heterozygous embryos indicated that there was a correlation between the level of SCL expression and the frequency of hematopoietic progenitor cells.

Currently little is known about the mechanisms regulating the homing and the early engraftment of prenatally transplanted hematopoietic cells due to the lack of a relevant functional assay. In this study, we have defined a reproducible kinetic profile of the homing and the early engraftment events in a murine model of prenatal stem cell transplantation. Light density mononuclear cells (LDMCs) from adult C57Pep3b and SJL/J marrow were transplanted by intraperitoneal (IP) injection into C57BL/6 fetuses (10(6) LDMCs/fetus) at 14 days of gestation. The fetuses were sacrificed at early time points (1.5 to 96 hours) after transplantation. Recipient fetal liver and cord blood were analyzed for donor cell frequency and donor cell phenotype by dual color flow cytometry. Pertinent findings included the following: (1) a triphasic kinetic profile exists after in utero hematopoietic stem cell (HSC) transplantation (homing of circulating donor cells, rapid reduction of donor cell frequency, and donor cell competitive equilibration); (2) homing to the fetal liver is nonselective and reflects the phenotypic profile of the donor population; and (3) the kinetics after the prenatal transplantation of congenic or fully allogeneic cells are identical. This model will facilitate a systematic analysis of the mechanisms that regulate the homing of prenatally transplanted hematopoietic cells.

We have developed a time-lapse camera system to follow the replication history and the fate of hematopoietic stem cells (HSC) at a single-cell level. Combined with single-cell culture, we correlated the early replication behavior with colony development after 14 days. The membrane dye PKH26 was used to monitor cell division. In addition to multiple, synchronous, and symmetric divisions, single-sorted CD34(+)/CD38(-) cells derived from fetal liver (FLV) also gave rise to a daughter cell that remained quiescent for up to 8 days, whereas the other daughter cell proliferated exponentially. Upon separation and replating as single cells onto medium containing a cytokine cocktail, 60.6% +/- 9.8% of the initially quiescent cells (PKH26 bright) gave rise again to colonies and 15.8% +/- 7.8% to blast colonies that could be replated. We have then determined the effects of various regulatory molecules on symmetry of initial cell divisions. After single-cell sorting, the CD34(+)/CD38(-) cells derived from FLV were exposed to flt3-ligand, thrombopoietin, stem cell factor (SCF), or medium containing a cytokine cocktail (with SCF, interleukin-3, interleukin-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin). Whereas mitotic rate, colony efficiency, and asymmetric divisions could be altered using various regulatory molecules, the asymmetric division index, defined as the number of asymmetric divisions versus the number of dividing cells, was not altered significantly. This observation suggests that, although lineage commitment and cell proliferation can be skewed by extrinsic signaling, symmetry of early divisions is probably under the control of intrinsic factors.

In this study, we examined the consequences of Fas deficiency on hematopoiesis in C57BL/6-lpr/lpr mice. We found a striking extramedullary increase in hematopoietic progenitor cells, comprising erythroid and nonerythroid lineages alike. These modifications preceded the lymphadenopathy, because early progenitors (colony-forming units-spleen [CFU-S] day 8) were already augmented in day-18 fetal livers of the lpr phenotype. Three weeks after birth, CFU-S increased in peripheral blood and spleen and colony-forming cells (CFU-C) began to accumulate 1 to 3 weeks later. Extramedullary myelopoiesis augmented progressively in Fas-deficient mice, reaching a maximum within 6 months. By then, mature and immature myeloid cells had infiltrated the spleen, the liver, and the peritoneal cavity. Similar changes occurred in C57BL/6-gld/gld mice, indicating that they resulted from Fas/FasL interactions. Medullary hematopoiesis was not significantly modified in adult mice of either strain. Yet, the incidence of CFU-S decreased after Fas cross-linking on normal bone marrow cells in the presence of interferon gamma, consistent with a regulatory function of Fas/FasL interactions in early progenitor cell development. These data provide evidence that Fas deficiency can affect hematopoiesis both during adult and fetal life and that these modifications occur independently from other pathologies associated with the lpr phenotype.

Required to supply nutrients and oxygen to the growing embryo, the vascular system is the first functional organ system to develop during vertebrate embryogenesis. Although there has been substantial progress in identifying the genetic cascade regulating vascular development, the initial stages of vasculogenesis, namely, the origin of vascular endothelial cells within the early embryo, remain unclear. To address this issue we constructed a fate map for specific vascular structures, including the aortic arches, endocardium, dorsal aorta, cardinal veins, and lateral abdominal veins, as well as for the red blood cells at the 16-cell stage and the 32-cell stage of Xenopus laevis. Using genetic markers to identify these cell types, our results suggest that vascular endothelial cells can arise from virtually every blastomere of the 16-cell-stage and the 32-cell-stage embryo, with different blastomeres preferentially, though not exclusively, giving rise to specific vascular structures. Similarly, but more surprisingly, every blastomere in the 16-cell-stage embryo and all but those in the most animal tier of the 32-cell-stage embryo serve as progenitors for red blood cells. Taken together, our results suggest that during normal development, both dorsal and ventral blastomeres contribute significantly to the vascular endothelial and red blood cell lineages., (Copyright 1999 Academic Press.)

We have previously shown that the HCA/ALCAM (CD166) glycoprotein, a member of the immunoglobulin family that mediates both homophilic and heterophilic cell-cell adhesion, via the CD6 ligand, is expressed at the surface of all of the most primitive CD38(-/lo), Thy-1(+), rho123(lo), CD34(+) hematopoietic cells in human fetal liver and fetal and adult bone marrow. In the present report we show that HCA is also expressed by subsets of stromal cells in the primary hematopoietic sites that sequentially develop in the human embryo and fetus, ie, the paraaortic mesoderm, liver, thymus, and bone marrow. Adult bone marrow stromal cells established in vitro, including those derived from Stro-1(+) progenitors and cells from immortalized cell lines, express HCA. In contrast, no HCA expression could be detected in peripheral lymphoid tissues, fetal spleen, and lymph nodes. HCA membrane molecules purified from marrow stromal cells interact with intact marrow stromal cells, CD34(+) CD38(-) hematopoietic precursors, and CD3(+) CD6(+) peripheral blood lymphocytes. Finally, low but significant levels of CD6 are here for the first time detected at the surface of CD34(+) rho123(med/lo) progenitors in the bone marrow and in mobilized blood from healthy individuals. Altogether, these results indicate that the HCA/ALCAM surface molecule is involved in homophilic or heterophilic (with CD6) adhesive interactions between early hematopoietic progenitors and associated stromal cells in primary blood-forming organs.

It is now accepted from studies in animal models that hematopoietic stem cells emerge in the para-aortic mesoderm-derived aorta-gonad-mesonephros region of the vertebrate embryo. We have previously identified the equivalent primitive hematogenous territory in the 4- to 6-week human embryo, under the form of CD34(+)CD45(+)Lin- high proliferative potential hematopoietic cells clustered on the ventral endothelium of the aorta. To characterize molecules involved in initial stem cell emergence, we first investigated the expression in that territory of known early hematopoietic regulators. We herein show that aorta-associated CD34(+) cells coexpress the tal-1/SCL, c-myb, GATA-2, GATA-3, c-kit, and flk-1/KDR genes, as do embryonic and fetal hematopoietic progenitors later present in the liver and bone marrow. Next, CD34(+)CD45(+) aorta-associated cells were sorted by flow cytometry from a 5-week embryo and a cDNA library was constructed therefrom. Differential screening of that library with total cDNA probes obtained from CD34(+) embryonic liver cells allowed the isolation of a kinase-related sequence previously identified in KG-1 cells. In addition to emerging blood stem cells, KG-1 kinase is also strikingly expressed in all developing endothelial cells in the yolk sac and embryo, which suggests its involvement in the genesis of both hematopoietic and vascular cell lineages in humans.

Translocations involving the human CBFA2 locus have been associated with leukemia. This gene, originally named AML1, is a human homologue of the Drosophila gene runt that controls early events in fly embryogenesis. To clarify the role of mammalian runt products in normal and leukemic hematopoiesis, we have studied their pattern of expression in mouse hematopoietic tissues in the adult and during ontogeny using an anti-runt box antiserum. In the adult bone marrow, we found expression of runt polypeptides in differentiating myeloid cells and in B lymphocytes. Within the erythroid lineage, runt expression is biphasic, clearly present in the erythroblasts of early blood islands and of the fetal liver, but absent in the adult. Biochemical analysis by Western blotting of fetal and adult hematopoietic populations shows several runt isoforms. At least one of them appears to be myeloid specific.

Hematopoiesis in the mouse conceptus begins in the visceral yolk (VYS), with primitive erythroblasts first evident in blood islands at the headfold stage (E8.0). VYS erythropoiesis is decreased or abrogated by targeted disruption of the hematopoietic transcription factors tal-1, rbtn2, GATA-1, and GATA-2. To better understand the potential roles of these genes, and to trace the initial temporal and spatial development of mammalian embryonic hematopoiesis, we examined their expression patterns, and that of betaH1-globin, in normal mouse conceptuses by means of in situ hybridization. Attention was focused on the 36-hour period from mid-primitive streak to early somite stages (E7.25 to E8.5), when the conceptus undergoes rapid morphologic changes with formation of the yolk sac and blood islands. Each of these genes was expressed in extraembryonic mesoderm, from which blood islands are derived. This VYS expression occurred in a defined temporal sequence: tal-1 and rbtn2 transcripts were detected earlier than the others, followed by GATA-2 and GATA-1, and then by betaH1-globin. Transcripts for all of these genes were present in VYS mesoderm cell masses at the neural plate stage (E7.5), indicating commitment of these cells to the erythroid lineage before the appearance of morphologically recognizable erythroblasts. By early somite stages (E8.5), GATA-2 mRNA expression is downregulated in VYS blood islands as terminal primitive erythroid differentiation proceeds. We conclude that primitive mammalian erythropoiesis arises during gastrulation through the ordered temporal expression of tal-1, rbtn2, GATA2, and GATA-1 in a subset of extraembryonic mesoderm cells. During the stages analyzed, tal-1 and rbtn2 expression was also present in posterior embryonic mesoderm, while GATA-1 and GATA-2 expression was evident in extraembryonic tissues of ectodermal origin.

In preliminary studies, we have analyzed the hematopoietic growth factor (HGF) requirement of hematopoietic progenitor cells (HPCs) purified from embryonic-fetal liver (FL) and grown in fetal calf serum-supplemented (FCS+) clonogenic culture. The key role of erythropoietin (Epo) for colony formation by early erythroid progenitors (burst-forming units-erythroid [BFU-E]) has been confirmed. Furthermore, in the absence of exogenous HGFs, FL monocytic progenitors (colony-forming unit monocyte [CFU-M]) generate large colonies exclusively composed of monocytes-macrophages; these colonies are absent in FCS- clonogenic culture. On this basis, we have investigated the role of all-trans retinoic acid (ATRA) and its isomer 9-cis RA in FL hematopoiesis. Both compounds modulate the growth of purified FL HPCs, which show a dose-dependent shift from mixed/erythroid/ monocytic to granulocytic colony formation. Studies on unicellular and paired daughter cell culture unequivocally indicate that the shift is mediated by modulation of the HPC differentiation program to the granulopoietic pathway (rather than RA-induced down-modulation of multipotent/ erythroid/monocytic HPC growth coupled with recruitment of granulocytic HPCs). ATRA and 9-cis RA also exert their effect on the proliferation of primitive HPCs (high-proliferative potential colony-forming cells [HPP-CFCs]) and putative hematopoietic stem cells (HSCs; assayed in Dexter-type long-term culture). High concentrations of either compound (1) drastically reduced the number of primary HPP-CFC colonies and totally abolished their recloning capacity and (2) inhibited HSC proliferation. It is crucial that these results mirror recent observations indicating that murine adult HPCs transduced with dominant negative ATRA receptor (RAR) gene are immortalized and show a selective blockade of granulocytic differentiation. Altogether, these results suggest that ATRA/9-cis RA may play a key role in FL hematopoiesis via a dual effect hypothetically mediated by interaction with the RAR/RXR heterodimer, ie, inhibition of HSC/ primitive HPC proliferation and induction of CFU-GEMM/ BFU-E/CFU-M shift from the multipotent/erythroid/monocytic to the granulocytic-neutrophilic differentiation program.

Differentiation of hematopoietic precursor cells results in the formation of clonally related descendent cells. Using the mosaic expression of beta-galactosidase in female mouse fetuses heterozygous for an X-linked lacZ transgene, we analyzed the clonal relationship of the hematopoietic progeny. The proportion of beta-galactosidase positive cells for different T- and B-lymphoid and myeloid cell populations was determined at different stages of fetal development. We found excellent correlations of the proportion of beta-galactosidase expressing cells for all hematopoietic lineages confirming that they share a common ancestry. Therefore, it was possible to estimate the number of common precursor cells (PC) based on binomial distribution and covariance analysis of pairs of different hematopoietic cell populations. Our results obtained from hematopoietic cells at 15.5 to 18.5 days of gestation indicated the presence of 15 to 18 lymphoid and 18 to 22 myeloid/lymphoid specific precursor cells. Statistical analysis of the precursor cell numbers showed a trend of increasing numbers that was highly significant. The precursor cell number was inversely related to maturity of the cell populations analyzed; ie, the lowest number of lymphoid and lymphoid/myeloid precursors was calculated when the most mature CD3+ T-cell population was used for comparison. Determination of PC numbers can therefore be used to assess the relative maturity and developmental potential of individual cell populations.

We examined long bones from 42 human embryos and fetuses whose gestational ages ranged from 6 to 28 weeks. Bone rudiment sections were stained using a panel of monoclonal antibodies directed at antigens expressed by hematopoietic cells, endothelial cells, smooth muscle cells, fibro-blasts, and stromal cells, to describe the events preceding and accompanying the onset of hematopoiesis in the diaphyseal region. Five distinct stages were identified. Stage I (6.6 to 8.5 gestational weeks [gw]) was that of entirely cartilaginous rudiments: chondrocytes were dilated and capillaries with CD34+ endothelial cells were observed in the perichondral limb mesenchyme. At stage II (8.5-9 gw) chondrolysis was actively proceeding; numerous CD68+ cells were observed, interspersed within the marrow cavity. Stage III (9 to 10.5 gw) was characterized by the development of the vascular bed in the absence of detectable hematopoiesis. At mid-diaphysis, specific structures that we named primary logettes were discernible; they consisted of small chambers of connective tissue, framed by a loose network of CD45-negative cells organized around an arteriole and limited from the surrounding sinus by a clearcut lining of CD34+ endothelial cells flanked on their abluminal side by alpha SM actin+ myoid cells. Stage IV (10.5-15 gw) was characterized by the onset of hematopoiesis. Hematopoietic cells were found exclusively in the primary logettes that had considerably increased in size. Logettes filled with hematopoietic cells were immersed within large and almost empty vascular sinuses. Logettes were attached by a short pedicle to connective tissue adjacent to bone/cartilage remaining formations; this tissue contained very rare hematopoietic cells. Logettes were few, usually less than 10 per long bone, and found solely in the diaphyseal area. Most hematopoietic cells found inside logettes were CD15+ myelocytes; rarely seen were glycophorin A+ immature erythroblasts and CD34+ nonendothelial cells. Hematopoietic cells within the logettes were in contact with alpha SM actin+ myoid cells and flattened endothelial-like (although consistently CD34-negative), aligned cells limiting small capillary lumina. Stage V (16 gw onward) was that of final organization of the long bones with areas of fully calcified bone and areas of dense hematopoiesis where logettes were no longer visible. This study shows three major features of incipient long bone hematopoiesis: 1) absence of CD34+ hematopoietic precursors before the onset of hematopoiesis and extreme rarity of those in the emerging blood-forming marrow, 2) predominance of granulopoiesis, and 3) exclusive development in specific structures organized by vascular cells. This study also suggests that CD68+ cells are instrumental in the chondrolysis process while vascular cells (endothelial and myoid cells) may be the critical microenvironment at the onset of hematopoiesis.

To elucidate the role of cytokine receptor signal transduction in T-cell development, we have investigated the expression pattern and biochemical characteristics of the murine Janus family tyrosine kinase, JAK3. Previous studies have shown that JAK3 is expressed in lymphoid and myeloid tumor cell lines and in a small number of lymphoid tissues. To further characterize JAK3 expression, we used a quantitative polymerase chain reaction approach to compare JAK3 mRNA levels at multiple stages of T-cell differentiation and in a broad range of mouse tissues. These studies, in conjunction with analyses of JAK3 protein expression, show that the highest levels of JAK3 are in adult, 2-week-old, and fetal thymus, followed by somewhat lower levels in bone marrow, spleen, fetal liver, and adult CD4-CD8- thymocytes. We also show that different forms of JAK3 mRNA arise by alternative splicing. Finally, our biochemical studies show that the JAK3 kinase domain, but not the pseudo-kinase domain, has tyrosine kinase activity and, furthermore, that JAK3 kinase activity is abolished by an amino acid substitution of the conserved lysine in the kinase domain (K851R). These studies show that JAK3 expression is profoundly skewed to hematopoietic and lymphoid precursor cells, strongly suggesting a role for JAK3 in hematopoiesis and T- and B-cell development.

Mice with a null mutation in the thrombopoietin (TPO) receptor c-Mpl were generated by gene targeting. c-mpl-deficient mice developed normally but were deficient in megakaryocytes and severely thrombocytopenic. The hematocrit and numbers of mature circulating leukocytes were normal in mpl-/- mice, as was the distribution of morphologically identifiable precursors in hematopoietic tissues. Bone marrow and spleen cells of adult mpl-/- mice lacked specific binding sites for TPO, were unresponsive to TPO in culture, and displayed a marked deficiency in progenitor cells with megakaryocytic potential. Significantly, total hematopoietic progenitor cell numbers were also reduced in mpl-/- mice including multipotential, blast cell, and committed progenitors of multiple lineages. The megakaryocyte deficiency was evident as early as 14 days of gestation in mpl-deficient mice, although reductions in progenitor cell numbers arose only later in development. The data suggest that the critical function of c-Mpl signalling in megakaryocytopoiesis is in maintenance of mature megakaryocyte numbers through control of progenitor cell proliferation and maturation. Moreover, our results also imply an important role for TPO and c-Mpl in the production of primitive pluripotent progenitor cells as well as progenitor cells committed to nonmegakaryocytic lineages.

The effects of a novel cytokine FLK2/FLT3 ligand (FL) on human fetal bone marrow-derived CD34+CD19+ pro-B cells were analyzed in a stromal-cell-independent, serum-deprived culture system. FL, like interleukin-3 (IL-3), synergized with IL-7 in promoting pro-B cell growth, and differentiation of these cells into CD34-CD19+clgM+slgM- pre-B cells, whereas a small proportion of these cells even differentiate into more mature slgM+ B cells. In contrast, KIT ligand (KL) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were ineffective in promoting IL-7-dependent pro-B cell growth and differentiation. Maximal levels of pro-B cell expansion, generally resulting in 15- to 30-fold increases in cellularity, were obtained in cultures supplemented with optimal doses of FL + IL-7 + IL-3. The addition of mouse bone marrow stromal cells further enhanced the proliferation and differentiation of pro-B cells obtained in the presence of these three cytokines. Under these conditions, cultures could be maintained for more than 4 weeks, and in general 40- to 50-fold increases in cell numbers were observed by 3 weeks of culture. The percentages of clgM+ and slgM+ B cells increased 1.5- to 3-fold and 2-fold, respectively, suggesting that stromal cells may provide additional costimulatory signals for human B-cell growth and differentiation that are different from IL-7, IL-3, and FL. Collectively, our results indicate that FL, in contrast to KL, strongly promotes long-term expansion and differentiation of human pro-B cells in the presence of IL-7 or in combination of IL-7 and IL-3, which is a novel property of this hematopoietic growth factor.

Recently, using immunohistochemical methods, we surprisingly found that endoplasmic reticulum glucose-6-phosphatase is present in human embryonic and fetal red blood cells (RBCs) but not in adult RBCs. The fact that an endoplasmic reticulum enzyme, whose major site of expression in adults is the liver, is present in human embryonic and fetal RBCs, particularly nucleated cells, indicated that it would be sensible to determine whether these cells also contain other endoplasmic reticulum enzyme systems normally found in adult liver. Therefore, we have studied the expression of other endoplasmic reticulum proteins and found that human embryonic and fetal RBC precursors contain other protein components of the glucose-6-phosphatase system, ie, the phosphate and glucose transport proteins as well as other enzymes (eg, uridine diphosphate-glucuronosyltransferases, cytochrome P450 isozymes, nicotinamide adenine dinucleotide phosphate cytochrome P450 oxidoreductase, and prostaglandin H synthase). In addition, we also found the predominantly cytosolic markers 15-hydroxyprostaglandin dehydrogenase, prostaglandins PGE2 and 13,14-dihydro-15-keto-PGE2. The expression of key enzymes that control glucose production, detoxification of endobiotics and xenobiotics, and the regulation of prostaglandin levels in embryonic and early fetal RBCs means that these cells may have an important role in protecting the developing conceptus before it establishes an efficient circulation and before all tissues fully express their normal complement of these enzymes.

Although the pluripotent hematopoietic stem cell can only be definitively identified by its ability to reconstitute the various mature blood lineages, a diversity of cell surface antigens have also been specifically recognized on this subset of hematopoietic progenitors. One such stem cell-associated antigen is the sialomucin CD34, a highly O-glycosylated cell surface glycoprotein that has also been shown to be expressed on all vascular endothelial cells throughout murine embryogenesis as well as in the adult. The functional significance of CD34 expression on hematopoietic progenitor cells and developing blood vessels is unknown. To analyze the involvement of CD34 in hematopoiesis, we have produced both embryonic stem (ES) cells and mice that are null for the expression of this mucin. Analysis of yolk saclike hematopoietic development in embryoid bodies derived from CD34-null ES cells showed a significant delay in both erythroid and myeloid differentiation that could be reversed by transfection of the mutant ES cells with CD34 constructs expressing either a complete or truncated cytoplasmic domain. Measurements of colony-forming activity of hematopoietic progenitor cells derived from yolk sacs or fetal livers isolated from CD34-null embryos also showed a decreased number of these precursor cells. In spite of these diminished embryonic hematopoietic progenitor numbers, the CD34-null mice developed normally, and the hematopoietic profile of adult blood appeared typical. However, the colony-forming activity of hematopoietic progenitors derived from both bone marrow and spleen is significantly reduced in adult CD34-deficient animals, and these CD34-deficient progenitors also appear to be unable to expand in liquid cultures in response to hematopoietic growth factors. Even with these apparent progenitor cell deficiencies, CD34-null animals showed kinetics of erythroid, myeloid, and platelet recovery after sublethal irradiation that are indistinguishable from wild-type mice. These data strongly suggest that CD34 plays an important role in the formation of progenitor cells during both embryonic and adult hematopoiesis. However, the hematopoietic sites of adult CD34-deficient mice may still have a significant reservoir of progenitor cells that allows for normal recovery after nonmyeloablative peripheral cell depletion.

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.

An early step in the formation of the extraembryonic and intraembryonic vasculature is endothelial cell differentiation and organization in blood islands and vascular structures. This involves the expression and function of specific adhesive molecules at cell-to-cell junctions. Previous work showed that endothelial cells express a cell-specific cadherin (vascular endothelial [VE]-cadherin, or 7B4/cadherin-5) that is organized at cell-to-cell contacts in cultured cells and is able to promote intercellular adhesion. In this study, we investigated whether VE-cadherin could be involved in early cardiovascular development in the mouse embryo. We first cloned and sequenced the mouse VE-cadherin cDNA. At the protein level, murine VE-cadherin presented 75% identity (90%, considering conservative amino acid substitutions) with the human homologue. Transfection of murine VE-cadherin cDNA in L cells induced Ca(++)-dependent cell-to-cell aggregation and reduced cell detachment from monolayers. In situ hybridization of adult tissues showed that the murine molecule is specifically expressed by endothelial cells. In mouse embryos, VE-cadherin transcripts were detected at the very earliest stages of vascular development (E7.5) in mesodermal cells of the yolk sac mesenchyme. At E9.5, expression of VE-cadherin was restricted to the peripheral cell layer of blood islands that gives rise to endothelial cells. Hematopoietic cells in the center of blood islands were not labeled. At later embryonic stages, VE-cadherin transcripts were detected in vascular structures of all organs examined, eg, in the ventricle of the heart, the inner cell lining of the atrium and the dorsal aorta, in intersomitic vessels, and in the capillaries of the developing brain. A comparison with flk-1 expression during brain angiogenesis revealed that brain capillaries expressed relatively low amounts of VE-cadherin. In the adult brain, the level of VE-cadherin transcript was further reduced. By immunohistochemistry, murine VE-cadherin protein was detected at cell-to-cell junctions of endothelial cells. Overall, these data demonstrate that VE-cadherin is an early, constitutive, and specific marker of endothelial cells. This distinguishes this molecule from other cadherins and suggests that its expression is associated with the early assembly of vascular structures.

To determine whether signaling via CD122 (interleukin-2 [IL-2]/IL-15 receptor beta-chain) plays a role in regulating the expansion and differentiation of lymphocyte precursors, we have characterized its expression and evaluated its ability to influence the activity of developing lymphoid cells. A significant fraction of Sca1+Lin- hematopoietic stem cells in day 12 fetal liver were found to be CD122+. CD122-mRNA+ and IL-2-mRNA+ cells were also localized in embryo sections within pharyngeal blood vessels adjacent to and surrounding the thymic analgen. This distribution is consistent with the migration of CD122+ progenitor cells from the liver to the developing thymus where a majority of Sca1+ intrathymic T-cell progenitors were CD122+. Analysis of CD122 expression in the day 12 fetal liver revealed that the majority of B220+ cells were CD122+. Furthermore, CD122 expression was restricted to the earliest B220+ cells (CD43+CD24-; prepro B cells; fraction A) that proliferate vigorously to IL-2 in the absence of any stromal cells, but not to IL-15. Consistent with a role for the IL-2/IL-2R pathway in lymphocyte development is the progressive loss of B cells seen in IL-2-deficient mice. Together, these observations suggest that CD122 plays a role in regulating normal lymphocyte development in vivo.