Search

The Barker hypothesis of 'foetal origin of adult diseases' has led to emphasize the concept of 'developmental programming', based on the crucial role of epigenetic factors. Accordingly, it has been demonstrated that parental adversity (before conception and during pregnancy) and foetal factors (i.e., hypoxia, malnutrition and placental insufficiency) permanently modify the physiological systems of the progeny, predisposing them to premature ageing and chronic disease during adulthood. Thus, an altered functionality of the endocrine, immune, nervous and cardiovascular systems is observed in the progeny. However, it remains to be understood whether the haematopoietic system itself also represents a portrait of foetal programming. Here, we provide evidence, reporting and discussing related theories, and results of studies described in the literature. In addition, we have outlined our opinions and suggest how it is possible to intervene to correct foetal mal-programming. Some pro-health interventions and recommendations are proposed, with the hope of guarantee the health of future generations and trying to combat the continuous increase in age-related diseases in human populations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Tunneling nanotubes (TNTs) are newly discovered tubular structures between two distant cells that facilitate the intercellular exchange of signals and components. Recent reports show that mesenchymal stem cells (MSCs) can rescue injured target cells and promote recovery from a variety of stresses via TNT-mediated mitochondrial transfer. In this study, we explored how TNTs form between bone marrow MSCs and endothelial cells (ECs) by using a human umbilical cord vein endothelial cell (HUVEC) model. TNT formation between MSCs and HUVECs could be induced by treating HUVECs with cytarabine (Ara-C), and human bone marrow mesenchymal stem cells (hBMMSCs) could transfer mitochondria to injured HUVECs through TNTs. Mitochondrial transfer from hBMMSCs to HUVECs via TNTs rescued the injured HUVECs by reducing apoptosis, promoting proliferation and restoring the transmembrane migration ability as well as the capillary angiogenic capacity of HUVECs. This study provides novel insights into the cell-cell communication between MSCs and ECs and supports the results of prior studies indicating that ECs promote hematopoietic regeneration. An improved understanding of MSC-EC cross-talk will promote the development of MSC-directed strategies for improving EC function and hematopoietic system regeneration following myelosuppressive and myeloablative injuries.

Circadian rhythms regulate over 40% of protein-coding genes in at least one organ in the body through mechanisms tied to the central circadian clock and to cell-intrinsic auto-regulatory feedback loops. Distinct diurnal differences in regulation of regeneration have been found in several organs, including skin, intestinal, and hematopoietic systems. Each regenerating system contains a complex network of cell types with different circadian mechanisms contributing to regeneration. In this review, we elucidate circadian regeneration mechanisms in the three representative systems. We also suggest circadian regulation of global translational activity as an understudied global regulator of regenerative capacity. A more detailed understanding of the molecular mechanisms underlying circadian regulation of tissue regeneration would accelerate the development of new regenerative therapies.

The major goal in regenerative medicine is to repair and restore injured, diseased or aged tissue function, thereby promoting general health. As such, the field of regenerative medicine has great translational potential in undertaking many of the health concerns and needs that we currently face. In particular, hematopoietic and vascular systems supply oxygen and nutrients and thus play critical roles in tissue development and tissue regeneration. Additionally, tissue vasculature serves as a tissue stem cell niche and thus contributes to tissue homeostasis. Notably, hematopoietic and vascular systems are sensitive to injury and subject to regeneration. As such, successful hematopoietic and vascular regeneration is prerequisite for efficient tissue repair and organismal survival and health. Recent studies have established that the interplay among the ETS transcription factor ETV2, vascular endothelial growth factor, and its receptor VEGFR2/FLK1 is essential for hematopoietic and vascular development. Emerging studies also support the role of these three factors and possible interplay in hematopoietic and vascular regeneration. Comprehensive understanding of the molecular mechanisms involved in the regulation and function of these three factors may lead to more effective approaches in promoting tissue repair and regeneration. Developmental Dynamics 246:318-327, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The GATA family of transcription factors consists of six proteins (GATA1-6) which are involved in a variety of physiological and pathological processes. GATA1/2/3 are required for differentiation of mesoderm and ectoderm-derived tissues, including the haematopoietic and central nervous system. GATA4/5/6 are implicated in development and differentiation of endoderm- and mesoderm-derived tissues such as induction of differentiation of embryonic stem cells, cardiovascular embryogenesis and guidance of epithelial cell differentiation in the adult.

Introduction and Objectives: The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2) transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. Targeted genome editing is an increasingly applied method for functional assessment of candidate genes. We therefore generated a stable klf2a mutant zebrafish and characterised its cardiovascular and haematopoietic development., Methods and Results: Using Transcription Activator-Like Effector Nucleases (TALEN) we generated a klf2a mutant (klf2ash317) with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC) development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl), a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17) in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants., Conclusions: The klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development.

As major immunological and hematological parameters evolve during the early period of life, laboratory data must be interpreted in relation to developmental changes. Wistar (WU) rats were sacrificed on PND2, 4, 7, 10, 14, 17 and 21. Peripheral blood, bone marrow, thymus samples and spleen cells were collected and a bronchoalveolar lavage (BAL) performed. Parameters of blood counts changed considerably between time points. IgM and IgG levels steadily increased. Spontaneous spleen cell proliferation was low before PND21, although mitogens had stimulatory effects above baseline. In the spleen, T-lymphocyte counts tripled by PND17 (mainly attributed to CD8(+) cytotoxic T-cells and CD4(+) T-helper cells). In peripheral blood an increase in B-lymphocytes to about 60% of the cell number was observed. In BAL fluid, macrophages represented 95-98% of the cells. In thymus architecture, lymphoblast migration was seen and epithelial structures appeared. The data presented will help to distinguish between maturational changes and treatment-related effects., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The animal body is composed of a variety of cells and extracellular matrices that are organized and orchestrated in a harmonized manner to support life. Therefore, the critical importance of a comprehensive understanding of the molecular network surrounding and integrating the cells is now emphasized. The CCN family is a novel group of matricellular proteins that interact with and orchestrate a number of extracellular signaling and matrix molecules to construct and maintain living tissues. This family comprises six distinct members in mammals, which are characterized by a unique and conserved modular structure. These proteins are not targeted to limited and specific receptors to execute specific missions, but manipulate a vast number of biomolecules in the network by serving as a molecular hub at the center. The unified nomenclature, CCN, originates from a simple acronym of the three classical members, which helps us to avoid having any preconception about their pleiotropic and anonymous functional nature. In this review, after a brief summary of the general molecular concepts regarding the CCN family, new aspects of each member uncovered by recent research are introduced, which represent, nevertheless, only the tip of the iceberg of the profound functionality of these molecules.

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.

Hematopoietic development is orchestrated by gene regulatory networks that progressively induce lineage-specific transcriptional programs. To guarantee the appropriate level of complexity, flexibility, and robustness, these networks rely on transcriptional and post-transcriptional circuits involving both transcription factors (TFs) and microRNAs (miRNAs). The focus of this review is on RUNX1 (AML1), a master hematopoietic transcription factor which is at the center of miRNA circuits necessary for both embryonic and post-natal hematopoiesis. Interference with components of these circuits can perturb RUNX1-controlled coding and non-coding transcriptional programs in leukemia.

ATP-dependent SWI/SNF-like BAF chromatin remodeling complexes are emerging as key regulators of embryonic and adult stem cell function. Particularly intriguing are the findings that specialized assemblies of BAF complexes are required for establishing and maintaining pluripotent and multipotent states in cells. However, little is known on the importance of these complexes in normal and leukemic hemopoiesis. Here we provide the first evidence that the actin-related protein BAF53a, a subunit of BAF complexes preferentially expressed in long-term repopulating stem cells, is essential for adult hemopoiesis. Conditional deletion of BAF53a resulted in multilineage BM failure, aplastic anemia, and rapid lethality. These severe hemopoietic defects originate from a proliferative impairment of BM HSCs and progenitors and decreased progenitor survival. Using hemopoietic chimeras, we show that the impaired function of BAF53a-deficient HSCs is cell-autonomous and independent of the BM microenvironment. Altogether, our studies highlight an unsuspected role for BAF chromatin remodeling complexes in the maintenance of HSC and progenitor cell properties.

Objective: The balance between proliferation and differentiation during hematopoietic development in the embryo is a complex process, the detailed molecular mechanisms of which remain to be fully characterized. The transcription factor Mxd4, a member of the Myc-Max-Mad network, was identified in a global gene expression profiling screen as being tightly regulated at the onset of hematopoietic lineage specification upon in vitro differentiation of mouse embryonic stem cells. Our study investigated the Mxd4 expression pattern at the onset of hematopoiesis and the biological relevance of its sharp and transient downregulation., Materials and Methods: To study the expression pattern and role of Mxd4 at the onset of hematopoiesis, the in vitro differentiation of embryonic stem cells was used as a model system. Gain of function assays were performed using a doxycycline-inducible embryonic stem cell system., Results: We show here that Mxd4 expression is transiently downregulated at an early stage of commitment to the hematopoietic lineage. Enforced expression of Mxd4 at this period of differentiation results in a defect in hematopoietic progenitor development, with impaired development of both primitive and definitive blood lineages. This effect is due to a severe decrease in cell proliferation, with an increased frequency of cells in the G(0)/G(1) phase of the cell cycle, alongside a reduced frequency of cells in the S phase., Conclusions: Together our results indicate that during embryonic hematopoietic differentiation Mxd4 is an important player in the regulation of blood progenitor proliferation, and suggest that downregulation of its expression might be required for a proliferative burst preceding lineage specification., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

During development hematopoietic stem cells (HSCs) expand in number and persist throughout life by undergoing self-renewing divisions. Nevertheless, the hematopoietic system does not escape the negative effects of aging, suggesting that self-renewal is not complete. A fundamental issue in stem cell biology relates to such age-dependent loss of stem cell activity. Both stem cell intrinsic factors and extrinsic factors associated with an aging micro-environment could contribute to aging of the hematopoietic system. Recently, changes in the clonal composition of the HSC compartment during aging have been put forward as a key factor. Here, we discuss these recent developments and speculate how they may be of clinical relevance., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: RUNX1 (also known as acute myeloid leukemia 1) is an essential regulator of hematopoiesis and has multiple isoforms arising from differential splicing and utilization of two promoters. We hypothesized that the rare Runx1c isoform has a distinct role in hematopoietic stem cells (HSCs)., Materials and Methods: We have characterized the expression pattern of Runx1c in mouse embryos and human embryonic stem cell (hESC)-derived embryoid bodies using in situ hybridization and expression levels in mouse and human HSCs by real-time polymerase chain reaction. We then determined the functional effects of Runx1c using enforced retroviral overexpression in mouse HSCs., Results: We observed differential expression profiles of RUNX1 isoforms during hematopoietic differentiation of hESCs. The RUNX1a and RUNX1b isoforms were expressed consistently throughout hematopoietic differentiation, whereas the RUNX1c isoform was only expressed at the time of emergence of definitive HSCs. RUNX1c was also expressed in the AGM region of E10.5 to E11.5 mouse embryos, the region where definitive HSCs arise. These observations suggested that the RUNX1c isoform may be important for the specification or function of definitive HSCs. However, using retroviral overexpression to study the effect of RUNX1 isoforms on HSCs in a gain-of-function system, no discernable functional difference could be identified between RUNX1 isoforms in mouse HSCs. Overexpression of both RUNX1b and RUNX1c induced quiescence in mouse HSCs in vitro and in vivo., Conclusions: Although the divergent expression profiles of Runx1 isoforms during development suggest specific roles for these proteins at different stages of HSC maturation, we could not detect an important functional distinction in adult mouse HSCs using our assay systems., (2010 ISEH-Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

The global obesity epidemic demands an improved understanding of the developmental and environmental factors regulating fat storage. Adipocytes serve as major sites of fat storage and as regulators of energy balance and inflammation. The optical transparency of developing zebrafish provides new opportunities to investigate mechanisms governing adipocyte biology, however zebrafish adipocytes remain uncharacterized. We have developed methods for visualizing zebrafish adipocytes in vivo by labeling neutral lipid droplets with Nile Red. Our results establish that neutral lipid droplets first accumulate in visceral adipocytes during larval stages and increase in number and distribution as zebrafish grow. We show that the cellular anatomy of zebrafish adipocytes is similar to mammalian white adipocytes and identify peroxisome-proliferator activated receptor gamma and fatty acid binding protein 11a as markers of the zebrafish adipocyte lineage. By monitoring adipocyte development prior to neutral lipid deposition, we find that the first visceral preadipocytes appear in association with the pancreas shortly after initiation of exogenous nutrition. Zebrafish reared in the absence of food fail to form visceral preadipocytes, indicating that exogenous nutrition is required for adipocyte development. These results reveal homologies between zebrafish and mammalian adipocytes and establish the zebrafish as a new model for adipocyte research.

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.

Developmental programs of blood and endothelium are closely correlated and remarkably conserved among species. To categorize the ontogeny relationship between hematopoietic and endothelial lineages, two putative models are presented here. In the yolk sac, hematopoietic and endothelial cells are more likely derived from a common precursor--the hemangioblast. By comparison, the hemogenic endothelium is proposed to characterize the generation of hematopoietic stem cells from mature endothelium in the P-Sp/AGM region. Furthermore, couples of molecules, including Scl, Flk-1 and Runx-1, are involved in formation and subsequent differentiation of the hemangioblast or hemogenic endothelium during embryonic hematopoiesis. In this article, the development of primitive and definitive hematopoiesis, two models associated with development of hematopoietic and vascular endothelial cells as well as molecules associated with development of hematopoietic and endotheliate cells were summaried.

HSCs differ during ontogeny in some important parameters, including anatomic site of residence and cell cycling characteristics. In this issue of the JCI, Bowie et al. show that postnatal HSCs as well as fetal liver HSCs in mice are active in the cell cycle at much higher rates than that of adult HSCs; however, this increased frequency of cycling abruptly ceases 4 weeks after birth (see the related article beginning on page 2808). The cycling postnatal HSCs expressed high levels of CXC chemokine ligand 12 (CXCL12, also known as stromal cell-derived factor 1 [SDF-1]), a chemokine previously implicated in stem cell trafficking to the marrow cavity and shown to be expressed by cells within the hematopoietic microenvironment. These cells also possessed an engraftment defect impeding reconstitution in irradiated recipient mice, which was reversible by pretransplant administration of antagonists of the CXCL12 receptor, CXCR4. Such agents are currently clinically available, suggesting that this approach could be used to improve stem cell transplantation and engraftment.

The regulation of HSC proliferation and engraftment of the BM is an important but poorly understood process, particularly during ontogeny. Here we show that in mice, all HSCs are cycling until 3 weeks after birth. Then, within 1 week, most became quiescent. Prior to 4 weeks of age, the proliferating HSCs with long-term multilineage repopulating activity displayed an engraftment defect when transiting S/G2/M. During these cell cycle phases, their expression of CXC chemokine ligand 12 (CXCL12; also referred to as stromal cell-derived factor 1 [SDF-1]) transiently increased. The defective engrafting activity of HSCs in S/G2/M was reversed when cells were allowed to progress into G1 prior to injection or when the hosts (but not the cells) were pretreated with a CXCL12 antagonist. Interestingly, the enhancing effect of CXCL12 antagonist pretreatment was exclusive to transplants of long-term multilineage repopulating HSCs in S/G2/M. These results demonstrate what we believe to be a new HSC regulatory checkpoint during development. They also suggest an ability of HSCs to express CXCL12 in a fashion that changes with cell cycle progression and is associated with a defective engraftment that can be overcome by in vivo administration of a CXCL12 antagonist.

Term newborns have a higher frequency of microbial infections than older children and adults. Extremely premature newborns (<28 weeks gestation) have an even higher frequency. Quantitative and qualitative differences in the development of the immune system have been identified as a partial explanation for the increase in the incidence of infectious sequelae in these two patient populations. A less studied population of patients is late preterm newborns that are 34 to 35 6/7 weeks gestation. In general, this subset of patients is frequently grouped with term newborns. However, recent studies have provided data suggesting a potential unrecognized risk to health in this population, including at least a clinical suspicion for an increased risk of sepsis. Although little specific data on the host-defense capability of the near-term newborn exist, recent advancements in developmental immunology provide a framework for understanding the mechanisms underlying the propensity of infections in the preterm, near-term, and term newborn.

The ontogeny of the teleost innate immune system was studied in carp using cellular, histological and quantitative molecular techniques. Carp myeloid cells first appeared ventro-lateral of the aorta at 2 days post fertilization (the start of hatching), and subsequently around the sinuses of the vena cardinalis (or posterior blood islet), head kidney and trunk kidney. In addition, the hematopoietic tissue around the sinuses of the vena cardinalis transformed into that of the trunk kidney, which is the first description of the ontogeny of the trunk kidney hematopoietic tissue in teleosts. The mAb's used in this study reacted with carp myeloid surface molecules that are already transcribed and processed from the first appearance of myeloid cells, and thus serve a significant role in unraveling ontogenetic processes of teleost immunology. Finally, this study associated the first appearance of myeloid cells with an immune response on the molecular level: 2 days post fertilization embryos responded to LPS injection with upregulation of interleukin 1-beta, inducible nitric oxide synthase and serum amyloid A, and down-regulation of complement factor 3 and alpha2-macroglobulin, implying a functional embryonic innate defense system.

All mature blood cells originate from a small population of self-renewing pluripotent hematopoietic stem cells (HSCs). The capacity to self-renew characterizes all stem cells, whether normal or neoplastic. Interestingly, recent studies suggest that self-renewal is essential for tumor cell maintenance, implicating that this process has therapeutic relevance. Unfortunately, the molecular bases for self-renewal of vertebrate cells remain poorly defined. This article will focus on the developmental mechanisms underlying fetal and adult HSC homeostasis. Specifically, distinctions between genetic programs regulating HSC specification (identity), self-renewal (in both fetal and adult) and differentiation/commitment will be discussed with a special emphasis on transcriptional and chromatin regulators.

This review explores the evolutionary origins of lymphocyte development by focusing on the transcription factors that direct mammalian lymphocyte development today. Gene expression data suggest that the programs to make lymphocytes involve the same transcription factor ensembles in all animals with lymphocytes. Most of these factors, GATA, Runx, PU.1/Spi, EBF/Olf, Ikaros, and Pax-2/5/8 family members, are also encoded in the genomes of animals without lymphocytes. We consider the functions of these factors in animals without lymphocytes in terms of discrete program components, which could have been assembled in a new way to create the lymphocyte developmental program approximately 500 My ago.

The development of the liver, bone marrow and spleen have been investigated in the stripe-faced dunnart. At birth, the liver was undergoing haematopoiesis but the level declined rapidly and by day 50 after birth the liver was histologically mature. Both the bone marrow and spleen were non-haematopoietic at birth but initiated haematopoiesis shortly thereafter. Bone marrow was initially detected at day 11 postpartum. By 57 days after birth, adipocytes had infiltrated the marrow and were abundant by day 60 after birth. Mitotic cells were observed in remaining areas of marrow until at least 170 days postpartum. The spleen at birth was undifferentiated, with trabeculae appearing by day 42. Red and white pulp areas became apparent by day 43 and were well defined by day 57 after birth. In summary, the pattern of the development of the liver, bone marrow and spleen in the stripe-faced dunnart were similar to that observed in eutherians and other metatherians studied to date.

Cryostat sections of bovine embryos of exactly known age (obtained from artificial insemination), ranging from 32 to 60 days post-insemination, were treated with a wide range of antibodies directed against cell surface antigens or lineage-specific factors in order to demonstrate different types of fetal blood cells and their precursors. An antibody specific to bovine c-kit (bk-1) stained not only presumptive haematopoietic stem cells in the dorsal aorta and the embryonic liver, but also a subpopulation of putative primordial germ cells in the gonadal anlage, the latter being further characterised by a positive labelling with the lectins STA, WFA and WGA and a histochemical reaction for alkaline phosphatase. The antibody against CD 45, commonly regarded as a pan-leukocyte marker, reacted in the bovine embryo with different types of blood cells, as well as with presumptive vasculogenetic cells and a subpopulation of putative primordial germ cells. CD 61 immunoreaction proved to be a useful tool for demonstrating megakaryocytopoiesis in the embryonic liver, in addition to the lumen of blood vessels and the mesonephros. Staining with BM-2 was restricted to a single population of medium-sized, round to oval cells, forming small groups within the parenchymal strands of the liver. Characterised furthermore by a U-shaped nucleus, this BM-2-positive cell type apparently represents a developmental stage in the granulopoietic lineage. B-lymphocytopoiesis in the bovine liver was detected with antibodies directed against WC-4 and IgM, but not until day 58 post-insemination. Using antibodies to CD 14, no positive results could be obtained in embryonic tissues, although anti-CD 14-positive macrophages were easily recognised in lymph nodes of adult bovines. The antibody against CD 68, however, identified two populations of primitive macrophages in our samples. One population was located in parenchymal strands of the embryonic liver, probably acting as nursing cells for haematopoietic foci, and the other was observed intravasally in the sinusoids of the liver, most probably representing primitive Kupffer cells.

Objective: STAT5A/5B-deficient mice are recognized to manifest defects in multiple cell types and tissues. In particular, the hematopoietic defects in these mice are widespread, affecting multiple lineages and multiple stages of development. Previous studies indicate that deficiencies intrinsic to hematopoietic cells contribute substantially to the observed defects. However, in light of the broad physiologic effects of STAT5 in the context of the organism outside the blood system, we wished to investigate the possibility of STAT5-dependent environmental influence of nonhematopoietic origin on hematopoietic development in these mice., Materials and Methods: We transplanted wild-type bone marrow into STAT5A/5B-deficient mice to determine the effects of loss of STAT5 in nonhematopoietic tissue on hematopoietic development., Results: We observed that transplantation of wild-type marrow completely corrects hematopoietic defects in STAT5A/5B-deficient recipient mice, including peripheral blood counts, bone marrow cellularity, and reductions in specific progenitor subsets., Conclusion: These results indicate that the important role of STAT5 in hematolymphoid development are mediated directly through effects on hematopoietic cells and not indirectly.

Objective: Cytokines bind high-affinity receptors expressed on hematopoietic cells to initiate signaling cascades that regulate differentiation, proliferation, and survival. Previous studies have established a role for STAT5 in transducing survival signals for hematopoietic progenitor cells in response to cytokines., Materials and Methods: To determine if constitutive expression of a member of the bcl-2 family of anti-apoptotic proteins could compensate for the loss of STAT5, we utilized combinatorial genetics to generate STAT5A/5B-deficient mice expressing a bcl-2 transgene., Results: Although bcl-2 expression restored peripheral blood counts to normal in STAT5A/5B(-/-) mice, we noted a striking failure of this transgene to correct defects in hematopoietic stem and progenitor cells., Conclusion: These data imply important effects of STAT5 in modulating hematopoietic cells in addition to promoting survival per se.

The zebrafish is firmly established as a genetic model for the study of vertebrate blood development. Here we have characterized the blood-forming system of adult zebrafish. Each major blood lineage can be isolated by flow cytometry, and with these lineal profiles, defects in zebrafish blood mutants can be quantified. We developed hematopoietic cell transplantation to study cell autonomy of mutant gene function and to establish a hematopoietic stem cell assay. Hematopoietic cell transplantation can rescue multilineage hematopoiesis in embryonic lethal gata1-/- mutants for over 6 months. Direct visualization of fluorescent donor cells in embryonic recipients allows engraftment and homing events to be imaged in real time. These results provide a cellular context in which to study the genetics of hematopoiesis.

An understanding of developmental hemostasis is pivotal for optimal prevention, diagnosis, and treatment of hemostatic problems during childhood. The development of microassays in the early 1980s enabled researchers to delineate age-dependent features of the coagulation system and to establish reference ranges for healthy children of all age groups, from premature infants to adolescents. Based on the results from these studies, the hemostatic system in the young can be described as evolving, and yet functional, since healthy fetuses, infants, and children do not suffer hemorrhagic nor thromboembolic complications spontaneously or in the presence of minor challenges. Plasma concentrations of most pro- and anticoagulant proteins are decreased throughout childhood but provide an effective hemostatic balance on a lower level compared with adults. The current article describes the development of pro- and anticoagulant systems throughout childhood.

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.

Careful study of the phylogeny and ontogeny of the three components of the immune system reveals that the macrophage, lymphatic, and hematopoietic systems originate independently of each other. Chronologically, the most ancient is the macrophage system, which arises in the coelomic cavity as mesenchymal ameboid cells having the properties to recognize self from non-self and to ingest foreign particles. The lymphatic system later develops from the endoderm of pharyngeal pouches, where the thymic anlage differentiates. The lymphocytes that originate here seed all lymphatic organs and retain the ability to divide and thereby form multiple colonies (lymphatic nodules) in the respiratory and digestive tract; further diversification of lymphocytes follows after confrontation with antigens. The last component of the immune system to appear is the hematopoietic system, which originates from the splanchnic mesoderm of the yolk sac as hematogenic tissue, containing hemangioblasts. The hematogenic tissue remains attached to the outer wall of the vitelline vessels, which provides an efficient mechanism for introducing the hematogenic tissue into the embryo. In an appropriate microenvironment, the hemangioblasts give rise to sinusoidal endothelium and to hemocytoblasts - the bone marrow stem cells for erythrocytes, myeloid cells, and megakaryocytes. The facts and opinions presented in this article are not in agreement with the currently accepted dogma that a common "hematolymphatic stem cell" localized in the marrow generates all of the cellular components of blood and the immune system.

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.

Objective: Studies on mice lacking the common receptor subunit gp130 reveal that activation of gp130-dependent signaling pathways is essential for normal fetal and adult hematopoiesis. However, the extent to which hematopoiesis is dependent upon activation of a particular gp130 signaling pathway, namely STAT1/3 or SHP2/MAPK, is unknown. This study examined the specific contribution of gp130-mediated STAT1/3 signaling to the regulation of hematopoiesis., Materials and Methods: Hematopoiesis was examined at various developmental stages in mice homozygous for a targeted carboxy-terminal truncation mutation in gp130 (gp130(delta)/(delta)) that deletes all STAT1/3 binding sites, thereby abolishing gp130-mediated STAT1/3 activation., Results: Adult gp130(delta)/(delta) mice have increased numbers of immature colony-forming unit spleen progenitor cells in the bone marrow and spleen, elevated numbers of committed myeloid progenitor cells in the spleen and peripheral blood, and leukocytosis. Increased progenitor cell production was observed in gp130(delta)/(delta) fetal livers from 14 days of gestation onward. In contrast, the circulating platelet count was reduced by 30% in gp130(delta)/(delta) mice, without any corresponding decrease in the number of bone marrow and splenic megakaryocytes. In liquid cultures, megakaryocytes from gp130(delta)/(delta) mice are smaller than those from wild-type mice and do not increase in size upon stimulation with interleukin-6 or interleukin-11. Administration of either interleukin-6 or interleukin-11 to gp130(delta)/(delta) mice failed to increase platelet numbers, despite an increase in the production of megakaryocytes., Conclusions: Collectively, these results reveal that gp130-mediated STAT1/3 activation is required to maintain the normal balance of hematopoietic progenitors during fetal and adult hematopoiesis. Furthermore, they suggest two distinct roles for gp130-mediated STAT1/3 activation in hematopoiesis, one restricting the production of immature hematopoietic progenitor cells and the other promoting the functional maturation of megakaryocytes to produce platelets.

Mobilization of hematopoietic stem and progenitor cells from the bone marrow into the circulation by repetitive, daily stimulations with G-CSF alone, or in combination with cyclophosphamide, is increasingly used clinically; however, the mechanism is not fully understood. Moreover, following mobilization stem cells also home back to the bone marrow, suggesting that stem cell release/mobilization and homing are sequential events with physiological roles. Previously, a role for cytokines such as G-CSF and SCF, and adhesion molecules such as VLA-4 and P/E selectins, was determined for stem cell mobilization. Recent results using experimental animal models and samples from clinical mobilization protocols demonstrate major involvement of chemokines such as stromal derived factor-1 (SDF-1) and IL-8, as well as proteolytic enzymes such as elastase, cathepsin G, and various MMPs in the mobilization process. These results will be reviewed together with the central roles of SDF-1 and CXCR4 interactions in G-CSF or G-CSF in combination with cyclophosphamide-induced mobilization. Furthermore, the central role of this chemokine in stem cell homing to the bone marrow as well as retention of undifferentiated cells within this tissue will also be discussed.

Objective: Hematopoietic stem cells (HSCs) and colony-forming progenitor cells (CFCs) are believed to migrate from liver to bone marrow (BM) around the time of birth, where they remain throughout the animal's life. Although in mice the spleen is also a hematopoietic organ, neither the origin nor the contribution of spleen HSCs to hematopoietic homeostasis has been assessed relative to that of BM HSCs. To investigate these issues we quantitated CFC and HSC activity in the spleen, BM, peripheral blood, and liver of the mouse during ontogeny., Methods: CFCs were assessed by clonogenic colony formation, and HSCs by long-term reconstituting ability., Results: CFCs gradually increased in the BM and decreased in the liver with age. Increased prevalence of CFCs in fetal and pup blood occurred at day (d) 12 postcoitus (pc) and during the period of d16 pc to 4d postbirth, corresponding to the times when hematopoietic cells migrate from the yolk sac and/or aorta-gonad-mesonephros (AGM) to the fetal liver and from the neonatal liver to the BM, respectively. In the spleen, CFCs displayed two peaks of activity at 2d and 14d-15d postbirth. Spleen HSCs also fluctuated during this time period. Neonatal splenectomy did not alter CFC or HSC frequencies in the BM, but CFCs increased in the livers of splenectomized mice., Conclusions: These data demonstrate that the liver may act as a site of extramedullary hematopoiesis in the neonate, especially in the absence of the spleen, and imply that the spleen, BM, and liver cooperatively contribute to hematopoietic homeostasis.

Aralar1 and citrin are members of the subfamily of calcium-binding mitochondrial carriers and correspond to two isoforms of the mitochondrial aspartate/glutamate carrier (AGC). These proteins are activated by Ca2+ acting on the external side of the inner mitochondrial membrane. Although it is known that aralar1 is expressed mainly in skeletal muscle, heart and brain, whereas citrin is present in liver, kidney and heart, the precise tissue distribution of the two proteins in embryonic and adult tissues is largely unknown. We investigated the pattern of expression of aralar1 and citrin in murine embryonic and adult tissues at the mRNA and protein levels. In situ hybridization analysis indicates that both isoforms are expressed strongly in the branchial arches, dermomyotome, limb and tail buds at early embryonic stages. However, citrin was more abundant in the ectodermal components of these structures whereas aralarl had a predominantly mesenchymal localization. The strong expression of citrin in the liver was acquired postnatally, whereas the characteristic expression of aralar1 in skeletal muscle was detected at E18 and that in the heart began early in development (E11) and was preferentially localized to auricular myocardium in late embryonic stages. Aralar1 was also expressed in bone marrow, T-lymphocytes and macrophages, including Kupffer cells in the liver, indicating that this is the major AGC isoform present in the hematopoietic system. Both aralar1 and citrin were expressed in fetal gut and adult stomach, ovary, testis, and pancreas, but only aralar1 is enriched in lung and insulin-secreting beta cells. These results show that aralar1 is expressed in many more tissues than originally believed and is absent from hepatocytes, where citrin is the only AGC isoform present. This explains why citrin deficiency in humans (type II citrullinemia) only affects the liver and suggests that aralar1 may compensate for the lack of citrin in other tissues.

To maintain self-renewal and multilineage differentiation capacity, hematopoietic stem cell (HSC) proliferation requires both symmetric and asymmetric cell divisions. We have applied a time-lapse camera system and our single-cell culture to correlate early replication behavior with short- and long-term function. Using five-dimensional flow cytometry to purify subpopulations of fetal liver (FLV), fetal bone marrow (FBM), umbilical cord blood (UCB), adult bone marrow (ABM), and mobilized peripheral blood (MPB), we studied the relationship between colony efficiency (CE) growth pattern and ontogenic age. The highest CE was found among HSC candidates from FLV, FBM, and UCB and the lowest from ABM. Relating the divisional behavior with functional readouts, we demonstrated that although mitotic rate, colony efficiency, and percent of asymmetric divisions all decreased with ontogenic age, the fraction of cells undergoing asymmetric divisions was consistently at 45%. After 10 days of culture, 60.6 +/- 9.8% of the PKH bright cells gave rise to colonies (15.8 +/- 7.8% dispersed) compared to 15.9 +/- 11.1% of the PKH dim cells (2.5 +/- 2.5% dispersed). In addition, the much more primitive Myeloid-Lymphoid Initiating Cells (ML-IC) are predominantly found in the PKH-bright population. Thus, primitive function of individual candidate HSCs closely related to their divisional behavior.

The in vitro differentiation of embryonic stem (ES) cells provides a powerful approach for studying the earliest events involved in the commitment of the hematopoietic and endothelial lineages. Using this model system, we have identified a precursor with the potential to generate both primitive and definitive hematopoietic cells as well as cells with endothelial characteristics. The developmental potential of this precursor suggests that it represents the in vitro equivalent of the hemangioblast, a common stem cell for both lineages. ES cells deficient for the transcription factor scl/tal-1 are unable to generate hemangioblasts, while those deficient for Runx1 generate reduced numbers of these precursors. These findings indicate that both genes play pivotal roles at the earliest stages of hematopoietic and endothelial development. In addition, they highlight the strength of this model system in studying the function of genes in embryonic development.

For more than a decade it was believed that hematopoietic stem cells express CD34. However, this dogma was recently challenged by the observation that stem cells of normal adult mice are CD34-. In order to clarify the controversy, we carried out systematic examination of stem cells by using C57BL/6 mice that are congenic for Ly-5. As reported previously, stem cells in the normal adult mice were CD34-. However, stem cells stimulated in vivo by 5-fluorouracil injection or in vitro by a combination of interleukin-11 and steel factor were CD34+. The activated CD34+ stem cells reverted to CD34- when the recipients' marrow achieved steady state. The majority of G-CSF-mobilized stem cells also were CD34+ and reverted to CD34- under steady-state conditions. Most recently, we examined the developmental changes of stem cell CD34 expression. In order to gain information on the total population of stem cells we prepared CD34+ and CD34- populations of mononuclear cells without prior enrichment and studied their engrafting potentials. All stem cells from perinatal to 5-week-old mice were CD34+. In 7-week-old mice CD34- stem cells began to emerge, and the majority of the stem cells were CD34- in the 10- and 20-week-old mice. An estimated 20% of the adult stem cells expressed CD34. These observations provide insight into the current controversy regarding CD34 expression by adult hematopoietic stem cells.

Transcription factor GATA-1 is essential for normal erythropoiesis. GATA-binding sites are consistently found in promoters or enhancers of genes expressed selectively in erythroid cells. To discover novel GATA-1-regulated genes, we searched for GATA-1-activated transcripts in G1E cells, an erythroid line derived from GATA-1(-) embryonic stem cells. By subtractive analysis, we identified a new ATP-binding cassette (ABC) transporter that is strongly and rapidly induced by GATA-1. This protein, named ABC-me (for ABC-mitochondrial erythroid), localizes to the mitochondrial inner membrane and is expressed at particularly high levels in erythroid tissues of embryos and adults. ABC-me is induced during erythroid maturation in cell lines and primary hematopoietic cells, and its overexpression enhances hemoglobin synthesis in erythroleukemia cells. The ABC proteins participate in diverse physiological processes by coupling ATP hydrolysis to the transport of a variety of substrates across cell membranes. We speculate that ABC-me, a newly identified erythroid-expressed ABC superfamily member, may mediate critical mitochondrial transport functions related to heme biosynthesis.

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.

The functional significance of CD95/Fas expressed by candidate hematopoietic stem cells (HSCs) from human fetal liver was studied by testing the effect of agonistic anti-CD95 monoclonal antibody (mAb) CH-11 and soluble CD95 ligand (sCD95L) on the growth of CD34(++)CD38(-)lineage cells in vitro. Candidate fetal HSCs exhibited a dose-dependent proliferative response to CH-11 as well as to sCD95L when combined with kit ligand (KL) + interleukin 3 (IL-3) under serum-deprived culture conditions. CH-11 mAb increased, in a synergistic fashion, the number of myeloid colony-forming unit culture (CFU-C) generated by candidate HSCs in liquid cultures with the cytokine combinations KL + IL-3, KL + granulocytemacrophage colony-stimulating factor, and KL + IL-6. CH-11 mAb and sCD95L also enhanced erythropoiesis supported by KL + IL-3 + erythropoietin (Epo). Furthermore, sCD95L was able to increase the number of megakaryocytes, granulocytes, and CD34- cells generated in the presence of KL + IL-3 + Epo + thrombopoietin. An analysis performed using Western blotting revealed that the membrane-bound CD95L (mCD95L) was expressed by both immature (total CD34+/++) and mature (CD34-) hematopoietic lin(-) FL cells. Among the CD34(++)lin(-)cells, both the freshly isolated CD38+ and CD38 subsets as well as CD95+ and CD95- cells constitutively expressed mCD95L, demonstrating that the CD95/CD95L system represents a paracrine and potentially autocrine regulator of early hematopoiesis. To study the role of the endogenously produced CD95L, we determined the effects of a neutralizing anti-CD95L NOK-1 on the growth of candidate HSCs. By blocking the endogenous CD95L with NOK-1 mAb, we observed an increase in CFU-C generated by candidate HSCs. We conclude that the endogenous CD95L has an inhibitory effect on fetal candidate HSCs, which can be blocked by sCD95L and CH-11 mAb.

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.

We used a radioactive in situ method to study expression of the RNA component of human telomerase (hTR) during normal human development and differentiation using archival tissues. In embryonic tissues, the highest and most uniform expression was present in undifferentiated neuroepithelium. Expression was stronger in immature epithelium than in accompanying immature mesenchyme. Differentiation of most tissues was accompanied by decreased or absent expression. Except for testis and adrenal, the adult pattern of expression was present by the 10th postnatal week. In adult tissues, high expression was present in the testis (primary spermatocytes and Sertoli cells), moderate expression was present in lymphoid follicles (germinal centers), and weak expression was present in epithelia (regenerative cells) but was absent in the nervous system and mesenchymal derived tissues. Expression in adult tissues was predominantly limited to dividing cells, although certain differentiated postmitotic cells expressed the hTR. Our studies demonstrate the complex interrelationship of hTR expression with human development, differentiation, and cell division.

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.

Stem cell antigen 2 (Sca-2) expression can distinguish the most immature T-lymphocyte precursors in the thymus from the hemopoietic stem cells. Sequence analysis of the Sca-2 protein showed that Sca-2 is a glycosylphosphatidylinositol (GPI) anchored molecule that shares some characteristics with the members of the Ly-6 multigene family, and that it is the same as the thymic shared antigen-1 (TSA-1). Here we extend these studies and critically reassess the expression of the Sca-2/TSA-1 antigen in hematopoietic tissues of adult and developing mice. With more sensitive methods we show that the distribution of Sca-2/TSA-1 differs from existing reports. We find especially high expression of Sca-2/TSA1 at day 14 of fetal development.

Megakaryocytes in fetal livers obtained from 30 water-balloon aborted normal fetuses of 3 to 6 months' gestation, in the bone marrow from the same 30 fetuses, and another 9 fetuses of 7 to 8 months' gestation and in the normal bone marrow of adults were analyzed by immunocytochemical staining for size and maturation stage distribution and by flow cytometry for ploidy distribution simultaneously. In human fetuses, megakaryocytes showed a shift during ontogenesis from smaller towards larger size and from less mature towards a more mature stage with advancement of gestation. This was accompanied by a significant progressive shift to higher ploidy. However, the proportions (78.64%) of hypoploidy (< or = 8N) megakaryocytes in bone marrow of 7-8 months' gestation fetuses was still much higher than that (33.32%) in human adults (p < 0.05), with the proportion of hyperploidy (> or = 16N) megakaryocytes lower than that (67.86%) in human adults. This result indicated that megakaryocyte polyploidization may be retarded or inhibited during development.

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.

Dtk (Tyro 3/Sky/Rse/Brt/Tif) belongs to a recently recognized subfamily of receptor tyrosine kinases that also includes Ufo (Axl/Ark) and Mer (Eyk). Ligands for Dtk and Ufo have been identified as protein S and the related molecule Gas6, respectively. This study examined expression of Dtk during ontogeny of the hematopoietic system and compared the pattern of expression with that of Ufo. Both receptors were abundantly expressed in differentiating embryonic stem cells, yolk sac blood islands, para-aortic splanchnopleural mesoderm, fractionated AA4+ fetal liver cells, and fetal thymus from day 14 until birth. Although Ufo was expressed at moderate levels in adult bone marrow, expression of Dtk in this tissue was barely detectable. In adult bone marrow subpopulations fractionated using counterflow centrifugal elutriation, immunomagnetic bead selection for lineage-depletion and FACS sorting for c-kit expression, very low levels of Dtk and/or Ufo were detected in some cell fractions. These results suggest that Dtk and Ufo are likely to be involved in the regulation of hematopoiesis, particularly during the embryonic stages of blood cell development.