RESUMEN
BACKGROUND: The cornerstone of the adult hematopoietic system and clinical treatments for blood-related disease is the cohort of hematopoietic stem cells (HSC) that is harbored in the adult bone marrow microenvironment. Interestingly, this cohort of HSCs is generated only during a short window of developmental time. In mammalian embryos, hematopoietic progenitor and HSC generation occurs within several extra- and intraembryonic microenvironments, most notably from 'hemogenic' endothelial cells lining the major vasculature. HSCs are made through a remarkable transdifferentiation of endothelial cells to a hematopoietic fate that is long-lived and self-renewable. Recent studies are beginning to provide an understanding of the biochemical signaling pathways and transcription factors/complexes that promote their generation. SCOPE OF REVIEW: The focus of this review is on the biochemistry behind the generation of these potent long-lived self-renewing stem cells of the blood system. Both the intrinsic (master transcription factors) and extrinsic regulators (morphogens and growth factors) that affect the generation, maintenance and expansion of HSCs in the embryo will be discussed. MAJOR CONCLUSIONS: The generation of HSCs is a stepwise process involving many developmental signaling pathways, morphogens and cytokines. Pivotal hematopoietic transcription factors are required for their generation. Interestingly, whereas these factors are necessary for HSC generation, their expression in adult bone marrow HSCs is oftentimes not required. Thus, the biochemistry and molecular regulation of HSC development in the embryo are overlapping, but differ significantly from the regulation of HSCs in the adult. GENERAL SIGNIFICANCE: HSC numbers for clinical use are limiting, and despite much research into the molecular basis of HSC regulation in the adult bone marrow, no panel of growth factors, interleukins and/or morphogens has been found to sufficiently increase the number of these important stem cells. An understanding of the biochemistry of HSC generation in the developing embryo provides important new knowledge on how these complex stem cells are made, sustained and expanded in the embryo to give rise to the complete adult hematopoietic system, thus stimulating novel strategies for producing increased numbers of clinically useful HSCs. This article is part of a Special Issue entitled Biochemistry of Stem Cells.
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Células Madre Hematopoyéticas/citología , Diferenciación Celular , HumanosRESUMEN
GATA-3 is one member of a growing family of related transcription factors which share a strongly conserved expression pattern in all vertebrate organisms. In order to elucidate GATA-3 function using a direct genetic approach, we have disrupted the murine gene by homologous recombination in embryonic stem cells. Mice heterozygous for the GATA3 mutation are fertile and appear in all respects to be normal, whereas homozygous mutant embryos die between days 11 and 12 postcoitum (p.c.) and display massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross aberrations in fetal liver haematopoiesis.
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Anomalías Múltiples/genética , Proteínas de Unión al ADN/fisiología , Marcación de Gen , Hematopoyesis Extramedular , Hígado/embriología , Malformaciones del Sistema Nervioso , Transactivadores/fisiología , Anomalías Múltiples/embriología , Animales , Células Cultivadas , Disostosis Craneofacial/embriología , Disostosis Craneofacial/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/anomalías , Muerte Fetal/etiología , Factor de Transcripción GATA2 , Factor de Transcripción GATA3 , Regulación del Desarrollo de la Expresión Génica , Genes Letales , Genotipo , Edad Gestacional , Células Madre Hematopoyéticas/metabolismo , Tamaño de la Camada , Ratones , Ratones Noqueados , Sistema Nervioso/embriología , Defectos del Tubo Neural/embriología , Defectos del Tubo Neural/genética , Transactivadores/genética , Factores de Transcripción/biosíntesisRESUMEN
Using an anti-idiotypic antibody previously characterized as specific for the hapten binding site of the 2,4-dinitrophenyl (DNP)-binding BALB/c myeloma protein MOPC-460, we have detected substantial amounts of this idiotype (Id-460) in the serum of normal mice. Whereas the idiotypic material in DNP-immune serum binds to DNP, the Id-460-positive material in normal mouse serum is not specific for DNP. The material in normal serum appears to be immunoglobulin. Furthermore, Id-460-positive, non-DNP-binding monoclonal immunoglobulins that completely inhibit our assay for Id-460 are repeatedly isolated when hybridomas are prepared from LPS-activated normal spleen cells. These data are interpreted in the context of Jerne's network hypothesis. It is our conclusion that the non-DNP-binding form of Id-460 is the inherited form and that this form establishes an idiotypic network favoring the production of anti-DNP bearing Id-460. Thus, the paradox of finding an inherited idiotype in the antibody response to the nonpathogen DNP may be resolved by proposing that the true form of Id-460 is specific for an environmental pathogen and that Id-460 dominance in the anti-DNP response is simply a consequence of idiotype-specific regulatory events preconditioned by Id-460-bearing immunoglobulin specific for antigenic determinants unrelated to DNP.
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Formación de Anticuerpos , Sitios de Unión de Anticuerpos , Dinitrobencenos/inmunología , Idiotipos de Inmunoglobulinas/genética , Nitrobencenos/inmunología , Animales , Vida Libre de Gérmenes , Cabras , Cobayas , Hibridomas/inmunología , Sueros Inmunes/farmacología , Lipopolisacáridos/farmacología , Activación de Linfocitos , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Ovalbúmina/inmunología , Conejos , RatasRESUMEN
After immunization of mice with 2,4-dinitrophenyl-ovalbumin (DNP-OVA), it was shown previously that strains having Igh-Va genes and able to express light chains of the Vk1 group produce high levels of anti-DNP antibody bearing an idiotype (Id-460) associated with the combining site of the BALB/c DNP-binding myeloma protein MOPC 460. Expression of Id-460 in serum is transient; Id-460 levels peak early in the response and are regulated independently of total anti-DNP antibody. In this paper, the transient dominance of Id-460 expression has been confirmed at the cellular level by inhibition of splenic anti-DNP plaque-forming cells (PFC) with rabbit anti-Id-460 antiserum. Id-460+ PFC can account for 52-91% of anti-DNP PFC early after secondary challenge with DNP-OVA. Furthermore, Id-460 is represented at these high levels in IgM, IgG, and IgG1, and IgG2a, the three isotypes tested in the PFC assay, as well as in IgE, as tested by passive cutaneous anaphylaxis. Thus, there is no preferential association of Id-460 with a given isotype. We conclude from these studies that Id-460 is a dominant idiotype in the anti-DNP antibody response of BALB/c mice to DNP-OVA. This dominance is expressed transiently and is independent of isotype. A further conclusion from these studies is that regulation of isotype expression is independent of the regulation of idiotype expression in this system. We would suggest that regulation of Id-460 expression involves Ig-dependent helper T cells specific for Id-460 that induce Id-460+ B cells and also activate suppressor T cells, both events occurring via idiotype-anti-idiotype interactions.
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Formación de Anticuerpos , Dinitrobencenos/inmunología , Genes Dominantes , Idiotipos de Inmunoglobulinas/genética , Nitrobencenos/inmunología , Animales , Células Productoras de Anticuerpos/inmunología , Técnica de Placa Hemolítica , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Conejos , Factores de TiempoRESUMEN
The genetic contro of the expression of an idiotype (Id-460) associated with the 2,4-dinitrophenyl (DNP)-binding BALB/c myeloma protein MOPC 460 was studied using congenic strains of mice. It was shown that the expression of high levels of Id-460 during secondary in vivo anti-DNP-ovalbumin responses was determined by genes governing immunoglobulin heavy-chain variable and kappa-light chain variable regions (V kappa). Appropriate alleles at both loci were required for the expression of Id-460. Genes in the major histocompatability complex and the X-linked immune deficiency gene found in strain CBA/N did not greatly affect Id-460 expression. The V kappa gene controlling Id-460 expression can be differentiated from Lyt-3, and it is the first instance in which expression of an idiotype subdivides the V kappa genes associated with the Lyt-3a allele. Although it is likely that the V kappa gene(s) involved are structural, the involvememt of a regulatory gene linked to the structural gene can not be excluded.
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Sitios de Unión de Anticuerpos/genética , Dinitrobencenos/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Idiotipos de Inmunoglobulinas/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Nitrobencenos/inmunología , Animales , Diversidad de Anticuerpos , Formación de Anticuerpos , Mapeo Cromosómico , Genes MHC Clase II , Complejo Mayor de Histocompatibilidad , Ratones , Proteínas de Mieloma/inmunologíaRESUMEN
Id-460+ immunoglobulins can be induced in vivo by immunization with dinitrophenyl (DNP) or P. pneumotropica and form two nonoverlapping groups of antibodies with respect to antigen binding specificity. In this study, using Id-460+ antibodies of differing antigen binding specificities, we compared on the molecular genetic level the five gene segment combinations (VH, DH, JH, VL, and JL) that encode the variable regions of these idiotype-positive immunoglobulins. The Id-460 determinant appears to be a conformational or combinatorial determinant encoded by VH460 and VK1 crosshybridizing genes. DH, JH, and JK gene segments appear to have no measurable effect upon expression of Id-460. Finally, antigen binding specificity does not appear to simply localize to any particular gene segment but may in part be the result of somatic mutation and/or VDJH junctional sequences, whose length correlates roughly with antigen binding specificity.
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Anticuerpos Monoclonales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Genes , Hibridomas/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Endogámicos BALB C , Plasmacitoma/inmunologíaRESUMEN
We have previously described an idiotype (Id460) that transiently dominates anti-2,4-dinitrophenyl (DNP) antibody responses of mice that possess the appropriate Igh-V and V kappa genotypes. Normal serum has significant levels of Id460 that does not bind DNP, and hybridomas derived from spleen cell fusions that produce monoclonal antibodies with these characteristics have been generated. Many of these monoclonal, Id460-positive antibodies bind the opportunistic mouse pathogen Pasteurella pneumotropica. P. pneumotropica induces a marked increase in serum Id460 titers without significantly increasing serum anti-DNP titers. Both normal serum and P. pneumotropica-induced Ig460-positive immunoglobulin specifically bind to P. pneumotropica. These results suggest that the normal serum Id460-positive immunoglobulin is induced by environmentally encountered antigens on P. pneumotropica. We propose that this naturally occurring Id460 activates antiidiotypic regulatory cells that in turn promote production of Id460-positive anti-DNP antibody following DNP-ovalbumin immunization. These data are compatible with those obtained in several other idiotypic systems that suggest that dominant idiotypes may be associated with antibodies that have been evolutionarily selected for expression because of their specificity for antigens on environmentally encountered pathogens.
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Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Dinitrofenoles/inmunología , Idiotipos de Inmunoglobulinas/genética , Infecciones por Pasteurella/inmunología , 2,4-Dinitrofenol , Absorción , Pruebas de Aglutinación , Animales , Anticuerpos Monoclonales/biosíntesis , Sitios de Unión de Anticuerpos , Epítopos/genética , Idiotipos de Inmunoglobulinas/inmunología , Ratones , Ratones Endogámicos BALB C , Pasteurella/inmunología , Pasteurella/metabolismo , Infecciones por Pasteurella/microbiología , ConejosRESUMEN
The hematopoietic system of vertebrates is derived from the mesodermal germ layer in early embryogenesis. Various animal models have been used for the study of hematopoiesis, from early stages in the visceral yolk sac or its analog, to the later stages where hematopoiesis is observed in intraembryonic areas surrounding the aorta, genital ridge and pro/mesonephros. Using the mouse as a model, we describe what is known about mammalian embryonic hematopoiesis and put it in the context of hematopoietic cell formation in avian, amphibian and fish embryos. Evolutionary comparisons and recent experimental evidence show that there are two embryonic sites of developing hematopoietic activity in the mouse before fetal liver hematopoiesis and suggest that, during ontogeny, two successive waves of hematopoietic activity may contribute to the blood system of the adult.
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Hematopoyesis Extramedular , Ratones/embriología , Animales , Aorta/embriología , Evolución Biológica , Linaje de la Célula , Desarrollo Embrionario y Fetal , Regulación del Desarrollo de la Expresión Génica , Genitales/embriología , Hígado/embriología , Hígado/fisiología , Mesodermo/fisiología , Mesonefro/embriología , Ratones/genética , Ratones/fisiología , Especificidad de la Especie , Factores de Transcripción/fisiología , Vertebrados/embriología , Vertebrados/genética , Vertebrados/fisiología , Saco Vitelino/fisiologíaRESUMEN
Understanding how the blood system is formed is an ongoing fundamental research challenge. Developmental biology has provided many insights into the molecules and processes that affect the formation of the blood tissues, both in health and disease. It is of particular interest for clinical transplantation therapies to understand how hematopoietic stem cells (HSCs)-the self-renewing purveyors of the adult blood system that produce over 10 different functionally specialized cell lineages and over 10(11) cells daily-are generated during embryonic stages. Recent successes to reprogram the fate of adult differentiated cells to pluripotency and to other cell lineages now highlight the importance of identifying the cells and molecules that affect the in vivo developmental initiation of rare and robust transplantable HSCs. The close association of the developing hematopoietic and vascular system, hematopoietic cell mobility through the circulation, and the essential role of the embryonic hematopoietic system in adult hematopoietic cell development make this a formidable study. This chapter reviews the advances, controversies, and current state of our knowledge of the growing field of hematopoietic development, with a special focus on the regulation of the natural transdifferentiation of endothelial cells to HSCs within the developing embryo.
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Reprogramación Celular , Endotelio/citología , Células Madre Hematopoyéticas/fisiología , Animales , Sangre , Técnicas de Cultivo de Célula , Embrión de Mamíferos/citología , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Humanos , RatonesRESUMEN
We have a developed a retroviral mediated molecular ablation method to specifically eliminate HIV Tat-expressing cells. This approach utilizes the Tat-inducible HIV-2 promoter and a conditional toxin gene. The Herpes Simplex Virus thymidine kinase gene product is toxic to mammalian cells only after treatment with Ganciclovir (GCV) or other nucleoside analogues. We demonstrate here that certain promoter modifications can decrease basal expression while retaining the ability to be transactivated. Furthermore, we show that a HIV-2 promoter thymidine kinase gene cassette transduced via retroviral vectors into tissue culture cells can specifically promote the ablation of HIV-Tat expressing cells in the presence GCV. We also show that there is a large differential in HIV-thymidine kinase gene transcription and lethal drug dose between Tat-expressing cells and Tat-negative cells.
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Productos del Gen tat/metabolismo , Genes tat/genética , VIH-2/genética , Regiones Promotoras Genéticas/genética , Timidina Quinasa/genética , Secuencia de Bases , Eliminación de Gen , Productos del Gen tat/genética , Humanos , Datos de Secuencia Molecular , Retroviridae/genética , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
The first definitive long-term repopulating hematopoietic stem cells (HSCs) emerge from and undergo rapid expansion in the embryonic aorta-gonad-mesonephros (AGM) region. To investigate the presumptive unique characteristics of the embryonic hematopoietic microenvironment and its surrounding tissues, we have generated stromal clones from subdissected day 10 and day 11 AGMs, embryonic livers (ELs) and gut mesentery. We here examine the ability of 19 of these clones to sustain extended long-term cultures (LTCs) of human CD34(+) umbilical cord blood (UCB) cells in vitro. The presence of in vitro repopulating cells was assessed by sustained production of progenitor cells (extended LTC-CFC) and cobblestone area-forming cells (CAFC). The embryonic stromal clones differed greatly in their support for human HSCs. Out of eight clones tested in the absence of exogenous cytokines, only one (EL-derived) clone was able to provide maintenance of HSCs. Addition of either Tpo or Flt3-L + Tpo improved the long-term support of about 50% of the tested clones. Cultures on four out of 19 clones, ie the EL-derived clone mentioned, two urogenital-ridge (UG)-derived clones and one gastrointestinal (GI)-derived clone, allowed a continuous expansion of primitive CAFC and CFU-GM with over several hundred-fold more CAFC(week6) produced in the 12th week of culture. This expansion was considerably higher than that found with the FBMD-1 cell line, which is appreciated by many investigators for its support of human HSCs, under comparable conditions. This stromal cell panel derived from the embryonic regions may be a powerful tool in dissecting the factors mediating stromal support for maintenance and expansion of HSCs.
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Embrión de Mamíferos/citología , Sangre Fetal/citología , Células Madre/citología , Células del Estroma/citología , Animales , Antígenos CD34/metabolismo , Aorta/embriología , Linaje de la Célula , Separación Celular , Células Clonales/citología , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Citocinas/metabolismo , Sistema Digestivo/embriología , Gónadas/embriología , Supervivencia de Injerto , Hematopoyesis , Humanos , Hígado/embriología , Mesenterio/embriología , Mesonefro/embriología , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/química , Células Madre/efectos de los fármacos , Células del Estroma/metabolismoRESUMEN
Transmembrane tyrosine kinase receptors are involved in cellular interactions which promote proliferation and differentiation of many cell types. To identify receptor tyrosine kinases important in embryonic hematopoietic cell development we have utilized the polymerase chain reaction (PCR) and degenerate oligonucleotides for isolation of such genes from mouse yolk sac and fetal liver. Sequence analysis of PCR amplified cDNAs from these hematopoietic sites of day 8 and 14 embryos, resulted in the isolation of nine tyrosine kinase and three serine/threonine kinase related clones. Two of these receptors, tek and flk-1, are expressed in both yolk sac and fetal liver and have been shown previously to be important for endothelial cell development. Two other clones, 9B4 and 9A2 appeared novel upon isolation but have been recently described as ryk and SK2 (rat homologue). Here we describe the twelve isolated kinases, the specific expression patterns of flk-1, tek and ryk kinases and their potential relationship to the development of the hematopoietic system.
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Sistema Hematopoyético/enzimología , Proteínas Tirosina Quinasas/genética , Animales , Médula Ósea/embriología , Médula Ósea/enzimología , Línea Celular , ADN Complementario , Expresión Génica , Hígado/embriología , Hígado/enzimología , Ratones , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Quinasas Receptoras/genética , Saco Vitelino/enzimologíaRESUMEN
Recent research on the ontogeny of the hematopoietic system in mammals has shown that a simple textbook steady-state hematopoietic hierarchy can not be strictly applied to the hematopoietic cells found within the embryo. During embryonic development, hematopoietic cells originate, migrate and differentiate in a number of distinct anatomical sites such as the yolk sac AGM region and liver and thus represent various classes of cells within diverse microenvironments. In this manuscript we review both cellular and molecular aspects of developmental hematopoiesis and present our current views on the numerous complex mechanisms underlying the establishment of definitive hematopoiesis.
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Hematopoyesis , Células Madre Hematopoyéticas/citología , Animales , Linaje de la Célula , Desarrollo Embrionario y Fetal , Humanos , Ratones , Saco Vitelino/citología , Saco Vitelino/embriologíaRESUMEN
The ability of the many cell types within the adult blood system to be constantly replenished and renewed from hematopoietic stem cells is an interesting problem in development and differentiation and has led to questions concerning how, when and where these stem cells for the adult hematopoietic system are generated within the embryo. During embryonic development many mature hematopoietic cells appear before adult-type hematopoietic stem cells thus the notion of a conventional hematopoietic hierarchy is challenged. Experiments probing the development of hematopoietic stem cells in the mouse embryo strongly suggest that at least two independent hematopoietic sites generate blood cells during development; the yolk sac, which produces the transient embryonic hematopoietic system, and the AGM (aorta-gonad-mesonephros) region, which initiates the long-lived adult hematopoietic system.
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Desarrollo Embrionario y Fetal , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Animales , Aorta/embriología , Gónadas/embriología , Mesonefro/fisiología , Ratones , Saco Vitelino/fisiologíaRESUMEN
Hematopoietic stem cells (HSCs) are at the foundation of the hematopoietic hierarchy and give rise to all blood lineages in the adult organism. A thorough understanding of the molecular, cellular, and developmental biology of HSCs is of fundamental importance, but is also clinically relevant for the advancement of cell replacement therapies and transplantation protocols in blood-related genetic disease and leukemias. While the major anatomical sites of hematopoiesis change during ontogeny, it was long believed that the developmental origin of the adult mammalian hematopoietic system was the yolk sac. However, current studies have shown that the first adult-type HSCs are autonomously generated in the intrabody portion of the mouse embryo, the aorta-gonads-mesonephros (AGM) region, and sublocalize to the dorsal aorta. HSCs are also found in the other large embryonic vessels, the vitelline and umbilical arteries. The intraluminal hematopoietic clusters along these vessels, together with the role of the Runx1 transcription factor in cluster and HSC formation and the HSC/endothelial/mesenchymal Runxl expression pattern, strongly suggest a vascular endothelial/mesenchymal origin for the first HSCs. Moreover, a transgenic mouse line expressing the GFP marker under the control of the Sca-1 transcriptional regulatory elements (GFP expression marks all HSCs) shows a clear localization of GFP-expressing cells to the endothelial cell layer of the dorsal aorta. Thus, highly enriched GFP-positive AGM HSCs will serve as a basis for the future examination of the cellular and molecular factors involved in the induction and expansion of adult HSCs.
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Linaje de la Célula , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Proteínas Proto-Oncogénicas , Animales , Diferenciación Celular , División Celular , Linaje de la Célula/genética , Linaje de la Célula/fisiología , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario y Fetal/genética , Desarrollo Embrionario y Fetal/fisiología , Humanos , Ratones , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The hematopoietic stem cell (HSC) is the prototype organ-regenerating stem cell (SC), and by far the most studied type of SC in the body. Currently, HSC-based therapy is the only routinely used SC therapy; however, advances in the field of embryonic SCs and induced pluripotent SCs may change this situation. Interest into in vitro generation of HSCs, including signals for HSC expansion and differentiation from these more primitive SCs, as well as advances in other organ-specific SCs, in particular the intestine, provide promising new applications for SC therapies. Here, we review the basic principles of different SC systems, and on the basis of the experience with HSC-based SC therapy, provide recommendations for clinical application of emerging SC technologies.
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Trasplante de Células Madre , Células Madre/citología , Adulto , Animales , Células de la Médula Ósea/citología , Ensayos Clínicos como Asunto/métodos , Modelos Animales de Enfermedad , Células Madre Embrionarias/citología , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/uso terapéutico , Células Madre Hematopoyéticas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Intestinos/citología , Ratones , Células Madre Neoplásicas/citología , Especificidad de Órganos , Selección de Paciente , Medicina Regenerativa/métodos , Trasplante de Células Madre/efectos adversos , Trasplante de Células Madre/métodos , Células Madre/clasificaciónRESUMEN
BACKGROUND: The Interleukin-1 (IL-1) signaling component TAK1 binding protein 2 (TAB2) plays a role in activating the NFkappaB and JNK signaling pathways. Additionally, TAB2 functions in the nucleus as a repressor of NFkappaB-mediated gene regulation. OBJECTIVE: To obtain insight into the function of TAB2 in the adult mouse, we analyzed the in vivo TAB2 expression pattern. MATERIALS AND METHODS: Cell lines and adult mouse tissues were analyzed for TAB2 protein expression and localization. RESULTS: Immunohistochemical staining for TAB2 protein revealed expression in the vascular endothelium of most tissues, hematopoietic cells and brain cells. While TAB2 is localized in both the nucleus and the cytoplasm in cell lines, cytoplasmic localization predominates in hematopoietic tissues in vivo. CONCLUSIONS: The TAB2 expression pattern shows striking similarities with previously reported IL-1 receptor expression and NFkappaB activation patterns, suggesting that TAB2 in vivo is playing a role in these signaling pathways.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Envejecimiento/fisiología , Regulación de la Expresión Génica , Proteínas Adaptadoras Transductoras de Señales/clasificación , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Médula Ósea/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Células Cultivadas , Cricetinae , Citoplasma/metabolismo , Isoenzimas/clasificación , Isoenzimas/genética , Isoenzimas/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Especificidad de Órganos , ARN Mensajero/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bazo/metabolismoRESUMEN
The adult hematopoietic system of mammals is a dynamic hierarchy of cells with the hematopoietic stem cell at its foundation. During embryonic development, the source and expansion potential of this cell remain unclear. Two sites of hematopoietic activity, the yolk sac and aorta-gonad-mesonephros (AGM) region, function in mouse ontogeny at the pre-liver stage of hematopoiesis. However, cellular interchange between these tissues obscures the embryonic site of hematopoietic stem cell generation. Here we present the results of a novel in vitro organ culture system demonstrating that, at day 10 in gestation, hematopoietic stem cells initiate autonomously and exclusively within the AGM region. Furthermore, we provide evidence for the in vitro expansion of hematopoietic stem cells within the AGM region. These results strongly suggest that the AGM region is the source of the definitive adult hematopoietic system, which subsequently colonizes the liver.
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Desarrollo Embrionario y Fetal/fisiología , Hematopoyesis/fisiología , Animales , Aorta/citología , Aorta/embriología , Ensayo de Unidades Formadoras de Colonias , Trasplante de Tejido Fetal , Edad Gestacional , Gónadas/citología , Gónadas/embriología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Hígado/citología , Hígado/embriología , Mesonefro/citología , Mesonefro/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Técnicas de Cultivo de Órganos , Reacción en Cadena de la Polimerasa , Saco Vitelino/citología , Saco Vitelino/embriologíaRESUMEN
The Sca-1 antibody recognizes antigens encoded by members of the Ly-6 multigene family. These antigens are expressed on fetal and adult hematopoietic stem cells, progenitor cells, mature activated T cells, and some nonhematopoietic cells and are most likely encoded by the Ly-6E.1 and Ly-6A.2 genes. Characterization and isolation of regulatory elements of Ly-6E.1 and A.2 genes that govern tissue-specific and high levels of expression in the cells of the hematopoietic system (particularly stem cells) are of considerable interest. To characterize the control elements of this gene, we have cloned a 30-kb fragment encoding a fully functional Ly-6E.1 gene and 13 kb of 5' and 13 kb of 3' flanking sequence. Transfection studies in murine erythroleukemia (MEL) cells show that a 14-kb BamHI fragment from this clone is sufficient to confer Ly-6E.1 gene expression at levels equivalent to those of the endogenous gene. By mapping regions of chromatin sensitive to DNase I digestion, we have located hypersensitive sites in the 5' and 3' regions of the gene in FDCP-1 cells, MEL cells, and various T-cell lines. The appearance of two 5' hypersensitive sites in hematopoietic cells correlates with Ly-6E.1 expression after gamma-interferon induction. We show that the presence of hypersensitive sites in the 5' and 3' regions corresponds to Sca-1 expression, and we also discuss the localization of putative regulatory control elements.