RESUMO
Hematopoietic stem cells (HSCs) are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors are implicated in regulating mouse HSC development, but which cells in the aorta-gonad-mesonephros (AGM) microenvironment produce these factors is unknown. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation were involved in the AGM HSC-generative microenvironment. CyTOF analysis of CD45+ AGM cells revealed predominance of two hematopoietic cell types, mannose-receptor positive macrophages and mannose-receptor negative myeloid cells. We show here that macrophage appearance in the AGM was dependent on the chemokine receptor Cx3cr1. These macrophages expressed a pro-inflammatory signature, localized to the aorta, and dynamically interacted with nascent and emerging intra-aortic hematopoietic cells (IAHCs). Importantly, upon macrophage depletion, no adult-repopulating HSCs were detected, thus implicating a role for pro-inflammatory AGM-associated macrophages in regulating the development of HSCs.
Assuntos
Diferenciação Celular , Desenvolvimento Embrionário , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Macrófagos/metabolismo , Animais , Biomarcadores , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Imunofluorescência , Imunofenotipagem , Inflamação/etiologia , Inflamação/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Células Mieloides/citologia , Células Mieloides/metabolismoRESUMO
Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here, we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modeling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modeling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Aorta/embriologia , Proteínas de Ligação ao Cálcio/genética , Divisão Celular , Células Progenitoras Endoteliais/fisiologia , Feminino , Hemangioblastos/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos TeóricosRESUMO
During vertebrate embryogenesis, hematopoietic stem cells (HSCs) arise in the aorta-gonads-mesonephros (AGM) region. We report here that blood flow is a conserved regulator of HSC formation. In zebrafish, chemical blood flow modulators regulated HSC development, and silent heart (sih) embryos, lacking a heartbeat and blood circulation, exhibited severely reduced HSCs. Flow-modifying compounds primarily affected HSC induction after the onset of heartbeat; however, nitric oxide (NO) donors regulated HSC number even when treatment occurred before the initiation of circulation, and rescued HSCs in sih mutants. Morpholino knockdown of nos1 (nnos/enos) blocked HSC development, and its requirement was shown to be cell autonomous. In the mouse, Nos3 (eNos) was expressed in HSCs in the AGM. Intrauterine Nos inhibition or embryonic Nos3 deficiency resulted in a reduction of hematopoietic clusters and transplantable murine HSCs. This work links blood flow to AGM hematopoiesis and identifies NO as a conserved downstream regulator of HSC development.
Assuntos
Fenômenos Fisiológicos Sanguíneos , Hematopoese , Células-Tronco Hematopoéticas/citologia , Animais , Embrião de Mamíferos/metabolismo , Embrião não Mamífero/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Peixe-ZebraRESUMO
Identifying signaling pathways that regulate hematopoietic stem and progenitor cell (HSPC) formation in the embryo will guide efforts to produce and expand HSPCs ex vivo. Here we show that sterile tonic inflammatory signaling regulates embryonic HSPC formation. Expression profiling of progenitors with lymphoid potential and hematopoietic stem cells (HSCs) from aorta/gonad/mesonephros (AGM) regions of midgestation mouse embryos revealed a robust innate immune/inflammatory signature. Mouse embryos lacking interferon γ (IFN-γ) or IFN-α signaling and zebrafish morphants lacking IFN-γ and IFN-Ï activity had significantly fewer AGM HSPCs. Conversely, knockdown of IFN regulatory factor 2 (IRF2), a negative regulator of IFN signaling, increased expression of IFN target genes and HSPC production in zebrafish. Chromatin immunoprecipitation (ChIP) combined with sequencing (ChIP-seq) and expression analyses demonstrated that IRF2-occupied genes identified in human fetal liver CD34(+) HSPCs are actively transcribed in human and mouse HSPCs. Furthermore, we demonstrate that the primitive myeloid population contributes to the local inflammatory response to impact the scale of HSPC production in the AGM region. Thus, sterile inflammatory signaling is an evolutionarily conserved pathway regulating the production of HSPCs during embryonic development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Transdução de Sinais , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Proliferação de Células/genética , Células Cultivadas , Citocinas/imunologia , Embrião de Mamíferos , Embrião não Mamífero , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Inflamação/genética , Inflamação/imunologia , Interferons/genética , Interferons/metabolismo , Camundongos , Peixe-Zebra/embriologiaRESUMO
Along with the aorta-gonad-mesonephros region, the head is a site of hematopoietic stem and progenitor cell (HS/PC) development in the mouse embryo. Macrophages are present in both these embryonic hemogenic sites, and recent studies indicate a functional interaction of macrophages with hematopoietic cells as they are generated in the aorta. Whereas brain macrophages or "microglia" are known to affect neuronal patterning and vascular circuitry in the embryonic brain, it is unknown whether macrophages play a role in head hematopoiesis. Here, we characterize head macrophages and examine whether they affect the HS/PC output of the hindbrain-branchial arch (HBA) region of the mouse embryo. We show that HBA macrophages are CD45+F4/80+CD11b+Gr1- and express the macrophage-specific Csf1r-GFP reporter. In the HBA of chemokine receptor-deficient (Cx3cr1-/-) embryos, a reduction in erythropoiesis is concomitant with a decrease in HBA macrophage percentages. In cocultures, we show that head macrophages boost hematopoietic progenitor cell numbers from HBA endothelial cells > twofold, and that the proinflammatory factor tumor necrosis factor-α is produced by head macrophages and influences HBA hematopoiesis in vitro. Taken together, head macrophages play a positive role in HBA erythropoiesis and HS/PC expansion and/or maturation, acting as microenvironmental cellular regulators in hematopoietic development.
Assuntos
Embrião de Mamíferos/embriologia , Eritropoese/fisiologia , Cabeça/embriologia , Células-Tronco Hematopoéticas/metabolismo , Macrófagos/metabolismo , Animais , Embrião de Mamíferos/citologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Células-Tronco Hematopoéticas/citologia , Macrófagos/citologia , Masculino , Camundongos , Camundongos KnockoutRESUMO
The hematopoietic system is one of the first complex tissues to develop in the mammalian conceptus. Of particular interest in the field of developmental hematopoiesis is the origin of adult bone marrow hematopoietic stem cells. Tracing their origin is complicated because blood is a mobile tissue and because hematopoietic cells emerge from many embryonic sites. The origin of the adult mammalian blood system remains a topic of lively discussion and intense research. Interest is also focused on developmental signals that induce the adult hematopoietic stem cell program, as these may prove useful for generating and expanding these clinically important cell populations ex vivo. This review presents a historical overview of and the most recent data on the developmental origins of hematopoiesis.
Assuntos
Linhagem da Célula , Embrião de Mamíferos/citologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Linhagem da Célula/genética , Eritrócitos/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , CamundongosRESUMO
The impact of nutritional status during fetal life on the overall health of adults has been recognized; however, dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs occurs during embryogenesis and is considered to be developmentally programmed. Secondary lymphoid organ formation depends on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells. Here we show that mouse fetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero, which pre-sets the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi cell differentiation was controlled by maternal retinoid intake and fetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, whereas RA receptors directly regulated the Rorgt locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.
Assuntos
Feto/imunologia , Imunidade Inata/imunologia , Efeitos Tardios da Exposição Pré-Natal/imunologia , Tretinoína/imunologia , Tretinoína/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Dieta , Feminino , Feto/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Tecido Linfoide/citologia , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/embriologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Tretinoína/administração & dosagem , Tretinoína/metabolismoRESUMO
Not all hematopoietic stem cells (HSCs) are alike. They differ in their physical characteristics such as cell cycle status and cell surface marker phenotype, they respond to different extrinsic signals, and they have different lineage outputs following transplantation. The growing body of evidence that supports heterogeneity within HSCs, which constitute the most robust cell fraction at the foundation of the adult hematopoietic system, is currently of great interest and raises questions as to why HSC subtypes exist, how they are generated and whether HSC heterogeneity affects leukemogenesis or treatment options. This Review provides a developmental overview of HSC subtypes during embryonic, fetal and adult stages of hematopoiesis and discusses the possible origins and consequences of HSC heterogeneity.
Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/citologia , Animais , Células da Medula Óssea/citologia , Linhagem da Célula/fisiologia , Células Endoteliais/citologia , Humanos , Nicho de Células-TroncoRESUMO
The Runx family of transcription factors (Runx1, Runx2, and Runx3) are highly conserved and encode proteins involved in a variety of cell lineages, including blood and blood-related cell lineages, during developmental and adult stages of life. They perform activation and repressive functions in the regulation of gene expression. The requirement for Runx1 in the normal hematopoietic development and its dysregulation through chromosomal translocations and loss-of-function mutations as found in acute myeloid leukemias highlight the importance of this transcription factor in the healthy blood system. Whereas another review will focus on the role of Runx factors in leukemias, this review will provide an overview of the normal regulation and function of Runx factors in hematopoiesis and focus particularly on the biological effects of Runx1 in the generation of hematopoietic stem cells. We will present the current knowledge of the structure and regulatory features directing lineage-specific expression of Runx genes, the models of embryonic and adult hematopoietic development that provide information on their function, and some of the mechanisms by which they affect hematopoietic function.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Regulação Leucêmica da Expressão Gênica , Hematopoese , Células-Tronco Hematopoéticas , Leucemia , Mutação , Proteínas de Neoplasias , Doença Aguda , Animais , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMO
The Gata2 transcription factor is a pivotal regulator of hematopoietic cell development and maintenance, highlighted by the fact that Gata2 haploinsufficiency has been identified as the cause of some familial cases of acute myelogenous leukemia/myelodysplastic syndrome and in MonoMac syndrome. Genetic deletion in mice has shown that Gata2 is pivotal to the embryonic generation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). It functions in the embryo during endothelial cell to hematopoietic cell transition to affect hematopoietic cluster, HPC, and HSC formation. Gata2 conditional deletion and overexpression studies show the importance of Gata2 levels in hematopoiesis, during all developmental stages. Although previous studies of cell populations phenotypically enriched in HPCs and HSCs show expression of Gata2, there has been no direct study of Gata2 expressing cells during normal hematopoiesis. In this study, we generate a Gata2Venus reporter mouse model with unperturbed Gata2 expression to examine the hematopoietic function and transcriptome of Gata2 expressing and nonexpressing cells. We show that all the HSCs are Gata2 expressing. However, not all HPCs in the aorta, vitelline and umbilical arteries, and fetal liver require or express Gata2. These Gata2-independent HPCs exhibit a different functional output and genetic program, including Ras and cyclic AMP response element-binding protein pathways and other Gata factors, compared with Gata2-dependent HPCs. Our results, indicating that Gata2 is of major importance in programming toward HSC fate but not in all cells with HPC fate, have implications for current reprogramming strategies.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Animais , Aorta/citologia , Aorta/embriologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Linhagem da Célula , Células Cultivadas , Técnicas de Reprogramação Celular , Fator de Transcrição GATA2/deficiência , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/fisiologia , Genes Reporter , Vetores Genéticos/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/fisiologia , Fígado/citologia , Fígado/embriologia , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transcriptoma , Transgenes , Artérias Umbilicais/citologia , Artérias Umbilicais/embriologiaRESUMO
Enhancers are the primary determinants of cell identity, and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the -8/+7/+9-kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/-8-kb enhancer targeted TIE2+/c-KIT+/CD41- endothelial cells that were enriched for hematopoietic potential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the 6 genes that were upregulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters, LRP2, a multiligand receptor, was the only gene that had not previously been associated with hematopoiesis. We show that LRP2 is indeed involved in definitive hematopoiesis and by doing so validate the use of reporter gene-coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.
Assuntos
Embrião de Mamíferos/metabolismo , Endoglina/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Hematopoese/fisiologia , Sondas Moleculares/metabolismo , Animais , Linhagem Celular , Embrião de Mamíferos/citologia , Endoglina/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Sondas Moleculares/genética , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismoRESUMO
Hematopoietic cell generation in the midgestation mouse embryo occurs through the natural transdifferentiation of temporally and spatially restricted set of hemogenic endothelial cells. These cells take on hematopoietic fate in the aorta, vitelline and umbilical arteries and appear as hematopoietic cell clusters that emerge from the vascular wall. Genetic and live imaging data have supported this. Recently, the embryonic head has been shown to contain fully functional hematopoietic stem cells (HSC). By lineage tracing, cerebrovascular specific endothelial cells were shown to contribute to the postnatal mouse hematopoietic system. Since Ly6aGFP is a marker of all HSCs, some hematopoietic cluster cells and hemogenic endothelial cells in the midgestation mouse aorta, we examine here whether embryonic head HSCs and vascular endothelial cells are positive for this marker. Whereas some head vasculature, single hematopoietic cells and all HSCs are Ly6aGFP expressing, we do not find clusters of hematopoietic cells emerging from the cerebrovasculature that are characteristic of endothelial-to-hematopoietic transition.
Assuntos
Antígenos Ly/análise , Cabeça/embriologia , Proteínas de Membrana/análise , Animais , Antígenos de Diferenciação/análise , Feminino , Proteínas de Fluorescência Verde , Células-Tronco Hematopoéticas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Haematopoietic stem cells (HSCs), responsible for blood production in the adult mouse, are first detected in the dorsal aorta starting at embryonic day 10.5 (E10.5). Immunohistological analysis of fixed embryo sections has revealed the presence of haematopoietic cell clusters attached to the aortic endothelium where HSCs might localize. The origin of HSCs has long been controversial and several candidates of the direct HSC precursors have been proposed (for review see ref. 7), including a specialized endothelial cell population with a haemogenic potential. Such cells have been described both in vitro in the embryonic stem cell (ESC) culture system and retrospectively in vivo by endothelial lineage tracing and conditional deletion experiments. Whether the transition from haemogenic endothelium to HSC actually occurs in the mouse embryonic aorta is still unclear and requires direct and real-time in vivo observation. To address this issue we used time-lapse confocal imaging and a new dissection procedure to visualize the deeply located aorta. Here we show the dynamic de novo emergence of phenotypically defined HSCs (Sca1(+), c-kit(+), CD41(+)) directly from ventral aortic haemogenic endothelial cells.
Assuntos
Aorta/citologia , Diferenciação Celular , Linhagem da Célula , Endotélio Vascular/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Aorta/embriologia , Aorta/cirurgia , Subunidade alfa 2 de Fator de Ligação ao Core/deficiência , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Dissecação , Embrião de Mamíferos/citologia , Células Endoteliais/citologia , Endotélio Vascular/embriologia , Feminino , Masculino , Camundongos , Microscopia Confocal , Fenótipo , GravidezRESUMO
Haematopoietic stem cells (HSCs) are the founder cells of the adult haematopoietic system, and thus knowledge of the molecular program directing their generation during development is important for regenerative haematopoietic strategies. Runx1 is a pivotal transcription factor required for HSC generation in the vascular regions of the mouse conceptus-the aorta, vitelline and umbilical arteries, yolk sac and placenta. It is thought that HSCs emerge from vascular endothelial cells through the formation of intra-arterial clusters and that Runx1 functions during the transition from 'haemogenic endothelium' to HSCs. Here we show by conditional deletion that Runx1 activity in vascular-endothelial-cadherin-positive endothelial cells is indeed essential for intra-arterial cluster, haematopoietic progenitor and HSC formation in mice. In contrast, Runx1 is not required in cells expressing Vav1, one of the first pan-haematopoietic genes expressed in HSCs. Collectively these data show that Runx1 function is essential in endothelial cells for haematopoietic progenitor and HSC formation from the vasculature, but its requirement ends once or before Vav is expressed.
Assuntos
Diferenciação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células Endoteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-vav/metabolismoRESUMO
Aging is a set of complex processes that occur temporally and continuously. It is generally a unidirectional progression of cellular and molecular changes occurring during the life stages of cells, tissues and ultimately the whole organism. In vertebrate organisms, this begins at conception from the first steps in blastocyst formation, gastrulation, germ layer differentiation, and organogenesis to a continuum of embryonic, fetal, adolescent, adult, and geriatric stages. Tales of the "fountain of youth" and songs of being "forever young" are dominant ideas informing us that growing old is something science should strive to counteract. Here, we discuss the normal life stages of the blood system, particularly the historical recognition of its importance in the early growth stages of vertebrates, and what this means with respect to progressive gain and loss of hematopoietic function in the adult.
RESUMO
An understanding of the mechanisms regulating embryonic hematopoietic stem cell (HSC) development would facilitate their regeneration. The aorta-gonad-mesonephros region is the site for HSC production from hemogenic endothelial cells (HEC). While several distinct regulators are involved in this process, it is not yet known whether macroautophagy (autophagy) plays a role in hematopoiesis in the pre-liver stage. Here, we show that different states of autophagy exist in hematopoietic precursors and correlate with hematopoietic potential based on the LC3-RFP-EGFP mouse model. Deficiency of autophagy-related gene 5 (Atg5) specifically in endothelial cells disrupts endothelial to hematopoietic transition (EHT), by blocking the autophagic process. Using combined approaches, including single-cell RNA-sequencing (scRNA-seq), we have confirmed that Atg5 deletion interrupts developmental temporal order of EHT to further affect the pre-HSC I maturation, and that autophagy influences hemogenic potential of HEC and the formation of pre-HSC I likely via the nucleolin pathway. These findings demonstrate a role for autophagy in the formation/maturation of hematopoietic precursors.
Assuntos
Hemangioblastos , Células-Tronco Hematopoéticas , Animais , Camundongos , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular , Embrião de Mamíferos , Hematopoese/genética , Fatores de Transcrição/metabolismo , Autofagia/genética , MesonefroRESUMO
Hematopoietic cell clusters in the aorta of vertebrate embryos play a pivotal role in the formation of the adult blood system. Despite their importance, hematopoietic clusters have not been systematically quantitated or mapped because of technical limitations posed by the opaqueness of whole mouse embryos. Here, we combine an approach to make whole mouse embryos transparent, with multicolor marking, to allow observation of hematopoietic clusters using high-resolution 3-dimensional confocal microscopy. Our method provides the first complete map and temporal quantitation of all hematopoietic clusters in the mouse embryonic vasculature. We show that clusters peak in number at embryonic day 10.5, localize to specific vascular subregions and are heterogeneous, indicating a basal endothelial to non-basal (outer cluster) hematopoietic cell transition. Clusters enriched with the c-Kit(+)CD31(+)SSEA1(-) cell population contain functional hematopoietic progenitors and stem cells. Thus, three-dimensional cartography of transparent mouse embryos provides novel insight into the vascular subregions instrumental in hematopoietic progenitor/stem cell development, and represents an important technological advancement for comprehensive in situ hematopoietic cluster analysis.
Assuntos
Vasos Sanguíneos/citologia , Embrião de Mamíferos/citologia , Células-Tronco Hematopoéticas/citologia , Coloração e Rotulagem/métodos , Animais , Aorta/embriologia , Vasos Sanguíneos/embriologia , Embrião de Mamíferos/irrigação sanguínea , Idade Gestacional , Imageamento Tridimensional/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Nicho de Células-Tronco/citologia , Artérias Umbilicais/citologia , Artérias Umbilicais/embriologia , Ducto Vitelino/citologia , Ducto Vitelino/embriologiaRESUMO
Many physiologic processes during the early stages of mammalian ontogeny, particularly placental and vascular development, take place in the low oxygen environment of the uterus. Organogenesis is affected by hypoxia inducible factor (HIF) transcription factors that are sensors of hypoxia. In response to hypoxia, HIFs activate downstream target genes - growth and metabolism factors. During hematopoietic system ontogeny, blood cells and hematopoietic progenitor/stem cells are respectively generated from mesodermal precursors, hemangioblasts, and from a specialized subset of endothelial cells that are hemogenic. Since HIFs are known to play a central role in vascular development, and hematopoietic system development occurs in parallel to that of the vascular system, several studies have examined the role of HIFs in hematopoietic development. The response to hypoxia has been examined in early and mid-gestation mouse embryos through genetic deletion of HIF subunits. We review here the data showing that hematopoietic tissues of the embryo are hypoxic and express HIFs and HIF downstream targets, and that HIFs regulate the development and function of hematopoietic progenitor/stem cells.
Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Animais , Medula Óssea/metabolismo , Hipóxia Celular , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Humanos , Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Knockout , Consumo de Oxigênio , Placenta/embriologia , Placenta/metabolismo , Gravidez , Nicho de Células-Tronco/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Hematopoietic cell clusters associated with the midgestation mouse aorta, umbilical and vitelline arteries play a pivotal role in the formation of the adult blood system. Both genetic and live-imaging data indicate that definitive hematopoietic progenitor/stem cells (visualized as clusters) are generated from hemogenic endothelium. A 3-dimensional (3-D) whole embryo immunostaining and imaging technique has allowed quantitation and cartographic mapping of intravascular hematopoietic clusters. During this period the vitelline artery is most extensively remodeled, and several reports have suggested that vitelline remodeling leads to extravascular hematopoietic cluster emergence. Whether the earliest definitive progenitors/stem cells are intra or extra vascular could influence the process by which these cells migrate to the next hematopoietic territory, the fetal liver. Hence, by 3-D imaging we more closely examined hematopoietic clusters in the vitelline and associated connected small vessels and show here that hematopoietic clusters (particularly large clusters) are intravascular during the period of vascular remodeling.