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1.
Sci Rep ; 12(1): 16974, 2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-36217016

RESUMO

Progress in the generation of Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro and ex vivo has been built on the knowledge of developmental hematopoiesis, underscoring the importance of understanding this process. HSPCs emerge within the embryonic vasculature through an Endothelial-to-Hematopoietic Transition (EHT). The transcriptional regulator Tal1 exerts essential functions in the earliest stages of blood development, but is considered dispensable for the EHT. Nevertheless, Tal1 is expressed with its binding partner Lmo2 and it homologous Lyl1 in endothelial and transitioning cells at the time of EHT. Here, we investigated the function of these genes using a mouse embryonic-stem cell (mESC)-based differentiation system to model hematopoietic development. We showed for the first time that the expression of TAL1 in endothelial cells is crucial to ensure the efficiency of the EHT process and a sustained hematopoietic output. Our findings uncover an important function of Tal1 during the EHT, thus filling the current gap in the knowledge of the role of this master gene throughout the whole process of hematopoietic development.


Assuntos
Células Endoteliais , Hematopoese , Animais , Diferenciação Celular/genética , Células Endoteliais/metabolismo , Endotélio , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismo
2.
Sci Adv ; 8(35): eabq5206, 2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36044572

RESUMO

Nucleic acid and histone modifications critically depend on the tricarboxylic acid (TCA) cycle for substrates and cofactors. Although a few TCA cycle enzymes have been reported in the nucleus, the corresponding pathways are considered to operate in mitochondria. Here, we show that a part of the TCA cycle is operational also in the nucleus. Using 13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei of HeLa cells. Proximity labeling mass spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Nuclear localization of the latter enzyme, which produces succinyl-CoA, changed from pluripotency to a differentiated state with accompanying changes in the nuclear protein succinylation. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus, warranting a revision of the canonical view on metabolic compartmentalization.

3.
Sci Rep ; 12(1): 12177, 2022 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-35842474

RESUMO

Hematopoietic stem cells (HSCs) are crucial for the continuous production of blood cells during life. The transplantation of these cells is one of the most common treatments to cure patient suffering of blood diseases. However, the lack of suitable donors is a major limitation. One option to get HSCs matching perfectly a patient is cellular reprogramming. HSCs emerge from endothelial cells in blood vessels during embryogenesis through the endothelial to hematopoietic transition. Here, we used single-cell transcriptomics analysis to compare embryonic and post-natal endothelial cells to investigate the potential of adult vasculature to be reprogrammed in hematopoietic stem cells. Although transcriptional similarities have been found between embryonic and adult endothelial cells, we found some key differences in term of transcription factors expression. There is a deficit of expression of Runx1, Tal1, Lyl1 and Cbfb in adult endothelial cells compared to their embryonic counterparts. Using a combination of gene expression profiling and gene regulatory network analysis, we found that endothelial cells from the pancreas, brain, kidney and liver appear to be the most suitable targets for cellular reprogramming into HSCs. Overall, our work provides an important resource for the rational design of a reprogramming strategy for the generation of HSCs.


Assuntos
Células Endoteliais , Transcriptoma , Adulto , Diferenciação Celular , Células Endoteliais/metabolismo , Endotélio , Perfilação da Expressão Gênica , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos
4.
Trends Genet ; 36(9): 632-634, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32631633

RESUMO

Gene expression is regulated by transcription factors (TFs) and chromatin modifiers such as histone acetyltransferases (HATs). Pessoa Rodrigues et al. revealed the role of the Males absent on the first (MOF) HAT in hematopoietic stem cell (HSC) differentiation into red blood cells. This work raises interesting questions about how MOF controls other hematopoietic differentiation processes.


Assuntos
Histona Acetiltransferases , Fatores de Transcrição , Células Sanguíneas , Diferenciação Celular
5.
Nat Commun ; 11(1): 586, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996681

RESUMO

The endothelial to haematopoietic transition (EHT) is the process whereby haemogenic endothelium differentiates into haematopoietic stem and progenitor cells (HSPCs). The intermediary steps of this process are unclear, in particular the identity of endothelial cells that give rise to HSPCs is unknown. Using single-cell transcriptome analysis and antibody screening, we identify CD44 as a marker of EHT enabling us to isolate robustly the different stages of EHT in the aorta-gonad-mesonephros (AGM) region. This allows us to provide a detailed phenotypical and transcriptional profile of CD44-positive arterial endothelial cells from which HSPCs emerge. They are characterized with high expression of genes related to Notch signalling, TGFbeta/BMP antagonists, a downregulation of genes related to glycolysis and the TCA cycle, and a lower rate of cell cycle. Moreover, we demonstrate that by inhibiting the interaction between CD44 and its ligand hyaluronan, we can block EHT, identifying an additional regulator of HSPC development.


Assuntos
Biomarcadores , Endotélio/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptores de Hialuronatos/metabolismo , Transcriptoma , Animais , Aorta , Artérias , Ciclo Celular , Ciclo do Ácido Cítrico/genética , Biologia Computacional , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação para Baixo , Glicólise/genética , Gônadas , Hematopoese/fisiologia , Receptores de Hialuronatos/sangue , Receptores de Hialuronatos/genética , Ácido Hialurônico , Mesonefro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Crescimento Transformador beta/metabolismo
6.
Sci Rep ; 9(1): 6414, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015568

RESUMO

In this study, we aimed to explore how cellular iron status affects embryonic haematopoiesis. For this purpose, we used a model of mouse embryonic stem cell differentiation into embryonic haematopoietic progenitors. We modulated the iron status by adding either the iron chelator Deferoxamine (DFO) for iron deficiency, or ferric ammonium citrate for iron excess, and followed the emergence of developing haematopoietic progenitors. Interestingly, we found that iron deficiency did not block the endothelial to haematopoietic transition, the first step of haematopoiesis. However, it did reduce the proliferation, survival and clonogenic capacity of haematopoietic progenitors. Surprisingly, iron deficiency affected erythro-myeloid progenitors significantly more than the primitive erythroid ones. Erythro-myeloid progenitors expressed less transferrin-receptor on the cell surface and had less labile iron compared to primitive erythroid progenitors, which could reduce their capacity to compete for scarce iron and survive iron deficiency. In conclusion, we show that iron deficiency could disturb haematopoiesis at an early embryonic stage by compromising more severely the survival, proliferation and differentiation of definitive haematopoietic progenitors compared to restricted erythroid progenitors.


Assuntos
Embrião de Mamíferos/metabolismo , Células Endoteliais/metabolismo , Hematopoese , Deficiências de Ferro , Animais , Apoptose , Linhagem Celular , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Células Endoteliais/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores da Transferrina/metabolismo
7.
Bio Protoc ; 9(1)2019 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-30652115

RESUMO

In vitro differentiation of mouse embryonic stem cells (mESCs) towards blood cells constitutes a well-established system to study the endothelial-to-hematopoietic transition (EHT) at the onset of blood development. Assessing the emergence of small non-adherent round blood cells in the culture without disturbing it is essential to evaluate the progression of EHT and also to test conditions potentially enhancing or repressing this process. Here, we describe how to quantify the formation of mouse hematopoietic progenitors during EHT in normal conditions or following over-expression of eight essential transcription factors using time-lapse microscopy and image analysis.

8.
Development ; 145(5)2018 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-29530939

RESUMO

During ontogeny, hematopoietic stem and progenitor cells arise from hemogenic endothelium through an endothelial-to-hematopoietic transition that is strictly dependent on the transcription factor RUNX1. Although it is well established that RUNX1 is essential for the onset of hematopoiesis, little is known about the role of RUNX1 dosage specifically in hemogenic endothelium and during the endothelial-to-hematopoietic transition. Here, we used the mouse embryonic stem cell differentiation system to determine if and how RUNX1 dosage affects hemogenic endothelium differentiation. The use of inducible Runx1 expression combined with alterations in the expression of the RUNX1 co-factor CBFß allowed us to evaluate a wide range of RUNX1 levels. We demonstrate that low RUNX1 levels are sufficient and necessary to initiate an effective endothelial-to-hematopoietic transition. Subsequently, RUNX1 is also required to complete the endothelial-to-hematopoietic transition and to generate functional hematopoietic precursors. In contrast, elevated levels of RUNX1 are able to drive an accelerated endothelial-to-hematopoietic transition, but the resulting cells are unable to generate mature hematopoietic cells. Together, our results suggest that RUNX1 dosage plays a pivotal role in hemogenic endothelium maturation and the establishment of the hematopoietic system.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Endotélio Vascular/fisiologia , Dosagem de Genes/fisiologia , Hemangioblastos/fisiologia , Hematopoese/genética , Células-Tronco Hematopoéticas/fisiologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout
9.
Elife ; 72018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29555020

RESUMO

Recent advances in single-cell transcriptomics techniques have opened the door to the study of gene regulatory networks (GRNs) at the single-cell level. Here, we studied the GRNs controlling the emergence of hematopoietic stem and progenitor cells from mouse embryonic endothelium using a combination of single-cell transcriptome assays. We found that a heptad of transcription factors (Runx1, Gata2, Tal1, Fli1, Lyl1, Erg and Lmo2) is specifically co-expressed in an intermediate population expressing both endothelial and hematopoietic markers. Within the heptad, we identified two sets of factors of opposing functions: one (Erg/Fli1) promoting the endothelial cell fate, the other (Runx1/Gata2) promoting the hematopoietic fate. Surprisingly, our data suggest that even though Fli1 initially supports the endothelial cell fate, it acquires a pro-hematopoietic role when co-expressed with Runx1. This work demonstrates the power of single-cell RNA-sequencing for characterizing complex transcription factor dynamics.


Assuntos
Perfilação da Expressão Gênica/métodos , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Análise de Célula Única/métodos , Fatores de Transcrição/genética , Animais , Análise por Conglomerados , Subunidades alfa de Fatores de Ligação ao Core/genética , Endotélio/citologia , Endotélio/embriologia , Endotélio/metabolismo , Redes Reguladoras de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Proto-Oncogênica c-fli-1/genética
10.
Sci Rep ; 6: 21518, 2016 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-26891705

RESUMO

The endothelial to haematopoietic transition (EHT) is a key developmental process where a drastic change of endothelial cell morphology leads to the formation of blood stem and progenitor cells during embryogenesis. As TGFß signalling triggers a similar event during embryonic development called epithelial to mesenchymal transition (EMT), we hypothesised that TGFß activity could play a similar role in EHT as well. We used the mouse embryonic stem cell differentiation system for in vitro recapitulation of EHT and performed gain and loss of function analyses of the TGFß pathway. Quantitative proteomics analysis showed that TGFß treatment during EHT increased the secretion of several proteins linked to the vascular lineage. Live cell imaging showed that TGFß blocked the formation of round blood cells. Using gene expression profiling we demonstrated that the TGFß signalling activation decreased haematopoietic genes expression and increased the transcription of endothelial and extracellular matrix genes as well as EMT markers. Finally we found that the expression of the transcription factor Sox17 was up-regulated upon TGFß signalling activation and showed that its overexpression was enough to block blood cell formation. In conclusion we showed that triggering the TGFß pathway does not enhance EHT as we hypothesised but instead impairs it.


Assuntos
Transdiferenciação Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Hematopoese , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Transdiferenciação Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcriptoma , Fator de Crescimento Transformador beta/farmacologia
11.
Nat Cell Biol ; 18(1): 21-32, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26619147

RESUMO

In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta-gonad-mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Hemangioblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Histona Desmetilases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Aorta/citologia , Aorta/embriologia , Diferenciação Celular/fisiologia , Embrião de Mamíferos/citologia , Hemangioblastos/citologia , Células-Tronco Hematopoéticas/citologia , Camundongos
12.
Blood ; 120(2): 314-22, 2012 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-22668850

RESUMO

Recent studies have established that during embryonic development, hematopoietic progenitors and stem cells are generated from hemogenic endothelium precursors through a process termed endothelial to hematopoietic transition (EHT). The transcription factor RUNX1 is essential for this process, but its main downstream effectors remain largely unknown. Here, we report the identification of Gfi1 and Gfi1b as direct targets of RUNX1 and critical regulators of EHT. GFI1 and GFI1B are able to trigger, in the absence of RUNX1, the down-regulation of endothelial markers and the formation of round cells, a morphologic change characteristic of EHT. Conversely, blood progenitors in Gfi1- and Gfi1b-deficient embryos maintain the expression of endothelial genes. Moreover, those cells are not released from the yolk sac and disseminated into embryonic tissues. Taken together, our findings demonstrate a critical and specific role of the GFI1 transcription factors in the first steps of the process leading to the generation of hematopoietic progenitors from hemogenic endothelium.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Hemangioblastos/citologia , Hemangioblastos/fisiologia , Hematopoese/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , 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/fisiologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Neovascularização Fisiológica , Gravidez , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Saco Vitelino/citologia , Saco Vitelino/embriologia , Saco Vitelino/fisiologia
13.
Blood ; 118(3): 594-7, 2011 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-21498670

RESUMO

Although the critical requirement for the transcription factor RUNX1/AML1 at the onset of hematopoietic development is well established, little is known about its transcriptional targets at this pivotal stage of blood development. Using microarrays, we identified the uncharacterized gene AI467606 as a gene whose expression level is dramatically reduced in the absence of RUNX1. We further demonstrated by chromatin immunoprecipitation and promoter assay a direct regulation of its transcription by RUNX1. Using a bacterial artificial chromosome transgenic approach, we established that AI467606 is expressed during the development of the hematopoietic system in vivo and in vitro and that its expression is detected within the CD41(+) population and marks definitive hematopoietic potential. Similarly, in the adult mouse, all hematopoietic cell lineages, except mature erythrocytes, express AI467606. Taken together, these findings indicate that AI467606 is a novel transcriptional target of RUNX1/AML1 at the onset of hematopoietic development that is extensively expressed within the hematopoietic system.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Hematopoese/fisiologia , Proteínas/genética , Proteínas/metabolismo , Transcrição Gênica/fisiologia , Fatores Etários , Animais , Medula Óssea/embriologia , Medula Óssea/fisiologia , Linhagem da Célula/fisiologia , Células Cultivadas , Cromossomos Artificiais Bacterianos/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Membrana , Camundongos , Dados de Sequência Molecular , Glicoproteína IIb da Membrana de Plaquetas/metabolismo
14.
Stem Cells ; 28(6): 1089-98, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20506544

RESUMO

During embryogenesis, the hematopoietic program is specified from the mesodermal germ layer through the formation of hemangioblast. This precursor gives rise to a hemogenic endothelium that later on matures to generate primitive and definitive hematopoietic precursors. A lack of specific cell surface markers to identify cells with discrete developmental potential is a major hurdle in the quest to further understand the cellular and molecular program governing blood formation. In the present study, we identify CD40 and Icam2, two markers typically associated with the adult immunological compartment, as expressed at the earliest stages of blood specification both in vitro and in vivo. Using in vitro serum-free culture conditions that support the efficient and directed differentiation of embryonic stem cells, we show that the sequential expression of CD40 and Icam2 delineate a transition in the acquisition of the blood potential from hemangioblast to hemogenic endothelium leading to the formation of primitive and definitive hematopoietic progenitors. CD40 is transiently expressed at the onset of blood development and marks first the hemangioblast then the hemogenic endothelium but is no longer expressed on fully committed hematopoietic precursors within the fetal liver. In contrast, Icam2 is first expressed on the hemogenic endothelium and its expression persists on fetal liver hematopoietic progenitors. Taken together, our data identify novel cell surface markers allowing us to further refine our understanding of the events marking progressive hematopoietic commitment from the mesoderm germ layer.


Assuntos
Antígenos CD40/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Cultivadas/citologia , Hematopoese , Mesoderma/metabolismo , Animais , Antígenos CD , Linhagem da Célula , Camundongos , Camundongos Endogâmicos ICR
15.
J Mol Med (Berl) ; 88(2): 167-72, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19856139

RESUMO

Understanding how blood cells are generated is important from a biological perspective but also has potential implications in the treatment of blood diseases. Such knowledge could potentially lead to defining new conditions to amplify hematopoietic stem cells (HSCs) or could translate into new methods to produce HSCs, or other types of blood cells, from human embryonic stem cells or induced pluripotent stem cells. Additionally, as most key transcription factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding their function during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia. In this review, we discuss our current understanding of the molecular and cellular mechanisms of blood development from the earliest hematopoietic precursor, the hemangioblast, a precursor for both endothelial and hematopoietic cell lineages.


Assuntos
Células Sanguíneas/citologia , Diferenciação Celular , Hemangioblastos/citologia , Animais , Células Sanguíneas/metabolismo , Linhagem da Célula , Hematopoese , Humanos , Leucemia/fisiopatologia , Camundongos , Fatores de Transcrição/metabolismo
16.
Blood ; 114(26): 5279-89, 2009 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-19858498

RESUMO

The transcription factor RUNX1/AML1 is a master regulator of hematopoietic development. Its spatiotemporal expression is tightly regulated during embryonic development and is under the control of 2 alternative promoters, distal and proximal. Despite the functional significance of Runx1, the relative and specific activities of these 2 promoters remain largely uncharacterized. To investigate these activities, we introduced 2 reporter genes under the control of the proximal and distal promoters in embryonic stem cell and transgenic mouse lines. Our study reveals that both in vitro and in vivo the proximal Runx1 isoform marks a hemogenic endothelium cell population, whereas the subsequent expression of distal Runx1 defines fully committed definitive hematopoietic progenitors. Interestingly, hematopoietic commitment in distal Runx1 knockout embryos appears normal. Altogether, our data demonstrate that the differential activities of the 2 Runx1 promoters define milestones of hematopoietic development and suggest that the proximal isoform plays a critical role in the generation of hematopoietic cells from hemogenic endothelium. Identification and access to the discrete stages of hematopoietic development defined by the activities of the Runx1 promoters will provide the opportunity to further explore the cellular and molecular mechanisms of hematopoietic development.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Desenvolvimento Embrionário/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Regiões Promotoras Genéticas/genética , Animais , Diferenciação Celular/genética , Técnicas de Cocultura , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Endotélio Vascular/citologia , Endotélio Vascular/embriologia , Citometria de Fluxo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Isoformas de Proteínas/genética
17.
Blood ; 114(2): 299-309, 2009 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-19339695

RESUMO

At the cellular level, development progresses through successive regulatory states, each characterized by their specific gene expression profile. However, the molecular mechanisms regulating first the priming and then maintenance of gene expression within one developmental pathway are essentially unknown. The hematopoietic system represents a powerful experimental model to address these questions and here we have focused on a regulatory circuit playing a central role in myelopoiesis: the transcription factor PU.1, its target gene colony-stimulating-factor 1 receptor (Csf1r), and key upstream regulators such as RUNX1. We find that during ontogeny, chromatin unfolding precedes the establishment of active histone marks and the formation of stable transcription factor complexes at the Pu.1 locus and we show that chromatin remodeling is mediated by the transient binding of RUNX1 to Pu.1 cis-elements. By contrast, chromatin reorganization of Csf1r requires prior expression of PU.1 together with RUNX1 binding. Once the full hematopoietic program is established, stable transcription factor complexes and active chromatin can be maintained without RUNX1. Our experiments therefore demonstrate how individual transcription factors function in a differentiation stage-specific manner to differentially affect the initiation versus maintenance of a developmental program.


Assuntos
Células Sanguíneas/metabolismo , Cromatina/genética , Cromatina/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Animais , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/deficiência , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Metilação de DNA , Camundongos , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Mensageiro/genética , Fatores de Tempo
18.
Methods Mol Biol ; 538: 317-34, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19277585

RESUMO

Embryonic Stem (ES) are pluripotent cells derived from the inner cell mass of blastocysts. ES cells differentiate in vitro into all kind of cells and the development of endothelial and hematopoietic cells from mouse ES cells has been especially established. As such, the in vitro differentiation of ES cells provides a powerful experimental model to study and determine the role of specific genes in the development of the hematopoietic system. Using this approach we have demonstrated the critical function of the transcription factor AML1/Runx1 at the onset of hematopoietic development (Blood 100:458-466, 2002; Blood 103:886-889, 2004). In this chapter, we will describe our protocols and methods for the culture of healthy ES cells, their effective differentiation toward hematopoiesis, and the quantitative analysis of their hematopoietic potential by replating or gene expression analyses.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Hematopoese/fisiologia , Animais , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Primers do DNA/química , Feminino , Fibroblastos/citologia , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase/métodos
19.
Nature ; 457(7231): 892-5, 2009 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-19182774

RESUMO

It has been proposed that during embryonic development haematopoietic cells arise from a mesodermal progenitor with both endothelial and haematopoietic potential called the haemangioblast. A conflicting theory instead associates the first haematopoietic cells with a phenotypically differentiated endothelial cell that has haematopoietic potential (that is, a haemogenic endothelium). Support for the haemangioblast concept was initially provided by the identification during mouse embryonic stem cell differentiation of a clonal precursor, the blast colony-forming cell (BL-CFC), which gives rise to blast colonies with both endothelial and haematopoietic components. Although recent studies have now provided evidence for the presence of this bipotential precursor in vivo, the precise mechanism for generation of haematopoietic cells from the haemangioblast still remains completely unknown. Here we demonstrate that the haemangioblast generates haematopoietic cells through the formation of a haemogenic endothelium intermediate, providing the first direct link between these two precursor populations. The cell population containing the haemogenic endothelium is transiently generated during BL-CFC development. This cell population is also present in gastrulating mouse embryos and generates haematopoietic cells on further culture. At the molecular level, we demonstrate that the transcription factor Tal1 (also known as Scl; ref. 10) is indispensable for the establishment of this haemogenic endothelium population whereas the core binding factor Runx1 (also known as AML1; ref. 11) is critical for generation of definitive haematopoietic cells from haemogenic endothelium. Together our results merge the two a priori conflicting theories on the origin of haematopoietic development into a single linear developmental process.


Assuntos
Hemangioblastos/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos ICR , Proteínas de Fusão Oncogênica/metabolismo
20.
J Immunol ; 174(4): 1980-8, 2005 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-15699126

RESUMO

The role of the thymus in T cell commitment of hemopoietic precursor is yet controversial. We previously identified a major T cell progenitor activity in precursor cells isolated from bone marrow-derived spleen colonies. In this study, we characterize the properties of these pre-T cells. We demonstrate that they have unique phenotype and can be generated in a total absence of any thymic influence. Indeed, even when studied at the single-cell level, extrathymic T cell-committed precursors express T cell-specific genes. Moreover, these cells are not committed to a particular T cell differentiation pathway because they can generate both extrathymic CD8alphaalpha+ intraepithelial lymphocytes and thymus-derived conventional thymocytes. We also compared these pre-T cells with fully T cell-committed thymic progenitors. When tested in vitro or by direct intrathymic transfer, these cells have a low clonogenic activity. However, after i.v. transfer, thymus repopulation is efficient and these precursors generate very high numbers of peripheral T cells. These results suggest the existence of extra steps of pre-T cell maturation that improve thymus reconstitution capacity and that can be delivered even after full T cell commitment. Consequently, our studies identify a source of extrathymic progenitors that will be helpful in defining the role of the thymus in the earliest steps of T cell differentiation.


Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Linfócitos T/citologia , Timo/citologia , Timo/imunologia , Transferência Adotiva , Animais , Complexo CD3/metabolismo , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Feminino , Transplante de Células-Tronco Hematopoéticas/métodos , Receptores de Hialuronatos/metabolismo , Imunofenotipagem , Injeções Intravenosas , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quimera por Radiação , Baço/citologia , Baço/imunologia , Timo/metabolismo , Timo/transplante
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