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1.
Br J Haematol ; 205(2): 580-593, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38887897

RESUMEN

The transcription factor GATA2 has a pivotal role in haematopoiesis. Heterozygous germline GATA2 mutations result in a syndrome characterized by immunodeficiency, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukaemia. Clinical symptoms in these patients are diverse and mechanisms driving GATA2-related phenotypes are largely unknown. To explore the impact of GATA2 haploinsufficiency on haematopoiesis, we generated a zebrafish model carrying a heterozygous mutation of gata2b (gata2b+/-), an orthologue of GATA2. Morphological analysis revealed myeloid and erythroid dysplasia in gata2b+/- kidney marrow. Because Gata2b could affect both transcription and chromatin accessibility during lineage differentiation, this was assessed by single-cell (sc) RNA-seq and single-nucleus (sn) ATAC-seq. Sn-ATAC-seq showed that the co-accessibility between the transcription start site (TSS) and a -3.5-4.1 kb putative enhancer was more robust in gata2b+/- zebrafish HSPCs compared to wild type, increasing gata2b expression and resulting in higher genome-wide Gata2b motif use in HSPCs. As a result of increased accessibility of the gata2b locus, gata2b+/- chromatin was also more accessible during lineage differentiation. scRNA-seq data revealed myeloid differentiation defects, that is, impaired cell cycle progression, reduced expression of cebpa and cebpb and increased signatures of ribosome biogenesis. These data also revealed a differentiation delay in erythroid progenitors, aberrant proliferative signatures and down-regulation of Gata1a, a master regulator of erythropoiesis, which worsened with age. These findings suggest that cell-intrinsic compensatory mechanisms, needed to obtain normal levels of Gata2b in heterozygous HSPCs to maintain their integrity, result in aberrant lineage differentiation, thereby representing a critical step in the predisposition to MDS.


Asunto(s)
Epigénesis Genética , Factor de Transcripción GATA2 , Heterocigoto , Pez Cebra , Animales , Factor de Transcripción GATA2/genética , Proteínas de Pez Cebra/genética , Células Eritroides/metabolismo , Células Eritroides/patología , Células Mieloides/metabolismo , Células Mieloides/patología , Eritropoyesis/genética , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Síndromes Mielodisplásicos/metabolismo
2.
Br J Haematol ; 200(1): 79-86, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36168923

RESUMEN

Severe congenital neutropenia (SCN) patients are prone to develop myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML). Leukaemic progression of SCN is associated with the early acquisition of CSF3R mutations in haematopoietic progenitor cells (HPCs), which truncate the colony-stimulating factor 3 receptor (CSF3R). These mutant clones may arise years before MDS/AML becomes overt. Introduction and activation of CSF3R truncation mutants in normal HPCs causes a clonally dominant myeloproliferative state in mice treated with CSF3. Paradoxically, in SCN patients receiving CSF3 therapy, clonal dominance of CSF3R mutant clones usually occurs only after the acquisition of additional mutations shortly before frank MDS or AML is diagnosed. To seek an explanation for this discrepancy, we introduced a patient-derived CSF3R-truncating mutation in ELANE-SCN and HAX1-SCN derived and control induced pluripotent stem cells and compared the CSF3 responses of HPCs generated from these lines. In contrast to CSF3R-mutant control HPCs, CSF3R-mutant HPCs from SCN patients do not show increased proliferation but display elevated levels of inflammatory signalling. Thus, activation of the truncated CSF3R in SCN-HPCs does not evoke clonal outgrowth but causes a sustained pro-inflammatory state, which has ramifications for how these CSF3R mutants contribute to the leukaemic transformation of SCN.


Asunto(s)
Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Ratones , Animales , Síndromes Congénitos de Insuficiencia de la Médula Ósea/genética , Leucemia Mieloide Aguda/diagnóstico , Mutación , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/complicaciones
3.
Blood ; 127(11): 1426-37, 2016 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-26834239

RESUMEN

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.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Animales , Aorta/citología , Aorta/embriología , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Linaje de la Célula , Células Cultivadas , Técnicas de Reprogramación Celular , Factor de Transcripción GATA2/deficiencia , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/fisiología , Genes Reporteros , Vectores Genéticos/genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/clasificación , Células Madre Hematopoyéticas/fisiología , Hígado/citología , Hígado/embriología , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transcriptoma , Transgenes , Arterias Umbilicales/citología , Arterias Umbilicales/embriología
4.
Circ Res ; 110(4): 578-87, 2012 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-22247485

RESUMEN

RATIONALE: The importance for Bmp signaling during embryonic stem cell differentiation into myocardial cells has been recognized. The question when and where Bmp signaling in vivo regulates myocardial differentiation has remained largely unanswered. OBJECTIVE: To identify when and where Bmp signaling regulates cardiogenic differentiation. METHODS AND RESULTS: Here we have observed that in zebrafish embryos, Bmp signaling is active in cardiac progenitor cells prior to their differentiation into cardiomyocytes. Bmp signaling is continuously required during somitogenesis within the anterior lateral plate mesoderm to induce myocardial differentiation. Surprisingly, Bmp signaling is actively repressed in differentiating myocardial cells. We identified the inhibitory Smad6a, which is expressed in the cardiac tissue, to be required to inhibit Bmp signaling and thereby promote expansion of the ventricular myocardium. CONCLUSION: Bmp signaling exerts opposing effects on myocardial differentiation in the embryo by promoting as well as inhibiting cardiac growth.


Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Células Madre Embrionarias/metabolismo , Corazón/embriología , Miocitos Cardíacos/metabolismo , Transducción de Señal , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Morfogenéticas Óseas/genética , Proliferación Celular , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Mutación , Proteína smad6/metabolismo , Proteínas de Dominio T Box/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética
5.
Dev Cell ; 14(2): 287-97, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18267096

RESUMEN

We have used high-resolution 4D imaging of cardiac progenitor cells (CPCs) in zebrafish to investigate the earliest left-right asymmetric movements during cardiac morphogenesis. Differential migratory behavior within the heart field was observed, resulting in a rotation of the heart tube. The leftward displacement and rotation of the tube requires hyaluronan synthase 2 expression within the CPCs. Furthermore, by reducing or ectopically activating BMP signaling or by implantation of BMP beads we could demonstrate that BMP signaling, which is asymmetrically activated in the lateral plate mesoderm and regulated by early left-right signals, is required to direct CPC migration and cardiac rotation. Together, these results support a model in which CPCs migrate toward a BMP source during development of the linear heart tube, providing a mechanism by which the left-right axis drives asymmetric development of the vertebrate heart.


Asunto(s)
Tipificación del Cuerpo , Movimiento Celular , Corazón/embriología , Miocardio/citología , Rotación , Células Madre/citología , Pez Cebra/embriología , Animales , Tipificación del Cuerpo/efectos de los fármacos , Proteínas Morfogenéticas Óseas/farmacología , Linaje de la Célula/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Glucuronosiltransferasa/metabolismo , Corazón/efectos de los fármacos , Humanos , Hialuronano Sintasas , Factores de Determinación Derecha-Izquierda , Mesodermo/citología , Mesodermo/efectos de los fármacos , Mutación/genética , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Células Madre/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Proteínas de Pez Cebra/metabolismo
6.
Front Cell Dev Biol ; 11: 1250827, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38033856

RESUMEN

The correct maintenance and differentiation of hematopoietic stem cells (HSC) in bone marrow is vital for the maintenance and operation of the human blood system. GATA2 plays a critical role in the maintenance of HSCs and the specification of HSCs into the different hematopoietic lineages, highlighted by the various defects observed in patients with heterozygous mutations in GATA2, resulting in cytopenias, bone marrow failure and increased chance of myeloid malignancy, termed GATA2 deficiency syndrome. Despite this, the mechanisms underlying GATA2 deficiency syndrome remain to be elucidated. The detailed description of how GATA2 regulates HSC maintenance and blood lineage determination is crucial to unravel the pathogenesis of GATA2 deficiency syndrome. In this review, we summarize current advances in elucidating the role of GATA2 in hematopoietic cell fate determination and discuss the challenges of modeling GATA2 deficiency syndrome.

7.
Blood Adv ; 7(10): 2082-2093, 2023 05 23.
Artículo en Inglés | MEDLINE | ID: mdl-36649572

RESUMEN

The first hematopoietic stem cells (HSCs) are formed through endothelial-to-hematopoietic transition (EHT) during embryonic development. The transcription factor GATA2 is a crucial regulator of EHT and HSC function throughout life. Because patients with GATA2 haploinsufficiency have inborn mutations, prenatal defects are likely to influence disease development. In mice, Gata2 haploinsufficiency (Gata2+/-) reduces the number and functionality of embryonic hematopoietic stem and progenitor cells (HSPCs) generated through EHT. However, the embryonic HSPC pool is heterogeneous and the mechanisms underlying this defect in Gata2+/- embryos remain unclear. Here, we investigated whether Gata2 haploinsufficiency selectively affects a cellular subset undergoing EHT. We showed that Gata2+/- HSPCs initiate, but cannot fully activate, hematopoietic programming during EHT. In addition, due to the reduced activity of the endothelial repressor Gfi1b, Gata2+/- HSPCs cannot repress endothelial identity to complete maturation. Finally, we showed that hematopoietic-specific induction of gfi1b could restore HSC production in gata2b-null (gata2b-/-) zebrafish embryos. This study illustrates the pivotal role of Gata2 in the regulation of the transcriptional network governing HSPC identity throughout the EHT.


Asunto(s)
Deficiencia GATA2 , Pez Cebra , Embarazo , Femenino , Animales , Ratones , Pez Cebra/metabolismo , Diferenciación Celular , Células Madre Hematopoyéticas/metabolismo , Factores de Transcripción/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo
8.
Blood Adv ; 6(5): 1537-1546, 2022 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-35114690

RESUMEN

Vaccination guidelines for patients treated for hematological diseases are typically conservative. Given their high risk for severe COVID-19, it is important to identify those patients that benefit from vaccination. We prospectively quantified serum immunoglobulin G (IgG) antibodies to spike subunit 1 (S1) antigens during and after 2-dose mRNA-1273 (Spikevax/Moderna) vaccination in hematology patients. Obtaining S1 IgG ≥ 300 binding antibody units (BAUs)/mL was considered adequate as it represents the lower level of S1 IgG concentration obtained in healthy individuals, and it correlates with potent virus neutralization. Selected patients (n = 723) were severely immunocompromised owing to their disease or treatment thereof. Nevertheless, >50% of patients obtained S1 IgG ≥ 300 BAUs/mL after 2-dose mRNA-1273. All patients with sickle cell disease or chronic myeloid leukemia obtained adequate antibody concentrations. Around 70% of patients with chronic graft-versus-host disease (cGVHD), multiple myeloma, or untreated chronic lymphocytic leukemia (CLL) obtained S1 IgG ≥ 300 BAUs/mL. Ruxolitinib or hypomethylating therapy but not high-dose chemotherapy blunted responses in myeloid malignancies. Responses in patients with lymphoma, patients with CLL on ibrutinib, and chimeric antigen receptor T-cell recipients were low. The minimal time interval after autologous hematopoietic cell transplantation (HCT) to reach adequate concentrations was <2 months for multiple myeloma, 8 months for lymphoma, and 4 to 6 months after allogeneic HCT. Serum IgG4, absolute B- and natural killer-cell number, and number of immunosuppressants predicted S1 IgG ≥ 300 BAUs/mL. Hematology patients on chemotherapy, shortly after HCT, or with cGVHD should not be precluded from vaccination. This trial was registered at Netherlands Trial Register as #NL9553.


Asunto(s)
COVID-19 , Hematología , Vacuna nCoV-2019 mRNA-1273 , COVID-19/prevención & control , Vacunas contra la COVID-19 , Humanos , SARS-CoV-2 , Vacunación
9.
Blood Adv ; 5(3): 829-842, 2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33560396

RESUMEN

Integrated molecular signals regulate cell fate decisions in the embryonic aortic endothelium to drive hematopoietic stem cell (HSC) generation during development. The G-protein-coupled receptor 56 (Gpr56, also called Adgrg1) is the most highly upregulated receptor gene in cells that take on hematopoietic fate and is expressed by adult bone marrow HSCs. Despite the requirement for Gpr56 in hematopoietic stem/progenitor cell (HS/PC) generation in zebrafish embryos and the highly upregulated expression of GPR56 in treatment-resistant leukemic patients, its function in normal mammalian hematopoiesis remains unclear. Here, we examine the role of Gpr56 in HS/PC development in Gpr56 conditional knockout (cKO) mouse embryos and Gpr knockout (KO) embryonic stem cell (ESC) hematopoietic differentiation cultures. Our results show a bias toward myeloid differentiation of Gpr56 cKO fetal liver HSCs and an increased definitive myeloid progenitor cell frequency in Gpr56KO ESC differentiation cultures. Surprisingly, we find that mouse Gpr97 can rescue Gpr56 morphant zebrafish hematopoietic generation, and that Gpr97 expression is upregulated in mouse Gpr56 deletion models. When both Gpr56 and Gpr97 are deleted in ESCs, no or few hematopoietic PCs (HPCs) are generated upon ESC differentiation. Together, our results reveal novel and redundant functions for these 2 G-protein coupled receptors in normal mammalian hematopoietic cell development and differentiation.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Pez Cebra , Animales , Diferenciación Celular , Hematopoyesis/genética , Células Madre Hematopoyéticas , Humanos , Ratones , Receptores Acoplados a Proteínas G/genética , Pez Cebra/genética
10.
Blood Adv ; 5(3): 775-786, 2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33560392

RESUMEN

Mutations in ELANE cause severe congenital neutropenia (SCN), but how they affect neutrophil production and contribute to leukemia predisposition is unknown. Neutropenia is alleviated by CSF3 (granulocyte colony-stimulating factor) therapy in most cases, but dose requirements vary between patients. Here, we show that CD34+CD45+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cell lines from patients with SCN that have mutations in ELANE (n = 2) or HAX1 (n = 1) display elevated levels of reactive oxygen species (ROS) relative to normal iPSC-derived HPCs. In patients with ELANE mutations causing misfolding of the neutrophil elastase (NE) protein, HPCs contained elevated numbers of promyelocyte leukemia protein nuclear bodies, a hallmark of acute oxidative stress. This was confirmed in primary bone marrow cells from 3 additional patients with ELANE-mutant SCN. Apart from responding to elevated ROS levels, PML controlled the metabolic state of these ELANE-mutant HPCs as well as the expression of ELANE, suggestive of a feed-forward mechanism of disease development. Both PML deletion and correction of the ELANE mutation restored CSF3 responses of these ELANE-mutant HPCs. These findings suggest that PML plays a crucial role in the disease course of ELANE-SCN characterized by NE misfolding, with potential implications for CSF3 therapy.


Asunto(s)
Elastasa de Leucocito/genética , Neutropenia , Proteínas Adaptadoras Transductoras de Señales , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Factor Estimulante de Colonias de Granulocitos , Humanos , Mutación , Neutropenia/congénito , Neutropenia/genética
11.
Blood Adv ; 5(13): 2687-2700, 2021 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-34170285

RESUMEN

The differentiation of hematopoietic stem cells (HSCs) is tightly controlled to ensure a proper balance between myeloid and lymphoid cell output. GATA2 is a pivotal hematopoietic transcription factor required for generation and maintenance of HSCs. GATA2 is expressed throughout development, but because of early embryonic lethality in mice, its role during adult hematopoiesis is incompletely understood. Zebrafish contains 2 orthologs of GATA2: Gata2a and Gata2b, which are expressed in different cell types. We show that the mammalian functions of GATA2 are split between these orthologs. Gata2b-deficient zebrafish have a reduction in embryonic definitive hematopoietic stem and progenitor cell (HSPC) numbers, but are viable. This allows us to uniquely study the role of GATA2 in adult hematopoiesis. gata2b mutants have impaired myeloid lineage differentiation. Interestingly, this defect arises not in granulocyte-monocyte progenitors, but in HSPCs. Gata2b-deficient HSPCs showed impaired progression of the myeloid transcriptional program, concomitant with increased coexpression of lymphoid genes. This resulted in a decrease in myeloid-programmed progenitors and a relative increase in lymphoid-programmed progenitors. This shift in the lineage output could function as an escape mechanism to avoid a block in lineage differentiation. Our study helps to deconstruct the functions of GATA2 during hematopoiesis and shows that lineage differentiation flows toward a lymphoid lineage in the absence of Gata2b.


Asunto(s)
Células Madre Hematopoyéticas , Pez Cebra , Animales , Diferenciación Celular , Factor de Transcripción GATA2/genética , Hematopoyesis , Ratones , Monocitos , Proteínas de Pez Cebra
12.
Front Genome Ed ; 2: 602182, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-34713225

RESUMEN

Inherited bone marrow failure syndromes (IBMFS) are monogenetic disorders that result in a reduction of mature blood cell formation and predisposition to leukemia. In children with myeloid leukemia the gene most often mutated is Gata binding protein 2 (GATA2) and 80% of patients with GATA2 mutations develop myeloid malignancy before the age of forty. Although GATA2 is established as one of the key regulators of embryonic and adult hematopoiesis, the mechanisms behind the leukemia predisposition in GATA2 haploinsufficiencies is ambiguous. The only curative treatment option currently available is allogeneic hematopoietic stem cell transplantation (allo-SCT). However, allo-SCT can only be applied at a relatively late stage of the disease as its applicability is compromised by treatment related morbidity and mortality (TRM). Alternatively, autologous hematopoietic stem cell transplantation (auto-SCT), which is associated with significantly less TRM, might become a treatment option if repaired hematopoietic stem cells would be available. Here we discuss the recent literature on leukemia predisposition syndromes caused by GATA2 mutations, current knowledge on the function of GATA2 in the hematopoietic system and advantages and pitfalls of potential treatment options provided by genome editing.

13.
Commun Biol ; 3(1): 71, 2020 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-32054973

RESUMEN

Gata2 is a key transcription factor required to generate Haematopoietic Stem and Progenitor Cells (HSPCs) from haemogenic endothelium (HE); misexpression of Gata2 leads to haematopoietic disorders. Here we deleted a conserved enhancer (i4 enhancer) driving pan-endothelial expression of the zebrafish gata2a and showed that Gata2a is required for HE programming by regulating expression of runx1 and of the second Gata2 orthologue, gata2b. By 5 days, homozygous gata2aΔi4/Δi4 larvae showed normal numbers of HSPCs, a recovery mediated by Notch signalling driving gata2b and runx1 expression in HE. However, gata2aΔi4/Δi4 adults showed oedema, susceptibility to infections and marrow hypo-cellularity, consistent with bone marrow failure found in GATA2 deficiency syndromes. Thus, gata2a expression driven by the i4 enhancer is required for correct HE programming in embryos and maintenance of steady-state haematopoietic stem cell output in the adult. These enhancer mutants will be useful in exploring further the pathophysiology of GATA2-related deficiencies in vivo.


Asunto(s)
Reprogramación Celular/genética , Secuencia Conservada , Endotelio/metabolismo , Elementos de Facilitación Genéticos , Factor de Transcripción GATA2/genética , Hematopoyesis/genética , Eliminación de Secuencia , Factores de Edad , Animales , Secuencia de Bases , Cromatina/genética , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Sitios Genéticos , Células Madre Hematopoyéticas/metabolismo , Pez Cebra
14.
Elife ; 92020 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-32367800

RESUMEN

Macrophages derive from multiple sources of hematopoietic progenitors. Most macrophages require colony-stimulating factor 1 receptor (CSF1R), but some macrophages persist in the absence of CSF1R. Here, we analyzed mpeg1:GFP-expressing macrophages in csf1r-deficient zebrafish and report that embryonic macrophages emerge followed by their developmental arrest. In larvae, mpeg1+ cell numbers then increased showing two distinct types in the skin: branched, putative Langerhans cells, and amoeboid cells. In contrast, although numbers also increased in csf1r-mutants, exclusively amoeboid mpeg1+ cells were present, which we showed by genetic lineage tracing to have a non-hematopoietic origin. They expressed macrophage-associated genes, but also showed decreased phagocytic gene expression and increased epithelial-associated gene expression, characteristic of metaphocytes, recently discovered ectoderm-derived cells. We further demonstrated that juvenile csf1r-deficient zebrafish exhibit systemic macrophage depletion. Thus, csf1r deficiency disrupts embryonic to adult macrophage development. Zebrafish deficient for csf1r are viable and permit analyzing the consequences of macrophage loss throughout life.


Immune cells called macrophages are found in all organs in the body. These cells are highly effective at eating and digesting large particles including dead cells and debris, and microorganisms such as bacteria. Macrophages are also instrumental in shaping developing organs and repairing tissues during life. Macrophages were, until recently, thought to be constantly replenished from cells circulating in the bloodstream. However, it turns out that separate populations of macrophages become established in most tissues during embryonic development and are maintained throughout life without further input. Previous studies of zebrafish, rodents and humans have shown that, when a gene called CSF1R is non-functional, macrophages are absent from many organs including the brain. However, some tissue-specific macrophages still persist, and it was not clear why these cells do not rely on the CSF1R gene while others do. Kuil et al. set out to decipher the precise requirement for the CSF1R gene in macrophage development in living zebrafish. The experiments used zebrafish that make a green fluorescent protein in their macrophages. As these fish are transparent, this meant that Kuil et al. could observe the cells within the living fish and isolate them to determine which genes are switched on and off. This approach revealed that zebrafish with a mutated version of the CSF1R gene make macrophages as embryos but that these cells then fail to multiply and migrate into the developing organs. This results in fewer macrophages in the zebrafish's tissues, and an absence of these cells in the brain. Kuil et al. went on to show that new macrophages did emerge in zebrafish that were about two to three weeks old. However, unexpectedly, these new cells were not regular macrophages. Instead, they were a new recently identified cell-type called metaphocytes, which share similarities with macrophages but have a completely different origin, move faster and do not eat particles. Zebrafish lacking the CSF1R gene thus lose nearly all their macrophages but retain metaphocytes. These macrophage-free mutant zebrafish constitute an unprecedented tool for further studies looking to discriminate the different roles of macrophages and metaphocytes.


Asunto(s)
Macrófagos/fisiología , Microglía/fisiología , Proteínas Tirosina Quinasas/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Proliferación Celular , Perfilación de la Expresión Génica , Macrófagos/metabolismo , Microglía/metabolismo , Proteínas Tirosina Quinasas Receptoras , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Pez Cebra/embriología , Proteínas de Pez Cebra/metabolismo
15.
Cell Rep Med ; 1(5): 100074, 2020 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-33205068

RESUMEN

Severe congenital neutropenia (SCN) patients treated with CSF3/G-CSF to alleviate neutropenia frequently develop acute myeloid leukemia (AML). A common pattern of leukemic transformation involves the appearance of hematopoietic clones with CSF3 receptor (CSF3R) mutations in the neutropenic phase, followed by mutations in RUNX1 before AML becomes overt. To investigate how the combination of CSF3 therapy and CSF3R and RUNX1 mutations contributes to AML development, we make use of mouse models, SCN-derived induced pluripotent stem cells (iPSCs), and SCN and SCN-AML patient samples. CSF3 provokes a hyper-proliferative state in CSF3R/RUNX1 mutant hematopoietic progenitors but does not cause overt AML. Intriguingly, an additional acquired driver mutation in Cxxc4 causes elevated CXXC4 and reduced TET2 protein levels in murine AML samples. Expression of multiple pro-inflammatory pathways is elevated in mouse AML and human SCN-AML, suggesting that inflammation driven by downregulation of TET2 activity is a critical step in the malignant transformation of SCN.


Asunto(s)
Transformación Celular Neoplásica/genética , Síndromes Congénitos de Insuficiencia de la Médula Ósea/genética , Síndromes Congénitos de Insuficiencia de la Médula Ósea/patología , Proteínas de Unión al ADN/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Mutación/genética , Neutropenia/congénito , Factores de Transcripción/genética , Animales , Línea Celular , Línea Celular Tumoral , Transformación Celular Neoplásica/patología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Células HEK293 , Humanos , Inflamación/genética , Inflamación/patología , Células K562 , Ratones , Neutropenia/genética , Neutropenia/patología , Transducción de Señal/genética
16.
Front Cell Dev Biol ; 6: 124, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30374440

RESUMEN

Hematopoiesis is an optimal system for studying stem cell maintenance and lineage differentiation under physiological and pathological conditions. In vertebrate organisms, billions of differentiated hematopoietic cells need to be continuously produced to replenish the blood cell pool. Disruptions in this process have immediate consequences for oxygen transport, responses against pathogens, maintenance of hemostasis and vascular integrity. Zebrafish is a widely used and well-established model for studying the hematopoietic system. Several new hematopoietic regulators were identified in genetic and chemical screens using the zebrafish model. Moreover, zebrafish enables in vivo imaging of hematopoietic stem cell generation and differentiation during embryogenesis, and adulthood. Finally, zebrafish has been used to model hematopoietic diseases. Recent technological advances in single-cell transcriptome analysis, epigenetic regulation, proteomics, metabolomics, and processing of large data sets promise to transform the current understanding of normal, abnormal, and malignant hematopoiesis. In this perspective, we discuss how the zebrafish model has proven beneficial for studying physiological and pathological hematopoiesis and how these novel technologies are transforming the field.

17.
J Exp Med ; 212(1): 93-106, 2015 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-25547674

RESUMEN

Hematopoietic stem cells (HSCs) are generated via a natural transdifferentiation process known as endothelial to hematopoietic cell transition (EHT). Because of small numbers of embryonal arterial cells undergoing EHT and the paucity of markers to enrich for hemogenic endothelial cells (ECs [HECs]), the genetic program driving HSC emergence is largely unknown. Here, we use a highly sensitive RNAseq method to examine the whole transcriptome of small numbers of enriched aortic HSCs, HECs, and ECs. Gpr56, a G-coupled protein receptor, is one of the most highly up-regulated of the 530 differentially expressed genes. Also, highly up-regulated are hematopoietic transcription factors, including the "heptad" complex of factors. We show that Gpr56 (mouse and human) is a target of the heptad complex and is required for hematopoietic cluster formation during EHT. Our results identify the processes and regulators involved in EHT and reveal the surprising requirement for Gpr56 in generating the first HSCs.


Asunto(s)
Transdiferenciación Celular/genética , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Células Madre Hematopoyéticas/metabolismo , Receptores Acoplados a Proteínas G/genética , Animales , Células CHO , Células COS , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Cricetulus , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Endoteliales/citología , Femenino , Ontología de Genes , Células Madre Hematopoyéticas/citología , Humanos , Hibridación in Situ , Ratones Endogámicos C57BL , Microscopía Confocal , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN/métodos , Regulación hacia Arriba
18.
Stem Cell Res ; 12(1): 24-35, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24141110

RESUMEN

Hypoxia affects many physiologic processes during early stages of mammalian ontogeny, particularly placental and vascular development. In the adult, the hypoxic bone marrow microenvironment plays a role in regulating hematopoietic stem cell (HSC) function. HSCs are generated from the major vasculature of the embryo, but whether the hypoxic response affects the generation of these HSCs is as yet unknown. Here we examined whether Hypoxia Inducible Factor1-alpha (HIF1α), a key modulator of the response to hypoxia, is essential for HSC development. We found hypoxic cells in embryonic tissues that generate and expand hematopoietic cells (aorta, placenta and fetal liver), and specifically aortic endothelial and hematopoietic cluster cells. A Cre/loxP conditional knockout (cKO) approach was taken to delete HIF1α in Vascular Endothelial-Cadherin expressing endothelial cells, the precursors to definitive hematopoietic cells. Functional assays show that HSC and hematopoietic progenitor cells (HPCs) are significantly reduced in cKO aorta and placenta. Moreover, decreases in phenotypic aortic hematopoietic cluster cells in cKO embryos indicate that HIF1α is necessary for generation and/or expansion of HPCs and HSCs. cKO adult BM HSCs are also affected under transplantation conditions. Thus, HIF1α is a regulator of HSC generation and function beginning at the earliest embryonic stages.


Asunto(s)
Hipoxia de la Célula/fisiología , Células Madre Hematopoyéticas/citología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Animales , Aorta/citología , Cadherinas/metabolismo , Separación Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Feto/citología , Trasplante de Células Madre Hematopoyéticas , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Hígado/citología , Ratones , Ratones Endogámicos C57BL , Placenta/citología , Embarazo , Trasplante Homólogo
19.
J Exp Med ; 211(12): 2411-23, 2014 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-25385755

RESUMEN

Hematopoietic stem cell (HSC) specification occurs in the embryonic aorta and requires Notch activation; however, most of the Notch-regulated elements controlling de novo HSC generation are still unknown. Here, we identify putative direct Notch targets in the aorta-gonad-mesonephros (AGM) embryonic tissue by chromatin precipitation using antibodies against the Notch partner RBPj. By ChIP-on-chip analysis of the precipitated DNA, we identified 701 promoter regions that were candidates to be regulated by Notch in the AGM. One of the most enriched regions corresponded to the Cdca7 gene, which was subsequently confirmed to recruit the RBPj factor but also Notch1 in AGM cells. We found that during embryonic hematopoietic development, expression of Cdca7 is restricted to the hematopoietic clusters of the aorta, and it is strongly up-regulated in the hemogenic population during human embryonic stem cell hematopoietic differentiation in a Notch-dependent manner. Down-regulation of Cdca7 mRNA in cultured AGM cells significantly induces hematopoietic differentiation and loss of the progenitor population. Finally, using loss-of-function experiments in zebrafish, we demonstrate that CDCA7 contributes to HSC emergence in vivo during embryonic development. Thus, our study identifies Cdca7 as an evolutionary conserved Notch target involved in HSC emergence.


Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Proteínas Nucleares/genética , Receptor Notch1/genética , Transcripción Genética , Animales , Aorta/embriología , Aorta/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular/genética , Línea Celular , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Células Madre Hematopoyéticas/citología , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/metabolismo , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Serrate-Jagged , Pez Cebra/embriología , Pez Cebra/genética
20.
J Exp Med ; 210(13): 2843-50, 2013 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-24297996

RESUMEN

Knowledge of the key transcription factors that drive hematopoietic stem cell (HSC) generation is of particular importance for current hematopoietic regenerative approaches and reprogramming strategies. Whereas GATA2 has long been implicated as a hematopoietic transcription factor and its dysregulated expression is associated with human immunodeficiency syndromes and vascular integrity, it is as yet unknown how GATA2 functions in the generation of HSCs. HSCs are generated from endothelial cells of the major embryonic vasculature (aorta, vitelline, and umbilical arteries) and are found in intra-aortic hematopoietic clusters. In this study, we find that GATA2 function is essential for the generation of HSCs during the stage of endothelial-to-hematopoietic cell transition. Specific deletion of Gata2 in Vec (Vascular Endothelial Cadherin)-expressing endothelial cells results in a deficiency of long-term repopulating HSCs and intra-aortic cluster cells. By specific deletion of Gata2 in Vav-expressing hematopoietic cells (after HSC generation), we further show that GATA2 is essential for HSC survival. This is in contrast to the known activity of the RUNX1 transcription factor, which functions only in the generation of HSCs, and highlights the unique requirement for GATA2 function in HSCs throughout all developmental stages.


Asunto(s)
Factor de Transcripción GATA2/fisiología , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/citología , Alelos , Animales , Apoptosis , Separación Celular , Supervivencia Celular , Citometría de Flujo , Eliminación de Gen , Ratones , Ratones Noqueados , Ratones Transgénicos , Células Madre
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