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1.
Development ; 151(3)2024 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-38240311

RESUMEN

In vertebrates, the central nervous system (CNS) harbours various immune cells, including parenchymal microglia, perivascular macrophages and dendritic cells, which act in coordination to establish an immune network to regulate neurogenesis and neural function, and to maintain the homeostasis of the CNS. Recent single cell transcriptomic profiling has revealed that the adult zebrafish CNS contains microglia, plasmacytoid dendritic cells (pDCs) and two conventional dendritic cells (cDCs), ccl35+ cDCs and cnn3a+cDCs. However, how these distinct myeloid cells are established in the adult zebrafish CNS remains incompletely defined. Here, we show that the Inhibitor of DNA binding 2a (Id2a) is essential for the development of pDCs and cDCs but is dispensable for the formation of microglia, whereas the Basic leucine zipper transcription factor ATF-like 3 (Batf3) acts downstream of id2a and is required exclusively for the formation of the cnn3a+ cDC subset. In contrast, the Zinc finger E-box-binding homeobox 2a (Zeb2a) promotes the expansion of microglia and inhibits the DC specification, possibly through repressing id2a expression. Our study unravels the genetic networks that govern the development of microglia and brain-associated DCs in the zebrafish CNS.


Asunto(s)
Microglía , Pez Cebra , Animales , Pez Cebra/genética , Diferenciación Celular/genética , Células Dendríticas/metabolismo , Encéfalo
2.
Development ; 149(19)2022 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-36178053

RESUMEN

The E26 transformation-specific or E-twenty-six (ETS) genes encode a superfamily of transcription factors involved in diverse biological processes. Here, we report the identification and characterization of a previously unidentified member of the ETS transcription factors, Spi2, that is found exclusively in the ray-finned fish kingdom. We show that the expression of spi2 is restricted to hemogenic endothelial cells (HECs) and to hematopoietic stem and progenitor cells (HSPCs) in zebrafish. Using bacteria artificial chromosome transgenesis, we generate a spi2 reporter line, TgBAC(spi2:P2a-GFP), which manifests the GFP pattern recapitulating the endogenous spi2 expression. Genetic ablation of spi2 has little effect on HEC formation and the endothelial-to-hematopoietic transition, but results in compromised proliferation of HSPCs in the caudal hematopoietic tissue (CHT) during early development and in severe myeloid lineage defect in adulthood. Epistatic analysis shows that spi2 acts downstream of runx1 in regulating HSPC development in the CHT. Our study identifies Spi2 as an essential regulator for definitive hematopoietic cell development and creates a TgBAC(spi2:P2a-GFP) reporter line for tracking HECs, HSPCs, myeloid cells and thrombocytes from early development to adulthood.


Asunto(s)
Hemangioblastos , Pez Cebra , Animales , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Hemangioblastos/metabolismo , Hematopoyesis/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
3.
Development ; 149(16)2022 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-35876681

RESUMEN

In vertebrates, hematopoietic stem and progenitor cells (HSPCs) are capable of self-renewal and continuously replenishing all mature blood lineages throughout life. However, the molecular signaling regulating the maintenance and expansion of HSPCs remains incompletely understood. Colony-stimulating factor 1 receptor (CSF1R) is believed to be the primary regulator for the myeloid lineage but not HSPC development. Here, we show a surprising role of Csf1rb, a zebrafish homolog of mammalian CSF1R, in preserving the HSPC pool by maintaining the proliferation of HSPCs. Deficiency of csf1rb leads to a reduction in both HSPCs and their differentiated progenies, including myeloid, lymphoid and erythroid cells at early developmental stages. Likewise, the absence of csf1rb conferred similar defects upon HSPCs and leukocytes in adulthood. Furthermore, adult hematopoietic cells from csf1rb mutants failed to repopulate immunodeficient zebrafish. Interestingly, loss-of-function and gain-of-function assays suggested that the canonical ligands for Csf1r in zebrafish, including Csf1a, Csf1b and Il34, were unlikely to be ligands of Csf1rb. Thus, our data indicate a previously unappreciated role of Csf1r in maintaining HSPCs, independently of known ligands.


Asunto(s)
Proteínas de Pez Cebra , Pez Cebra , Animales , Diferenciación Celular/fisiología , Hematopoyesis/genética , Células Madre Hematopoyéticas/fisiología , Mamíferos , Pez Cebra/genética , Proteínas de Pez Cebra/genética
4.
Proc Natl Acad Sci U S A ; 119(13): e2119051119, 2022 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-35333649

RESUMEN

SignificanceHematopoietic stem cells (HSCs) are generated from specialized endothelial cells, called hemogenic endothelial cells (HECs). It has been debated whether HECs and non-HSC-forming conventional endothelial cells (cECs) arise from a common precursor or represent distinct lineages. Moreover, the molecular basis underlying their distinct fate determination is poorly understood. We use photoconvertible labeling, time-lapse imaging, and single-cell RNA-sequencing analysis to trace the lineage of HECs. We discovered that HECs and cECs arise from a common hemogenic angioblast precursor, and their distinct fate is determined by high or low dosage of Etv2, respectively. Our results illuminate the lineage origin and a mechanism on the fate determination of HECs, which may enhance the understanding on the ontogeny of HECs in vertebrates.


Asunto(s)
Hemangioblastos , Hematopoyesis , Animales , Diferenciación Celular , Endotelio Vascular
5.
Proc Natl Acad Sci U S A ; 119(39): e2203273119, 2022 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-36122226

RESUMEN

Microglia are the central nervous system (CNS)-resident macrophages involved in neural inflammation, neurogenesis, and neural activity regulation. Previous studies have shown that naturally occurring neuronal apoptosis plays a critical role in regulating microglial colonization of the brain in zebrafish. However, the molecular signaling cascades underlying neuronal apoptosis-mediated microglial colonization and the regulation of these cascades remain undefined. Here, we show that basic leucine zipper (b-Zip) transcription factors, Mafba and Mafbb, two zebrafish orthologs of mammalian MAFB, are key regulators in neuronal apoptosis-mediated microglial colonization of the brain in zebrafish. We document that the loss of Mafba and Mafbb function perturbs microglial colonization of the brain. We further demonstrate that Mafba and Mafbb act cell-autonomously and cooperatively to orchestrate microglial colonization, at least in part, by regulating the expression of G protein-coupled receptor 34a (Gpr34a), which directs peripheral macrophage recruitment into the brain through sensing the lysophosphatidylserine (lysoPS) released by the apoptotic neurons. Our study reveals that Mafba and Mafbb regulate neuronal apoptosis-mediated microglial colonization of the brain in zebrafish via the lysoPS-Gpr34a pathway.


Asunto(s)
Microglía , Pez Cebra , Animales , Encéfalo/fisiología , Quimiotaxis , Factores de Transcripción Maf , Mamíferos/metabolismo , Microglía/metabolismo , Proteínas Oncogénicas , Factores de Transcripción/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra
7.
Adv Exp Med Biol ; 1442: 137-157, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38228963

RESUMEN

Hematopoiesis is a complex process that tightly regulates the generation, proliferation, differentiation, and maintenance of hematopoietic cells. Disruptions in hematopoiesis can lead to various diseases affecting both hematopoietic and non-hematopoietic systems, such as leukemia, anemia, thrombocytopenia, rheumatoid arthritis, and chronic granuloma. The zebrafish serves as a powerful vertebrate model for studying hematopoiesis, offering valuable insights into both hematopoietic regulation and hematopoietic diseases. In this chapter, we present a comprehensive overview of zebrafish hematopoiesis, highlighting its distinctive characteristics in hematopoietic processes. We discuss the ontogeny and modulation of both primitive and definitive hematopoiesis, as well as the microenvironment that supports hematopoietic stem/progenitor cells. Additionally, we explore the utility of zebrafish as a disease model and its potential in drug discovery, which not only advances our understanding of the regulatory mechanisms underlying hematopoiesis but also facilitates the exploration of novel therapeutic strategies for hematopoietic diseases.


Asunto(s)
Enfermedades Hematológicas , Pez Cebra , Animales , Pez Cebra/genética , Hematopoyesis , Células Madre Hematopoyéticas , Proteínas de Pez Cebra
8.
J Biol Chem ; 295(26): 8846-8856, 2020 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-32398256

RESUMEN

Microglia are tissue-resident macrophages in the central nervous system (CNS) that play essential roles in the regulation of CNS development and homeostasis. Yet, the genetic networks governing microglia development remain incompletely defined. Here, we report the identification and characterization of a microglia-defective zebrafish mutant wulonghkz12 (wulhkz12 ) isolated from an ethylnitrosourea (ENU)-based genetic screen. We show that wulhkz12 mutants harbors a missense point mutation in the gene region encoding the PRY/SPRY domain of the tripartite-motif family protein 35-28 (trim35-28) gene. Time-lapse imaging revealed that the loss of Trim35-28 function causes lytic necrosis of microglial precursors/peripheral macrophages, as indicated by cytoplasmic swelling and membrane rupture of these precursors and accompanied by neutrophil infiltration and systemic inflammation. Intriguingly, the lytic necrosis of microglial precursors in trim35-28-deficient mutants appeared to depend neither on the canonical pyroptotic nor necroptotic pathways, as inhibition of the key component in each pathway could not rescue the microglia phenotype in trim35-28-deficient mutants. Finally, results from tissue-specific rescue experiments suggested that Trim35-28 acts cell-autonomously in the survival of microglial precursors. Taken together, the findings of our study reveal Trim35-28 as a regulatory protein essential for microglia development.


Asunto(s)
Microglía/citología , Células-Madre Neurales/citología , Pez Cebra/embriología , Animales , Microglía/fisiología , Mutación Missense , Necroptosis , Células-Madre Neurales/metabolismo , Neurogénesis , Pez Cebra/genética
9.
Development ; 143(12): 2103-10, 2016 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-27151951

RESUMEN

Hematopoietic stem and progenitor cells (HSPCs) are capable of self-renewal and replenishing all lineages of blood cells throughout life and are thus crucial for tissue homeostasis. However, the mechanism regulating HSPC development is still incompletely understood. Here, we isolate a zebrafish mutant with defective T lymphopoiesis and positional cloning identifies that Rpc9, a component of DNA-directed RNA polymerase III (Pol III) complex, is responsible for the mutant phenotype. Further analysis shows that rpc9 deficiency leads to the impairment of HSPCs and their derivatives in zebrafish embryos. Excessive apoptosis is observed in the caudal hematopoietic tissue (CHT; the equivalent of fetal liver in mammals) of rpc9(-/-) embryos and the hematopoietic defects in these embryos can be fully rescued by suppression of p53 Thus, our work illustrates that Rpc9, a component of Pol III, plays an important tissue-specific role in HSPC maintenance during zebrafish embryogenesis and might be conserved across vertebrates, including mammals.


Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , ARN Polimerasa III/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proliferación Celular , Supervivencia Celular , Embrión no Mamífero/metabolismo , Técnicas de Silenciamiento del Gen , Hematopoyesis , Mutación/genética , Fenotipo , Linfocitos T/citología , Proteína p53 Supresora de Tumor/metabolismo
10.
Blood ; 129(4): 509-519, 2017 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-27940477

RESUMEN

Macrophages are key components of the innate immune system and play pivotal roles in immune response, organ development, and tissue homeostasis. Studies in mice and zebrafish have shown that tissue-resident macrophages derived from different hematopoietic origins manifest distinct developmental kinetics and colonization potential, yet the genetic programs controlling the development of macrophages of different origins remain incompletely defined. In this study, we use zebrafish, where tissue-resident macrophages arise from the rostral blood island (RBI) and ventral wall of dorsal aorta (VDA), the zebrafish hematopoietic tissue equivalents to the mouse yolk sac and aorta-gonad-mesonephros for myelopoiesis, to address this issue. We show that RBI- and VDA-born macrophages are orchestrated by distinctive regulatory networks formed by the E-twenty-six (Ets) transcription factors Pu.1 and Spi-b, the zebrafish ortholog of mouse spleen focus forming virus proviral integration oncogene B (SPI-B), and the helix-turn-helix DNA-binding domain containing protein Irf8. Epistatic studies document that during RBI macrophage development, Pu.1 acts upstream of Spi-b, which, upon induction by Pu.1, partially compensates the function of Pu.1. In contrast, Pu.1 and Spi-b act in parallel and cooperatively to regulate the development of VDA-derived macrophages. Interestingly, these two distinct regulatory networks orchestrate the RBI- and VDA-born macrophage development largely by regulating a common downstream gene, Irf8. Our study indicates that macrophages derived from different origins are governed by distinct genetic networks formed by the same repertoire of myeloid-specific transcription factors.


Asunto(s)
Linaje de la Célula/inmunología , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Macrófagos/inmunología , Proteínas Proto-Oncogénicas/inmunología , Transactivadores/inmunología , Pez Cebra/inmunología , Secuencia de Aminoácidos , Animales , Aorta/citología , Aorta/crecimiento & desarrollo , Aorta/inmunología , Diferenciación Celular , Linaje de la Célula/genética , Embrión no Mamífero , Humanos , Inmunidad Innata , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/inmunología , Macrófagos/citología , Mesonefro/citología , Mesonefro/crecimiento & desarrollo , Mesonefro/inmunología , Ratones , Especificidad de Órganos , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/inmunología , Transducción de Señal , Transactivadores/genética , Saco Vitelino/citología , Saco Vitelino/crecimiento & desarrollo , Saco Vitelino/inmunología , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo
11.
Blood ; 128(3): 415-26, 2016 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-27268086

RESUMEN

Neutrophils are the key effectors for generating innate immunity in response to pathogenic infection and tissue injury in vertebrates. Dysregulation of neutrophil development and function is known to associate with various human disorders. Yet, the genetic network that orchestrates lineage commitment, differentiation, and maturation of neutrophils remains incompletely defined. Here, we present an in vivo study to delineate the genetic program underlying neutrophil development during zebrafish embryonic myelopoiesis. We show that loss of c-Myb function has no effect on macrophages but severely impairs neutrophil terminal differentiation, resulting in the accumulation of neutrophils with unsegmented nuclei and scant granule. This neutrophilic defect, which resembles the neutrophil-specific granule deficiency (SGD) caused by the mutations in CCAAT/enhancer-binding protein ε (C/EBPε) in humans, is attributed, at least in part, to the downregulation of the granule protein transcription. Likewise, genetic inactivation of Cebp1, the zebrafish functional homolog of mammalian C/EBPε, also leads to a similar SGD-like phenotype in zebrafish. Genetic epistasis and biochemical analysis further reveals that c-Myb and Cebp1 act in parallel and cooperatively to control neutrophil differentiation by directly regulating granule protein gene transcription. Our study indicates that c-MYB is an intrinsic master regulator for neutrophil terminal differentiation and a potential target in SGD patients.


Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular/fisiología , Neutrófilos/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas Potenciadoras de Unión a CCAAT/genética , Neutrófilos/citología , Proteínas Proto-Oncogénicas c-myb/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética
12.
Blood ; 125(19): 2974-84, 2015 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-25778530

RESUMEN

Isocitrate dehydrogenase 1 mutation (IDH1-R132H) was recently identified in acute myeloid leukemia with normal cytogenetics. The mutant enzyme is thought to convert α-ketoglutarate to the pathogenic 2-hydroxyglutarate (2-HG) that affects DNA methylation via inhibition of ten-eleven translocation 2. However, the role of wild-type IDH1 in normal hematopoiesis and its relevance to acute myeloid leukemia is unknown. Here we showed that zebrafish idh1 (zidh1) knockdown by morpholino and targeted mutagenesis by transcription activator-like effector nuclease might induce blockade in myeloid differentiation, as evident by an increase in pu.1 and decrease in mpo, l-plastin, and mpeg1 expression, and significantly reduce definitive hematopoiesis. Morpholino knockdown of zidh2 also induced a blockade in myeloid differentiation but definitive hematopoiesis was not affected. The hematopoietic phenotype of zidh1 knockdown was not rescuable by zidh2 messenger RNA, suggesting nonredundant functions. Overexpression of human IDH1-R132H or its zebrafish ortholog resulted in 2-HG elevation and expansion of myelopoiesis in zebrafish embryos. A human IDH1-R132H-specific inhibitor (AGI-5198) significantly ameliorated both hematopoietic and 2-HG responses in human but not zebrafish IDH1 mutant expression. The results provided important insights to the role of zidh1 in myelopoiesis and definitive hematopoiesis and of IDH1-R132H in leukemogenesis.


Asunto(s)
Embrión no Mamífero/metabolismo , Hematopoyesis/fisiología , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Mutación/genética , Mielopoyesis/fisiología , Pez Cebra/crecimiento & desarrollo , Animales , Animales Modificados Genéticamente , Bencenoacetamidas/farmacología , Western Blotting , Células Cultivadas , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Cromatografía de Gases y Espectrometría de Masas , Glutaratos/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Imidazoles/farmacología , Técnicas para Inmunoenzimas , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Mutagénesis Sitio-Dirigida , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Pez Cebra/genética , Pez Cebra/metabolismo
13.
Mol Cell ; 33(6): 692-703, 2009 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-19328064

RESUMEN

The cytoplasmic domains of UNC5 are responsible for its netrin-mediated signaling events in axonal migrations, blood vessel patterning, and apoptosis, although the molecular mechanisms governing these processes are unknown. To provide a foundation for the elucidation of the UNC5-mediated signaling mechanism, we determined the crystal structure of the cytoplasmic portion of UNC5b. We found that it contains three distinctly folded domains, namely ZU5, UPA, and death domain (DD). These three domains form a structural supramodule, with ZU5 binding to both UPA and DD, thereby locking the ZU5-UPA-DD supramodule in a closed conformation and suppressing its biological activities. Release of the closed conformation of the ZU5-UPA-DD supramodule leads to the activation of the receptor in the promotion of apoptosis and blood vessel patterning. Finally, we provide evidence showing that the supramodular nature of UNC5 ZU5-UPA-DD is likely to be shared by the ankyrin and PIDD families of scaffold proteins.


Asunto(s)
Apoptosis/fisiología , Citoplasma/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Animales , Células Cultivadas , Cromatografía de Afinidad , Cristalografía por Rayos X , Técnica del Anticuerpo Fluorescente , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Fenotipo , Unión Proteica , Conformación Proteica , Ratas , Receptores de Superficie Celular/genética , Transducción de Señal , Transfección , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo
14.
Development ; 140(19): 3977-85, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24046317

RESUMEN

Recent studies have shown that nascent hematopoietic stem cells (HSCs) derive directly from the ventral aortic endothelium (VAE) via endothelial to hematopoietic transition (EHT). However, whether EHT initiates from a random or predetermined subpopulation of VAE, as well as the molecular mechanism underlying this process, remain unclear. We previously reported that different zebrafish stem cell leukemia (scl) isoforms are differentially required for HSC formation in the ventral wall of the dorsal aorta. However, the exact stage at which these isoforms impact HSC development was not defined. Here, using in vivo time-lapse imaging of scl isoform-specific reporter transgenic zebrafish lines, we show that prior to EHT scl-ß is selectively expressed in hemogenic endothelial cells, a unique subset of VAE cells possessing hemogenic potential, whereas scl-α is expressed later in nascent HSCs as they egress from VAE cells. In accordance with their expression, loss-of-function studies coupled with in vivo imaging analysis reveal that scl-ß acts earlier to specify hemogenic endothelium, which is later transformed by runx1 into HSCs. Our results also reveal a previously unexpected role of scl-α in maintaining newly born HSCs in the aorta-gonads-mesonephros. Thus, our data suggest that a defined hemogenic endothelial population preset by scl-ß supports the deterministic emergence of HSCs, and unravel the cellular mechanisms by which scl isoforms regulate HSC development.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hemangioblastos/citología , Hemangioblastos/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células Cultivadas , Femenino , Hibridación in Situ , Masculino , Microscopía Confocal , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteína 1 de la Leucemia Linfocítica T Aguda , Pez Cebra , Proteínas de Pez Cebra/genética
15.
J Immunol ; 192(12): 5998-6008, 2014 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-24835391

RESUMEN

During inflammation, the proper inflammatory infiltration of neutrophils is crucial for the host to fight against infections and remove damaged cells and detrimental substances. IL-1ß and NADPH oxidase-mediated reactive oxygen species (ROS) have been implicated to play important roles in this process. However, the cellular and molecular basis underlying the actions of IL-1ß and ROS and their relationship during inflammatory response remains undefined. In this study, we use the zebrafish model to investigate these issues. We find that, similar to that of NADPH oxidase-mediated ROS signaling, the Il-1ß-Myd88 pathway is required for the recruitment of neutrophils, but not macrophages, to the injury-induced inflammatory site, whereas it is dispensable for bacterial-induced inflammation. Interestingly, the Il-1ß-Myd88 pathway is independent of NADPH oxidase-mediated ROS signaling and critical for the directional migration, but not the basal random movement, of neutrophils. In contrast, the NADPH oxidase-mediated ROS signaling is required for both basal random movement and directional migration of neutrophils. We further document that ectopic expression of Il-1ß in zebrafish induces an inflammatory disorder, which can be suppressed by anti-inflammatory treatment. Our findings reveal that the Il-1ß-Myd88 axis and NADPH oxidase-mediated ROS signaling are two independent pathways that differentially regulate neutrophil migration during sterile inflammation. In addition, Il-1ß overexpressing Tg(hsp70:(m)il-1ß_eGFP;lyz:DsRed2)hkz10t;nz50 transgenic zebrafish provides a useful animal model for the study of chronic inflammatory disorder and for anti-inflammatory drug discovery.


Asunto(s)
Movimiento Celular/inmunología , Interleucina-1beta/inmunología , Neutrófilos/inmunología , Transducción de Señal/inmunología , Proteínas de Pez Cebra/inmunología , Pez Cebra/inmunología , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Interleucina-1beta/genética , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , NADPH Oxidasas/genética , NADPH Oxidasas/inmunología , Neutrófilos/patología , Especies Reactivas de Oxígeno/inmunología , Transducción de Señal/genética , Proteínas de Pez Cebra/genética
16.
Development ; 139(23): 4321-9, 2012 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-23132242

RESUMEN

In vertebrates, establishment of the hematopoietic stem/progenitor cell (HSPC) pool involves mobilization of these cells in successive developmental hematopoietic niches. In zebrafish, HSPCs originate from the ventral wall of the dorsal aorta (VDA), the equivalent of the mammalian aorta-gonad-mesonephros (AGM). The HSPCs subsequently migrate to the caudal hematopoietic tissue (CHT) for transitory expansion and differentiation during the larval stage, and they finally colonize the kidney, where hematopoiesis takes place in adult fish. Here, we report the isolation and characterization of a zebrafish mutant, tango(hkz5), which shows defects of definitive hematopoiesis. In tango(hkz5) mutants, HSPCs initiate normally in the AGM and subsequently colonize the CHT. However, definitive hematopoiesis is not sustained in the CHT owing to accelerated apoptosis and diminished proliferation of HSPCs. Positional cloning reveals that tango(hkz5) encodes SUMO1-activating enzyme subunit 1 (Sae1). A chimera generation experiment and biochemistry analysis reveal that sae1 is cell-autonomously required for definitive hematopoiesis and that the tango(hkz5) mutation produces a truncated Sae1 protein (ΔSae1), resulting in systemic reduction of sumoylation. Our findings demonstrate that sae1 is essential for the maintenance of HSPCs during fetal hematopoiesis in zebrafish.


Asunto(s)
Hematopoyesis/genética , Células Madre Hematopoyéticas/fisiología , Sistema Hematopoyético/embriología , Proteína SUMO-1/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Aorta/embriología , Apoptosis/genética , Diferenciación Celular , Línea Celular , Movimiento Celular , Proliferación Celular , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Células HEK293 , Células Madre Hematopoyéticas/citología , Humanos , Morfolinos/genética , Mutación , Proteína SUMO-1/genética , Sumoilación , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética
17.
Proc Natl Acad Sci U S A ; 109(43): 17388-93, 2012 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-23012428

RESUMEN

Processive movements of unconventional myosins on actin filaments generally require motor dimerization. A commonly accepted myosin dimerization mechanism is via formation of a parallel coiled-coil dimer by a stretch of amino acid residues immediately carboxyl-terminal to the motor's lever-arm domain. Here, we discover that the predicted coiled-coil region of myosin X forms a highly stable, antiparallel coiled-coil dimer (anti-CC). Disruption of the anti-CC either by single-point mutations or by replacement of the anti-CC with a parallel coiled coil with a similar length compromised the filopodial induction activity of myosin X. We further show that the anti-CC and the single α-helical domain of myosin X are connected by a semirigid helical linker. The anti-CC-mediated dimerization may enable myosin X to walk on both single and bundled actin filaments.


Asunto(s)
Miosinas/química , Secuencia de Aminoácidos , Animales , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Homología de Secuencia de Aminoácido
18.
Biophys J ; 107(10): 2436-43, 2014 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-25418312

RESUMEN

In this study, we demonstrate a noninvasive imaging approach based on multimodal nonlinear optical microscopy to in vivo image the responses of immune cells (neutrophils) to the tissue injury and bacterial infection in a zebrafish model. Specifically, the second harmonic generation from myosin thick filaments in sarcomere enabled a clear visualization of the muscle injury and infection. Two-photon excited fluorescence was used to track the behavior of the neutrophils that were transgenically labeled by red fluorescent protein. The corresponding reduced nicotinamide adenine dinucleotide (NADH) two-photon excited fluorescence images revealed a detailed morphological transformation process of individual neutrophils during muscle tissue injury and bacterial infection. The analysis of time-resolved NADH signals from the neutrophils provided important biological insights of the cellular energy metabolism during the immune responses. We found a significant increase of free/protein-bound NADH ratios in activated neutrophils in bacterial-infected tissue. In this study, we also discovered that, under 720 nm excitation, two wild-type strains (DH5? and BL21) of bacteria Escherichia coli emitted distinct endogenous fluorescence of double-peak at ?450 and ?520 nm, respectively. We demonstrated that the double-peak fluorescence signal could be used to differentiate the E. coli from surrounding tissues of dominant NADH signals, and to achieve label-free tracking of E. coli bacteria in vivo.


Asunto(s)
Inmunidad Innata , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Animales , Embrión no Mamífero/citología , Embrión no Mamífero/inmunología , Embrión no Mamífero/microbiología , Metabolismo Energético/inmunología , Escherichia coli/aislamiento & purificación , Escherichia coli/fisiología , Músculo Esquelético/citología , Músculo Esquelético/inmunología , Miosinas/metabolismo , NAD/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Transducción de Señal/inmunología , Pez Cebra/inmunología , Pez Cebra/microbiología
19.
Development ; 138(4): 619-29, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21228005

RESUMEN

The hallmark of vertebrate definitive hematopoiesis is the establishment of the hematopoietic stem/progenitor cell (HSPC) pool during embryogenesis. This process involves a defined ontogenic switching of HSPCs in successive hematopoietic compartments and is evolutionarily conserved from teleost fish to human. In zebrafish, HSPCs originate from the ventral wall of the dorsal aorta (VDA), from which they subsequently mobilize to an intermediate hematopoietic site known as the caudal hematopoietic tissue (CHT) and finally colonize the kidney for adult hematopoiesis. Despite substantial understanding of the ontogeny of HSPCs, the molecular basis governing migration, colonization and maintenance of HSPCs remains to be explored fully. Here, we report the isolation and characterization of two zebrafish mutants, rumba(hkz1) and samba(hkz2), that are defective in generating definitive hematopoiesis. We find that HSPC initiation in the VDA and subsequent homing to the CHT are not affected in these two mutants. However, the further development of HSPCs in the CHT is compromised in both mutants. Positional cloning reveals that Rumba is a novel nuclear C2H2 zinc-finger factor with unknown function and samba encodes an evolutionarily conserved protein that is homologous to human augmin complex subunit 3 (HAUS3). Furthermore, we show that these two factors independently regulate cell cycle progression of HSPCs and are cell autonomously required for HPSC development in the CHT. Our study identifies Rumba and Haus3 as two essential regulators of HSPC maintenance during zebrafish fetal hematopoiesis.


Asunto(s)
Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Células COS , Chlorocebus aethiops , Regulación del Desarrollo de la Expresión Génica , Mutación , Proteínas de Pez Cebra/genética
20.
Blood ; 119(22): 5239-49, 2012 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-22493295

RESUMEN

Proper cell fate choice in myelopoiesis is essential for generating correct numbers of distinct myeloid subsets manifesting a wide spectrum of subset-specific activities during development and adulthood. Studies have suggested that myeloid fate choice is primarily regulated by transcription factors; however, new intrinsic regulators and their underlying mechanisms remain to be elucidated. Zebrafish embryonic myelopoiesis gives rise to neutrophils and macrophages and represents a promising system to derive new regulatory mechanisms for myeloid fate decision in vertebrates. Here we present an in vivo study of cell fate specification during zebrafish embryonic myelopoiesis through characterization of the embryos with altered Pu.1, Runx1 activity alone, or their combinations. Genetic analysis shows that low and high Pu.1 activities determine embryonic neutrophilic granulocyte and macrophage fate, respectively. Inactivation and overexpression of Runx1 in zebrafish uncover Runx1 as a key embryonic myeloid fate determinant that favors neutrophil over macrophage fate. Runx1 is induced by high Pu.1 level and in turn transrepresses pu.1 expression, thus constituting a negative feedback loop that fashions a favorable Pu.1 level required for balanced fate commitment to neutrophils versus macrophages. Our findings define a Pu.1-Runx1 regulatory loop that governs the equilibrium between distinct myeloid fates by assuring an appropriate Pu.1 dosage.


Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/biosíntesis , Regulación del Desarrollo de la Expresión Génica/fisiología , Mielopoyesis/fisiología , Proteínas Proto-Oncogénicas/biosíntesis , Transactivadores/biosíntesis , Proteínas de Pez Cebra/biosíntesis , Pez Cebra/embriología , Animales , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Macrófagos/citología , Macrófagos/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismo , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética
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