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1.
Curr Opin Hematol ; 28(1): 43-49, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33264224

RESUMEN

PURPOSE OF REVIEW: Myeloid cells contribute to immune response to infection and tissue regeneration after injury as well as to the developmental induction of the hematopoietic system overall. Here we review recent uses of zebrafish to advance the study of myeloid biology in development and disease. RECENT FINDINGS: Recent studies have made use of advanced imaging and genetic strategies and have highlighted key concepts in myeloid cell behavior. These include immune-cell cross-talk and subpopulation response in infection and regeneration, and tightly regulated inflammatory and tissue remodeling behaviors in development. SUMMARY: These new findings will shape our understanding of the developmental origins of immune populations as well as their specific cellular behaviors at all stages of infection, regeneration, and myeloid neoplasms.


Asunto(s)
Infecciones/patología , Leucemia Mieloide/patología , Células Mieloides/patología , Pez Cebra , Animales , Modelos Animales de Enfermedad , Hematopoyesis , Humanos , Infecciones/inmunología , Leucemia Mieloide/inmunología , Células Mieloides/inmunología , Regeneración , Pez Cebra/embriología , Pez Cebra/fisiología
2.
bioRxiv ; 2024 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-38895208

RESUMEN

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

3.
Dev Cell ; 58(12): 1037-1051.e4, 2023 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-37119815

RESUMEN

The hematopoietic niche is a supportive microenvironment composed of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses in zebrafish, we define a conserved gene expression signature and cis-regulatory landscape that are unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code that involves members of the Ets, Sox, and nuclear hormone receptor families and is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance, and division of HSPCs in vivo. These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo, and for effective therapies to modulate the endogenous niche.


Asunto(s)
Nicho de Células Madre , Factores de Transcripción , Animales , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Endoteliales/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Regulación de la Expresión Génica
4.
Science ; 377(6613): 1413-1419, 2022 09 23.
Artículo en Inglés | MEDLINE | ID: mdl-36137040

RESUMEN

Tissue-specific stem cells persist for a lifetime and can differentiate to maintain homeostasis or transform to initiate cancer. Despite their importance, there are no described quality assurance mechanisms for newly formed stem cells. We observed intimate and specific interactions between macrophages and nascent blood stem cells in zebrafish embryos. Macrophage interactions frequently led to either removal of cytoplasmic material and stem cell division or complete engulfment and stem cell death. Stressed stem cells were marked by surface Calreticulin, which stimulated macrophage interactions. Using cellular barcoding, we found that Calreticulin knock-down or embryonic macrophage depletion reduced the number of stem cell clones that established adult hematopoiesis. Our work supports a model in which embryonic macrophages determine hematopoietic clonality by monitoring stem cell quality.


Asunto(s)
Apoptosis , Calreticulina , Comunicación Celular , Hematopoyesis Clonal , Células Madre Hematopoyéticas , Macrófagos , Animales , Calbindina 2/genética , Calbindina 2/fisiología , Calreticulina/genética , Calreticulina/metabolismo , Hematopoyesis Clonal/genética , Hematopoyesis Clonal/fisiología , Embrión no Mamífero , Células Madre Hematopoyéticas/fisiología , Macrófagos/fisiología , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiología
5.
Zebrafish ; 17(5): 354-357, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32749922

RESUMEN

Collecting large numbers of rare cells for high-throughput molecular analysis remains a technical challenge, primarily due to limitations in existing technologies. In developmental biology this has impeded single-cell analysis of primordial organs, which derive from few cells. In this study, we share novel transgenic lines for rapid cell enrichment from zebrafish embryos using human surface antigens for immunological binding and magnetic sorting. As proof of principle, we tagged, enriched, and performed single-cell RNA sequencing on nascent hematopoietic stem/progenitor cells and endothelial cells from early embryos. Our method is a quick, efficient, and cost-effective approach to a previously intractable problem.


Asunto(s)
Embrión no Mamífero/embriología , Embriología/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente/embriología , Desarrollo Embrionario , Células Endoteliales/química , Células Madre Hematopoyéticas/química , Humanos , Fenómenos Magnéticos , Análisis de la Célula Individual/instrumentación
6.
Sci Rep ; 9(1): 18613, 2019 12 09.
Artículo en Inglés | MEDLINE | ID: mdl-31819086

RESUMEN

Recent advances in CRISPR/Cas gene editing technology have significantly expanded the possibilities and accelerated the pace of creating genetically engineered animal models. However, CRISPR/Cas-based strategies designed to precisely edit the genome can often yield unintended outcomes. Here, we report the use of zygotic CRISPR/Cas9 injections to generate a knock-in GFP reporter mouse at the Gdf11 locus. Phenotypic and genomic characterization of founder animals from these injections revealed a subset that contained the correct targeting event and exhibited GFP expression that, within the hematopoietic system, was restricted predominantly to lymphoid cells. Yet, in another subset of founder mice, we detected aberrant integration events at the target site that dramatically and inaccurately shifted hematopoietic GFP expression from the lymphoid to the myeloid lineage. Additionally, we recovered multiple Gdf11 deletion alleles that modified the C-terminus of the GDF11 protein. When bred to homozygosity, most of these alleles recapitulated skeletal phenotypes reported previously for Gdf11 knockout mice, suggesting that these represent null alleles. However, we also recovered one Gdf11 deletion allele that encodes a novel GDF11 variant protein ("GDF11-WE") predicted to contain two additional amino acids (tryptophan (W) and glutamic acid (E)) at the C-terminus of the mature ligand. Unlike the other Gdf11 deletion alleles recovered in this study, homozygosity for the Gdf11WE allele did not phenocopy Gdf11 knockout skeletal phenotypes. Further investigation using in vivo and in vitro approaches demonstrated that GDF11-WE retains substantial physiological function, indicating that GDF11 can tolerate at least some modifications of its C-terminus and providing unexpected insights into its biochemical activities. Altogether, our study confirms that one-step zygotic injections of CRISPR/Cas gene editing complexes provide a quick and powerful tool to generate gene-modified mouse models. Moreover, our findings underscore the critical importance of thorough characterization and validation of any modified alleles generated by CRISPR, as unintended on-target effects that fail to be detected by simple PCR screening can produce substantially altered phenotypic readouts.


Asunto(s)
Alelos , Proteínas Morfogenéticas Óseas/genética , Sistemas CRISPR-Cas , Eliminación de Gen , Edición Génica , Factores de Diferenciación de Crecimiento/genética , Animales , Femenino , Genes Reporteros , Ingeniería Genética , Genoma , Ácido Glutámico/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Hematopoyéticas/metabolismo , Homocigoto , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Células Mieloides/metabolismo , Fenotipo , Dominios Proteicos , Triptófano/metabolismo
7.
Blood Adv ; 2(21): 3063-3069, 2018 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-30425071

RESUMEN

Each stem cell resides in a highly specialized anatomic location known as the niche that protects and regulates stem cell function. The importance of the niche in hematopoiesis has long been appreciated in transplantation, but without methods to observe activity in vivo, the components and mechanisms of the hematopoietic niche have remained incompletely understood. Zebrafish have emerged over the past few decades as an answer to this. Use of zebrafish to study the hematopoietic niche has enabled discovery of novel cell-cell interactions, as well as chemical and genetic regulators of hematopoietic stem cells. Mastery of niche components may improve therapeutic efforts to direct differentiation of hematopoietic stem cells from pluripotent cells, sustain stem cells in culture, or improve stem cell transplant.


Asunto(s)
Células Madre Hematopoyéticas/citología , Nicho de Células Madre , Pez Cebra/crecimiento & desarrollo , Animales , Diferenciación Celular , Quimiocinas CXC/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Hematopoyesis , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Proteínas de Pez Cebra/metabolismo
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