Mediator Subunit 12 Is Required for Neutrophil Development in Zebrafish

Granulocyte colony-stimulating factor (GCSF) and its receptor (GCSFR), also known as CSF3 and CSF3R, are required to maintain normal neutrophil numbers during basal and emergency granulopoiesis in humans, mice and zebrafish. Previous studies identified two zebrafish CSF3 ligands and a single CSF3 receptor. Transient antisense morpholino oligonucleotide knockdown of both these ligands and receptor reduces neutrophil numbers in zebrafish embryos, a technique widely used to evaluate neutrophil contributions to models of infection, inflammation and regeneration. We created an allelic series of zebrafish csf3r mutants by CRISPR/Cas9 mutagenesis targeting csf3r exon 2. Biallelic csf3r mutant embryos are viable and have normal early survival, despite a substantial reduction of their neutrophil population size, and normal macrophage abundance. Heterozygotes have a haploinsufficiency phenotype with an intermediate reduction in neutrophil numbers. csf3r mutants are viable as adults, with a 50% reduction in tissue neutrophil density and a substantial reduction in the number of myeloid cells in the kidney marrow. These csf3r mutants are a new animal model of human CSF3R-dependent congenital neutropenia. Furthermore, they will be valuable for studying the impact of neutrophil loss in the context of other zebrafish disease models by providing a genetically stable, persistent, reproducible neutrophil deficiency state throughout life.

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“…These mutants include, among many others: cloche/npas4 2223, alk8 24, med12 25, prpf8 26, plcg1 27 and runx1 28.…”

Section: Discussionmentioning

confidence: 99%

A GCSFR/CSF3R zebrafish mutant models the persistent basal neutrophil deficiency of severe congenital neutropenia

Pazhakh

Clark

Keightley

et al. 2017

Sci Rep

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“…However, little is known about the role of MED12 (or any other Mediator subunit) in adult stem cell and specifically HSC function. The first indication of a function for MED12 in hematopoiesis came from zebrafish studies, showing a potential role in myelopoiesis (Keightley et al, 2011). The recent identification of MED12 mutations in cancer, including leukemia (KÄmpjÄrvi et al, 2016), further supports this notion.…”

Section: Introductionmentioning

confidence: 99%

MED12 Regulates HSC-Specific Enhancers Independently of Mediator Kinase Activity to Control Hematopoiesis

et al. 2016

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SUMMARY Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis.

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“…Zebrafish also appear to possess a full component of cells of the adaptive immune system. Recombination Activating Gene ( rag ) expressing T-lymphocytes are found in the thymus as early as 3.5dpf by in situ hybridization 33–35 ; B-cells have similarly been reported, although their site of maturation remains controversial 36,37 and Natural Killer cells are also thought to exist 38,39 . Recent studies have indicated B cells possess similar function including proliferation in response to antigen exposure 37 .…”

Section: Introductionmentioning

confidence: 99%

Oceans of opportunity: Exploring vertebrate hematopoiesis in zebrafish

Carroll

North

2014

Experimental Hematology

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Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell (HSPC) biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor and effector cell emergence, expansion and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell-types can be identified and characterized. Further, a myriad of transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of HSPC biology during development, in response to infection or injury, or in the setting of hematological malignancy, continues to deepen, zebrafish will remain essential for exploring the spatio-temporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease.

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Mediator Subunit 12 Is Required for Neutrophil Development in Zebrafish

Cited by 20 publications

References 43 publications

A GCSFR/CSF3R zebrafish mutant models the persistent basal neutrophil deficiency of severe congenital neutropenia

A GCSFR/CSF3R zebrafish mutant models the persistent basal neutrophil deficiency of severe congenital neutropenia

MED12 Regulates HSC-Specific Enhancers Independently of Mediator Kinase Activity to Control Hematopoiesis

Oceans of opportunity: Exploring vertebrate hematopoiesis in zebrafish

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