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Characterization of a weak allele of zebrafish cloche mutant.

ABSTRACT: Hematopoiesis is a complicated and dynamic process about which the molecular mechanisms remain poorly understood. Danio rerio (zebrafish) is an excellent vertebrate system for studying hematopoiesis and developmental mechanisms. In the previous study, we isolated and identified a cloche(172) (clo(172)) mutant, a novel allele compared to the original cloche (clo) mutant, through using complementation test and initial mapping. Here, according to whole mount in-situ hybridization, we report that the endothelial cells in clo(172) mutant embryos, although initially developed, failed to form the functional vascular system eventually. In addition, further characterization indicates that the clo(172) mutant exhibited weaker defects instead of completely lost in primitive erythroid cells and definitive hematopoietic cells compared with the clo(s5) mutant. In contrast, primitive myeloid cells were totally lost in clo(172) mutant. Furthermore, these reappeared definitive myeloid cells were demonstrated to initiate from the remaining hematopoietic stem cells (HSCs) in clo(172) mutant, confirmed by the dramatic decrease of lyc in clo(172)runx1(w84x) double mutant. Collectively, the clo(172) mutant is a weak allele compared to the clo(s5) mutant, therefore providing a model for studying the early development of hematopoietic and vascular system, as well as an opportunity to further understand the function of the cloche gene.

SUBMITTER: Ma N

PROVIDER: S-EPMC3223178 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Characterization of a weak allele of zebrafish cloche mutant.

Ma Ning N Huang Zhibin Z Chen Xiaohui X He Fei F Wang Kun K Liu Wei W Zhao Linfeng L Xu Xiangmin X Liao Wangjun W Ruan Hua H Luo Shenqiu S Zhang Wenqing W

PloS one 20111123 11

Hematopoiesis is a complicated and dynamic process about which the molecular mechanisms remain poorly understood. Danio rerio (zebrafish) is an excellent vertebrate system for studying hematopoiesis and developmental mechanisms. In the previous study, we isolated and identified a cloche(172) (clo(172)) mutant, a novel allele compared to the original cloche (clo) mutant, through using complementation test and initial mapping. Here, according to whole mount in-situ hybridization, we report that th ...[more]

PMID: 22132109

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Project description:BACKGROUND: Zebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants). RESULTS: Differential counts of peripheral blood leukocytes (PBL) showed that rag1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes. Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte-like cells and comprised 7% of the gated cells in the rag1 mutants and 26% in the wild-types. Reverse transcriptase polymerase chain reaction (RT-PCR) assays demonstrated rag1 mutant kidney hematopoietic tissue expressed mRNA encoding Non-specific Cytotoxic cell receptor protein-1 (NCCRP-1) and Natural Killer (NK) cell lysin but lacked T cell receptor (TCR) and immunoglobulin (Ig) transcript expression, while wild-type kidney hematopoietic tissue expressed NCCRP-1, NK lysin, TCR and Ig transcript expression. CONCLUSION: Our study demonstrates that in comparison to wild-type zebrafish, rag1 mutants have a significantly reduced lymphocyte-like cell population that likely includes Non-specific cytotoxic cells (NCC) and NK cells (and lacks functional T and B lymphocytes), a similar macrophage/monocyte population, and a significantly increased neutrophil population. These zebrafish have comparable leukocyte populations to SCID and rag 1 and/or 2 mutant mice, that possess macrophages, natural killer cells and neutrophils, but lack T and B lymphocytes. Rag1 mutant zebrafish will provide the platform for remarkable investigations in fish and innate immunology, as rag 1 and 2 mutant mice did for mammalian immunology.

Dataset Information

Characterization of a weak allele of zebrafish cloche mutant.

Publications

Characterization of a weak allele of zebrafish cloche mutant.

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