No abstract

Mast cells (MCs) are well-known for their role in allergic reactions and inflammation, but their developmental origin is controversial. In addition, abnormal clonal proliferation of MCs, referred to as systemic mastocytosis (SM), may be associated with acute myeloid leukemia, portending a poor outcome. Mutations in the C-KIT tyrosine kinase have been identified in SM, that can be potentially targeted by small molecule tyrosine kinase inhibitors. The zebrafish is a robust model organism for studying hematopoiesis and leukemogenesis, and has an inherent capacity to accommodate genetic and chemical modifier screens. Thus, this system holds potential for elucidating MC lineage and for use in high-throughput screening of targeted therapeutics in MC diseases. MCs have not been previously described in zebrafish. We have identified putative MCs in adult zebrafish gill and intestine containing eosinophilic granules that stain with peroxidase acid shift (PAS) and toluidine blue, as well as with carboxypeptidase A5 (cpa5), a zebrafish homologue of the mammalian MC specific enzyme, CPA1. Electron microscopic analysis demonstrates a striking morphologic resemblance to mammalian MCs with abundant homogeneous dense granules. Classical functional studies reveal degranulation of these cells and increased histamine production upon stimulation with compound 48/80 and stimulated IgE. Whole mount in situ hybridization experiments on zebrafish embryos demonstrate cpa5 expression in a population of blood cells at 28 hours post-fertilization co-localizing with the early myeloid marker, pu.1, the granulocytic marker, mpo, and monocytic markers, l-plastin and lysozyme C. These data point to the existence of a zebrafish MC equivalent and suggests that this lineage arises at the level of the granulocyte/monocyte progenitor. Ongoing characterization of these cells will provide further insight into the mechanisms underlying MC development. Furthermore, a zebrafish cpa5 promoter element has been cloned and is being used to generate transgenic zebrafish lines. These transgenic zebrafish will provide a valuable tool in identifying new effective therapeutic agents targeting MCs in allergic and immune responses, as well as in their contribution to leukemic progression.

Mast cells (MCs), tissue counterparts to mammalian basophils, are known for their role in allergic reactions, inflammation and cancer progression, yet their developmental origin remains controversial due to limitations studying these cells in traditional animal models. The zebrafish provides a highly efficient system for studying vertebrate MC development. All other major hematopoietic lineages have zebrafish counterparts and the fundamental genetic mechanisms controlling hematopoiesis are well conserved. We are the first to identify zebrafish MCs in the gill and intestine. These cells demonstrate classic MC phenotypes including prominent metachromatic granules following staining with toluidine blue and positive immunohistochemical reactions to antibodies against human tryptase and C-KIT. Electron microscopy demonstrates a striking morphologic resemblance to mammalian MCs. Functional studies using the stimulating agent, Compound 48/80 or formalin-killed Aeromonas result in MC degranulation and increased blood levels of key mediators, such as tryptase. These cells also express carboxypeptidase A5 (cpa5), a zebrafish homologue of the human mast cell-specific CPA enzymes. Cpa5 expression in zebrafish embryonic blood cells begins at 24 hours post fertilization and co-localizes with a number of established granulocytic and monocytic markers suggesting that MCs arise from a common granulocyte/monocyte progenitor. Morpholino knockdown studies have demonstrated that the transcription factors gata-2 and pu.1, but not gata-1 are necessary for early MC development. Interestingly, friend of gata-1 (fog-1) may also be required, but in a gata-1 dependent manner. Ongoing morpholino and mutant rescue studies will further establish in vivo the contribution that these transcription factors make to vertebrate MC development. Finally, we have cloned a cpa5 promoter element and shown it can drive expression of the green fluorescent protein in early zebrafish MCs providing a means for generating transgenic zebrafish lines to model human MC diseases for use in high throughput small molecule modifier screens.