The Identification and Characterization of Zebrafish Mast Cells.

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.