Mast cells (MCs) play critical roles in allergy and inflammation, yet their development remains controversial due to limitations posed by traditional animal models. The zebrafish provides a highly efficient system for studying vertebrate hematopoiesis. We have identified zebrafish MCs in the gill and intestine, which resemble their mammalian counterparts both structurally and functionally. Carboxypeptidase A5 (cpa5), a MCspecific enzyme, is expressed in zebrafish blood cells beginning at 24 hours post fertilization (hpf). At 28 hpf, colocalization is observed with pu.1, mpo, l-plastin, and lysozyme C, but not fms or cepb␣, identifying these early MCs as a distinct myeloid population arising from a common granulocyte/ monocyte progenitor. Morpholino "knockdown" studies demonstrate that transcription factors gata-2 and pu.1, but not gata-1 or fog-1, are necessary for early MC development. These studies validate the zebrafish as an in vivo tool for studying MC ontogeny and function with future capacity for modeling human MC diseases. (Blood. 2008;112:2969-2972) IntroductionMast cells (MCs) play central roles in allergic and inflammatory reactions. 1,2 Stimulation of cell-surface receptors, such as C-KIT and the high-affinity IgE receptor, 1,2 results in the release of mediators from cytoplasmic granules, including tryptase and histamine. 2 MC number and function are regulated by their development, proliferation, migration, and survival. 1 Barriers to understanding these processes include accessibility and imaging limitations posed by traditional animal models. The zebrafish has proven itself to be a robust and highly conserved model for studying vertebrate hematopoiesis. 3 Here, we provide the first evidence that the zebrafish possesses MC equivalents that share structural and functional characteristics with their mammalian counterparts. Furthermore, we demonstrate the utility of the zebrafish as an in vivo tool in dissecting the contribution of transcription factors to MC development. MethodsZebrafish were maintained, bred, and developmentally staged according to Westerfield. 4 Use of zebrafish in this study was approved by the Dalhousie University Animal Care Committee. Zebrafish gills and intestine were fixed in 10% neutral buffered formalin, and standard staining procedures were applied ( Figure 1A-F). Immunohistochemistry was facilitated by antigen retrieval ( Figure 1I,J). For electron microscopy, tissues were fixed overnight in 2% glutaraldehyde in 0.1 M caccodylate and postfixed in 1% osmium tetroxide. Thin sections (90 nm) were stained in 25% uranyl acetate in methanol and lead citrate.Bromophenol blue and 10 g compound 48/80 or saline were injected intraperitoneally, and blood was extracted by cardiac puncture after 2.5 minutes. Tryptase activity was measured in plasma spectrophotometrically at 415 nm by the release of p-nitroanilide from N-benzoyl-DL-argininep-nitroanilide (BAPNA), a tryptase substrate.Digoxogenin-or fluorescein isothiocyanate (FITC)-labeled antisense mRNA probes for zebrafish carboxypep...

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Fluorescence-activated cell sorting (FACS) of whole mount in situ hybridization (WISH) labelled haematopoietic cell populations in the zebrafishOver the last decade, the zebrafish (Danio rerio) has secured its place as a robust model for studying vertebrate haematopoiesis (Berman et al, 2003;Carradice & Lieschke, 2008). More recently, the zebrafish has emerged as an in vivo platform for investigating immune function and host-pathogen interactions (Meeker & Trede, 2008). While a number of technical advances including whole mount in situ hybridization (WISH), morpholino gene 'knockdown', and transgenesis have greatly enriched the ability to study blood cells in the zebrafish, a significant void has been the paucity of antibodies that recognize cell surface receptors, limiting the ability to perform fluorescence activated cell sorting (FACS) (Berman et al, 2003;Carradice & Lieschke, 2008). FACS serves as the cornerstone of mammalian haematopoiesis and immunology research, permitting the separation and identification of specific cell populations. Flow cytometry has been adapted to the zebrafish with the recognition that blood cell populations from the kidney marrow (zebrafish bone marrow equivalent) can be sorted on the basis of cell size and granularity (forward and side scatter, respectively) (Traver et al, 2003). The development of transgenic lines expressing fluorescently labelled markers has subsequently enabled FACS in zebrafish (Traver et al, 2003;Hsu et al, 2004). However, despite this progress, FACS has remained relatively restricted, necessitating a transgenic line driving a fluorescent protein under a specific promoter to isolate a particular cell population.In our recent paper characterizing the mast cell lineage in the zebrafish (Dobson et al, 2008), we devised a novel approach to cell sorting in the zebrafish, combining the fluorescent labelling of cells using whole mount RNA in situ hybridization (WISH) together with FACS, by gating on the wavelength of fluorescein isothiocyanate (FITC). This report expands on these studies, demonstrating the utility of this technique for separating out a number of haematopoietic cell lineages based on their RNA expression patterns. MethodsFITC-labelled antisense mRNA probes for zebrafish l-plastin, myeloperoxidase (mpo), gata-1 and be3-globin were synthesized according to the published literature (Detrich et al, 1995 SummaryThe zebrafish is a robust animal model for studying vertebrate haematopoiesis and immune cell interactions. However, fluorescence activated cell sorting (FACS) has been limited due to a paucity of available functional zebrafish antibodies. We have developed a technique combining FACS with whole mount in situ hybridization (WISH) that enables the sorting and examining of fixed zebrafish blood cell populations at different stages of embryonic development, providing the opportunity to correlate RNA expression data with cellular morphology.