Carboxypeptidase A5 identifies a novel mast cell lineage in the zebrafish providing new insight into mast cell fate determination

Abstract

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...

Characterization of the mononuclear phagocyte system in zebrafish

“…Purified mhc2dab:GFP ϩ cells from the gut demonstrated similar morphologic characteristics to mast cells described previously in the zebrafish. 46 Whereas the exact nature of these cells needs to be validated, the expression of mhc2 has been described previously in mammalian mast cells. 47 The cd45:DsRed transgenic line that we describe in this study marks the majority of leukocytes.…”

The Proprotein Convertase Subtilisin/Kexin FurinA Regulates Zebrafish Host Response against Mycobacterium marinum

“…As in mammals, the cells of the zebrafish immune system include lymphocytes, neutrophils, and macrophages (49), as well as dendritic cells (50), eosinophils (51,52), human mast cell-like cells (53), and natural killer cells (54). Our flow cytometric analyses of furinA td204e/ϩ mutant fish kidneys (the primary site of hematopoiesis in fish) showed normal numbers of lymphocytes, blood cell precursors, and erythrocytes, but low granulocyte counts, indicating that FurinA promotes granulopoiesis.…”

The zebrafish reveals dependence of the mast cell lineage on Notch signaling in vivo

“…[4][5][6] All of the major hematopoietic cellular lineages studied to date have zebrafish counterparts, and the fundamental genetic mechanisms that control hematopoiesis are well conserved. 4,7,8 We first described a mast cell counterpart in the zebrafish 9 and subsequently showed conserved roles of these cells in adaptive and innate responses to inflammatory stimuli. 10 Zebrafish carboxypeptidase a5 (cpa5) was identified as a zebrafish mast cell-specific marker, and gata2 and pu.1 were found to be the key transcription factors required for early mast cell lineage commitment in keeping with studies in mammalian systems.…”

“…10 Zebrafish carboxypeptidase a5 (cpa5) was identified as a zebrafish mast cell-specific marker, and gata2 and pu.1 were found to be the key transcription factors required for early mast cell lineage commitment in keeping with studies in mammalian systems. 9,11,12 The Notch signaling pathway is a critical regulator of cell fate determination conserved through evolution. Aberrant Notch signaling is associated with a wide range of human disorders from developmental syndromes to cancer.…”