Using the Zebrafish As a Tool for Modeling Systemic Mastocytosis,

Abstract 3208

The zebrafish system provides many advantages in investigating intricate molecular pathways regulating vertebrate blood cell development and disease in vivo. We have been exploiting these assets to elucidate normal mast cell (MC) function and previously described the structural and functional characteristics of zebrafish MC equivalents (Dobson et al, Blood 2008, Da'as et al, Dev Comp Imm 2011). We have used this knowledge to develop transgenic zebrafish models of systemic mastocytosis (SM). SM is a pre-leukemic myeloproliferative disease that results from perturbed MC development and proliferation. Our recent studies have suggested that zebrafish MCs are uniquely dependent on Notch pathway signaling in contrast to that observed for other myeloid cell populations. Whole mount in situ hybridization (WISH) studies on the zebrafish Notch signaling mutant, mindbomb and notch1b “morphant” embryos both displayed decreased to absent carboxypeptidase A5 (cpa5) positive mast cells by WISH. Furthermore, wild type embryos treated with Compound E (CpdE), a γ-secretase inhibitor that inhibits Notch signaling, showed a similar phenotype. Given the role for Notch signalling in normal MC development, we wanted to see if driving the Notch pathway would result in a phenotype reminiscent of SM. Through a heat-shock inducible Gal4-UAS based system; we now demonstrate that over-expression of Notch signalling results in increased cpa5 positive mast cells in embryos as observed by WISH at 30, 36 and 48 hours post fertilization (hpf). Importantly, we were able to inhibit this increase and even reduce MC numbers below baseline levels in a dose-dependent manner using CpdE. Concurrently, we have established a transgenic zebrafish model of SM that ubiquitously expresses the human c-KIT D816V mutation under the zebrafish β-actin promoter. Beginning at 9 months of age, adult fish develop a number of skin and visceral lesions, many of which have been found to contain an abundance of MCs as identified by toluidine blue staining and tryptase immunohistochemistry. Transgenic embryos lack a developmental phenotype but demonstrate evidence of decreased phospho-histone H3 (pH3) signaling, suggesting additional mutations are required to progress to SM. In support of this G2/M arrest phenotype, microarray studies conducted on transgenic embryos revealed upregulation of p53 and cyclin G1. These studies have provided new insights into the role of Notch signaling in MC development and the opportunity to use the zebrafish as an in vivo model to identify and evaluate novel therapeutic strategies in MC diseases.