Abstract

Mast cells originate from mammalian myeloid hematopoiesis and exert a crucial role in inflammation and allergies. In recent years, studies have reported that carboxypeptidase A5 ( cpa5 ) can serve as a marker for zebrafish mast cells. However, the absence of systematic experimental validation limited understanding of the origin, molecular characteristics, and function of mast cells. To address this gap, we initially confirmed zebrafish mast cell like cells in hematopoietic and peripheral tissues via staining and cytomorphological identification. Subsequently, spatiotemporal analysis and Tg( cpa5 :GFP) sorting showed cpa5 mainly in mature neutrophils, rarely in mast cell like cells. Finally, gene knockout experiments revealed genetic compensation between cpa1 and cpa5 within the same gene family, and we further generated cpa1 −/− cpa5 −/− double mutant zebrafish. Experiment showed that in cpa1 −/− cpa5 −/− , the number of neutrophils was reduced while their functional properties remained intact; the number of mast cell like cells increased but their functions were impaired. This study revises the current understanding of cpa5 as a specific mast cell marker, explores its role in neutrophils and mast cells development, and thereby offers novel insights into zebrafish immune system.