Diverse Epithelial Lymphocytes in Zebrafish Revealed Using a Novel Scale Biopsy Method.

Abstract

Zebrafish (Danio rerio) are a compelling model for studying lymphocytes because zebrafish and humans have similar adaptive immune systems, including their lymphocytes. Antibodies that recognize zebrafish proteins are sparse, so many investigators use transgenic, lymphocyte-specific fluorophore-labeled lines. Human and zebrafish lymphocyte types are conserved, but many aspects of zebrafish lymphocyte biology remain uninvestigated, including lymphocytes in peripheral tissues, like epidermis. This study is, to our knowledge, the first study to focus on zebrafish epidermal lymphocytes, using scales. Obtaining zebrafish blood via nonlethal methods is difficult; scales represent a source to longitudinally sample live fish. We developed a novel biopsy technique, collecting scales to analyze epithelial lymphocytes from several transgenic lines. We imaged scales via confocal microscopy and demonstrated multiple lymphocyte types in scales/epidermis, quantifying them flow cytometrically. We profiled gene expression of scale, thymic, and kidney-marrow (analogous to mammalian bone marrow) lymphocytes from the same animals, revealing B- and T-lineage signatures. Single-cell quantitative real-time PCR and RNA sequencing show not only canonical B and T cells but also novel lymphocyte populations not described previously. To validate longitudinal scale biopsies, we serially sampled scales from fish treated with dexamethasone, demonstrating epidermal lymphocyte responses. To analyze cells functionally, we employed a bead-ingestion assay, showing that thymic, marrow, and epidermal lymphocytes have phagocytic activity. In summary, we establish a novel, nonlethal technique to obtain zebrafish lymphocytes, providing the first quantification, expression profiling, and functional data from zebrafish epidermal lymphocytes.