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

Abstract

Zebrafish are a compelling model to study lymphocytes because zebrafish and humans have similar adaptive immune systems. Human and zebrafish lymphocyte types are conserved, but many aspects of zebrafish lymphocyte biology remain uninvestigated, including lymphocytes in peripheral tissues, like epidermis. Here, we report the first study focused on zebrafish scale epidermal lymphocytes. Zebrafish scales represent a source to longitudinally sample live fish. We developed a novel biopsy technique, collecting scales to analyze epithelial lymphocytes from several fluorescently-labeled 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 marrow lymphocytes from the same animals, revealing B- and T-lineage signatures. Single-cell qRT-PCR and RNA sequencing (scRNAseq) 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 (DXM), demonstrating epidermal lymphocyte responses. To analyze cells functionally, we employed a bead-ingestion assay, showing thymic, marrow, and epidermal lymphocytes have phagocytic activity. In summary, we establish a non-lethal technique to obtain zebrafish lymphocytes, providing the first quantification, expression profiling, and functional data from epidermal lymphocytes in the zebrafish model. SummaryThis study describes a new biopsy method to acquire zebrafish lymphocytes for ex vivo studies, without euthanasia. Expression profiles of individual lymphocytes from multiple zebrafish transgenic lines reveal diverse lymphocyte populations, including novel cells expressing genes of both the B- and T-lineages.