Diverse Epithelial Lymphocytes in Zebrafish Revealed Using a Novel Scale Biopsy Method
Zebrafish (Danio rerio) are a compelling model to study lymphocytes because zebrafish and humans have similar adaptive immune systems, including their lymphocytes. Antibodies that recognize zebrafish proteins are sparse, so many investigators utilize 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. Here, we report the first study focused on zebrafish epidermal lymphocytes, using scales. Obtaining zebrafish blood via non-lethal 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 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 (scRNA-seq) 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 novel, non-lethal technique to obtain zebrafish lymphocytes, providing the first quantification, expression profiling, and functional data (DXM responses and phagocytosis) from epidermal lymphocytes in the zebrafish model. Overall design: This experimental study aimed to investigate the gene expression profiles of individual lymphocytes from the zebrafish lck:GFP transgenic line by using single-cell RNA sequencing (scRNA-seq) analysis. We performed scRNA-seq on GFPhi thymocytes and GFPlo scale/marrow cells from lck:GFP fish which mark different lymphocyte populations. scRNA-seq data reveal diverse thymic, scale, and marrow lymphocyte populations for subsequent transcriptomic analysis and isolation of specific cell types.