Single-Cell Analyses Reveal Early Thymic Progenitors and Pre-B Cells in the Zebrafish

The zebrafish has proven to be a valuable model organism for studying hematopoiesis, but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single-cell RNA sequencing on ~70,000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus, elucidated B and T cell developmental trajectories, and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic-like cell populations and provided evidence in support of the existence of a pre-B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations, a total of 7 adult zebrafish (3-5 months post-fertilization) and 21 juvenile zebrafish (4 weeks post-fertilization) were studied. More specifically, kidney marrows only were dissected from 3 adult GESTALT zebrafish, paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish, and thymi only were dissected from 2 adult Tg(lck:eGFP) zebrafish. Thymi only were dissected and pooled from 21 juvenile Tg(lck:eGFP) zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation, filtering, and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics (lymphoid and progenitor fraction; granulocyte fraction) were collected in preparation for 10x single-cell RNA sequencing. A single 10x lane was used for each adult sample, whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.