Single Cell Atlas Reveals Abundant Innate Lymphoid Cells in Larval Zebrafish

Innate Lymphoid cells (ILCs) are a group of tissue resident lymphocytes that participate in immune defense and tissue homeostasis. The existence and ontogeny of ILCs during zebrafish embryonic development are not defined. During embryogenesis, hematopoietic stem cells (HSCs) and HSC-independent Progenitor Cells (HPCs) form contemporaneously and produce overlapping and distinct immune cell types. To delineate when ILCs emerge and if they originate exclusively from HSCs or HPCs, we performed lineage tracing of the emerging hematopoietic system during larval development followed by single cell RNA sequencing of HSC and HPC progeny. To determine if the ILC-like cells are derived from the definitive hematopoietic wave, we did lineage tracing and scRNA-seq experiment in runx1W84X mutants and illustrated that all ILC-like clusters are Runx1-dependent. We confirmed that the transcriptionally detected larval ILC-like cells are Rag-independent and Il2rg-dependent. Lastly, embryonic ILCs are functional because they respond to an immune system stimulation with a viral mimic. The work provides fundamental knowledge on the early establishment of immune hierarchies and opens the zebrafish model to the exploration of ILC origination and function. Experiment (1): for lineage tracing experiment, HSPCs were labled with mCherry at 3 timepoints in development: either at 1, 2, or 3 dpf. The differentiated progeny of labled HSPCs were sorted out from whole zebrafish using FACS at one-day-post labelling, 6 dpf, and 10 dpf. Experiment (2): for Runx1 mutants experiment, early-HSPCs (1dpf) derived mCherry cells were isolated from sibling controls or homozygous runx1(W84X) mutants at 6 dpf from whole zebrafish. Experiment (3): for rag1/R848 experiment, mCherry cells derived from early-HSPCs (1dpf) were isolated at 8 dpf from slc24a5 crispant controls, rag1 crispants and from slc24a5 and rag1 crispants treated with R848 agonist.