Dynamic Changes in Lymphocyte Populations Establish Zebrafish as a Thymic Involution Model

The thymus is the site of T lymphocyte development and T cell education to recognize foreign, but not self, antigens. B cells also reside and develop in the thymus, although their function(s) are less clear. During 'thymic involution,' a process of lymphoid atrophy and adipose replacement linked to sexual maturation, thymic cells decline. However, thymic B cells decrease far less than T cells, such that B cells comprise ~1% of neonatal thymocytes, but up to ~10% in adulthood in humans. All jawed vertebrates possess a thymus, and we and others have shown that zebrafish (Danio rerio) also have thymic B cells. Here, we investigated the precise identities of zebrafish thymic T and B cells and how they change with involution. We assessed the timing and specific details of zebrafish thymic involution using multiple lymphocyte-specific, fluorophore-labeled transgenic lines, quantifying changes in thymic lymphocytes pre- vs. post-involution. Our results prove that, as in humans, zebrafish thymic B cells increase relative to T cells post-involution. We also performed RNA sequencing (RNA-seq) on D. rerio thymic and marrow lymphocytes of four novel double-transgenic lines, identifying distinct populations of immature T and B cells. Collectively, this is the first comprehensive analysis of zebrafish thymic involution, demonstrating its similarity to human involution, and establishing the highly genetically-manipulatable zebrafish model as a template for involution studies. Overall design: This experimental study aimed to investigate the gene expression profiles of lymphocytes in zebrafish using bulk RNA sequencing (RNA-seq) analysis. Specifically, four different double-transgenic zebrafish lines were utilized, each expressing specific fluorescent markers to label distinct cell populations. The double-transgenic lines included rag2:RFP × cd79a:GFP, rag2:RFP × cd79b:GFP, cd79a:GFP × lck:mCherry, and cd79b:GFP × lck:mCherry. These transgenic lines allowed for the identification and isolation of specific cell types for subsequent transcriptomic analysis.