Zebrafish Immune Cell Development and Diversity in Health and Disease

Abstract

Hematopoiesis, the process by which all blood cells are generated, is directed by transcription factors and signaling pathways that are highly conserved between zebrafish and humans. In healthy individuals, hematopoietic stem cells are tightly regulated to appropriately self-renew and differentiate into committed progenitors that will in turn give rise to the large diversity of blood cells. In hematologic disease, the production, maintenance, destruction, and/or function of a subset or multiple subsets of blood cells goes awry leading to clinical manifestations related to the affected cell type(s). This dysregulation is commonly the result of inherited or acquired genetic mutations that alter cellular biology. While much work has been done to identify the genes and environmental stimuli implicated across the gamut of hematologic disorders, the mechanisms by which these factors cause disease are less completely understood. Animal models including the zebrafish have proven useful for investigating these mechanisms in greater detail. To transcriptionally characterize adult zebrafish hematopoiesis, I performed single-cell RNA sequencing (scRNA-seq) on zebrafish thymi and kidney marrows. These data allowed me to identify hematopoietic stem and progenitor cells (HSPCs) and their progeny and to explore lymphocyte developmental trajectories. I defined novel cell types and states, including two dendritic-like cell populations and a pre-B cell state and transcriptionally characterized early thymic progenitors and thymic epithelial cells. I then built upon this work to study hematopoiesis in dysregulated states. Specifically, I employed zebrafish models of hoxa9a and hoxb4a overexpression, two genes capable of expanding hematopoietic stem cells with varying degrees of malignant potential, to investigate hematopoietic output and clonality. I found that both hoxa9a and hoxb4a overexpression expand HSPCs in embryogenesis and that stable overexpression of hoxa9a leads to strong and robust myeloid expansion in adulthood. By scRNA-seq, I observed global transcriptional changes with hoxa9a overexpression, including the upregulation of numerous cellular stress response genes. Through establishing and employing a transcriptional atlas of zebrafish hematopoiesis, I expanded what is known about zebrafish immune cell development and diversity, gained insight into cellular changes that occur with hoxa9a overexpression, and demonstrated the utility of this framework for studying hematopoiesis in health and disease.