Lymph nodes and other secondary lymphoid organs play critical roles in immune surveillance and immune activation in mammals, but the deep internal locations of these organs make it challenging to image and study them in living animals. Here, we describe a previously uncharacterized external immune organ in the zebrafish ideally suited for studying immune cell dynamics in vivo, the axillary lymphoid organ (ALO). This small, translucent organ has an outer cortex teeming with immune cells, an inner medulla with a mesh-like network of fibroblastic reticular cells along which immune cells migrate, and a network of lymphatic vessels draining to a large adjacent lymph sac. Noninvasive high-resolution imaging of transgenically marked immune cells can be carried out in ALOs of living animals, which are readily accessible to external treatment. This newly discovered tissue provides a superb model for dynamic live imaging of immune cells and their interaction with pathogens and surrounding tissues, including blood and lymphatic vessels.

The thymus is the site of T lymphocyte development and T cell education to recognize foreign, but not self, Ags. B cells also reside and develop in the thymus, although their functions are less clear. During “thymic involution,” a process of lymphoid atrophy and adipose replacement linked to sexual maturation, thymocytes decline. However, thymic B cells decrease far less than T cells, such that B cells comprise ∼1% of human neonatal thymocytes but up to ∼10% in adults. All jawed vertebrates possess a thymus, and we and others have shown zebrafish (Danio rerio) also have thymic B cells. In this article, 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 the changes in thymic T- and B-lymphocytes pre- versus postinvolution. Our results prove that, as in humans, zebrafish thymic B cells increase relative to T cells postinvolution. We also performed RNA sequencing 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, to our knowledge, 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.

Despite advancements in the diagnosis and treatment of acute lymphoblastic leukemia (ALL), a need for improved strategies to decrease morbidity and improve cure rates in relapsed/refractory ALL still exists. Such approaches include the identification and implementation of novel targeted combination regimens, and more precise upfront patient risk stratification to guide therapy. New curative strategies rely on an understanding of the pathobiology that derives from systematically dissecting each cancer’s genetic and molecular landscape. Zebrafish models provide a powerful system to simulate human diseases, including leukemias and ALL specifically. They are also an invaluable tool for genetic manipulation, in vivo studies, and drug discovery. Here, we highlight and summarize contributions made by several zebrafish T-ALL models and newer zebrafish B-ALL models in translating the underlying genetic and molecular mechanisms operative in ALL, and also highlight their potential utility for drug discovery. These models have laid the groundwork for increasing our understanding of the molecular basis of ALL to further translational and clinical research endeavors that seek to improve outcomes in this important cancer.

Zebrafish (Danio rerio) are a compelling model for studying lymphocytes because zebrafish and humans have similar adaptive immune systems, including their lymphocytes. Antibodies that recognize zebrafish proteins are sparse, so many investigators use 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. This study is, to our knowledge, the first study to focus on zebrafish epidermal lymphocytes, using scales. Obtaining zebrafish blood via nonlethal 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 transgenic 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 kidney-marrow (analogous to mammalian bone marrow) lymphocytes from the same animals, revealing B- and T-lineage signatures. Single-cell quantitative real-time PCR and RNA sequencing 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, demonstrating epidermal lymphocyte responses. To analyze cells functionally, we employed a bead-ingestion assay, showing that thymic, marrow, and epidermal lymphocytes have phagocytic activity. In summary, we establish a novel, nonlethal technique to obtain zebrafish lymphocytes, providing the first quantification, expression profiling, and functional data from zebrafish epidermal lymphocytes.

Precursor-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common pediatric cancer, but there are no useful zebrafish pre-B ALL models. We describe the first highly-penetrant zebrafish pre-B ALL, driven by human MYC . Leukemias express B lymphoblast-specific genes and are distinct from T cell ALL (T-ALL)—which these fish also develop. Zebrafish pre-B ALL shares in vivo features and expression profiles with human pre-B ALL, and these profiles differ from zebrafish T-ALL or normal B and T cells. These animals also exhibit aberrant lymphocyte development. As the only robust zebrafish pre-B ALL model and only example where T-ALL also develops, this model can reveal differences between MYC -driven pre-B vs. T-ALL and be exploited to discover novel pre-B ALL therapies. Statement of significance: We describe the first robust zebrafish pre-B ALL model in MYC-transgenic animals known to develop T-ALL, revealing the only animal model with both human ALL types. We also describe aberrant multi-lineage lymphopoiesis. This powerful system can be used to study MYC -driven leukemogenesis and discover new pre-B ALL targeted therapies.

MFGE8 is a major exosome (EV) protein known to mediate inflammation and atherosclerosis in type 2 diabetes mellitus (T2DM) in animal studies. However, its role in the pathophysiology of human T2DM, obesity, and cardiovascular disease has been controversial and less explored. Earlier, we reported a rare Asian Indian population-specific missense variant (rs371227978; Arg148His) in the MFGE8 associated with increased circulating Mfge8 and T2DM. Here, we have further investigated the role of Mfge8 with T2DM risk in additional Asian Indians (n=4917) and Europeans and other multiethnic cohorts from UK Biobank (UKBB) (n= 455,808) and the US (n=1150). We also evaluated the exposure of Mfge8-enriched human EVs (with and without mutation) in zebrafish (ZF) for their impact on the cardiometabolic organ system. Most individual carriers of Arg148His variant not only had high circulating Mfge8 but also showed a positive significant correlation with glucose (r= 0.42; p = 4.9x10-04), while the non-carriers showed a negative correlation of Mfge8 with glucose (r = -0.38; p = 0.001) in Asian Indians. However, the same variant was monomorphic in Europeans and other ethnic groups of UKBB and US cohorts. Even in the absence of Arg148His variant, serum Mfge8 correlated significantly with blood glucose both in Caucasians (r = 0.56; p = 8.4x10-10) and individuals from other ethnicities (African Americans, Hispanics, and others) (r = 0.31; p = 5.8x10-4). Since Mfge8 is an EV marker, the exposure of Mfge8-enriched human EVs to ZF larvae rapidly developed fatty liver disease and heart hypertrophy and exhibited redundant growth with poor muscular architecture with and without the high-fat diet (HFD), while the control group fishes developed fatty liver disease and heart hypertrophy only after the HFD feeding. With strong evidence from animal studies supporting the role of Mfge8 in obesity, insulin resistance, and the development of atherosclerosis in T2DM, the current research suggests that circulating Mfge8 may be a potential marker for preventing or reducing the risk of T2DM and cardiovascular disease.