Background: How different immune cell compartments contribute to a successful immune response is central to fully understanding the mechanisms behind normal processes such as tissue repair and the pathology of inflammatory diseases. However, the ability to observe and characterize such interactions, in real-time, within a living vertebrate has proved elusive. Recently, the zebrafish has been exploited to model aspects of human disease and to study specific immune cell compartments using fluorescent reporter transgenic lines. A number of blood-specific lines have provided a means to exploit the exquisite optical clarity that this vertebrate system offers and provide a level of insight into dynamic inflammatory processes previously unavailable.

We have generated novel transgenic lines that brightly mark the lymphatic system of zebrafish using the lyve1 promoter. Facilitated by these new transgenic lines, we generated a map of zebrafish lymphatic development up to 15 days post-fertilisation and discovered three previously uncharacterised lymphatic vessel networks: the facial lymphatics, the lateral lymphatics and the intestinal lymphatics. We show that a facial lymphatic vessel, termed the lateral facial lymphatic, develops through a novel developmental mechanism, which initially involves vessel growth through a single vascular sprout followed by the recruitment of lymphangioblasts to the vascular tip. Unlike the lymphangioblasts that form the thoracic duct, the lymphangioblasts that contribute to the lateral facial lymphatic vessel originate from a number of different blood vessels. Our work highlights the additional complexity of lymphatic vessel development in the zebrafish that may increase its versatility as a model of lymphangiogenesis.

Evidence suggests the bactericidal activity of mitochondria-derived reactive oxygen species (mROS) directly contributes to killing phagocytozed bacteria. Infection-responsive components that regulate this process remain incompletely understood. We describe a role for the mitochondria-localizing enzyme encoded by Immunoresponsive gene 1 (IRG1) during the utilization of fatty acids as a fuel for oxidative phosphorylation (OXPHOS) and associated mROS production. In a zebrafish infection model, infection-responsive expression of zebrafish irg1 is specific to macrophage-lineage cells and is regulated cooperatively by glucocorticoid and JAK/STAT signaling pathways. Irg1-depleted macrophage-lineage cells are impaired in their ability to utilize fatty acids as an energy substrate for OXPHOS-derived mROS production resulting in defective bactericidal activity. Additionally, the requirement for fatty acid β-oxidation during infection-responsive mROS production and bactericidal activity toward intracellular bacteria is conserved in murine macrophages. These results reveal IRG1 as a key component of the immunometabolism axis, connecting infection, cellular metabolism, and macrophage effector function.

Runx transcription factors determine cell fate in many lineages. Maintaining balanced levels of Runx proteins is crucial, as deregulated expression leads to cancers and developmental disorders. We conducted a forward genetic screen in zebrafish for positive regulators of runx1 that yielded the cohesin subunit rad21. Zebrafish embryos lacking Rad21, or cohesin subunit Smc3, fail to express runx3 and lose hematopoietic runx1 expression in early embryonic development. Failure to develop differentiated blood cells in rad21 mutants is partially rescued by microinjection of runx1 mRNA. Significantly, monoallelic loss of rad21 caused a reduction in the transcription of runx1 and of the proneural genes ascl1a and ascl1b, indicating that downstream genes are sensitive to Rad21 dose. Changes in gene expression were observed in a reduced cohesin background in which cell division was able to proceed, indicating that cohesin might have a function in transcription that is separable from its mitotic role. Cohesin is a protein complex essential for sister chromatid cohesion and DNA repair that also appears to be essential for normal development through as yet unknown mechanisms. Our findings provide evidence for a novel role for cohesin in development, and indicate potential for monoallelic loss of cohesin subunits to alter gene expression.

Inflammatory bowel disease (IBD) results from dysfunctional interactions between the intestinal immune system and microbiota, influenced by host genetic susceptibility. Because a key feature of the pathology is intestinal epithelial damage, potential disease factors have been traditionally analyzed within the background of chemical colitis models in mice. The zebrafish has greatly complemented the mouse for modeling aspects of disease processes, with an advantage for high content drug screens. Larval zebrafish exposed to the haptenizing agent trinitrobenzene sulfonic acid (TNBS) displayed impaired intestinal homeostasis and inflammation reminiscent of human IBD. There was a marked induction of pro-inflammatory cytokines, the degradative enzyme mmp9 and leukocytosis. Enterocolitis was dependent on microbiota and Toll-like receptor signaling, that can be ameliorated by antibiotic and anti-inflammatory drug treatments. This system will be useful to rapidly interrogate in vivo the biological significance of the IBD candidate genes so far identified and to carry out pharmacological modifier screens. Developmental Dynamics 240:288-298,