Zebrafish as a Model to Study Chemokine Function

Abstract

Zebrafish have emerged as a powerful model organism to study embryo morphogenesis. Due to their optical clarity, they are uniquely suited for time-lapse imaging studies, providing insights into the dynamic processes underlying tissue formation and cell migration. These studies have been tremendously facilitated by the availability of transgenic zebrafish lines, labelling distinct embryonic structures, individual cells, or even subcellular structures, such as the nucleus. Zebrafish studies have revealed that the migration of several different cell types in the embryo is controlled by chemokines, small vertebrate-specific proteins. Here, we report methods to analyze the expression pattern of a given chemokine and its receptor in transgenic zebrafish using fluorescent in situ hybridization in combination with an anti-green fluorescent protein (GFP) antibody staining. We furthermore illustrate how to image migrating cell populations using time-lapse microscopy in double-transgenic embryos. We show how to investigate cell number and direction of migration by using a nuclear-localized GFP. The combination of this transgene with a membrane-targeted red fluorescent protein allows for the simultaneous determination of changes in cell shape, such as the formation of filopodial extensions. We exemplify this by describing how a mutation in the chemokine receptor cxcr4a affects endothelial cell migration and blood vessel formation. Finally, we provide a method to perform fluorescent angiography to monitor blood vessel perfusion in chemokine receptor mutants.

Endothelial Notch signalling limits angiogenesis via control of artery formation

“…Whole mount FISH combined with EGFP antibody staining was performed in Tg(kdrl:EGFP) s843 line as described previously 67 . Double FISH was performed in the Tg (dll4:gal4) mu106 ; (UAS:GFP) nkuasgfp1a embryos as described earlier 68 .…”

Chemokine Receptors and Phagocyte Biology in Zebrafish

“…The zebrafish model has been successfully used to study how chemokine signaling networks determine macrophage and neutrophil functions and to ascribe these receptors a role in immunity, inflammation, and cancer models (4,13,16,22,(40)(41)(42). It is a powerful vertebrate model well-suited for noninvasive in-vivo imaging given its optical transparency at early embryonic and larval stages.…”

Spatiotemporal endothelial cell – pericyte association in tumors as shown by high resolution 4D intravital imaging

“…Depicting angiogenesis by classical histology provides a static image and is often not sufficient for a correct interpretation of kinetics as such spatiotemporal complexity requires successive observations in a 4D (XYZ spatial + T, time dimension) intravital manner. A major technological development which improved possibilities for longitudinal cellular investigation in vivo is the introduction of fluorescent proteins to the genome of animals, most often mice and zebrafish 24 – 26 .…”