Methods for Studying Developmental Angiogenesis in Zebrafish

Abstract

Angiogenesis is a very complicated biological process in which the inter-communication between various cell types in a time-dependent manner is crucial for correct formation and perfusion of new vessels to expand the developing vasculature. Zebrafish have turned out to be incredibly well suited for the study of developmental angiogenesis, as the embryo develop outside of the mother, is transparent and develop very fast enabling highly dynamic visualizations (such as video time-lapse recordings) of the entire developing vasculature. Such a unique advantage of this model has enabled researchers to delineate the spatiotemporal involvement of signaling factors important for the formation and maintenance of tip cells, cues important for vascular patterning and new modes of blood vessel and lumen formation that could not have been delineated in rodent or other models. Here we will thoroughly describe how to use embryonic zebrafish models in studies of developmental angiogenesis. We will describe how to interfere genetically with the process by injection of morpholinos or mRNA into the embryo which would lead to positive or negative regulation of particular genes under investigations. We will also address how to establish gradients of angiogenic factors in non-vascularized areas of the embryo by injection of recombinant growth factors or cells in the perivitelline space, which will then lead to ectopic angiogenesis toward the stimuli – a process that can give information related to the angiogenic properties of compounds. Finally we will go through how to draw maximal benefit from such investigations by confocal imaging. This chapter will end with a trouble-shooting section in which common problems and solutions will be discussed, helping you – the reader – to as smoothly as possible get started on your research on developmental angiogenesis in the zebrafish embryo.