Development of an ImageJ-based method for analysing the developing zebrafish vasculature

Abstract

Zebrafish with fluorescently labelled blood vessels provide an excellent model for studying angiogenesis. Most commonly the growth of the intersegmental blood vessels is investigated in response to compounds or manipulation of gene expression and analysed using manual methods, typically scoring the connectivity of these blood vessels to the dorsal longitudinal anastomotic vessel. Such methods are laborious and best suited to time points after the connectivity of these vessels has been established. By contrast, reported image processing-based methods are difficult to implement and often depend on specialist software. This study aimed to develop and evaluate a computational method using the freely available ImageJ software to quantify the development of intersegmental blood vessel formation. This methodology enabled rapid analysis of vascular development. The outputs of total vessel length and number of junctions best documented defective vascular development at differing levels of severity and gave comparable results to the frequently used manual approach of calculating the percentage connectivity of the intersegmental vessels. This ImageJ-based analysis method allowed objective quantitation of vascular network formation in zebrafish enabling a free, straightforward and rapid approach to determine the effect of novel compounds or genetic manipulation of the angiogenic process. Keywords Zebrafish -angiogenesis -vessel quantitation IntroductionThe zebrafish ( Danio rerio) is a powerful and widely used model organism for the study of vascular development and angiogenesis. Features such as the embryo's rapid and external development, small size, optical transparency, fluorescent tagging of vessels and methods to modify its gene expression all contribute to its utility in studying vessel development [ 1]. This vertebrate model shares both anatomical features and similar molecular mechanisms of vessel development observed in higher vertebrates making this a valuable tool for understanding gene function in both normal human development and angiogenesis dependent pathologies [ 2,3].The widespread use of zebrafish in studying the effects of genes or compounds in vessel development were used in conjunction with confocal fluorescence microscopy to generate high resolution vascular images, which were then subjected to processing and analysis using the Analyze Skeleton 2D/3D plugin of ImageJ. This computational analysis method was tested by using images generated via a developmental time course as well as images of embryos with varying levels of vascular disruption produced either pharmacologically or by translation blocking morpholino oligonucleotides. Of the generated analysis outputs, total vessel length and junction number best allowed differentiation between the varying phenotypes. This method allowed objective quantitation of vascular network formation in zebrafish enabling a more rapid and systematic approach to determine the effect of novel compounds or genetic manipulation of the angiogenic process. Methods Z...

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