Laser-scanning velocimetry: A confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae

This technique, called laser-scanning velocimetry, was used to quantify the effects of pharmacological, developmental, and genetic modifiers of cardiac function. Laser-scanning velocimetry was applied to analyze the cardiovascular effects of morpholino knockdown of osmosensing scaffold for MEKK3 (OSM), which when mutated causes the human vascular disease cerebral cavernous malformations. OSM-deficient embryos had a constricted aortic arch and markedly increased peak cell velocity, a characteristic indicator of aortic stenosis.These data validate laser-scanning velocimetry as a quantitative tool to measure cardiovascular performance for pharmacological and genetic analysis in zebrafish, which requires no specialized equipment other than a laser-scanning confocal microscope.The zebrafish Danio rerio are widely used in genome-wide screens to identify genes that regulate the development and function of the vertebrate cardiovascular system [1-3]. Zebrafish are powerful model organisms to define genes relevant to human cardiovascular disease because of their similarity in genetic complexity to mammals, the ease of selective gene knockdown, and their suitability for large-scale mutagenesis screens [4,5]. A beating heart and functional circulatory system forms within 26 hours of fertilization, and within 48 hours the heart develops into a two-chambered conformation, complete with functional valves. Because of their transparency, severe morphological abnormalities in cardiac development are easily observed, but the functional consequences of these defects are difficult to quantify. Methods are needed for functional analysis of cardiovascular defects that can be utilized by any investigator studying zebrafish cardiovascular physiology. Using an unmodified, commercially-available laser-scanning confocal microscope, we have developed a novel, non-invasive method of measuring cardiac performance in zebrafish embryos that provides measurements similar to those obtained with Doppl