title: In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences.
abstract: The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments. In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.
authors.affiliation: School of Biosciences, The University of Melbourne; Australian Regenerative Medicine Institute (ARMI), Monash University.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Dudczig
authors.affiliation: Australian Regenerative Medicine Institute (ARMI), Monash University.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Currie
authors.affiliation: The David J Apple Center for Vision Research, Department of Ophthalmology, Heidelberg University; lucia.poggi@med.uni-heidelberg.de.
authors.forename: Lucia
authors.initials: L
authors.lastname: Poggi
authors.affiliation: School of Biosciences, The University of Melbourne; Australian Regenerative Medicine Institute (ARMI), Monash University; patricia.jusuf@unimelb.edu.au.
authors.forename: Patricia R
authors.initials: PR
authors.lastname: Jusuf
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002454:Cell Differentiation; D002465:Cell Movement; D002678:Chimera / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D008845:Microinjections / Q000379:methods; D023421:Models, Animal; D009024:Morphogenesis / Q000235:genetics*; D012160:Retina / Q000196:embryology* / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics