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ToxSci Advance Access originally published online on January 24, 2006
Toxicological Sciences 2006 90(2):529-538; doi:10.1093/toxsci/kfj115
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© The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Four-Dimensional Imaging and Quantification of Gene Expression in Early Developing Zebrafish (Danio rerio) Embryos

Richard M. K. Yu*,1, Chun Chi Lin*,1, P. K. Chan*, Elly Suk Hen Chow*,2, Margaret B. Murphy*, Barbara P. Chan{dagger}, Ferenc Müller{ddagger}, Uwe Strähle{ddagger} and S. H. Cheng*,3

* Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Hong Kong; {dagger} Medical Engineering Program, Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong; and {ddagger} Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, 76021 Karlsruhe, Germany

Received October 31, 2005; accepted January 17, 2006

Four-dimensional (4D) imaging is a powerful tool for studying three-dimensional (3D) changes in an organism through time. Different imaging systems for obtaining 3D data from in vivo specimens have been developed but usually involved large and expensive machines. We successfully used a simple inverted compound microscope and a commercially available program to study and quantify in vivo changes in sonic hedgehog (shh) expression during early development in a green fluorescence protein (GFP) transgenic zebrafish (Danio rerio) line. We applied the 4D system to study the effect of 100 µM cadmium exposure on shh expression. In control zebrafish embryos, shh:GFP expression was detected at about 9 h post-fertilization (hpf) and increased steadily in the next 7 h, peaking at about 17 hpf and decreasing in the following 4 h. In the same time period, different shh expression volumes were observed in cadmium-treated and control embryos. Embryos affected by cadmium-exposure demonstrated a down-regulation in shh expression. The number of GFP-expressing cells measured by flow cytometry decreased, and expression of neurogenin-1, a downstream target of the shh signaling pathway, was down-regulated, providing additional supporting data on the effects of cadmium on shh. In summary, we demonstrated the setup of a 4D imaging system and its application to the quantification of gene expression.

Key Words: zebrafish embryo; green fluorescence protein; four-dimensional imaging; cadmium; sonic hedgehog.


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