Four-dimensional imaging and quantification of gene expression in early developing zebrafish (Danio rerio) embryos.
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 microM 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.
Yu
Richard M K
RM
Department of Biology and Chemistry, City University of Hong Kong, Hong Kong. bhcheng@cityu.edu.hk
Lin
Chun Chi
CC
Chan
P K
PK
Chow
Elly Suk Hen
ES
Murphy
Margaret B
MB
Chan
Barbara P
BP
Müller
Ferenc
F
Strähle
Uwe
U
Cheng
S H
SH
D000818:Animals; D030801:Animals, Genetically Modified; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D002104:Cadmium / Q000633:toxicity*; D004625:Embryo, Nonmammalian / Q000187:drug effects; D047108:Embryonic Development / Q000187:drug effects*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D049452:Green Fluorescent Proteins; D053823:Hedgehog Proteins; D008856:Microscopy, Fluorescence / Q000379:methods; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*
D051792:Basic Helix-Loop-Helix Proteins; D002104:Cadmium; D049452:Green Fluorescent Proteins; D053823:Hedgehog Proteins; D009419:Nerve Tissue Proteins; D015534:Trans-Activators; D029961:Zebrafish Proteins; C486337:neurog1 protein, zebrafish