In vivo imaging of zebrafish retinal cells using fluorescent coumarin derivatives.
BACKGROUND
The zebrafish visual system is a good research model because the zebrafish retina is very similar to that of humans in terms of the morphologies and functions. Studies of the retina have been facilitated by improvements in imaging techniques. In vitro techniques such as immunohistochemistry and in vivo imaging using transgenic zebrafish have been proven useful for visualizing specific subtypes of retinal cells. In contrast, in vivo imaging using organic fluorescent molecules such as fluorescent sphingolipids allows non-invasive staining and visualization of retinal cells en masse. However, these fluorescent molecules also localize to the interstitial fluid and stain whole larvae.


RESULTS
We screened fluorescent coumarin derivatives that might preferentially stain neuronal cells including retinal cells. We identified four coumarin derivatives that could be used for in vivo imaging of zebrafish retinal cells. The retinas of living zebrafish could be stained by simply immersing larvae in water containing 1μg/ml of a coumarin derivative for 30 min. By using confocal laser scanning microscopy, the lamination of the zebrafish retina was clearly visualized. Using these coumarin derivatives, we were able to assess the development of the zebrafish retina and the morphological abnormalities induced by genetic or chemical interventions. The coumarin derivatives were also suitable for counter-staining of transgenic zebrafish expressing fluorescent proteins in specific subtypes of retinal cells.


CONCLUSIONS
The coumarin derivatives identified in this study can stain zebrafish retinal cells in a relatively short time and at low concentrations, making them suitable for in vivo imaging of the zebrafish retina. Therefore, they will be useful tools in genetic and chemical screenings using zebrafish to identify genes and chemicals that may have crucial functions in the retina.
Watanabe
Kohei
K
Department of Molecular and Cellular Pharmacology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan.
Nishimura
Yuhei
Y
Oka
Takehiko
T
Nomoto
Tsuyoshi
T
Kon
Tetsuo
T
Shintou
Taichi
T
Hirano
Minoru
M
Shimada
Yasuhito
Y
Umemoto
Noriko
N
Kuroyanagi
Junya
J
Wang
Zhipeng
Z
Zhang
Zi
Z
Nishimura
Norihiro
N
Miyazaki
Takeshi
T
Imamura
Takeshi
T
Tanaka
Toshio
T
D000818:Animals; D001812:Blood-Brain Barrier / Q000502:physiology; D003374:Coumarins / Q000737:chemistry / Q000493:pharmacokinetics; D004730:Endothelium, Vascular / Q000502:physiology; D005456:Fluorescent Dyes / Q000737:chemistry / Q000493:pharmacokinetics; D007150:Immunohistochemistry; D008845:Microinjections; D009474:Neurons / Q000473:pathology; D012160:Retina / Q000166:cytology* / Q000473:pathology; D012164:Retinal Diseases / Q000235:genetics / Q000473:pathology; D017948:Retinal Rod Photoreceptor Cells / Q000502:physiology; D019108:Tight Junctions / Q000502:physiology; D014466:Ultraviolet Rays; D015027:Zebrafish / Q000502:physiology*
D003374:Coumarins; D005456:Fluorescent Dyes