title: In vivo imaging of zebrafish retinal cells using fluorescent coumarin derivatives.
abstract: 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.
authors.affiliation: Department of Molecular and Cellular Pharmacology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Watanabe
authors.forename: Yuhei
authors.initials: Y
authors.lastname: Nishimura
authors.forename: Takehiko
authors.initials: T
authors.lastname: Oka
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Nomoto
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Kon
authors.forename: Taichi
authors.initials: T
authors.lastname: Shintou
authors.forename: Minoru
authors.initials: M
authors.lastname: Hirano
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Shimada
authors.forename: Noriko
authors.initials: N
authors.lastname: Umemoto
authors.forename: Junya
authors.initials: J
authors.lastname: Kuroyanagi
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Wang
authors.forename: Zi
authors.initials: Z
authors.lastname: Zhang
authors.forename: Norihiro
authors.initials: N
authors.lastname: Nishimura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Miyazaki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Imamura
authors.forename: Toshio
authors.initials: T
authors.lastname: Tanaka
mesh_terms: 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*
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes