In vivo imaging of zebrafish retinal cells using fluorescent coumarin derivatives

<p>Abstract</p> <p>Background</p> <p>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. <it>In vitro </it>techniques such as immunohistochemistry and <it>in vivo </it>imaging using transgenic zebrafish have been proven useful for visualizing specific subtypes of retinal cells. In contrast, <it>in vivo </it>imaging using organic fluorescent molecules such as fluorescent sphingolipids allows non-invasive staining and visualization of retinal cells <it>en masse</it>. However, these fluorescent molecules also localize to the interstitial fluid and stain whole larvae.</p> <p>Results</p> <p>We screened fluorescent coumarin derivatives that might preferentially stain neuronal cells including retinal cells. We identified four coumarin derivatives that could be used for <it>in vivo </it>imaging of zebrafish retinal cells. The retinas of living zebrafish could be stained by simply immersing larvae in water containing 1 μg/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.</p> <p>Conclusions</p> <p>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 <it>in vivo </it>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.</p

The use of fluorescent indoline dyes for side population analysis.

Dye efflux assay evaluated by flow cytometry is useful for stem cell studies. The side population (SP) cells, characterized by the capacity to efflux Hoechst 33342 dye, have been shown to be enriched for hematopoietic stem cells (HSCs) in bone marrow. In addition, SP cells are isolated from various tissues and cell lines, and are also potential candidates for cancer stem cells. However, ultra violet (UV) light, which is not common for every flow cytometer, is required to excite Hoechst 33342. Here we showed that a fluorescent indoline dye ZMB793 can be excited by 488-nm laser, equipped in almost all the modern flow cytometers, and ZMB793-excluding cells showed SP phenotype. HSCs were exclusively enriched in the ZMB793-excluding cells, while ZMB793 was localized in cytosol of bone marrow lineage cells. The efflux of ZMB793 dye was mediated by ATP binding cassette (ABC) transporter Abcg2. Moreover, staining properties were affected by the side-chain structure of the dyes. These data indicate that the fluorescent dye ZMB793 could be used for the SP cell analysis

<i>In Vivo</i> Detection of Mitochondrial Dysfunction Induced by Clinical Drugs and Disease-Associated Genes Using a Novel Dye ZMJ214 in Zebrafish

Mitochondrial dysfunction has been implicated in various drug-induced toxicities and genetic disorders. Recently, the zebrafish has emerged as a versatile animal model for both chemical and genetic screenings. Taking advantage of its transparency, various <i>in vivo</i> fluorescent imaging methods have been developed to identify novel functions of chemicals and genes in zebrafish. However, there have not been fluorescent probes that can detect mitochondrial membrane potential in living zebrafish. In this study, we identified a novel cyanine dye called ZMJ214 that detects mitochondrial membrane potential in living zebrafish from 4 to 8 days post fertilization and is administered by simple immersion. The fluorescence intensity of ZMJ214 in zebrafish was increased and decreased by oligomycin and FCCP, respectively, suggesting a positive correlation between ZMJ214 fluorescence and mitochondrial membrane potential. <i>In vivo</i> imaging of zebrafish stained with ZMJ214 allowed for the detection of altered mitochondrial membrane potential induced by the antidiabetic drug troglitazone and the antiepileptic drug tolcapone, both of which have been withdrawn from the market due to mitochondrial toxicity. In contrast, pioglitazone and entacapone, which are similar to troglitazone and tolcapone, respectively, and have been used commercially, did not cause a change in mitochondrial membrane potential in zebrafish stained with ZMJ214. Live imaging of zebrafish stained with ZMJ214 also revealed that knock-down of slc25a12, a mitochondrial carrier protein associated with autism, dysregulated the mitochondrial membrane potential. These results suggest that ZMJ214 can be a useful tool to identify chemicals and genes that cause mitochondrial dysfunction <i>in vivo.</i

Självklar mångfald - oklar strategi

På uppdrag av Kultur Skåne har Malmö högskola genomfört en kartläggning av mångfaldsarbetet vid scenkonstinstitutionerna i Region Skåne. Studien har ett enomgripande mångfaldsperspektiv och har inneburit att ur ett kultursociologiskt perspektiv kartlägga arbetet med mångfald på regionens dans-, teater- och musikinstitutioner. Syftet har varit att få en överblick över hur mångfaldsarbetet ser ut vad gäller såväl organisation, rekrytering, produktion, som publikarbete. Författarna arbetar på Enheten för kompetensutveckling och utvärdering på Malmö högskola, och rapporten SJÄLVKLAR MÅNGFALD – OKLAR STRATEGI är en rapport från enhetens rapportserie Malmö högskolas utvärderingsrapporter