Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The zebrafish is unique in its intrinsic ability to regenerate many different tissue types, including the RPE. This protocol can be used to identify molecular pathways that drive RPE regeneration and RPE disease-related mechanisms. Versatility is a primary advantage of this methodology.

In addition to studying RPE regeneration, this protocol can be utilized to examine RPE degenerative processes and the effects of RPE damage on adjacent tissues in the eye. Mammals, including humans, are unable to repair large RPE injuries resulting from trauma or degenerative disease. Using zebrafish as a tool to discover RPE pro-regenerative factors is a significant step towards promoting mammalian RPE regeneration.

To prepare a fresh 10 millimolar MTZ solution, five days post-fertilization, add MTZ powder to system water without PTU and mix thoroughly by vigorous shaking for one hour at 37 degrees Celsius. Cool 10 millimolar MTZ solution for one hour at room temperature on a tabletop rotator or shaker. To screen zebrafish larvae from the rpe65a:nsfB-eGFP transgene, separate transgenic eGFP positive larvae from non-transgenic eGFP negative larvae using a fluorescent stereo microscope with a 488 nanometer excitation laser.

Wake screened larvae up immediately by pipetting directly into a Petri dish with fresh 1.5X PTU without tricaine. Upon completion of screening, further separate the eGFP positive larvae into two groups of Petri dishes, one group to receive MTZ treatment and one group to be the unablated control. Then ablate the retinal pigment epithelium by removing 1.5X PTU from the ablated treatment dishes and add the freshly made 10 millimolar MTZ solution.

Also remove 1.5X PTU from the unablated control dishes and add fresh system water without PTU. Remove the 10 millimolar MTZ solution after exactly 24 hours and add fresh system water without PTU. Change the fresh system water without PTU on the MTZ negative dishes.

Screen eGFP positive larvae on four days post-fertilization. The eGFP is visible four days post-fertilization, but appears dimmer than signal intensity on the fifth day. Place eGFP positive larvae into six-well plates instead of Petri dishes at a density of not more than 10 larvae per well for pharmacological treatment.

Designate separate six-well plates for ablated and unablated larvae. Determine the volume of 15 micromolar IWR-1 or volume matched DMSO vehicle control pre-treatments needed and aliquot 1.5X PTU into conical tubes accordingly. Add IWR-1 stock to 1.5X PTU for a final concentration of 15 micromolar IWR-1.

Add a matched volume of DMSO stock to 1.5X PTU. Mix well by vortexing and visually confirm the dissolution of compounds. Remove 1.5X PTU from the eGFP positive larvae in six-well plates and add five milliliters per well of freshly made 0.06%DMSO or 15 micromolar IWR-1 treatments.

Ablate the RPE the next day. On five days post-fertilization, determine the volume of pharmacological and vehicle control treatments needed for both unablated and ablated six-well plates and aliquot appropriate volumes of either fresh system water without PTU or 10 millimolar MTZ solution into conical tubes. Add IWR-1 and DMSO stock solutions to respective conical tubes as performed previously.

Mix well by vortexing and visually confirm the dissolution of compounds. Remove 0.06%DMSO and 15 micromolar IWR-1 pre-treatments in 1.5X PTU from designated unablated and ablated six-well plates. Replenish with the appropriate fresh system water without PTU and MTZ solution treatments.

Remove 0.06%DMSO and 15 micromolar IWR-1 treatments in 10 millimolar MTZ solution after exactly 24 hours and replenish with treatments in fresh system water without PTU. Replenish 0.06%DMSO and 15 micromolar IWR-1 treatments in fresh system water without PTU on the unablated six-well plate. Monitor the success and extent of ablation in vivo using transmitted light illumination on a stereo microscope for larval maintenance post-genetic ablation.

To generate the RPE region of interest or ROI using Fiji, open an eight bit TIFF image, reorient the image so that the dorsal side is up and distal is left by choosing image, transform, and flip horizontally. For the last command, choose the option that best suits the directionality needed for that image. Start the ROI manager by choosing analyze, tools, ROI manager.

Use image, zoom, and toggle between the DAPI and Brightfield channels. Use keyboard shortcuts for efficiency. Identify the point at which the apical side of the RPE is adjacent from the tip of the outer limiting membrane and use this anatomical landmark as the ROI starting point.

Create the RPE ROI with the polygon selections tool in the Fiji toolbar using both the DAPI and Brightfield image channels and the image zoom function to identify apical and basal RPE boundaries. Bring the dorsal and ventral ends of the ROI to a sharp point rather than blunting or rounding off. Add the ROI by clicking on add in the ROI manager.

Save the ROI file by choosing more and save within the ROI manager. Double click on the rpegen. m file to open in the editor pane.

Under the user defined variable section of the rpegen. m file, enter the directory locations for folders containing the roi files, the tiff image files and where output files should be saved. Enter the group name for the mat file to be exported and the location of the Brightfield channel in the TIFF image stack.

Run the script by clicking on the run button in the editor menu at the top of MATLAB. After saving the MAT file, a three panel figure will appear and also be saved as a PDF to the output directory for each image run. Wait until these quality control PDFs have been saved to the output folder and the last figure has disappeared.

Open the individual PDFs and verify that all ROIs match the Brightfield images. Double click on the rpegen_permplot. m file to open in a new editor tab.

Under section one-user defined variables, enter the directory location for the output folder containing the MAT files from running the rpegen. m script. Enter each MAT filename to be loaded.

Run this section of the script by clicking on the run section button in the editor menu. In section two, enter the names of the two groups for a statistical comparison in the data A and data B variables and designate the number of permutation simulation repetitions in the reps variable. Only 2, 000 reps were used for this demo to decrease processing time.

Run this section of the script by clicking on the run section button in the editor menu. Run heat map figure and group results and P-value sections independently using the run section button. Whole mount damage of a five-day post-fertilization larva showed bright transgene expression in the RPE at the time of screening for genetic ablation.

Transverse cryosection of unablated six-day post-fertilization larva showed transgene expression was restricted to mature RPE cells with the brightest expression confined to the central 2/3 of the RPE. Arrowheads indicate apical microvilli. Transverse cryosection of an ablated one-day post-injury larva revealed disruption of eGFP positive cell morphology.

Arrows indicate pyknotic nuclei. Whole mount damages of unablated seven-day post-fertilization larvae showed RPE pigmentation throughout the eye and ablated two-day post-injury larvae showed an ablation zone absent pigment in the central RPE. A plot generated using RpEGEN showed similarity between the unablated nine-day post-fertilization DMSO and IWR-1 groups across the length of the RPE.

The plot showed overall lighter pixel intensity in the ablated four-day post-injury DMSO and IWR-1 groups, which appeared lightest in the ablated IWR-1 when compared to any other treatment group. Differences in central RPE pigmentation were significant when comparing ablated IWR-1 treated larvae to ablated DMSO treated controls. Using this zebrafish ablation paradigm, we have been able to identify several molecular signaling pathways, such as wind signaling which we've shown here, that are critical regulators of RPE regeneration.