Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

This protocol is an amalgamation of various techniques that would enable the researchers to delineate the role of candidate gene in the melanocyte biology. It is a holistic approach towards achieving a logical inference. By combining various methods, confounding factors can be avoided and it can help researchers gain a substantial result.

To begin the analysis, immobilize the 48 HPF embryos with 0.016%tricaine. Mount the embryos using a few milliliters of 1.5 to 2%methyl cellulose in a Petri dish. Pick the embryos with a Pasteur pipette and place them in methyl cellulose to restrict movement during imaging.

For optimal lateral or dorsal imaging, adjust their position with a magnification of more than 5X. Place the Petri dish under the microscope. Using a manipulator, adjust the fish so that all five melanocyte embryonic stripes are visible concurrently.

Using the acquisition software, capture the images. If the fish awakens, place it into tricaine water until it stabilizes. Using the open tool in ImageJ software, open the image to be quantified.

Select the freehand shape tool to outline the area for analysis. Select the set measurements option, click on the mean gray value and then area. To calculate the mean gray value for the selected area, click on M or go to analyze and select measure.

Based on the stage of interest, transfer the embryos to a Petri dish containing 0.6 milligrams per milliliter Pronase. Using a Pasteur pipette, transfer the dechorionated embryos to a Petri dish containing plain embryo water after five to 10 minutes. With a glass Pasteur pipette, collect and transfer around 100 embryos to a two milliliter microcentrifuge tube.

Discard the medium and add 200 microliters of ice cold deyolking Ringers solution. Place the tube on ice and mix the content around 20 times with a pipette to dissolve the yoke. Centrifuge the tubes at 100 G for one minute at four degrees Celsius twice, and discard the supernatant.

Transfer the deyolked embryos using a one milliliter pipette in a Petri dish containing 10 milliliters of trypsin solution. Mix the solution once or twice using a one milliliter pipette to decrease the aggregation. Pass the trypsinized suspension through a 70 micrometer strainer placed on a 50 milliliter conical tube to collect a single cell suspension.

Wash the Petri dishes with the trypsinized suspension to remove the adhered cells from the surface. For counting the fluorescently labeled cells using a flow cytometer, after creating a new experiment folder, draw a forward versus side scatter plot. Make a histogram for the intensity of fluorescein isothiocyanate.

Load the wild type cells first to set the forward and side scatter gates and the FITC threshold. Afterward, load the cells isolated from transgenic FTYRP GFP line embryos to count the melanocytes. Mount the embryos in 1.5 to 2%methyl cellulose in a petri dish.

Place it under the microscope and adjust it in the desired orientation using a pipette tip. Using the acquisition software, acquire the images. For embryos less than 24 HPF, using 10X magnification, capture the whole image, while for embryos more than 24 HPF, acquire multiple scan fields and subsequently assemble them.

After performing the assay, the Brightfield imaging after 48 hours revealed the presence of all five pigmented melanophore stripes. At the 48 HPF stage, the number of lateral melanophores was calculated and it was found that the histone variant h2afv morphants demonstrated a reduced number of melanophores as compared to the control. The mean gray value was measured for CA14 and h2afv morphants, which revealed that the values were higher for the variants than the control morphants.

By employing a sodium hydroxide-based spectrophotometric absorption method, melanin content was quantified where the CA14 morphants showed less content than the control morphants. Fluorescence imaging was performed to evaluate morpholino-based alteration for different stages of zebrafish melanophore development. The number of melanophores in CA14 and h2afv morphants was analyzed using FACS.

It was observed that the number of melanophores in CA14 remained unchanged, while they were significantly reduced in h2afv compared to the control morphant. The mean fluorescence intensity per area was also calculated, demonstrating that the h2afv morphants show a considerable decrease in the value relative to control morphants. While adjusting the fish before imaging, make sure you can visualize all five melanophore stripes.

You need to tilt the fish a little to distinguish between the two lateral stripes. After establishing the role of a candidate gene in melanocyte development, we can eliminate the gene in a tissue-specific way using CRISPR technology. That will be a more targeted approach.