Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Summary

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

Abstract

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes.

Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

Introduction

While the use of melanin for photoprotection has evolved several times across the animal kingdom, vertebrates have seemingly perfected the process. Dedicated pigment-producing cells with an elaborate machinery to synthesize and contain melanin are conserved from fish to humans¹. However, the outcome of pigmentation is dramatically varied, ranging in the color to recipience and presents as vivid patterns on integuments, the skin, and hair². Despite the diversity, the repertoire of genes involved in pigmentation response is strikingly conserved. The core components of the melanin-synthesizing machinery, such as the key melanin-synthesizing enzymes, the components of the melanosomes, and the upstream connectivity to the signaling pathway, remain essentially identical across organisms. Subtle genetic differences bring about dramatic changes in the patterns of pigmentation observed across species³. Hence, a reverse genetic approach in a lower vertebrate organism, the zebrafish (Danio rerio), offers an excellent opportunity to decipher the involvement of genes in rendering the pigmented state⁴.

Zebrafish embryos develop from a single-celled fertilized zygote to a larva within a span of ~24 h post fertilization (hpf)⁵. Strikingly, the melanocyte-equivalent cells-the melanophores-are large cells that are present in the dermis and are conspicuous due to the dark melanin content⁶. These neural crest-derived cells emanate ~11 hpf and begin to pigment ~24 hpf^6,7. Conserved gene expression modules have enabled the identification of key factors that orchestrate melanocyte functions and led to the development of transgenic fluorescent reporter lines Tg(sox10:GFP), Tg(mitfa:GFP), and Tg(ftyrp1:GFP)^8,9,10 that label selective stages of melanocyte development. Using these transgenic fish lines enables the interrogation of cell biology of melanocytes at the organismal level in the tissue context with appropriate cues according to the developmental timelines. These reporters complement pigment-based quantitation of melanocytes and enable a distinct assessment of melanocyte numbers irrespective of melanin content.

This article provides a detailed protocol for deciphering the biology of melanocytes by assessing two critical parameters, namely melanin content and melanocyte numbers. While the former is a common functional readout emanating from a hypopigmentation response, the latter is associated with a reduction in the specification or survival of melanocytes and is often associated with genetic or acquired depigmentation conditions. The overall strategy of this reverse genetic screen is to silence select genes using a morpholino and investigate the melanocyte-specific outcomes. Melanin content is analyzed using image-based quantitation of mean grey values followed by confirmation using a melanin content assay. The number of melanocytes at various stages of maturation is analyzed using image-based quantitation and further confirmed using FACS analysis. Here, the screening protocol is demonstrated using two candidate genes, namely carbonic anhydrase 14, involved in melanogenesis, and a histone variant H2AFZ.2 involved in the specification of melanocytes from the neural crest precursor population. While the former alters melanin content and not the melanocyte numbers, the latter alters the number of specified melanocytes and, consequently, the melanin content in the embryo. In all, this method provides a detailed protocol to identify the role of a candidate gene in pigmentation and distinguish its role in controlling melanocyte numbers versus melanin content.

Protocol

Zebrafish experiments were performed in strict accordance with the institutional animal ethics approval (IAEC) of the CSIR-Institute of Genomics and Integrative Biology (IGIB), India (Proposal No 45a). All efforts were made to minimize animal suffering.

1. Injecting morpholino into zebrafish embryos

1. Using a standard needle puller, draw very sharp and closed-tipped pipets.
2. Load the solution containing morpholino into the micropipettes using a microloader tip and insert it into the microinjector apparatus. Tighten the screw properly to lock the micropipette.
   NOTE: Dosage standardization of morpholinos is necessary. The appropriate dosage has a survival rate of >70% and a specific phenotype at 24 hpf.
3. Cut the tip of the micropipette using fine forceps and calibrate it¹¹.
4. To calibrate, inject 1 volume of morpholino solution into the capillary tube from the micropipette, keeping the injecting time at 1 s. Repeat this five times. Using the standard, 1 mm = 30 nL volume, find the volume injected in 5 s by keeping the capillary tube against a measuring scale. Using the above information, calculate the time (in seconds) required to inject 1-3 nL per injection¹².
   ​NOTE: The standard, 1 mm = 30 nL, is specific to the microinjector pressure settings and diameter of the capillary used for calibration. Ideally, the injection must be made into the yolk-cell interface, which is formed within 15-20 min after fertilization. To facilitate multiple injections, development can be slightly delayed by maintaining embryos at a lower temperature (18 °C). However, this should be kept to a minimum and not extended beyond 30 min due to the compounding effect of lower temperature on gene expression changes.
5. Mount the embryos on an agarose-cast Petri dish (60 mm). Stack the embryos tightly within the ridges. Using fire-polished glass pipettes, align them in the proper orientation for injection.
6. Under a microscope, use manipulators to bring the micropipette closer to the embryo and inject inside the yolk-cell interface by pressing the footswitch.
7. Inject all the embryos similarly; collect them in a Petri dish containing fresh embryo water medium and incubate at 28 °C.
8. Check the injected embryos after 6-8 h. Remove all the dead embryos identifiable due to their high opacity and keep changing the embryo water at least once per day to avoid infection.

2. Pigmentation analysis

1. Preparation for counting lateral midline melanophores in 3 dpf zebrafish embryos
   1. Treat embryos with 0.016% neuro-muscular anesthetic to immobilize them.
   2. To mount the embryos for imaging, add a few milliliters of 1.5-2% methylcellulose in the Petri dish (60 mm) so that it forms a thin layer. Using a Pasteur pipette, pick embryos and gently position them in methylcellulose to restrict further movement during imaging.
   3. Adjust their position for optimal lateral (dorsal stripe, ventral stripe, yolk stripe, and mid-line melanophores) or dorsal (melanophores on the dorsal part of the head) imaging. For better resolution of adjacent melanophores, image at higher magnification (>5x)
2. Brightfield imaging
   NOTE: Brightfield imaging is performed using a stereomicroscope with 8-10x magnification.
   1. Place the Petri dish containing the zebrafish embryos under the microscope. Using a manipulator, adjust the fish in such orientation so that all five melanocyte embryonic stripes are visible (dorsal, ventral, two lateral, yolk) simultaneously (Figure 1C). Quickly capture images using the acquisition software. In case where the animal regains movement before it is imaged, re-immerse it in anesthetic and proceed with imaging once it is sufficiently immobilized.
   2. Repeat this with all the fish and ensure that magnification remains the same for every image.
3. Calculating mean gray value using ImageJ software
   1. Take dorsal and lateral images of 2 dpf zebrafish embryos.
   2. Open the image to be quantified in ImageJ using the Open tool. Use the freehand shape tool to outline the area for analysis. Go to the Set measurements option and select Mean grey value | area. Press M (or Analyze | Measure) to calculate the mean grey value for the selected area.
   3. Keeping the area to be analyzed constant, calculate the mean grey area for every animal separately.
   4. Plot a bar graph with the acquired data (Figure 2F).
4. Melanin content assay
   1. At 2 dpf, use a glass Pasteur pipette to collect ~25 zebrafish embryos.
   2. Perform manual dechorionation using 1 mL insulin needles and add them to 1.5 mL microcentrifuge tubes.
   3. Discard the embryo medium carefully and add 1 mL of ice-cold lysis buffer (20 mM sodium phosphate (pH 6.8), 1% Triton X-100, 1 mM PMSF, 1 mM EDTA) with protease inhibitor cocktail and prepare protein lysates by sonication.
   4. Dissolve the lysates in 1 mL of 1 N NaOH and incubate the samples at 100 °C for 50 min in a water bath. Vortex it intermittently to homogenize the lysates completely.
   5. Take absorbance readings of the samples at 490 nm using a spectrophotometer.
   6. Calculate the melanin content by comparing the sample absorbance to a standard curve of known concentrations of synthetic melanin (Figure 2G).

3. Melanophore count

NOTE: Fluorescence analysis in transgenic Zebrafish embryos can be done by two methods: 1) counting GFP-positive cells; 2) measuring fluorescence intensity.

1. Preparation for FACS-based counting of GFP-positive cells in early zebrafish embryos
   1. Collect 200-250 ftyrp:GFP embryos and wash them in a strainer using plain embryo water. As a negative control, process GFP-negative, stage-matched wild-type (Assam Wildtype) embryos¹².
   2. Based on the stage of interest, transfer the embryos to a Petri dish containing 0.6 mg/mL pronase. After 5-10 min, using a Pasteur pipette, transfer the dechorionated embryos to a fresh Petri dish containing plain embryo water. Using a glass Pasteur pipette, collect ~100 embryos and transfer them to a 2 mL microcentrifuge tube.
   3. Discard the medium carefully and add 200 µL of ice-cold Ringer's solution (deyolking buffer). Keeping the tube on ice, pipette the contents up and down ~20 times until the yolk is dissolved. Centrifuge the tubes at 100 × g for 1 min in a tabletop centrifuge at 4 °C. Centrifuge again and carefully discard the supernatant.
   4. Using a 1 mL pipette, transfer the deyolked embryos to a Petri dish containing 10 mL of trypsin solution (cell dissociation buffer). Perform it in multiple Petri dishes to avoid overcrowding of the embryos and inefficient trypsinization. Use a 1 mL pipette, mix the solution containing the embryo bodies once or twice to decrease aggregation.
   5. Incubate the Petri dishes at room temperature for 15 min (<24 hpf embryos) or 30 min (24-30 hpf embryos). Aspirate and dispense the suspension occasionally using a 1 mL pipette to aid the disintegration of cells.
   6. During the incubation period, initialize the flow cytometer machine for cell counting.
   7. Place a 70 µm cell strainer above a 50 mL conical tube and pass the trypsinized suspension through the strainer to obtain a single-cell suspension. Wash the Petri dishes with the same suspension a few times to remove cells adhered to the surface.
   8. Spin the samples at 450 × g, 4 °C for 5 min in a swinging bucket rotor.
   9. Discard the supernatant and resuspend the pellet in 1mL of ice-cold 1x phosphate-buffered saline (PBS).
   10. Spin again at 450 × g, 4 °C for 5 min and discard the supernatant.
   11. Finally, resuspend the pellet in PBS + 5% fetal bovine serum and keep it on ice until the FACS run.
2. Preparing the flow cytometer
   1. Switch on the flow cytometer and initiate start-up.
      ​NOTE: Before switching on the machine, empty the waste bin and fill the sheath fluid tank.
   2. Normalize the flow of the stream for a 70 µm (or 85 µm) nozzle. Leave it undisturbed for 10-15 min if it is unstable.
3. Counting fluorescently labeled cells using a flow cytometer
   1. Initialize a new experiment folder and make a scatter plot of forward scatter versus side scatter and a histogram of fluorescein isothiocyanate (FITC) intensity.
   2. Load the wild-type cells first to set the forward and side scatter gates (to exclude doublets and debris) and the FITC threshold.
   3. After the gates are set, remove the sample and load the cells isolated from Tg(ftyrp1:GFP) embryos to count melanocytes.
      NOTE: Here, as ftyrp1:GFP specifically labels mature melanocytes, the number of GFP-positive cells under the FITC gate will correspond to the number of melanocytes.
   4. Repeat the above step with morpholino-injected samples to estimate and compare the number of melanocytes with the control.
4. Measuring fluorescence intensity in zebrafish embryos
   1. Using a glass Pasteur pipette, collect 100-120 embryos and transfer them to a Petri dish containing plain embryo water.
   2. At ~10-12 hpf, transfer the embryos to 0.003% 1-phenyl-2-thiourea to inhibit pigmentation.
   3. Perform manual dechorionation using insulin needles for imaging <48 hpf embryos.
   4. To immobilize embryos, treat them with 0.016% tricaine.
   5. To mount embryos for imaging, put a few mL of 1.5-2% methylcellulose in the Petri dish (60 mm) so that it forms a thin layer. Add the embryos to methylcellulose to restrict any further movement during imaging. Place the Petri dish containing the samples under the microscope. Using a pipette tip, adjust the animal in the desired orientation.
   6. Acquire images using the acquisition software. For embryos <24 hpf, capture the whole embryo at once under 10x magnification. For >24 hpf stages, acquire multiple scan-fields and subsequently assemble (stitch) them together.
   7. Repeat this with all the fish and ensure that the setting for aquisition remains constant throughot the experiment.
5. Quantification
   1. Analyze the image further using ImageJ software.
   2. Open the image to be quantified in ImageJ using the Open tool.
   3. Use the freehand shape tool to outline the area for analysis (Figure 3E).
   4. Press M (or Analyze | Measure) to acquire a selected area intensity measurement.
   5. Keeping the area to be analyzed constant, calculate the mean intensity per area for every animal separately.

Results

The workflow described in Figure 1 was used to perform morpholino-based genetic perturbation at the zebrafish one-cell stage. Pigmentation analysis was performed using various methods, as mentioned below. To illustrate the representative results, standardized volumes of antisense morpholino targeting h2afv and ca14 genes were injected in the yolk or one-cell stage of the zebrafish embryo. The initial phenotyping based on brightfield imaging was performed at 48 hours post fe...

Discussion

Pigmentation phenotype is often manifested as alterations in the content of melanin or the number of pigment-bearing melanocytes. The method described herein allows the dissection of this dichotomy and permits qualitative as well as quantitative assessment of melanin content and the number of melanophores per embryo, irrespective of the melanin content. The high fecundity of zebrafish, visible nature of pigmented melanocytes, and lack of melanosome transfer enable the dissection of melanocyte biology using this reverse g...

Materials

Name	Company	Catalog Number	Comments
1.5 mL Microtubes	Axygen	MCT-150-A	For preparing MO solution
2 mL Microtubes	Axygen	MCT-200-C	For washing steps in FACS protocol
Agarose	Sigma-Aldrich	A-9539-500G	For microinjection
BD FACSAria II	BD Biosciences	NA	For cell sorting
Capillary tube	Drummond	1-000-0010
Corning cell strainer	Corning	CLS431751	For making single cell suspension
DMEM High Glucose Media	Sigma-Aldrich	D5648	FACS protocol
Ethyl 3-aminobenzoate methanesulfonate (Tricaine)	Sigma-Aldrich	E10521-50G	to immobilize ZF for imaging
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)	Sigma-Aldrich	E5134	For cold lysis buffer
FACS tubes	BD-Biosciences	342065	FACS protocol
Fetal bovine serum (FBS)	Invitrogen	10270	FACS protocol
Graphpad prism Software	Graphstats Technologies	NA	For data representation
ImageJ Software	National Institute of health	NA	For image analysis
Insulin Syringes (1 mL)	DispoVan	NA	For manual dechorionation
Melanin, Synthetic	Sigma-Aldrich	M8631	For melanin content assay
Methylcellulose	Sigma-Aldrich	M7027-250G	to immobilize ZF for imaging
Microloader tips	Eppendorf	5242956003	For microinjection
Morpholino	Gene-tools	NA	For knock-down experiments
N-Phenylthiourea (PTU)	Sigma-Aldrich	P7629	to inhibit melanin formation
Needle puller	Sutter Instrument	P-97	For microinjection
Nunc 15 mL Conical Sterile Polypropylene Centrifuge Tubes	Thermo Fisher Scientific	339650	FACS protocol
Petridish (60 mm)	Tarsons	460090	For embryo plates
Phenylmethylsulphonyl fluoride	Sigma-Aldrich	10837091001	For cold lysis buffer
Phosphate buffer saline (PBS)	HiMedia	TL1099-500mL	For washing cells
Pronase	Sigma-Aldrich	53702	For dechorionation
Protease inhibitor cocktail	Sigma-Aldrich	P8340	For cold lysis buffer
Sheath fluid	BD FACSFlowTM	342003	FACS protocol
Sodium phosphate	Merck	7558-79-4	Cold lysis buffer
Triton X-100	Sigma-Aldrich	T9284-500ML	For cold lysis buffer
TrypLE	Gibco	1677119	For trypsinization