We acknowledge the funding support from the Council for Scientific and Industrial Research vide project MLP2008 and the Department of Science and Technology for the project GAP165 for supporting the work presented in this manuscript. We thank Jeyashri Rengaraju and Chetan Mishra for their help with experiments.

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
<ol>
	<li>Using a standard needle puller, draw very sharp and closed-tipped pipets.</li>
	<li>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 &#62;70% and a specific phenotype at 24 hpf.</li>
	<li>Cut the tip of the micropipette using fine forceps and calibrate it11.</li>
	<li>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. 
	&#8203;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 &#176;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.</li>
	<li>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.</li>
	<li>Under a microscope, use manipulators to bring the micropipette closer to the embryo and inject inside the yolk-cell interface by pressing the footswitch.</li>
	<li>Inject all the embryos similarly; collect them in a Petri dish containing fresh embryo water medium and incubate at 28 &#176;C.</li>
	<li>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.</li>
</ol>
2. Pigmentation analysis
<ol>
	<li>Preparation for counting lateral midline melanophores in 3 dpf zebrafish embryos
	<ol>
		<li>Treat&#160;embryos with 0.016% neuro-muscular anesthetic to immobilize them.</li>
		<li>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.</li>
		<li>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 (&#62;5x)</li>
	</ol>
	</li>
	<li>Brightfield imaging 
	NOTE: Brightfield imaging is performed using a stereomicroscope with 8-10x magnification.
	<ol>
		<li>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.</li>
		<li>Repeat this with all the fish&#160;and ensure that magnification remains the same for every image.</li>
	</ol>
	</li>
	<li>Calculating mean gray value using ImageJ software
	<ol>
		<li>Take dorsal and lateral images of 2 dpf zebrafish embryos.</li>
		<li>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.</li>
		<li>Keeping the area to be analyzed constant, calculate the mean grey area for every animal separately.</li>
		<li>Plot a bar graph with the acquired data (Figure 2F).</li>
	</ol>
	</li>
	<li>Melanin content assay
	<ol>
		<li>At 2 dpf, use a glass Pasteur pipette to collect ~25 zebrafish embryos.</li>
		<li>Perform manual dechorionation using 1 mL insulin needles and add them to 1.5 mL microcentrifuge tubes.</li>
		<li>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.</li>
		<li>Dissolve the lysates in 1 mL of 1 N NaOH and incubate the samples at 100 &#176;C for 50 min in a water bath. Vortex it intermittently to homogenize the lysates completely.</li>
		<li>Take absorbance readings of the samples at 490 nm using a spectrophotometer.</li>
		<li>Calculate the melanin content by comparing the sample absorbance to a standard curve of known concentrations of synthetic melanin (Figure 2G).</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Preparation for FACS-based counting of GFP-positive cells in early zebrafish embryos
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Discard the medium carefully and add 200 &#181;L of ice-cold Ringer&#39;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 &#215; g for 1 min in a tabletop centrifuge at 4 &#176;C. Centrifuge again and carefully discard the supernatant.</li>
		<li>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.</li>
		<li>Incubate the Petri dishes at room temperature for 15 min (&#60;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.</li>
		<li>During the incubation period, initialize the flow cytometer machine for cell counting.</li>
		<li>Place a 70 &#181;m 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.</li>
		<li>Spin the samples at 450 &#215; g, 4 &#176;C for 5 min in a swinging bucket rotor.</li>
		<li>Discard the supernatant and resuspend the pellet in 1mL of ice-cold 1x phosphate-buffered saline (PBS).</li>
		<li>Spin again at 450 &#215; g, 4 &#176;C for 5 min and discard the supernatant.</li>
		<li>Finally, resuspend the pellet in PBS + 5% fetal bovine serum and keep it on ice until the FACS run.</li>
	</ol>
	</li>
	<li>Preparing the flow cytometer
	<ol>
		<li>Switch on the flow cytometer and initiate start-up. 
		&#8203;NOTE: Before switching on the machine, empty the waste bin and fill the sheath fluid tank.</li>
		<li>Normalize the flow of the stream for a 70 &#181;m (or 85 &#181;m) nozzle. Leave it undisturbed for 10-15 min if it is unstable.</li>
	</ol>
	</li>
	<li>Counting fluorescently labeled cells using a flow cytometer
	<ol>
		<li>Initialize a new experiment folder and make a scatter plot of forward scatter versus side scatter and a histogram of fluorescein isothiocyanate (FITC) intensity.</li>
		<li>Load the wild-type cells first to set the forward and side scatter gates (to exclude doublets and debris) and the FITC threshold.</li>
		<li>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.</li>
		<li>Repeat the above step with morpholino-injected samples to estimate and compare the number of melanocytes with the control.</li>
	</ol>
	</li>
	<li>Measuring fluorescence intensity in zebrafish embryos
	<ol>
		<li>Using a glass Pasteur pipette, collect 100-120 embryos and transfer them to a Petri dish containing plain embryo water.</li>
		<li>At ~10-12 hpf, transfer the embryos to 0.003% 1-phenyl-2-thiourea to inhibit pigmentation.</li>
		<li>Perform manual dechorionation using insulin needles for imaging &#60;48 hpf embryos.</li>
		<li>To immobilize embryos, treat them with 0.016% tricaine.</li>
		<li>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.</li>
		<li>Acquire images using the acquisition software. For embryos &#60;24 hpf, capture the whole embryo at once under 10x magnification. For &#62;24 hpf stages, acquire multiple scan-fields and subsequently assemble (stitch) them together.</li>
		<li>Repeat this with all the fish and ensure that the setting for aquisition remains constant throughot the experiment.</li>
	</ol>
	</li>
	<li>Quantification
	<ol>
		<li>Analyze the image further using ImageJ software.</li>
		<li>Open the image to be quantified in ImageJ using the Open tool.</li>
		<li>Use the freehand shape tool to outline the area for analysis (Figure 3E).</li>
		<li>Press M (or Analyze | Measure) to acquire a selected area intensity measurement.</li>
		<li>Keeping the area to be analyzed constant, calculate the mean intensity per area for every animal separately.</li>
	</ol>
	</li>
</ol>

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All authors declare no conflict of interest.

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 genetic approach that is amenable to medium-throughput screening.
The choice of morpholino-based silencing emanates from the ease of silencing and the ability to test multiple candidates parallelly. There are certain limitations of using a morpholino-based silencing approach, and guidelines for using and interpreting these valuable tools have been described previously13. Essentially, the validation of phenotype upon knockdown using multiple MOs provides a tangible solution to this problem. An alternate strategy is the use of CRISPR-based genetic perturbation. The penetrance of the phenotype is limited due to the inherently lower frequency of genetic events and could be compounded by heterogeneous mutations. That limits its use for reverse genetic screening14.
Phenotype assessment of melanocyte-selective perturbations often results in decreased pigment content due to altered melanogenic response. However, there are several genes that perturb the specification of melanocytes and hence cause a reduction in pigmentation due to a decrease in overall melanocyte numbers. A systematic dissection of the two scenarios is essential to ascribe the molecular function of the gene in pigmentation. The measurement of melanin content is compounded by the complex nature of melanin polymer. Of the two available methods, namely melanin solubilization by NaOH and HPLC-based detection of stable melanin oxidation products 1H-pyrrole-2,3,5-tricarboxylic acid&#160;and pyrrole-2,3- dicarboxylic acid, the former method can be routinely performed in a laboratory setting while the latter requires specific standards and sophisticated instrumentation15.
Additionally, the number of embryos required by the NaOH method is also higher due to several steps involved in the detection. NaOH-based hydrolysis solubilizes melanin and enables spectrophotometric quantitation. While this method is robust and can be adopted for a medium-throughput screen (10-15 candidates at a time), xanthine present in xanthophores is a potential interfering substance due to its inherent absorption in the 400-450 nm range16. Hence, a concordance between image analysis-based mean grey value determination and melanin content assay must be carried out to confirm the reduction in the melanin content.
PTU offers an easy tool to reduce the pigmentation and visualize the otherwise transparent embryo. The use could possibly result in feedback changes and invoke alterations in melanocytes. However, we have observed minimal changes in the expression of pigmentation genes (Supplemental Figure S2).
The counting of melanocytes is easiest in the lateral mid line and the head region, as the melanocytes are relatively distinctly observable in these regions (Supplemental Figure S1). Lateral line melanophores arise from the melanocyte stem cell population that resides close to the dorsal root ganglia18. Imaging this population has several advantages such as fixed number per embryo and clear distinction. However, the two contralateral lines need to be imaged by tilting the embryo slightly; else the two merge due to the transparent nature of the embryo, making it difficult to count (Figure 1C versus Figure 2C).
Zebrafish melanophores are distinct from the pigmented cells of the eye that are derived from neuro-ectoderm. These are distinguishable by their positioning within the embryo and are evident in the eye versus the body pattern. In the FACS-based experiment, the eye-derived cells can be distinguished by their size as they are significantly smaller than the melanophores and can be gated using size or scatter criteria.
Estimates of melanocytes at different maturation stages using transgenic lines are easily carried out using image-based quantitation. Quantitation of the number of lateral-line melanophores is robust as their number varies in a window between 2 dpf and 5 dpf on each side of the embryo17. However, the number of lateral-line melanophores is determined by the migration and establishment of a dorsal root ganglion-associated stem cell pool18. Hence, any alterations in these processes can lead to a similar observation. To circumvent this confounding factor, FACS-based estimation of all melanophores is a tangible solution.
Alternately, quantitation of head melanophores can be performed as delineated in Supplemental Figure S1 as a surrogate for melanophore numbers. It is interesting to note that the alterations in steady-state levels of melanocyte numbers could signify two opposite functions for the candidate gene in question. It could either result in decreased specification of melanocytes from neural-crest precursors or cause melanocyte-specific death and need to be addressed using methods such as the tunnel assay. Variations such as microinjections followed by live-cell imaging would enable monitoring other melanocyte-specific phenotypes such as migration and patterning. Of interest to pigment-cell researchers would be the process of melanosome transfer, which is not operative in the fish system. However, concerted melanosome movement in melanophores can be studied with alterations in live-cell imaging described herein with modifications. In all, the detailed protocol presented in this video article would enable pigment cell biology researchers to query candidate genes and assess their role in melanocyte biology.

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 hpf6,7. 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.

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

reverse genetic approach to identify regulators of pigmentation using zebrafish

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.

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 fertilization (hpf), when all the five pigmented melanophore stripes (dorsal, ventral, yolk, two lateral) were evident.
Figure 2A,B represent an approach to analyze pigmentation by calculating the number of lateral melanophores per embryo. Lateral melanophores were manually counted at the 48 hpf stage by zooming in on the lateral region of the tilted zebrafish. An alternative method to quantify the pigmented melanophores (Supplemental Figure S1) involves the manual counting of the head melanophores by imaging the dorsal view of the zebrafish.
The melanin content per embryo is calculated by measuring the mean gray value, keeping the region of interest constant with the help of ImageJ software. Figure 2C-F show that the mean gray value of ca14 and h2afv morphants was higher than in control morphants. As the mean gray value is inversely proportional to melanin content, knockdown of ca14 and h2afv genes led to a decrease in melanin content per embryo. Another robust method to quantify the melanin content is a NaOH-based spectrophotometric absorption method. As shown in Figure 2G, the total melanin content in ca14&#160;morphants was less than in control morphants.
Fluorescent imaging revealed two different stages of zebrafish melanophore development: early specified melanophores (mitfa:gfp) and differentiating melanophores (ftyrp:gfp). The morpholino-based alteration was evaluated by fluorescent imaging (Figure 3A and Figure 3C). To further validate these observations, the frequency of GFP-positive cells was calculated using FACS. The knockdown of h2afv but not ca14 significantly reduced the number of melanophores with respect to control(Figure 3A-D). Moreover, a reduction in the mean fluorescence intensity/area can be calculated using ImageJ software, keeping the region of interest constant. H2afv morphants show a significant decrease in the mean fluorescent intensity/area relative to control morphants (Figure 3E,F).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62955/62955fig01v2.jpg" /> 
Figure 1: Workflow for a reverse genetic approach to identify regulators of melanocyte biology using zebrafish. (A) Flowchart depicting the experimental workflow starting from microinjection to evaluating pigmentation by various methods. (B) Microinjection needle holder and micromanipulator, along with a dissecting microscope, is a typical setup for microinjection of one-cell stage zebrafish embryos. (C) Embryonic melanocyte patterning in zebrafish at 3 dpf showing all five stripes: dorsolateral, two lateral midlines (pointed by four red arrows), ventral stripe, and yolk stripe. (D) To study the pigmentation outcome upon morpholino injection, lateral midline melanophores can be counted under a stereomicroscope.&#160;Brightfield image of a dorsal region at 2 dpf; melanin content can be quantified by measuring mean grey value. (E) The mean gray values are inversely proportional to the melanin content of the embryo. Melanin-filled melanophores at 2 dpf in wild-type embryos. (F) Melanin content assay can be performed to estimate the melanin production within these melanophores. Transgenic zebrafish labeling the cells expressing TYRP1 protein at 2 dpf.&#160;(G)&#160;Fluorescently labeled cells can be quantified using FACS; scale bar = 100 &#181;m. Transgenic zebrafish labeling the cells expressing Mitfa protein at 1 dpf. Meanfluorescence intensity per animal can be measured using ImageJ software. Scale bars = 100 &#181;m. Abbreviations: dpf = days post fertilization; FACS = fluorescence-activated cell sorting. <a href="https://www.jove.com/files/ftp_upload/62955/62955fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62955/62955fig02v2.jpg" /> 
Figure 2: Counting lateral midline melanophores, measuring mean gray value, and melanin content assay. Brightfield microscopic images of the lateral view of morphants were used to quantify melanin content using ImageJ software. (A) Control and Histone variant h2afv (h2afv) morphants at 3 dpf; on the right are zoomed-in trunk regions of these embryos depicting melanocyte numbers. (B) The number of melanophores in h2afv morphants is drastically reduced relative to control, n &#62; 10 each in 3 biological replicates. (C) Representative image of control vs carbonic anhydrase 14 morphants at 2 dpf. (D) Melanin quantification of ca14 vs control morphants, n &#62; 10 across 3 biological replicates. (E) Lateral view of h2afv vs control morphants at 2 dpf. (F) Melanin content quantification of h2afv vs control morphants. Red rectangles represent ROI chosen for ImageJ-based analysis. (G) Melanin content assay performed on morpholino-injected embryos for quantifying the total melanin content. Mean of three independent experiments &#177; SEM.; **P &#60; 0.01, **** P &#60; 0.0001. Student&#39;s t-test; error bars are mean &#177; standard error of the mean (SEM). Scale bars = 100 &#181;m. Abbreviations: MO = morpholino; dpf = days post fertilization; ROI = region of interest. <a href="https://www.jove.com/files/ftp_upload/62955/62955fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62955/62955fig03v2.jpg" /> 
Figure 3: Quantifying fluorescently labeled cells using FACS and measuring mean fluorescence intensity. (A) Fluorescence images of ca14 and control morphant embryos at 2 dpf from Tg (ftyrp1: GFP) line, where the differentiating melanocytes are marked by GFP expression. (B) Number of melanophores in ca14 and control morphant embryos at 2 dpf remain unchanged, n = 3 biological replicates. (C) Fluorescence images at 2 dpf of h2afv and control morphants from Tg(ftyrp1:GFP) line. (D) The number of melanophores in h2afv morphants is significantly reduced compared to the control morphant embryos at 2 dpf, n = 3 biological replicates. (E) Fluorescence images of control and h2afv morphant embryos at 36 hpf from Tg(mitfa:GFP) with labeled melanocytes. (F) MFI per animal is calculated using ImageJ software and represented as a bar graph depicting individual data sets. MFI is significantly reduced in h2afv morphants compared to control. Red rectangle represents ROI chosen for ImageJ software-based analysis. n = 3 biological replicates; ***P &#60; 0.001, ****P &#60; 0.0001. Student&#39;s t-test; error bars are mean &#177; standard error of the mean (SEM); scale bars = 100 &#181;m. Abbreviations: MO = morpholino; FACS = fluorescence-activated cell sorting; GFP = green fluorescent protein; dpf = days post fertilization; ROI = region of interest; MFI = mean fluorescence intensity. <a href="https://www.jove.com/files/ftp_upload/62955/62955fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure S1: Quantitation of head melanophores. (A)&#160;Dorsal view of h2afv and control morphants at 48 hpf showing head melanophores. Red box represents the ROI. Keeping the area of interest constant, the number of head melanophores can be quantified manually. (B) Number of head melanophores significantly reduced upon h2afv knockdown. Bars represent geometric mean with 95% CI of the number of head melanophores per embryo with &#62;30 embryos each. Scale bars = 100 &#181;m. Abbreviations: MO = morpholino; CI = confidence interval; hpf = hours post fertilization; ROI = region of interest. <a href="https://www.jove.com/files/ftp_upload/62955/Supplementary Figure.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure S2: Microarray of human primary melanocytes treated with PTU. Heat map shows the expression of pigmentation genes (mitf, tyrosinase, dct, mc1r) upon treatment in human primary melanocytes. Abbreviations: PTU = 1-phenyl-2-thiourea; tyr = tyrosinase. <a href="https://www.jove.com/files/ftp_upload/62955/supplementary figure 2..pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish at JoVE.com

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

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.

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from ...

reverse-genetic-approach-to-identify-regulators-pigmentation-using

Nanyang Technological University

Research

JoVE Journal

Biology

2.2K Views.  CSIR-Institute of Genomics and Integrative Biology. 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. 

Video: Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

Genetics

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Developmental Biology

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Medicine

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

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

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

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

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Summary

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