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>

<ol>
	<li>Kelsh, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+evolution+of+pigment+patterns+in+fish.">Genetics and evolution of pigment patterns in fish.</a> Pigment Cell Research. 17 (4), 326-336 (2004).</li><li>Jablonski, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+human+skin+and+skin+color.">The evolution of human skin and skin color.</a> Annual Review of Anthropology. 33, 585-623 (2004).</li><li>Hoekstra, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics,+development+and+evolution+of+adaptive+pigmentation+in+vertebrates.">Genetics, development and evolution of adaptive pigmentation in vertebrates.</a> Heredity. 97 (3), 222-234 (2006).</li><li>Quigley, I. K., Parichy, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pigment+pattern+formation+in+zebrafish:+a+model+for+developmental+genetics+and+the+evolution+of+form.">Pigment pattern formation in zebrafish: a model for developmental genetics and the evolution of form.</a> Microscopy Research and Technique. 58 (6), 442-455 (2002).</li><li>Meyers, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+development+of+a+vertebrate+model+organism.">Zebrafish: development of a vertebrate model organism.</a> Current Protocols Essential Laboratory Techniques. 16 (1), 19 (2018).</li><li>Kelsh, R. N., Schmid, B., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+melanophore+development+in+zebrafish+embryos.">Genetic analysis of melanophore development in zebrafish embryos.</a> Developmental Biology. 225 (2), 277-293 (2000).</li><li>Rocha, M., Singh, N., Ahsan, K., Beiriger, A., Prince, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+development:+insights+from+the+zebrafish.">Neural crest development: insights from the zebrafish.</a> Developmental Dynamics. 249 (1), 88-111 (2020).</li><li>Carney, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+role+for+Sox10+in+specification+of+neural+crest-derived+sensory+neurons.">A direct role for Sox10 in specification of neural crest-derived sensory neurons.</a> Development. 133 (23), 4619-4630 (2006).</li><li>Curran, K., Raible, D. W., Lister, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxd3+controls+melanophore+specification+in+the+zebrafish+neural+crest+by+regulation+of+Mitf.">Foxd3 controls melanophore specification in the zebrafish neural crest by regulation of Mitf.</a> Developmental Biology. 332 (2), 408-417 (2009).</li><li>Zou, J., Beermann, F., Wang, J., Kawakami, K., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fugu+tyrp1+promoter+directs+specific+GFP+expression+in+zebrafish:+tools+to+study+the+RPE+and+the+neural+crest-derived+melanophores.">The Fugu tyrp1 promoter directs specific GFP expression in zebrafish: tools to study the RPE and the neural crest-derived melanophores.</a> Pigment Cell Research. 19 (6), 615-627 (2006).</li><li>Xu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+into+zebrafish+embryos.">Microinjection into zebrafish embryos.</a> Methods in Molecular Biology. 127, 125-132 (1999).</li><li>Hermanson, S., Davidson, A. E., Sivasubbu, S., Balciunas, D., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleeping+Beauty+transposon+for+efficient+gene+delivery.">Sleeping Beauty transposon for efficient gene delivery.</a> Methods in Cell Biology. 77, 349-362 (2004).</li><li>Stainier, D. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+morpholino+use+in+zebrafish.">Guidelines for morpholino use in zebrafish.</a> PLoS Genetics. 13 (10), 1007000 (2017).</li><li>Wu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+progress+of+CRISPR/Cas9-mediated+gene+editing+in+generating+mouse/zebrafish+models+of+human+skeletal+diseases.">The progress of CRISPR/Cas9-mediated gene editing in generating mouse/zebrafish models of human skeletal diseases.</a> Computational and Structural Biotechnology Journal. 17, 954-962 (2019).</li><li>Affenzeller, S., Frauendorf, H., Licha, T., Jackson, D. J., Wolkenstein, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+eumelanin+and+pheomelanin+markers+in+diverse+biological+samples+by+HPLC-UV-MS+following+solid-phase+extraction.">Quantitation of eumelanin and pheomelanin markers in diverse biological samples by HPLC-UV-MS following solid-phase extraction.</a> PLoS One. 14 (10), 0223552 (2019).</li><li>Fernandes, B., Matam&#225;, T., Guimar&#227;es, D., Gomes, A., Cavaco-Paulo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+quantification+of+melanin.">Fluorescent quantification of melanin.</a> Pigment Cell &amp; Melanoma Research. 29 (6), 707-712 (2016).</li><li>Hultman, K. A., Johnson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+direct-developing+and+stem+cell-derived+melanocytes+to+the+zebrafish+larval+pigment+pattern.">Differential contribution of direct-developing and stem cell-derived melanocytes to the zebrafish larval pigment pattern.</a> Developmental Biology. 337 (2), 425-431 (2010).</li><li>Mort, R. L., Jackson, I. J., Patton, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+melanocyte+lineage+in+development+and+disease.">The melanocyte lineage in development and disease.</a> Development. 142 (4), 620-632 (2015).</li></ol>

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 g...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 FACSFlowTM</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 fe...

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 ...

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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

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

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 ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.