Research support was provided by a UCR Graduate Division Fellowship to SAB, a NRSA T32 Training Program Fellowship (T32ES018827) to SAB, and a National Institutes of Health grant (R01ES027576) and USDA National Institute of Food and Agriculture Hatch Project (1009609) to DCV.

Adult breeders were handled and treated in accordance with an Institutional Animal Care and Use Committee (IACUC)-approved animal use protocol (#20180063) at the University of California, Riverside.
1. Zebrafish embryo collection and chemical exposure
<ol>
	<li>Add in-tank breeding traps to tanks containing sexually mature and reproductively viable adult male and female zebrafish. Add at least three traps per 6 L tank at least 12 h prior to collection at ~9:00 AM, which is the approximate time of fertilization and spawning of eggs within tanks.</li>
	<li>Prepare a fresh exposure solution on the morning of collection. The exposure solution is prepared using a 5 mL pipette and adding 5.0 mL of particulate-free system water to a 60 mm glass Petri dish.</li>
	<li>Then, use a 10 &#181;L glass microcapillary pipette to transfer 10 &#181;L of vehicle (dimethyl sulfoxide, or DMSO) or chemical stock solution to system water in the glass dish.</li>
	<li>Swirl the glass Petri dish to ensure the stock solution dissipates. Add another 5.0 mL of system water to the glass Petri dish. Swirl the glass Petri dish to homogenize the exposure solution.</li>
	<li>Repeat for each treatment group in replicates of four to yield four replicates per treatment.</li>
	<li>After exposure solutions have been prepared, cap the glass dishes with glass lids to minimize evaporation.</li>
	<li>While removing the breeding traps from each tank, carefully allow the water within each trap to drain out into the tank before removing from the tanks. Make sure the traps stay right-side up.</li>
	<li>Transfer the breeding traps to the lab sink and rinse them using a squirt bottle filled with reverse osmosis (RO) water into a fish net previously soaked in 10% bleach and rinsed with water. Rinse the embryos until all debris is cleared and only clean embryos remain.</li>
	<li>Transfer the embryos from the fish net using an RO water-filled squirt bottle into a 100 mm plastic Petri dish. Randomly sort 50 live embryos at 0.75 h post-fertilization (hpf) and remove excess RO water.</li>
	<li>Using a microspatula, place sorted embryos into the treatment solution. All embryos must be within vehicle or treatment solution no earlier than 9:45 AM. Swirl the glass dishes to ensure the embryos are evenly coated within the treatment solution.</li>
	<li>After four replicate dishes (N = 50 per replicate) have been set up for each treatment group, place the glass lid on top of the exposure dishes and transfer all dishes into a temperature-controlled incubator set at 28 &#176;C until 2 hpf, 4 hpf, 6hpf, 8 hpf, or 10 hpf.</li>
	<li>Prepare fresh 4% paraformaldehyde (PFA) at least 4 h prior to exposure termination and store at 4 &#176;C.
	<ol>
		<li>Turn on the hotplate to 115 &#176;C, make 20 mL of 1x phosphate-buffered saline (PBS) (2 mL of 10x PBS + 18 mL of RO water) in a 50 mL centrifuge tube, and weight out 800 mg of paraformaldehyde.</li>
		<li>Within a chemical fume hood, transfer 1x PBS solution to a 250 mL Erlenmeyer flask, add 800 mg of paraformaldehyde, and then add 2 &#181;L of 10 N NaOH. Place a thermometer inside the flask, place the flask on the hot plate, and watch the temperature while stirring.</li>
		<li>When the temperature reaches 62-65 &#176;Cremove the 4% PFA from the hot plate and allow it to cool to room temperature (RT). Store the 4% PFA at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Once the exposure duration has ended, remove the embryos from the incubator and transfer all living embryos to a 1.5 mL microcentrifuge tube. Aspirate residual exposure solution using a 1 mL pipette. Do not aspirate any embryos.</li>
	<li>Add 500 &#181;L of chilled 4% PFA to a 1.5 mL microcentrifuge tube and mix the embryos in 4% PFA by inverting several times. Allow the embryos to fix in 4% PFA overnight. 
	&#8203;NOTE: It is not recommended to allow embryos to stay in PFA for longer than 12 h.</li>
</ol>
2. Dechorionation of embryos
<ol>
	<li>The following day, aspirate off the 4% PFA, resuspend the embryos in 1x PBS, and gently pipette up and down for 30 s. If needed, stop the protocol at this step and store the embryos in 1x PBS at 4 &#176;C for up to 48 h.</li>
	<li>Using a plastic transfer pipette, carefully transfer fixed embryos to a RO water-filled glass dish and gently swirl for 30 s. Repeat this step one more time.</li>
	<li>Use syringe needles under 5.0x magnification using a standard stereomicroscope to manually dechorionate all embryos. Do this by using the needle tip to puncture the chorion and gently peel it away from the yolk and cell mass4.</li>
	<li>Once the embryos have been dechorionated, use a glass microcapillary pipette to transfer up to 25 intact embryos into one immunochemistry (IHC) basket and place the IHC basket into a 96-well plate. Use a 1 mL pipette to aspirate the RO water from each well.</li>
</ol>
3. Immunohistochemistry using 5-mC-specific antibody
<ol>
	<li>Resuspend the embryos in 500 &#181;L of blocking buffer (1x PBST + 2% sheep serum + 2 mg/mL bovine serum albumin) per well, and wrap the plate with parafilm and aluminum foil to protect them from light. Incubate at 4 &#176;C for 4 h on an orbital shaker (100 rpm).</li>
	<li>Remove the parafilm wrap and aluminum and use a 1 mL pipette to aspirate the blocking buffer from each well. Replace it with 500 &#181;L of a 1:100 dilution of monoclonal mouse anti-5-mC antibody in the blocking buffer. Incubate all embryos with primary antibodies except for a subset of vehicle control embryos which will be incubated with the blocking buffer to account for background noise. Be careful not to disrupt the integrity of embryos, nor to aspirate embryos during this step.</li>
	<li>Rewrap the plate in parafilm and aluminum foil and allow the plate to incubate at 4 &#176;C overnight on an orbital shaker (100 rpm).</li>
	<li>The following day, remove the primary antibody solution from each well and replace it with 1x PBS + 0.1% Tween-20 (1x PBST). Wash each well three times with 1x PBST for 15 min per wash on an orbital shaker (100 rpm).</li>
	<li>Replace 1x PBST with a 1:500 dilution of goat anti-mouse IgG antibody in blocking buffer and incubate all embryos with the secondary antibody. Let the plate incubate at 4 &#176;C overnight on an orbital shaker (100 rpm).</li>
	<li>The following day, remove the secondary antibody and wash the residual solution three times with 1x PBST for 15 min per wash on an orbital shaker (100 rpm). 
	NOTE: The protocol can be stopped here for up to 48 h if the IHC baskets are stored in clean wells filled with 1x PBS.</li>
	<li>Use a 1 mL pipette to aspirate 1x PBST from each well and place the IHC basket into a glass Petri dish filled with RO water. Under a standard stereomicroscope, randomly sort intact embryos into a 96-well plate, resulting in one embryo per well.</li>
	<li>Using a 1 mL pipette, remove all RO water from individual wells and replace it with 200 &#181;L of 1x PBS. Centrifuge the plate for 3 min at 1 x g.</li>
</ol>
4. Automated imaging of embryos within 96-well plates
<ol>
	<li>Image the embryos with a 2x objective under transmitted light and FITC using a high-content screening system (Table of Materials). 
	NOTE: A fluorescent image of each embryo was captured automatically using the above-mentioned magnification and a FITC filter19.
	<ol>
		<li>Select Autofocus to ensure the focus of the camera is on the bottom of the plate and offset by the bottom thickness.</li>
		<li>Select a FITC wavelength with an exposure duration of 100 ms, and set the Autofocus to laser with a Z-offset. Observe and confirm proper image acquisition in several wells prior to commencing plate acquisition. 
		NOTE: The instrument automatically acquires images from all selected wells containing embryos. A 96-well plate required approximately 30 min for image acquisition and data storage. For the duration of the acquisition period, the internal temperature within the imaging system was maintained at RT.</li>
	</ol>
	</li>
	<li>Following image acquisition, carry out data analysis using the high-content screening system and a custom automated image analysis procedure. Select each embryo on the 96-well plate to be analyzed for total area and integrated intensity of fluorescence. 
	NOTE: The analysis included providing images that contained overlays of data points from the analysis.</li>
	<li>Then, tabulate the data points and export them from the high-content screening system by going under Measure and selecting Open Data Log to a spreadsheet. The plate acquisition and data analysis portions are depicted in Figure 1.</li>
</ol>
5.&#160;Data analysis
<ol>
	<li>Upload the exported spreadsheet into a computer program where the deployer (dplyr) package is used to sort, summate data for each well, and filter by well number. The writexl package is then used to export the summated data into a spreadsheet. The 5mC program code is shown in Supplemental File 1.</li>
</ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+existence+of+5-hydroxymethylcytosine+and+5-formylcytosine+in+both+DNA+and+RNA+in+mammals.">The existence of 5-hydroxymethylcytosine and 5-formylcytosine in both DNA and RNA in mammals.</a> Chemical Communications. 52 (4), 737-740 (2016).</li><li>Huang, W., 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=Formation+and+determination+of+the+oxidation+products+of+5-methylcytosine+in+RNA.">Formation and determination of the oxidation products of 5-methylcytosine in RNA.</a> Chemical Science. 7 (8), 5495-5502 (2016).</li><li>Avila-Barnard, S., 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=Tris(1,3-dichloro-2-propyl)+phosphate+disrupts+the+trajectory+of+cytosine+methylation+within+developing+zebrafish+embryos.">Tris(1,3-dichloro-2-propyl) phosphate disrupts the trajectory of cytosine methylation within developing zebrafish embryos.</a> Environmental Research. 211, 113078 (2022).</li><li>Dasgupta, S., Cheng, V., Volz, D. 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M., Muegge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+in+early+development.">DNA methylation in early development.</a> Molecular Reproduction and Development. 77 (2), 105-113 (2010).</li><li>Wossidlo, M., 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=5-Hydroxymethylcytosine+in+the+mammalian+zygote+is+linked+with+epigenetic+reprogramming.">5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming.</a> Nature Communications. 2 (1), 1-8 (2011).</li><li>Rottach, A., Leonhardt, H., Spada, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation-mediated+epigenetic+control.">DNA methylation-mediated epigenetic control.</a> Journal of Cellular Biochemistry. 108 (1), 43-51 (2009).</li><li>Egger, G., Liang, G., Aparicio, A., Jones, P. 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V. C., Bourc'his, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diverse+roles+of+DNA+methylation+in+mammalian+development+and+disease.">The diverse roles of DNA methylation in mammalian development and disease.</a> Nature Reviews Molecular Cell Biology. 20 (10), 590-607 (2019).</li><li>Yuan, B. -. F., Feng, Y. -. 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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

During this protocol, there are a few steps that are critical. First, when dechorionating embryos, it is important to point the needle away from the tissue of the embryo/yolk sac/cell mass, as these portions of the developing embryo are very fragile and easy to puncture. Second, when transferring labeled embryos to individual wells, use a glass pipette to transfer embryos as they will adhere to a plastic pipette. Third, when performing whole-mount IHC, ensure that the plate is protected from light. Finally, after completing the whole-mount IHC protocol, allow the plate to incubate in 1x PBS at 4 &#176;C overnight before imaging, as this will minimize autofluorescence that may interfere with imaging. 
If there are embryos that have been severed during dechorionation or IHC, exclude these embryos from the remainder of the protocol. If no fluorescence is detected, this may be solved by incubating for longer (up to 16 h). Since this is a 5-mC-specific antibody, both DNA and RNA may be labeled. A general understanding of spatial localization is important. 
There are some limitations in this method. First, it is a non-targeted technique, meaning it will not provide the exact quantity of 5-mC, but rather the relative abundance based on the total area and integrated intensity of fluorescence. In addition, this protocol has only been tested on embryos prior to segmentation, so if testing later stages of development, some additional optimization may be needed. Furthermore, since the method is IHC-based, it may be susceptible to non-specific binding; therefore, it is not certain to only be staining 5-mC localized to DNA, but may also be staining 5-mC localized to RNA as well. Lastly, optimization of the data analysis threshold may be needed depending on the chemical and stringency of the conditions that are preferred. 
Overall, this method provides quick and cost-efficient detection of 5-mC across multiple stages of development and chemical concentrations3. It therefore provides an alternative to cost-prohibitive bisulfite sequencing-based approaches. By offering this protocol, investigators can use this method to quickly screen chemicals and assess how the abundance of 5-mC may be affected during early embryonic development. In addition, this method may be utilized as a prescreening tool to identify the concentration range, period of development, and/or window of sensitivity in which the chemical of interest affects 5-mC abundance. Alternatively, this same method may be utilized for a different biomarker and antibody, albeit further optimization is needed. By utilizing this method, an investigator can quickly, efficiently, and cost-effectively screen and identify a chemical that alters the relative abundance of 5-mC within zebrafish embryos prior to investing in labor-intensive bisulfite sequencing-based approaches. However, this method is zebrafish-specific, and further research is needed to determine whether 5-mC can be detected in situ within early embryos of other model organisms.

Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine1,2. Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a widely used flame retardant in the United States that has been previously demonstrated to alter the trajectory of cytosine methylation following early embryonic exposure from 0.75 hours post-fertilization (hpf) through early gastrulation (6 hpf)3,4,5,6,7,8. Within vertebrates, 5-mC and its modified derivatives are critical for regulating early embryonic development9. Fertilization of an embryo triggers demethylation of parental DNA, followed by maternal mRNA degradation, zygotic genome activation, and remethylation of the zygotic genome9. Biologically relevant processes that utilize cytosine methylation include histone modification, recruitment of transcriptional machinery, RNA methylation, epigenetic reprogramming, and determination of chromatin structure10,11. Cytosine methylation is also conserved among vertebrate species, underscoring the importance of understanding and investigating how aberrant cytosine methylation may affect the trajectory of an organism&#39;s development11. Furthermore, in utero development is sensitive to maternal exposure and has the potential to cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks12,13,14.
Long stretches of cytosine-guanine pairs, or CpG islands, have been the primary foci of investigators that aim to characterize the dynamics of cytosine methylation across the genome15,16,17. Bisulfite-based strategies such as whole-genome bisulfite sequencing, reduced representation bisulfite sequencing, and bisulfite amplicon sequencing represent the gold standard for interrogating cytosine methylation at base-pair resolution. However, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. In addition, sequencing-based approaches do not provide information about spatial localization, which is critical for understanding potentially affected cell types and areas within a developing embryo. Similarly, global DNA methylation assays such as methylation-dependent restriction analysis, 5-mC enzyme-linked immunoassays (ELISAs), and 5-methyl-2&#39;-deoxycytidine (5-mC) liquid chromatography-mass spectrometry (LC-MS) rely on cell or tissue homogenates and, as such, preclude the ability to monitor the localization and magnitude of cytosine methylation over space and time within intact specimens12,18.
Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be widely used as physiologically intact models to uncover xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available antibodies specific to 5-mC, the protocol below describes a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry (IHC), automated high-content imaging, and efficient data processing using programming language prior to statistical analysis.
To current knowledge, this method is the first to monitor 5-mC within intact zebrafish embryos. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

<table><tbody><tr><td>1.5-mL microcentrifuge tubes</td><td>Fisher Scientific</td><td>540225</td><td></td></tr><tr><td>10-&amp;micro;L glass microcapillary pipette</td><td>Fisher Scientific</td><td>211762B</td><td></td></tr><tr><td>100-mm plastic Petri dish</td><td>Fisher Scientific</td><td>08757100D</td><td></td></tr><tr><td>10x phosphate-buffered saline&amp;nbsp;</td><td>Fisher Scientific</td><td>BP399500</td><td></td></tr><tr><td>1-mL pipette&amp;nbsp;</td><td>Fisher Scientific</td><td>13690032</td><td></td></tr><tr><td>250-mL Erlenmeyer flask</td><td>Fisher Scientific</td><td>FB501250</td><td></td></tr><tr><td>5-mL pipette</td><td>Fisher Scientific</td><td>13690033</td><td></td></tr><tr><td>60-mm glass petri dishes with lids</td><td>Fisher Scientific</td><td>08747A</td><td></td></tr><tr><td>96-well plate</td><td>Fisher Scientific</td><td>720089</td><td></td></tr><tr><td>AlexaFluor 488-conjugated goat anti-mouse IgG antibody&amp;nbsp;</td><td>Fisher Scientific</td><td>A21121</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Fisher Scientific</td><td>BP67110</td><td></td></tr><tr><td>DMSO</td><td>Fisher Scientific</td><td>BP2311</td><td></td></tr><tr><td>Hotplate&amp;nbsp;</td><td>Fisher Scientific</td><td>1110016SH</td><td></td></tr><tr><td>In-tank breeding traps</td><td>Aquatic Habitats</td><td>N/A</td><td>This product is no longer available following acquisition of Aquatic Habitats by Pentair.&amp;nbsp; Investigators can use standard off-system breeding tanks available from multiple vendors.</td></tr><tr><td>ImageXpress Micro XLS Widefield High-Content Screening System</td><td>Molecular Devices</td><td>N/A</td><td>Any high-content screening system equipped with transmitted light and FITC filter will be suitable.</td></tr><tr><td>Immunochemistry (IHC) basket</td><td>N/A</td><td>N/A</td><td>Manufactured in-house using microcentrifuge tubes with conical portion removed and bottom fitted with mesh, sized for 24- or 48-well plates.</td></tr><tr><td>MetaXpress 6.0.3.1658&amp;nbsp;</td><td>Molecular Devices</td><td>N/A</td><td>Any software capable of quantifying total area and integrated intensity of fluorescence will be suitable.</td></tr><tr><td>Microspatula</td><td>Fisher Scientific</td><td>2140115</td><td></td></tr><tr><td>Monoclonal mouse anti-5-mC antibody</td><td>Millipore Sigma</td><td>MABE146</td><td></td></tr><tr><td>NaOH</td><td>Fisher Scientific</td><td>BP359-500</td><td></td></tr><tr><td>Orbital shaker&amp;nbsp;</td><td>Fisher Scientific</td><td>50998290</td><td></td></tr><tr><td>Parafilm&amp;nbsp;</td><td>Fisher Scientific</td><td>1337412</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Fisher Scientific</td><td>18612139</td><td></td></tr><tr><td>Plastic transfer pipette</td><td>Fisher Scientific</td><td>1368050</td><td></td></tr><tr><td>Rstudio</td><td>RStudio</td><td>N/A</td><td>RStudio is open-source software and can be downloaded at https://www.rstudio.com.</td></tr><tr><td>Sheep serum</td><td>Millipore Sigma</td><td>S3772-5ML</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>10450103</td><td></td></tr><tr><td>Temperature-controlled incubator&amp;nbsp;</td><td>Fisher Scientific</td><td>PR505755L</td><td></td></tr><tr><td>Tween-20&amp;nbsp;</td><td>Fisher Scientific</td><td>P7949-500ML</td><td></td></tr></tbody></table>

rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

The overall aim of this protocol is to determine whether a treatment affects the relative abundance of 5-mC by assessing the total area and relative intensity of fluorescence within fixed and labeled zebrafish embryos. After completing the protocol, a fluorescence stereomicroscope can be used to first determine whether the whole-mount IHC was successful. When labeled embryos are observed under a FITC or GFP filter, a positive result is indicated by a positive FITC signal within the embryo, whereas a negative result is indicated by the absence of fluorescence within control embryos. Using a high-content screening system, these results can also be confirmed during image acquisition using a FITC filter. In addition, during data analysis, the custom module will identify and quantify the total area and integrated intensity of fluorescence. Representative images of successful results are shown in Figure 1, where acquired images were measured successfully by the custom module, and individual data points (shown as blue overlay) were acquired for both total area and integrated intensity.
During data extraction, the threshold stringency within the custom module is an additional variable that needs to be optimized to maximize the signal-to-noise ratio and increase the probability of detecting a significant treatment-specific difference in 5-mC abundance. During optimization, this final threshold provided the most significant separation between medians of control and treatment groups (e.g., the largest signal-to-noise ratio). Representative results at varying custom module thresholds that impact signal-to-noise ratios are presented in Figure 2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64190/64190fig01.jpg" /> 
Figure 1: A flow diagram providing a graphical representation of the protocol for exposure and in situ detection of 5-methylcytosine for 6 hpf zebrafish embryos. The direction of the flow diagram is provided with black arrows. Exposures occurred in replicates of four replicate dishes per treatment and 50 embryos per replicate dish. Abbreviations: cm = cell mass; ys = yolk sac. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64190/64190fig01large.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/64190/64190fig02.jpg" /> 
Figure 2: Optimization of the custom module. Panels A-D show optimization of the custom module by assessing the&#160;median and distribution of 5-mC-specific total area and integrated intensity within zebrafish embryos at 6 hpf. (A,B) Different thresholds tested are listed in the legend to the right (difference of 100 between each threshold) and are ordered by stringency, where 1500 and 2500 represent the least and most stringent thresholds tested, respectively. (C,D) Different thresholds tested are listed in the legend to the right (difference of 250 between each threshold) and ordered by stringency, where 1500 and 2500 represent the least and most stringent thresholds tested, respectively. The (*) in C,D denotes thresholds that are significantly different from vehicle-treated embryos (p &#60; 0.05). For A,B, all thresholds tested were significant from the vehicle control. The x-axis denotes exposure to either vehicle (0.1% DMSO) or 0.78 &#181;M TDCIPP (positive control). The y-axis denotes relative fluorescence. Panel A displays the total area of 5-mC detected within embryos as a function of treatment, whereas Panel B displays the integrated intensity of 5-mC within that same area. One embryo is represented by a single data point for a total of N = 96 for each treatment group. All exposures were performed in replicates of four dishes per treatment with 50 embryos per glass dish. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64190/64190fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1: 5mC program code. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64190/64190-R2-Supplemental File 1.docx" target="_blank">Please click here to download this File.</a>

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Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos.

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical ...

rapid-efficient-spatiotemporal-monitoring-normal-aberrant-cytosine

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

1.3K Views.  University of California, Riverside. This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos. 

Video: Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Genetics

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Chemistry

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

Refined Multiview Light Sheet Microscopy Protocol for Zebrafish Embryos

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

Author Spotlight: Strategies for Mounting Zebrafish Embryos for High-Resolution Multiview Light-Sheet Microscopy &#8212; Techniques for Imaging and Image Reconstruction

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Biology

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 ...

Elucidating Microglia Behavior in Zebrafish Optic Tectum Development

Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and function. Externally developing zebrafish embryos possess optical transparency, which along with well-characterized transgenic reporter lines that fluorescently label microglia, make zebrafish an ideal vertebrate model for such studies. In this paper, we take advantage of the unique features of the zebrafish model to visualize the dynamics of microglia cells in vivo and under physiological conditions. We use confocal microscopy to record a timelapse of microglia cells in the optic tectum of the zebrafish embryo and then, extract tracking data using the IMARIS 10.0 software to obtain the cells' migration path, mean speed, and distribution in the optic tectum at different developmental stages. This protocol can be a useful tool to elucidate the physiological significance of microglia behavior in various contexts, contributing to a deeper characterization of these highly motile cells.

Author Spotlight: Using Zebrafish to Explore Microglia Migration During Brain Development

We demonstrate a method that takes advantage of fast scanning confocal microscopy to perform live imaging of microglia cells in the developing zebrafish optic tectum, allowing for the analysis of the dynamics of these cells in vivo.

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

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Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Metabolic Profile Analysis of Zebrafish Embryos

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

Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo