We would like to thank the staff in Biomedical Services in Oxford and Birmingham for excellent zebrafish husbandry. T.D. was funded by a Wellcome Trust Chromosome and Developmental Biology PhD Scholarship (#WT102345/Z/13/Z). R.M. and M.K. were funded by the British Heart Foundation (BHF IBSR Fellowship FS/13/50/30436) and are thankful for their generous support. R.M. acknowledges support from the BHF Centre of Research Excellence (RE/13/1/30181), Oxford.

Procedures involving animal subjects are regulated by the Animals (Scientific Procedures) Act 1986 and have been approved by the Home Office and the local Animal Welfare and Ethical Review Body.
1. Image ISH-stained embryos
<ol>
	<li>Prepare a glycerol solution (50%&#8211;80% in 1x PBS buffer) and mix to homogenize the solution (e.g., leave in a roller for at least 5 min). This solution can be kept for months at room temperature.</li>
	<li>After in situ hybridization14,15,16,17, transfer embryos to the glycerol solution with a 3 mL Pasteur pipette and leave to settle for at least 5 min. If imaging embryos older than 24 hpf (hours post fertilization), they may be bleached as described18.
	<ol>
		<li>Prepare and label enough PCR tubes to transfer ISH-stained embryos after imaging.</li>
	</ol>
	</li>
	<li>Add 100% glycerol to the bottom of the well in a glass depression slide with a 3 mL Pasteur pipette.</li>
	<li>Using a 3 mL Pasteur pipette, transfer a single ISH-stained embryo to the glass slide and orient as required under a stereomicroscope equipped with a digital camera and bottom and top illumination. 
	NOTE: Use gel loading pipette tips to position the embryos for imaging, but other tools (e.g., forceps, dissecting needle) will be equally adequate.</li>
	<li>Using the first embryo, adjust the illumination and exposure time at the desired magnification. Use these conditions for ALL of the embryos in the same experiment (i.e., if imaging 40 embryos from a heterozygous mutant incross, make sure the illumination, exposure time and magnification are the same for all).</li>
	<li>Image as many embryos as required. Label each image with a unique number. After imaging, transfer the embryo to a PCR tube/plate labelled with the same number. 
	NOTE: Images should be saved as TIF files, but other formats are also adequate.
	<ol>
		<li>If required, remove excess glycerol in the PCR tubes/plates. 
		NOTE: At this point, the embryos can be stored in the PCR tubes for several weeks at room temperature.</li>
	</ol>
	</li>
</ol>
2. Extract DNA and genotype the ISH-stained embryos
NOTE: Here, use a reliable and inexpensive method to isolate genomic DNA based on the HoTSHOT method19 with a DNA extraction efficiency of 95%-100%3.
<ol>
	<li>After the imaging is complete, add 40-75 &#181;L of alkaline lysis buffer (e.g., HoTSHOT) to each tube.</li>
	<li>Incubate at 95 &#176;C for approximately 30 min and cool the tubes to 4 &#176;C before adding an equal volume of neutralization buffer. An overnight incubation at 4 &#176;C may improve PCR efficiency. 
	NOTE: At this point, the genomic DNA can be used for genotyping or stored at -20 &#176;C until it is required.</li>
	<li>Genotype samples with an appropriate method (e.g., HRMA, RFLP)3,20,21 as required for the mutation of interest.</li>
	<li>Note the genotype corresponding to each sample (e.g., using a spreadsheet software).</li>
</ol>
3. Quantify the pixel intensity of ISH-stained embryos (image analysis using Fiji software)
<ol>
	<li>To quantify the in situ hybridization (ISH) staining signal intensity, convert all images to 8-bit grayscale as described3. If the images were saved as .TIF files, use a Fiji macro for batch conversion3. Alternatively, convert images in other formats (e.g., .JPG) to .TIF using appropriate software and then convert to 8-bit grayscale using Fiji. For convenience, here is the step-by-step procedure that was published previously3, with some alterations.</li>
	<li>Open images in Fiji and invert the image with Edit &#62; Invert. Then change the image type to 8-bit (Image &#62; Type &#62; 8-bit).</li>
	<li>Using the polygon selection tool, draw the region of interest (ROI) manually on the image around the region containing the signal.</li>
	<li>Press t to open the ROI manager. Use the ROI manager&#39;s Measure command to measure the intensity of the ROI. Copy the mean value from the Results window to a spreadsheet software.</li>
	<li>Move the same ROI, ensuring the same size and shape as the original region, to a region of the zebrafish not containing any staining. Repeat step 3.4 to measure the background.</li>
	<li>To obtain the mean pixel intensity of the ISH signal, subtract the mean intensity value of the background region from that of the stained region for each embryo.</li>
	<li>Assign each intensity value to a genotype (from step 2.3).</li>
</ol>
4. Analyze the results with appropriate statistical tests
<ol>
	<li>Plot all the values on one Q-Q plot to identify any deviations from normal distribution. 
	NOTE: Normal distribution can also be verified with the Kolmogorov-Smirnov test or the Shapiro-Wilk test. However, for large sample sizes there is a high risk of false positives in these tests.</li>
	<li>If there are strong deviations from normal distribution, transform all the values (using ln or sqrt functions) to make sure they are normally distributed before proceeding.</li>
	<li>Analyze the differences between the values (transformed if necessary) assigned to each genotype (wt vs. heterozygote vs. mutant) with 2-tailed ANOVA with 95% confidence levels, accounting for the equality of variances with a Levene&#39;s test and Welch correction. For pairwise comparisons between each pair of genotypes, use Tukey&#39;s (equal variances) or Games-Howell (unequal variances) post-hoc test.
	<ol>
		<li>If the values are not normally distributed despite the transformation, use a non-parametric test (Kruskall-Wallis) to analyze the differences between ranked values and a post-hoc Dunn&#39;s multiple comparisons test with Bonferroni correction for pairwise comparisons.</li>
	</ol>
	</li>
	<li>Plot the untransformed values (from step 3.6) as dot plots for the best representation of the results.</li>
</ol>

<ol>
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K., 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=CRISPRz:+a+database+of+zebrafish+validated+sgRNAs.">CRISPRz: a database of zebrafish validated sgRNAs.</a> Nucleic Acids Research. 44 (D1), D822-D826 (2016).</li><li>Dobrzycki, T., Krecsmarik, M., Bonkhofer, F., Patient, R., Monteiro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimised+pipeline+for+parallel+image-based+quantification+of+gene+expression+and+genotyping+after+in+situ+hybridisation.">An optimised pipeline for parallel image-based quantification of gene expression and genotyping after in situ hybridisation.</a> Biology Open. 7 (4), bio031096 (2018).</li><li>Bresciani, E., 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=CBFbeta+and+RUNX1+are+required+at+2+different+steps+during+the+development+of+hematopoietic+stem+cells+in+zebrafish.">CBFbeta and RUNX1 are required at 2 different steps during the development of hematopoietic stem cells in zebrafish.</a> Blood. 124 (1), 70-78 (2014).</li><li>Gore, A. V., 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=Epigenetic+regulation+of+hematopoiesis+by+DNA+methylation.">Epigenetic regulation of hematopoiesis by DNA methylation.</a> Elife. 5, e11813 (2016).</li><li>Fan, 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=Tissue-Specific+Gain+of+RTK+Signalling+Uncovers+Selective+Cell+Vulnerability+during+Embryogenesis.">Tissue-Specific Gain of RTK Signalling Uncovers Selective Cell Vulnerability during Embryogenesis.</a> PLoS Genetics. 11 (9), e1005533 (2015).</li><li>Wen, B., 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=GATA5+SUMOylation+is+indispensable+for+zebrafish+cardiac+development.">GATA5 SUMOylation is indispensable for zebrafish cardiac development.</a> Biochimica et Biophysica Acta. 1861 (7), 1691-1701 (2017).</li><li>Espin-Palazon, R., 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=Proinflammatory+signaling+regulates+hematopoietic+stem+cell+emergence.">Proinflammatory signaling regulates hematopoietic stem cell emergence.</a> Cell. 159 (5), 1070-1085 (2014).</li><li>Peterkin, T., Gibson, A., Patient, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redundancy+and+evolution+of+GATA+factor+requirements+in+development+of+the+myocardium.">Redundancy and evolution of GATA factor requirements in development of the myocardium.</a> Developmental Biology. 311 (2), 623-635 (2007).</li><li>Genthe, J. 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L., 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=Runx1+is+required+for+zebrafish+blood+and+vessel+development+and+expression+of+a+human+RUNX1-CBF2T1+transgene+advances+a+model+for+studies+of+leukemogenesis.">Runx1 is required for zebrafish blood and vessel development and expression of a human RUNX1-CBF2T1 transgene advances a model for studies of leukemogenesis.</a> Development. 129 (8), 2015-2030 (2002).</li><li>Butko, E., 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=Gata2b+is+a+restricted+early+regulator+of+hemogenic+endothelium+in+the+zebrafish+embryo.">Gata2b is a restricted early regulator of hemogenic endothelium in the zebrafish embryo.</a> Development. 142 (6), 1050-1061 (2015).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Lleras Forero, L., 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=Segmentation+of+the+zebrafish+axial+skeleton+relies+on+notochord+sheath+cells+and+not+on+the+segmentation+clock.">Segmentation of the zebrafish axial skeleton relies on notochord sheath cells and not on the segmentation clock.</a> Elife. 7, (2018).</li><li>Jowett, T., Yan, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+fluorescent+in+situ+hybridization+to+zebrafish+embryos.">Double fluorescent in situ hybridization to zebrafish embryos.</a> Trends in Genetics. 12 (10), 387-389 (1996).</li><li>Thisse, C., Thisse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+in+situ+hybridization+to+whole-mount+zebrafish+embryos.">High-resolution in situ hybridization to whole-mount zebrafish embryos.</a> Nature Protocols. 3 (1), 59-69 (2008).</li><li>Narayanan, R., Oates, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+mRNA+by+Whole+Mount+in+situ+Hybridization+and+DNA+Extraction+for+Genotyping+of+Zebrafish+Embryos.">Detection of mRNA by Whole Mount in situ Hybridization and DNA Extraction for Genotyping of Zebrafish Embryos.</a> Bio-protocol. , e3193 (2019).</li><li>Monteiro, R., Pouget, C., Patient, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gata1/pu.1+lineage+fate+paradigm+varies+between+blood+populations+and+is+modulated+by+tif1gamma.">The gata1/pu.1 lineage fate paradigm varies between blood populations and is modulated by tif1gamma.</a> EMBO JOURNAL. 30 (6), 1093-1103 (2011).</li><li>Truett, G. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+method+of+genotyping+zebrafish+mutants.">A rapid and efficient method of genotyping zebrafish mutants.</a> Developmental Dynamics. 238 (12), 3168-3174 (2009).</li><li>Jin, H., 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=Definitive+hematopoietic+stem/progenitor+cells+manifest+distinct+differentiation+output+in+the+zebrafish+VDA+and+PBI.">Definitive hematopoietic stem/progenitor cells manifest distinct differentiation output in the zebrafish VDA and PBI.</a> Developement. 136 (4), 647-654 (2009).</li><li>Stainier, D. Y. 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A., 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=Genetic+compensation+triggered+by+mutant+mRNA+degradation.">Genetic compensation triggered by mutant mRNA degradation.</a> Nature. 568 (7751), 193-197 (2019).</li><li>Meier, 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=Novel+binding+partners+of+Ldb1+are+required+for+haematopoietic+development.">Novel binding partners of Ldb1 are required for haematopoietic development.</a> Development. 133 (24), 4913-4923 (2006).</li><li>Kettleborough, R. 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The authors declare they have no competing financial interests.

A few factors should be considered when using this method to quantify gene expression. The imaging conditions must be maintained throughout the experiment (for example illumination, exposure times and embryo positioning) to reduce variability between measurements. A critical point is to avoid overstaining of the samples, as differences in staining between samples may be masked. For example, the decrease in VegfA expression in the absence of Eto2 in Xenopus laevis embryos30 could only be detected by carefully monitoring staining over a 24 hour period. Thus, it is good practice to empirically determine adequate staining levels for each gene that best represent its expression, without reaching saturation. Overstaining will also artificially increase the background pixel intensity in the converted 8-bit grayscale images and skew the quantitation results. In extreme cases, the background level in embryonic tissues might be higher than the ISH signal in the selected ROI and these samples should be excluded from the analysis. A similar phenomenon was observed when we tested the suitability of the unstained yolk for background correction (Figure 4). After inversion and conversion to 8-bit the darker pixels in the yolk region become brighter than the ISH signal in the embryo and render the background corrected values negative. Thus, avoid the use of the yolk for background correction. Measuring the background signal in pigmented areas in the embryo (e.g., the eyes or the dorsal part of the trunk from 26/28 hpf onwards) will equally skew the quantitation results and should also be avoided. There are protocols available for bleaching zebrafish embryos, either before or after ISH18 and bleaching embryos older than 24 hpf before imaging is recommended.
Because this method relies on measuring the pixel intensity in a defined area against a background pixel intensity in an equivalent non-stained area, it is not appropriate for quantitation of ubiquitous or near-ubiquitously expressed genes as is. Instead, it is well suited for measuring expression of genes with a spatially restricted distribution where an area for measuring background pixel intensity can be readily identified. Our additional analysis now suggests that using a smaller area (3-4x smaller) for background correction yields similar outcomes to using an equivalent area to that of the ROI. This extends the applicability of the method to genes expressed in wider spatial domains (and thus requiring larger ROIs for intensity measurements), as long as one can use clearly unstained areas of the embryo for background correction.
Finally, we suggest that the genotyping be performed in parallel or after the image quantitation to minimize experimenter bias. Asking a second experimenter to repeat the quantitation on anonymized samples and compare with the first set of measurements will also help reduce experimenter bias. If the images to be quantified are from a comparison between treatments that does not require genotyping (e.g., wild type vs chemical inhibitor or wild type vs. MO knockdown), the experimenter performing the measurements should be blinded to the identity of the sample.

The introduction of genome editing technologies (ZFN, TALENs and more recently, CRISPR/Cas9) has led to a massive increase in the number of laboratories around the world that make use of these systems to study the function of specific genes in vivo. Zebrafish in particular are amenable to genetic manipulation and many mutants have been generated in the recent past1,2. For developmental biologists, one of the most common methods to assess the phenotypical consequences of gene mutations in embryonic development is in situ hybridization (ISH). In the absence of obvious morphological defects that separate homozygous mutants from their wild type or heterozygous siblings, it is essential to be able to correctly identify different genotypes accurately.
Classical ISH relies on qualitative analyses of signal intensities to derive conclusions about regulatory interactions between the gene of interest and selected marker genes. Although useful, these analyses suffer from technical variation and may be biased by researcher expectations. Thus, a method was developed to quantify gene expression after imaging ISH-stained embryos, without prior knowledge of the corresponding genotype. This was followed by an efficient DNA extraction and genotyping that allowed us to quantitatively correlate genotype with gene expression3. While the genotyping of embryos post ISH has been used before4,5, image-based quantification of ISH patterns has not been widely used apart from a couple of studies6,7. The most popular alternatives rely on visual scoring or counting of ISH-stained cells8,9,10, both prone to poor reproducibility and researcher bias. This method is particularly useful to study changes in genes with expression patterns that are spatially restricted, such as runx1 or gata2b, both expressed in a restricted subset of aortic floor cells called the haemogenic endothelium11,12.
Here, we aim to provide a practical guide to the implementation of the quantification by image analysis using Fiji13, as well as the DNA extraction and genotyping protocol. This is meant to illustrate visually our previously published method3. Our method allows an accurate representation of the variation in gene expression detected by ISH and an unbiased assignment of gene expression levels to specific genotypes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL PCR tubes (8-strips with lids)</td>
			<td>StarLab</td>
			<td>A1402-3700</td>
			<td>96-well plates are equally appropriate for sample handling but beware of cross contamination between samples</td>
		</tr>
		<tr>
			<td>3 mL Pasteur pipettes</td>
			<td>Alpha Laboratories</td>
			<td>LW4114</td>
			<td></td>
		</tr>
		<tr>
			<td>Cavity slides</td>
			<td>Brand</td>
			<td>BR475535-50EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera (Qimaging Micropublisher 5.0)</td>
			<td>Qimaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Microloader tips</td>
			<td>Eppendorf</td>
			<td>10289651</td>
			<td>the tips are used to orient the embryos for imaging in glycerol</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F3000 Fiber Optic Cold Light Source</td>
			<td>Photonic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism 8.01</td>
			<td>GraphPad Software, Inc.</td>
			<td></td>
			<td>we prefer to use Graphpad, but other statistics software packages are also suitable (e.g. SigmaPlot or SPSS)</td>
		</tr>
		<tr>
			<td>HotSHOT alkaline lysis buffer</td>
			<td></td>
			<td>25 mM NaOH, 0.2 mM disodium EDTA, pH 12</td>
			<td></td>
		</tr>
		<tr>
			<td>HotSHOT neutralization buffer</td>
			<td></td>
			<td>Tris HCl 40 mM, pH 5</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X) pH 7.4</td>
			<td>Thermofisher</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope with illumination stand (Nikon SMZ800N)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Thermofisher</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

genotyping and quantification of in situ hybridization staining in zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Here, we describe the practical application of the pipeline for image quantitation and embryo genotyping as published elsewhere3. The workflow for the method is shown in Figure 1. To illustrate how to use this method, ISH was performed for dnmt3bb.1 in 33 hpf embryos from a runx1W84X/+ 22 incross (Figure 2). 130 embryos were imaged using the same illumination conditions as detailed in the protocol and labeling them with a unique number. After imaging, each embryo was transferred to a PCR tube for genotyping. At this point, the image analysis was performed to attribute a pixel intensity value to each image. The genotype was then assigned to its corresponding image and the pixel intensity values grouped according to their genotype for statistical analysis. A decrease in dnmt3bb.1 expression was detected in runx1W84X/W84X mutants (Figure 2A,B)3, in agreement with previous observations5. Interestingly, runx1W84X/+ heterozygous embryos showed no significant differences in dnmt3bb.1 expression (Figure 2A,B) compared to its wild type siblings, suggesting that one copy of Runx1 is sufficient to maintain dnmt3bb.1 expression at appropriate levels.
Many zebrafish mutants fail to show an embryonic phenotype that can otherwise be detected using other loss of function technologies like morpholino oligonucleotides (MOs). This discrepancy can be attributed to a number of causes including off-target effects, maternal protein compensation, a hypomorphic allele23 or the recently discovered phenomenon of genetic compensation24,25,26,27. In this example, we asked whether runx1 expression was reduced or lost in lmo4auob100 mutants since previously published data using an lmo4a MO suggested that runx1 is decreased in lmo4a morphants28. Here, the analysis revealed no significant differences in runx1 expression between wild type and lmo4auob100 homozygous mutants3 (Figure 3A,B). Further analysis by single embryo qPCR showed that there was a small but significant decrease in runx1 expression in lmo4auob100 mutants (Figure 3C). Thus, it is possible that image quantification might not be able to detect small differences in expression levels. Alternatively, the lack of difference between genotypes that we detected is real and the qPCR experiments are detecting changes in runx1 expression in other tissues like the telenchephalon where both lmo4a and runx1 are expressed. Researchers should always verify their results with an independent method like qPCR, but ideally enriching for the tissue of interest by flow cytometry, for example.
In rare instances where the ISH has high background (Figure 3D), the pixel intensity value of this area is so high that subtraction from the signal value produces a negative number and in such instances those embryos would be excluded from the analysis. In our experience, this occurred in about 0.4% of the runx1-probed embryos3 but may vary between experiments, probes or batches of reagents. Although that might be a limitation of the method, the low frequency of high background is very unlikely to influence the overall results.
To test the effect of selecting different areas for background corrections, we first measured the pixel intensity of the runx1 ISH signal in 28 hpf embryos, using different regions for background corrections (Figure 4). Four different regions were selected: two in the trunk region (R1, and R2), one in the yolk region (unstained, but likely to accumulate background staining) and a smaller area anterior to the ROI (R4, Figure 4B). Measuring pixel intensity in these regions showed a relatively stable difference in intensity between ROI and either background area (Figure 4C). However, R3 always showed very high values (above those in the ROI). After inversion and conversion to 8-bit, the yolk region appears very bright and thus is not suitable for use as a background correction. R2 was closer to the ROI but contained some ISH signal, and using it for correction decreased the mean pixel intensity when compared with either R1 (located further dorsally, away from the ISH signal) or R4. Thus, either R1 or R4 are appropriate areas that can be used for background correction (despite the area of R4 being smaller than that of R1). Next, we wished to compare how using R1 or R4 affected the outcomes when comparing runx1 expression. For this, we incrossed dll4+/- heterozygotes29 and analyzed runx1 expression in randomly selected wild type and dll4-/-embryos (Figure 4E). Although using R1 or R4 for background correction affected individual values, the mean pixel intensities within the same genotype were not significantly different (Figure 4E). Moreover, comparing runx1 expression still yields similar mean intensity values between genotypes using either R1 or R4 areas as background correction (&#181;R1=16.3 and &#181;R4=18.2, respectively). Taken together, we concluded that although the choice of background area is important, the main criteria is that it does not include yolk regions (prone to accumulation of background staining) and that it should not contain any (specific) staining that might skew the pixel intensity values of the background.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59956/59956fig1a.jpg" /> 
Figure 1: Workflow of the parallel image quantitation and genotyping protocol. Embryos collected from an incross of fish heterozygous for a mutant allele are probed for the measured gene with a standard ISH protocol. After imaging, genomic DNA is extracted using the HotSHOT protocol by adding the lysis buffer directly to the embryo in a 0.2 mL PCR tube, followed by a 30 min incubation at 95 &#176;C. This DNA is used for genotyping of the embryos by PCR, PCR and restriction fragment length polymorphism (RFLP), KASP assays or any other appropriate method. In parallel, the images for each embryo are inverted and converted to 8-bit greyscale. ROIs of identical shape and size containing the ISH signal (yellow) and background (blue) are manually selected and measured. The measurements, assigned to corresponding genotypes, are statistically analyzed. Figure adapted from Dobrzycki et al.3 <a href="https://www.jove.com/files/ftp_upload/59956/59956fig01large.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/59956/59956fig02.jpg" /> 
Figure 2: Image quantitation in runx1 mutants reveals reduced levels of dnmt3bb.1 expression by ISH. (A) Example images of ISH in 33 hpf wild type (blue), runx1+/W84X (green) and runx1W84X/W84X (orange) embryos, showing dnmt3bb.1 expression in the dorsal aorta. (B) Pixel intensity values of dnmt3bb.1 mRNA in runx1W84X/W84Xembryos (n=36) are significantly decreased compared to wild types (n=32) and heterozygotes (n=62) (ANOVA, p &#60; 0.001). The coefficients of variation are 24%, 22% and 21% for wild type, heterozygote and mutant groups, respectively. Blue, green and orange data point correspond to the example images from panel A. The bars represent mean &#177; s.d. ***p&#60;0.001 (Games-Howell post-hoc test). Figure adapted from Dobrzycki et al.3 <a href="https://www.jove.com/files/ftp_upload/59956/59956fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59956/59956fig03.jpg" /> 
Figure 3: Measuring runx1 expression levels by ISH in lmo4auob100mutants. (A) Representative images of ISH for runx1 in 28 hpf wild type (blue), heterozygous (green) and lmo4auob100/uob100 (orange) embryos, showing the expression in the dorsal aorta. (B) Quantification of the runx1 mRNA signal, detected by ISH, from 28 hpf wild type (n=15), heterozygous lmo4a+/- (het) (n=34) and lmo4auob100/uob100 mutant (n=18) embryos from one clutch shows no significant difference in runx1 pixel intensity among the different genotypes (ANOVA,&#62; p 0.6). Blue, green and orange data point correspond to the example images from panel A. The bars represent mean &#177; s.d. (C) Boxplots displaying normalized runx1 mRNA levels (2-&#916;Ct) in single wild type (blue; n=12) and lmo4auob100/uob100 (mut, orange; n=12) embryos, measured by qRT-PCR, showing decreased levels of runx1 in the mutants compared to wild type. *p &#60; 0.05 (t test). (D) Example of an ISH experiment on a 28 hpf embryo (stained for runx1, yellow arrowheads) showing high background. Figure adapted from Dobrzycki et al.3 <a href="https://www.jove.com/files/ftp_upload/59956/59956fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59956/59956fig04.jpg" /> 
Figure 4: Effect of background intensity correction on measurement outcomes. (A) Representative image of runx1 ISH staining in a wild type embryo at 28 hpf. (B) Same image after inversion and conversion to 8-bit. The region of interest (ROI) is highlighted in green and four different areas used for background correction (R1-R4) are highlighted in yellow. (C) Raw pixel intensity measurements in all regions shown in panel B. Note the intensity in R3 (yolk) is consistently higher than the actual ISH signal in the ROI (n=11). (D) Runx1 expression levels in the ROI using R1, R2 and R4 background areas. Areas for ROI, R1, R2 and R3~28500 pixels; R4~8500 pixels. Note that R3 background was not used for this comparison as the background correction (ROI-R3) consistently yielded negative values. (E) Runx1 expression levels in wild type and dll4-/- mutants using either R1 or R4 for background correction (n=10 for each sample). Statistical analysis in panels D and E was performed using a non-parametric Kruskal-Wallis test, assuming that the pixel intensity values are not normally distributed. <a href="https://www.jove.com/files/ftp_upload/59956/59956fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

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9.3K Views.  University of Oxford. Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

Video: Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...