Name: analysis of congenital heart defects in mouse embryos using qualitative and quantitative histological methods
Uploaded: 2020-03-10T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods at JoVE.com

The Aguirre Lab is supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under award number K01HL135464 and by the American Heart Association under award number 19IPLOI34660342.

All animals used in the experiments referenced in this paper were treated using the animal care guidelines of the Michigan State University Institutional Animal Care and Use Committee (IACUC).
1. Timed mating of C57BL6/J mice for embryo production
<ol>
	<li>Once mice have reached breeding age (6-8 weeks), put them together in harem breeding format (i.e., two females per one male). Set them up for breeding sometime in the afternoon or evening. 
	NOTE: Mice often breed within 1 h after li...

<ol>
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E., Kaplan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+incidence+of+congenital+heart+disease.">The incidence of congenital heart disease.</a> Journal of the American College of Cardiology. 39 (12), 1890-1900 (2002).</li><li>Fahed, A. C., Gelb, B. D., Seidman, J. G., Seidman, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+congenital+heart+disease:+The+glass+half+empty.">Genetics of congenital heart disease: The glass half empty.</a> Circulation Research. 112 (4), 707-720 (2013).</li><li>Pelech, A. N., Broeckel, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+etiologies+of+congenital+heart+diseases.">Toward the etiologies of congenital heart diseases.</a> Clinics in Perinatology. 32 (4), 825-844 (2005).</li><li>Zaidi, S., Brueckner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+Genomics+of+Congenital+Heart+Disease.">Genetics and Genomics of Congenital Heart Disease.</a> Circulation Research. 120 (6), 923-940 (2017).</li><li>Kenny, L. 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=Transplantation+in+the+single+ventricle+population.">Transplantation in the single ventricle population.</a> Annals of Cardiothoracic Surgery. 7 (1), 152-159 (2018).</li><li>Navaratnam, D., 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=Exercise-Induced+Systemic+Venous+Hypertension+in+the+Fontan+Circulation.">Exercise-Induced Systemic Venous Hypertension in the Fontan Circulation.</a> The American Journal of Cardiology. 117 (10), 1667-1671 (2016).</li><li>De Leval, M. R., Deanfield, J. 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D., 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=Lower+rate+of+selected+congenital+heart+defects+with+better+maternal+diet+quality+:+a+population-based+study.">Lower rate of selected congenital heart defects with better maternal diet quality : a population-based study.</a> Archives of Disease in Childhood - Fetal and Neonatal Edition. 101 (1), F43-F49 (2016).</li><li>Knowles, R. 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=Ethnic+and+socioeconomic+variation+in+incidence+of+congenital+heart+defects.">Ethnic and socioeconomic variation in incidence of congenital heart defects.</a> Archives of Disease in Childhood. 102 (6), 496-502 (2017).</li><li>Feng, 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=Maternal+Folic+Acid+Supplementation+and+the+Risk+of+Congenital+Heart+Defects+in+Offspring+:+A+Meta-Analysis+of+Epidemiological+Observational+Studies.">Maternal Folic Acid Supplementation and the Risk of Congenital Heart Defects in Offspring : A Meta-Analysis of Epidemiological Observational Studies.</a> Scientific Reports. 17 (5), 8506 (2015).</li><li>Rhinn, M., Dolle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+signalling+during+development.">Retinoic acid signalling during development.</a> Development. 139 (5), 843-858 (2012).</li><li>Liu, 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=Circulating+retinoic+acid+levels+and+the+development+of+metabolic+syndrome.">Circulating retinoic acid levels and the development of metabolic syndrome.</a> The Journal of Clinical Endocrinology &amp; Metabolism. 101 (February), 2015 (2016).</li><li>Kurian, 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=Identification+of+novel+long+noncoding+RNAs+underlying+vertebrate+cardiovascular+development.">Identification of novel long noncoding RNAs underlying vertebrate cardiovascular development.</a> Circulation. 131 (14), 1278-1290 (2015).</li><li>Aguirre, A., Sancho-Martinez, I., Izpisua Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+toward+heart+regeneration:+Stem+cells+and+beyond.">Reprogramming toward heart regeneration: Stem cells and beyond.</a> Cell Stem Cell. 12 (3), 275-284 (2013).</li><li>Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+or+breaking+the+heart:+from+lineage+determination+to+morphogenesis.">Making or breaking the heart: from lineage determination to morphogenesis.</a> Cell. 126 (6), 1037-1048 (2006).</li><li>Heyne, G. 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=A+simple+and+reliable+method+for+early+pregnancy+detection+in+inbred+mice.">A simple and reliable method for early pregnancy detection in inbred mice.</a> Journal of the American Association for Laboratory Animal Science. 54 (4), 368-371 (2015).</li><li>. Stage Definition Available from: <a target="_blank" href="https://www.emouseatlas.org/emap/ema/theiler_stages/StageDefinition/stagedefinition.html">https://www.emouseatlas.org/emap/ema/theiler_stages/StageDefinition/stagedefinition.html</a> (1998)</li><li>Part 4-Measure Areas using Thresholding. Science Education Resource Center Available from: <a target="_blank" href="https://serc.carleton.edu/eet/measure_sat2/part_4.html">https://serc.carleton.edu/eet/measure_sat2/part_4.html</a> (2017)</li><li>. Examples of Image Analysis Using ImageJ Available from: <a target="_blank" href="https://imagej.nih.gov/ij/docs/pdfs/examples.pdf">https://imagej.nih.gov/ij/docs/pdfs/examples.pdf</a> (2007)</li><li>MacIver, D. H., Adeniran, I., Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+ejection+fraction+is+determined+by+both+global+myocardial+strain+and+wall+thickness.">Left ventricular ejection fraction is determined by both global myocardial strain and wall thickness.</a> IJC Heart and Vasculature. 1 (7), 113-118 (2015).</li><li>Towbin, J. 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This protocol explores the techniques involved in the analysis of cardiac development in embryonic hearts. Some limitations of this method are the required physical dexterity for the preparatory techniques, which may require practice, and skill with microscope imaging. If the slices obtained at the cryostat are messy, the hematoxylin and eosin staining will not be clear, or if the images taken at the microscope have poor lighting, then the method used with ImageJ will not work. A limitation of the threshold feature of Im...

CHDs are the most common type of birth defect in humans and are the leading cause of birth defect-related deaths1,2,3,4,5,6. Although about 90% of newborn children survive CHD, it is frequently associated with significant morbidity and medical interventions over the years, imposing a heavy burden on the patients&#39; lives and the healthcare system7,8,9,10. Outside of purely genetic factors, the causes of CHD are poorly understood4. Unidentified causes account for ~56-66% of all CHD cases according to the American Heart Association and other sources2,3,4,11. Well-known factors include genetic mutations, CNVs, de novo single nucleotide variants, and aneuploidy. It is suspected that environmental and dietary factors are also important sources contributing to CHD, as suggested by epidemiological studies linking maternal lifestyle2,12, economic deprivation, and race13, and by research into dietary factors such as folic acid11,14 and the bioactive lipid retinoic acid15,16. Investigating the mechanisms and causes of CHD and other cardiovascular defects is important to develop preventive strategies and novel therapeutic options1,4,17,18,19.
The developing mouse is a cornerstone model for studying CHD in mammals. However, some of the methods and analyses employed, such as dissections preserving heart morphology, analysis of developmental stages, and identification of CHD-associated defects, can be daunting for scientists that are new to the analysis of murine hearts. The goal of the methods described in this protocol is to offer qualitative and quantitative guidelines for these processes. Thus, in this protocol we explain how to perform timed matings to produce embryos of the desired gestational stage, dissect pregnant females for intact heart recovery (including associated tissues such as the outflow tract), heart fixation and preparation for cryostat sectioning, basic histology methods, quantitative analyses of common heart defects, and qualitative analysis of heart muscle compaction, a common precursor phenotype to some types of CHD.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL Conical Tube(s)</td>
			<td>Fisher Scientific</td>
			<td># 1495970C</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J Mice</td>
			<td>Jackson Labs</td>
			<td>C57BL/6J - stock 000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin Staining Jars (x6)</td>
			<td>VWR Scientific</td>
			<td># 25457-006</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips 24X50MM #1.5</td>
			<td>VWR Scientific</td>
			<td># 48393-241</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat - Leica CM3050S</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Dish(s)</td>
			<td>Fisher Scientific</td>
			<td># 50930381</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 - Fine Forceps (x2)</td>
			<td>Fine Science Tools</td>
			<td># 11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y Solution</td>
			<td>Millipore Sigma</td>
			<td># HT110116-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol (Pure, 200 proof)</td>
			<td>Fisher Scientific</td>
			<td># BP2818-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Millipore Sigma</td>
			<td># E9884-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt</td>
			<td>Millipore Sigma</td>
			<td># 03989-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td># 14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Stereo Microscope Leica M165 FC</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Millipore Sigma</td>
			<td># 410225-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td># 11052-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism 8 Software</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>ImageJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher Scientific</td>
			<td># 06666A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#39;s hematoxylin solution</td>
			<td>Millipore Sigma</td>
			<td># MHS16-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tip(s) - p200</td>
			<td>Fisher Scientific</td>
			<td># 02707448</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel Software</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>VWR Scientific</td>
			<td># 102094-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus CkX53 Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paint Brushes (at least 2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>VWR Scientific</td>
			<td># 0215014601</td>
			<td>Make into 4% solution (dissolved in PBS)</td>
		</tr>
		<tr>
			<td>Pasteur pipette(s)</td>
			<td>Fisher Scientific</td>
			<td># 13-711-7M</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td># 15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td># 70011044</td>
			<td>Dilute from 10x to 1x before using</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Mettler Toledo</td>
			<td># MS1602TS</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Mettler Toledo</td>
			<td># MS105</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle #3</td>
			<td>VWR Scientific</td>
			<td># 10161-918</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blades</td>
			<td>VWR Scientific</td>
			<td># 21909-612</td>
			<td></td>
		</tr>
		<tr>
			<td>Square Mold</td>
			<td>VWR Scientific</td>
			<td># 100500-224</td>
			<td>For OCT molds</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Millipore Sigma</td>
			<td># S9378-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Slides</td>
			<td>Fisher Scientific</td>
			<td># 1255015</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td># 14002-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Disposable Microtome Blades</td>
			<td>VWR Scientific</td>
			<td># 25608-964</td>
			<td></td>
		</tr>
		<tr>
			<td>Travel Scale</td>
			<td>Acculab</td>
			<td>VIC 5101</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Millipore Sigma</td>
			<td>214736-1L</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of congenital heart defects in mouse embryos using qualitative and quantitative histological methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

The muscle compaction index was compared between hearts developing under two different environments, a control and an experimental group. These protocols were used to analyze the compaction of muscle tissue quantitatively, which permitted statistical analysis. Muscle compaction was shown to be significantly reduced in the experimental hearts relative to the embryos that developed in nonexperimental conditions.
Embryos were disca...

Watch this Scientific Journal Video about Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods at JoVE.com

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...

This protocol standardizes new tissue analysis techniques so that they can be more widely used by other groups who also study congenital heart defects. As both qualitative and quantitative analysis techniques have their pros and cons, this methodology uses both to address the widest range of experimental questions. These techniques can have a positive impact on cardiology diagnostic methods.For example, pathologists could apply our microscopic cardiac tissue analysis techniques towards diagnosing human heart disease from biopsy samples. This protocol can be used for developmental cardiology or general cardiology research. However, these methods also provide various useful techniques that can be applied towards any tissue analysis.To collect embryonic day 15 to 15.5 hearts, secure the limbs of a pregnant dam and starting around the urethra up to the sternum, carefully make an I-shaped incision along the torso. Using forceps in a scooping motion, gently lift the uterus out of the abdominal cavity grasping the superficial tissues of the uterus as necessary with fine forceps to aid in the extraction. Use scissors to release the uterus from the abdomen.After soaking the uterus in ice-cold PBS, pin the organ into a dissection dish. Using scissors, cut the uterus into individual sections each containing a separate embryo. Using two pairs of fine forceps, gently peel the embryos out of the uterine tissue and place them in to a new Petri dish containing four degrees Celsius PBS-EDTA.To collect the hearts, place the dish under a dissecting microscope and use two pairs of fine forceps to position the embryo into the supine position. After stabilizing access to the chest, use the sharp point of a second pair of fine forceps to make an incision along the sternum of the embryo extending between the clavicles to the navel. Making very fine and superficial incisions while gradually working toward the inside of the chest cavity, open the abdomen of the embryo.When the heart just becomes visible, use a pair of forceps to squeeze the torso slightly coddled to the rib cage to peel the ribs open. Using the sides of one pair of forceps, gently grab the pulmonary blood vessels without separating the tissue and pull the heart out of the cavity. Clean the heart in fresh PBS-EDTA for one to two minutes before fixing the tissue in 4%Paraformaldehyde for 45 to 60 minutes.Then, wash the heart three times for 5 to 10 minutes per wash in PBS supplemented with glycine. Before storing the hearts in PBS at four degrees Celsius. To prepare the heart for cryosectioning, place the samples into a tube of 30%sucrose and PBS at four degrees Celsius for 24 to 40 hours.When the hearts have sunk to the bottom of the tube, use a Pasteur Pipette with a modified tip to carefully transfer each heart unto a piece of lint free wipe. After a brief air-drying, place each sample into individual optimal cutting temperature molds in the desired orientations for sectioning. Submerge the tissues in optimal cutting temperature medium without bubbles.Then, store the molds at 20 degrees Celsius for up to a few days before freezing the tissues at 80 degrees Celsius for at least 24 hours before cryosectioning. To obtain cryostat sections of the samples, use fresh optimal temperature cutting medium to attach the tissue block of interest to the chuck of the cryostat and allow the medium to freeze until it is opaque white. Insert a new blade and adjust the distance between the tissue block and the blade to allow sections to be obtained.Trim the block in 10 micrometer slices until the point of interest is reached. When the tissue can be observed, use a small flat paintbrush to gently pull the slice against the stand in a continuous cutting motion. Once the tissue has been completely cut through, use the detail brush to pat the slice down flat against the stage.Hold the section in place for approximately 20 to 30 seconds before removing the detailed brush and finishing the slice. Use both paintbrushes to gently flatten the slice against the stage preventing any rolling and hold the tissue section against the stage. After 20 to 30 seconds, flip the section and flatten it against the stage again before placing an electrostatically charged slide close enough for the slice to be attracted and adhere to the slide without having to touch the slide to the stage.To evaluate the heart tissue sections for the presence of common heart defects, using a microscope equipped with a camera, obtain images at 5 to 10X and 40X magnifications. For cardiac muscle compaction analysis, open a tissue of interest in an appropriate image analysis software program and set the image to 8-bit. To set the threshold of the image, click Image, Adjust, and Threshold and move the top bar to adjust the threshold until only the pixels for the background are selected.Next, use the polygon selections tool to draw a region of interest around the tissue then click Analyze, Set Measurements, Area, and Limit to threshold. Click Analyze and Measure to measure the area of the highlighted pixels to obtain the value of the area of the negative space. To measure the area of the entire region of interest, click Analyze, Set Measurements, and deselect Limit to threshold.Then, select Analyze and Measure to measure the entire area of the selected region of interest. To calculate the Area of muscle tissue, subtract the Area of Negative Space from the Area of the Region of Interest. Then divide the Area of muscle tissue by the Area of the Region of Interest to calculate the muscle compaction and X.The staining of tissue slices from harvested embryonic hearts, provides enough contrast to make out the tissue edges, but is not so dark as to make the cell features indistinguishable.In this representative analysis, the muscle compaction was observed to be significantly reduced in the experimental hearts relative to the embryos that developed under non-experimental conditions. These samples are valuable and require a long time to create. During necropsies and the cryostat sectioning specifically, make sure to be intentional about every action you perform.Preserving the tissues in Paraformaldehyde maintains the antigen activity permitting the samples to be used for tests like Aminofluorescein staining which help identify pathological causes found during the morphology assessment. In our lab, we obtained quantitative representation of experimental treatments which can help us to identify the degree to which different treatments affect our samples. Paraformaldehyde can fix any tissue that it touches.Take care to always wear gloves to minimize exposed skin and to work in a film hood whenever working with this chemical.

analysis-congenital-heart-defects-mouse-embryos-using-qualitative

Research

JoVE Journal

Developmental Biology

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...