The use of mice for these studies is necessary as mice have four-chambered hearts that can mimic human CHDs. Mice were provided veterinary care and housed in the institution&#39;s Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited animal care facility. Strict protocols were followed to minimize the mice&#39;s discomfort, stress, pain, and injury. Mice were euthanized using CO2 gas, which is acceptable for small rodents according to the American Veterinary Medical Association Guidelines on Euthanasia. The studies on mice in this manuscript were carried out with an approved IACUC protocol at the University of Pittsburgh.
1. Fetal echocardiogram
NOTE: An echocardiogram is a powerful tool for identifying cardiovascular malformation and extracardiac defects in mice. Due to the small size of the mouse embryos (about 1-2 mm at midgestation, 3.5 mm at birth), ultrahigh-frequency echocardiographic equipment with ultrasound biomicroscopy (UBM) is required. UBM provides different high-frequency (30-50 MHz) probes with a small imaging window (15 mm x 14 mm) that provides the resolution (30 &#181;m axial x 68 &#181;m lateral) to visualize one mouse fetus at a time. A 40 MHz transducer provides high-resolution images to identify cardiovascular phenotypes7.
<ol>
	<li>Turn on the echocardiogram machine and select the program Cardiology. 
	NOTE: The following protocol can be used for any mouse background from embryonic day (E)14.5 to 19.5.</li>
	<li>Anesthetize the desired mouse in an anesthetic induction chamber. Induce anesthesia using a concentration of 4% isoflurane and medical oxygen at a flow rate of 1 L/min and reduce it to 2%-3% for maintenance.</li>
	<li>Place the mouse on the imaging platform quickly. The imaging platform has heated steel to keep the mouse warm during the procedure. Put the mouth and nose of the mouse into the anesthetic nose cone. Secure the limbs with tape to avoid movement. Monitor the heart rate to ensure that it stays between 400-450 bpm.</li>
	<li>Monitor the temperature using a rectal thermometer probe and make sure it rests at 37 &#176;C &#177; 0.5 &#176;C. Monitor the breathing to avoid hypoxia. Keep a gentle force on the probe to prevent harm. 
	NOTE: A heat lamp can be set above the mouse to prevent hypothermia while under and to recover from anesthesia. Petrolatum ophthalmic ointment can be used as a lubricant to avoid dry eyes.</li>
	<li>Remove the fur from the thorax and abdomen using depilatory cream. Apply the cream and wait 3 min before removing. Clean the area with 70% ethanol. Ethanol works better than water as a shaving lubricant.</li>
	<li>Warm up ultrasound gel to a normal body temperature. Apply the ultrasound gel generously and place the transducer on the abdomen to orient it in a horizontal plane and identify the bladder on the screen. Once the bladder is identified, scan cranially from the bladder and look for the fetus. Measure the crown-to-rump length to determine the gestational age10 (Figure 1 and Table 1). 
	NOTE: Change transducer positions to visualize different planes, including the transverse four-chamber, sagittal, and frontal/coronal imaging planes7 (Figure 1).</li>
	<li>Use the color Doppler to analyze blood flow from the heart.</li>
	<li>Put the mouse back in the cage if the embryos have not reached the intended stage. Otherwise, prepare the mouse for harvest. 
	NOTE: Ensure that the mouse is already awake and recovering well from anesthesia before putting it back into the cage.</li>
	<li>Turn off isoflurane and oxygen, clean the work area, and turn off the machine. 
	NOTE: It is important to remove the gel from the transducer.</li>
</ol>
2. Necropsy
NOTE: Once abnormal cardiac phenotypes are suspected using fetal echocardiography, fetuses are collected and fixed via full-body submersion in the fixative solution: either 10% buffered formalin phosphate or 4% paraformaldehyde (PFA). Inspect the sample&#39;s external and internal morphology, looking for macroscopic anatomical abnormalities or malformations.
<ol>
	<li>Prepare the mouse.
	<ol>
		<li>If the mouse is an adult, euthanize the mouse using a standard CO2 protocol. Use forceps or dissecting scissors to make incisions (about 3 cm) in the lateral thorax and abdomen to allow penetration of the fixative into the internal organs. 
		NOTE: The sample should be fixed for at least 24 h before necropsy if the embryo is older than E14.5.</li>
	</ol>
	</li>
	<li>Analyze the exterior of the body.
	<ol>
		<li>Set up the software to save the pictures with a name, including the sample&#39;s identification, the microscope magnification, and the content of the picture. 
		NOTE: Magnification of 1.0x to 3.2x should be adequate for imaging most structures in E14.5 mice or older.</li>
		<li>Place the mouse on the plate under the stereomicroscope lens. Fill the plate with phosphate-buffered saline (PBS) to completely cover the sample to prevent dehydration and reflection in the pictures. Adjust the magnification so that the screen includes the whole embryo, and then take a picture of both the left and right sides of the embryo. 
		NOTE: The bottom of the plate should be coated in paraffin, silicon, or another such substrate to facilitate pinning.</li>
		<li>Pin the sample through its throat, facing up, and take another picture. 
		NOTE: Orient the pin slightly upward to ensure no important structures of the thoracic cavity&#160;are pierced.</li>
	</ol>
	</li>
	<li>Analyze the chest.
	<ol>
		<li>While lifting the skin in the middle of the neck with forceps, cut the skin toward both the armpits with the scissors before cutting the skin along the median axis toward the tail. Then, cut the skin from the umbilicus to the legs. Pin the sample through its wrists and ankles. 
		NOTE: Be careful to cut only the skin. It is recommended to hold the scissor&#39;s blades horizontally or angled upward.</li>
		<li>To break away the connective tissue, lift the skin with one pair of forceps while holding the underlying tissue in place with the other pair. Pin the sample through the skin to help expose the chest and the abdomen (Figure 2). 
		NOTE: Too much stretching while pinning, cutting, or scraping may result in tissue damage.</li>
		<li>Take a photo of the exposed muscles, and then gently scrape away the muscles to expose the ribs.</li>
		<li>Take a picture of the exposed ribs. Separate the ribs from the diaphragm and cut the ribs on both sides as far as possible on the lateral axis toward the neck.</li>
		<li>Expose the heart by removing the cut ribs. Take a picture of the heart.</li>
		<li>Remove the thymus by peeling it up with one pair of forceps. Use the other pair of forceps to stabilize the base of the thymus to avoid tearing any underlying vessels. Take pictures of the heart and the great vessels. 
		NOTE: Using pins to stretch some adjacent structures may help get better imaging views. Additionally, take separate photos focused on the great arteries, heart, and other structures of interest as they may not be in focus together.</li>
	</ol>
	</li>
	<li>Analyze the abdomen.
	<ol>
		<li>Pull the diaphragm to remove it and expose the liver. Take a picture.</li>
		<li>Pin back the liver to expose the stomach and pancreas. Take a picture.</li>
		<li>Cut the esophagus. Remove the colon and the intestines by pulling them out with forceps. Cut just above the liver and remove it to reveal the kidneys and adrenal glands. Take a picture. 
		NOTE: The removed organs should be kept in the fixative solution.</li>
	</ol>
	</li>
	<li>Isolate the thorax for the ECM analysis.
	<ol>
		<li>Cut the lower thorax along a straight line between the liver and the lungs. Cut the head off high enough to not cut the branchings of the carotid arteries.</li>
		<li>Gently remove the lateral ribs while maintaining the dorsal ribs and the spine. Peel up and scrape the dorsal fat away.</li>
		<li>Detach the thorax from the rest of the body and place it in a 10% buffered formalin phosphate solution.</li>
	</ol>
	</li>
</ol>
3. Embedding
<ol>
	<li>Decant the fixative into an appropriate hazardous waste bottle. Wash the samples with 1x PBS for 15 min three times.</li>
	<li>Use increasing concentrations of ethanol, and xylene to dehydrate the samples. The duration of all the following steps depends on the stage of the embryos. Please refer to Table 2 for details. 
	NOTE: Caution should be taken when changing solutions to avoid damaging the samples. The optimal parameters for sample processing can be empirically adjusted. Xylene will dissolve certain plastics; glass instruments and containers should be used.</li>
	<li>Replace xylene with paraffin for the desired duration. Leave the bottles in a 65 &#176;C incubator for the appropriate duration (Table 2).</li>
	<li>Use fresh paraffin to embed the samples in the desired position. 
	&#8203;NOTE: It is recommended to orient the sample into the middle of the paraffin block, with its dorsal side facing the top and the posterior side facing the front of the block. When orienting the sample, remember that the sample is embedded with the block facing upside down.</li>
</ol>
4. Episcopic confocal microscopy (ECM)
NOTE: After appropriate embedding, embryos undergo image collection serially via ECM for histopathology analysis. Individual slides can be recovered from the microtome for further studies.
<ol>
	<li>Take out the paraffin block from the -20 &#176;C freezer and remove the metal molds.</li>
	<li>Use a razor blade to trim the wax on the edges and the back of the cassette. Cut the wax surrounding the sample until it is encased in a small square of wax attached to the cassette. 
	NOTE: Use extreme caution while handling blades.</li>
	<li>Use the metal lever to clamp the paraffin block against the slicing stage of the microtome. Select the MAN (manual) function in Run Mode, raise the surface of the paraffin close to the blade, and run a few slides to ensure that the blade contacts the paraffin block.</li>
	<li>Open the LAS AF application and select MatrixScreener. Select Single Regular Matrix and load the appropriate previously saved template. Click on the Quick LUT button to switch to white and orange ombre.</li>
	<li>Select Set Up Jobs and drag the 405 nm visible laser to the maximum. Match the left edge of the spectrum block to the 405 nm line and drag the right edge to the 800 nm line. Mark the Pinhole option and start the Live view.</li>
	<li>Adjust the position of the laser projection to center the specimen on the screen and adjust the Zoom knob to ~20x. To optimize the resolution, set the gain to 1,250 V and maximize the blue area using the focus knob; then, reset the gain to approximately 750 V for imaging. 
	NOTE: Specific values may vary depending on the condition of the embryo.</li>
	<li>Switch the cutting method to Auto, set the thickness to around 50 &#181;m, and run the slides. Stop cutting when the lungs and airway are in view.</li>
	<li>Choose a cutting thickness between 8-10 &#181;m and stop the Live view. Open Microtome Communicator to start imaging. Ensure the temporary storage folder is empty before collecting images.</li>
	<li>Stop cutting when no additional heart structure is visualized. Close the Microtome Communicator application, and export the temporary file into one .tiff image series via the image processing software for 3D reconstructions later.</li>
</ol>
5. Three-dimensional (3D) reconstruction
NOTE: The purpose of 3D reconstruction is to process a 2D image stack from ECM imaging into 3D videos in the coronal, sagittal, and transverse orientations and to use the 3D videos for diagnosis of the structural and anatomical abnormalities in the samples.
<ol>
	<li>Open the ECM image stack in the image processing software.
	<ol>
		<li>Drag and drop image the files into the image processing software. Flip the ECM images horizontally by selecting Image &#62; Transform &#62; Flip Horizontally in the menu bar.</li>
		<li>Save the flipped image and close the image processing software.</li>
	</ol>
	</li>
	<li>Import the ECM image stack in the DICOM viewing software.
	<ol>
		<li>Drag and drop the horizontally flipped ECM images into the DICOM viewing software. Make sure there is a light blue border around the sample list, or images may be added to an existing sample folder.</li>
		<li>Select the links or the files to copy into the database when the pop-up window appears. A new sample will appear in the sample list with the same name as the file copied into the DICOM viewing software.</li>
		<li>Click on the newly added file to open it.</li>
	</ol>
	</li>
	<li>Perform 3D reconstruction.
	<ol>
		<li>Once the file is opened, click on the 2D/3D Reconstruction Tools menu from the toolbar and select 3D MPR.</li>
		<li>For Pixel X Resolution and Pixel Y Resolution, input the image resolution by giving the zoom used during ECM imaging. For the Slice Interval, input the slice thickness used to cut during ECM imaging. 
		NOTE: The camera resolution changes depending on the camera&#39;s zoom and may differ from camera to camera.</li>
	</ol>
	</li>
	<li>Use the different tools from the Tools tab on the left side to adjust image stacks as desired.
	<ol>
		<li>Use the WW/WL tool to adjust Window Width and Window Level. Click and drag the tool on the image upward to decrease image brightness and downward to increase it. Click and drag the tool on the image rightward to decrease image contrast and leftward to increase it. 
		NOTE: The optimal WW/WL settings for one structure may be suboptimal for another. For this reason, creating separate videos to view different structures is recommended.</li>
		<li>Use the Pan tool to drag the images into the desired positions. Use the Zoom tool to enlarge or shrink the image as desired and the Rotate tool to rotate the image as desired. 
		NOTE: Enlarging images may cause decreased image quality. Be cautious when rotating images, as doing so may cause the axes to flip.</li>
	</ol>
	</li>
	<li>Click and drag the colored axis of the first panel. Notice how rotating this axis changes the orientation of the other two panels. Rotate the three panels&#39; axes until the three panels represent coronal, sagittal, and transverse views of the sample. 
	NOTE: While reorienting the samples, maintain correct anterior/posterior orientation.</li>
	<li>Generate videos.
	<ol>
		<li>Once all three panels are properly positioned, oriented, and brightened, click on the panel representing the coronal view.</li>
		<li>Click on Movie Export on the right side of the menu bar. Click on Batch and drag the From and To sliders to encompass the entire region of interest. For Interval, select the Same as Thickness option. Save the video indicating the view&#39;s orientation.</li>
		<li>Review the video to see whether the structures of interest can be adequately identified. If not, use the tools (step 5.4) to readjust the videos as needed and re-save the video.</li>
	</ol>
	</li>
	<li>Repeat step 5.6 for transverse and sagittal views. Diagnose the samples using reconstructed videos. 
	NOTE: Carefully watch the videos in each orientation to make a complete assessment of whether the sample exhibits any anatomical abnormalities or disease phenotypes (Figure 3).</li>
</ol>

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The authors declare no conflicts of interest in this manuscript.

Genetically modified mice have been used to understand the pathomechanisms of congenital heart defects. The protocols we provide in this study attempt to streamline and standardize the process of assessing murine fetal heart defects. However, there are critical steps to note during the protocol. Mouse embryos grow significantly during each day of gestation, and the correct time to harvest a mouse can be determined by performing a fetal echocardiogram accurately. The fetal echocardiogram can be used to screen the fetal cardiovascular pathology. A 2D image allows for the identification of abnormal anatomy and cardiac function, with the assistance of color Doppler to examine the blood flow and detect any communications between the chambers of the heart or abnormal outflow and inflow tracts. To determine the CHD diagnosis, fetuses are scanned from embryonic day E14.5 when the outflow tract septation and cardiac chamber formation are completed. Scanning at earlier stages might reflect the developmental delay.
Histopathology is the standard to characterize CHDs8&#160;by using a microtome followed by optical microscope visualization. The major disadvantage of the standard method is the lack of an intuitive 3D display of the cardiovascular structures for diagnosis and the limitation in the lack of providing different views of the samples. An ECM histology is the most time-efficient method to characterize cardiac defects. If ECM is unavailable, a microtome may be used to section the embryo and determine cardiac phenotypes. Other options for obtaining cardiac imaging are performing high-throughput, high-resolution magnetic resonance imaging (MRI) or computed tomography (CT). One key aspect of MRI or CT is that multiple embryos can be imaged simultaneously; however, even after an ultrasound, CT, or MRI phenotyping, histopathology is required to confirm any CHD diagnosis.
Using ECM imaging for histopathology, the embryo is serially imaged after each cut using a laser scanning confocal microscope mounted over the automatic sliding microtome. The individual images collected from ECM allow for subsequent 3D reconstructions and allow the samples to be digitally resliced in any imaging plane without re-taking the images8,11. Such an operation enables a comprehensive assessment of the cardiac anatomy, which can be used in different developmental stages. Furthermore, individual slides could be recovered from the microtome while gathering the data from the ECM equipment. Although ECM histopathology is the gold standard to assess any structural cardiac anomaly, there are limitations with the availability of the equipment, software, and running time per embryo. The running time to section an embryonic heart can vary from 1-3 h per sample, depending on its size. Due to the complexity of the equipment, researchers must regularly check on the computer to make sure that the region of interest inside the field is being recorded. Researchers must also check the sample to ensure no paraffin wax shavings obstruct the sample from the laser scanner.
ECM and subsequent 3D reconstruction provide a complete high-resolution assessment of any structural cardiac defect regardless of the embedding plan of the specimen. These protocols have helped to successfully diagnose a range of CHDs and allowed us to understand the cardiac embryogenesis in murine models and pathologies related to genetic mutations.

Congenital heart diseases (CHDs) are the most common neonatal birth defect1,2, affecting about 0.8%-1.7% of neonates and resulting in significant neonatal mortality and morbidity3. A genetic etiology is strongly indicated with CHDs4,5. Genetically modified mouse models have been used widely to understand the complexity of CHDs and the mechanisms that cause them due to the mice having four-chamber hearts and comparable cardiac developmental DNA sequences in mouse and human fetuses6. Identifying the phenotype of the mouse mutants is the fundamental first step in characterizing the function of the targeted gene. Mouse models expressing gene dosage effects, in which a single genetic mutation can result in a spectrum of cardiac defects that mimic human CHDs, are important for understanding the complexity of CHDs and the mechanisms that cause them.
This article outlines a pipeline to characterize cardiac phenotypes in mouse models. The applied methods utilize fetal echocardiogram7, followed by necropsy and ECM histopathology7,8, which can display the detailed anatomy of developing murine cardiac phenotypes. A fetal echocardiogram is a noninvasive modality that allows direct visualization of multiple embryos with reasonable imaging resolution. In addition, a fetal echocardiogram provides a quick determination of the total number of embryos in a litter, their developing stages, and the relative orientation and location in the uterine horn. Using a spectral Doppler/color flow, abnormal embryos can be identified based on the structure, the hemodynamic disturbance, the growth restriction, or the development of&#160;hydrops. Since a fetal echocardiogram study is a noninvasive technique, it can be used to scan on multiple days and to observe the changes in hemodynamics or cardiac morphology. Obtaining high-quality imaging of fetal echocardiograms requires practice and skill, as specific heart defects may be missed due to a lack of experience and knowledge. Because of this, a more definitive analysis of cardiac morphology may be obtained through a combination of necropsy and ECM histopathology. Necropsy provides direct visualization of the arch structure, the relative relationships of the aorta and pulmonary artery, the size of the ventricles and atria, the position of the heart relative to the chest, and the bronchopulmonary structures. However, interior features such as the heart valves and wall thickness may be difficult to assess through necropsy alone. Thus, ECM histopathology is recommended for a conclusive diagnosis. ECM histopathology is a high-resolution visualization technique that allows for both 2D and 3D reconstruction of the image stack9. These images are obtained through serial Episcopic fluorescent imaging of a paraffin-embedded sample as it is thinly sectioned at a consistent interval by an automatic microtome. Unlike classical histology, images are captured as a section before it is cut from the block such that all images are captured within the same reference frame. Because of this, the 2D image stack produced by ECM histopathology may easily and reliably be reconstructed in three dimensions. This is done using a DICOM viewer, which allows 3D visualization of the images in the three anatomical planes: coronal, sagittal, and transverse. From these high-resolution 3D reconstructions, a definitive cardiac diagnosis may be made. The application of these three different visualization modalities, either individually or in combination, can provide accurate characterizations of structural heart defects in mouse embryos.

<table><tbody><tr><td>1x phosphate-buffered saline solution (PBS), PH7.4</td><td>Sigma Aldrich</td><td>P3813</td><td></td></tr><tr><td>1.5 mL Eppendorf tubes (or preferred vial for tissue storage)</td><td>SealRite</td><td>1615-5599</td><td></td></tr><tr><td>10% buffered formalin phosphate solution</td><td>Fisher Chemical</td><td>SF100-4</td><td></td></tr><tr><td>100% Ethanol</td><td>Decon Laboratories</td><td>2701</td><td></td></tr><tr><td>16% paraformaldehyde (PFA) fixative&amp;nbsp;</td><td>ThermoScientific</td><td>28908</td><td>4% working concentration freshly prepared in 1x PBS at 4 &amp;deg;C</td></tr><tr><td>50 mL tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>6&amp;ndash;12 Well plate or 20 mL vial&amp;nbsp; for embryo storage</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>Dissecting microscope&amp;nbsp;</td><td>Leica</td><td>MDG36</td><td></td></tr><tr><td>Dissecting Pins (A1 or A2 grade)</td><td>F.S.T</td><td>26002-15</td><td></td></tr><tr><td>Dissecting Plate&amp;nbsp;</td><td>F.S.T</td><td>FB0875713</td><td>Petri dish with paraffin base</td></tr><tr><td>Embedding molds</td><td>Sakura</td><td>4133</td><td></td></tr><tr><td>Extra narrow scissors (10.5 cm)</td><td>F.S.T</td><td>14088-10</td><td>1&amp;ndash;2 pairs&amp;nbsp;</td></tr><tr><td>Fiji application/Image J</td><td>NIH</td><td></td><td>Fiji.sc</td></tr><tr><td>Fine tip (0.05 mm x 0.01 mm) Dissecting Forceps (11 cm)</td><td>F.S.T</td><td>11252-00</td><td>2 Pairs</td></tr><tr><td>Hot forceps&amp;nbsp;</td><td>F.S.T</td><td>11252-00</td><td>For orientation of embryos</td></tr><tr><td>Industrial Marker for Wax Blocks&amp;nbsp;</td><td>Sharpie</td><td>2003898</td><td>Formatted for labratory use</td></tr><tr><td>Jenoptik ProgRes C14plus Microscope Camera&amp;nbsp;</td><td>Jenoptik</td><td>017953-650-26</td><td></td></tr><tr><td>Jenoptik ProgRess CapturePro acquisition software</td><td>Jenoptik</td><td></td><td>jenoptik.com</td></tr><tr><td>Large glass beaker&amp;nbsp;</td><td>Fisher Scientific</td><td>S111053</td><td>For melting paraffin</td></tr><tr><td>Leica M165 FC binocular microscope (16.5:1 zoom optics)</td><td>Leica</td><td>M165 FC</td><td></td></tr><tr><td>OsiriX MD Version 12.0</td><td>OsiriX</td><td></td><td>osirix-viewer.com&amp;nbsp;</td></tr><tr><td>Paraplast embedding paraffin wax</td><td>Millipore Sigma</td><td>1003230215</td><td></td></tr><tr><td>Small glass beaker</td><td>Fisher Scientific</td><td>S111045</td><td></td></tr><tr><td>Small, perforated spoon (14.5 cm)</td><td>F.S.T</td><td>10370-17</td><td></td></tr><tr><td>Straight Vannas Scissors (4&amp;ndash;8 mm)</td><td>F.S.T</td><td>15018-10</td><td>A pair</td></tr><tr><td>Vevo2100 ultrahigh-frequency ultrasound biomicroscope</td><td>FUJIFILM VisualSonics Inc.</td><td>Vevo2100</td><td></td></tr><tr><td>Xylene</td><td>Fisher Chemical</td><td>UN1307</td><td></td></tr></tbody></table>

a pipeline to characterize structural heart defects in the fetal mouse

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe CHD died before adulthood, with the maximum mortality during the first week of life. Remarkable advances in surgical techniques, diagnostic approaches, and medical management have led to marked improvements in outcomes. To address the critical research needs of understanding congenital heart defects, murine models have provided an ideal research platform, as they have very similar heart anatomy to humans and short gestation rates. The combination of genetic engineering with high-throughput phenotyping tools has allowed for the replication and diagnosis of structural heart defects to further elucidate the molecular pathways behind CHDs. The use of noninvasive fetal echocardiography to screen the cardiac phenotypes in mouse models coupled with the high fidelity of Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) histopathology with three-dimensional (3D) reconstructions enables a detailed view into the anatomy of various congenital heart defects. This protocol outlines a complete workflow of these methods to obtain an accurate diagnosis of murine congenital heart defects. Applying this phenotyping protocol to model organisms will allow for accurate CHD diagnosis, yielding insights into the mechanisms of CHD. Identifying the underlying mechanisms of CHD provide opportunities for potential therapies and interventions.

The mouse embryos with significant hemodynamic defects were noted to be embryonic lethal. A wide variety of CHDs can be identified through the high output, noninvasive fetal echocardiogram using different views (Figure 1).
Septal defects: The most common CHDs are septal defects such as a ventricular septal defect (VSD), an atrioventricular septal defect (AVSD), and an atrial septal defect (ASD)1. VSD or AVSD can be easily visualized using 2D images and color flow images. Blood flow across the ventricles or between the atria and the ventricles can be easily identified (Figure 3). ASD is difficult to distinguish with patent foramen ovale in a fetus.
Outflow tract anomalies: As shown in Figure 3, the flow in the main pulmonary artery is across the ascending aorta flow in normal embryos. In embryos with a double outlet of the right ventricle (DORV), both great arteries can be seen arising from the right ventricle. DORV is also associated with VSD (Figure 3D) and can be identified using color flow. However, given the small size of the embryos, DORV sometimes cannot be reliably distinguished from the overriding of the aorta, pulmonary atresia, or persistent truncus arteriosus (PTA). In PTA, only one outflow tract flow can be seen in the fetal echocardiogram (Figure&#160;3E). The detailed arch structure and main pulmonary artery can be observed using necropsy (Figure 2E,F).
Necropsy can quickly diagnose the situs status in the chest and abdomen and the cardiac position relative to the chest (either levocardia (Figure 2C-E) or dextrocardia). Outflow tract structures and the relative size of both atria and ventricles can be easily visualized (Figure 2E,F).
ECM histopathology is the gold standard technique for assessing any structural cardiac anomaly8,11,12. It provides an unparalleled resolution and detail to the structures of embryos. Three-dimensional reconstruction using different planes and views can easily identify the relationship between the great arteries and ventricles (Figure 3) and the defect between the ventricles and atria.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64582/64582fig01v2.jpg" /> 
Figure 1: The locations for measuring the crown-rump length in 2D echocardiogram images of embryos to determine the gestational age. 2D echocardiogram images of embryos at (A) E11.5, (B) E12.5, (C,D) E13.5-E14.5. (D) The diseased embryo is smaller than its (C) sibling and is noted to have a &#34;mushy&#34; appearance with hydrops (arrow). (A-C) Live embryos demonstrate distinct organs. Representative VB-mode with color images of E14.5 heart in a coronal 4-chamber view tilted anteriorly to image the (E) outflow tracts demonstrate intact ventricular septum with the normal relationship of great arteries, which is confirmed with ECM in the (E&#39;) coronal view. (F) Representative sagittal view of E14.5 heart demonstrates the left ventricular and right ventricular outflow tracts with the ascending aorta (AO) pointed cranially and the pulmonary artery pointed posteriorly (toward the spine), which is confirmed with ECM in the (F&#39;) sagittal view. (G) Representative transverse view of the left ventricle (LV) and right ventricle (RV), which is confirmed with ECM in the (G&#39;) transverse view. (H) A transverse view at the base of the heart in ECM demonstrates a distinct and separated aortic valve (AV) and pulmonary valve (PV). PV is anterior to AV. (H&#39;) Representative ECM transverse view image of persistent truncus arteriosus (PTA) demonstrates the left pulmonary artery and right pulmonary artery arising posteriorly from the undivided persistent truncal artery, with the left carotid artery arising anteriorly and cranially from the persistent truncal artery. Abbreviations: A: anterior, AO: aorta, AV: aortic valve, Cd: caudal, Cr: cranial, DAO: descending aorta, L: left, LA: left atrium, LCA: left carotid artery, LPA: left pulmonary artery, LV: left ventricle, P: posterior, PA: pulmonary artery, PTA: persistent truncus arteriosus, R: right, RA: right atrium, RPA: right pulmonary artery, RV: right ventricle. Scale bar: 0.5 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64582/64582fig01largev2.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/64582/64582fig02.jpg" /> 
Figure 2: Representative images of a necropsy to observe cardiovascular abnormalities. (A) An embryo pinned to a black-background dissecting dish with no obvious phenotypes. The image shows the gross anatomy of the head, digitals, chest cavity, and abdomen. (B) The dermis is removed from the pup, revealing submandibular glands, ribcage, and abdomen. (C) The ribcage is lifted, revealing the gross anatomy of the chest cavity, including the thymus, heart, lungs, and diaphragm. (D) A zoomed view of the chest with the ribcage removed. (E) The thymus is removed, revealing great vessels and trachea. (F) A zoomed view of great vessels. Abbreviations: AAO: ascending aorta, D: diaphragm, LA: left atrium, LCA: left carotid artery, LSVC: left superior vena cava, LV: left ventricle, P: pericardium, RA: right atrium, Rb: ribs, RCA: right carotid artery, RL: right lung, RSVC: right superior vena cava, RV: right ventricle, S: sternum, SCA: right subclavian artery, SMG: submandibular glands, T: trachea, Th: thymus. Scale bar: 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64582/64582fig02large.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/64582/64582fig03v2.jpg" /> 
Figure 3: Representative fetal echocardiogram (Echo) and images from episcopic confocal microscope (ECM). (A) Echo demonstrates an intact ventricular septum without an interventricular shunt and normal related great arteries in the normal control, confirmed by (A&#39;) ECM at E14.5-15.5 stage. (B) Ultrasound detection of an atrioventricular septal defect (AVSD). Echo imaging in the 4-chamber view demonstrates the communication between LA, RA, LV, and RV, confirmed by (B&#39;) ECM at the E14.5 stage. (C) Ultrasound diagnosis of the ventricular septal defect (VSD) with color flow demonstrates the flow across LV and RV, confirmed by (C&#39;) ECM at the E16.5 stage. (D) Echo and (D&#39;) ECM demonstrate DORV with a VSD between LV and RV with side-by-side great arteries (aorta is right to the pulmonary artery) at the E14.5 stage. (E) Ultrasound diagnosis of persistent truncus arteriosus (PTA) demonstrates communication between LV and RV with a single outflow tract overriding both ventricles (PTA). (E&#39;) This pathology is confirmed by ECM at the E14.5 stage. Scale bar: 0.5 mm. AO: aorta, AVSD: atrioventricular septal defect, Cd: caudal, Cr: cranial, L: left, LA: left atrium, LV: left ventricle, PA: pulmonary artery, PTA: persistent truncus arteriosus, R: right, RA: right atrium, RV: right ventricle, VSD: ventricular septal defect. Scale bar: 0.5 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64582/64582fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stage</td>
			<td>Crown-to-Rump Length (mm)</td>
			<td>Fetus Area (mm2)</td>
			<td>Heart Area (mm2)</td>
			<td>Heart Area/Fetus Area</td>
		</tr>
		<tr>
			<td>E12.5</td>
			<td>7.9 &#177; 0.8 (n = 77)</td>
			<td>23.2 &#177; 5 (n = 77)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E13.5</td>
			<td>10.5 &#177; 0.9 (n = 92)</td>
			<td>40.9 &#177; 7 (n = 92)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E14.5</td>
			<td>12.5 &#177; 0.9 (n = 101)</td>
			<td>57.6 &#177; 8 (n = 101)</td>
			<td>2.9 &#177; 0.5 (n = 70)</td>
			<td>0.050 &#177; 0.004 (n = 70)</td>
		</tr>
		<tr>
			<td>E15.5</td>
			<td>14.1 &#177; 0.5 (n = 134)</td>
			<td>71.4 &#177; 6 (n = 134)</td>
			<td>3.8 &#177; 0.4 (n = 87)</td>
			<td>0.053 &#177; 0.004 (n = 87)</td>
		</tr>
		<tr>
			<td>E16.5</td>
			<td>15.4 &#177; 0.6 (n = 112)</td>
			<td>82.7 &#177; 6 (n = 112)</td>
			<td>4.9 &#177; 0.5 (n = 87)</td>
			<td>0.058 &#177; 0.007 (n = 87)</td>
		</tr>
		<tr>
			<td>E17.5</td>
			<td>16.6 &#177; 0.4 (n = 211)</td>
			<td>96.9 &#177; 7 (n = 211)</td>
			<td>6.1 &#177; 0.6 (n = 146)</td>
			<td>0.063 &#177; 0.004 (n = 146)</td>
		</tr>
		<tr>
			<td>E18.5</td>
			<td>17.7 &#177; 0.6 (n = 139)</td>
			<td>112.1 &#177; 8 (n = 139)</td>
			<td>7.1 &#177; 0.8 (n = 93)</td>
			<td>0.063 &#177; 0.005 (n = 93)</td>
		</tr>
		<tr>
			<td>E19.5</td>
			<td>18.7 &#177; 0.7 (n = 57)</td>
			<td>126.7 &#177; 8 (n = 57)</td>
			<td>7.7 &#177; 0.7 (n = 36)</td>
			<td>0.062 &#177; 0.005 (n = 36)</td>
		</tr>
	</tbody>
</table>
Table 1: Developmental profile of Fetal Growth.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>E14.5&#160;</td>
			<td>E16.5&#160;</td>
			<td>E18.5&#160;</td>
			<td>Newborn&#160;</td>
		</tr>
		<tr>
			<td>70% Ethanol&#160;</td>
			<td>1 h</td>
			<td>1.5 h</td>
			<td>3 h&#160;</td>
			<td>4 h</td>
		</tr>
		<tr>
			<td>95% Ethanol&#160;</td>
			<td>35 min&#160;</td>
			<td>45 min&#160;</td>
			<td>1 h</td>
			<td>1 h</td>
		</tr>
		<tr>
			<td>95% Ethanol&#160;</td>
			<td>35 min&#160;</td>
			<td>45 min&#160;</td>
			<td>1 h</td>
			<td>1 h</td>
		</tr>
		<tr>
			<td>100% Ethanol&#160;</td>
			<td>15 min&#160;</td>
			<td>15 min&#160;</td>
			<td>30 min&#160;</td>
			<td>6 min&#160;</td>
		</tr>
		<tr>
			<td>Xylene 1</td>
			<td>20 min</td>
			<td>30 min</td>
			<td>40 min</td>
			<td>30 min</td>
		</tr>
		<tr>
			<td>Xylene 2</td>
			<td>20 min</td>
			<td>30 min</td>
			<td>40 min</td>
			<td>30 min</td>
		</tr>
		<tr>
			<td>Wax 1</td>
			<td>20 min</td>
			<td>20 min</td>
			<td>20 min</td>
			<td>30 min</td>
		</tr>
		<tr>
			<td>Wax 2</td>
			<td>20 min</td>
			<td>20 min</td>
			<td>20 min</td>
			<td>30 min</td>
		</tr>
		<tr>
			<td>Wax 3</td>
			<td>Overnight</td>
			<td>Overnight</td>
			<td>Overnight</td>
			<td>Overnight</td>
		</tr>
	</tbody>
</table>
Table 2: Protocol for embryo embedding based on embryonic days.

Watch this Scientific Journal Video about A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse at JoVE.com

A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse

This article details murine congenital heart disease (CHD) diagnostic methods using fetal echocardiography, necropsy, and Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) followed by three-dimensional (3D) reconstruction.

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe ...

a-pipeline-to-characterize-structural-heart-defects-in-the-fetal-mouse

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

1.7K Views.  University of Pittsburgh. This article details murine congenital heart disease (CHD) diagnostic methods using fetal echocardiography, necropsy, and Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) followed by three-dimensional (3D) reconstruction.

Video: A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse

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 ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

Medicine

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 ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the surgical and instrumental expertise required to accurately quantify the structure and organization across multiple length scales have stunted its advancement. This work provides a detailed description of the murine dissection, en bloc staining, sample processing, and correlative imaging procedures for depicting the heart valve at different length scales. Hydrostatic transvalvular pressure was used to control the temporal heterogeneity by chemically fixing the heart valve conformation. Micro-computed tomography (&#181;CT) was used to confirm the geometry of the heart valve and provide a reference for the downstream sample processing needed for the serial block face scanning electron microscopy (SBF-SEM). High-resolution serial SEM images of the extracellular matrix (ECM) were taken and reconstructed to provide a local 3D representation of its organization. &#181;CT and SBF-SEM imaging methods were then correlated to overcome the spatial variation across the pulmonary valve. Though the work presented is exclusively on the pulmonary valve, this methodology could be adopted for describing the hierarchical organization in biological systems and is pivotal for the structural characterization across multiple length scales.

Here, we describe a correlative workflow for the excision, pressurization, fixation, and imaging of the murine pulmonary valve to determine the gross conformation and local extracellular matrix structures.

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the ...

Engineering

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.

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 ...

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse

Summary

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Chapters in this video

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Murine Fetal Echocardiography

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

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve

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

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

Cell Labeling and Injection in Developing Embryonic Mouse Hearts