The authors acknowledge Dr. Kenzo Ivanovitch for previous work on this method and the group of Dr. Shigenori Nonaka (National Institutes of Natural Sciences, Japan) for providing the initial expertise on embryo mounting. This study was supported by Grant PGC2018-096486-B-I00 from the Spanish Ministerio de Ciencia e Innovaci&#243;n and Grant H2020-MSCA-ITN-2016-722427 from the EU Horizon 2020 program to MT and Grant 1380918 from the FEDER Andaluc&#237;a 2014-2020 Operating Program to JND. MS was supported by a La Caixa Foundation PhD fellowship (LCF/BQ/DE18/11670014) and The Company of Biologists travelling fellowship (DEVTF181145). The CNIC is supported by the Spanish Ministry of Science and the ProCNIC Foundation.

All animal procedures were approved by the CNIC Animal Experimentation Ethics Committee, by the Community of Madrid (Reference PROEX 220/15) and conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201/2005.
This protocol includes the use of two males from the fluorescent transgenic mouse line reporting NOTCH activity Tg(CBF:H2BVenus,+)18. See the Table of Materials for details about materials, animals, and equipment used in this protocol.
1. Tools and holder customization
<ol>
	<li>Cut 1 cm long tungsten wire pieces and sharpen them to 0.02-0.05 mm diameter using any of the simple methods available, for example, submerging the tip of the wire in a saturated sodium nitrite solution19.</li>
	<li>Prepare elbowed glass capillaries.
	<ol>
		<li>Place the midpoint of a standard 1.0 mm glass capillary over a Bunsen lighter for 3-6 s, pulling slowly from both ends until the capillary turns thin and flexible. At that point, break the capillary in half and heat it briefly to produce a 90&#176; bend 2 cm from the tip.</li>
	</ol>
	</li>
	<li>Saw a piece of polymethyl methacrylate measuring approximately 7 mm long, 4 mm wide, and 2 mm thick (Figure 3D). 
	NOTE: Polymethyl methacrylate sheets can be either obtained from recycled laboratory equipment or ordered new.</li>
	<li>Hold the methacrylate piece using a bench vise. Next, drill custom-sized holes transversally through the whole piece (Figure 3D). 
	NOTE: Typically, E6.5 to E7.5 mouse embryos require using 0.2-0.5 mm diameter drills.
	<ol>
		<li>Rotate the drill slowly while applying low but constant pressure. If a drill breaks inside the holder, discard the piece, as the metal cannot be taken out.</li>
	</ol>
	</li>
	<li>Use a fine file to smooth the holder&#39;s edges and adjust its size. Additionally, create a mild slope on the edges of the holder to facilitate embryo positioning (Figure 3D).</li>
	<li>Place the finished holder in a dish with distilled water to rinse it.</li>
	<li>Check the holder under the stereomicroscope to see if the holes contain drilling dust. If dust is present, use tungsten needles to remove it.</li>
	<li>To sterilize the holder, place it in a conical tube full of distilled water and sonicate it for 20 min with a minimum 20 W power output.</li>
</ol>
2. Media preparation
<ol>
	<li>To heat-inactivate the complement system, leave two 500 &#956;L aliquots of commercial or homemade20 rat serum at 56 &#176;C for 30 min. 
	NOTE: For a detailed protocol on how to prepare rat serum, see21.</li>
	<li>In a cell culture hood, prepare two aliquots of the medium for culturing the embryos in the microscope. For each, mix 490 &#956;L of DMEM for live-cell imaging, 500 &#956;L of inactivated rat serum, and 10 &#956;L&#160;of penicillin-streptomycin to obtain a final concentration of 50 &#181;g/mL penicillin and 50 &#181;g/mL streptomycin22. 
	NOTE: The volume of culture medium depends on the time and number of embryos to be cultured. As a rule of thumb, optimal growth requires a minimum of 200 &#956;L per embryo per day in E7.0 embryos.</li>
	<li>Using a 2 mL syringe, filter the medium through a 0.22 &#181;m pore size membrane filter into a new tube and place it lid-open in a cell culture incubator at 37 &#176;C and 7% CO2 for 1 h before embryo culture23.</li>
	<li>Prepare the dissection medium by adding to a 500 mL of DMEM supplemented with L-glutamine bottle the following: 50 mL of fetal bovine serum, 10 mL of 25 mM HEPES-NaOH (pH 7.2), and 10 mL of penicillin and streptomycin (50 mg/mL).</li>
	<li>Next, separate it into 50 mL aliquots, place three aliquots in a 37 &#176;C bath and store the rest at 4 &#176;C for up to 3 months.</li>
</ol>
3. Embryo dissection
<ol>
	<li>Euthanize an E6.75-E7.5 pregnant mouse.</li>
	<li>Extract the uterus and place it on a dry and clean paper wipe. Then, cut the uterus, inserting the tip of fine scissors from the mesometrial side and sliding the blade along to expose the deciduae and transfer them to the dissection medium. For a detailed protocol, see24.</li>
	<li>Dissect the embryos, keeping the ectoplacental cone intact (Figure 3A,B). For a detailed dissection method, refer to25.</li>
	<li>Then, peel off the Reichert&#39;s membrane, leaving part of it on the extraembryonic region (Figure 3B). 
	NOTE: Fine forceps are essential for this step. Inexperienced users can dissect embryos on 2% agarose gel-coated dishes to avoid bending the forceps&#39; tips against the dish surface (Figure 3C).</li>
	<li>To keep the dissection medium warm, replace it every 10 min. Moreover, dissect deciduae in groups of 3-4 while keeping the rest in dissection medium placed in a 37 &#176;C bath. Immediately place the dissected embryos in culture medium.</li>
	<li>Select the embryos with the desired fluorescence features using a fluorescence stereomicroscope and place them in the tube containing culture medium in the cell culture incubator.</li>
	<li>If the embryos are at E6.5 to E7.0 stages, close the lid and leave the embryos to recover for 2 h before imaging. 
	&#8203;NOTE: E6.5 to E7.0 embryos are sensitive. They can also be placed directly under the microscope but preculturing allows for selection of the ones that best recover from dissection, increasing the chance of success.</li>
</ol>
4. Microscope preparation
<ol>
	<li>Turn on all microscope components, including lasers, computer, and software. Turn on the temperature controllers and set to 37 &#176;C 3 h in advance to ensure equilibration.</li>
	<li>If using an immersion detection objective, clean it using a residue-free disposable wipe. Dip the objective&#39;s tip in double-distilled water using a 60 mm dish and leave it until the acquisition starts.</li>
	<li>Turn on the CO2 controller and set it to 7%.</li>
	<li>Place a drop of high-vacuum silicon grease on a 35 mm dish with glass coverslip bottom (14 mm diameter), place the holder on top of the drop, and press gently to immobilize it.</li>
	<li>Under a stereomicroscope, add culture medium to fill the glass-bottom portion of the dish. While doing so, aim the stream to the holder&#39;s holes to prevent formation of air bubbles in them.</li>
	<li>If the bubbles remain, use the glass capillary prepared in step 1.2, connected to a silicone tube with a mouthpiece, to suck the bubbles out gently. 
	&#8203;NOTE: The elbowed end of the capillary will facilitate access through the holes.</li>
</ol>
5. Embryo mounting
<ol>
	<li>Transfer 2-3 embryos that were left to recover in the incubator to the dish with the holder. Leave the rest in the incubator as a backup.</li>
	<li>Set the embryos in the holes by using a pair of forceps and a tapered tungsten needle. Use the needle to hook the embryo by the ectoplacental cone and insert it into a size-matching hole. To immobilize the embryo, rotate the cone by 90-120&#176; so that the Reichter&#39;s membrane adheres to the hole walls (Figure 3C). For an alternative description, see26.</li>
	<li>Carefully move the dish containing the embryos and the holder to the microscope plate (Figure 3F,G). 
	NOTE: If not moved steadily, the embryo can move out of the holder.</li>
	<li>Lower the objective until a liquid meniscus is formed at the medium-objective interface.</li>
	<li>Locate the embryo using transmitted light under the microscope binocular and place the embryo in focus.</li>
	<li>Mount the incubation chamber and seal it around the objective using tape (Figure 3F).</li>
	<li>Using a Live Capture at the microscope control software, adjust the Laser Output and Gain levels. 
	NOTE: For two-photon acquisition of green fluorescent protein (GFP) and Tdtomato reporters, 30% output laser power at 980 nm and 70% gain on non-descanned detectors were used in this protocol (Figure 3G).</li>
	<li>To avoid evaporation, cover the medium with paraffin oil by dripping it slowly on the objective. 
	&#8203;NOTE: This is the last point where additional embryos can be mounted in the holder. Once covered with paraffin oil, one would have to clean the holder and replace the medium to do so. For alternative mounting methods see27.</li>
</ol>
6. Image acquisition
<ol>
	<li>Set up time-lapse recording. Set a 5-10 min time interval with 3-5 &#181;m z-space between stacks. Leave a blank space on top of the z-stack to anticipate embryo growth, a minimum of 50 &#181;m, adding 30 &#181;m for every hour the acquisition will not be supervised.</li>
	<li>If available, enable the Auto-Save option to allow supervision of the acquisition from a personal computer to allow the adjustment of the z-stack size, if needed, to fit the area of interest within focus.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64273/64273fig03.jpg" /> 
Figure 3: Live imaging tools and setup. (A) Dissecting mouse embryos with an agarose-coated dish under the stereomicroscope. (B) Diagram of the steps to dissect mouse embryos for live imaging. (C) Embryo positioning in the holder. (D) Embryo holder design and features. (F) Incubator chamber around the immersion objective. (G) Diagram of final setup for time-lapse acquisition. Scale bar = 500 &#181;m (A). <a href="https://www.jove.com/files/ftp_upload/64273/64273fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have no conflicts of interest to disclose.

Early heart progenitors organize in a primitive heart tube that starts beating while it is still forming. Understanding how this process takes place is key to pinpoint the wide spectrum of congenital heart defects to specific morphogenetic events. For that, live imaging offers an opportunity to study normal and defective embryonic development with increased temporal resolution. This is especially useful to study early cardiac progenitor cells as they transition quickly through multiple differentiation and migration behav...

The heart forms early during embryogenesis to start pumping nutrients to the whole embryo, while it continues developing1. In mouse embryos, one and a half days after the initiation of gastrulation, a rudimentary heart organ assembles at the anterior pole2,3. By Early Streak (ES) stage, cardiac progenitors in the epiblast ingress through the primitive streak to the nascent mesodermal layer4,5,6 and start migrating to the anterior pole, where they differentiate to form the primitive heart tube. Throughout this process, early heart progenitors undergo cell rearrangements, shape transformations, and differentiation, in addition to migration7 (Figure 1).
Early cardiac progenitors have attracted researchers for nearly a century due to their remarkable ability to differentiate and build a functional organ simultaneously. Over the last two decades, clonal analysis and conditional knockout models have shown that early heart development implicates distinct cell sources in a highly dynamic process8,9,10. However, the 3D structure of the primitive heart tube and the dynamic nature of its morphogenesis make it challenging to study (Figure 1), and we are far from understanding its full complexity11.
To study these dynamic cellular processes, live imaging methods now offer an unprecedented detail7,12,13,14. In the mouse model, live approaches have been key to interrogating developmental topics that are difficult to address by static analysis7,13,15. While long-term ex vivo culture and robust microscope setups are advancing fast16,17, few researchers have the expertise to successfully image live embryos. Although paper-based publications provide enough technical details to reproduce live imaging experiments, some skills and tricks are hard to grasp without visual examples or peer-to-peer assistance. To accelerate this learning process and spread the use of live imaging among laboratories, we assembled a video protocol (Figure 2) that gathers the necessary skills to perform live imaging on gastrulating mouse embryos.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64273/64273fig01.jpg" /> 
Figure 1: Early differentiation of cardiac progenitor cells in the mouse embryo from the onset of gastrulation to the stage preceding primitive heart tube formation. Cardiac progenitor cells ingress the mesoderm soon after the start of gastrulation, migrating to the opposite side of the embryo. Morphological and embryonic day (E) stage are written on top of the diagrams. Dashed arrows depict the migration trajectory of primitive heart tube progenitors during gastrulation. This figure was adapted from11. Abbreviations: ES = Early Streak; MS = Middle Streak; EHF = Early Head Fold. <a href="https://www.jove.com/files/ftp_upload/64273/64273fig01large.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/64273/64273fig02.jpg" /> 
Figure 2: Workflow diagram for live imaging of early heart progenitors. <a href="https://www.jove.com/files/ftp_upload/64273/64273fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>#55 Forceps</td><td>Dumont&amp;nbsp;</td><td>11295-51</td><td></td></tr><tr><td>35 mm Dish with glass coverslip bottom 14 mm Diameter</td><td>Mattek</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>35 mm vise table&amp;nbsp;</td><td>Grandado</td><td>SKU 8798771617573</td><td></td></tr><tr><td>50 mL tubes</td><td>BD Falcon</td><td>352070</td><td></td></tr><tr><td>Distilled water</td><td></td><td></td><td></td></tr><tr><td>DMEM - Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco</td><td>11966025</td><td>&amp;nbsp;with L-Glutamine, without Glucose, without Na Pyruvate</td></tr><tr><td>Fetal Bovine Serum</td><td>Invitrogen</td><td>10438-026</td><td></td></tr><tr><td>Fluorescent reporter transgenic mice (Tg(CBF:H2BVenus,+)&amp;nbsp;</td><td>JAX</td><td></td><td></td></tr><tr><td>Fluorobrite DMEM</td><td>ThermoFisher</td><td>A1896701&amp;nbsp;</td><td>DMEM for live-cell imaging</td></tr><tr><td>High-vacuum silicone grease</td><td>Dow Corning</td><td>Z273554-1EA</td><td></td></tr><tr><td>Holder for wires</td><td>Perlen Pressen</td><td>pwb1</td><td></td></tr><tr><td>LSM 780 Upright microscope</td><td>Zeiss&amp;nbsp;</td><td></td><td></td></tr><tr><td>MaiTai Deepsee far red pulsed-laser tuned at 980 nm</td><td>Spectra-Physics</td><td></td><td></td></tr><tr><td>Non Descanned Detectors equipped with the filter sets&lt;br/&gt; cyan-yellow (BP450-500/BP520-560), green-red (BP500-520/BP570-610) and yellow-red (BP520-560/BP645-710)</td><td>Zeiss&amp;nbsp;</td><td></td><td></td></tr><tr><td>Obj: 20x water dipping 1.0 NA, long working distance</td><td>Zeiss&amp;nbsp;</td><td></td><td></td></tr><tr><td>P1000 and P200 pipettes</td><td></td><td></td><td></td></tr><tr><td>Paraffin Oil</td><td>Nidacon</td><td>VNI0049</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Invitrogen</td><td>15070-063</td><td>(the final concentration should be 50 &amp;mu;g/mL penicillin and 50 &amp;mu;g/mL streptomycin)</td></tr><tr><td>Petri dishes 35 mm x 10 mm</td><td>BD Falcon</td><td>351008</td><td></td></tr><tr><td>Pipette tips</td><td></td><td></td><td></td></tr><tr><td>Polymethyl methacrylate&amp;nbsp;</td><td></td><td></td><td>Reused from old laboratory equipment</td></tr><tr><td>Rat Serum culture embryo, male rats SPRAGUE DAWLEY RjHan SD</td><td>Janvier Labs</td><td>9979</td><td></td></tr><tr><td>Set of 160 mm fines</td><td>RS PRO</td><td>541-6933</td><td></td></tr><tr><td>Standard 1.0 mm glass capillaries</td><td>Anima Lab</td><td>&amp;nbsp;1B100F-3</td><td></td></tr><tr><td>Sterile 0.22 &amp;mu;m syringe filter</td><td>Corning</td><td>431218</td><td></td></tr><tr><td>Sterile 5 mL syringe</td><td>Fisher Scientific</td><td>15809152</td><td></td></tr><tr><td>Tungsten needles</td><td></td><td></td><td></td></tr><tr><td>Ultrasonic homogeniser (sonicator)</td><td>Bandelin</td><td>BASO_17021</td><td></td></tr></tbody></table>

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 used the protocol to visualize NOTCH signaling activation in early cardiac progenitors about to differentiate to endothelial cells during primitive heart tube morphogenesis. For that, we crossed wild-type C57BL/6-N mice with Tg(CBF:H2BVenus,+) mice18 to obtain embryos reporting NOTCH activity through yellow fluorescent protein Venus. At E7.5, Venus fluorescence is present throughout the neural ectoderm, with a few positive nuclei at the splanchnic and extraembryonic mesoderm. After 4 h, endothe...

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Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

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 ...

live-imaging-of-early-cardiac-progenitors-in-the-mouse-embryo

Research

JoVE Journal

Developmental Biology

1.3K Views.  Centro Nacional de Investigaciones Cardiovasculares (CNIC). Dit protocol maakt het mogelijk om muizenembryo's in beeld te brengen terwijl ze zich ontwikkelen. Dit maakt het mogelijk om vroege cardiogenese in detail te bestuderen. Net als het maken van stilstaande beelden van nepembryo's, geeft live beeldvorming volledige toegang tot het dynamische proces in de ontwikkeling van embryo's.De vaardigheden die nodig zijn voor het beeldvorming van het leven zijn moeilijk te begrijpen uit publicatie alleen tekst. Kijken naar een andere persoon die het...

Video: Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

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 ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neuroscience

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

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

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 ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

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