This work was supported by grant R00HL122360 from the National Institutes of Health, National Heart, Lung, and Blood Institute (NHLBI).

All methods described adhere to animal care guidelines of The University of North Carolina at Chapel Hill.
1. Preparation of micro-injection pipettes
<ol>
	<li>Pull glass capillaries using a micropipette puller. For some injections, needle beveling is recommended as it provides an extremely sharp surface devoid of structural impurities. To do this, polish the end of a pulled needle on a beveling wheel at an angle of 45&#176; for 15&#8722;20 min. 
	NOTE: Exact settings for pulling will vary based on the puller being used. The final inner diameter of the bevel should be between 20&#8722;40 &#181;m.</li>
	<li>Coat the inner and external surfaces of the glass capillary with silicone. First dip the needles in the siliconizing agent to coat the external surface of the needle and then backload the siliconizing agent solution into each micropipette to coat the inner surface. 
	NOTE: Coating of the glass capillaries should be done 24 h before the implantation experiment. Coating the glass capillaries with silicone provides a chemically inert surface to the glass. If the capillaries are left untreated, the cell suspension generated in later steps will adhere to the glass and plug the needle. Therefore, coating is necessary and vital to the success of the method. 
	CAUTION: The siliconizing agent is a ready-made commercially available mixture of heptane and 1,7-dichloro-1,1,3,3,5,5,7,7-octamethyltetrasiloxane (Table of Materials). It is extremely flammable and acutely toxic. Always handle with proper PPE inside a fume hood.
	<ol>
		<li>To backload the needle, load ~5&#8722;10 &#181;L of the siliconizing agent into a micro-injection pipette tip (Table of Materials). Place the micro-injection tip in the wide end of the pulled glass capillary and position the tip as far down as possible (close to the glass needle tip). Eject the siliconizing agent while slowly removing the loading pipette in order to minimize air bubbles in the needle.</li>
		<li>Leave the siliconizing agent in the glass needle for 10 min, remove by aspirating with a new loading pipette and allow needles dry overnight in a fume hood.</li>
	</ol>
	</li>
	<li>The morning of the experiment, rinse the glass capillaries with deionized water following the procedure in step 1.2 and allow to dry for 3&#8722;4 h.</li>
</ol>
2. Preparation of solutions
<ol>
	<li>Prepare 5 mL of trypsin neutralizing solution by supplementing 4.2 mL of Dulbecco&#39;s modified Eagle&#39;s medium and Ham&#39;s nutrient mixture F12 (DMEM/F12) with 750 &#181;L of Fetal Bovine Serum (FBS) and 50 &#181;L of Penicillin/Streptomycin. Store at 37 &#176;C until use.</li>
	<li>Prepare 5 &#181;M labeling dye solution by pipetting 5 &#181;L of 1 mM stock labeling dye (in dimethyl sulfoxide [DMSO], Table of Materials) into 995 &#181;L of Hank&#39;s balanced salt solution (HBSS). Vortex for 1 min and store at 37 &#176;C until use.</li>
	<li>Prepare fresh paraformaldehyde (PFA) by combining 10 mL of 32% PFA stock solution with 62 mL of molecular biology grade water and 8 mL of 10x Dulbecco&#39;s phosphate-buffered saline (DPBS). The final concentration is 4% PFA in 1x DPBS.</li>
</ol>
3. Preparation of host embryos
<ol>
	<li>Incubate fertile chicken eggs in a horizontal orientation in a humidified incubator at 38 &#176;C until Hamburger and Hamilton (HH) Stage 1910. 
	NOTE: The stage chosen for manipulation is flexible and entirely dependent upon the aims of each individual experiment.</li>
	<li>Score the &#34;flat&#34; end of the egg shell along the egg equator using angled forceps to make a small puncture &#62;1 mm in diameter. Insert an 18 G needle with attached 10 mL syringe through the puncture and remove ~5 mL of albumin. 
	NOTE: This anatomical location is external to the &#34;air cell&#34; inside the egg and keeps albumin from leaking once the puncture is made. This step is recommended as it &#34;drops&#34; the embryo away from the egg shell, preventing potential damage in the subsequent steps.</li>
	<li>Apply transparent tape to the top of the egg shell. Score with angled forceps and cut a ~2.5 cm window using curved tenotomy scissors.</li>
	<li>Inspect and stage the embryo based on criteria established by Hamburger and Hamilton10 and seal the puncture from step 3.2 with transparent tape. 
	NOTE: Here, stage HH 19 embryos are used which have 37&#8722;40 somites extending into the tail bud. The puncture should not be sealed until after the window is opened along the top of the egg (step 3.3).</li>
	<li>Inject ~200 &#181;L of India Ink/HBSS mixture (1:5) beneath the embryo using a 32 G needle with attached 1 mL syringe. 
	NOTE: India ink provides visual contrast between the embryo and the yolk beneath. Alternative dyes such as neutral red or commercially available cyan fluorescent protein (CFP) or blue fluorescent protein (BFP) fluorescence filter sets can be used to improve contrast.</li>
	<li>Add 1 mL of HBSS dropwise onto the embryonic disc and seal windowed shells with paraffin film. Place eggs back in the humidified incubator until ready for injection.</li>
</ol>
4. Isolation of donor tissue
<ol>
	<li>Incubate donor fertile chicken eggs in a humidified incubator at 38 &#176;C until Stage HH 19 (or desired stage).</li>
	<li>Remove the embryo from the egg and place in a 100 mm x 15 mm petri dish containing sterile HBSS at room temperature (RT).</li>
	<li>Surgically microdissect atrial donor tissue from each embryo by first isolating the entire embryonic heart from the embryo and then by isolating the atria from the heart using forceps, tenotomy scissors, and microspatula under a stereo dissecting microscope. Pool in a sterile 1.5 mL microcentrifuge tube containing 1 mL of HBSS on ice.</li>
	<li>Once all donor tissue has been collected, pellet the tissue by centrifugation at 1000 x g for 5 min at 4 &#176;C in a fixed-angle microcentrifuge.</li>
</ol>
5. Trypsin digestion of donor tissue
<ol>
	<li>Resuspend cell pellets in 1 mL of prewarmed 0.05% trypsin-EDTA and incubate at 37 &#176;C for 15 min in a shaking heat block at 300 rpm. Alternatively, use a water bath with periodical agitation of the sample.</li>
	<li>Pipette the digestion solution up and down to break up any remaining tissue, and pellet as in step 4.4.</li>
	<li>Resuspend the pellet in 1 mL of the trypsin neutralizing solution and centrifuge as in step 4.4.</li>
	<li>Resuspend the cells in 400 &#181;L of red fluorescent labeling dye solution and incubate at 37 &#176;C for 20 min in a heat block. Alternatively, use a water bath.</li>
	<li>Once the labeling reaction is finished, pellet the cells as in step 4.4 and wash with 1 mL of HBSS (number of wash steps can be varied between 1 and 3).</li>
	<li>Resuspend the labeled, pelleted cells at a concentration of ~50,000 cells/&#181;L, which generally results in a 5&#8722;10 &#181;L working volume depending on the total cell yield. 
	NOTE: Cell concentrations below ~50,000 cells/&#181;L can result in poor injection efficiency.</li>
</ol>
6. In-vivo injection
<ol>
	<li>Backload the cell suspension into a silicone treated glass capillary pipette following the procedures in step 1.2. Mount the pipette into the pressure microinjector apparatus.</li>
	<li>Remove host embryos from humidified incubator and place in an egg holder underneath the fluorescent stereo dissecting microscope.
	<ol>
		<li>Open the vitelline membrane using sterile fine forceps and make a small incision (~0.5&#8722;1.0 mm in length) in the pericardium. Additional manipulation/dissection may be needed depending on target region for injection.</li>
	</ol>
	</li>
	<li>Position the microinjector such that the tip of the microinjection needle penetrates the target tissue.
	<ol>
		<li>Pressure inject cells, and use the fluorescent label to determine that implanted cells are present in desired tissue. For typical injections, apply single pulses less than 0.5 s in duration ranging from 100-400 hectopascals in pressure. 
		NOTE: Pulse length and absolute pressure will vary depending on the number of cells to be injected and can be modified to suit individual needs.</li>
		<li>Retract the microinjector apparatus and remove the egg from the holder after pressure injecting.</li>
	</ol>
	</li>
	<li>Add 1 mL of warm HBSS dropwise onto the embryo, seal eggs using transparent packing tape, and incubate in the humidified incubator at 38 &#176;C for 24 h post implantation.</li>
</ol>
7. Isolation and analysis
<ol>
	<li>Isolate host embryos in RT HBSS using forceps, tenotomy scissors, and microspatula similar to step 4.3, and fix in 4% PFA overnight at 4 &#176;C with gentle rocking.</li>
	<li>Wash embryos 3 x 5 min in HBSS at RT with gentle rocking, and store in HBSS at 4 &#176;C for further downstream analysis (microscopy, immunohistochemistry, in-situ hybridization, etc.).</li>
</ol>

<ol>
	<li>Guo, Y. X., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Mosaics+for+Greater+Precision+in+Cardiovascular+Research.">Genetic Mosaics for Greater Precision in Cardiovascular Research.</a> Circulation Research. 123, 27-29 (2018).</li><li>Guo, Y. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Cardiac+Myocyte+Maturation+Using+CASAAV,+a+Platform+for+Rapid+Dissection+of+Cardiac+Myocyte+Gene+Function+In+Vivo.">Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo.</a> Circulation Research. 120 (12), 1874-1888 (2017).</li><li>Rugh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experimental embryology; techniques and procedures. 3rd edition. , (1962).</li><li>Slack, J. M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essential developmental biology. 3rd edition. , (2013).</li><li>Reinecke, H., Zhang, M., Bartosek, T., Murray, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival,+integration,+and+differentation+of+cardiomyocyte+grafts:+a+study+in+normal+and+injured+rat+hearts.">Survival, integration, and differentation of cardiomyocyte grafts: a study in normal and injured rat hearts.</a> Circulation. 100, 193-202 (1999).</li><li>Rojas, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+purified+iPSC-derived+cardiomyocytes+in+myocardial+infarction.">Transplantation of purified iPSC-derived cardiomyocytes in myocardial infarction.</a> PLOS ONE. 12, 0173222 (2017).</li><li>Zhang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+Grafting+for+Cardiac+Repair:+Graft+Cell+Death+and+Anti-Death+Strategies.">Cardiomyocyte Grafting for Cardiac Repair: Graft Cell Death and Anti-Death Strategies.</a> Journal of Molecular and Cellular Cardiology. 33 (5), 907-921 (2001).</li><li>Bressan, M., Liu, G., Mikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+mesodermal+cues+assign+avian+cardiac+pacemaker+fate+potential+in+a+tertiary+heart+field.">Early mesodermal cues assign avian cardiac pacemaker fate potential in a tertiary heart field.</a> Science. 340 (6133), 744-748 (2013).</li><li>Bressan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+myocardial-endocardial+interactions+pattern+the+delay+in+atrioventricular+junction+conduction.">Reciprocal myocardial-endocardial interactions pattern the delay in atrioventricular junction conduction.</a> Development. 141 (21), 4149-4157 (2014).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88 (1), 49-92 (1951).</li></ol>

The authors have nothing to disclose.

The ability to define how microenvironmental conditions impact cardiac cell fate specification and lineage stabilization is fundamental to creating a compressive understanding of congenital heart disease as well as to developing efficient protocols for proper maturation of stem cell or somatic cell reprograming-based cardiomyocytes. The protocol outlined above gives investigators the ability to directly assay cardiac cell development under altered in vivo conditions, allowing for cell autonomous maturation processes to be separated from paracrine/juxtacrine and/or hemodynamic cues. When combined with high resolution imaging, genetic analysis, and physiological assays, this technique can serve as a powerful complement to existing transgenic models.
Form a technical stand point, the protocol presented here relies on efficient isolation, labeling, and precise implantation of donor heart cells into host embryonic tissues. The use of a microinjection system greatly aids in the targeting of the donor cells and allows for successful implantation without the need for creating a large engraftment site in the host tissue. Some operational skill is required to perform this technique however, as reduced viability can result if the injection needle is not carefully placed in the target tissue (causing rupture of the heart or local vasculature). Care and thought should also be given to the isolation and labeling steps. Over digestion of the donor tissue can lead to poor implantation efficiency, and transient labeling techniques can limit the time window over which donor cells can be tracked (as cell division can dilute the label).
This technique is highly modifiable and can be adapted for a variety of purposes. For example, donor cells from a large range of tissues and stages can be isolated (though optimization of the enzymatic digestion is required) and can similarly be injected into a variety of host tissues across different stages of development. Similarly, the labeling approach can be modified to track cells across different temporal windows, including the use of fluorescent inorganic semiconductor nanocrystals for longer transient labeling and implantation of quail cells or cells from green fluorescent protein transgenic (GFP+) donor embryos11 for permeant labeling.
While we currently use this technique for avian implantation studies, we feel that it could be used for a large range of chimeric studies in the future. For example, genetically altered cardiac cells from transgenic organisms could be isolated and microinjected into the avian heart using a very similar protocol. Furthermore, cells differentiated into cardiomyocytes from stems cells or via somatic cell reprograming approaches could be microinjected into the embryonic heart to evaluate their integration into the tissue and/or maturation under in vivo biomechanical conditions.

Cardiac developmental research has benefitted enormously from the advent of germline transgenic model systems which have identified many of the gene regulatory networks that pattern different cell lineages and functional domains in the heart. However, identifying how these gene networks interact with and respond to microenvironmental conditions, including paracrine/juxtacrine signals and biophysical inputs (stretch, strain, hemodynamic flow), can be challenging. As such, it is not always easy to determine whether a cellular phenotype arises as a direct consequence of a genetic perturbation or as a secondary result of an adaption to changes in cardiac biomechanics or higher order tissue composition1,2.
Grafting experiments, which have classically been used to address concepts of fate specification, commitment, induction, and competence3,4, would represent an ideal approach to circumvent some of the challenges inherent in defining cell autonomous versus environmental influence in the heart. Unfortunately, heart contractions make standard grafting approaches difficult. Rapid movement of the tissue often prevents grafted cells from adhering to the heart and large tissue punctures (normally required for grafting) frequently lead to heart failure and embryonic lethality5,6,7. Therefore, we have developed a pressure-based, microinjection system for precision cellular implantation into the developing chick heart, circumventing the technical hurdles of tissue grafting described previously8,9. Using this technique, individual or small groups of cardiac cells isolated from a donor embryo can be microinjected into a variety of regions of a host embryonic heart eliminating the need for extensive host preparation and the large tissue insults that arise using standard grafting techniques. The microinjection needles used for these implantation studies have an outer diameter of ~30&#8722;40 &#181;m, which means that the needle can be placed directly in the target tissue (i.e., can penetrate the embryonic myocardial wall) and cells can be focally delivered with minimal damage to the surrounding tissue. The protocol can be used to perform a variety of isotopic, heterotopic, isochoric, and heterochronic manipulations, providing a rapid, flexible, and low-cost approach to directly examine classical experimental embryological paradigms in the developing four-chambered heart.
In the protocol outlined below, we label donor cells with a cell permeant fluorescent dye, which allows for the success of a microinjection experiment to be monitored in real-time and the location of engrafted cells to be documented without the need for any additional staining. However, it should be noted that this approach is best suited for short term experiments (approximately 48 h) as the fluorescent dye can be lost through cell division. Alternative approaches can be used for longer term experiments.
While we are presenting this technique in the context of cardiac development, we have used it to great effect for cell implantation experiments into the mesoderm, head, limbs, and somites. As such the basic approach described below is highly tractable and can be used in a variety of organ systems.

<table border="0" cellpadding="0" cellspacing="0" style="width:1133px;" width="1133">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">1 mL Insulin Syringe</td>
			<td style="width:257px;">BD</td>
			<td style="width:268px;">329654</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:441px;">1.7 mL Microtubes, Clear</td>
			<td style="width:257px;">Genesee Scientific</td>
			<td style="width:268px;">24-282</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">10 ml Syringe</td>
			<td style="width:257px;">BD</td>
			<td style="width:268px;">305482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1000ul Reach Barrier Tip Racked, Sterile</td>
			<td style="width:257px;">Genesee Scientific</td>
			<td style="width:268px;">24-430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">15 mL Centriguge Tubes, Racked</td>
			<td style="width:257px;">Genesee Scientific</td>
			<td style="width:268px;">28-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">1588 Genesis Hova-Bator Incubator</td>
			<td style="width:257px;">GQF</td>
			<td>813927021221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18G x 1 1/2 Needle</td>
			<td style="width:257px;">BD</td>
			<td style="width:268px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200ul Barrier Tip Low Binding, Racked, Sterile</td>
			<td style="width:257px;">Genesee Scientific</td>
			<td style="width:268px;">24-412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">32G x 1/2&#34; Needle</td>
			<td style="width:257px;">TSK Steriject Air-Tite</td>
			<td style="width:268px;">TSK3213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Alchohol Wipes 70%</td>
			<td style="width:257px;">Thermo Fisher Scientific</td>
			<td>19015744</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Angled Forceps</td>
			<td style="width:257px;">Fine Scientific Tools</td>
			<td style="width:268px;">11260-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Backloading Tips</td>
			<td style="width:257px;">Eppendorf</td>
			<td style="width:268px;">930001007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Black India Ink</td>
			<td>KOH-I-NOOR</td>
			<td style="width:268px;">3084-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTracker Green CMF</td>
			<td style="width:257px;">Thermo Fisher Scientific</td>
			<td>C7025</td>
			<td>1 mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTracker Red CMTPX</td>
			<td style="width:257px;">Thermo Fisher Scientific</td>
			<td>C34552</td>
			<td>1 mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Centrifuge</td>
			<td style="width:257px;">Eppendorf</td>
			<td style="width:268px;">5424R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Commercial Grade Packing Tape</td>
			<td style="width:257px;">Staples</td>
			<td>2619001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Curved Tenotomy Scissors</td>
			<td style="width:257px;">Fine Scientific Tools</td>
			<td style="width:268px;">14067-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">DMEM/F12</td>
			<td style="width:257px;">Thermo Fisher Scientific</td>
			<td style="width:268px;">11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">DMSO, anhydrous</td>
			<td style="width:257px;">Thermo Fisher Scientific</td>
			<td style="width:268px;">D12345</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS (10X), no calcium, no magnesium</td>
			<td style="width:257px;">Thermo Fisher Scientific</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Femtojet 4i</td>
			<td style="width:257px;">Eppendorf</td>
			<td style="width:268px;">5252000021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Fetal Bovine Serum</td>
			<td style="width:257px;">Thermo Fisher Scientific</td>
			<td style="width:268px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Hatching Eggs</td>
			<td style="width:257px;">Pilgrim&#39;s Hatchery</td>
			<td style="width:268px;">--</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">HBSS, calcium, magnesium, no phenol red</td>
			<td style="width:257px;">Thermo Fisher Scientific</td>
			<td style="width:268px;">14025-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Injectman 4</td>
			<td style="width:257px;">Eppendorf</td>
			<td>5192000027D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Micromanipulator</td>
			<td style="width:257px;">Leica Microsystems&#160;</td>
			<td style="width:268px;">--</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Parafilm</td>
			<td style="width:257px;">SIGMA</td>
			<td>P6543-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 32% in aqueous solution, EM Grade</td>
			<td style="width:257px;">VWR</td>
			<td>100496-496</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Penicillin/Streptomycin</td>
			<td style="width:257px;">Thermo Fisher Scientific</td>
			<td style="width:268px;">15140-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Petri Dish</td>
			<td style="width:257px;">Genesee Scientific</td>
			<td style="width:268px;">32-107</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:441px;">Pipette Grinder</td>
			<td>Narishige</td>
			<td>EG-44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Pipette Puller</td>
			<td style="width:257px;">HEKA</td>
			<td style="width:268px;">PIP 6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Scotch Transparent Tape</td>
			<td style="width:257px;">Staples</td>
			<td style="width:268px;">487909</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Sigmacote</td>
			<td style="width:257px;">SIGMA</td>
			<td>SL2-25ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Stereo Microscope</td>
			<td style="width:257px;">Leica</td>
			<td style="width:268px;">--</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">ThermoMixer C</td>
			<td style="width:257px;">Eppendorf</td>
			<td>5382000023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Thin Wall Glass Capillaries</td>
			<td style="width:257px;">World Precision Instruments</td>
			<td>TW100F-4&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Transfer Pipette</td>
			<td style="width:257px;">Thermo Fisher Scientific</td>
			<td style="width:268px;">273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:257px;">Thermo Fisher Scientific</td>
			<td>25300-054</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

After 24 h incubation, the heart and surround tissue of host embryos were isolated, photographed (Figure 1A), and processed for immunofluorescent analysis. In this example, donor atrial myocytes were microinjected into the proepicardium of a similar staged host embryo. The host embryo was then stained with the muscle marker (MF20 green) and 4&#39;,6-diamidino-2-phenylindole (DAPI; blue). Injected cells (red) are clearly visible (Figure 1B). Possible adjustments to consider if cells are not visible include: donor tissue was over digested (cells would be unable to attach), labeling dye solution was too dilute, cells were over-washed, or multiple cell divisions resulted in loss of the label.
To confirm that the injected cells in this example were myocardial, we optically sectioned this embryo using a confocal microscope (Figure 1C-E). The only MF20 positive cells within the proepicardium (PE) are the fluorescent red positive cells that were focally implanted.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59267/59267fig1.jpg" /> 
Figure 1: Representative images of embryos isolated 24 hours post injection. (A) Low magnification brightfield image of the trunk region of an E3.5 (HH Stage 19) chick embryo. (B) Merged image showing injected cells (red), cardiomyocytes (green), and DAPI. Cells were isolated from the atria and microinjected into the proepicardium. (C) High magnification confocal imaging showing labeled cells in the core of the proepicardium. (D) High magnification confocal imaging confirming CT Red labeled cells are cardiomyocytes. (E) Three-dimensional (3D) reconstruction of injected cells from panels D and E. At, atria; OFT, outflow tract; PE, proepicardium; Vt, ventricle; MF20, Myosin 4. <a href="https://www.jove.com/files/ftp_upload/59267/59267fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo at JoVE.com

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

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 ...

microinjection-based-system-for-vivo-implantation-embryonic

Research

JoVE Journal

Developmental Biology

6.1K Views.  University of North Carolina at Chapel Hill. 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.

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

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

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

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

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

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

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

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...