We thank Dean Li for sharing his laboratory's protocol for aortic SMC isolation. Funding support was provided by the National Institutes of Health (R21NS088854, R01HL125815, and R01HL133016 to D.M.G), the American Heart Association (Grant-in-Aid 14GRNT19990019 to D.M.G.), and Yale University (Brown-Coxe Fellowship to A.M. and startup funds to D.M.G.).

All mouse protocols are approved by the Institutional Animal Care and Use Committee at Yale University.
1. In Vivo Embryonic Fate Mapping and Clonal Analysis
Note: We have used these approaches widely to evaluate the origins of cells and their clonal architecture in development and disease models7,8,9,10.
<ol>
	<li>Set up mating...

<ol>
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In contrast to the extensive investigations of the murine aorta and its major branches in adult pathological conditions, such as models of atherosclerosis, less is known regarding the morphogenesis and the pathogenesis of the embryonic and perinatal aorta. Here, we focus on the embryonic/perinatal aorta, specifically the SMCs, and provide protocols to study the aorta through in vivo, tissue explant, and SMC isolation approaches. These complimentary approaches provide the investigator with diverse approaches to s...

The circulatory systems of multicellular organisms function to deliver nutrients and oxygen to cells that are not in contact with the external environment and to remove waste products and carbon dioxide from these cells. In vertebrates, the primary circulatory system consists of the heart, which pumps blood through a series of blood vessels. The walls of large blood vessels, such as arteries and veins, consist of three layers: i) the intima, or inner layer of endothelial cells; ii) the media, or middle layer of alternating circumferentially elongated smooth muscle cells SMCs and elastic lamellae; and iii) the adventitia, or outer layer of connective tissue and fibroblasts. The vast majority of studies in vascular biology focus on endothelial cells, investigating the formation of new endothelial cell-lined tubes through angiogenesis. In comparison, SMCs receive relatively little attention. However, SMCs are a critical cell type in the construction of the normal arterial wall and in vascular pathologies.
The aorta is the largest-caliber artery in the body, receiving the cardiac output from the left ventricle of the heart. It is afflicted by diverse human diseases, including atherosclerosis, aneurysm, and dissection. In adult organisms, the aorta and its major branches are intensely studied in models of vascular disease. For instance, high fat diet fed mice that are null for the gene encoding the low-density lipoprotein receptor or apolipoprotein E, develop atherosclerosis, and recent fate mapping studies indicate that pre-existing SMCs give rise to multiple cell types in the atherosclerotic plaque1. In aortic aneurysms, pathological changes include SMC apoptosis and extracellular matrix remodeling2,3.
Substantially less is known regarding SMC morphogenesis and pathogenesis during the embryonic and perinatal periods. Here, we provide protocols for studying embryonic and perinatal aortic SMCs in vivo, in tissue explants and in isolated cells. For instance, the first section of the protocol delineates fate mapping and clonal analysis in embryonic mice. Cre recombinase expressed under the control of a cell-specific promoter facilitates the marking of specific cells and their progeny4,5,6; however, temporal control of cell-specific labeling can be challenging during embryonic development in mice. In this context, with embryos expressing the conditional CreER under a promoter active in SMCs (e.g.,Myh11 or Acta2) and a Cre reporter, we provide methods for injecting tamoxifen or its active metabolite 4-OH-tamoxifen in pregnant dams and for analyzing the labeled cells in embryos or postnatal offspring. Furthermore, in contrast to fate mapping studies, which predominately utilize Cre reporters with a single reporter fluorophore1,7, clonal analysis is substantially enhanced with multi-color Cre reporters.
The second and third sections of protocol describe methods for isolating and culturing embryonic aortic explants and aortic SMCs from neonates, respectively. These approaches allow for the manipulation of signaling pathways, specifically in aortic explants or SMCs, and for analyzing the direct effects of pharmacological agents. Thus, the role of specific genes in the tissue of interest can be screened in a far more rapid fashion than through traditional genetic manipulations in mice. In addition, the isolated SMC studies facilitate the analysis of cell migration and adhesion, which are technically limited in vivo.
Finally, the fourth protocol section delineates the placement of a subcutaneous osmotic mini-pump loaded with pharmacological agents in pregnant (or non-pregnant) mice. This method facilitates the analysis of the effect on embryonic development caused by agents that require continuous infusion because of rapid metabolism. The alternative of frequent injections is not practical for many agents and should be avoided, as it may cause significant discomfort in the pregnant dam.

<table width="1061">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Tamoxifen</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:111px;">T5648</td>
			<td style="width:412px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Corn oil</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:111px;">C-8267</td>
			<td>Vehicle for tamoxifen</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">4-OH-tamoxifen</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:111px;">H7904</td>
			<td>Active metabolite of tamoxifen</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Progesterone</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:111px;">P8783-5G</td>
			<td>Use at half the concentration of tamoxifen</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">OCT compound</td>
			<td style="width:216px;">Sakura tissue tek</td>
			<td style="width:111px;">4583</td>
			<td>For making cryoblocks</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Cryomolds</td>
			<td style="width:216px;">Polysciences inc</td>
			<td style="width:111px;">18986</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">DAPI</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:111px;">D9542</td>
			<td>IHC staining of nucleus, final concentration 5 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy3 directly conjugated anti-SMA antibody</td>
			<td>Sigma</td>
			<td>A2547</td>
			<td>IHC staining of SMA, final dilution 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Anti-CD31 antibody</td>
			<td style="width:216px;">BD Pharmingen</td>
			<td style="width:111px;">550274</td>
			<td>IHC staining of GFP, final concentration 0.006 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Anti-GFP antibody</td>
			<td style="width:216px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">A-11121</td>
			<td>IHC staining of CD31, final concentration 0.0016 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibody goat anti-rabbit, Alexa 647</td>
			<td style="width:216px;">Life Technologies</td>
			<td>a21244</td>
			<td>IHC staining, final concentration 0.004 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibody goat anti-rabbit, Alexa 488</td>
			<td style="width:216px;">Life Technologies</td>
			<td>a11008</td>
			<td>IHC staining, final concentration 0.004 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">DMEM</td>
			<td style="width:216px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">10567-014</td>
			<td>For cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">FBS</td>
			<td style="width:216px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">10437028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Anti-integrin beta3 blocking antibody</td>
			<td style="width:216px;">BD Biosciences</td>
			<td style="width:111px;">553343</td>
			<td>Clone 2C9.G2, final concentration 0.02 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase</td>
			<td>Worthington Biochemical Corp</td>
			<td>44H14977A</td>
			<td>For digesting aorta</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elastase</td>
			<td>Worthington Biochemical Corp</td>
			<td>34K15139</td>
			<td>For digesting aorta</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Antibiotic-antimycotic (100X)</td>
			<td style="width:216px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">15240062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Recombinant human FGF</td>
			<td style="width:216px;">Promega</td>
			<td style="width:111px;">G5071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Recombinant human EGF</td>
			<td style="width:216px;">Promega</td>
			<td style="width:111px;">G5021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Penicillin/streptomycin (10,000 U/ml)</td>
			<td style="width:216px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Amphotericin B</td>
			<td style="width:216px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">15290026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Tissue culture plates</td>
			<td style="width:216px;">Corning</td>
			<td style="width:111px;">CLS430165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:323px;height:21px;">Alzet osmotic mini-pump</td>
			<td style="width:216px;">Durect Corporation</td>
			<td style="width:111px;">2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECLIPSE 80i Upright Fluorescent Microscope</td>
			<td style="width:216px;">Nikon </td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"> TCS SP5</td>
			<td style="width:216px;">Leica</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Branson Sonifier 450</td>
			<td style="width:216px;">VWR</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Myh11-CreERT2 mice</td>
			<td style="width:216px;">The Jackson Laboratory </td>
			<td>19079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acta2-CreERT2 mice</td>
			<td style="width:216px;"></td>
			<td style="width:111px;"></td>
			<td>Obtained from lab of Dr. Pierre Chambon and Daniel Metzger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROSA26R-CreERT2 mice</td>
			<td style="width:216px;">The Jackson Laboratory </td>
			<td style="width:111px;">8463</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROSA26R(mTmG/mTmG) mice</td>
			<td style="width:216px;">The Jackson Laboratory </td>
			<td> 026862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROSA26R(EYFP/EYFP) mice</td>
			<td style="width:216px;">The Jackson Laboratory </td>
			<td> 006148 </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROSA26R(Confetti/Confetti) mice</td>
			<td style="width:216px;">The Jackson Laboratory </td>
			<td>13731</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROSA26R(Rb/Rb) mice</td>
			<td style="width:216px;">Lab of Dr. Irv Weissman</td>
			<td style="width:111px;"></td>
			<td style="width:412px;">Obtained from lab of Dr. Irv Weissman</td>
		</tr>
	</tbody>
</table>

using in vivo and tissue and cell explant approaches to study the morphogenesis and pathogenesis of the embryonic and perinatal aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

In a representative clonal analysis of SMCs in embryos mutant for Eln (the gene encoding the extracellular matrix protein elastin), Eln(+/-), Acta2-CreERT2 mice were mated to Eln(+/-) mice also carrying the multi-color ROSA26R(Rb/Rb) reporter. As described in step 1, plugs were checked, pregnant dams were induced with a single tamoxifen injection (1.5 mg) at E12.5, and they were sacrificed at E18.5. Embryos were harves...

Watch this Scientific Journal Video about Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta at JoVE.com

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

The overall goal of these procedures is to study the formation of the embryonic and perinatal aortic walls during normal development as well as, perturbations in this process during disease. This method can help answer key questions in the vascular development field such as, what is the source of the cells that contribute to the embryonic and perinatal vascular medium. The main advantage of these techniques is that they allow the evaluation of the smooth muscle pathogenesis and morphogenesis that occur during the embryonic and perinatal stages of development.The implication of these techniques stem towards the therapy of vascular diseases such as, supravalvular aortic stenosis of periodic disease that causes excessive sumo cell production. The mini pump placement procedure will be performed by Dr.Jiasheng Zhang, a research scientist directing a microsurgery core and Yale University. Begin by setting up mating pairs between two to six month old mice with Cre ER and mice with a cre reporter.Using a metal probe, check for vaginal plugs each morning, separating the female from the male when a plug is detected. For early embryonic induction, inject tamoxifen intraperitoneally into the pregnant damp. To mark cells in the mid to late gestational period and to trace their fate or clonality in the post-natal mouse, inject progesterone into the intraperitoneal cavity of the pregnant damp incompetently with tamoxifen in a one to two dose ratio.To harvest the embryos on the appropriate embryonic date, cleanse the abdomen of the pregnant damp with 70%ethanol and use scissors to open the abdomen to remove the uterus. Collect the embryos from the uterus using forceps and a stereo microscope to carefully separate them from the placenta and yolk sac. Fix the embryos in 4%paraformaldehyde at four degrees celsius for two hours.Followed by three washes in PBS to remove the excess fixative. After the third wash, incubate the embryos in 30%sucrose in PBS, in individual 15 milliliter tubes until the embryos sink. Next, fill plastic freezing molds with optimum cutting temperature compound up to two thirds of the maximum volume and place one embryo vertically in each mold.After 10 minutes at room temperature, freeze the tissues upright in a dry ice 70%ethanol bath and set the cryostat temperature to 22 degrees celsius. Section the tissue blocks to generate 10 to 20 micrometer thick transverse tissue slices through the entirety of each aorta, capturing each section on a glass microscope slide as they are obtained. When all of the samples have been acquired, dry the slides at room temperature for 30 minutes prior to 80 degree celsius storage.To stain the tissue samples, thaw the slides at room temperature, protect it from light to avoid fluorophore bleaching and wash the slides in 0.1%PBS twin 20. For clonal analysis, stain the tissues with an appropriate nuclear dye and directly image the other flurophores of the rainbow cre reporter on a fluorescent microscope. For fate mapping, with the mTmG cre reporter, visualize the GFP expression by direct imaging with or without immunostaining.To culture and explant an embryonic aorta, place the embryo in the supine position and secure the extended limbs to a dissection board under a stereo microscope. Using scissors, make a vertical incision from the abdomen through the sternum to the upper thorax. Dissect away the finest tracheal lungs, esophagus, liver and intestine.Use forceps to gently pull the heart ventrally, using scissors to dissect the aorta away from the dorsal aspect of the thoracic and abdominal cavities. Cut the aorta at the proximal root and the distal abdominal positions to release it and place the harvested aorta in sterile ice cold PBS in a cell culture hood. After a brief wash, transfer the aorta to one well of a 24 well plate, containing DMEM medium supplemented with 0.5%PBS and place the plate in a cell culture incubator for up to 24 hours.At the end of the incubation, wash the aorta two times with PBS and fix the tissue in 4%paraformaldehyde for 20 minutes. After three more PBS washes, transfer the aorta to a 1.5 milliliter tube containing 30%sucrosine PBS until the tissue sinks. For perinatal aorta smooth muscle cell isolation, use a 23 gauge needle to puncture the left ventricle of a postnatal day 0.5 mouse and use plastic tubing to connect the needle to a sterile PBS containing syringe, held vertically in an elevated position, so the PBS flows into the left ventricle by gravity.When the liver blanches, use forceps to remove the adventitial tissue on the outside of the aorta and harvest the aorta as just demonstrated. Dissociate the aortic tissue in 500 microliters of digestion solution with manual shaking every five to 10 minutes. After 45 minutes, transfer the tube to a sterile cell culture hood and use a 200 microliter pipette to triturate the tissue slurry until a single cell suspension has been obtained.Transfer the cells into a 15 milliliter conical tube and then, add two milliliters of DMEM and collect the cells by centrifugation. Resuspend the pellet in three milliliters of smooth muscle cell culture medium and seed the cells onto a 35 millimeter culture plate. Then, place the plate into a 37 degree celsius incubator, refreshing the cell culture medium every three days.To place a subcutaneous osmotic mini pump, begin by shaving the lower back of an anesthetized mouse with clippers and place the mouse in the prone position on a heated surgical plate. Scrub the back with betadine and isopropyl alcohol and place the mouse under a stereo microscope. Then, use a scalpel to make a 0.5 to one centimeter horizontal skin incision approximately three to five centimeters rostral to the base of the tail, without injuring the underlying muscle.Insert a hemostat into the incision and open and close the hemostat jaws to create a subcutaneous pocket. Insert an osmotic mini pump filled with the agent of interest into the pocket and use 6 o non-absorbable sutures to close the incision. Then, allow the mouse to recover with monitoring until the achievement of sternal recumbency.Embryos mutant for the gene encoding the extracellular matrix protein, elastin accumulate excess smooth muscle cells in the inner layers of the aorta, beginning after embryonic day 15.5. Clonal analysis of the smooth muscle cells with the multicolor cre reporter during this process, indicates that the cells are of multiple colors and thus, are polyclonal. Immunostaining explanted aorta sections reveals that elastin mutant embryonic day 15.5 aortas become hyper muscular and stenodic within 18 hours of culture, a process that is attenuated by the addition of an anti antegrin beta three blocking antibody.Smooth muscle cells isolated from the aorta of wild type mice that post natal day 0.5 express smooth muscle cell markers but not CD31. Continuous infusion of an integrin beta three inhibitor into pregnant damps beginning on embryonic day 13.5, significantly attenuates aortic stenosis and hyper muscularization in mice mutant for the extracellular matrix protein, elastin. But do not alter the aortas of wild type mice.Once mastered, the osmotic mini pump placement technique can be completed in less than 10 minutes if it is performed properly. Following these procedures, these approaches can also be extrapolated to study smooth muscle coded, non-vascular such as, tissue of gastrointestinal tract or the lung areas. After watching this video, you should have a good understanding of how to conduct in-vivo embryonic fit mapping and clonal analysis, culture explanted embryonic aortas, perinatal aortic smooth muscle cells and explant subcutaneous osmotic mini pumps.Don't forget that working with mice, tamoxifen and a fixed reducer can be hazardous and that precautions such as, wearing gloves and a lab coat and taking extra care with should always be taking while performing these procedures.

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Developmental Biology

8.4K Views.  Yale University School of Medicine. The overall goal of these procedures is to study the formation of the embryonic and perinatal aortic walls during normal development as well as, perturbations in this process during disease. This method can help answer key questions in the vascular development field such as, what is the source of the cells that contribute to the embryonic and perinatal vascular medium. The main advantage of these techniques is that they allow the evaluation of the smooth muscle pathogenesis and morphogenesis ...