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 between mice with a CreER and mice with a Cre reporter. 
	NOTE: A CreER is used for SMC marking; Myh11-CreERT2 or Acta2-CreERT2 mice11,12 is commonly used for this purpose. We have used ROSA26R-CreERT2 mice13 for broadly marking embryonic tissues.
	<ol>
		<li>For clonal analysis, use a multi-color Cre reporter (e.g., Confetti or Rainbow [Rb] mice)8,14,15 and for fate mapping, use a single-color Cre reporter (e.g., ROSA26R-YFP mice16) or the double-color Cre reporter ROSA26R-mTomato-mGFP (mTmG)17. 
		NOTE: This breeding scheme uses adult mice that are 2 - 6 months old (generally ~30 g) and will generate embryos with a CreER and a Cre reporter.</li>
	</ol>
	</li>
	<li>Check for vaginal plugs in the morning using a metal probe and consider noon on the day of plug detection as embryonic day (E) 0.5.</li>
	<li>Separate the female from the male when a plug is detected.</li>
	<li>Prepare 4-OH-tamoxifen and tamoxifen working solutions.
	<ol>
		<li>For 4-OH-tamoxifen, dissolve 5 mg into 50 &#181;L of 100% ethanol, vortex for 2 min and then add 450 &#181;L of corn oil. On ice, sonicate at output level 2 for 10 s cycles with resting between cycles until sample cools down. After sample is completely dissolved (usually ~ 4 - 5 cycles of sonication), take 200 &#181;L of sonicated solution and dissolve in 1,800 &#181;L of corn oil to make final working solution (4-OH-tamoxifen, 1 mg/mL).</li>
		<li>For tamoxifen, dilute to 50 mg/mL in 100% ethanol and vortex until it is dissolved. Dilute in corn oil to make the final working solution (tamoxifen, 10 mg/mL) and stir at 45 &#176;C to ensure that it is completely dissolved.</li>
	</ol>
	</li>
	<li>For early embryonic induction, inject 4-OH-tamoxifen intraperitoneally into a pregnant dam.
	<ol>
		<li>Use 4-OH-tamoxifen injections (up to ~150 &#181;g per pregnant mouse, or roughly 5 mg/kg bodyweight) at E5.5 and thereafter as they yield viable dam, embryos, and pups.</li>
		<li>Use tamoxifen at doses of 0.5 - 1.5 mg (or 17 - 50 mg/kg) for dams pregnant with embryos at ~E9 and thereafter. Use a 30G needle for all injections. 
		&#160;Note: Filtration through a 0.22 &#181;m filter is typically used to sterilize all mixtures prior to injection. &#160;</li>
	</ol>
	</li>
	<li>For clonal analysis, use intraperitoneal injections of high doses of 4-OH-tamoxifen (e.g., 5 mg/kg) or tamoxifen (e.g., 50 mg/kg) to mark multiple cells.
	<ol>
		<li>To mark individual cells, titrate down the 4-OH-tamoxifen or tamoxifen dose such that in the tissue of interest (i.e., the aorta), almost all embryos analyzed (as described below at the end of this section) have either no cells labeled or only cells of a single color; this is the threshold dose.</li>
	</ol>
	</li>
	<li>To mark cells in the mid-late gestational period and to trace their fate or clonality in the postnatal mouse, inject progesterone into the intraperitoneal cavity of the pregnant dam concomitantly with tamoxifen in a 1:2 (progesterone:tamoxifen) dose ratio. 
	NOTE: Tamoxifen doses are 17 - 50 mg/kg and progesterone doses are 8.5 - 25 mg/kg (i.e., half of the tamoxifen doses). The progesterone injection may delay labor by ~1 - 2 days.</li>
	<li>Euthanize the dam pregnant with embryos of a desired age using an open drop method, where the dam is placed in a receptacle containing cotton or gauze soaked with undiluted isoflurane. Confirm death by opening the chest cavity to induce pneumothorax, by removing the vital organs, and/or by performing cervical dislocation.</li>
	<li>Cleanse the abdomen with 70% ethanol. Use scissors to cut open the abdomen and dissect out the uterus. Remove the embryos from uterus and carefully separate the embryos from the placenta and yolk sac using forceps.</li>
	<li>Euthanize the embryos at E15 or older by performing either cervical dislocation or decapitation with surgical scissors or a sharp blade. 
	NOTE: Embryos younger than E15 rapidly die following euthanasia of the mother and/or the removal of the embryos from mother.</li>
	<li>Place the embryos in ice-cold PBS and cut a small piece of tail using scissors to genotype for the CreER and Cre reporter.</li>
	<li>Fix the embryos in 4% paraformaldehyde at 4 &#176;C for 2 h. Wash the embryos three times in PBS and then incubate the embryos in 30% sucrose in PBS in 15-mL tubes, waiting until they sink, which may take up to two days.</li>
	<li>Fill plastic freezing molds up to 2/3 of maximum level with optimum cutting temperature (OCT) compound. Place each embryo vertically in the mold, entirely submerged in OCT. Incubate for 10 min at room temperature (RT) to minimize bubbles.</li>
	<li>Place freezing tissue blocks upright in a dry ice, 70% ethanol bath to freeze. Store the blocks at -80 &#176;C.</li>
	<li>Set the cryostat temperature to -22 &#176;C and cut blocks to generate transverse sections through the entire aorta, 10 - 20 &#181;m thick. Dry the slides at RT for 30 min prior to storing them at -80 &#176;C.</li>
	<li>Thaw the slides at RT, protected from light to avoid the bleaching of fluorophores. Wash the slides with PBS-Tween20 0.1%.
	<ol>
		<li>For clonal analysis, stain the slides for nuclei (DAPI, 5 mg/mL) and directly image other fluorophores in the Rainbow Cre reporter (Cerulean: 433-nm excitation max, 475-nm emission max; mOrange: 548 nm, 562 nm; mCherry: 587 nm, 610 nm).
		<ol>
			<li>Use the following filters for detecting fluorophores with an upright fluorescent microscope: DAPI (excitation max/bandwidth 350/50 nm, emission max/bandwith 460/50 nm), Cerulean (excitation 436/20 nm, emission 480/40 nm), texas red (excitation 560/40 nm, emission 630/75 nm) and custom filter to separate mCherry from mOrange (excitation 577/10 nm, emission 630/50 nm).</li>
		</ol>
		</li>
		<li>For fate mapping with the mTmG Cre reporter, detect GFP (484 nm, 510 nm), either by directly imaging or by using immunostaining. For imaging with an upright fluorescent microscope use a GFP filter (excitation 470/40 nm, emission 525/50 nm).</li>
		<li>For immunostaining, incubate sections with primary antibodies overnight at 4 &#176;C, wash with 0.1% Triton X-100 in PBS and then incubate with secondary antibodies for 1 h. Use anti-CD31 (final concentration 0.0016 mg/mL), anti-GFP (0.006 mg/mL) and Cy3-directly conjugated anti-SMA (1:500 final dilution) primary antibodies and use secondary antibodies directly conjugated to fluorophores (1:500 final dilution) (see the Table of Materials).</li>
	</ol>
	</li>
	<li>Image slides with an upright fluorescent microscope (magnification: 4X - 20X) or confocal microscope (10X -63X). For high magnification, use a confocal imaging 63x oil objective with a numerical aperture of 1.4-0.6 and a frame size of 1,024 x 1,024 pixels.</li>
</ol>
2. Explant Embryonic Aorta Culture
Note: This approach was previously used to evaluate the role of integrin beta3 in stenosis of the explanted Eln(-/-) embryonic aorta9,18.
<ol>
	<li>Euthanize a timed-pregnant dam at E15.5 by excess isoflurane inhalation, as per step 1.8, above.</li>
	<li>Harvest and euthanize the embryos, as per steps 1.9 - 1.10, above.</li>
	<li>Position the embryo supinely and pin the extended limbs to the dissection board. Visualize with a dissection stereoscope and use scissors to cut a vertical incision from the abdomen through the sternum to the upper thorax. Dissect away the thymus, trachea, lungs, esophagus, liver, and intestine.</li>
	<li>Gently pull the heart ventrally using forceps and use scissors to dissect the aorta away from the dorsal aspect of the thoracic and abdominal cavities. To release the aorta, cut it at the proximal root and the distal abdominal positions.</li>
	<li>Place the aorta in ice-cold sterile PBS in a cell culture hood. Transfer the aorta to a 24-well plate and culture it in DMEM with 0.5% FBS for up to 24 h at 37 &#176;C. 
	NOTE: The culture medium can be supplemented with a blocking antibody (e.g., anti-integrin &#945;v&#946;3 antibody at 0.02 mg/mL9; see the Table of Materials) or a pharmacological agent.</li>
	<li>Wash the aorta twice with PBS and then fix it with 4% paraformaldehyde for 20 min.</li>
	<li>Wash three times with PBS and transfer the aorta to a 1.5-mL tube with 30% sucrose in PBS. After the aorta sinks, make frozen tissue blocks, cut sections, and immunostain, as described in steps 1.13 - 1.15, above.</li>
</ol>
3. SMC Isolation from the Perinatal Aorta
Note: This approach is currently being used to compare the biology of aortic SMCs isolated from Eln(-/-) and wildtype perinatal mice.
<ol>
	<li>Euthanize a murine pup at postnatal day (P) 0.5, either by cervical dislocation or by decapitation with surgical scissors or a sharp blade, as described in step 1.10, above.</li>
	<li>Perform step 2.3 and then, after opening the thorax and abdomen, puncture left ventricle with 23G needle. Use plastic tubing to connect the needle to sterile PBS-containing syringe held vertically at an elevated position so that PBS flows into the left ventricle by gravity. Allow infusion of sterile PBS into left ventricle until the liver blanches.
	<ol>
		<li>Use forceps to remove adventitial tissue on the outside of the aorta and dissect the aorta as described in step 2.4.</li>
	</ol>
	</li>
	<li>Digest the aorta in a single solution of DMEM (500 &#181;L) supplemented with 225 U/mL collagenase, 2.25 U/mL elastase, and 1x antibiotic-antimycotic for 45 min at 37 &#176;C. Manually shake the tube every 5 - 10 min.</li>
	<li>In the sterile cell culture hood, titurate the digested tissue by pipetting up and down with a 200-&#181;L pipette to generate a single-cell suspension. Transfer the single-cell suspension to a 15-mL tube, add 2 mL of DMEM, and centrifuge at 920 x g for 5 min at 25&#176;C.</li>
	<li>Discard the supernatant. Resuspend the cell pellet in 3 mL of SMC culture medium (DMEM containing 10% FBS supplemented with 1 &#181;g/mL rhFGF, 10 &#181;g/mL rhEGF, 100 U/mL penicillin/streptomycin, and 2.5 &#181;g/mL Amphotericin B). Transfer to a 35-mm culture plate. 
	NOTE: Upon initial isolation, ~400,000 cells are obtained from a single P0.5 wildtype aorta; ~300,000 of these cells are SMCs.</li>
	<li>Culture the cells at 37 &#176;C and change to fresh SMC culture medium every 3 days. Using standard techniques with trypsin, passage the cells when confluent. For experiments, use cells from passages 3 - 7.</li>
</ol>
4. Subcutaneous Osmotic Mini-pump Placement in Pregnant (or Non-pregnant) Mice
Note: This approach was previously used to continuously deliver a pharmacological agent (e.g., integrin &#946;3 and &#946;5 inhibitor cilengitide) to embryos in utero9. The pump was inserted in the pregnant dam at E13.5 and maintained until parturition.
<ol>
	<li>Anesthetize the mice with 2 - 5% isoflurane in oxygen (flow rate of 1 L/min) for induction and 1-3% for maintenance. Use the toe-pinch reflex to monitor the level of anesthesia, and adjust the anesthetic agent as needed. Administer subcutaneous buprenorphine (0.05 - 0.1 mg/kg) for analgesia.</li>
	<li>Shave the lower back with clippers. Use sterile drapes, gloves, and instruments and maintain the mice on a heating surgical plate during surgery.</li>
	<li>Place each mouse in prone position and scrub the back with betadine followed by isopropyl alcohol. Approximately 3 - 5 cm rostral to the base of the tail in the lower back, use a scalpel to make a horizontal skin incision 0.5 - 1.0 cm in length without injuring the underlying muscle.</li>
	<li>Insert a hemostat into the incision and create a subcutaneous pocket rostrally for pump implantation by opening and closing hemostat jaws.</li>
	<li>Fill an osmotic mini-pump with the agent of choice, as per the manufacturer&#39;s guidelines (see the Table of Materials). Insert the filled pump into the pocket and close the incision with clips or 6-0 non-absorbable sutures. Ensure that the total surgical time is less than 10 min.</li>
	<li>Post-operatively, monitor the mice every 15 min until they have recovered from sternal recumbency. Administer subcutaneous buprenorphine (0.05 - 0.1 mg/kg) every 6 - 12 h for 48 h. When the mice have fully recovered from general anesthesia, monitor them daily. Remove the clips or sutures 7 - 10 days post-surgery.</li>
</ol>

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The authors have nothing to disclose.

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 study the embryonic/perinatal aorta.
Clonal analysis is a very powerful approach that allows one to investigate the behavior of individual cells. We have recently used this approach to help identify specialized SMCs in the pulmonary vasculature10. It is critical to recognize that, in an individual mouse carrying a multi-color Cre reporter, there is a chance that cells of the same color could derive from more than one recombination event. Thus, clonal analysis with a multi-color reporter necessitates the evaluation of numerous mice. Because tamoxifen in the mid-late gestational period can interfere with parturition, for experiments that mark cells in the mid-late gestational period and trace them postnatally, progesterone and tamoxifen should be injected concomitantly. Similar to protocols in the embryo, fate mapping and clonal analysis of cells in the adult mouse can be undertaken with the intraperitoneal injection of tamoxifen in the adult.
Clonal analysis and fate mapping of SMCs uses mice carrying transgenes with the expression of Cre recombinase under the control of promoters with enhanced activity in the smooth muscle. Here, we describe protocols using mice carrying conditional transgenes Myh11-CreERT2 or Acta2-CreERT2. Myh11 is the most specific marker of SMCs19; however, it is downregulated in the Eln(-/-) aorta9. Acta2 is expressed in SMCs, including those of the Eln-null aorta, but it is not as specific because it is also expressed in other cell types. If these conditional transgenes were &#34;leaky&#34; (i.e., induce recombination in the absence of tamoxifen), it might be problematic, as the experiments would not delineate the timing of cell marking. The leakiness of these transgenes was analyzed by assessing beta-galactosidase activity in mice carrying a lacZ-based Cre reporter and either Myh11-CreERT2 or Acta2-CreERT2 following vehicle treatment (i.e., in the absence of tamoxifen induction)11,12. Based on this analysis, these transgenes were considered not leaky.
Hypermuscularization characterizes multiple vascular pathologies in humans. For instance, supravalvular aortic stenosis (SVAS) is a devastating congenital condition that is characterized by the obstruction of large and medium-sized arteries due to excess SMCs. SVAS results from the loss of function of one allele of the ELN gene and occurs as an isolated entity or, more commonly, as part of Williams-Beuren syndrome20,21,22,23,24. Eln(-/-) mutant mice phenocopy many aspects of the aortic phenotype of SVAS18,21,22. Aortic explants from Eln(-/-) embryos rapidly become occluded during culture, whereas explants from wildtype embryos remain patent9,18. The aortic explant approach facilitates screening for agents that attenuate stenosis in elastin aortopathy9. However, this approach does not take into account the mechanical or physical forces that the aorta is subject to while in the body. Thus, positive hits in aortic explant screening should be assessed with in vivo Eln mutant mouse models.
The SMC isolation protocol is a rapid and robust method to obtain SMCs from the perinatal murine aorta. A similar approach can be extrapolated to isolate SMCs from adult mice. Because of its relatively large size, the adult aorta can be opened longitudinally and the adventitia and endothelial cells dissected away. To evaluate the purity of the cells isolated from the perinatal or adult aorta, it is important to check for the expression of SMC markers (e.g., SMMHC, smoothelin, SM22&#945;, and SMA) and the lack of expression of endothelial cell markers (e.g., CD31 and vascular-endothelial cadherin). A potential alternative isolation approach is to use flow cytometry to isolate fluorescently labeled SMCs from the dissected aorta. Mice carrying a fluorescent Cre reporter and Myh11-CreERT2, Acta2-CreERT2, or Tgln-Cre can be used to fluorescently label SMCs11,12,25,26,27. Alternatively, existing transgenic mice with the SMA promoter driving the expression of a fluorophore (i.e., GFP, mCherry, or RFP) can be used28,29,30.
Implanted osmotic mini-pumps are used to provide the continuous delivery of pharmacological agents to laboratory animals. We have implanted mini-pumps in pregnant mice at E13.5 to deliver agents to developing embryos during the rest of the gestational period9. To reach the embryos, such agents must be able to pass the blood-placental barrier. In general, mini-pumps are very well tolerated and, given the low rate of infection, routine antibiotics are not recommended. Wound dehiscence is rare; however, if a dehiscence greater than 4 mm is detected, the mouse should be re-anesthetized and the wound cleaned and closed again with a surgical suture.
This work describes an armamentarium of approaches that can be used to study the normal formation of the embryonic and perinatal aortic wall during development, as well as after perturbations in this process during disease. In addition, these protocols can be applied to study the maintenance and disease of the adult aorta. Approaches can be extrapolated to other blood vessels, as well as to smooth muscle-coated non-vascular structures, such as the gastrointestinal tract or the lung airways. Finally, similar techniques can be used to study cell types beyond SMCs, such as endothelial cells or fibroblasts, as well as the interplay between these cells types and SMCs.

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 harvested, fixed, frozen, and genotyped. Transverse descending aortic cryosections were cut. Sections from Eln(+/-), Acta2-CreERT2, ROSA26R(Rb/+) embryos indicate that the excess inner layer SMCs that accumulate in Eln-null mice (starting after E15.5) are marked by multiple colors (Figure 1) and hence derive from multiple alpha-smooth muscle actin (SMA)+ cells that are present at ~E12.5. In the E12.5 aorta, SMA+ cells are limited to the tunica media.
To generate Eln(-/-) embryonic aortas for explants, male and female Eln(+/-) mice were mated. Using methods described in step 2, plugs were checked and pregnant Eln(+/-) dams were sacrificed at E15.5. The aortas were isolated from Eln(-/-) embryos and cultured for 0 or 18 h in the presence of an anti-integrin &#946;3 blocking antibody or an IgG1 control. Aortas were then fixed, frozen, and cryosectioned, and cryosections were stained for SMA (SMC marker), CD31 (endothelial cell marker), and nuclei (DAPI) (Figure 2). The results demonstrate that within 18 h of culturing, the E15.5 Eln(-/-) aorta becomes hypermuscular and stenotic. This process is attenuated by an anti-integrin &#946;3 blocking antibody.
SMCs were isolated from the aorta of wildtype mice at P0.5. As described in step 3, SMCs were isolated from the dissected neonatal aorta by enzyme digestion and were cultured. Cells were passaged, and passage 3 cells were stained for SMC markers (i.e., SMA, smooth muscle myosin heavy chain (SMMHC), or transgelin (also known as SM22&#945;)), CD31, and nuclei (DAPI) (Figure 3). Most of the cultured cells express SMC markers but not CD31.
To evaluate whether pharmacological inhibition of integrin &#946;3 can attenuate hypermuscularization and stenosis of the Eln(-/-) aorta in vivo, we continuously infused the inhibitor cilengitide because of its short half-life in plasma. Eln(+/&#8722;) males and females were mated, and, as described in step 4, osmotic mini-pumps loaded with cilengitide were implanted in pregnant dams at E13.5. At P0.5, pups were euthanized and genotyped, and their aortas were analyzed. Cilengitide treatment significantly attenuates aortic stenosis and hypermuscularization in Eln(-/-) mice (Figure 4) without altering the wildtype aorta9. Thus, anti-integrin &#946;3 is a potentially promising noninvasive approach to treat elastin aortopathy.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56039/56039fig1.jpg" /> 
Figure 1. Excess SMCs in the Eln(-/-) Aorta Derive from Multiple Pre-existing SMCs. Dams pregnant with Eln(-/-), Acta2-CreERT2 embryos also carrying the multicolor Cre reporter ROSA26R(Rb/+) were induced with a single intraperitoneal injection of tamoxifen (1.5 mg) at E12.5. Dams were sacrificed at E18.5, and transverse sections of the descending aortas of the embryos were analyzed, with staining for DAPI and the direct fluorescence of Rb colors, Cerulean (Cer), mCherry (mCh), and mOrange (mOr). Excess SMCs accumulate in the inner aspect of the Eln-null aorta after E15.518. The excess inner-layer SMCs include cells of multiple colors (asterisks), indicating polyclonality. Lu, aortic lumen. Scale bar, 10 &#181;m. Reprinted from Misra et al., 20169. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56039/56039fig1large.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/56039/56039fig2.jpg" /> 
Figure 2. Anti-&#945;v&#946;3 Integrin Blockade Attenuates Hypermuscularization and Stenosis of Eln(-/-) Aortic Explant. At E15.5, pregnant dams were euthanized and the aortas of Eln(-/-) embryos were harvested. Isolated aortas were either fixed immediately or cultured in the presence of an isotype control IgG1 or an integrin &#945;v&#946;3 blocking antibody for 18 h prior to fixation. Fixed aortas were stained for CD31, SMA, and nuclei (DAPI). Lu, aortic lumen. Scale bar, 100 &#181;m. Reprinted from Misra et al., 20169. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56039/56039fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56039/56039fig3.jpg" /> 
Figure 3. Isolated and Cultured Aortic SMCs from Neonatal Mice Express Smooth Muscle Markers. SMCs were isolated from P0.5 pups and were cultured. Cells from the third passage were fixed and stained for CD31 (EC marker), nuclei (DAPI), and an SMC marker (either SMA, SM22&#945;, or SMMHC, as indicated). This analysis indicates that ~90% of the cultured cells express SMC markers, and none were observed to express CD31. Scale bar, 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56039/56039fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56039/56039fig4.jpg" /> 
Figure 4. Continuous Infusion of Cilengitide In Vivo Attenuates Eln(-/-) Aortic Hypermuscularization and Stenosis. Male and female Eln(+/-) mice were crossed. The integrin &#946;3 inhibitor cilengitide or vehicle (PBS) was continuously infused in pregnant dams using osmotic mini-pumps starting at E13.5. Transverse sections at P0.5 were stained for SMA (red), CD31 (white), and nuclei (DAPI, blue). Lu, aortic lumen. Scale bar, 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56039/56039fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Developmental Biology

8.4K Views.  Yale University School of Medicine. 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.

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

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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 ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

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 ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

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

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Chapters in this video

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Live Imaging of Mouse Secondary Palate Fusion

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

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

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

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay