This work was supported by the Deutsche Forschungsgemeinschaft SFB688 (to A.G.).

Mouse breeding was approved by the Regierung Unterfranken, and all analyses were carried out in strict accordance with all German and European Union applicable laws and regulations concerning care and use of laboratory animals.
1. Coating of Microtiter Plates with &#945;4&#946;1 Integrin for Ex Utero Allantois Explant Culture
<ol>
	<li>Reconstitute the lyophilized murine &#945;4&#946;1 integrin at 200 &#181;g/mL in sterile phosphate-buffered saline (PBS). Dilute to a final concentration of 10 &#181;g/mL in pre-warmed (37 &#176;C) PBS. Pipette 20 &#181;L/well of the diluted &#945;4&#946;1 integrin solution into the required number of wells of a microtiter plate (1 allantois/well). 
	NOTE: Avoid the formation of air bubbles when coating the wells of the microtiter plate.</li>
	<li>Incubate the microtiter plates for 60 min at 37 &#176;C by placing them in a humidified tissue culture incubator.</li>
	<li>Carefully aspirate the supernatant with a pipette. Use gentle suction and incline the plate to avoid touching the well bottom with the pipette tip. Wash wells twice by adding 50 &#181;L of pre-warmed (37 &#176;C) culture medium such as Dulbecco&#39;s Modified Eagle Medium (DMEM), and remove the washing medium by gentle suction.</li>
	<li>Critical step: Block free binding sites by adding 50 &#181;L/well of 0.1% (w/v) bovine serum albumin (BSA)/DMEM and incubate for &#8805; 30 min at 37 &#176;C by placing the microtiter plates in a humidified tissue culture incubator. 
	NOTE: Allantois explants can attach to various surfaces in the presence of serum. It is therefore imperative to block non-specific binding sites, after coating the wells with integrin, and before adding the dissected allantois for explant culture in serum-containing medium (see step 4.4).</li>
	<li>Keep the coated wells in the blocking solution at 37 &#176;C until needed in step 4.4.</li>
</ol>
2. Uterus Dissection from a Pregnant Mouse at Dpc 8
NOTE: Reduce false-positive pregnancy rates by weight gain discrimination15.
<ol>
	<li>Sacrifice the mouse by cervical dislocation. Disinfect the coat in the abdominal area by spraying it with 70% (v/v) ethanol. 
	NOTE: Wear gloves when handling mice. Euthanasia by cervical dislocation without anesthetics prevents chemical contamination of the allantois, which may interfere with allantois attachment.</li>
	<li>Open the abdominal skin by making a small incision at the midline with scissors. Use gloved fingers to hold the skin above and below the incision, and pull the skin apart towards the chest and tail of the mouse.</li>
	<li>Grasp the peritoneum with forceps. With the other hand, make a V-shaped incision from anterior towards posterior using scissors, and move the tissue away to expose the abdominal cavity. Use the forceps to push the intestines to one side in order to gain access to the uterus.</li>
	<li>Locate the two uterine horns. Hold the cervix (the caudal segment) with forceps and cut the ligaments with fine scissors. Then detach both uterine horns by severing the oviducts with&#160;scissors.</li>
	<li>Use forceps to transfer the uterus into a sterile 10 cm dish containing sterile, room temperature (RT) PBS. Remove the adipose tissue, vessels, and nerves surrounding the uterine horns with fine forceps and/or scissors (Figure 1A).</li>
</ol>
3. Embryo Dissection
NOTE: Perform the following steps under a stereomicroscope equipped with an 8:1 zoom range.
<ol>
	<li>Separate the embryos by cutting in between the implantation sites with scissors. Grasp the muscular uterine lining of the uterus buds with forceps and pull it apart to expose the decidua (Figure 1B).</li>
	<li>Using forceps, transfer the uterus buds into a new 10 cm dish containing sterile, RT PBS.</li>
	<li>Fix the decidua around the embryo with the tips of forceps.</li>
	<li>Make an incision on the anti-mesometrial side of the decidua with fine forceps&#160;(Figure 1C), and peel the decidua away with forceps to expose the embryo (Figure 1D). 
	NOTE: The mesometrial pole of the decidua is highly vascularized and can be wider at its base than the anti-mesometrial part, which contains the embryo and appears paler.</li>
	<li>Dissect the embryo with forceps (Figure 1E, F).</li>
	<li>Using a micro-spoon, transfer the embryo into a drop of sterile PBS contained in a sterile 3.5 cm dish.</li>
	<li>Transfer the embryo into a fresh drop of PBS in the same 3.5 cm dish using a micro-spoon, and dissect the embryo from the yolk sac using forceps (Figure 1G, H).</li>
	<li>Optionally, if needed (e.g., for genotyping by polymerase chain reaction), transfer the yolk sac into a sterile tube by aspirating it with ~ 5 &#181;L PBS into a large orifice 200 &#181;L pipette tip. Store at -20 &#176;C. 
	NOTE: Maternal tissue (i.e., the parietal yolk sac including Reichert&#39;s membrane and the ectoplacental cone) that may contaminate the yolk sac preparation can lead to erroneous genotyping results. Removal of these tissues is easiest prior to the embryo dissection from the yolk sac.</li>
</ol>
4. Allantois Dissection and Ex Utero Culture on Immobilized &#945;4&#946;1 Integrin
<ol>
	<li>Cut the allantois from its basal insertion site at the posterior end of the embryo using fine forceps (Figure 1H).</li>
	<li>Optionally, if developmental staging is required, count the somite pairs of the embryo by visual inspection using a microscope equipped with phase contrast optics and a 5&#215; and 10&#215; objective. 
	NOTE: The embryo can be extended with forceps to facilitate somite counting. Chorioallantoic attachment and fusion occur between E 8.0 and E 9.0 in embryos with &#8805; 6 somite pairs. Turning begins in embryos with 6-8 somite pairs.</li>
	<li>Pipette 40 &#181;L/well of DMEM supplemented with 10% (v/v) fetal bovine serum, L-glutamine, and antibiotics in the required number of pre-coated microtiter plate wells (see Section 1).</li>
	<li>Collect the floating allantois by aspirating it with ~ 5 &#181;L PBS into a large orifice of a 200 &#181;L pipette tip. Transfer 1 allantois/well.</li>
	<li>Incubate the allantoides by placing the microtiter plates in a humidified tissue culture incubator (37 &#176;C, 5% CO2) for e.g., 6, 12, and 24 h. In order to prevent interference with allantois attachment, do not move the microtiter plates in between these time points.</li>
	<li>At each time point, score allantois attachment (e.g., no attachment/floating allantois; partially or fully attached allantois; fully spread allantois) using a microscope equipped with phase contrast or differential interference contrast optics and a 5&#215; or 10&#215; objective (Figure 2). 
	NOTE: Representative examples of freshly isolated and fully attached allantoides are shown in Figure 2. Ex utero, the distal tip of allantoides isolated from wildtype C57BL/6J mice attaches within 4-6 h (this is referred to as partial attachment of the allantois), and full attachment of the entire allantois is achieved within ~ 12 h of culture. By 18-24 h, the explant has flattened and assumed a circular shape (referred to as fully spread allantois) with a CD31-positive vascular plexus9,16.</li>
</ol>
5. Confirmatory Staining of the Explanted Allantois for the Endothelial Cell Marker CD31
<ol>
	<li>Aspirate the culture medium of fully spread explants and fix the cells by adding 40 &#181;L/well of 4% (w/v) paraformaldehyde/PBS. 
	NOTE: Paraformaldehyde is toxic. Wear gloves and safety goggles to avoid skin and eye contact.</li>
	<li>Incubate for 10 min at RT, then aspirate the supernatant. Wash three times by adding 40 &#181;L/well of PBS, and remove the washing medium by gentle suction.</li>
	<li>Permeabilize the cells and block non-specific binding sites by adding 40 &#181;L/well of 0.1% (v/v) Triton X-100/0.1% (w/v) BSA/10% (v/v) normal goat serum in PBS for 30 min at RT. Wash twice by adding 40 &#181;L/well of PBS, and remove the washing medium by gentle suction. 
	NOTE: The protocol can be paused overnight at this point.</li>
	<li>Stain cells by adding 20 &#181;L/well of primary anti-CD31 antibodies diluted 1:50 in 0.1% (w/v) BSA/PBS overnight at 4 &#176;C. Wash three times by adding 40 &#181;L/well of PBS, and remove the washing medium by gentle suction.</li>
	<li>Detect bound primary antibodies by adding 20 &#181;L/well of fluorescently-labeled secondary goat anti-rat antibodies diluted 1:200 in 0.1% (w/v) BSA/PBS for 1 h at RT. Protect microtiter plates from light by wrapping in aluminum foil or by placing in a dark chamber. Wash three times by adding 40 &#181;L/well of PBS, and remove the washing medium by gentle suction.</li>
	<li>Embed cells by adding 20 &#181;L/well of mounting medium. Incubate for ~ 1 h at RT until the mounting medium has solidified. Store microtiter plates at RT or at 4 &#176;C, protected from light.</li>
	<li>Image the vascular plexus of the allantois explants under a fluorescence microscope equipped with a 5&#215; or 10&#215; objective and suitable fluorescence filters. 
	NOTE: Representative images of the CD31-positive vascular plexus of a fully spread allantois are shown in Figure 3. Other markers for the vascular plexus, such as antibodies directed against Flk-1, Flt, Tie-1 or Tie-2 can be used as well16.</li>
</ol>

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M., Temkin, R., Gifford, S., McHugh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+murine+allantois+by+allantoic+explants.">Study of the murine allantois by allantoic explants.</a> Dev Biol. 233 (2), 347-364 (2001).</li><li>Hou, W., Sarikaya, D. P., Jerome-Majewska, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+culture+of+pre-placental+tissues+reveals+that+the+allantois+is+required+for+maintained+expression+of+Gcm1+and+Tpbpalpha.">Ex vivo culture of pre-placental tissues reveals that the allantois is required for maintained expression of Gcm1 and Tpbpalpha.</a> Placenta. 47, 12-23 (2016).</li><li>Zeigler, B. 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=The+allantois+and+chorion,+when+isolated+before+circulation+or+chorio-allantoic+fusion,+have+hematopoietic+potential.">The allantois and chorion, when isolated before circulation or chorio-allantoic fusion, have hematopoietic potential.</a> Development. 133 (21), 4183-4192 (2006).</li><li>Zaidel-Bar, R., Itzkovitz, S., Ma'ayan, A., Iyengar, R., Geiger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+atlas+of+the+integrin+adhesome.">Functional atlas of the integrin adhesome.</a> Nature cell biology. 9 (8), 858-867 (2007).</li></ol>

The authors have nothing to disclose.

In the presence of serum, which is a rich source of pro-adhesive extracellular matrix molecules such as fibronectin, allantois explants will readily attach to various plastic, glass, or filter surfaces9,16. Thus, if the aim of the performed assay is to specifically investigate the effect of a genetic modification or any other treatment on allantois attachment to immobilized &#945;4&#946;1, it is critical to block non-specific binding sites (e.g., with bo...

The placenta is indispensable for embryonic growth and development in the uterine environment. It constitutes the interface for oxygen, metabolite, and nutrient exchange between the fetal and maternal circulation, and also functions as an endocrine and immunological organ. Any endogenous or exogenous insult that impairs placental development can lead to intrauterine growth retardation, fetal loss, or pregnancy complications, both in mice and in humans1.While the number of targeted mouse mutations that cause abnormal placental physiology continues to increase2, with 219 genotypes currently listed on the Mouse Genome Informatics (MGI) website (http://www.informatics.jax.org/vocab/ mp_ontology/MP:0010038), many of these phenotypes are not well understood from a mechanistic point of view. In addition to genetic alterations, small molecule drugs, monoclonal antibodies, toxins, pathogens, excess metabolites, or various environmental agents may affect placenta development and function3,4,5,6,7. Yet, simple in vitro assays that model critical steps in placenta development are scarce.
Murine placental development is initiated in early midgestation, when the allantois (the developmental precursor of the umbilical cord) emerges from the posterior end of the embryo as a bud that grows toward the chorion8. Chorioadhesive cells in the outer layer of the allantoic bud mediate the attachment and spreading of the allantois on the surface of the chorion (chorioallantoic &#34;fusion&#34;). The chorion subsequently folds into villi into which the vasculature of the allantois grows to form the labyrinth, the inner placental layer where nutrients and oxygen are exchanged between closely juxtaposed maternal blood sinusoids and embryonic vessels9,10.
The attachment of the allantois to the chorion is the initial and critical step in labyrinth formation, and defects in this process are among the most common causes of embryonic lethality in midgestation1. Although a number of mouse mutants have been described where chorioallantoic attachment fails to occur10, and the MGI database currently lists 108 mouse mutants that are characterized by abnormal chorioallantoic fusion (http://www.informatics.jax.org/vocab/mp_ontology/MP:0002824), the intercellular interaction between the vascular cell adhesion molecule-1 (VCAM-1, expressed on the allantoic mesoderm) and &#945;4&#946;1 integrin (expressed on the chorionic mesothelium, which is derived from extraembryonic mesoderm) appears to be indispensable for chorioallantoic attachment and fusion11,12,13. Immunohistochemical staining of whole-mount wild-type embryos has shown that VCAM-1 is expressed within the distal two-thirds of the allantoic stalk at approximately embryonic day (E) 7.513 (1-4 somite stage), and that its expression remains high during chorioallantoic attachment and -fusion11,12. Failure of allantoic attachment to the chorion is typically detected by histological analyses of uterus buds. However, allantois sizes are physiologically highly variable in between and even within litters of the same developmental stage14, and the allantois can only attach to the chorion when it has reached a sufficient size to make physical contact. Reflecting this natural variability, chorioallantoic attachment takes place sometime between ~ E 8.0 and E 9.0 in utero, and a statistically reliable evaluation of this process by histology is therefore dependent on the analysis of a large number of conceptuses to obtain enough specimens at the appropriate developmental stage.
Here, we describe a method to assess allantoic attachment ex utero that is less dependent on allantois size. We demonstrate the dissection of mouse embryos and their allantoides from the uteri of pregnant mice ~ 8 days post coitum (dpc; dpc 0.5 designates the detection of a vaginal plug), and the subsequent culture of allantois explants on immobilized &#945;4&#946;1 integrins. This method enables a rapid, functional evaluation of the attachment and spreading behavior of multiple allantois explants in parallel and at different successive time points. The protocol may be used to screen for the effects of targeted mutations, drugs, or various environmental factors on allantois attachment ex vivo.

<table><tbody><tr><td>C57BL/6J wildtype mice</td><td>The Jackson Laboratory</td><td>Stock No:&amp;nbsp;000664</td><td></td></tr><tr><td>70 % (v/v) Ethanol</td><td>VWR International</td><td>930031006</td><td></td></tr><tr><td>Standard pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td>Forceps used to open the abdominal cavity.</td></tr><tr><td>Micro dissecting forceps</td><td>Fine Science Tools</td><td>11254-20</td><td>Dumont # 5 medical biology forcepts with fine, sharp tip.&amp;nbsp;</td></tr><tr><td>Fine scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>14094-11</td><td>Straight blades.</td></tr><tr><td>Spring scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>15012-12</td><td>Noyes spring scissors with 14 mm blades for the dissection of uterus buds.</td></tr><tr><td>Microspoon</td><td>Carl Roth</td><td>AT18.1</td><td>5 mm Spoon diameter.</td></tr><tr><td>Microtiter plates</td><td>ibidi</td><td>81501</td><td>We use uncoated, sterile angiogenesis slides (internal volume 50 &amp;micro;L) with a hydrophobic surface that is not tissue-culture treated to reduce non-&amp;#945;4&amp;#946;1-mediated binding to plastic.</td></tr><tr><td>10 cm dish</td><td>Nunc</td><td>150350</td><td>Sterile tissue culture dishes.</td></tr><tr><td>3.5 cm dish</td><td>Nunc</td><td>150288</td><td>Sterile tissue culture dishes.</td></tr><tr><td>Large orifice pipette tips&amp;nbsp;</td><td>Biozym</td><td>VT0140X</td><td>Low binding pipette tips, 200 &amp;micro;L.</td></tr><tr><td>&amp;#945;4&amp;#946;1 integrin</td><td>R&amp;amp;D Systems</td><td>6054-A4</td><td>Murine &amp;#945;4&amp;#946;1 integrin recombinantly expressed and purified from a Chinese hamster ovary cell line.</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS)</td><td>Sigma Aldrich</td><td>D8537</td><td>Without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;.</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>PAN Biotech</td><td>P04-03600</td><td>The formulation contains 4.5 g/L glucose, 110 mg/L sodium pyruvate and 3.7 g/L NaHCO&lt;sub&gt;3&lt;/sub&gt; but no L-glutamine, which is added separately.</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco (ThermoFisher Scientific)</td><td>15140-122</td><td>100 x (10,000 U/mL Penicillin and 10,000 &amp;micro;g/mL streptomycin) stock solution.&amp;nbsp;Prepare and store aliquots at -20 &amp;deg;C to avoid freeze/thaw.</td></tr><tr><td>L-Glutamine</td><td>PAN Biotech</td><td>P04-80100</td><td>100 x (200 mM) Stock solution. Prepare and store aliquots at -20 &amp;deg;C to avoid freeze/thaw.</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Biochrom</td><td>S 0115</td><td>We use heat-inactivated FBS (heated to 56 &amp;deg;C for 30 min with mixing to inactivate complement).</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma Aldrich</td><td>A7030</td><td>We use protease- and fatty acid-free albumin. Prepare a 0.1 % (w/v) solution in DMEM. Sterile filter the solution through a disposable cell culture filter with a 0.22 &amp;micro;m pore size and low protein-binding membrane, and store aliquots at -20 &amp;deg;C.</td></tr><tr><td>&lt;em&gt;para&lt;/em&gt;-Formaldehyde</td><td>Carl Roth</td><td>0335.2</td><td>To make a formaldehyde solution in PBS, weigh &lt;em&gt;para&lt;/em&gt;-formaldehyde powder in a ventilated hood, and add it to PBS preheated to ca. 60 &amp;deg;C in a beaker on a stir plate. Add 1 N NaOH dropwise until solution clears. Let solution cool to room temperature, and filter through 0.22 mm filter syringe. Adjust pH with diluted HCl to ca. 6.9, and adjust to final volume with PBS. We aliquot and freeze the solution, but it can also be stored at 4 &amp;deg;C for approximately four weeks.&amp;nbsp;</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100</td><td>Use wide-orifice pipette tips to handle undiluted Triton X-100.</td></tr><tr><td>Normal goat serum</td><td>Sigma Aldrich</td><td>G9023</td><td>To block non-specific binding of antibodies in immunofluorescence analyses.</td></tr><tr><td>&amp;#945;-CD31 Antibodies</td><td>BD Biosciences</td><td>550274</td><td>Purified rat anti-mouse monoclonal antibodies, clone MEC 13.3.</td></tr><tr><td>Secondary goat anti-rat antibodies</td><td>Thermo Fisher</td><td>A-11006</td><td>Goat anti-rat IgG (heavy and light chains), cross-adsorbed, fluorescently labeled with Alexa Fluor 488. Other fluorescent labels are also possible.</td></tr><tr><td>Mowiol 4-88</td><td>Sigma Aldrich</td><td>81381</td><td>Mounting medium for cytochemistry. MW ~31,000</td></tr><tr><td>Labovert&amp;nbsp;</td><td>Leitz</td><td>Labovert&amp;nbsp;</td><td>Inverted phase contrast microscope equipped with a 4 x and 10 x objective for somite pair counting. Other phase contrast microscopes (such as those that are routinely used in tissue culture) are also suitable.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>M80</td><td>Leica Microsystems</td><td>M80</td><td>Stereomicroscope with 8 : 1 zoom range (yielding a magnification between 7.5 x and 60 x) for embryo and allantois dissection.</td></tr><tr><td>DM4000B</td><td>Leica Microsystems</td><td>DM4000B</td><td>Microscope system for histology with LED illumination. We use HCX PL FLUOTAR 5 x/0.15 and HC PL FLUOTAR 10 x/0.30 objectives. Other microscopes equipped phase contrast and fluorescence optics can be used to score allantois attachment.&amp;nbsp;</td></tr><tr><td>Digital camera</td><td>JVC</td><td>KY-F75U</td><td>3-CCD digital capture camera attached to the DM4000B LED microscope. We use Diskus software (Hilgers) to operate both the microscope and the camera.</td></tr><tr><td>Confocal microscope</td><td>Leica Microsystems</td><td>SP5</td><td>Confocal microscope for higher-resolution imaging of endothelia in the vascular plexus of allantois explants. Immunofluorescence images were taken with a 40 x objective.</td></tr></tbody></table>

dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized &#945;4&#946;1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

This protocol describes a method to isolate and explant murine allantoides ex vivo, and details the qualitative assessment of allantois attachment to α4β1 integrin, a critical process during in vivo chorioallantoic fusion. The representative results shown in Figure 1A-H demonstrate the successive steps of allantois isolation starting from the pregnant uterus. Maternal tissues that surround the uterine horn...

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Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

We describe an in vitro assay to model chorioallantoic attachment, the first step in placenta formation. The protocol demonstrates the dissection and explant culture of murine allantoides on immobilized &#945;4&#946;1 integrin. Allantois attachment is evaluated microscopically at pre-determined time points.

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

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Research

JoVE Journal

Medicine

7.8K Views.  University of Würzburg. We beschrijven een in vitro assay model chorion gehechtheid, de eerste stap in de vorming van de placenta. Het protocol blijkt de dissectie en explant cultuur van lymfkliertest allantoides op geïmmobiliseerdet α4β1 integrine. Allantois bijlage wordt microscopisch beoordeeld op vooraf bepaalde tijdstippen.

Video: Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

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.

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.

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 ...

Developmental Biology

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Immunology and Infection

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However, because of their small body size, some structures, such as the oviduct with a diameter of 200-400 &#956;m, have proven to be relatively difficult to study except by immunohistochemistry. Recently, immunohistochemical studies have uncovered more complex differences in oviduct segments than were previously recognized; thus, the oviduct is divided into four functional segments with different ratios of seven distinct epithelial cell types. The different embryological origins and ratios of the epithelial cell types likely make the four functional regions differentially susceptible to disease. For example, precursor lesions to serous intraepithelial carcinomas arise from the infundibulum in mouse models and from the corresponding fimbrial region in the human fallopian tube. The protocol described here details a method for microdissection to subdivide the oviduct in such a way to yield a sufficient amount and purity of RNA necessary for downstream analysis such as reverse transcription-quantitative PCR (RT-qPCR) and RNA sequencing (RNAseq). Also described is a mostly non-enzymatic tissue dissociation method appropriate for flow cytometry or single cell RNAseq analysis of fully differentiated oviductal cells. The methods described will facilitate further research utilizing the murine oviduct in the field of reproduction, fertility, cancer, and immunology.

A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition, non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells.

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Biology

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Establishing Villous and Decidual Organ Cultures as Ex Vivo Models of Human Maternal-Fetal Interface

Source: Rizzuto, G. A., et al. <a href="https://app.jove.com/v/54237">Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface.</a> J. Vis. Exp. (2016).
This video demonstrates the establishment of primary human placental villous and decidual organ cultures in ex vivo conditions. These models are well-suited for studying pathogenesis at the human maternal-fetal interface.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

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