The authors are grateful to Isabel Alcobia for the critical reading of the manuscript, to M&#225;rio Henriques for video narration and to Vitor Proa from the Histology Service of the Instituto de Histologia e Biologia do Desenvolvimento, Faculdade de Medicina de Lisboa, Universidade de Lisboa, for technical support. We are particularly indebted to Paulo Caeiro and Hugo Silva from the Unidade de audiovisuais (Audiovisual Unit), Faculdade de Medicina de Lisboa, Universidade de Lisboa for their outstanding commitment to the production of this video. We acknowledge Leica Microsystems for kindly providing a stereoscope equipped with a video system and to Interaves - Sociedade Agro-Pecu&#225;ria, S.A for contributing with quail fertilized eggs. This work was supported by Faculdade de Medicina de Lisboa, Universidade de Lisboa (FMUL).

All these experiments follow the animal care and ethical guidelines of the Centro Acad&#233;mico de Medicina de Lisboa.
1. Fertilized quail and chicken egg incubation
<ol>
	<li>Place fertilized eggs of Japanese quail (Coturnix coturnix japonica) in a 38 &#176;C humidified incubator for 3 days. Incubate the eggs (egg blunt end) facing up in the air chamber. 
	NOTE: The humidified environment is achieved by placing a water container at the bottom of the incubator.</li>
	<li>Incubate fertilized eggs of chicken (Gallus gallus) for 2.5 days in a 38 &#176;C humidified incubator. Incubate the eggs in a horizontal position and mark the upper side using a piece of charcoal to identify the embryo location. 
	NOTE: Start with 40 quail eggs and 60 chicken eggs when establishing this experiment.</li>
</ol>
2. Isolation of quail endoderm containing the prospective domain of the thymic rudiment
NOTE: Use a horizontal laminar flow hood and sterilized instruments and materials for egg manipulation procedures in sterile conditions.
<ol>
	<li>Remove the embryonic region containing the presumptive territory of thymic rudiment, the pharyngeal arch region containing the 3rd and 4th arches (3/4PAR), as described7,8.
	<ol>
		<li>Fill a large borosilicate glass bowl (100 mm x 50 mm; 100 cm3) with 60 mL of cold phosphate-buffered saline solution (PBS).</li>
		<li>With the help of curved scissors, tap and cut a circular hole in the shell of a quail egg that has been incubated for 3 days. Make the hole on the opposite side of the egg blunt and transfer the yolk (with the embryo) to the bowl with cold PBS.</li>
		<li>Remove the embryo from the yolk by cutting the vitelline membrane externally to extra-embryonic vessels using curved scissors.</li>
		<li>With the help of thin forceps, transfer the embryo to a small bowl (60 mm x 30 mm; 15 cm3) filled with 10 mL of cold PBS.</li>
		<li>With a skimmer, move the embryo to a 100 mm Petri dish with a black base (see Table of Materials) containing 10 mL of cold PBS and place it under a stereomicroscope.</li>
		<li>Dissect the 3/4PAR, as previously described8.</li>
		<li>Aspirate the 3/4PAR and transfer to a glass dish three-quarters filled with cold PBS using a 2 mL sterile Pasteur pipette.</li>
	</ol>
	</li>
	<li>Isolate the endoderm containing the presumptive territory of thymic rudiment (the 3/4PP endoderm) by enzymatic digestion with pancreatin.
	<ol>
		<li>With the help of spatula and thin forceps, transfer the 3/4PAR to a glass dish three-quarters filled with cold pancreatin (8 mg/mL; 1:3 dilution of 25 mg/mL with cold PBS).</li>
		<li>Incubate for 1 h on ice for enzymatic digestion. 
		NOTE: The time of enzymatic digestion depends of the stage of development (Table 1).</li>
		<li>Place the glass dish under the stereomicroscope (40x-60x magnification) to isolate the endoderm from the 3/4PAR. 
		NOTE: Keep all surfaces and solutions cold during this procedure. Change to a new cold pancreatin solution if taking a long time to dissect the tissues (&#62;15 min). As an illumination source, use LED lights incorporated in the stereomicroscope or in the optic fibers, considering the limited heat load.</li>
		<li>To isolate the endoderm from the surrounding tissues, use two stainless steel microscalpels in pin holders. 
		NOTE: Use microscalpels with a diameter between 0.1 mm and 0.2 mm and nickel pin holders with a jaw opening diameter of 0 mm to 1 mm.
		<ol>
			<li>First remove the neural tube and mesoderm attached to the dorsal surface of the pharyngeal endoderm.</li>
			<li>With the dorsal side up, carefully detach and remove the mesenchyme between the pharyngeal arches and expose the pharyngeal pouches. Perform this procedure on both sides of the 3/4PAR.</li>
			<li>Remove the heart tube and the mesenchyme surrounding the anterior pouches.</li>
			<li>With the ventral side up, cut the ectoderm of the 2nd and 3rd pharyngeal arches and carefully remove the mesenchyme attached to the pouches. Repeat this procedure on the other side of the 3/4PAR. At this stage the thyroid rudiment should be visible.</li>
			<li>Remove any remaining mesenchymal cells attached to the pharyngeal endoderm with the two microscalpels.</li>
			<li>Make a transversal cut between the 2nd and 3rd PP, dissociating the pharyngeal endoderm containing the 3rd and 4th pouches from the anterior part of the endoderm having the thyroid rudiment and 2nd pharyngeal pouch.</li>
			<li>With the help of spatula and thin forceps, transfer the isolated 3/4PP endoderm to a glass dish three-quarters filled with 100% cold fetal bovine serum (FBS).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Keep the glass dish with the isolated tissues on ice during the preparation of in vitro assay. Alternatively, the isolated tissues can be three-dimensionally preserved and in situ analyzed for gene-expression.</li>
</ol>
3. Isolation of chicken somatopleura mesoderm
NOTE: Perform egg manipulation procedures in sterile conditions using a horizontal laminar flow hood and sterilized instruments and materials.
<ol>
	<li>Remove the embryonic territory containing the somatopleura mesoderm at the level of somites 19-24 (ss19-24).
	<ol>
		<li>Remove the chicken egg from the incubator after 2.5 days of incubation.</li>
		<li>With curved scissors, open a small hole in the shell. Insert a needle and aspirate 2 mL of albumin with a 10 mL syringe to lower albumin volume inside the egg and prevent damage of the embryo (located below the marked region of the shell). Discard the aspirated albumin.</li>
		<li>Cut a circular hole (up to two-thirds of the top surface area) in the marked region of the shell using curved scissors.</li>
		<li>Cut the vitelline membrane externally to the extraembryonic vessels while holding the embryo with thin forceps.</li>
		<li>Under a stereomicroscope, place the embryo in a 100 mm Petri dish with a black base containing 10 mL of cold PBS. 
		NOTE: Use a stereomicroscope from this point forward for progressive magnification of microsurgery procedures.</li>
		<li>Use four thin insect pins to hold the embryo to the bottom of the plate. Place the pins in the extraembryonic region forming a square shape.</li>
		<li>Perform two cuts between the somites 19 and 24 transversely to the embryo axis and crossing all embryo territory, using wecker eye scissors.</li>
		<li>Release the embryo section, ss19-24, by cutting marginal embryonic edges.</li>
		<li>Aspirate the ss19-24 tissues and transfer to a glass dish three-quarters filled with cold PBS using a 2 mL sterile Pasteur pipette.</li>
	</ol>
	</li>
	<li>Isolate the lateral mesoderm from somatopleura region (ss19-24) by enzymatic digestion with pancreatin (8 mg/mL; 1:3 dilution of 25 mg/mL with cold PBS).
	<ol>
		<li>With the help of spatula and thin forceps, transfer the ss19-24 tissues to a glass dish three-quarters filled with cold pancreatin solution.</li>
		<li>Incubate for 30 min on ice for enzymatic digestion.</li>
		<li>Under the stereomicroscope, isolate the mesoderm from the surrounding tissues using two microscalpels in a holder. 
		NOTE: Keep all surfaces and solutions cold during this procedure. Change to a new cold pancreatin solution if taking a long time to dissect the tissues (&#62;10 min.). As an illumination source, use LED lights incorporated in the stereomicroscope or in the optic fibers, considering the limited heat load.</li>
		<li>During mesoderm isolation, first remove the ectoderm at the surface followed by careful detachment of the ventrally located splancnopleura tissues.</li>
		<li>Release the right lateral mesoderm of the somatopleura by cutting it in a parallel motion to the neural tube.</li>
		<li>Repeat the mesoderm separation of the left side of the embryo. 
		NOTE: Make slow microscalpel movements during this procedure. The exposed extra-cellular matrix proteins stick to tissues and instruments preventing fluid movements.</li>
		<li>With the help of spatula and thin forceps transfer the isolated mesoderm to a glass dish three-quarters filled with cold FBS.</li>
	</ol>
	</li>
	<li>Keep the glass dish with the isolated tissues on ice during the preparation of in vitro assay.</li>
</ol>
4. In vitro organotypic assay: heterospecific association of quail 3/4PP endoderm and chicken somatopleura mesoderm
<ol>
	<li>Prepare the culture medium with RPMI-1640 medium supplemented with 10% FBS and 1% Pen/Strep3,5.</li>
	<li>Place a metal grid in a 35 mm Petri dish with 5 mL of culture medium. 
	NOTE: Remove the excess of liquid to level the medium surface with the top of the grid.</li>
	<li>With the help of thin forceps, dip a membrane filter into the culture medium and then place it on the top of the grid to have one surface in contact with air. 
	NOTE: One-quarter of the membrane area (with 13 mm diameter) is adequate for the tissue association.</li>
	<li>Under the stereomicroscope, associate the isolated tissues on the top of the membrane filter. First transfer the 3/4PP endoderm (step 2) from the glass dish by gentle sliding with the help of a transplantation spoon (or spatula) and thin forceps. Repeat this procedure for the isolated mesoderm (step 3). 
	NOTE: With the help of a microscalpel, mix the tissues to maximize its association.</li>
	<li>Carefully place the associated tissues in a humidified incubator at 37 &#176;C with 5% CO2 for 48 h. Cultured tissues can be grafted onto the chorioallantoic membrane (CAM) . 
	NOTE: Ectopic organ formation in the CAM was previously detailed8.</li>
</ol>

<ol>
	<li>Le Douarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nogent+Institute--50+years+of+embryology.">The Nogent Institute--50 years of embryology.</a> The International Journal of Developmental Biology. 49 (2-3), 85-103 (2005).</li><li>Le Douarin, N. M., Teillet, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+migration+of+neural+crest+cells+to+the+wall+of+the+digestive+tract+in+avian+embryo.">The migration of neural crest cells to the wall of the digestive tract in avian embryo.</a> Journal of Embryology and Experimental Morphology. 30 (1), 31-48 (1973).</li><li>Le Douarin, N. M., Jotereau, F. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+of+cells+of+the+avian+thymus+through+embryonic+life+in+interspecific+chimeras.">Tracing of cells of the avian thymus through embryonic life in interspecific chimeras.</a> Journal of Experimental Medicine. 142 (1), 17-40 (1975).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.+1951.">A series of normal stages in the development of the chick embryo. 1951.</a> Developmental Dynamics. 195 (4), 231-272 (1992).</li><li>Neves, H., Dupin, E., Parreira, L., Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Bmp4+signalling+in+the+epithelial-mesenchymal+interactions+that+take+place+in+early+thymus+and+parathyroid+development+in+avian+embryos.">Modulation of Bmp4 signalling in the epithelial-mesenchymal interactions that take place in early thymus and parathyroid development in avian embryos.</a> Developmental Biology. 361 (2), 208-219 (2012).</li><li>Takahashi, Y., Bontoux, M., Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelio--mesenchymal+interactions+are+critical+for+Quox+7+expression+and+membrane+bone+differentiation+in+the+neural+crest+derived+mandibular+mesenchyme.">Epithelio--mesenchymal interactions are critical for Quox 7 expression and membrane bone differentiation in the neural crest derived mandibular mesenchyme.</a> Embo Journal. 10 (9), 2387-2393 (1991).</li><li>Figueiredo, 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=Notch+and+Hedgehog+in+the+thymus/parathyroid+common+primordium:+Crosstalk+in+organ+formation.">Notch and Hedgehog in the thymus/parathyroid common primordium: Crosstalk in organ formation.</a> Developmental Biology. 418 (2), 268-282 (2016).</li><li>Figueiredo, M., Neves, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+Approach+to+Explore+Early-and+Late-stages+of+Organ+Formation+in+the+Avian+Model:+The+Thymus+and+Parathyroid+Glands+Organogenesis+Paradigm+Video+Link.">Two-step Approach to Explore Early-and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm Video Link.</a> Journal of Visualuzed Experiments. , (2018).</li><li>Nehls, 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=Two+genetically+separable+steps+in+the+differentiation+of+thymic+epithelium.">Two genetically separable steps in the differentiation of thymic epithelium.</a> Science (New York, N.Y.). 272 (5263), 886-889 (1996).</li><li>Morahan, G., 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+nu+gene+acts+cell-autonomously+and+is+required+for+differentiation+of+thymic+epithelial+progenitors.">The nu gene acts cell-autonomously and is required for differentiation of thymic epithelial progenitors.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (12), 5742-5746 (1996).</li><li>Jerome, L. A., Papaioannou, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DiGeorge+syndrome+phenotype+in+mice+mutant+for+the+T-box+gene,+Tbx1.">DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1.</a> Nature Genetics. 27 (3), 286-291 (2001).</li><li>Nie, X., Brown, C. B., Wang, Q., Jiao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+Bmp4+from+the+Tbx1+expression+domain+causes+abnormal+pharyngeal+arch+artery+and+cardiac+outflow+tract+remodeling.">Inactivation of Bmp4 from the Tbx1 expression domain causes abnormal pharyngeal arch artery and cardiac outflow tract remodeling.</a> Cells Tissues Organs. 193 (6), 393-403 (2011).</li><li>Zou, D., 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=Patterning+of+the+third+pharyngeal+pouch+into+thymus/parathyroid+by+Six+and+Eya1.">Patterning of the third pharyngeal pouch into thymus/parathyroid by Six and Eya1.</a> Developmental Biology. 293 (2), 499-513 (2006).</li><li>Davey, M. G., Tickle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chicken+as+a+model+for+embryonic+development.">The chicken as a model for embryonic development.</a> Cytogenetic and Genome Research. 117 (1-4), 231-239 (2007).</li><li>Nowak-sliwinska, P., Segura, T., Iruela-arispe, M. L., Angeles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chicken+chorioallantoic+membrane+model+in+biology,+medicine+and+bioengineering.">The chicken chorioallantoic membrane model in biology, medicine and bioengineering.</a> Angiogenesis. 17 (4), 779-804 (2014).</li><li>Uematsu, E., 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=Use+of+in+ovo+chorioallantoic+membrane+engraftment+to+culture+testes+from+neonatal+mice.">Use of in ovo chorioallantoic membrane engraftment to culture testes from neonatal mice.</a> Comparative Medicine. 64 (4), 264-269 (2014).</li><li>Martyn, I., Kanno, T. Y., Ruzo, A., Siggia, E. D., Brivanlou, A. 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The authors have nothing to disclose.

The embryonic tissue isolation procedure detailed here was improved from previous techniques to produce quail-chicken chimeric embryos in different biological contexts3,5,6.
This approach is suitable to isolate pure embryonic tissues without requiring genetic manipulation or the use of tissue-specific markers, which are often undetermined, limiting the use of genetically modified animal models. It can...

In the early 1970s, an elegant quail-chicken chimera system was developed by Le Douarin, opening new avenues to understand the role of cell migration and cellular interactions during development1,2. The model was devised on the premise that cell exchange between the two species would not significantly disturb embryogenesis, later confirmed when used to study numerous developmental processes, including the formation of the nervous and the hematopoietic systems1. Taking the latter as an example, the cyclic waves of hematopoietic progenitors colonizing the thymic epithelial rudiment was first observed using the quail-chicken chimera system3. For that, the prospective territory of the thymus, the endoderm of the third and fourth pharyngeal pouches (3/4PP), was mechanically and enzymatically isolated from quail (q) embryos at 15 to 30-somite stage [embryonic day (E) 1.5- E2.5]. These stages correspond to chicken Hamburger and Hamilton4 (HH) - stages 12-17. The isolation procedures started with the use of trypsin to enzymatically dissociate the endoderm from the attached mesenchyme. The isolated endoderm was grafted into the somatopleura region of a host chicken (c) embryo at&#160;E3-E3.5 (HH-stages 20-21). This heterologous mesenchyme was considered &#34;permissive&#34; to thymic epithelium development contributing also to the organ formation3. Afterward, successive waves of chicken host blood-borne progenitor cells infiltrated the quail donor thymic epithelial counterpart contributing to thymus formation in the host embryo3.
More recently, a modified version of this approach was also proven to be important for studying epithelial-mesenchymal interactions during early-stages of thymus formation5. In this respect, the tissues involved in the formation of the ectopic thymus in chimeric embryos3 were isolated, both from donor and host embryos, and associated ex vivo. An improved protocol was used to isolate the quail 3/4PP endoderm (E2.5-E3) and the chicken somatopleura mesoderm (E2.5-E3). Briefly, embryonic tissues were isolated by microsurgery and subject to in vitro pancreatin digestion. Also, the conditions of enzymatic digestion, temperature and time of incubation were optimized according to tissue-type and developmental stage (Table 1).
Next, the isolated tissues were associated in an organotypic in vitro system for 48 h, as previously reported5,6. The in vitro association of tissues mimics the local cellular interactions in the embryo, overcoming some restrictions of the in vivo manipulation. This system is particularly useful to study&#160;cellular interactions in complex morphogenic events, such as the development of the pharyngeal apparatus.
The contribution of each tissue in thymus histogenesis, as well as the ability of the heterospecific association to generate a thymus can be further explored using the CAM methodology, previously detailed5,7,8. Succinctly, the cultured tissues were grafted onto the CAM of cE8 embryos and allowed to develop in ovo for 10 days. Then, thymus formation was evaluated by morphological analysis in the harvested explants. As in the classical quail-chicken studies3, the quail thymic epithelium was colonized by hematopoietic progenitor cells (HPCs) derived from the chicken embryo, which was later shown to contribute to organ development9,10. The HPCs migrated from the embryo to the ectopic chimeric thymus through the highly vascularized CAM5,7,8. Quail derived thymic epithelium can be identified by immunohistochemistry using species-specific antibodies (i.e., QCPN- MAb Quail PeriNuclear), overcoming the need for tissue-specific molecular markers.
This experimental method, as the two-step approach reported in previous publication8, allows the modulation of signalling pathways by regular administration of pharmacological agents during in vitro and in ovo development. Also, explants can be harvested at any time-point of the course of the experiment8.
Lastly, the isolation protocol here detailed allows the preservation of the natural properties and 3D-architecture of embryonic tissues, particularly useful for detailing in situ gene-expression patterns of embryonic territories otherwise inaccessible by conventional methods. In addition, transcriptome analysis approaches, including RNA-seq or microarrays, can also be applied in isolated tissues without requiring genetic markers while providing a tissue-specific high throughput &#34;omics&#34; analysis.

<table border="0" cellpadding="0" cellspacing="0" style="width:1279px;" width="1277">
	<tbody>
		<tr height="24">
			<td height="24" style="height:25px;width:379px;">Chicken fertilized eggs (Gallus gallus)</td>
			<td style="width:306px;">Pintobar, Portugal</td>
			<td style="width:149px;"></td>
			<td style="width:445px;">Poultry farm&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:379px;">Quail fertilized eggs (Coturnix coturnix)</td>
			<td>Interaves, Portugal</td>
			<td></td>
			<td style="width:445px;">Bird farm&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">15 mL PP centrifuge tubes</td>
			<td>Corning</td>
			<td>430052</td>
			<td style="width:445px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">50 mL PP centrifuge tubes</td>
			<td>Corning</td>
			<td>430290</td>
			<td style="width:445px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">60 x 20 mm pyrex dishes</td>
			<td>Duran group</td>
			<td style="width:149px;">21 755 41</td>
			<td style="width:445px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">100 x 20 mm pyrex dishes</td>
			<td>Duran group</td>
			<td style="width:149px;">21 755 48</td>
			<td style="width:445px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">Metal grid</td>
			<td>Goodfellows</td>
			<td style="width:149px;"></td>
			<td style="width:445px;">fine meshed&#160; stainless steel grid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">Membrane filter</td>
			<td>Millipore</td>
			<td style="width:149px;">DTTP01300</td>
			<td style="width:445px;">0.6 mm Isopore membrane filter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">Petri dish, 35 x 10 mm</td>
			<td>Sigma-Aldrich</td>
			<td style="width:149px;">P5112&#160;</td>
			<td style="width:445px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">60 x 30 mm pyrex bowls (Small size)</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:445px;">from supermarket&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">100 x 50 mm pyrex bowls (Large size)</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:445px;">from supermarket&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">Transfer pipettes</td>
			<td>Samco Scientific, Thermo Fisher Scientific</td>
			<td>2041S</td>
			<td style="width:445px;">2 mL plastic pipet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">Glass pasteur pipette</td>
			<td>Normax</td>
			<td style="width:149px;">5426015</td>
			<td style="width:445px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">Clear plastic tape</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:445px;">from supermarket&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:379px;">Cytokeratin (pan; acidic and basic, type I and II cytokeratins), clone Lu-5</td>
			<td>BMA Biomedicals</td>
			<td>T-1302</td>
			<td style="width:445px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">Fetal Bovine Serum</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td></td>
			<td style="width:445px;">Standart FBS</td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:379px;">Pancreatin</td>
			<td>Sigma-Aldrich</td>
			<td>P-3292</td>
			<td style="width:445px;">Prepare a 25 mg/mL solution according to manufacturer&#39;s instructions; centrifuge and filter prior to aliquote and store at -20&#186;C. Aliquots can be kept frozen for several years.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:379px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
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			<td height="20" style="height:20px;width:379px;">Insect pins&#160;</td>
			<td>Fine Science Tools</td>
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			<td>Fine Science Tools</td>
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			<td style="width:445px;">Transplantation spoon</td>
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			<td>Fine Science Tools</td>
			<td style="width:149px;">26002-20</td>
			<td style="width:445px;">0.2 mm Stainless steel microscalpel</td>
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			<td height="20" style="height:20px;width:379px;">Minutien Pins</td>
			<td>Fine Science Tools</td>
			<td style="width:149px;">26002-10</td>
			<td style="width:445px;">0.1 mm Stainless steel microscalpel</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:379px;">Moria Nickel Plated Pin Holder</td>
			<td>Fine Science Tools</td>
			<td style="width:149px;">26016-12</td>
			<td style="width:445px;">Nickel plated pin holder</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:379px;">Moria Perforated Spoon</td>
			<td>Fine Science Tools</td>
			<td style="width:149px;">10370-17</td>
			<td style="width:445px;">Skimmer</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:379px;">Wecker Eye Scissor</td>
			<td>Fine Science Tools</td>
			<td style="width:149px;">15010-11</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:379px;">Camera</td>
			<td>Leica Microsystems&#160;</td>
			<td style="width:149px;">MC170 HD</td>
			<td style="width:445px;"></td>
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			<td height="20" style="height:20px;width:379px;">Microscope</td>
			<td>Leica Microsystems&#160;</td>
			<td style="width:149px;">DM2500</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:379px;">NanoZoomer&#160;S360 Digital&#160;slide scanner&#160;</td>
			<td>Hamamatsu Photonics</td>
			<td style="width:149px;">C13220-01</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:379px;">Stereoscope</td>
			<td>Leica Microsystems&#160;</td>
			<td style="width:149px;">Leica M80</td>
			<td style="width:445px;"></td>
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isolation of embryonic tissues and formation of quail-chicken chimeric organs using the thymus example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

The protocol details a method to isolate avian embryonic tissues to be used in several cellular and developmental biology technical approaches. This method was previously employed to study epithelial-mesenchymal interactions during early stages of thymus formation5. Herein, new results are shown in Figure 1 and Figure 2, using similar approaches.
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Watch this Scientific Journal Video about Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example at JoVE.com

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

isolation-embryonic-tissues-formation-quail-chicken-chimeric-organs

Research

JoVE Journal

Developmental Biology

7.3K Views.  Universidade de Lisboa. This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs. 

Video: Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

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

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

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

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

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

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

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 ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

Summary

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Chapters in this video

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

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Isolation of Murine Embryonic Hemogenic Endothelial Cells

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

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

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs