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>

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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. 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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><tbody><tr><td>Chicken fertilized eggs (&lt;em&gt;Gallus gallus&lt;/em&gt;)</td><td>Pintobar, Portugal</td><td></td><td>Poultry farm&amp;nbsp;</td></tr><tr><td>Quail fertilized eggs (&lt;em&gt;Coturnix coturnix&lt;/em&gt;)</td><td>Interaves, Portugal</td><td></td><td>Bird farm&amp;nbsp;</td></tr><tr><td>15 mL PP centrifuge tubes</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>50 mL PP centrifuge tubes</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>60 x 20 mm pyrex dishes</td><td>Duran group</td><td>21 755 41</td><td></td></tr><tr><td>100 x 20 mm pyrex dishes</td><td>Duran group</td><td>21 755 48</td><td></td></tr><tr><td>Metal grid</td><td>Goodfellows</td><td></td><td>fine meshed&amp;nbsp; stainless steel grid</td></tr><tr><td>Membrane filter</td><td>Millipore</td><td>DTTP01300</td><td>0.6 mm Isopore membrane filter</td></tr><tr><td>Petri dish, 35 x 10 mm</td><td>Sigma-Aldrich</td><td>P5112&amp;nbsp;</td><td></td></tr><tr><td>60 x 30 mm pyrex bowls (Small size)</td><td></td><td></td><td>from supermarket&amp;nbsp;</td></tr><tr><td>100 x 50 mm pyrex bowls (Large size)</td><td></td><td></td><td>from supermarket&amp;nbsp;</td></tr><tr><td>Transfer pipettes</td><td>Samco Scientific, Thermo Fisher Scientific</td><td>2041S</td><td>2 mL plastic pipet</td></tr><tr><td>Glass pasteur pipette</td><td>Normax</td><td>5426015</td><td></td></tr><tr><td>Clear plastic tape</td><td></td><td></td><td>from supermarket&amp;nbsp;</td></tr><tr><td>Cytokeratin (pan; acidic and basic, type I and II cytokeratins), clone Lu-5</td><td>BMA Biomedicals</td><td>T-1302</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Invitrogen, Thermo Fisher Scientific</td><td></td><td>Standart FBS</td></tr><tr><td>Pancreatin</td><td>Sigma-Aldrich</td><td>P-3292</td><td>Prepare a 25 mg/mL solution according to manufacturer&#039;s instructions; centrifuge and filter prior to aliquote and store at -20&amp;ordm;C. Aliquots can be kept frozen for several years.</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen, Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>GIBCO, Thermo Fisher Scientific</td><td>10010023</td><td></td></tr><tr><td>QCPN antibody</td><td>Developmental Studies Hybridoma Bank</td><td>QCPN</td><td></td></tr><tr><td>RPMI 1640 Medium, GlutaMAX Supplement&amp;nbsp;</td><td>GIBCO, Thermo Fisher Scientific</td><td>61870010</td><td></td></tr><tr><td>Bluesil RTV141A/B Silicone Elastomer 1.1Kg Kit</td><td>ELKEM/Silmid</td><td>RH141001KG</td><td>To prepare the back base for petri dish</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-30&amp;nbsp;</td><td>Thin forceps</td></tr><tr><td>Extra fine Bonn scissors, curved</td><td>Fine Science Tools</td><td>14085-08</td><td>Curved scissors</td></tr><tr><td>Insect pins&amp;nbsp;</td><td>Fine Science Tools</td><td>26001-30</td><td>0.3 mm Stainless steel pin</td></tr><tr><td>Micro spatula&amp;nbsp;</td><td>Fine Science Tools</td><td>10087-12</td><td>Transplantation spoon</td></tr><tr><td>Minutien Pins</td><td>Fine Science Tools</td><td>26002-20</td><td>0.2 mm Stainless steel microscalpel</td></tr><tr><td>Minutien Pins</td><td>Fine Science Tools</td><td>26002-10</td><td>0.1 mm Stainless steel microscalpel</td></tr><tr><td>Moria Nickel Plated Pin Holder</td><td>Fine Science Tools</td><td>26016-12</td><td>Nickel plated pin holder</td></tr><tr><td>Moria Perforated Spoon</td><td>Fine Science Tools</td><td>10370-17</td><td>Skimmer</td></tr><tr><td>Wecker Eye Scissor</td><td>Fine Science Tools</td><td>15010-11</td><td></td></tr><tr><td>Camera</td><td>Leica Microsystems&amp;nbsp;</td><td>MC170 HD</td><td></td></tr><tr><td>Microscope</td><td>Leica Microsystems&amp;nbsp;</td><td>DM2500</td><td></td></tr><tr><td>NanoZoomer&amp;nbsp;S360 Digital&amp;nbsp;slide scanner&amp;nbsp;</td><td>Hamamatsu Photonics</td><td>C13220-01</td><td></td></tr><tr><td>Stereoscope</td><td>Leica Microsystems&amp;nbsp;</td><td>Leica M80</td><td></td></tr></tbody></table>

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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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

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Neuroscience

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 ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Creating Avian Forebrain Chimeras to Assess Facial Development

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the strengths of this model system is that the effect of, and interaction among, tissues can be directly assessed in chimeric embryos. We have previously shown that signals from the forebrain contribute to facial morphogenesis by regulating the shape of the expression domain of Sonic hedgehog (SHH) in the Frontonasal Ectodermal Zone (FEZ). In this article, the method of generating the forebrain chimeras and provide illustrations of the outcomes of these experiments is described.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of basal forebrain&#160;during craniofacial development.

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Biology

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

In Ovo Intravascular Injection in Chicken Embryos

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

Isolation of Preadipocytes from Broiler Chick Embryos

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. Chicken embryos provide the opportunity to isolate preadipocytes from the earliest stage of adipose development. This primary cell can be used to identify factors influencing preadipocyte proliferation and adipogenic differentiation, making them a valuable model for studies related to childhood obesity and control of excess fat deposition in poultry. The rapid growth of postnatal adipose tissue effectively wastes feed by allocating it away from muscle growth in broiler chickens. Therefore, methods to understand the earliest stages of adipose tissue development may provide clues to regulate this tendency and identify ways to limit adipose expansion early in life. The present study was designed to develop an efficient method for isolation, primary culture, and adipogenic differentiation of preadipocytes isolated from developing adipose tissue of commercial broiler (meat-type) chick embryos. The procedure has been optimized to yield cells with high viability (~98%) and increased capacity to differentiate into mature adipocytes. This simple method of embryonic preadipocyte isolation, culture, and differentiation supports functional analyses of fat growth and development in early life.

The present protocol describes a simple method for isolating preadipocytes from adipose tissue in broiler embryos. This method enables isolation with high yield, primary culture, and adipogenic differentiation of preadipocytes. Oil Red O staining and lipid/DNA stain measured the adipogenic ability of isolated cells induced with differentiation media.

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

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

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Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

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

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Creating Avian Forebrain Chimeras to Assess Facial Development

Xenotransplantation of Human Stem Cells into the Chicken Embryo

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

In Ovo Intravascular Injection in Chicken Embryos

Isolation of Preadipocytes from Broiler Chick Embryos

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development