Name: Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example (Video) | JoVE Journal
Uploaded: 2019-02-16T14:00:00.000+00:00
Duration: 14 min 23 s
Description: Learn microsurgical isolation of quail and chicken embryonic tissues for gene expression studies. This method enables high-resolution analysis of developmental processes and chimeric organ formation.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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/pubmed?term=((The+Nogent+Institute--50+years+of+embryology%5BTitle%5D)+AND+%22The+International+Journal+of+Developmental+Biology%22%5BJournal%5D)">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/pubmed?term=((The+migration+of+neural+crest+cells+to+the+wall+of+the+digestive+tract+in+avian+embryo%5BTitle%5D)+AND+%22Journal+of+Embryology+and+Experimental+Morphology%22%5BJournal%5D)">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/pubmed?term=((Tracing+of+cells+of+the+avian+thymus+through+embryonic+life+in+interspecific+chimeras%5BTitle%5D)+AND+%22Journal+of+Experimental+Medicine%22%5BJournal%5D)">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/pubmed?term=((A+series+of+normal+stages+in+the+development+of+the+chick+embryo.+1951%5BTitle%5D)+AND+%22Developmental+Dynamics%22%5BJournal%5D)">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/pubmed?term=((Modulation+of+Bmp4+signalling+in+the+epithelial-mesenchymal+interactions+that+take+place+in+early+thymus+and+parathyroid+development+in+avian+embryos%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">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/pubmed?term=((Epithelio--mesenchymal+interactions+are+critical+for+Quox+7+expression+and+membrane+bone+differentiation+in+the+neural+crest+derived+mandibular+mesenchyme%5BTitle%5D)+AND+%22Embo+Journal%22%5BJournal%5D)">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/pubmed?term=((Notch+and+Hedgehog+in+the+thymus%2Fparathyroid+common+primordium%3A+Crosstalk+in+organ+formation%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">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/pubmed?term=((Two-step+Approach+to+Explore+Early-and+Late-stages+of+Organ+Formation+in+the+Avian+Model%3A+The+Thymus+and+Parathyroid+Glands+Organogenesis+Paradigm+Video+Link%5BTitle%5D)+AND+%22Journal+of+Visualuzed+Experiments%22%5BJournal%5D)">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/pubmed?term=((Two+genetically+separable+steps+in+the+differentiation+of+thymic+epithelium%5BTitle%5D)+AND+%22Science+%28New+York%2C+N.Y.%29%22%5BJournal%5D)">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/pubmed?term=((The+nu+gene+acts+cell-autonomously+and+is+required+for+differentiation+of+thymic+epithelial+progenitors%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((DiGeorge+syndrome+phenotype+in+mice+mutant+for+the+T-box+gene%2C+Tbx1%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">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/pubmed?term=((Inactivation+of+Bmp4+from+the+Tbx1+expression+domain+causes+abnormal+pharyngeal+arch+artery+and+cardiac+outflow+tract+remodeling%5BTitle%5D)+AND+%22Cells+Tissues+Organs%22%5BJournal%5D)">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/pubmed?term=((Patterning+of+the+third+pharyngeal+pouch+into+thymus%2Fparathyroid+by+Six+and+Eya1%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">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/pubmed?term=((The+chicken+as+a+model+for+embryonic+development%5BTitle%5D)+AND+%22Cytogenetic+and+Genome+Research%22%5BJournal%5D)">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/pubmed?term=((The+chicken+chorioallantoic+membrane+model+in+biology%2C+medicine+and+bioengineering%5BTitle%5D)+AND+%22Angiogenesis%22%5BJournal%5D)">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/pubmed?term=((Use+of+in+ovo+chorioallantoic+membrane+engraftment+to+culture+testes+from+neonatal+mice%5BTitle%5D)+AND+%22Comparative+Medicine%22%5BJournal%5D)">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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-organization+of+a+human+organizer+by+combined+Wnt+and+Nodal+signaling%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Self-organization of a human organizer by combined Wnt and Nodal signaling.</a> Nature. 558 (7708), 132-135 (2018).</li>
</ol>

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

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.
<p class="jove_content" fo:keep-together.within-page="1"...

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

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.

Xenotransplantation of Human Stem Cells into the Chicken Embryo

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

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

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.

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

This article describes a method to generate chimeric embryos that are designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.<br...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

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.

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

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.

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

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

Creating Avian Forebrain Chimeras to Assess Facial Development

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.

Isolation of Preadipocytes from Broiler Chick Embryos

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

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.

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

Learn microsurgical isolation of quail and chicken embryonic tissues for gene expression studies. This method enables high-resolution analysis of developmental processes and chimeric organ formation.

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

Explore More Videos