This work is supported by NIH NIAMS grant R37 AR 060306, R01 AR 047364, and RO1 AR078050. The work is also supported by a collaborative research contract between USC and China Medical University in Taiwan. We thank the USC BISC 480 Developmental Biology 2023 class for successfully testing this avian skin culture protocol during several lab modules.

1. Chicken skin explant culture (Figure 1)
<ol>
	<li>Incubate fertilized chicken eggs in a humidified incubator at 38 &#176;C and stage them according to Hamburger and Hamilton14.
	<ol>
		<li>At stage 28 (~E5.5), the limb&#39;s second digit and third toe are longer than the others; three digits and four toes are distinct.</li>
		<li>At stage 29 (~E6), the wing is bent at the elbow; the second digit is distinctly longer than the others.</li>
		<li>At stage 30 (~E6.5), two dorsal feather bud rows to either side of the spinal cord are visible at the brachial level and three rows are seen at the level of the legs.</li>
		<li>At stage 31 (~E7), there are ~7 rows of feathers at the jumbo-sacral levels.</li>
		<li>At stage 32, eleven or more rows of feather buds are present at the level of the legs.</li>
	</ol>
	</li>
	<li>Place a stage 28-32 chicken embryo in a 60 mm Petri dish containing Hanks&#39;s buffered saline solution (HBSS) or phosphate-buffered saline (PBS).
	<ol>
		<li>Dissect out the embryo dorsal skin between the lower neck to the tail region. Use scissors to remove the head from the neck. Gently pull the limb buds to position the ventral side of the body downwards with the appendages spreading outwards.</li>
		<li>Make an anterior to posterior incision in the skin along the two flanking sides of the embryo body using watchmaker&#39;s forceps or sharp spring scissors. Be sure to stabilize the body with a pair of forceps by pinching the limb buds longitudinally. Gently peel the skin from the neck to the tail region or from the tail to the neck using watchmaker&#39;s forceps.</li>
	</ol>
	</li>
	<li>Gently remove the subcutaneous tissues that remain attached to the peeled skin and smooth the edges of the skin with the sharp spring scissors.</li>
	<li>Add 2 mL/well of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Penicillin/streptomycin solution (diluted 1:100) to a well in a 6-well dish. Once the medium and antibiotics have been added, place a sterile tissue culture insert inside the well, such that the media is on the exterior of the culture insert.</li>
	<li>Transfer the skin with a spatula to the culture insert with the epidermis face up and the mesenchyme face down on the insert membrane. To ensure the skin lays flat without any creases, pull the skin onto the spatula in HBSS. Slide the skin off the spatula with the liquid to facilitate the transfer of the skin without folding.</li>
	<li>Remove excess HBSS from the culture insert with a 200 &#181;L pipette. Leave a thin layer of HBSS inside the culture insert to keep the explant semi-wet to provide an air-liquid interface.</li>
	<li>Incubate the skin explant cultures at 37 &#176;C using 5% CO2 and 95% air. Change the medium every 2 days. 
	&#8203;NOTE: This procedure has been published4. This culture system can also be used with the skin of other birds such as quail or finch skin explant cultures.</li>
</ol>
2. Chicken skin epithelial-mesenchymal recombination (Figure 2)
<ol>
	<li>Incubate fertilized chicken eggs in a humidified incubator at 38 &#176;C and stage the embryos according to Hamburger and Hamilton14 (section 1.1).</li>
	<li>Prepare 2x calcium-magnesium-free (CMF) saline with 0.25% ethylenediaminetetraacetic acid (EDTA) buffer.
	<ol>
		<li>Make 10x CMF buffer (NaCl (1.37 M) 80 g, KCl (0.04 M) 3 g, NaH2PO4 (0.004 M) 0.5 g, KH2PO4 (2 M) 0.25 g, NaHCO3 (0.12 M) 10 g, glucose (0.1 M) 20 g in 1,000 mL of distilled water. Adjust pH to 7.3. To make 100 mL of 2x CMF with 0.25% EDTA buffer, the EDTA should be dissolved in 80 mL of distilled water first, then add 20 mL of 10x CMF buffer to make 2x CMF with 0.25% EDTA working solution.</li>
	</ol>
	</li>
	<li>Dissect stage 30-32 chicken embryo dorsal skins in HBSS as described above (section 1.2) and incubate them in 2x CMF saline with 0.25% EDTA on ice for 15-20 min.</li>
	<li>Carefully separate the epithelium and mesenchyme using watchmaker&#39;s forceps. Transfer the separated epithelium and mesenchyme to a clean dish containing HBSS.</li>
	<li>Recombine the epithelium with the mesenchyme by first placing the mesenchyme on a Petri dish containing HBSS, then place the epithelium on top of the mesenchyme. Place the epithelium along the same anterior-posterior axis as the mesenchyme or turn the epithelium 90o from the anterior-posterior axis of the mesenchyme to determine which component regulates different aspects of skin morphogenesis.</li>
	<li>Transfer the recombined skins to culture inserts in 6-well culture dishes for growth and remove excess HBSS around the recombined skin.</li>
	<li>Place 2 mL of DMEM culture medium with 10% FBS in the well outside the insert. Place a thin layer of DMEM inside the insert chamber to keep the explant semi-wet to provide an air-liquid interface.</li>
	<li>Incubate the skin recombinants at 37 &#176;C in a mixture of 5% CO2 and 95% air. Change the medium every 2 days. Document phenotypic changes in initial skin phases of skin development using time-lapse photography with a camera mounted on a dissecting microscope. 
	NOTE: This procedure has been published11.</li>
</ol>
3. Chicken skin reconstitution (Figure 3)
<ol>
	<li>Incubate fertilized chicken eggs in a humidified incubator at 38 &#176;C and stage the embryos according to Hamburger and Hamilton14 (section 1.1).</li>
	<li>Dissect stage 30-33 chicken embryo dorsal skins in HBSS. Incubate the skins in 2x CMF saline with 0.25% EDTA on ice for 15-20 min as described above (section 2.3).</li>
	<li>Carefully separate the epithelium and mesenchyme using watchmaker&#39;s forceps. Transfer the separated epithelium and mesenchyme to HBSS on ice. 
	NOTE: Be careful not to damage the basal layer of the epithelium when separating the epithelium from the mesenchyme.</li>
	<li>Collect the separated mesenchyme in a 15 mL centrifuge tube and incubate with 0.1% collagenase/trypsin solution made in PBS in a 37 &#176;C water bath for 10-15 min.</li>
	<li>Dissociate the skin mesenchyme with a Pasteur pipet to make a single-cell suspension. Add DMEM with 10% FBS to stop the digestion.</li>
	<li>Pellet and centrifuge the cells at 233 &#215; g for 5 min. Resuspended the cells with culture medium at a concentration of 2 &#215; 107 cells/mL. 
	NOTE: At this step, the mesenchymal cells can also be resuspended in virus-containing medium for 2-3 h on ice for gene transduction.</li>
	<li>Place a cell culture insert in one well of a 6-well dish. Drop 10 &#181;L of 2 &#215; 107 cells/mL mesenchymal cells on the tissue culture insert. Place 2 ml of DMEM culture medium with 10% FBS in the well outside of the insert. Incubate the plated mesenchymal cells at 37 &#176;C using a 5% CO2 and 95% air incubator for 1 h to allow the cells to settle on the insert membrane.</li>
	<li>Transfer the intact epithelium on top of the plated mesenchymal droplet with the epithelium basal cell layer facing the mesenchymal cells using a 1 mL pipet. 
	NOTE: When transferring epithelium to the mesenchymal droplet, the mesenchymal cells should not be disturbed.</li>
	<li>Flatten the intact epithelium over the mesenchyme to make the reconstituted skin explant. Leave a thin layer of the medium on the insert to keep the reconstituted skin explant semi-wet to provide an air-liquid interface. Incubate the reconstituted skin explant at 37 &#176;C in a 5% CO2 and 95% air incubator. Change the medium every 2 days. 
	NOTE: This procedure has been published13.</li>
</ol>

A Comprehensive Review of Avian Embryonic Skin Explant Culture Protocols

<ol><li>Lucas, A. M., Stettenheim, P. R. Avian anatomy: Integument part I and part II. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLucas%2C+AM+%22Agriculture+Handbook%22">Agriculture Handbook</a>. 362, Agricultural Research Service, U.S. Department of Agriculture, Washington D.C.. (1972).</li>
<li>Sengel, P. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSengel%2C+P+%22In+Morphogenesis+of+Skin%2C+l-277%22">In Morphogenesis of Skin, l-277</a>. , Cambridge Univ. Press. New York. (1976).</li>
<li>Dhouailly, D., Wilehm Roux, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formation+of+cutaneous+appendages+in+dermo-epidermal+recombinations+between+reptiles%2C+birds+and+mammals%5BTitle%5D)+AND+%22Archives+of+Developmental+Biology%22%5BJournal%5D)">Formation of cutaneous appendages in dermo-epidermal recombinations between reptiles, birds and mammals.</a> Archives of Developmental Biology. 177 (4), 323-340 (1975).</li>
<li>Jiang, T. -X., Chuong, C. -M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+feather+morphogenesis%3A+I.+Analyses+with+antibodies+to+Adhesion+Molecules+Tenascin%2C+N-CAM+and+Integrin%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Mechanism of feather morphogenesis: I. Analyses with antibodies to Adhesion Molecules Tenascin, N-CAM and Integrin.</a> Developmental Biology. 150 (1), 82-98 (1992).</li>
<li>Li, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shaping+organs+by+a+Wnt+%2F+Notch+%2F+non-muscle+myosin+module+which+orients+feather+bud+elongation%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%2C+USA%22%5BJournal%5D)">Shaping organs by a Wnt / Notch / non-muscle myosin module which orients feather bud elongation.</a> Proceedings of the National Academy of Sciences, USA. 110 (16), E1452-E1461 (2013).</li>
<li>Li, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+oscillations+coordinate+feather+mesenchymal+cell+movement+by+SHH+dependent+modulation+of+gap+junction+networks%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Calcium oscillations coordinate feather mesenchymal cell movement by SHH dependent modulation of gap junction networks.</a> Nature Communications. 9 (1), 5377(2018).</li>
<li>Ting-Berreth, S. A., Chuong, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Local+delivery+of+TGF+beta2+can+restore+epithelium+dependent+organization+of+mesenchymal+condensation+during+skin+appendage+morphogenesis%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Local delivery of TGF beta2 can restore epithelium dependent organization of mesenchymal condensation during skin appendage morphogenesis.</a> Developmental Biology. 179 (2), 347-359 (1996).</li>
<li>Widelitz, R. B., Jiang, T. -X., Noveen, A., Chen, C. -W. J., Chuong, C. -M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FGF+induces+new+feather+buds+from+developing+avian+skin%5BTitle%5D)+AND+%22Journal+of+Investigation+Dermatology%22%5BJournal%5D)">FGF induces new feather buds from developing avian skin.</a> Journal of Investigation Dermatology. 107 (6), 797-803 (1996).</li>
<li>Rawles, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+interactions+in+scale+and+feather+development+as+studies+in+dermal-epidermal+recombinations%5BTitle%5D)+AND+%22Journal+of+Embryology+and+Experimental+Morphology%22%5BJournal%5D)">Tissue interactions in scale and feather development as studies in dermal-epidermal recombinations.</a> Journal of Embryology and Experimental Morphology. 11, 765-789 (1963).</li>
<li>McAleese, S. R., Sawyer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correcting+the+phenotype+of+the+epidermis+from+chick+embryos+homozygous+for+the+gene+scaleless+%28sc%2Fsc%29%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Correcting the phenotype of the epidermis from chick embryos homozygous for the gene scaleless (sc/sc).</a> Science. 214 (4524), 1033-1034 (1981).</li>
<li>Chuong, C. M., Widelitz, R. B., Ting-Berreth, S., Jiang, T. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+events+during+avian+skin+appendage+regeneration%3A+dependence+on+epithelial-mesenchymal+interaction+and+order+of+molecular+reappearance%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">Early events during avian skin appendage regeneration: dependence on epithelial-mesenchymal interaction and order of molecular reappearance.</a> J Invest Dermatol. 107 (4), 639-646 (1996).</li>
<li>Jiang, T. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+feather+orientations+changed+by+electric+current%5BTitle%5D)+AND+%22iScience%22%5BJournal%5D)">Global feather orientations changed by electric current.</a> iScience. 24 (6), 102671(2021).</li>
<li>Jiang, T. X., Jung, H. S., Widelitz, R. B., Chuong, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-organization+of+periodic+patterns+by+dissociated+feather+mesenchymal+cells+and+the+regulation+of+size%2C+number+and+spacing+of+primordia%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Self-organization of periodic patterns by dissociated feather mesenchymal cells and the regulation of size, number and spacing of primordia.</a> Development. 126 (22), 4997-5009 (1999).</li>
<li>Hamburger, V., Hamilton, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+series+of+normal+stages+in+the+development+of+the+chick+embryo%5BTitle%5D)+AND+%22Journal+of+Morphology%22%5BJournal%5D)">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 188, 49-92 (1951).</li>
<li>Chen, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conservation+of+early+odontogenic+signaling+pathway+in+Aves%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%2C+USA%22%5BJournal%5D)">Conservation of early odontogenic signaling pathway in Aves.</a> Proceedings of the National Academy of Sciences, USA. 97 (18), 10044-10049 (2000).</li>
<li>Chuong, C. -M., Ting, S. A., Widelitz, R. B., Lee, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+Skin+Morphogenesis%3A+II.+Retinoic+acid+gradient+modulates+axis+orientation+and+phenotypes+of+skin+appendages%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Mechanism of Skin Morphogenesis: II. Retinoic acid gradient modulates axis orientation and phenotypes of skin appendages.</a> Development. 115 (3), 839-852 (1992).</li>
<li>Noveen, A., Jiang, T. -X., Chuong, C. -M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+kinase+A+and+protein+kinase+C+modulators+have+reciprocal+effects+on+mesenchymal+condensation+during+skin+appendage+morphogenesis%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Protein kinase A and protein kinase C modulators have reciprocal effects on mesenchymal condensation during skin appendage morphogenesis.</a> Developmental Biology. 171 (2), 677-693 (1995).</li>
<li>Wu, X. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-assembly+of+biological+networks+via+adaptive+patterning+revealed+by+avian+intradermal+muscle+network+formation%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%2C+USA%22%5BJournal%5D)">Self-assembly of biological networks via adaptive patterning revealed by avian intradermal muscle network formation.</a> Proceedings of the National Academy of Sciences, USA. 116 (22), 10858-10867 (2019).</li>
</ol>

The authors have no conflicts of interest to declare.

Tissue recombination provides an assay to explore the unique contributions of the epithelium and mesenchyme. In chickens, feathers begin to develop at embryonic day 7 (E7) while scales begin at E9. When E9 scale mesenchyme is recombined with E7 feather epithelium, the recombined tissue forms scales, and when E7 feather mesenchyme is recombined with E9 scale epithelium feathers are formed11. These studies have demonstrated that the mesenchyme controls the pattern formation spacing and organ identity. Of course, the epithelium must be competent to be able to receive and interpret the mesenchymal signals appropriately. Competence only exists in the epithelium for a short time interval. In contrast to the above results, surprisingly when the chicken oral mucosa and aboral E5 epithelium are recombined with E8 trunk mesenchyme, the aboral epithelium forms feathers; however, the oral mucosa forms multiple tooth-like appendages15. This shows that while the skin trunk mesenchyme directs the aboral epithelium to make feathers, the oral epithelium does not have the ability to make feathers and can only make teeth. Oral epithelium in this study may already have been committed to forming teeth at E5. In situ hybridization, RNAscope, and immunostaining can be used to confirm molecular expression in the recombined explants to verify the identity of tissues that form. Using this assay, perturbations can be specifically directed to the epithelium or mesenchyme.
Tissue reconstitution resets cells within the mesenchyme to a noncommitted state. E7-E8 feather skin has begun to form mesenchymal condensations in the dorsal tract before the skin is dissociated and the mesenchyme is dissociated to single cells. By labeling epithelial or mesenchymal cells in the primordial bud or interbud regions, the dermal cells can be found inside or outside of buds without regard to their previous location. At low mesenchymal cell density, the reconstituted skin explants form few normal-sized feather buds. As the mesenchymal cell density increases, the number of buds increases until a maximum packing density is achieved13. These data indicate that the cells are reprogrammed through loss of contact with their initial neighbors and undergo a new round of pattern formation. These cultures also develop condensations and feather primordia. Interestingly, buds form simultaneously across the skin as opposed to the progressive propagation of bud formation seen in skin explants and recombined skin explants. This method affords the observation and manipulation of early-stage skin cellular and molecular interactions. Molecular expression can be assessed in culture using promoter-reporter assays. Reconstitution between transgenic or knockout lines can help to further analyze the roles of molecules in the skin patterning process. Viral transduction is enhanced in the dissociated mesenchymal cells and can often produce an increased perturbation.
While each of these experimental approaches can help investigators to explore cellular and molecular events that take place during skin morphogenesis. We frequently use each of these approaches and see which one provides the best insights. Findings can then be explored further under other culture conditions or using intact embryos.
Limitations of these approaches 
Each of these three assays provides highly reproducible results. However, as these assays may respond to the same perturbation to different degrees, it is often worthwhile to perturb conditions using all three assays if no alterations are initially encountered. Although the skin explant culture provides a good model for early skin pattern formation and skin appendage development, the explant can only be cultured for ~1 week. This precludes their use in later skin appendage development, for example, the process for dermal papilla formation and follicle morphogenesis although procedures are being improved to culture these explants longer. To date, at early times after making reconstituted skin explants the cells are loosely attached to their substrates, making analyses that use several washes, such as in situ hybridization, difficult. These cultures bind their substrates more firmly by 24 h at which condensations have formed throughout the culture.
Avian embryonic skin provides an excellent model to study the development of skin appendages. Skin explant cultures have the advantage of enabling the manipulation of the culture conditions in which the ex vivo skin organ cultures grow. It also enables long-term time-lapse imaging of development to see the propagation of buds from the midline to lateral edges over time. In addition, the culture can be started using different embryonic stages to explore different events in feather patterning and growth. For periodic pattern formation, skin can be cultured starting at E6 and cultured for 4 days. For feather follicle morphogenesis, the culture can be started at E8 and cultured for 4 days. It is difficult to dissect skin at stages prior to E6. With explant cultures, global perturbations can be made by adding growth factors or inhibitors to the skin explant culture medium8,16,17. Perturbing the skin locally can be achieved by placing protein-coated beads on the skin explant or by virally transducing cells to modulate gene expression8,16,17. Explant cultures facilitate the imaging of collective cell behavior such as calcium activities6 and the application of biophysical forces such as tissue tension18 or electric currents12. These methods offer great opportunities to provide new insights into the factors that regulate periodic organ patterning.

Avian embryo skin development is an excellent model for studying the mechanisms of morphogenesis because of the distinct patterns and the accessibility to microsurgery and manipulation1,2. However, evaluating cellular and molecular events in intact tissues can be difficult because the presence of extraneous tissues can complicate microscopic observations. Furthermore, the ability to manipulate gene expression to test their role in skin morphogenesis is not always a simple task. We find we can test gene functions using retroviral transduction with a higher success rate using skin explant models. Here we discuss the advantages of three skin explant models that have been developed.
Avian embryonic skin culture is a powerful system to assess cell behavior, gene regulation, and function during skin feather bud development3,4,5,6. It allows for the evaluation of the molecular mechanisms of feather bud development through the global addition of growth factors placed in the culture media or their local release from growth factor-coated beads. Developmental regulatory genes can also be manipulated using viral gene transduction of intact or dominant negative forms for functional studies evaluating their roles in specific morphogenetic events 7,8.
Avian epithelial-mesenchymal recombination culture enables investigators to determine the contributions of each skin component during the early stages of skin morphogenesis. Rawles&#39; use of this approach revealed that interactions between the mesenchyme and epithelium are essential to forming skin appendages9. The mesenchyme can form condensations and the epithelium is needed to induce and maintain mesenchymal condensation formations2. Later, this approach was used to assess why Scaleless chickens fail to form feathers. The defect was discovered to be in mesenchyme10. Dhouailly performed tissue epithelial-mesenchymal recombination studies in embryos from different species. These studies provided developmental and evolutionary insights into epithelial-mesenchymal communications that promote skin morphogenesis3.
This study was used to better understand factors that control feather growth. The method also improves the visualization of cellular and molecular events involved in skin patterning that take place during feather initiation, development, and elongation along the anterior-posterior axis. When the epithelium is separated from the mesenchyme and the two components are then recombined, new interactions re-establish skin patterning. This approach allows us to evaluate mesenchymal inducing signals and epithelial competence molecules that enable the epidermis to respond to the mesenchymal signals11. The subsequent downstream molecular expression that is required for feather bud development and pattern formation can also be examined. These studies have established that the location of buds is controlled by the mesenchyme. Rotation of the epithelium 90o before recombination with the mesenchyme demonstrates that the direction of feather bud elongation is controlled by the epithelium. This method was essential for us to study the molecular mechanism regulating feather bud orientation12.
Avian skin reconstitution culture, in which the skin mesenchyme is dissociated to single cells before plating at high cell density and overlaid with intact epithelium, resets dermal cells to a primordial state. The explant then self-organizes to form a new periodic pattern independent of the previous cues13. This skin reconstitution model can be used to study the initial processes of feather periodic patterning. We used this approach to explore how modulating the ratio of mesenchymal cells to a single piece of epithelium can influence the size or number of feather buds. The number of buds was found to increase but not the size of buds as the ratio of mesenchymal cells increased. Another advantage to this approach is that mesenchymal cell viral transduction shows higher efficiency than in the other two culture conditions and can produce more obvious phenotypes.

<table><tbody><tr><td>6-well culture dish&amp;nbsp;</td><td>Falcon</td><td>REF 353502</td><td>Air-Liquid Interface (ALI) Cultures &amp;nbsp;</td></tr><tr><td>Cell culture inset&amp;nbsp;</td><td>Falcon</td><td>REF 353090.</td><td>0.4 &amp;micro;m Transparent PET Membrane</td></tr><tr><td>Collagenase Type 1</td><td>Worthington Biochemical</td><td>LS004196</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium&amp;nbsp;</td><td>Corning</td><td>10-013-CV</td><td>4.5 g/L glucose</td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ThermoFisher</td><td>16140-071</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Hanks&amp;rsquo;s buffered saline solution</td><td>Gibco</td><td>14170-112</td><td>No calcium, no magnesium</td></tr><tr><td>Penicillin/streptomycin&amp;nbsp;</td><td>Gibco</td><td>15-140-122</td><td></td></tr><tr><td>Pogassium phosphate monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P5379</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>Sodium chloride (Nacl)</td><td>EMD&amp;nbsp;</td><td>CAS 7647-14-5</td><td></td></tr><tr><td>Sodium phosphate monobasic (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S0751</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>27250-042</td><td></td></tr></tbody></table>

using avian skin explants to study tissue patterning and organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

Skin explant cultures 
Feather bud development from ex vivo skin organ cultures can directly be observed under the microscope. Using the skin explant culture model of chicken stage 30&#160;dorsal skin, the placodes are visible along the midline. The morphogenetic front then gradually propagates laterally toward the skin periphery with the formation of new feather primordia. These feather primordia will develop into short feather buds after 2 days in culture and long feather buds after 4 days in culture (Figure 1).
Skin recombination cultures 
For skin recombination, when epithelia and mesenchyme are recombined, the original placodes disappear. New placodes will appear shortly after recombination and develop to form short feather buds and long feather buds after 2 and 4 days in culture, respectively. If the epithelium is rotated 90&#176; relative to the mesenchyme, the orientation of the elongating buds will be determined by the epithelium (Figure 2).
Skin reconstitution cultures 
For skin reconstitution, the ex vivo organ cultures appear homogeneous at first, and then dermal condensations with even spacing form simultaneously after 1 day in culture. It should be noted that the number of feather buds is dependent on the number of mesenchymal cells. Lower mesenchymal numbers induced fewer buds of a similar size to form11. Short feather buds will form after 2-3 days in culture and long feather buds will form after 4-5 days in culture (Figure 3).
Figure 4&#160;shows summary diagrams of ex vivo skin organ culture, skin recombination organ culture, and skin reconstitution organ culture.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65580/65580fig01.jpg" /> 
Figure 1: Ex vivo skin organ culture. Stage 32 chicken embryo skin is dissected in HBSS and cultured in 6-well culture inserts (T0, at time 0) for 2 and 4 days. The feather primordia develop into short feather buds after 2 days in culture and long feather buds after 4 days in culture. Dermal placodes are indicated by the white arrow. Note the buds in the midline are more mature than those on both lateral sides. This method has been modified from Jiang and Chuong4. Scale bar = 500 &#181;m. Abbreviation: HBSS = Hank&#39;s buffered saline solution. <a href="https://www.jove.com/files/ftp_upload/65580/65580fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65580/65580fig02.jpg" /> 
Figure 2: Ex vivo skin recombination organ culture. Stage 32 chicken embryo skin is dissected in HBSS, and epithelium and mesenchymal are separated in 2x CMF buffer. Skin epithelium and mesenchyme are recombined with or without rotation and cultured for 2 days and 4 days. The new placodes develop into short and long feather buds after 2- and 4-days in culture. If the epithelium is rotated 90&#176; relative to the mesenchyme, the orientation of the new buds is determined by the epithelium12. This method has been modified from Chuong et al.11. Scale bar = 500 &#181;m. Abbreviation: CMF = calcium-magnesium-free. <a href="https://www.jove.com/files/ftp_upload/65580/65580fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65580/65580fig03.jpg" /> 
Figure 3: Ex vivo skin reconstitution organ culture. Stage 32 chicken embryo skin is dissected, and epithelium and mesenchyme are separated in 2x CMF buffer. The mesenchyme is dissociated into single cells by 0.1% collagenase and trypsin and pelleted at high cell density on a culture insert. The dermal cell pellet is reconstituted with an intact epithelium (T0, at time 0) and cultured for 6 h, 2 days, and 5 days. The explants appear homogeneous at first (6 h) and then dermal condensations with even spacing form simultaneously after 1 day in culture. Short feather buds will form after 2-3 days in culture and long feather bud form after 4-5 days in culture. This method has been modified from Jiang et al.13. Scale bar = 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65580/65580fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65580/65580fig04.jpg" /> 
Figure 4: Diagrams of ex vivo skin organ culture models. The skin initiates at E6.5 as a single layer of epithelium overlaying mesenchymal cells. This is depicted at the top of the three explant methods. (A) Ex vivo skin organ culture. The skin is plated intact on top of a culture insert at the air: media interface at 37 &#176;C in a 5% CO2 and 95% air incubator. (B)&#160;Ex vivo skin recombination organ culture. Skin epithelium is separated from the mesenchyme and then recombined before plating onto the cell culture insert at the air: media interface at 37 &#176;C in a 5% CO2 and 95% air incubator. (C) Ex vivo skin reconstitution organ culture. Skin epithelium is separated from the mesenchyme. The mesenchyme is then dissociated into a single-cell suspension and the mesenchymal cells are pelleted in a centrifuge. The mesenchymal cells are then resuspended at a concentration of 2 &#215; 107 cells/mL and 10 &#181;L of the suspension placed onto the culture insert and then overlaid with an intact piece of epidermis. The culture is incubated at the air: media interface at 37 &#176;C in a 5% CO2 and 95% air incubator. <a href="https://www.jove.com/files/ftp_upload/65580/65580fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure S1: Transduction of mesenchyme cells with GFP-expressing virus. Dissociated mesenchymal cells (2 &#215; 107 cells/mL) were incubated for 3 h on ice with &#62;107 infectious units/mL replication-competent avian sarcoma virus expressing Green Fluorescent Protein (GFP). The mesenchymal cells were then used to form reconstituted skin organ cultures as described in protocol section 3 above and photographed 24 h later. The data show that ~40% of mesenchymal cells were labeled with GFP. <a href="https://www.jove.com/files/ftp_upload/65580/Supplementary Figure 1 - July 13 2023.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Using Avian Skin Explants to Study Tissue Patterning and Organogenesis at JoVE.com

Author Spotlight: Insight into the Current Experimental Avian Skin Explant Methodologies

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

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

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1.1K Views.  Keck School of Medicine of University of Southern California. 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. 

Video: Author Spotlight: Insight into the Current Experimental Avian Skin Explant Methodologies

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 ...

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.

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 ...

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 ...

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 ...

Biology

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

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.

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

Explant Culture for Developmental Studies

Explant culture is a technique in which living cells or tissues are removed from an embryo for continued development outside of the organism. This ex vivo approach allows researchers to manipulate and observe developing tissues in ways that are not possible in vivo. Once established, explant culture is frequently used to understand the role of genes and signaling molecules in organogenesis.
This video will first introduce the basic principles of explant culture and demonstrate a protocol to isolate and grow explanted mammalian tissues. Common genetic and molecular methods of manipulating explant cultures will then be discussed. Finally, the viewers will learn about how explant techniques are currently being applied to study organ development.

Explant Culture for Developmental Studies and Morphogenesis

Explant culture is a technique in which living cells or tissues are removed from an embryo for continued development outside of the organism. This ex vivo ...

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An Introduction to Organogenesis

Organogenesis is the process by which organs arise from one of three germ layers during the later stages of embryonic development. Researchers studying organogenesis want to better understand the genetic programs, cell-cell interactions, and mechanical forces involved in this process. Ultimately, scientists hope to use this knowledge to create therapies and artificial organs that will help treat human diseases.
This video offers a comprehensive overview of organogenesis, including historical highlights starting with breakthrough studies done in the 1800s. Next, key questions asked by developmental biologists are introduced, followed by a discussion of how tissue transplantations, imaging, and in vitro culture techniques can be used to answer these queries. Finally, we describe how these methods are currently being employed in developmental biology laboratories.

Organogenesis, Fate Mapping and Tissue Engineering

Organogenesis is the process by which organs arise from one of three germ layers during the later stages of embryonic development. Researchers studying ...

Transplantation Studies

Many developmental biologists are interested in the molecular signals and cellular interactions that induce a group of cells to develop into a particular tissue. To investigate this, scientists can use a classic technique known as transplantation, which involves tissue from a donor embryo being excised and grafted into a host embryo. By observing how transplanted tissues develop in host environments, scientists have started to dissect the molecular pathways underlying development.
In this video, we first look at the role of cellular interactions in development, and move on to a basic transplantation protocol. Finally, some specific developmental studies utilizing this technique are discussed, which examine the effect of tissue transplantation on the fate of donor and host tissue.

Transplantation Studies: Techniques and Applications

Many developmental biologists are interested in the molecular signals and cellular interactions that induce a group of cells to develop into a particular ...

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

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Explore More Videos

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

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

Creating Avian Forebrain Chimeras to Assess Facial Development

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

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

Explant Culture for Developmental Studies

An Introduction to Organogenesis

Transplantation Studies