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 lon...

A Comprehensive Review of Avian Embryonic Skin Explant Culture Protocols

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	<li>Lucas, A. M., Stettenheim, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+anatomy:+Integument+part+I+and+part+II.">Avian anatomy: Integument part I and part II.</a> Agriculture Handbook. 362, (1972).</li><li>Sengel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In Morphogenesis of Skin, l-277. , (1976).</li><li>Dhouailly, D., Wilehm Roux, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+cutaneous+appendages+in+dermo-epidermal+recombinations+between+reptiles,+birds+and+mammals.">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. -. 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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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+organs+by+a+Wnt+/+Notch+/+non-muscle+myosin+module+which+orients+feather+bud+elongation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+oscillations+coordinate+feather+mesenchymal+cell+movement+by+SHH+dependent+modulation+of+gap+junction+networks.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+delivery+of+TGF+beta2+can+restore+epithelium+dependent+organization+of+mesenchymal+condensation+during+skin+appendage+morphogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+induces+new+feather+buds+from+developing+avian+skin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+interactions+in+scale+and+feather+development+as+studies+in+dermal-epidermal+recombinations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correcting+the+phenotype+of+the+epidermis+from+chick+embryos+homozygous+for+the+gene+scaleless+(sc/sc).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+events+during+avian+skin+appendage+regeneration:+dependence+on+epithelial-mesenchymal+interaction+and+order+of+molecular+reappearance.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+feather+orientations+changed+by+electric+current.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+of+periodic+patterns+by+dissociated+feather+mesenchymal+cells+and+the+regulation+of+size,+number+and+spacing+of+primordia.">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/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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+early+odontogenic+signaling+pathway+in+Aves.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Skin+Morphogenesis:+II.+Retinoic+acid+gradient+modulates+axis+orientation+and+phenotypes+of+skin+appendages.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+A+and+protein+kinase+C+modulators+have+reciprocal+effects+on+mesenchymal+condensation+during+skin+appendage+morphogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+biological+networks+via+adaptive+patterning+revealed+by+avian+intradermal+muscle+network+formation.">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>

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 identit...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-well culture dish&#160;</td>
			<td>Falcon</td>
			<td>REF 353502</td>
			<td>Air-Liquid Interface (ALI) Cultures &#160;</td>
		</tr>
		<tr>
			<td>Cell culture inset&#160;</td>
			<td>Falcon</td>
			<td>REF 353090.</td>
			<td>0.4 &#181;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&#8217;s modified Eagle&#8217;s medium&#160;</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&#8217;s buffered saline solution</td>
			<td>Gibco</td>
			<td>14170-112</td>
			<td>No calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pogassium phosphate monobasic (KH2PO4)</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 (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (Nacl)</td>
			<td>EMD&#160;</td>
			<td>CAS 7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (NaH2PO4)</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.

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

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The research objective of my research team is to understand tissue development and the regeneration. This goal is particularly important in the stem cell research today. We hope to understand the principles how a organ form.And this knowledge is not only help us to understand the nature, but also can be applied to help regenerative medicine. The challenge we face is to understand the molecular and the cellular process involved in these morphogenetic processes. So therefore, we need to have a explant culture system to analyze molecular and cellular processes carefully because it's not possible to do this in vivo.To fill in this knowledge gap, scientists have developed different ways of organoid cultures. In the organoid culture, we will be able to take collective cell movement to see how cells interact with each other. We are also able to manipulate the cells so they will express different molecules to test the function of the molecules.The unique approach we use is the chicken skin explant culture. Chicken skin is used because they are easily accessible from the egg and they form distinct feather buds, arranged the in hexagonal pattern as a readout for tissue patterns. There are three ways we do the culture, as you will see.One is to hold the explant culture, allow us to take the movies or deliver molecules to perturb the system. Second, to study the contribution of epicilia or mesenzyme, we can take them apart and put them back. Third, to further challenge the ability of each cells, we dissociate them into the single cells and test how they come back to reorganize into the tissue patterns.Through these approaches, we have been able to gain new understanding toward the tissue patterning.

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 day...

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

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.

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

using-avian-skin-explants-to-study-tissue-patterning-and-organogenesis

Research

JoVE Journal

Developmental Biology

Chicken Skin Explant Culture for Organogenesis Study

To start, incubate fertilized chicken eggs in a humidified incubator at 38 degrees Celsius. Then, place a stage 28 to 32 chicken embryo in a 60-millimeter Petri dish filled with HBSS. After dissecting out the dorsal embryo skin and head, gently pull the limb buds to reposition the ventral side of the body downwards.Now, along the two sides of the embryo body, make an anterior to posterior incision. Grip the limb buds longitudinally with a pair of forceps to stabilize the body. Use a watchmaker's forceps to carefully peel the skin from the neck down to the tail region.Use sharp scissors to delicately remove any remaining subcutaneous tissues still attached to the peeled skin and smooth the skin edges. Next, add two milliliters of supplemented DMEM to each well of a six-well dish. After adding antibiotics to the wells, place a sterile tissue culture insert inside the well, ensuring the medium surrounds the exterior of the culture insert.Then move the skin onto a spatula while immersed in HBSS to prevent creasing and folding. Once done, transfer the excised skin to a dish containing HBSS. Slowly slide the skin off the spatula onto the culture insert without folding.Using a 200-microliter pipette, remove any excess HBSS from the insert. Finally, incubate the skin explant cultures at 37 degrees Celsius with a blend of 5%carbon dioxide and 95%air. Replace the medium every two days.The feather primordia developed into short feather buds after two days in culture. and long feather buds developed after four days in culture. The dermal placodes were more mature in the midline than at the sides.

Chicken Skin Epithelial-Mesenchymal Recombination Culture

To begin, incubate fertilized chicken eggs in a humidified incubator. Next, prepare two times CMF saline with 0.25%EDTA buffer. Then peel the skin off the chicken embryos in HBSS solution, and incubate them in two times CMF-EDTA solution on ice for 15 to 20 minutes.Use a pair of watchmakers forceps to carefully separate the epithelium and mesenchyme. Then transfer the isolated epithelium and mesenchyme to a single clean dish containing HBSS. For recombination, place the mesenchyme on a Petri dish containing HBSS, then place the epithelium on it, aligning it along the mesenchyme's anterior/posterior axis.Alternatively, rotate the epithelium by 90 degrees from the mesenchymal anterior posterior axis. Next, transfer the recombined skins to the culture inserts in six well culture dishes. Remove any excess HBSS surrounding the recombined skin.After adding DMEM containing 10%FBS to the exterior well of the insert, pipette a thin layer of DMEM into the insert chamber, ensuring the explant remains semi hydrated. Finally, place the skin recombinants in an incubator at 37 degrees in an environment of 5%carbon dioxide, and 95%air. Monitor the phenotypic changes in the initial skin development phases using time-lapse photography, with a mounted dissecting microscope.New placodes appeared shortly after recombination, and developed into short feather buds two days post-recombination. Long feather buds developed in four days. When the epithelium was rotated by 90 degrees, the orientation of the new buds was determined by the epithelium.

Tissue Studies by Culturing Reconstituted Chicken Skin 

To start, incubate fertilized chicken eggs in a humidified incubator at 38 degrees Celsius. Now dissect stage 30 to 33 chicken embryo dorsal skins in HBSS. Then incubate the skins in two times calcium and magnesium free saline with 0.25%EDTA for 15 to 20 minutes.Carefully separate the epithelium and the MESENCHYME using watchmaker's forceps and move the separated material to HBSS, placed on ice. Collect the separated mesenchyme into a 15 milliliter centrifuge tube and add two milliliters of 0.1%collagenase trypsin made in PBS. Then incubate the solution in a water bath.Next, use a pasteur pipette to disperse the skin mesenchyme into a single cell suspension. Then add eight milliliters of DMEM containing 10%fetal bovine serum to halt digestion. Centrifuge the cells at 233G for five minutes.Then resuspend the pellet in culture medium. Next place a cell culture insert in a well of a six well dish, drop a 10 microliter droplet of the mesenchymal cells onto the insert. In the well outside of the insert, pipette two milliliters of DMEM with 10%fetal bovine serum.Incubate the plated cells at 37 degrees in an incubator set with 5%carbon dioxide and 95%air for one hour. Using a one milliliter pipette, transfer the intact epithelium on top of the plated mesenchymal droplet. Flatten the intact epithelium over the mesenchyme to form the reconstituted skin explant.Leave a thin layer of medium on the insert to keep the skin explant semi wet. Providing an air/liquid interface incubate the reconstituted skin explant at 37 degrees in an incubator with 5%carbon dioxide and 95%air environment. The ex vivo organ cultures appear homogeneous at first short feather buds formed after two to three days in culture and long feather buds formed after four to five days in culture.

658 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. 

A Comprehensive Review of Avian Embryonic Skin Explant Culture Protocols

概要