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. <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. -. 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=Mechanism+of+feather+morphogenesis:+I.+Analyses+with+antibodies+to+Adhesion+Molecules+Tenascin,+N-CAM+and+Integrin.">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/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>

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 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.

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

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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Developmental Biology

1.0K 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

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

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

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

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

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

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 ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. The high transfection efficiency permits at least 4 distinct mRNAs to be co-transfected with ~87% efficiency. Most of the electroporated mRNAs are degraded during the first 2 h post-electroporation, permitting time-sensitive experiments to be carried out in the developing embryo. Finally, we describe how to dynamically image live embryos electroporated with mRNAs that encode various subcellular targeted fluorescent proteins.

We report messenger RNA (mRNA) electroporation as a method that permits fast and efficient expression of multiple proteins in the quail embryo model system. This method can be used to fluorescently label cells and record their in vivo movements by time-lapse microscopy shortly after electroporation.

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...