Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

Summary

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.

Abstract

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.

Introduction

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 manipulation^1,2. 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 development^3,4,5,6. 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' 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 90^o 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.

Protocol

1. Chicken skin explant culture (Figure 1)

1. Incubate fertilized chicken eggs in a humidified incubator at 38 °C and stage them according to Hamburger and Hamilton¹⁴.
   1. At stage 28 (~E5.5), the limb's second digit and third toe are longer than the others; three digits and four toes are distinct.
   2. At stage 29 (~E6), the wing is bent at the elbow; the second digit is distinctly longer than the others.
   3. 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.
   4. At stage 31 (~E7), there are ~7 rows of feathers at the jumbo-sacral levels.
   5. At stage 32, eleven or more rows of feather buds are present at the level of the legs.
2. Place a stage 28-32 chicken embryo in a 60 mm Petri dish containing Hanks's buffered saline solution (HBSS) or phosphate-buffered saline (PBS).
   1. 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.
   2. Make an anterior to posterior incision in the skin along the two flanking sides of the embryo body using watchmaker'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's forceps.
3. Gently remove the subcutaneous tissues that remain attached to the peeled skin and smooth the edges of the skin with the sharp spring scissors.
4. Add 2 mL/well of Dulbecco's modified Eagle'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.
5. 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.
6. Remove excess HBSS from the culture insert with a 200 µL pipette. Leave a thin layer of HBSS inside the culture insert to keep the explant semi-wet to provide an air-liquid interface.
7. Incubate the skin explant cultures at 37 °C using 5% CO₂ and 95% air. Change the medium every 2 days.
   ​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.

2. Chicken skin epithelial-mesenchymal recombination (Figure 2)

1. Incubate fertilized chicken eggs in a humidified incubator at 38 °C and stage the embryos according to Hamburger and Hamilton¹⁴ (section 1.1).
2. Prepare 2x calcium-magnesium-free (CMF) saline with 0.25% ethylenediaminetetraacetic acid (EDTA) buffer.
   1. 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.
3. 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.
4. Carefully separate the epithelium and mesenchyme using watchmaker's forceps. Transfer the separated epithelium and mesenchyme to a clean dish containing HBSS.
5. 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 90^o from the anterior-posterior axis of the mesenchyme to determine which component regulates different aspects of skin morphogenesis.
6. Transfer the recombined skins to culture inserts in 6-well culture dishes for growth and remove excess HBSS around the recombined skin.
7. 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.
8. Incubate the skin recombinants at 37 °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.

3. Chicken skin reconstitution (Figure 3)

1. Incubate fertilized chicken eggs in a humidified incubator at 38 °C and stage the embryos according to Hamburger and Hamilton¹⁴ (section 1.1).
2. 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).
3. Carefully separate the epithelium and mesenchyme using watchmaker'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.
4. Collect the separated mesenchyme in a 15 mL centrifuge tube and incubate with 0.1% collagenase/trypsin solution made in PBS in a 37 °C water bath for 10-15 min.
5. Dissociate the skin mesenchyme with a Pasteur pipet to make a single-cell suspension. Add DMEM with 10% FBS to stop the digestion.
6. Pellet and centrifuge the cells at 233 × g for 5 min. Resuspended the cells with culture medium at a concentration of 2 × 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.
7. Place a cell culture insert in one well of a 6-well dish. Drop 10 µL of 2 × 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 °C using a 5% CO₂ and 95% air incubator for 1 h to allow the cells to settle on the insert membrane.
8. 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.
9. 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 °C in a 5% CO₂ and 95% air incubator. Change the medium every 2 days.
   NOTE: This procedure has been published¹³.

Representative Results

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 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° 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 shows summary diagrams of ex vivo skin organ culture, skin recombination organ culture, and skin reconstitution organ culture.

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 µm. Abbreviation: HBSS = Hank's buffered saline solution. Please click here to view a larger version of this figure.

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° 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 µm. Abbreviation: CMF = calcium-magnesium-free. Please click here to view a larger version of this figure.

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 µm. Please click here to view a larger version of this figure.

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 °C in a 5% CO₂ and 95% air incubator. (B) 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 °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 × 10⁷ cells/mL and 10 µL 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 °C in a 5% CO₂ and 95% air incubator. Please click here to view a larger version of this figure.

Supplemental Figure S1: Transduction of mesenchyme cells with GFP-expressing virus. Dissociated mesenchymal cells (2 × 10⁷ cells/mL) were incubated for 3 h on ice with >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. Please click here to download this File.

Discussion

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 medium^8,16,17. Perturbing the skin locally can be achieved by placing protein-coated beads on the skin explant or by virally transducing cells to modulate gene expression^8,16,17. 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.

Materials

Name	Company	Catalog Number	Comments
6-well culture dish	Falcon	REF 353502	Air-Liquid Interface (ALI) Cultures
Cell culture inset	Falcon	REF 353090.	0.4 µm Transparent PET Membrane
Collagenase Type 1	Worthington Biochemical	LS004196
Dulbecco’s modified Eagle’s medium	Corning	10-013-CV	4.5 g/L glucose
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)	Sigma-Aldrich	E5134
Fetal bovine serum	ThermoFisher	16140-071
Glucose	Sigma-Aldrich	G8270
Hanks’s buffered saline solution	Gibco	14170-112	No calcium, no magnesium
Penicillin/streptomycin	Gibco	15-140-122
Pogassium phosphate monobasic (KH2PO4)	Sigma-Aldrich	P5379
Potassium chloride (KCl)	Sigma-Aldrich	P9333
Sodium bicarbonate (NaHCO3)	Sigma-Aldrich	S6014
Sodium chloride (Nacl)	EMD	CAS 7647-14-5
Sodium phosphate monobasic (NaH2PO4)	Sigma-Aldrich	S0751
Trypsin	Gibco	27250-042