Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

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 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.

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....

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 lon.......

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 day.......

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.......

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.