Introduction

The zebrafish is a widespread vertebrate model for research in developmental biology. In 5 days, the transparent embryos progress from their first cell division to a fully developed larvae (1). There is much information about regulatory genes that control the development of many zebrafish organs. However, most of the molecular and cellular events participating in skin development are unexplored to date. During early zebrafish development, epithelial cells surrounding the embryo became flattened and arranged as a monolayer known as periderm. It is unknown whether the periderm will later form the epidermis (2). Once the epidermis appears, it is formed solely by two layers of cells, which are separated from the subepidermal space by a basal membrane. Eventually more cell layers are added to the epidermis and the subepidermal space thickens by collagen deposition. Collagen is produced by cells from both the epidermis and the dermis (3). In adult zebrafish, three layers form the epidermis: (i) the superficial stratum, (ii) the intermediate stratum and (iii) the basal layer. The intermediate stratum has been suggested to be the source for stem cells that maintain cell renewal.

The skin epidermis is a protective barrier against harmful conditions in the environment; for that reason it has the remarkable ability to regenerate itself, but for the same reason when affected by disease, it could carry significant health problems. Many human epidermal diseases have been described, like the fragile skin disorder, epidermolysis, the Hay-Wells syndrome, and the Striate palmoplantar keratoderma, which are known to be produced by mutations in the plakophilin-1, keratin-5, p63 and desmoglein-1 genes, respectively (4). Yet, for the majority of epidermal diseases, no responsible genes have been identified.

Mutagenic screenings in zebrafish have successfully identified the faulty genes from mutants that are models to study human disease, like cancer (5), muscular dystrophy (6), or skin hypopigmentation (7). Zebrafish skin mutants have been also described, like penner, in which a mutation of the hemidesmosome protein pen/lgl2 leads to hyperproliferation and ectopic localization of epidermal cells (8). Some other examples are the goose pimple (gsp), dandruff (ddf), and bouillabaisse (bob) mutants, sharing the same phenotype of having loose, round epidermal cells. However, the affected genes for these three mutants are not known (9).

Identifying novel epidermis mutants through genetic screening will reveal new molecular events of epidermis development (10). Histology and electron microscopy are the most common methods used to study epidermal cell development in zebrafish. These techniques are laborious and time-consuming and therefore not suitable for large-scale screenings in zebrafish. A faster method is required in which the epidermis from multiple larvae can be easily analyzed at the tissue and subcellular level. Based on the flat-mounts and viewing chambers routinely used by the zebrafish community (11), we designed a simple glass-slide chamber in which epidermal tissue from multiple zebrafish larvae is captured by a poly-L-lysine-treated slide. The obtained epidermis tissue remain as a flattened layer, which then can be treated with fluorescent compounds labeling the cell membrane, nuclei, and the Golgi apparatus. Following this technique, epidermal cells from 15 to 75 larvae can be prepared for analysis in 2–4 h, respectively. We also describe an alternate procedure in which epidermal cells can be treated for fluorescent immunostaining.

Materials and Methods

Epidermis Freeze-Crack

All experiments were carried out with the zebrafish wild-type strain TAB-14, which was maintained at 28° in a recirculation system (AquaticHabitats, Apopka, FL, USA) using standard techniques (12). Embryos were obtained by natural crosses and staged according to Kimmel et al. (1). The zebrafish larvae used were maintained at 28.5°C during 5 days postfertilization (dpf) (Figure 1A). Prior to the freeze-crack, two different types of glass slides were prepared. The first was holding slides, in which small, round coverslips (1.2 cm diameter; Propper Laboratory Products, Long Island City, NY, USA) were glued closely together with a separation of 0.3–0.6 mm between them. They were arrayed in a triangular shape (see Figure 1B), a rhomboidal shape (see Supplementary Figure S2A available online at www.BioTechniques.com), or in rows (see Supplementary Figure S2, C and E) in order to hold 3, 5, 8, and 15 larvae, respectively.