Zebrafish keratocyte explants to study collective cell migration and reepithelialization in cutaneous wound healing

• Published in: Journal of visualized experiments 2015-02 (96)
• Main Authors: Rapanan, Jose L, Pascual, Agnes S, Uppalapati, Chandana K, Cooper, Kimbal E, Leyva, Kathryn J, Hull, Elizabeth E
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Animals; Cell Movement - physiology; Developmental Biology; Epithelial-Mesenchymal Transition; Keratinocytes - cytology; Keratinocytes - metabolism; Tissue Culture Techniques - methods; Wound Healing - physiology; Zebrafish
• ISSN: 1940-087X; 1940-087X
• DOI: 10.3791/52489

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.