Determining the influence of the in-vivo environment on the wound response of keratocytes in the zebrafish epidermis

Actin-based cell motility is essential for animal development and regeneration. Much of our understanding of the mechanisms by which animal cells crawl through their environment derives from careful study of a particular cell type: the keratocyte, an epidermal cell of fish and amphibians. When isolated from fish scales, individual keratocytes move rapidly and are capable of moving consistently in one direction for minutes or hours. In the second chapter of this thesis, I contribute to the understanding of the mechanics of keratocyte migration, presenting evidence that characteristic wrinkles in the keratocyte lamellipodium can be explained by a physical model, in which the lamellipodium behaves as an elastic sheet under compression from myosin contractility. Although the keratocyte has fascinated cell biologists for nearly a century, the physiological relevance of its remarkable migratory abilities has been unclear, in part because keratocyte migration has not been systematically explored in vivo. During my PhD, I developed zebrafish larvae as a model system to investigate the movement of keratocytes in their native tissue context. Keratocytes, which are referred to as basal cells in vivo, form the lower layer of a bilayered epidermis, and react rapidly to injury, polarizing their actin cytoskeleton towards the wound site, migrating towards it, and reforming an epithelial layer in only 15-20 minutes. By studying the movement of these cells in vivo during wound healing, I have identified unexpected cellular behaviors arising from the interaction of the keratocyte with its tissue environment. First, I discovered that keratocytes in vivo are profoundly sensitive to the ionic balance of the surrounding extracellular fluid. Changes in that composition rapidly induce a coordinated wound response extending several hundred microns away from the wound. I suggest that keratocytes sense shifts the composition of their surroundings through electric fields generated by ion transport across the epidermis. In support of this hypothesis, I demonstrate control over keratocyte migration in vivo using applied electric fields, in the absence of other wound cues. This study supports a physiological rationale for the previously observed sensitivity of isolated keratocytes to applied electric fields in culture. Second, I discovered that as the migratory phase of the wound response comes to a close, keratocytes separate from each other in a wave starting at the wound site and propagating through the tissue up to 300 microns. Through a combination of quantitative live imaging, electron microscopy, and serial section histology, I characterize this separation and show that cells still remain connected by thin tethers containing cell-cell adhesions. I suggest that forcing of fluid through the epidermis by compression at the wound site could cause separation, which may be promoted by myosin contractility. This thesis opens up new possibilities for deepening the connection between the quantitative mechanistic understanding of cell movement in culture and the physiologically relevant cell movement through complex environments in living animals, with keratocytes forming a bridge between these two domains

Advisor: Dunn Alexander Robert
Feldman Jessica L.
Talbot William S.