zebrafish-keratocyte-explants-to-study-collective-cell-migration

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

Midwestern University

Lidocaine injection for local anesthesia is a common podiatric medical procedure. We tested the hypothesis that injection of bacteriostatic saline solution containing 0.9% benzyl alcohol before the lidocaine infiltration can reduce the burning caused by lidocaine injection.

Reducing the pain of local 1% lidocaine infiltration with a preceding bacteriostatic saline injection: a double-blind prospective trial.

The control of collective cell migration of zebrafish keratocyte sheets in explant culture is of interest for cell migration and epithelial wound healing and depends on the gene expression profile. In a zebrafish genome array, ∼17.5% of the probe sets were differentially expressed greater than two-fold (p≤0.003) between 1 and 7 days of explant culture. Among the differentially expressed genes were a variety of wound healing-related genes and many of the biomarkers for epithelial-mesenchymal transition (EMT), including a switch from keratin and E-cadherin to vimentin and N-cadherin expression and several EMT-related transcription factors were found to be differentially expressed. Supporting evidence for EMT is seen in both morphological change and rearrangement of the actin cytoskeleton and in expression of cadherins during explant culture with a visible disassembly of the cell sheet. TGFβ1 and TNFα expression were analyzed by qPCR at various time points and peak differential expression of both cytokines occurred at 3 days, indicating that the EMT process is ongoing under conditions routinely used in the study of fish keratocyte motility. These data establish that an EMT process is occurring during zebrafish keratocyte explant culture and support the use of this system as a wound healing model.

Zebrafish keratocyte explant cultures as a wound healing model system: differential gene expression & morphological changes support epithelial-mesenchymal transition.

Fish keratocytes are an established model in single cell motility but little is known about their collective migration. Initially, sheets migrate from the scale at ~145 μm/h but over the course of 24h the rate of leading edge advance decreases to ~23 μm/h. During this period, leader cells retain their ability to migrate rapidly when released from the sheet and follower cell area increases. After the addition of RGD peptide, leader cell lamellae are lost, altering migratory forces within the sheet, resulting in rapid retraction. Leader and follower cell states interconvert within minutes with changes in cell-cell adhesions. Leader cells migrate as single cells when they detach from the leading edge and single cells appear to become leader cells if they rejoin the sheet. Follower cells rapidly establish leader cell morphology during closing of holes formed during sheet expansion and revert to follower cell morphology after hole-closure. Inhibition of Rho associated kinase releases leader cells and halts advancement of the leading edge suggesting an important role for the intercellular actomyosin cable at the leading edge. In addition, the presence of the stationary scale orients direction of sheet migration which is characterized by a more uniform advance of the leading edge than in some cell line systems. These data establish fish keratocyte explant cultures as a collective cell migration system and suggest that cell-cell interactions determine the role of keratocytes within the migrating sheet.

Article	Year
Reducing the pain of local 1% lidocaine infiltration with a preceding bacteriostatic saline injection: a double-blind prospective trial. Journal of the American Podiatric Medical Association\| PubMed ID: 21622634	2011
Zebrafish keratocyte explant cultures as a wound healing model system: differential gene expression & morphological changes support epithelial-mesenchymal transition. Experimental cell research\| PubMed ID: 23588205	2013
Collective cell migration of primary zebrafish keratocytes. Experimental cell research\| PubMed ID: 24973510	2014

Kimbal E. Cooper

Biography

JoVE Journal Publications

Other Publications