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Observation of Dynamic Cellular Migration of the Medial Edge Epithelium of the Palatal Shelf in vitro.

ABSTRACT: Palatal fusion is a critical step during palatogenesis. In this fusing interface, the epithelial sheets need to be removed in order to achieve mesenchymal continuity. Epithelial cellular migration is one of the possible mechanisms, and live imaging of the labeled epithelium could provide direct evidence for it. However, the removal of medial edge epithelium (MEE) between the bilateral processes takes place in the middle of the dorso-ventral axis of the palatal shelf, and thus it is challenging to capture the cellular behavior directly. Here, we evaluate cellular behavior of MEE cells using a live imaging technique with a mouse model which expresses GFP under the promoter of Keratin14 (K14-GFP) and unpaired palatal shelf culture. Using this approach, we successfully obtained live images of epithelial behavior and detected epithelial cell migration on the surface of the secondary palatal shelf without touching of the opposing shelf. Additionally, the pattern of epithelial elimination resulted in oval-shaped exposed mesenchyme, which recapitulated the situation during secondary palate fusion in vivo. Detailed image processing revealed that most of the MEE migrated in an outward direction at the boundary regions as the oval shape of the exposed mesenchyme expanded. The migration was preceded by the bulging of MEE, and disappearance of GFP signals was not evident in bulging or migrating MEE at the boundary regions. Furthermore, the MEE migration and the subsequent mesenchymal exposure were disturbed by application of ROCK inhibitor. Together, these findings indicated that epithelial cell migration contributed importantly to the MEE removal and the subsequent exposure of the underlying mesenchyme. Furthermore, they indicated that the migration of epithelial cells was regulated in a time- and space-specific manner, since unpaired palatal shelf culture exhibited these cellular behaviors even in the absence of the opposing shelf. Altogether, present data indicated that this new experimental system combining live imaging with GFP-labeled epithelium mice and unpaired palatal shelf culture enabled direct visualization of cellular migration of MEE in vitro and could be a powerful tool to investigate its cellular and molecular mechanisms.

SUBMITTER: Aoyama G

PROVIDER: S-EPMC6562562 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Observation of Dynamic Cellular Migration of the Medial Edge Epithelium of the Palatal Shelf <i>in vitro</i>.

Aoyama Gozo G Kurosaka Hiroshi H Oka Ayaka A Nakatsugawa Kohei K Yamamoto Sayuri S Sarper Safiye Esra SE Usami Yu Y Toyosawa Satoru S Inubushi Toshihiro T Isogai Yukako Y Yamashiro Takashi T

Frontiers in physiology 20190606

Palatal fusion is a critical step during palatogenesis. In this fusing interface, the epithelial sheets need to be removed in order to achieve mesenchymal continuity. Epithelial cellular migration is one of the possible mechanisms, and live imaging of the labeled epithelium could provide direct evidence for it. However, the removal of medial edge epithelium (MEE) between the bilateral processes takes place in the middle of the dorso-ventral axis of the palatal shelf, and thus it is challenging t ...[more]

PMID: 31244674

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Project description:Endothelial cell migration plays a crucial role in achieving corneal clearance after corneal transplantation when using smaller-sized endothelial grafts to increase the donor pool. In this study we investigated how different strategies of Quarter-Descemet Membrane Endothelial Keratoplasty (Quarter-DMEK) limbal graft edge modification influence peripheral endothelial cell migration in an in vitro culture environment. For this study, 15 Quarter-DMEK grafts, prepared from 7 corneas deemed ineligible for transplantation but with intact and viable endothelial cells, were embedded in a cooled biocompatible, thermoresponsive matrix for culture. The limbal edge of ten Quarter-DMEK grafts were modified, either by using a small diameter punch or by peripheral radial cuts. All Quarter-DMEK grafts showed substantial collective endothelial cell migration from the radial cut graft edges, as observed by light microscopy at standardized time intervals. Grafts were retrieved from the polymer matrix after the two-week culture for immunohistochemistry analyses of the newly formed cell monolayers; this demonstrated the presence of tightly packed and viable cells that showed higher migratory ability at the leading edge. Peripheral endothelial cell migration, however, was not triggered by increasing cell exposure to free space through surgical modifications of the far periphery. Our data suggest that alterations in the far peripheral area of Quarter-DMEK grafts were insufficient to triggering cell migration from the limbal graft edge. This may be due to transient-amplifying cells that reside in the far periphery and which lack cytokinetic directional cues. Understanding the migration capacity of the peripheral endothelium could unlock cells' therapeutic potential which are, at present, routinely discarded from transplantation. Encouraging peripheral cell migration may also improve clinical outcomes from Quarter-DMEK, but a more effective solution is required prior to clinical implementation of modified grafts.

Dataset Information

Observation of Dynamic Cellular Migration of the Medial Edge Epithelium of the Palatal Shelf in vitro.

Publications

Observation of Dynamic Cellular Migration of the Medial Edge Epithelium of the Palatal Shelf <i>in vitro</i>.

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