Observation of Dynamic Cellular Migration of the Medial Edge Epithelium of the Palatal Shelf in vitro
Observation of Dynamic Cellular Migration of the Medial Edge Epithelium of the Palatal Shelf in vitro
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DOI：
10.3389/fphys.2019.00698
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发表时间：
2019-06-06
影响因子：
4
通讯作者：
Yamashiro, Takashi
中科院分区：
文献类型：
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作者：
Aoyama, Gozo;Kurosaka, Hiroshi;Yamashiro, Takashi
腭裂融合是腭裂发生过程中的关键步骤。在这个融合界面中，需要去除上皮片以实现间充质的连续性。上皮细胞迁移是可能的机制之一，对标记上皮细胞的实时成像可以为其提供直接证据。然而，两侧突起之间的内侧缘上皮(MEE)的去除发生在腭架背腹轴的中部，因此直接捕捉细胞的行为是具有挑战性的。在这里，我们使用活成像技术评估MEE细胞的细胞行为，该技术使用在角蛋白14(K14-GFP)启动子和未配对的腭架培养下表达GFP的小鼠模型。利用这种方法，我们成功地获得了上皮行为的实时图像，并在没有接触到相对的腭板的情况下，检测到了上皮细胞在次级腭板表面的迁移。此外，上皮清除的模式导致了椭圆形裸露的间充质，这重现了体内二次腭裂融合的情况。详细的图像处理显示，随着暴露的间充质椭圆形的扩张，大部分MEE在边界区域向外迁移。MEE的迁移先于MEE的隆起，在边界区肿胀或迁移的MEE中GFP信号消失不明显。此外，岩石缓蚀剂的应用还干扰了MEE的迁移和随后的间质暴露。综上所述，这些发现表明，上皮细胞的迁移对MEE的去除和随后暴露下的间质具有重要作用。此外，他们指出，上皮细胞的迁移是以时间和空间特有的方式调节的，因为未配对的腭架培养即使在没有相对的搁架的情况下也表现出这些细胞行为。总之，目前的数据表明，这个新的实验系统结合了GFP标记的小鼠上皮细胞和未配对的腭架培养，能够直接显示MEE细胞在体外的迁移，可能成为研究其细胞和分子机制的有力工具。
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 dorsoventral 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.