Supplementary Material for: Spatiotemporal Localization of Periostin and Its Potential Role in Epithelial-Mesenchymal Transition during Palatal Fusion

The medial epithelial seam (MES) between the palatal shelves degrades during palatal fusion to achieve the confluence of palatal mesenchyme. Cellular mechanisms underlying the degradation of MES have been proposed, such as apoptosis, epithelial-mesenchymal transition (EMT) and migration of medial edge epithelia (MEE). Extracellular matrix components have been shown to play an important role in EMT in many model systems. Periostin (also known as osteoblast-specific factor-2) is a secreted mesenchymal extracellular matrix component that affects the ability of cells to migrate and/or facilitates EMT during both embryonic development and pathologic conditions. In this study, we evaluated the spatiotemporal expression patterns of periostin during mouse palatal fusion by in situ hybridization and immunofluorescence. Periostin mRNA and protein were present in the palatal mesenchyme, the protein being distributed in a fine fibrillar network and in the basement membrane, but absent from the epithelium. During MES degradation, the protein was strongly expressed in the basement membrane underlying the MES and in some select MEE. Confocal microscopic analysis using an EMT marker, twist1, and an epithelial marker, cytokeratin 14, provided evidence that select MEE were undergoing EMT in association with periostin. Moreover, the major extracellular matrix molecules in basement membrane, laminin and collagen type IV were degraded earlier than periostin. The result is that select MEE establish interactions with periostin in the mesenchymal extracellular matrix, and these new cell-matrix interactions may regulate MEE transdifferentiation during palatal fusion.

腭突（palatal shelves）之间的内侧上皮接缝（medial epithelial seam, MES）在腭融合过程中发生降解，以实现腭间间充质的汇合。介导MES降解的细胞机制已被学界提出，包括细胞凋亡、上皮-间充质转化（epithelial-mesenchymal transition, EMT）以及内侧边缘上皮（medial edge epithelia, MEE）的迁移。已有研究证实，细胞外基质组分在诸多模型系统的EMT进程中发挥关键调控作用。骨膜蛋白（periostin，又称成骨细胞特异性因子-2，osteoblast-specific factor-2）是一种分泌型间充质细胞外基质组分，可在胚胎发育与病理状态下影响细胞迁移能力，并促进EMT进程。本研究通过原位杂交与免疫荧光技术，分析了骨膜蛋白在小鼠腭融合过程中的时空表达模式。结果显示，骨膜蛋白的mRNA与蛋白产物均表达于腭间充质内；其蛋白以细密纤维网络形式分布，并定位于基底膜中，而上皮组织中无其表达。在MES降解阶段，该蛋白在MES下方的基底膜及部分特定内侧边缘上皮中呈强阳性表达。研究人员借助EMT标志物twist1与上皮标志物细胞角蛋白14（cytokeratin 14）开展共聚焦显微镜分析，证实部分特定内侧边缘上皮正发生与骨膜蛋白协同的上皮-间充质转化。此外，基底膜中的主要细胞外基质分子层粘连蛋白（laminin）与IV型胶原蛋白（collagen type IV）的降解时间早于骨膜蛋白。综上，部分特定内侧边缘上皮可与间充质细胞外基质中的骨膜蛋白建立相互作用，这种全新的细胞-基质相互作用或可调控腭融合过程中内侧边缘上皮的转分化进程。