The endoplasmic reticulum (ER) is a site where protein folding and posttranslational modifications occur, but when unfolded or misfolded proteins accumulate in the ER lumen, an unfolded protein response (UPR) occurs. A UPR activates ER-stress signalling genes, including inositol-requiring enzyme-1 (Ire1), activating transcription factor 6 (Atf6), and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (Perk), to maintain homeostasis. The involvement of ER stress molecules in metabolic disease and hard tissue matrix formation has been established; however, an understanding of the role of ER-stress signalling molecules in tooth development is lacking. The aims of this study are to define the stage-specific expression patterns of ER stress-related molecules and to elucidate their putative functions in the organogenesis of teeth. This study leverages knowledge of the tissue morphology and expression patterns of a range of signalling molecules during tooth development. RT-qPCR, in situ hybridization, and immunohistochemistry analyses were performed to determine the stage-specific expression patterns of ER-stress-related signalling molecules at important stages of tooth development. RT-qPCR analyses showed that Atf6 and Perk have similar expression levels during all stages of tooth development; however, the expression levels of Ire1 and its downstream target X-box binding protein (Xbp1) increased significantly from the cap to the secretory stage of tooth development. In situ hybridization results revealed that Atf6 and Xbp1 were expressed in cells that form the enamel knot at cap stage and ameloblasts and odontoblasts at secretory stage in stage-specific patterns. In addition, Atf6, Ire1, and Xbp1 expression exhibited distinct localization patterns in secretory odontoblasts and ameloblasts of PN0 molars. Overall, our results strongly suggest that ER-stress molecules are involved in tooth development in response to protein overload that occurs during signaling modulations from enamel knots at cap stage and extracellular matrix secretion at secretory stage.

内质网（ER）是发生蛋白质折叠和翻译后修饰的位点，但是当未折叠或错误折叠的蛋白质积聚在ER内腔中时，就会发生未折叠的蛋白质反应（UPR）。UPR激活ER应激信号基因，包括需要肌醇的酶1（Ire1），激活转录因子6（Atf6）和双链RNA激活的蛋白激酶样内质网激酶（Perk），以维持体内平衡。已经确定ER应激分子参与代谢疾病和硬组织基质形成；然而，缺乏对内质网应激信号分子在牙齿发育中的作用的了解。这项研究的目的是确定内质网应激相关分子的阶段特异性表达模式，并阐明其在牙齿器官发生中的假定功能。这项研究利用了牙齿发育过程中一系列信号分子的组织形态和表达模式的知识。进行了RT-qPCR，原位杂交和免疫组织化学分析，以确定在牙齿发育的重要阶段ER应激相关信号分子的阶段特异性表达模式。RT-qPCR分析表明，Atf6和Perk在牙齿发育的各个阶段都有相似的表达水平；然而，Ire1及其下游靶标X-box结合蛋白（Xbp1）的表达水平从帽顶到牙齿发育的分泌阶段显着增加。原位杂交结果表明，Atf6和Xbp1在细胞中表达，这些细胞在帽形成阶段形成釉质结，在成分泌细胞的成釉细胞和成牙本质细胞以阶段特异性模式表达。此外，Atf6，Ire1和Xbp1表达在PN0磨牙的分泌性成牙本质细胞和成釉细胞中表现出不同的定位模式。总体而言，我们的研究结果强烈表明，内质网应激分子参与了牙齿的发育，以响应蛋白质的过载，该过程在帽状阶段的釉质结和分泌阶段的细胞外基质分泌进行信号传导调节期间发生。Xbp1的表达在PN0磨牙的分泌性成牙本质细胞和成釉细胞中表现出独特的定位模式。总体而言，我们的研究结果强烈表明，内质网应激分子参与了牙齿发育，以响应蛋白质过量，蛋白质过量发生在盖帽阶段的牙釉质结的信号传导调节和分泌阶段的细胞外基质分泌。Xbp1的表达在PN0磨牙的分泌性成牙本质细胞和成釉细胞中表现出独特的定位模式。总体而言，我们的研究结果强烈表明，内质网应激分子参与了牙齿的发育，以响应蛋白质的过载，该过程在帽状阶段的釉质结和分泌阶段的细胞外基质分泌进行信号传导调节期间发生。