Expression Patterns and Subcellular Localization of Carbonic Anhydrases Are Developmentally Regulated during Tooth Formation

Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA) but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for future single or multiple gene targeting strategies aiming at deciphering the function of CAs during odontogenesis.

碳酸酐酶（Carbonic Anhydrases, CAs）在诸多生理过程中发挥核心作用，越来越多的研究证据表明其参与了包括癌症在内的多种疾病进程。目前学界对碳酸酐酶在牙齿不同细胞中的表达情况尚不清楚，更遑论其在牙发生（odontogenesis）过程中的表达模式。为阐明碳酸酐酶在牙发生过程中的作用，本研究采用免疫组织化学（immunohistochemistry）、组织化学（histochemistry）及原位杂交（in situ hybridization）技术，首次揭示了小鼠体内8种碳酸酐酶此前未被报道的动态分布模式。最显著的研究结果包括：CAII/Car2不仅表达于成熟阶段成釉细胞（ameloblasts, MA），还表达于乳头层、牙乳头间充质、成牙本质细胞以及马拉色上皮剩余（epithelial rests of Malassez）。本研究发现，这类上皮剩余在切牙周围形成了蕾丝状网络结构——而此前学界仅认为其存在于磨牙中。所有被检测的碳酸酐酶均由成釉细胞表达；其中CAIV、CAIX及CARPXI蛋白在皱褶型成釉细胞的皱褶膜中显著富集，而在平滑型成釉细胞中则呈均匀分布。尽管CAIV、CAVI/Car6、CAIX、CARPXI及CAXIV均在所有成牙本质细胞中表达，但CAIII的分布呈现出显著的不对称性：其几乎仅局限于磨牙牙根以及切牙牙根类似结构中的成牙本质细胞。值得注意的是，在牙发生从启动到接近完成的整个阶段以及其他多种组织中，CAXIII主要定位于细胞内颗粒/囊泡中；本研究证实，这类囊泡与溶酶体相关膜蛋白1、2（lysosome-associated membrane protein 1/2, LAMP-1/2）阳性囊泡存在共定位，提示CAXIII定位于溶酶体内。本研究证实，发育中牙齿内的碳酸酐酶表达并不局限于参与生物矿化的细胞，这提示其还参与了其他生物学过程。最后，本研究还发现了碳酸酐酶表达的全新位点，包括发育中的脑、眼、嗅上皮、黑素母细胞、舌、脊索、髓核以及皮脂腺。本研究为未来旨在阐明碳酸酐酶在牙发生过程中功能的单基因或多基因靶向策略提供了重要的理论依据。