Pang Liping, Zhang Zhichun, Shen Yan, Cheng Zhenchao, Gao Xuejun, Zhang Bo, Wang Xiaoyan, Tian Hua
Department of Cariology and Endodontology & National Clinical Research Center for Oral Disease & Beijing Key Laboratory of Digital Stomatology, School and Hospital of Stomatology, Peking University, Beijing, PR China.
Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking Universiy, Beijing, PR China.
PeerJ. 2020 Feb 19;8:e8515. doi: 10.7717/peerj.8515. eCollection 2020.
Tricho-dento-osseous (TDO) syndrome is an autosomal dominant disorder characterized by anomalies in hair, teeth and bone (OMIM190320). Various mutations of Distal-Less 3 () gene are found to be responsible for human TDO. The aim of this study was to investigate effects of on tooth and bone development using a zebrafish model.
The mutant zebrafish lines were established using the gene targeting tool transcription activator-like effector nuclease (TALEN). Micro-computed tomography was used to render the three-dimensional skeletal structures of mutant fishes. The pharyngeal bone along with connected teeth was isolated and stained by Alizarine Red S, then observed under stereomicroscope. Scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) were used to examine the tooth surface morphology and mineral composition. Quantitative real-time PCR was used to analyze gene expression.
A moderate curvature of the spine toward the dorsal side was found at the early larval stages, appearing in 86 out of 100 larvae in group as compared to 3 out of 99 in the group. At the adult stage, three of the thirty homozygotes exhibited prominent abnormal curvature in the spine. SEM revealed morphological surface changes in pharyngeal teeth enameloid, accompanied by a decrease in the mineral content detected by EDS. Furthermore, specific secretory calcium-binding phosphoprotein (SCPP) genes, including , , , , and were significantly downregulated in mutants.
The findings of this study suggest that is critical for enamel mineralization and bone formation in zebrafish. Moreover, the discovery of the downregulation of SCPP genes in mutants sheds new light on the molecular mechanisms underlying TDO syndrome.
毛发-牙齿-骨（TDO）综合征是一种常染色体显性疾病，其特征为毛发、牙齿和骨骼异常（在线人类孟德尔遗传数据库编号190320）。研究发现，远端缺失3（DLX3）基因的各种突变是导致人类TDO的原因。本研究旨在利用斑马鱼模型研究DLX3对牙齿和骨骼发育的影响。
使用基因靶向工具转录激活样效应核酸酶（TALEN）建立DLX3突变斑马鱼品系。采用显微计算机断层扫描技术构建突变鱼的三维骨骼结构。分离出咽骨及其相连的牙齿，用茜素红S染色，然后在体视显微镜下观察。利用扫描电子显微镜（SEM）和能谱仪（EDS）检测牙齿表面形态和矿物质组成。采用定量实时聚合酶链反应分析基因表达。
在幼体早期阶段发现脊柱向背侧有中度弯曲，DLX3组100尾幼体中有86尾出现这种情况，而对照组99尾幼体中只有3尾出现。在成体阶段，30尾DLX3纯合子中有3尾脊柱出现明显异常弯曲。SEM显示咽牙釉质的形态表面发生变化，同时EDS检测到矿物质含量降低。此外，在DLX3突变体中，包括Spp1、Spp2、Enam、Ambn和Mmp20在内的特定分泌型钙结合磷蛋白（SCPP）基因显著下调。
本研究结果表明，DLX3对斑马鱼的釉质矿化和骨形成至关重要。此外，DLX3突变体中SCPP基因下调的发现为TDO综合征的分子机制提供了新的线索。
