成骨细胞特异性转录因子osterix在斑马鱼颅颌面发育早期时空表达特征的初步研究

Expression of osterix gene in the early stage of cranio-maxillofacial development in zebrafish

目的探讨成骨细胞特异性转录因子osterix基因在斑马鱼颅颌面发育早期的表达特征，为进一步研究其在颅颌面骨骼和牙齿发育早期的分子调控机制奠定基础。方法PCR扩增osterix基因的特异性基因片段，制备地高辛标记osterix基因的反义mRNA探针和正义mRNA探针；通过整胚原位杂交技术检测osterix基因在斑马鱼颅颌面骨骼与牙齿发育早期的转录分布，进一步比较osterix基因在颅颌面骨骼发育中的矿化表达特征；通过茜素红染色突出osterix基因在牙齿发育中的矿化表达特点。结果成功合成osterix基因的反义和正义mRNA探针；反义探针杂交检测显示：在受精后3～4dosterix基因开始在斑马鱼颅颌面膜内成骨部位表达，在受精后5—6dosterix基因在牙齿特异性表达；正义探针杂交未检测到osterix基因的表达信号。osterix基因在早期尚未矿化的骨基质以及初代牙齿（3V^1、5V^1）、第一颗替换牙齿（4V^2）的牙基质中高度表达。结论提示osterix基因可能在斑马鱼早期未矿化骨基质转化为晚期成熟的矿化骨基质过程中，以及牙齿冠部牙基质发育矿化过程中发挥一定作用。

Objective To investigate the expression patterns of osterix in the early stage of cranio- maxillofacial developmental in zebrafish and to prepare for a further research of osterix gene in bone and tooth development. Methods The osterix templates were amplified by PCR to generate DIG labeled antisense and sense probes. Whole mount in situ hybridization was used to analyze the expression patterns of osterix in the early stage cranio-maxillofacial development of zebrafish. The expression patterns of osterix gene in mineralization progresses of cranial and maxillofacial bones were compared. The osterix gene expression in tooth development and mineralization was highlighted by alizarin red staining. Results Specific DIG labeled probes of osterixwere synthesized successfully. The whole mount in situ hybridization showed that the osterix expression was in the intramembranous ossification at 3 days post fertilization（dpf） and 4 dpf. The specific osterix expression in tooth at 5 dpf and 6 dpf were also observed. The sense probe served as a negative control. Osterix expressed in the unmineralized early bone matrix, the tooth matrix of the primary tooth（3V^1, 5V^1） and the first replacement tooth（4V^2）. Conclusions Our findings showed that osterix might play roles in the process of the early mineralized bone matrix changing into the late mature mineralized bone matrix and the process of development and mineralization of tooth crown matrix.