Department of Anatomy, School of Life Dentistry at Tokyo, The Nippon Dental University, Tokyo, Japan.
Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, Japan.
Ann Anat. 2022 Feb;240:151868. doi: 10.1016/j.aanat.2021.151868. Epub 2021 Nov 23.
During tooth development, Wnt5a, a member of the noncanonical Wnt ligand, is expressed prominently in the dental mesenchyme. However, the spatiotemporal profiles of Wnt5a protein production and distribution in tooth germs are largely unknown, which impairs elucidation of the Wnt5a-mediated regulatory mechanism of tooth development.
We performed analyzes of the spatiotemporal expression of Wnt5a in embryonic tooth germs (E11.5-E18.5) by using in situ hybridization and immunohistochemistry in parallel. The developmental stages of the embryonic tooth germs were determined by HE staining. In order to compare the spatiotemporal distribution patterns of Wnt5a mRNA-expressing cells and those of Wnt5a protein-expressing cells, serial frontal sections of paraffinized mouse embryo heads were used for the analyzes. When needed, the immunohistochemistry images were subjected to digital detection analysis of Wnt5a immunostaining signal using the WinROOF 2018 Ver. 4.19.0 image processing software program.
Throughout the developmental process, cells expressing Wnt5a mRNA were found in various tissues including the dental follicle, dental papilla, inner and outer enamel epithelium, stratum intermediate, and stellate reticulum. However, odontoblasts differentiating and polarizing at E18.5 were the only cells representing an accumulation of Wnt5a protein in the apical region of the odontoblast process. The Wnt5a protein was undetectable in undifferentiated mesenchymal cells as well as any other cells positive for Wnt5a mRNA.
Differentiating odontoblasts execute Wnt5a expression, the mode of which is distinct from that executed by the other cells constituting tooth germ. Change of the mode of Wnt5a expression begins to take place in the mesenchymal cells by E18.5, starting the elongation of the cytoplasmic process.
在牙齿发育过程中，非经典 Wnt 配体成员 Wnt5a 在牙间质中大量表达。然而，牙胚中 Wnt5a 蛋白产生和分布的时空特征在很大程度上是未知的，这阻碍了对 Wnt5a 介导的牙齿发育调控机制的阐明。
我们通过原位杂交和免疫组织化学平行分析，研究了 Wnt5a 在胚胎牙胚（E11.5-E18.5）中的时空表达。胚胎牙胚的发育阶段通过 HE 染色确定。为了比较 Wnt5a mRNA 表达细胞和 Wnt5a 蛋白表达细胞的时空分布模式，我们使用石蜡包埋的小鼠胚胎头部连续额切片进行分析。当需要时，使用 WinROOF 2018 Ver.4.19.0 图像处理软件程序对免疫组织化学图像进行 Wnt5a 免疫染色信号的数字检测分析。
在整个发育过程中，在包括牙囊、牙乳头、内釉上皮和外釉上皮、中间层和星状网状层在内的各种组织中都发现了表达 Wnt5a mRNA 的细胞。然而，只有在 E18.5 分化和极化的成牙本质细胞在成牙本质细胞突起的顶端区域积累 Wnt5a 蛋白。在未分化的间充质细胞以及任何其他表达 Wnt5a mRNA 的细胞中都检测不到 Wnt5a 蛋白。
分化的成牙本质细胞执行 Wnt5a 表达，其模式与构成牙胚的其他细胞不同。Wnt5a 表达模式的改变始于 E18.5 的间充质细胞，开始伸长细胞质突起。
