Abstract
Tooth morphogenesis is a highly conserved process that occurs under very strict genetic control with the dentition of an organism developing as a result of reciprocal interactions between the oral epithelium and the underlying neural crest derived mesenchymal cells. In order to gain a deeper understanding of the mechanisms that underlie tooth growth and development, the use of optical projection tomography (OPT) as a method for capturing particular gene expression pattern was used and the developing dentition of the mouse were reconstructed using three dimensional (3-D) data reconstruction. This project covered the time window between embryonic days 11.5 and 14.5 in the mouse (Mus musculus) which corresponds to the time frame of early development in the tooth. 3D domains of gene expression were visualized using marker genes, Keratin 5, Sonic Hedgehog (Shh), Pitx2 and Pax9 first by whole mount in situ hybridization and then viewed using special software that allow the generation of 3-D data. This allowed us to reconstruct the developing dentition based on four gene expressions. A complete 3-D reconstruction of the dental epithelium was achieved with K5; Pitx2 allowed us to reconstruct the oral ectoderm exclusively whereas the 3-D analysis of prospective tooth germs was made possible by mapping the expression of Shh in all three axes. Pax9/ Pitx2 double gene expression made it possible to create a 3-D model of both the epithelium and mesenchyme thereby showing the extent to which those two tissues interact. In the OPT models, considerable details such as the shape the epithelium adopts in the developing incisor tooth could be viewed in three dimensions. Even at a minimum resolution, 3-D anatomical details and expression of genes deep into the anatomy of the mouse could be identified and visualised. The OPT technology and the generated models along with all the accompanying technologies provided a powerful approach to the study and analysis of gene expression during early odontogenesis.
Highlights
Tooth development involves complex changes in shape and structure over time
Mouse embryos at stages E11.5, E12.5, E13.5 and E14.5 were used to allow us to reconstruct the development of the tooth in its early stages and there were used to map the expression of Keratin 5, Pax 9, Pitx 2 and Sonic Hedgehog (Shh)
Complete epithelial reconstruction was possible with Keratin5, selective tooth germ reconstruction with epithelial marker Shh, oral ectodermal reconstruction with Pitx2 and mesenchymal and epithelial reconstruction was made from Pax9+Pitx2 double in situ hybridisation specimens
Summary
Tooth development involves complex changes in shape and structure over time. During a period of approximately a week (Embryonic day 11.5 to Embryonic day 18.5 in the mouse) the dental region of the mammalian embryo is home to one of the most complex forms of organogenesis.The first visible indication of odontogenesis is the thickening of the oral ectoderm to form a structure known as the dental lamina Gritli-Linde et al [1] which subsequently buds into the underlying mesenchyme. Tooth development involves complex changes in shape and structure over time. During a period of approximately a week (Embryonic day 11.5 to Embryonic day 18.5 in the mouse) the dental region of the mammalian embryo is home to one of the most complex forms of organogenesis. The first visible indication of odontogenesis is the thickening of the oral ectoderm to form a structure known as the dental lamina Gritli-Linde et al [1] which subsequently buds into the underlying mesenchyme. The third and most dramatic stage of tooth development is the bell stage It starts at E16.5 with the epithelium invaginating further into the mesenchyme and it eventually completely encloses the condensing mesenchyme at the late bell stage (E18) Thesleff and Sharpe [2]
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