Tracking diphyodont development in miniature pigs in vitro and in vivo

Abnormalities of tooth number in humans, such as agenesis and supernumerary tooth formation, are closely related to diphyodont development. There is an increasing demand to understand the molecular and cellular mechanisms behind diphyodont development through the use of large animal models, since they are the most similar to the mechanism of human tooth development. However, attempting to study diphyodont development in large animals remains challenging due to large tooth size, prolonged growth stage and embryo manipulation. Here, we characterized the expression of possible genes for diphyodont development and odontogenesis of an organoid bud from single cells of tooth germs in vitro using Wzhishan pig strain (WZSP). Following this, we used a method of ectopic transplantation of tooth germs at cap stage to dynamically track diphyodont development of tooth germs in mouse subrenal capsules to overcome the restrictions in pig embryos. The results showed that pig tooth germ at cap stage could restore diphyodont development and maintain efficient long-term survival and growth in mouse subrenal capsules, which is suitable for future manipulation of large mammalian tooth development. Our pilot study provided an alternative for studying diphyodont development in large mammals, which will further promote the use of pig as a diphyodont model similar to humans for craniofacial development study.