Amelogenin P2 Assembly in Enamel Development In Vivo

Objectives: Self-assembly of the extracellular matrix protein amelogenin is believed to play an essential role in regulating the growth and organization of enamel crystals during enamel formation. Peptide 2 (P2), residues #8-21 of full-length human amelogenin, was reported to be a critical assembly domain for nanoribbons in vitro. The objective of the investigation is to identify the functions of P2 in vivo model.
Methods: A mouse model (Amelogenin-P2^-/-) was generated using CRISPR/Cas9 genome editing technique to site-specifically delete the P2 region (residues #8-18) in Exon 3 from amelogenin genome. Histology, immunohistochemistry, Micro-CT and SEM analyses were used to investigate the effects of P2 deletion on ameloblasts and the enamel matrix.

Results: PCR and DNA sequencing showed successful deletions of P2 region in Exon 3 from mouse genome.The erupted tooth enamel showed rough and thin enamel in Amelogenin-P2^-/- mice. Histological and immunohistochemistry analyses indicated that secretory ameloblasts were significant shorter in length lacking apical Tomes’ processes, and the underlying enamel matrix was barely detectable. Micro-CT and SEM analyses showed that the enamel of mouse teeth was only 5–20um thick, hypomineralized, and non-prismatic as compared to the WT controls. These results indicated a faster and earlier removal of amelogenin from secretory enamel matrix, corresponding to our in vitro findings that amelogenin protein mutants with P2 deletion were liable to be digested by enamel proteinases.
Conclusions: The P2 assembly domain of amelogenin plays an essential role in tooth enamel development, which may be related to secretory ameloblast morphogenesis, proper secretory matrix formation and its degradation.