Dual Role of Amelogenin during craniofacial development

Introduction: The amelogenins comprise 90% of the developing extracellular-enamel-matrix-proteins and play a major role in the biomineralization and structural organization of enamel. Amelogenins were also detected in postnatal calcifying mesenchymal tissues, and in several non mineralizing tissues. Amelogenin isoforms were suggested to have signaling activity; to produce ectopically chondrogenic and osteogenic-like tissue. Recently, some amelogenin isoforms were found to bind to the cell surface receptors; LAMP-1, LAMP-2 and CD63, and subsequently localize to the perinuclear region of the cell. The recombinant amelogenin protein alone brought about regeneration of the tooth supporting tissues: cementum, periodontal-ligament and alveolar-bone, through recruitment of progenitor cells and mesenchymal stem cells.

Objective: In this study we focused on the spatio-temporal expression of amelogenin in different tissues of the developing embryonic mouse craniofacial complex. We also characterized amelogenin induced cell movement in the developing tooth bud.

Methods: CD1 mice embryos aged E10.5-E18.5 were sacrificed. RNA was isolated, reverse-transcribed, followed by PCR and sequencing. Western blot analysis was preformed on total proteins. Paraffin sections of embryos were stained for amelogenin presence. Application of amelogenin (rHAM+) beads together with DiI, on E13.5 and E14.5 embryonic mandibular slice cultures and on embryonic tooth germ was preformed.

Results: We show that amelogenin is expressed in various tissues of the developing mouse embryonic craniofacial complex, and is already expressed at E10.5, long before the initiation of tooth formation. Amelogenin protein expression was detected in the tooth germ already at E13.5, much earlier than previously reported (E19). Application of amelogenin (rHAM+) beads together with DiI, on E13.5 and E14.5 embryonic mandibular slice cultures revealed recruitment of mesenchymal cells.

Conclusion: The present results strongly indicate that amelogenin has an important role in many tissues of the cranio-facial complex during mouse embryonic development and differentiation, and might be a multifunctional protein.