Regulation of proliferation in developing human tooth germs by MSX homeodomain proteins and cyclin-dependent kinase inhibitor p19^INK4d

Abstract

Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19^INK4d. p19^INK4d induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19^INK4d by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19^INK4d in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19^INK4d throughout the investigated period indicates that p19^INK4d plays active role during human tooth development. Furthermore, comparison of expression domains of p19^INK4d with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.

KEYWORDS:

• cell cycle
• development
• human tooth germ
• MSX1
• MSX2
• p19^INK4d
• proliferation

ABBREVIATION

MSX1	=	Muscle Segment Homeodomain Protein 1
MSX2	=	Muscle Segment Homeodomain Protein 2
p19INK4d	=	Ink4 Family Cyclin-dependent Kinase Inhibitor p19
phospho Rb	=	phosphorylated Retinoblastoma Protein

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed.

Acknowledgements

The authors wish to thank Mrs. Marica Maretic, mag.chem.ing., for expert technical assistance.

FUNDING

This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia (grant no. 021-2160528-0507, principal investigator prof. Mirna Saraga-Babic, MD, PhD).

AUTHOR CONTRIBUTIONS

Darko Kero designed the study, performed selection of primary antibodies and immunofluorescent staining of tissue sections (MSX1/MSX2/p19^INK4d), acquired, interpreted and processed data, assessed the literature review reports and wrote the manuscript. Katarina Vukojevic performed double immunofluorescent staining (Cyclin A2/phospho Rb), acquired and interpreted data, reviewed part of the literature (cell cycle regulation) and wrote Materials and Methods and Results sections of the manuscript. Petra Stazic performed double immunofluorescent staining (Ki67/p19^INK4d), acquired and interpreted data, reviewed part of the literature (p19^INK4d and INK4 CDK cell cycle inhibitors/ functional studies) and wrote part of Results section of the manuscript. Danijela Sundov performed single immunofluorescent staining (MSX1/MSX2), reviewed part of the literature (MSX1 and MSX2 in organogenesis and odontogenesis), and wrote Result section of the manuscript. Snjezana Mardesic Brakus performed double immunofluorescent staining (Ki67/p19^INK4d), and reviewed part of the literature (regulation of proliferation in organogenesis and odontogenesis). Mirna Saraga Babic designed the study, provided inputs on staining procedures and data interpretation, revised and co-edited the manuscript with Darko Kero.