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miRNA and tooth development

Final Report Summary - MIRNATOOTH (miRNA and tooth development)

Project context and objectives

Prior to the start of my postdoctoral training in Prof. Thesleff’s laboratory, research on micro-RNAs (miRNAs) had become more and more extensive on ectodermal organs, but there was nothing concerning tooth development and renewal.

My first objective was to analyse the micro-ribonucleic acid (miRNA) pathway during tooth formation and tooth renewal (the mouse incisor contains a stem cell niche involved in the continuously growing tooth).

Work performed

In order to achieve the first part of this objective, I had to learn and adapt miRNA methods to the tooth context. After total RNA extraction and by means of microarray profiling, I found 15 tooth-enriched miRNAs. The profiling studies exhibited a dynamic regulation of miRNA expression that reflected the involvement of different miRNAs at different stages of tooth formation. I established a set of miRNAs involved during morphogenesis, another during cell differentiation and a third one during renewal. I validated their expression via in situ hybridisation. The expression pattern of tooth-enriched miRNAs exhibited mainly an epithelial expression, reflecting an involvement in ameloblast differentiation and enamel knot regulation (tooth signalling centre). To check the involvement of the miRNA pathway during the tooth formation, I analysed the expression of factors involved in miRNA silencing: Drosha and Dicer-1, Ribonucleases (RNases) involved in miRNA maturation; Ago-1 to -4 and GW-182, factors forming the RISC (RNA-induced silencing complex); Staufen-1 and -2, involved in the silencing of miRNA targets. By means of quantitative polymerase chain reaction (qPCR), I have shown the dynamic of the miRNA pathway factor expressions. This result was consistent with the miRNA profiling: an increasing involvement of miRNA involvement from the tooth induction to the morphogenesis, then a slight decrease during the cell differentiation, but reinforcement in the stem cell niche, reflecting an important role in the stem cell regulation.

To study the role of miRNA regulation in tooth formation, I have studied the mouse tooth phenotype of the epithelial Dicer-1 knockout. Dicer-1 is a cytoplasmic RNase necessary for the miRNA processing. Whereas no gross defect was observed at the end of embryonic development, the erupted teeth (incisor and molar) exhibited various defects. The overall shape of the molar was conserved, but the cusp patterning was perturbed, the crown size was different and some extra cusps appeared. The pattern was completely modified on the second and third molar. The resulting molar phenotype can be compared to the mouse field (Apodemus) cusp pattern; this is considered as an evolutionary ancestor of the common mouse lab. It is then probable that miRNA genetic network regulation shaped the tooth during evolution. Concerning the incisor, the morphology was normal until the stem cell niche started to produce new ameloblast in order to insure the tooth growth and renewal. The epithelial Dicer-1 mutation led to an over-proliferation of the pre-ameloblast, disturbing the tooth homeostasis. Epithelial grooves appeared on the labial side of the tooth, and defects in the ameloblast differentiation occurred.

Main result

In order to determine the genes that were regulated by the tooth enriched miRNAs, I designed a database, where for each gene known to be involved during the tooth formation I gathered the miRNAs that were able to regulate it. This database is freely available on the bite-it website (see http://bite-it.helsinki.fi/ for more information). I succeeded in associating the different miRNA to genes already known to be involved in the tooth genetic network, opening a door to further studies concerning the link between the tooth-enriched miRNAs and the predicted targets, providing more knowledge about ameloblast differentiation from a stem cell, the first step to tooth bioengineering.