Project description:The tooth is derived from both the ectoderm and the neural crest (ectomesenchyme). It is often used as a model for studies of the basic mechanisms of organ development, including differentiation, cellular interaction, morphogenesis, and mineralization of extracellular matrices. In mouse, tooth development begins at embryonic day 11.5 (E11.5) by thickening of the dental epithelium, whereas mineralization of enamel and dentin in first molars starts at postnatal day 0 (P0). Tooth development entails a multistep and complex process of gene expression. Studies with genetically modified mice have contributed substantially to our understanding of genes that regulate tooth development and morphology. MicroRNAs (miRNA) are a class of non-coding RNAs that regulate gene expression at a post-transcriptional level, and are considered as important regulatory molecules during foetal development. By binding to target mRNAs, miRNA induce mRNA decay or translation repression. Recent bioinformatic predictions of miRNA targets in vertebrates indicate that hundreds of miRNAs are responsible for regulation of expression of up to 30% of the human protein-coding genes. Recent experiments has verified that miRNAs can regulate expression of hundreds of mRNAs in cultured cells. Previous study has also shown that microarrays can be used to detect physiologically relevant effects of miRNAs on expression of mRNAs in vertebrates; miRNAs have been shown to alter expression of a greater number of transcripts than previously appreciated, primarily through interaction with 3M-4UTRs. Understanding the biological function of miRNAs require knowledge of their mRNA targets. Bioinformatic approaches have been used to predict mRNA targets, among which both transcription factors and pro-apoptopic genes were prominent candidates. The precise molecular function of miRNAs remains largely unknown and a better understanding may require loss-of-function studies in vivo. It has been shown that intravenous administration of a novel class of chemically engineered oligonucleotides termed M-^SantagomirsM-^T resulted in a marked reduction of corresponding miRNA levels in most tissues. This resulted in upregulated expression of hundreds of genes predicted to be repressed by miR-122 because these genes had a miR-122 recognition motif in their 3M-4UTR region. Paradoxically, antagomir treatment also revealed a significant number of downregulated genes that may be activated by miR-122. The mechanism by which miRNAs may activate gene expression in vivo is unknown. However, it may involve an indirect effect (the suppression of a transcriptional repressor), or alternatively, miRNA may have a direct effect on gene activation (e.g. chromatin remodeling). Studies of microRNA expression profiles of the developing murine molar tooth germ have shown these to be highly dynamic, some miRNAs being abundantly expressed during the early development of the molar tooth germ, while others are highly expressed only at later developmental stages. A recent study also suggested that miRNAs modulate tooth morphogenesis and ameloblast differentiation, perhaps largely by fine tuning conserved signaling networks. The function of microRNA-214 (miR-214) is unknown, however, it has been related to normal growth and skeletal development in mice. In tooth germs miR-214 is more highly expressed during later developmental stages. It was selected as a target for silencing using anti-miR-214. Here we report effects from injections of 1-100 pmol of anti-miR-214 close to developing first mandibular molar in newborn mice. At 24 hrs, or more, after injection significant changes in gene expression, and in levels of several correspondingly encoded proteins, were found in the tooth germ.

Project description:Previous studies have suggested that Bmp4 is a key Msx1-dependent mesenchymal odontogenic signal for driving tooth morphogenesis through the bud-to-cap transition. Whereas the bud stage tooth developmental arrest in Msx1-/- mutant mice was accompanied by reduction in mesenchymal Bmp4 mRNA expression, we show that depleting functional Bmp4 mRNAs in the tooth mesenchyme, through neural crest-specific gene inactivation in Bmp4f/f;Wnt1Cre mice, caused mandibular molar developmental arrest at the bud stage but allowed maxillary molars and incisors to develop to mineralized teeth. We show that the Wnt inhibitors Dkk2 and Wif1 were much more abundantly expressed in the mandibular than maxillary molar mesenchyme in wildtype embryos and that Dkk2 expression was significantly unregulated in the tooth mesenchyme in Bmp4f/f;Wnt1Cre embryos. In addition, expression of Osr2, which encodes a zinc finger protein that antagonizes Msx1-mediated activation of odontogenic mesenchyme, is significantly upregulated in the molar mesenchyme in Bmp4f/f;Wnt1Cre embryos. Msx1 heterozygosity enhanced maxillary molar developmental defects whereas Osr2 heterozygosity rescued mandibular first molar morphogenesis in Bmp4f/f;Wnt1Cre mice. Moreover, in contrast to complete lack of supernumerary tooth initiation in Msx1-/-Osr2-/- mutant mice, Osr2-/-Bmp4f/f;Wnt1Cre compound mutant mice exhibit formation and subsequent arrest of supernumerary tooth germs that correlated with down regulation of Msx1 expression in the tooth mesenchyme. Taken together, our data indicate that, while reduction in mesenchymal Bmp4 expression alone could not account for the tooth bud arrest phenotype in Msx1-/- mutant mice, Bmp4 signaling synergizes with Msx1 and antagonizes Osr2 to activate mesenchymal odontogenic activity to drive tooth morphogenesis and sequential tooth formation. E13.5 mouse embryos tooth germs were microdissected by laser capture microdissection (LCM), and the mandibular molar and maxillary molar were separated. 3 pairs of control and mutant samples were pooled for the RNA extraction.

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Mus musculus

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