Mechanisms responsible for tooth morphogenesis in vertebrates

About This Grant

Abstract Misshapen teeth are highly common in humans. In most instances, they are due to genetic mutations in genes controlling the morphogenesis of teeth. This is the case for the Runx2 gene, which once mutated leads to cleidocranial dysplasia (CCD) an autosomal dominant genetic syndrome presenting with peg-like teeth in humans. While many factors have been identified in the process of tooth morphogenesis, currently, little is known about how cell signaling participates in establishing organ shape during odontogenesis. Our previous work has identified the signaling molecule retinoic acid (RA) to be one of the main actors of tooth induction in fish and recent data suggest that RA also plays a role during tooth morphogenesis. Fish and zebrafish, in particular, are good models to study genetics, cell and molecular biology, and organogenesis of the tooth in vertebrates. The objective of the proposed studies is to understand the roles played by retinoic acid during tooth morphogenesis in different fish species, to develop a fish model of CCD, to clarify the role played by Runx2 during tooth morphogenesis, and to understand its link with RA signaling. Finally, this project proposes to identify novel genes implicated in tooth morphogenesis and to study their function by gene knock-outs in zebrafish. By exposing fish embryos and larvae to exogenous RA and RA inhibitor during tooth morphogenesis we will be able to understand the mechanism of action of RA signaling during tooth morphogenesis in fish. Our preliminary data identified that the levels of RA in different cells of the tooth germ are controlled by the timing and level of expression of the RA degrading enzyme cyp26b1 in a subset of cells of the developing tooth germ. Modifying the onset of cyp26b1 expression in the tooth germ will, therefore, change the level of RA available in the tooth germ ultimately modifying the shape of the tooth. We will study the cis-regulatory changes responsible for evolutionary changes in the timing of cyp26b1 expression during tooth morphogenesis between two closely related fish species, the zebrafish and the mountain minnow, that bear dramatically different shape of teeth in adults and during embryonic development. We have in hands the zebrafish runx2b (the zebrafish ortholog of the human Runx2 gene that is expressed in the tooth germ) loss-of-function mutant. This mutant will be used to perform a phenotypic analysis of tooth morphogenesis and to understand the relationship between RA signaling and Runx2 expression during tooth morphogenesis. To identify novels genes playing a role during tooth morphogenesis, we will select by cell sorting, tooth germ cells exposed to exogenous RA signaling and compare their transcriptome to control developing tooth cells at the same developmental stage. The resulting genes differentially expressed will be subjected to phenotypic analysis by gene knock-out using the CRISPR/cas9 technology. This work will reveal more about how RA and genes it regulates, including Runx2, controls tooth morphogenesis in development, diseases (cleidocranial dysplasia) and evolution.