The expected overall impact of this project is to identify developmental and genetic mechanisms underlying tooth formation and replacement. Teeth have classically been used as a model to study organogenesis, as teeth, like most organs, develop through reciprocal epithelial-mesenchymal interactions. Furthermore, 30 percent of people worldwide over the age of 65 have no natural teeth. Thus, knowledge of the developmental and genetic basis of tooth formation and replacement has relevance both for understanding organogenesis, as well as for understanding how teeth can be regenerated in vitro and ultimately in vivo. Although genetic studies in mice and humans have identified signaling pathways involved in tooth development, less is known about genetic mechanisms regulating tooth replacement. Fish replace their teeth constantly throughout adult life, and offer powerful systems for genetic analysis. Here, natural variation in tooth number in the threespine stickleback fish (Gasterosteus aculeatus) is leveraged as a new model system to learn how genes regulate tooth number and tooth replacement. Different stickleback populations adapted to different diets exhibit dramatic heritable changes in tooth number. Two different, independently derived freshwater populations have evolved major increases in tooth number compared to ancestral marine fish. In both high-toothed populations, the tooth number increase arises late in development through an accelerated tooth replacement rate. The different forms can be crossed in the lab, enabling detailed forward genetic analyses to map factors controlling the changes in tooth number. New genome editing methods allow functional tests of genes and cis-regulatory elements of interest. A cis-regulatory allele of the Bone Morphogenetic Protein 6 (Bmp6) gene is associated with evolved tooth gain in one high-toothed population. Pharmacological and genetic data suggest BMP signaling and Bmp6 positively regulate primary tooth number, but inhibit tooth replacement. To test hypotheses about the developmental and genetic bases of tooth formation and replacement, three specific aims are proposed. First, we will test whether BMP signaling and Bmp6 regulate dental stem cell quiescence during tooth replacement by BrdU and vital dye pulse-chase labeling, gene expression, and pharmacological experiments. Second, we will identify upstream regulators of two Bmp6 enhancers and determine enhancer and regulator functions during tooth development and replacement by pharmacological, transgenic, and genome editing experiments. Third, we will identify the genetic basis of evolved tooth gain in an independently derived high-toothed freshwater population with a distinct developmental genetic basis by a combination of genetic mapping, genome editing, and gene expression experiments. Together these results will shed new light on developmental and genetic mechanisms underlying tooth replacement.

Public Health Relevance

PUBLIC HEALTH RELEVANCE: This research will provide fundamental knowledge of how genes control tooth formation and tooth replacement. This knowledge will help efforts to engineer tooth formation in vitro and ultimately regenerate teeth in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE021475-09
Application #
9720678
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Stein, Kathryn K
Project Start
2011-03-01
Project End
2021-06-30
Budget Start
2019-07-01
Budget End
2020-06-30
Support Year
9
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94710
Erickson, Priscilla A; Baek, Jiyeon; Hart, James C et al. (2018) Genetic Dissection of a Supergene Implicates Tfap2a in Craniofacial Evolution of Threespine Sticklebacks. Genetics 209:591-605
Cleves, Phillip A; Hart, James C; Agoglia, Rachel M et al. (2018) An intronic enhancer of Bmp6 underlies evolved tooth gain in sticklebacks. PLoS Genet 14:e1007449
Hart, James C; Miller, Craig T (2017) Sequence-Based Mapping and Genome Editing Reveal Mutations in Stickleback Hps5 Cause Oculocutaneous Albinism and the casper Phenotype. G3 (Bethesda) 7:3123-3131
Ellis, Nicholas A; Miller, Craig T (2016) Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton. J Vis Exp :
Erickson, Priscilla A; Glazer, Andrew M; Killingbeck, Emily E et al. (2016) Partially repeatable genetic basis of benthic adaptation in threespine sticklebacks. Evolution 70:887-902
Erickson, Priscilla A; Ellis, Nicholas A; Miller, Craig T (2016) Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks. J Vis Exp :
Ellis, Nicholas A; Donde, Nikunj N; Miller, Craig T (2016) Early development and replacement of the stickleback dentition. J Morphol 277:1072-83
Erickson, Priscilla A; Cleves, Phillip A; Ellis, Nicholas A et al. (2015) A 190 base pair, TGF-? responsive tooth and fin enhancer is required for stickleback Bmp6 expression. Dev Biol 401:310-23
Glazer, Andrew M; Killingbeck, Emily E; Mitros, Therese et al. (2015) Genome Assembly Improvement and Mapping Convergently Evolved Skeletal Traits in Sticklebacks with Genotyping-by-Sequencing. G3 (Bethesda) 5:1463-72
Ellis, Nicholas A; Glazer, Andrew M; Donde, Nikunj N et al. (2015) Distinct developmental genetic mechanisms underlie convergently evolved tooth gain in sticklebacks. Development 142:2442-51

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