Fishing for Mineralized Craniofacial and Tooth Mutants
The zebrafish, Danio rerio, exhibits significant nucleotide and amino acid sequence identity, and chromosomal organization, or synteny, to higher vertebrates including mouse and man. Strengths of the zebrafish including high fecundity, extra-utero fertilization, transparency and rapid growth, facilitate studies of vertebrate craniofacial development. Zebrafish are well adapted for a variety of molecular and genetic techniques including cell transplantations, fate mapping, and microinjection approaches, facilitating analyses that are difficult to perform in mammals. In particular, the ability to perform saturation mutagenesis screens in zebrafish makes them extremely useful for identifying genes acting within a particular developmental pathway, including that of craniofacial and tooth development. The fact that zebrafish continuously regenerate teeth throughout their lives, makes them a unique and valuable model to study signaling pathways regulating this process. We are performing an ongoing forward genetic, chemical mutagenesis screen to identify mineralized craniofacial and tooth mutants in the zebrafish. We anticipate the identification of novel genes required for craniofacial and tooth development and regeneration, whose functional characterization will eventually facilitate the design and implementation of clinically relevant therapies to treat a variety of human craniofacial dysplasias, including edentulism.
Figure 1. Strategy for forward genetic chemical mutagenesis approach to identify genes regulating mineralized craniofacial and tooth development and regeneration. A typical F3 mutagenesis screen is being performed, using wild type and reporter lines. Mineralized tissue defects are identified using Alcian blue and Alizarin Red stains.
ALK8 Signaling in Primary and Replacement Tooth Formation
Based on the fact that TGFβ signaling is critical for craniofacial development, early efforts in my laboratory focused on the isolation and characterization of zebrafish TGFβ family member receptors. These studies resulted in the identification of the novel type I receptor activin like kinase 8, alk8. Functional studies of constitutively active and dominant negative Alk8 isoforms revealed roles for alk8 in neural crest cell formation and mediolateral specification, early dorsoventral patterning of the embryo, and in later developmental events including zebrafish primary and replacement tooth development, and craniofacial cartilage and osteoblast differentiation. The laf/alk8 mutant is a valuable model for replacement tooth development, since heterozygous laf/alk8 animals are viable and fertile, but exhibit a replacement tooth phenotype. We anticipate that studies of laf/alk8, and other zebrafish mutants, will reveal molecular targets that may eventually facilitate replacement tooth induction in humans.
Characterizing the Zebrafish Dental Stem Cell Niche
We are also working to identify signaling pathways regulating dental stem cell maintenance and differentiation in zebrafish primary and replacement tooth development, for comparison to ongoing studies in mouse and humans.
Figure 2. Do similar genes regulate primary and replacement tooth in mice, humans, and zebrafish? Mouse incisor: The continuously erupting incisor model has been used to identify genes regulating dental epithelial progenitor cell differentiation. Human adult tooth bud: Abbreviations: DE, dental epithelium; df, dental follicle; dl, dental lamina; do, dental organ; dp, dental papilla; IDE, inner dental epithelium; ODE, outer dental epithelium; ST, stellate reticulum; TA, transit amplifying cells.
Mammalian Tooth Tissue Engineering
In collaboration with Dr Joseph Vacanti, MD, Chief of Surgery at Massachusetts General Hospital, Harvard Medical School, we are investigating approaches to bioengineer biological tooth substitutes using state-of-the-art tissue engineering techniques. Our studies use cultured human, pig, and rat tooth bud cells seeded onto biodegradable polyester scaffolds and implanted in animal hosts, to generate bioengineered tooth tissues. To date, we have succeeded in generating small bioengineered tooth crowns composed of organized dental pulp, odontoblasts, dentin, ameloblasts, enamel, and cementum. We are currently working to generate full-sized tooth structures of specified size and shape, using a variety of scaffold materials and designs. We anticipate that these studies will likely reveal additional applications for bioengineered dental tissues, including therapies using bioengineered reparative dentin and enamel.
Figure 3. Three dimensional Dental Tissue Engineering Models. The upper panels and the lower left panel show 3-D Hydrogel scaffolds. The lower right panel shows natural pig tooth tissues. Abbreviations: d, dentin; e, enamel; pu, pulp.