The mammalian dentition is a premier model for understanding how organs form. In particular, it exemplifies the recursive and widely deployed developmental strategy of inductive epithelial-mesenchymal (E-M) tissue interactions, by which signals are sequentially and reciprocally exchanged between interacting epithelium and mesenchyme, resulting in the progressive differentiation of each tissue. In the prior grant period, we developed important new methods for reconstructing the gene regulatory network (GRN) that underlies early molar tooth formation, and we identified a unique E-M Wnt-Bmp4 feedback circuit that controls key steps in this process. In addition to providing a mechanism for the pivotal roles of Bmp and Wnt in odontogenesis, this Wnt-Bmp4 feedback circuit can explain several specific properties of E-M organogenesis, including phasic E-M expression dynamics and the coupling of E-M development. Thus, the reconstruction of this odontogenic GRN affords a systems level understanding of how vertebrate organs form. We hypothesize that this GRN can be further developed to enable the efficient manipulation of organ fate, with profound significance for regenerative medicine. We will test this hypothesis by pursuing three Specific Aims.
In Aim 1, we will significantly improve the version 1.0 odontogenic GRN that controls early murine molar tooth development from initiation- to cap-stage. We will obtain new gene expression data from unique fluorescently labeled dental epithelial and mesenchymal progenitor cell populations, compare these profiles to our existing datasets, and use this data to add a spatial resolution parameter to the odontogenic GRN. We will also obtain microRNA data via RNA-Seq, functionally validate selected miRNAs, and integrate critical miRNA regulators into the GRN.
In Aim 2, we will perform DNase-Seq and ChIP-Seq analysis on uninduced and induced dental mesenchyme, using ChIP-Seq for histone modifications (H3K4me1, H3K4me3, H3K27me3, H3K27ac), co-activator marks (p300), and specific TFs that occupy key nodal points in the existing odontogenic GRN (e.g., Msx1, Pax9). We will also assay epigenetic marks in Msx1 and Hdac3 mutant mice. New bioinformatic methods will be developed to integrate this data into the existing GRN. Lastly, in Aim 3, we will validate the new version 2.0 GRN both in vitro and in vivo. In vitro, we will perturb tooth germ explant cultures and primary dental mesenchymal cells in defined ways, and we will compare the gene expression responses to those observed in endogenous dental tissues. We will also seek to validate the odontogenic GRN in vivo, by activating odontogenesis in the mouse oral cavity genetically and via treatment with specific factors identified from the GRN. These experiments hold the potential to provide the most complete GRN for any vertebrate organ, and will identify key nodal points for intervention. Ideally, this work will provide an instructional template for defined organ replacement.
This project will employ genomic and computational approaches to construct a circuit diagram that explains how the mouse embryonic molar tooth - a powerful model for organogenesis - forms. We will significantly improve our original 'circuit diagram'for tooth formation by incorporating new and different types of data, making it more accurate and powerful. We will also test the validity of this new circuit diagram in explanted tooth germs, in cultured cells, and in mice.
|Rebustini, Ivan T (2017) A Functional MicroRNA Screening Method for Organ Morphogenesis. Curr Protoc Cell Biol 74:19.19.1-19.19.17|
|Rebustini, Ivan T; Vlahos, Maryann; Packer, Trevor et al. (2016) An integrated miRNA functional screening and target validation method for organ morphogenesis. Sci Rep 6:23215|
|Campas, Otger; Mammoto, Tadanori; Hasso, Sean et al. (2014) Quantifying cell-generated mechanical forces within living embryonic tissues. Nat Methods 11:183-9|
|Nusinow, David P; Kiezun, Adam; O'Connell, Daniel J et al. (2012) Network-based inference from complex proteomic mixtures using SNIPE. Bioinformatics 28:3115-22|
|Jumlongras, Dolrudee; Lachke, Salil A; O'Connell, Daniel J et al. (2012) An evolutionarily conserved enhancer regulates Bmp4 expression in developing incisor and limb bud. PLoS One 7:e38568|
|Kuraguchi, Mari; Wang, Xiu-Ping; Bronson, Roderick T et al. (2006) Adenomatous polyposis coli (APC) is required for normal development of skin and thymus. PLoS Genet 2:e146|
|Alkuraya, Fowzan S; Saadi, Irfan; Lund, Jennifer J et al. (2006) SUMO1 haploinsufficiency leads to cleft lip and palate. Science 313:1751|
|Jumlongras, Dolrudee; Lin, Jenn-Yih; Chapra, Anas et al. (2004) A novel missense mutation in the paired domain of PAX9 causes non-syndromic oligodontia. Hum Genet 114:242-9|
|Ma, Liang; Liu, Jian; Wu, Tobey et al. (2003) 'Cyclic alopecia' in Msx2 mutants: defects in hair cycling and hair shaft differentiation. Development 130:379-89|
|Gritli-Linde, Amel; Bei, Marianna; Maas, Richard et al. (2002) Shh signaling within the dental epithelium is necessary for cell proliferation, growth and polarization. Development 129:5323-37|
Showing the most recent 10 out of 17 publications