Tissue specific regeneration requires the function of progenitor cells that can differentiate to repair diseased or injured tissues. Affected tissues can contain stem cells and their growth and patterning are controlled by transcription factors and signaling pathways. Stem cells derived from stem cell niches contribute to the regeneration of mature tissue types in many different organs, including the trachea, lungs and teeth, amongst others. These niches are formed in developing embryos, and must be maintained throughout life by symmetric cellular divisions that produce daughter pluripotent stem cells. Another equally important behavior of the cells in a stem cell niche is the production of differentiated daughter cells by asymmetric cell division, which then take the place of damaged cells in regenerative organs, in order to allow the organ to continue to function. However, the roles of specific microRNAs (miRs) in these processes are unclear. miRs have become appreciated as playing a role in stem cell differentiation. The ability to coopt miR expression in order to reprogram and control the differentiation of naive cells into different cell types will be an important tool required to create artificial organs and repair diseased tissues, saving millions of lives and public dollars. We hypothesize that miR-26b is acting in part to regulate the stem cell niche of the lower and upper incisor by regulating Lef-1 expression, and that this mechanism may be common to many tissues that house stem cell niches. Thus, the absence of miR-26b expression prior to E14.5 allows Lef-1 expression in the oral epithelium (placode) and LaCL (stem cell niche) during development. However, after E14.5, miR-26b expression in the dental epithelium restricts Lef-1 expression and then other factors (Sox2) regulate stem cell maintenance. In order to determine how Lef-1 and miR-26b is affecting gene expression during development and cells we will pursue three aims: 1) to use our murine models to understand in vivo stem cell proliferation and differentiation by Lef-1 and miR-26b OE; and use miR-26b OE to rescue the Lef-1 OE phenotype and define the role of miR-26b OE in dental development; 2) validate molecular interactions between miR-26b, Lef- 1 and known factors involved in DESC proliferation and differentiation; 3) isolate the dental epithelial cells from WT, Lef-1 OE, miR-26b OE and rescue mice to identify new genetic pathways using RNA-Seq, ChIP-Seq. and Single cell Seq.
This project will determine how stem cell niches within the craniofacial/dental complex are activated by Lef-1 and compartmentalized by miR-26b expression. Stem cells within these compartments will be isolated and the epigenetic, miRNA and gene expression signatures identified. The knowledge gleaned from these experiments will be applied to reprogramming adult oral epithelial cells to different types of dental and oral stem cell progenitors capable of repairing and regenerating specific craniofacial and dental tissues affected by disease, injury or genetic anomalies.
