Mason Sweat, Yan Sweat, Wenjie Yu, Dan Su, Riley J. Leonard, Steven L. Eliason, Brad A. Amendt

The murine lower incisor ectodermal organ contains a single epithelial stem cell (SC) niche that provides epithelial progenitor cells to the continuously growing rodent incisor. The dental stem cell niche gives rise to several cell types and we demonstrate that the miR-200 family regulates these cell fates. The miR-200 family is highly enriched in the differentiated dental epithelium and absent in the stem cell niche. In this study, we inhibited the miR-200 family in developing murine embryos using new technology, resulting in an expanded epithelial stem cell niche and lack of cell differentiation. Inhibition of individual miRs within the miR-200 cluster resulted in differential developmental and cell morphology defects. miR-200 inhibition increased the expression of dental epithelial stem cell markers, expanded the stem cell niche and decreased progenitor cell differentiation. RNA-seq. identified miR-200 regulatory pathways involved in cell differentiation and compartmentalization of the stem cell niche. The miR-200 family regulates signaling pathways required for cell differentiation and cell cycle progression. The inhibition of miR-200 decreased the size of the lower incisor due to increased autophagy and cell death. New miR-200 targets demonstrate gene networks and pathways controlling cell differentiation and maintenance of the stem cell niche. This is the first report demonstrating how the miR-200 family is required for in vivo progenitor cell proliferation and differentiation. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: Current microRNA (miR) inhibition methods cannot be used to study in vivo developmental stem cell processes. CRISPR-Cas genome editing cannot specifically knockout a miR within a cluster. Furthermore, not all miRs, especially within introns can be targeted by the CRISPR method without affecting gene expression. ES cells have been profiled for miR expression; however, cell-based assays using oligonucleotides targeting miRs are not specific and are toxic. The authors developed a highly specific, effective miR inhibitor that can be used to knockdown miRs during embryonic development to determine their effect on stem cells, cell proliferation, and differentiation. The authors show that the miR-200 family acts to compartmentalize an ectodermal stem cell niche by regulating progenitor cell differentiation during development.