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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., The LaCL stem cell niche does not express miR‐200, which regulates Sox2 expression. Progenitor cells exiting the stem cell niche start expressing miR‐200 to promote differentiation and the specification of cell fates for mature tooth development. miR‐200 therefore acts to compartmentalize the stem cell niche

Epithelial signaling centers control epithelial invagination and organ development, but how these centers are specified remains unclear. We report that Pitx2 (the first transcriptional marker for tooth development) controls the embryonic formation and patterning of epithelial signaling centers during incisor development. We demonstrate using Krt14Cre/Pitx2flox/flox (Pitx2cKO) embryos, and Rosa26CreERT/Pitx2flox/flox mice that loss of Pitx2 delays epithelial invagination, decreases progenitor cell proliferation, and dental epithelium cell differentiation. Developmentally, Pitx2 regulates formation of the Sox2+ labial cervical loop (LaCL) stem cell niche in concert with two signaling centers, the initiation knot (IK) and enamel knot (EK). The loss of Pitx2 disrupted the patterning of these two signaling centers resulting in tooth arrest at E14.5. Mechanistically, Pitx2 transcriptional activity and DNA binding is inhibited by Sox2, and this interaction controls gene expression in specific Sox2 and Pitx2 co-expression progenitor cell domains. We demonstrate new transcriptional mechanisms regulating signaling centers by Pitx2, Sox2, Lef-1 and Irx1.

In this study, we investigated the role of the transcription factor Six2 in palate development. Six2 was selected using the SysFACE tool to predict genes from the 2p21 locus, a region associated with clefting in humans by GWAS, that are likely to be involved in palatogenesis. We functionally validated the predicted role of Six2 in palatogenesis by showing that 22% of Six2 null embryos develop cleft palate. Six2 contributes to palatogenesis by promoting mesenchymal cell proliferation and regulating bone formation. The clefting phenotype in Six2(−/−) embryos is similar to Pax9 null embryos, so we examined the functional relationship of these two genes. Mechanistically, SIX2 binds to a PAX9 5’ upstream regulatory element and activates PAX9 expression. In addition, we identified a human SIX2 coding variant (p.Gly264Glu) in a proband with cleft palate. We show this missense mutation affects the stability of the SIX2 protein and leads to decreased PAX9 expression. The low penetrance of clefting in the Six2 null mouse combined with the mutation in one patient with cleft palate underscores the potential combinatorial interactions of other genes in clefting. Our study demonstrates that Six2 interacts with the developmental gene regulatory network in the developing palate.

Molar incisor hypomineralization (MIH) is an enamel condition characterized by lesions ranging in color from white to brown which present rapid caries progression, and mainly affects permanent first molars and incisors. These enamel defects usually occur when there are disturbances during the mineralization or maturation stage of amelogenesis. Both genetic and environmental factors have been suggested to play roles in MIH’s development, but no conclusive risk factors have shown the source of the disease. During head and neck development, the interferon regulatory factor 6 (IRF6) gene is involved in the structure formation of the oral and maxillofacial regions, and the transforming growth factor alpha (TGFA) is an essential cell regulator, acting during proliferation, differentiation, migration and apoptosis. In this present study, it was hypothesized that these genes interact and contribute to predisposition of MIH. Environmental factors affecting children that were 3 years of age or older were also hypothesized to play a role in the disease etiology. Those factors included respiratory issues, malnutrition, food intolerance, infection of any sort and medication intake. A total of 1,065 salivary samples from four different cohorts were obtained, and DNA was extracted from each sample and genotyped for nine different single nucleotide polymorphisms. Association tests and logistic regression implemented in PLINK were used for analyses. A potential interaction between TGFA rs930655 with all markers tested in the cohort from Turkey was identified. These interactions were not identified in the remaining cohorts. Associations (p

The mechanisms that regulate post-natal growth of the craniofacial complex and that ultimately determine the size and shape of our faces are not well understood. Hippo signaling is a general mechanism to control tissue growth and organ size, and although it is known that Hippo signaling functions in neural crest specification and patterning during embryogenesis and before birth, its specific role in postnatal craniofacial growth remains elusive. We have identified the transcription factor FoxO6 as an activator of Hippo signaling regulating neonatal growth of the face. During late stages of mouse development, FoxO6 is expressed specifically in craniofacial tissues and FoxO6-/- mice undergo expansion of the face, frontal cortex, olfactory component and skull. Enlargement of the mandible and maxilla and lengthening of the incisors in FoxO6-/- mice are associated with increases in cell proliferation. In vitro and in vivo studies demonstrated that FoxO6 activates Lats1 expression, thereby increasing Yap phosphorylation and activation of Hippo signaling. FoxO6-/- mice have significantly reduced Hippo Signaling caused by a decrease in Lats1 expression and decreases in Shh and Runx2 expression, suggesting that Shh and Runx2 are also linked to Hippo signaling. In vitro, FoxO6 activates Hippo reporter constructs and regulates cell proliferation. Furthermore PITX2, a regulator of Hippo signaling is associated with Axenfeld-Rieger Syndrome causing a flattened midface and we show that PITX2 activates FoxO6 expression. Craniofacial specific expression of FoxO6 postnatally regulates Hippo signaling and cell proliferation. Together, these results identify a FoxO6-Hippo regulatory pathway that controls skull growth, odontogenesis and face morphology., Author summary The basic question of how human faces develop, undergo morphogenesis and grow after birth to define our final characteristic shape has been studied from the earliest days of comparative vertebrate developmental research. While many studies have shown the factors and mechanisms that contribute to the cells and tissues of the face during embryology, fewer studies have determined mechanisms that promote face growth after birth and into childhood. In our quest to understand developmental mechanisms of facial growth we used murine gene expression and bioinformatics analyses combined with human 3D facial variations and genome-wide association studies to identify genes and variants controlling post-natal face growth. Bioinformatics analyses of mouse craniofacial gene expression identified FoxO6 as a transcription factor expressed at late stages of face development. Ablation of FoxO6 in the mouse resulted in specific anterior growth of the mouse face. The increased FOXO6 expression activated Hippo signaling to reduce face growth. These data indicate that changes in FOXO6 expression control face growth during early childhood.