bCe Inhibiting FGFR2b in the dental epithelium fully (D) or partially (E) rescued the supernumerary tooth phenotype in the mice Table?2 The timing and effects of inhibiting FGFR2b in rescuing the supernumerary tooth formation in tests. were collected at E12.5 and subjected to cryosection for Bifeprunox Mesylate fluorescence assay. On the same cryosections, Sox2-expressing cells were labeled by immunofluorescence using anti-Sox2 antibody and EGFP-conjugated secondary antibody. The Cre activity indicated by Tomato fluorescence (red) was strongly present in Bifeprunox Mesylate the dental epithelium (arrows) and oral epithelium, as well as SQSTM1 nasal mucosa and palatal epithelium. The antibody-labeled Sox2-expressing cells (green) mostly overlapped with CreER active cells (red) and showed yellow around the merged channel. CreER-active cells showed overall broader range than the antibody-labeled Sox2(+) cells in the dental epithelium (especially in the distal side) and nasal mucosa, indicating that the efficiency of Sox2-CreER was strong enough for deleting floxed alleles from the Sox2-expressing cells. n, nose; p, palate; m, mandible. B To determine the regulatory manner of GAGs on Sox2(+) cell homeostasis, we inactivated from Sox2(+) lineage using injection at E11.5 and E12.0. mice did not recapitulate the replacement tooth phenotype, suggesting that GAGs regulate the homeostasis of Sox2(+) cells in a nonautonomous manner. 12915_2020_813_MOESM4_ESM.jpg (339K) GUID:?2EC54A76-3941-4317-9AA8-CAD7D74081C6 Additional file 5: Physique S5. WNT signaling was not changed in the around the coronal sections Bifeprunox Mesylate of lower incisors showed no differences between the around the coronal sections of lower incisors showed no differences between the on E12.5 mandibles showed no differences between the incisors of around the coronal sections of lower incisors showed no differences between the and was not changed in the and on the coronal sections of lower incisors showed no differences between the and between the back to the normal size. Scale bars, 250?m. 12915_2020_813_MOESM8_ESM.jpg (185K) GUID:?6C52C8EC-58B7-4EF5-8C29-FC8F5C3589E4 Additional file 9: Figures S9A-S9B. Fig. S9A-[GAGs did not show synergistic effects on FGF10-FGFR2b signaling]. Fig. S9B-[GAGs did not show inhibitory effects on FGF10-FGFR2b signaling]. HS and CS did not show significant synergistic or inhibitory effects on FGF10-FGFR2b signaling in BaF3 cells. A BaF3 cells expressing FGFR2b were cultured in RPMI 1640 media supplemented with 1000 pM FGF10 and 0C5?ng/ml GAGs (HS/HS2S/HS6S/CSA/heparin) for 45?h. Heparin (positive control) showed significant synergistic effects on FGFR2b signaling (results in embryonic death at E13.5 [11], we generated a in the dental epithelium led to supernumerary incisors that were formed in a manner similar to replacement tooth formation [12], uncovering a previously unknown function of GAGs in the control of tooth number. Our results demonstrate that this FAM20B-catalyzed GAGs control the tooth number in mice by modulating the commitment of dental epithelial stem/progenitor cells through a mechanism involving the restriction of FGFR2b signaling at the initial stage of tooth development. Our findings provide novel insights into the molecular mechanism regulating tooth number and renewal in mice that may shed light on other GAG-mediated signaling events during organogenesis. Results GAG deficiency in the dental epithelium leads to supernumerary incisors in mice It has been long known that proteoglycans are important molecules regulating signaling pathways during organogenesis. Decades ago, Thesleff et al. reported the expression of proteoglycans in developing murine teeth [13], and subsequent studies have identified multiple proteoglycans in both the dental epithelium and dental mesenchyme at various embryonic stages [6, 7, 14C16]. However, dissecting their mechanistic roles in tooth development has been challenging, because mice lacking individual proteoglycans or a particular type of GAGs did not show overt tooth phenotypes [17]. To explore this issue, we generated in the dental epithelium (mice. F, G The ectopic thickening of dental epithelium formed an extended dental lamina (black arrows) at the mesial-lingual side of native enamel organs and developed into a novel enamel organ (white arrows and dashed lines) at the end of the extended lamina. H At E18.5, the extended dental lamina disappeared, and the extra enamel organs developed into supernumerary incisors (white arrow) at the mesial-lingual side of native teeth. ICL In contrast, disrupting GAGs in the.