Expression patterns of CXCR4 and CXCL12 mRNAs and proteins in developing incisors. a, b An incisor germ and the inner enamel epithelium. Arrow indicates the apical bud. The dotted line indicates the border between IEE and the apical bud. c, d Quantitation of relative CXCR4 and CXCL12 mRNA levels in the apical bud and developing incisors (**, p<0.01). e–n Expression patterns of CXCR4 (e, g, i, k, m) and CXCL12 (f, h, j, l, n) mRNAs were analyzed by in situ hybridization. The dotted line indicates the outline of the epithelium. In E14 incisors, CXCR4 mRNA was detected on the labial and lingual sides of the IEE (e, arrows and an arrowhead), whereas CXCL12 mRNA was detected in the mesenchyme surrounding the enamel organ (f, arrows). At E16, CXCR4 and CXCL12 mRNA expression patterns became restricted to the apical end of the incisor (g, h, arrows). In E18 and PN4 incisors, strong CXCR4 expression was detected in BE to TA (arrows) of the apical bud (i, k, m). A few cells in the SR and OEE expressed CXCR4 mRNA (m, arrowheads). CXCL12 expression was detected in the dental follicle (arrows) and dental papilla (arrowheads) around the apical bud (j, l, n). o–r Immuno histochemistry of CXCR4 and CXCL12 proteins in 6 week incisors. o CXCR4 signals existed in the TA (o, arrow). The signal was detected from the OEE region (p, arrowhead) to the end of TA (arrowhead). q CXCL12 signals in the mesenchyme were adjacent to the apical bud (q, arrowheads). The intense signals were detected in the lateral side of the apical bud (r, arrow) and dental follicle (r, arrow heads). Scale bar 100 μm. Significant differences between samples were determined by Tukey’s test. **p<0.01. ab apical bud, LA labial side, LI lingual side, OEE outer enamel epithelium, BE basal epithelium, TA transit-amplifying cells, SR stellate reticulum

CXCR4 ^−/− mutant incisors contain epithelial cell aggregations in the apical bud. a–e CXCR4 ^−/− and CXCR4^+/+ heads, mandibulars and incisor germs. f Relative length of the lower incisors of CXCR4 ^−/− and CXCR4^+/+ mice (**p<0.01). g–n Hematoxylin and eosin staining of mandibular incisor sections from E18.5 CXCR4^−/− and CXCR4^+/+ mouse incisors. Boxes in (g, j) are shown in higher magnification in subsequent panels (h, k). i A transverse section of a CXCR4 ^−/− apical bud and l controls. Epithelial cell aggregation was observed in CXCR4 ^−/− apical buds (h arrow, i). Scale bars (a, b) 500 μm, (d, g) 100 μm, (h, i) 50 μm (m) 20 μm. IEE inner enamel epithelium, BE basal epithelium, SR stellate reticulum, OEE outer enamel epithelium, DP dental pulp, Od odontoblasts, De dentin, Am ameloblasts

CXCR4/CXCL12 signaling promotes dental epithelial cell morphology and motility. a–c Phase contrast images show mHAT9a cells in CXCL12-treated, untreated (control) and Fgf2-treated culture after 24 h. Spreading cells were present around the colonies within the CXCL12-containing medium (a), while not significant in control medium (b, c). d Expression of CXCR4 mRNA in mHAT9a cells transfected with vectors expressing either CXCR4 shRNA or negative-control shRNA. e–g Phase-contrast images. CXCR4 KD cells formed dense colonies (e). Spreading cells were present around the margins of control colonies (f, arrows). CXCL12-treated CXCR4 KD cells formed dense colonies (g). h CXCL12 significantly induced migration by mHAT9a cells after 24 h incubation. i The motility of CXCR4 KD cells was suppressed. Data are expressed as mean ± SEM; n=3. Significant differences between samples were determined by Tukey’s test. **p<0.01. *p<0.05. Scale bars 50 μm. NC mHAT9a cells expressing negative-control shRNA, KD mHAT9a cells expressing CXCR4 shRNA

Epithelial cell proliferation is suppressed in the CXCR4 mutant apical bud. a CXCR4/Ki-67 (i.e., proliferating) double staining of the apical bud. Arrows indicate CXCR4/Ki-67-double-positive cells in the TA. Arrowheads indicate the double-positive cells in the SR. b, c Ki-67 staining of CXCR4 ^−/− apical bud (b) and control (c). Arrows indicate Ki-67-positive cells in the TA. Arrowheads indicate Ki-67-positive cells in the SR. d In E18.5 CXCR4^−/− mutant mice, Ki-67-positive cell number was significantly reduced in the apical bud. e In CXCR4 KD mHAT9a cells, proliferation was inhibited, but the impact of CXCL12 was small. Scale bar 50 μm. Data are expressed as mean ± SEM, n=3 (b) and n=4 (c). Significant differences between samples were determined by Tukey’s test. **p<0.01