Fig. 5. Localization of proliferating cells at the initiation of HERS formation. BrdU labeling analysis of mutant incisors grown under kidney capsules (A-C) and of the formation of HERS in in vitro cultures (D,E). (A-C) Small amounts of labels are detected in the dental epithelium and in the surrounding mesenchyme at the apical end. (B,C) Higher magnification of BrdU immunostaining at the labial and lingual sides. BrdU-positive cells are visible in the outer layer (arrowheads) and in the inner layer (red arrows) of the epithelial sheath. Black arrows indicate BrdU-positive papilla cells. An asterisk indicates artificial space separating the inner and outer layers of the epithelial sheath formed when making sections. (D,E) BrdU staining of the mouse molar germ cultures at PN d3 and d5. The number of BrdU-positive cells (red arrows) in the IEE and the inner layer of HERS is much smaller than the number of positive cells in the OEE and the outer layer (arrowheads). (F,G) BrdU-labeled cells in the inner and outer layers and dental papilla cells in mutant incisors (F), and in vitro cultures of molar germs of PN d3 and d5 (G). More BrdU-positive cells are visible in the outer layer than in the inner layer on either side (F). Comparing the OEE/stellate reticulum (sr) and the IEE in molar germ at PN d3, or comparing the outer and inner layers at PN d5, showed similar results as those found in the mutant incisors. The total number of BrdU-positive cells increased at PN d5. Data are presented as mean±s.d. Significant differences in the mitotic indices between two groups (with error bars) were calculated using a Student's t-test (asterisks indicate P<0.01). The broken red line indicates the border between the epithelium and the mesenchyme, which is identified by the CK14 immunostaining of serial sections. Scale bars: 100 µm in A; 50 µm in B-E.