Figure 2. Several signaling factors are mutually coordinated in HERS development. (a) At PN10, an immunopositive reaction for PCNA was observed, not only in many HERS cells, but also in the cells of the dental pulp and dental follicle. (b) At PN18, some HERS cells showed an immunopositive reaction. However, the number of immunopositive cells decreased compared with that at PN10. Moreover, clusters of 3-4 PCNA-positive cells could be seen in the HERS. (c) At PN10, no TUNEL-positive cells could be detected in the HERS. In the dental pulp and dental follicle, a few cells showed a positive reaction. (d) At PN18, although the numbers of IEE and OEE cells decreased dramatically, a small number of HERS cells showed a TUNEL-positive reaction. (e) In the underside view of the specimen, DiI labeling could be observed on the base (orange dotted line) and end (yellow dotted line) of the HERS (arrows) after 3 days of culture. (f) In this section, the HERS is indicated as a yellow dotted line. The DiI-labeled cells did not move during tooth root formation. The blue line shows dentin. (g) At PN10, an immunopositive reaction for laminin beta-3 localized in HERS cells. The dental pulp and dental follicle cells show a negative reaction. (h) At PN10, syndecan-1 immunoreactivity could be observed in the HERS cells, although the cells of the dental pulp and dental follicle did not show a positive reaction. (i) Bmp-2 was detected in the HERS at PN10. Positive signals were also observed in the cells of the dental pulp and dental follicle. (j) At PN10, Bmp-4 was expressed in the HERS. However, the expression was weak compared with that of the Bmp-2. (k) Compared with the mesenchyme, there was strong expression for Msx-2 in the HERS. Black dotted line, HERS; F, dental follicle, P, dental pulp. Bars: a-d, 15 µm; e, 100 µm; f, 50 µm; g-k, 15 µm.