Figure 2.

Ameloblasts and stratum intermedium cells initially differentiate in Msx2^−/−. Genotypes are indicated. Sagittally sectioned upper incisors at P9 (incisal edge side to right). (A–D, K, L) In situ hybridization. (E–J, M–X) Immunohistochemistry. (A, B) Sonic hedgehog (Shh) expression is detected in preameloblasts (pAB) in both genotypes. (C–J) Enamel proteins such as Ameloblastin (Ambn), Amelogenin (Amel), and Enamelin (Enam) are expressed in ameloblasts (AB) at the secretory stage in both genotypes. (K, L) Msx1 is not significantly upregulated in Msx2^−/−. (M–V) Immunoreactivity for Sox2 and Notch1 is positive in the apical bud (ApB, arrow in M, N) and stratum intermedium (SI) cells (arrows in O, P, S, T) adjacent to pAB in both genotypes. Compared with wild-type (WT), SI cells neighboring AB partially lose immunoreactivity for Sox2 and Notch1 in Msx2^−/− (asterisks in R, V), and some Sox2-positive cells detach from the original layer (arrows in R). Note that the outer enamel epithelium (OEE) is already expanded when SI cells are normally differentiated in Msx2^−/− (double-headed arrow in P, T). (W, X) Compared with WT, desmosome marker Desmoplakin is partially attenuated between AB and SI cells of Msx2^−/− (asterisks in X). D, dentin; DF, dental follicle; DP, dental pulp; OB, odontoblast. Bars: 25 µm in A for A–L, 50 µm in M for M and N, and 25 µm in O for O–X.