PERP is expressed in developing teeth and is regulated by P63. (A,G) Cartoon of the cap and bell stages during molar development. At the cap stage, the enamel organ (EO) forms and surrounds the dental papilla (DP). At the bell stage, the EO begins to differentiate into the inner enamel epithelium (IEE), which generates the future ameloblasts, as well as the stratum intermedium (SI), stellate reticulum (SR), and the outer enamel epithelium (OEE). (B,H) H&E staining of coronal sections of molars at E14.5 and E16.5. (C–E,I–K) Double immunofluorescence staining and merge of PERP and desmoplakin (DSP). Higher magnification images show colocalization of PERP and DSP in the EO at E14.5 (C′–E′) and in the SI, SR and IEE at E16.5 (I′–K′). (F,F′,L,L′) Immunofluorescence staining of P63 shows overlapping expression profile with that of PERP and DSP. (M) Luciferase assays in ameloblast-like LS8 cell line using empty vector (vector), the Perp promoter and intron containing a P63-responsive element (PerpLuc-wt), or a construct with a mutated P63-responsive element (PerpLuc-mut). (M′) Inset shows the endogenous expression of Tp63 and Perp in LS8 cells detected by PCR with (+RT) or without (−RT) reverse transcription. (N) qPCR analysis showing relative expression levels of the various P63 isoforms, N-terminal transcriptional activation (TAp63), N-terminal truncated (ΔNP63) or pan p63. **P<0.01.

PERP localization in developing incisors at P7. (A) H&E staining of the lower incisor and mandible in sagittal section. (A′) Cartoon of the mandible. (B–E) H&E staining of the lower incisor. (F–I) PERP immunofluorescence staining at presecretory, secretory, transitional and mature stages. (J–M) DSP immunofluorescence staining during amelogenesis. (N–Y) High-magnification views of double immunofluorescence staining of PERP and DSP show colocalization at ameloblast–SI interface. En, enamel; Am, ameloblasts; SI, stratum intermedium.

SEM analysis of teeth at P2. (A,C) Low-magnification images of the enamel surface of the first lower molars from wild-type (Perp^+/+) and Perp-null (Perp^−/−) mice. (B,D) Higher-magnification images of the boxed areas.

H&E staining of developing mandibular incisors and maxillary molars in wild-type (Perp^+/+) and Perp-null (Perp^−/−) mice at P7 in sagittal sections. (A–D) Low-magnification images of the lower incisor and 1st upper molar. (A′–D′) Higher-magnification images of the boxed areas. Dentin (Dn) and enamel (En) are denoted by yellow and red arrowheads, respectively. Areas of defective enamel are denoted by red asterisks in the incisors and molars of Perp-null mice. P, proximal; D, distal.

Displacement of ameloblasts from the SI. (A,B) H&E staining of the transitional stage in sagittal sections of incisors at P7. Detached cells between ameloblasts and the enamel matrix in Perp-null mice are indicated (red arrowheads). (C,D) DAPI staining of adjacent sections shows the presence of the detached cells (red arrowheads). (E–H) Detached cells express ameloblast-specific proteins such as ameloblastin (AMBN, red arrowheads) and amelogenin (AMEL; red arrowheads). Matrix vesicles present in both wild-type and Perp-null teeth are indicated (yellow arrowheads). En, enamel; Am, ameloblasts; SI, stratum intermedium.

Transmission electron microscope (TEM) analysis of desmosomes at the ameloblast-SI interface in wild-type (Perp^+/+) and Perp-null (Perp^−/−) mice at P2. (A–D) Native (A,B) and contrast-enhanced (C,D) TEM images show greater separation between the secretory ameloblasts and the SI (yellow arrowheads) and between the cells in the SI (red arrowheads), but not between ameloblasts (blue arrowheads), in Perp-null mice compared with the wild type. (E,F) Representative desmosomes from wild-type and Perp-null mice. (G) Reduced number and size of desmosomes at the ameloblast–SI interface in Perp-null mice compared with the wild type. Am, ameloblasts; SI, stratum intermedium. **P<0.01.

Relative expression of genes involved in amelogenesis. (A) Heat map depiction of candidate genes involved in amelogenesis from the microarray comparison of first lower molars of wild-type and Perp-null mice at P0. Percentage expression changes relative to wild-type are displayed in brackets and any changes >1.75-fold are denoted with an asterisk (*). Red and green represent high and low expression, respectively. (B) qPCR analysis shows significant downregulation of Ambn, Enam, Mmp20, Klk4 and Perp in teeth of Perp-null mice, whereas Amel, Tuft1 and Odam are unaffected. No or little expression of Amtn is shown by microarray and qPCR analyses. Ambn, ameloblastin; Amel, amelogenin; Enam, enamelin; Tuft1, tuftelin 1; Odam, odontogenic ameloblast-associated (or Apin); Mmp20, matrix metalloproteinase 20, Klk4, kallikrein 4; Amtn, amelotin. **P<0.01.

Knockdown of PERP in LS8 cells using siRNA. (A) The knockdown of PERP with siRNA (PERPkd) relative to scrambled control was demonstrated by western blot analysis. (B) Ambn, Amel, Enam, Klk4 and Perp were significantly downregulated in PERPkd LS8 cells. There was little or no expression of Odam and Amtn. qPCR results were normalized to Gapdh as an internal control and expression levels are relative to scrambled controls. **P<0.01.

Identification of additional genes regulated by PERP. (A) qPCR was used to confirm differentially expressed genes determined by microarray analysis. Dmkn, Krt83, Sost and Lama2 were significantly downregulated in teeth of Perp-null mice. (B) Dmkn and Sost were also downregulated in LS8 cells with PERP knockdown (PERPkd), whereas Krt83 and Lama2 were unchanged. qPCR results were normalized to Gapdh as an internal control and expression levels are relative to wild-type or scrambled controls. Dmkn, dermokine; Krt83, keratin-83, Sost, sclerostin; Lama2, laminin α2. **P<0.01.