Differential expression between "DSP-only" and DSP-PP523 transcripts in rat molar teeth

Abstract

Objective: To compare the expression patterns of two multiple transcripts derived from DSP-PP gene during tooth development. One is DSP-only transcript (i.e. does not encode PP) and the other is DSP-PP₅₂₃ transcript, a main DSP-PP transcript.

Design: Unique antisense and sense riboprobes were generated from DSP-only and DSPPP₅₂₃ cDNAs for in situ studies to examine DSP-only and DSP-PP₅₂₃ transcript expression in developing molars. Paraffin-embedded sections (5-7μ m) from embryonic 20day, postnatal 2, 3 and 6days were deparaffined and hydrated. Tissues were prehybridized, then hybridized with DSP-only and DSP-PP₅₂₃ anti-sense (AS) or sense (S) Digoxigenin labeled-riboprobes overnight, and washed. Anti-Digoxigenin antibodies conjugated to alkaline phosphatase were used to detect the presence of bound riboprobes by color reaction with NBT/BCIP. Stro-1 antibody was used for immunohistochemical analysis of Stro-1 protein expression in rat molars.

Results: We found that unlike the DSP-PP₅₂₃ transcript, the DSP-only transcript does not express in the entire polarized mature odontoblasts but is expressed in the areas subjacent to the mature odontoblast layer. In addition, DSP-only transcript is expressed in the dental pulp. Interestingly, Stro-1 protein, a stem cell marker, was also identified in the areas subjacentto odontoblasts and in dental pulp.

Conclusion: Differential expression of DSP-only and DSP-PP₅₂₃ transcripts suggest that these two kinds of transcripts may play different roles during dentinogenesis. DSP-PP₅₂₃ transcript is expressed in mature odontoblasts, which actively participates in dentin formation. DSP-only transcript might have a different function.

Keywords: DSP-PP(523) transcript; DSP-only transcript; Differential expression; a cell layer subjacent to odontoblast layer.

Fig. 1.

Diagram of rat DSP-PP₅₂₃ and DSP-only transcripts and riboprobe generation. A. The DSP-PP₅₂₃ cDNA is comprised of a 17 amino acid Leader sequence (designated as L), DSP₄₃₀, PP₅₂₃ coding sequences and 3′ noncoding sequence (). DSP-only cDNA is comprised of a Leader sequence (17 amino acids; L), DSP₃₀₇ and a short 3′noncoding sequence (). B. Sequence homology comparison between DSP-PP₅₂₃ and DSP-only cDNAs. 286 amino acids are identical to the published rat DSP₄₃₀ sequence from DSP-PP₅₂₃ cDNA. The BglI site (nucleotide position 825) is indicated by an arrow (↓) in both DSP-PP523 and DSP-only cDNA. The DSPonly sequence shares sequence homology () with the 3′ noncoding sequence of DSP-PP₅₂₃ (as shown by connected brackets).

Fig. 2.

No DSP-PP523 and DSP-only transcript expression in molar teeth of 20-day-old rat embryo. A: Hematoxylin & Eosin staining of embryonic 20 day molar. B: DSPPP523-AS riboprobe detected no DSP-PP523 transcript expression in 20 day rat embryonic molar. C:DSP-only unique AS riboprobe detected no DSP-only transcript expression in rat embryonic 20 day molar.

Fig. 3.

DSP-PP₅₂₃ and DSP-only transcript expression pattern in molar teeth of postnatal 3 day molars. Panels A-C represent DSP-PP₅₂₃ transcript expression in molar tooth. A. In situ hybridization with DSP-PP₅₂₃ AS riboprobe. 100× magnification. B. Enlarged white left box (from Fig. 3A). C. Higher magnification of white right box (from Fig. 3A). A big arrow represents the area of preodontoblasts. Panels D-F represent DSP-only transcript expression in molar tooth. D. In situ hybridization with DSPonly AS riboprobe shows that the DSP-only transcript is expressed at the base of the odontoblasts and in the dental pulp. E. Higher magnification of left box (from Fig. 3D). F. Higher magnification of right box (from Fig. 3D). Small arrows represent DSP-only mRNA expression in dental pulp in Fig. 3E and F. A big arrow represents the area of preodontoblasts (Fig. 3F). G. In situ hybridization with DSP-PP₅₂₃ S riboprobe: no signal. H. In situ hybridization with DSP-only S riboprobe: no signal.

Fig. 4.

DSP-PP₅₂₃ transcript and Stro-1 protein expression in postnatal 6 day molar tooth in rat. A. H & E staining of 6 day molar tooth. B. DSP-PP₅₂₃ mRNA expression in preameloblasts and odontoblasts as well as in osteoblasts (indicated by arrows). C. Strol-1 protein expression in day 6 molar. Arrows (→) represent Stro-1 positive cells in dental pulp. Harrow arrows () represent Stro-1 positive staining at the base areas of odontoblasts. AB: alveolar bones. A: ameloblasts. DP: dental pulp. O: odontoblasts. PA: preameloblasts. D. Only the secondary antibody (mouse primary IgM antibodies to human Stro-1) was applied to the tissues as a negative control.

Fig. 5.

Stro-1 staining of postnatal 6 day rat molar. A. H & E staining of 6 day first molar. B. Stro-1 antibodies were used for immunohistochemical analysis. Stro-1 was detected in the base of the odontoblasts (as indicated by arrows) and in the dental pulp (represented by arrow heads). White color dash line represents the border between odontoblast layer and dental pulp. C. Stro-1 and Hematoxylin staining of 6 day first molar. Stro-1 and Hematoxylin stained sections clearly show the odontoblasts and the Stro-1 positive staining is located at the base of the odontoblasts. 1000× magnification. DP: dental pulp. O: odontoblasts. D: dentin. E: enamel. D. Only the secondary antibody (mouse primary IgM antibodies to human Stro-1) was applied to the tissues as a negative control.