In the rat incisor, the layer of ameloblasts contains the entire sequence of the cell developmental stages. From the apical toward the incisal end these stages are classified regionally into presecretory, secretory, transition, and maturation. Secretory ameloblasts produce enamel matrix proteins such as amelogenin, and at the maturation stage the ameloblasts reabsorb the matrix protein and transport calcium to the maturing enamel. In addition to their fundamental role in enamel formation, the rat incisor ameloblasts incorporate iron and deposite it into the surface layer of the mature enamel.

All living cells require iron for various biological reactions, particularly those involving redox regulation, but iron itself is a potentially toxic oxidant which can cause severe cellular damage. Intracellularly, therefore, iron must be detoxified and its bioavailability controlled by a metal-binding protein such as ferritin. In rat ameloblasts, electron-microscopic studies have revealed that iron-containing pigment accumulates in the form of ferritin particles during the maturation stage. The particles first appear free in the cytoplasm, then gradually become confined to the ferritin-containing vesicles with the progression of the cell developmental stages. Finally, the iron is secreted from the ameloblasts to the enamel at the end of maturation, presumably through the process of lysosomal digestion of ferritin.

The amount of ferritin protein in cells is under the stringent regulation of translation in response to the cellular iron state such that the iron supply significantly enhances the biosynthesis of ferritin. In ameloblasts, iron is incorporated into the cells at the very early maturation stage. Specifically, iron entry and the appearance of ferritin particles are clearly correlated throughout the developmental course of ameloblasts.

As preparation for the rapid increase in the amount of ferritin in response to the rise in cellular iron content, ferritin mRNA must preaccumulate in the cells before iron entry. We have now examined the timing of ferritin mRNA expression during the development of ameloblasts by in situ hybridization and RT-PCR. The mRNA expression pattern was compared with the localization of its translation products as detected immunohistochemically.

Male Wistar rats (5 weeks old) were fed standard laboratory chow (Oriental Yeast, Tokyo, Japan) and given tap water ad libitum. The maxillary incisors were dissected by the method of Josephsen (1974). The enamel organ on the tooth surface was scraped out with a scalpel and immediately frozen in liquid nitrogen. Enamel organs from eight maxillary incisors (total wet wt, about 100 mg) were combined for use for total RNA isolation or for protein extraction. The organ is composed of a layer of columnar ameloblasts, a single layer of stratum intermedium cells, papillary layer cells, and a few mesenchymal cells. For regional fractionation, three segments of each enamel organ were separately collected as described in Fig. 5(a). Each length was measured with dial calipers. For each analysis, the segmental enamel organs from eight maxillary incisors were collected separately. The approximate wet weight of the prepared segments 1, 2 and 3 [see Fig. 5(a) below] was 40, 40 and 20 mg, respectively.

Fig. 5. RT-PCR for detection of ferritin mRNA in the three segments of the enamel organ. (a) Diagram of rat maxillary incisor showing the regional segmentation of the enamel organ for total RNA isolation. Segment 1, 0–5 mm from the apical end of the incisor; segment 2, 5–10 mm from the apical end; and segment 3, 10 mm from the apical end to the incisal end of the enamel organ. (b) Ethidium bromide staining of ferritin/GAPDH and amelogenin RT-PCR products obtained from the total RNA in each of the three segments of the enamel organ.

A piece of each rat tissue (calvaria, kidney, thymus, liver, heart, and spleen) was excised and immediately frozen in liquid nitrogen. The total RNA from each tissue and enamel organ was isolated with a guanidine/isothiocyanate solution, TRIzol (Gibco BRL, Tokyo, Japan), according to the manufacturer’s instruction. The total RNA (15 ng) was reverse-transcribed into cDNA and amplified in a final volume of 50 ?l using the RNA PCR kit (TaKaRa, Osaka, Japan) with specific primer sets. The primers were: ferritin-H sense primer, 5?-ATGACCACCGCGTCTCC-3? (bases 169–186 in exon 1; Murray et al., 1987); ferritin-H antisense primer, 5?-TAGCTCTCATCA CCGTGTC-3? (bases 1047–1066 in exon 4); ferritin-L sense primer, 5?-ATGACCTCTCAGATTCGT-3? (bases 1–18 in cDNA; Leibold et al., 1984); ferritin-L antisense primer, 5?-GTCGTGCTTCAGAGTGAG-3? (bases 529–546 in cDNA); amelogenin sense primer, 5?-TCTTGTTTGCCTGCCTCCTG-3? (bases 39–58 in cDNA; Bonass et al., 1994); amelogenin antisense primer, 5?-CTTGGTCTTGTCTGTCGCTG-3? (bases 579–598 in cDNA); GAPDH sense primer, 5?-ATGTCGTGGAGTCTACTGGC-3? (bases 346–365 in cDNA; Fort et al., 1985); and GAPDH antisense primer, 5?-TGACCTTGCCCACAGCCTTG-3? (bases 707–726 in cDNA). Ex Taq (TaKaRa), a Taq polymerase with high fidelity, was used at the PCR step. One cycle of PCR consisted of 1 min at 94°C, 1 min at 62°C, and 1 min at 72°C; a total of 21 cycles was performed using a programmed temperature-control system (TaKaRa PCR Thermal Cycler MP).

The PCR products were subjected to 1.5% agarose gel electrophoresis and stained with 1 ?g/ml ethidium bromide for 5 min at room temperature. The DNA bands in the gel were quantified with a fluorescent densitometer, equipped with a Cooled CCD camera (Series 200, Photometrics, AZ) and IP Lab gel software (Signal, VA), essentially following the method described by Wang et al. (1989). The RT-PCR products of ferritin-H (543 bp), ferritin-L (546 bp), GAPDH (381 bp) and amelogenin (560 bp) from liver or enamel organ were gel- purified and cloned into the pBluescript KS^± vector (Stratagene, CA). The identity of each PCR product was confirmed by DNA sequencing. The sequence of ferritin-H or -L PCR product corresponds to the entire coding region for each protein, while that of GAPDH or amelogenin PCR product corresponds to the middle part of the respective cDNA.

BamHI sequence was attached at the 5? end of each ferritin-H and -L cDNA, and HindIII sequence was attached at the 3? end of the cDNA, by a PCR-mediated standard DNA manipulation. Each construct was inserted into the BamHI/HindIII site of the His6-tag-introducing vector, pQE9 (Quiagen, CA) to produce recombinant histidine-tagged ferritin H-chain (His6–ferritin H) and L-chain (His6–ferritin L). As a result, the amino acid sequence flanked to the authentic N-terminal polypeptide was the following: Met-Arg-Gly-Ser-His-His-His-His-His-His-Gly-Ser. The estimated molecular weight of each His6–ferritin H and His6–ferritin L was 22,380 and 21,370 Da, respectively. The recombinant proteins were produced in M15 strain Escherichia coli and purified with Ni²⁺·nitrilo-tri-acetic acid resin (Quiagen, CA).

Each ferritin-H and ferritin-L cDNA flanked by BamHI and HindIII sites was subcloned into the pBluescript KS⁺ vector and used as a template for cRNA production. DIG-11-UTP-labelled single-strand antisense and sense RNA probes were prepared with a DIG-RNA Labelling Kit (Boehringer Mannheim, Germany), using either T7 or T3 RNA polymerase according to the manufacturer’s instruction. The specificity of these probes was confirmed by filter hybridization. Each probe reacts only with the corresponding reverse-strand RNA (data not shown).

Rats were killed under ether anaesthesia and fixed with 4% paraformaldehyde in 0.1 M sodium phosphate (pH 7.4)/150 mM NaCl by cardiac perfusion. The maxilla was excised and further fixed overnight at 4°C. After being demineralized with 10% EDTA in diethylpyrocarbonate-treated H₂O (pH 7.4) at 4°C for 10 days, these samples were embedded in paraffin and sectioned (5 ?m thickness).