Advances in Dental Research

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Expression Patterns of RNAs for Amelin and Amelogenin in Developing Rat Molars and Incisors C.D. Fong, L. Hammarström, C. Lundmark, T. Wurtz and I. Slaby ADR 1996 10: 195 DOI: 10.1177/08959374960100021301 The online version of this article can be found at: http://adr.sagepub.com/content/10/2/195

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EXPRESSION PATTERNS OF R N A S FOR AMELIN AND AMELOGENIN IN DEVELOPING RAT MOLARS AND INCISORS

I

C D . FONG L. HAMMARSTROM

C. LUNDMARK T. WURTZ I. SLABY

Karolinska Institute Center for Oral Biology P.O. Box 4064 S-14104 Huddinge, Sweden Adv Dent Res 10(2): 195-200, November, 1996

Abstract—We have recently identified a novel RNA sequence in ameloblasts, coding for amelin (Cerny et al, 1996). In the present paper, its expression has been compared with that of amelogenin in developing incisors and molars of rats, by means of in situ hybridization of paraffin sections. The RNAs for both amelin and amelogenin were highly expressed in secretory ameloblasts. The expression of RNA for amelogenin gradually decreased in the post-secretory ameloblasts. In contrast, the RNA expression for amelin remained high in post-secretory ameloblasts up to the stage of fusion between dental and oral epithelia at the time of tooth eruption. We suggest that amelin might be involved in the mineralization of enamel or in the attachment of ameloblasts to the enamel surface. The whole-mount in situ hybridization procedure is described for the first time in dental research. It proved to be a useful method and confirmed the results of the conventional in situ hybridization.

Key words: Amelin, amelogenin, ameloblasts, in situ hybridization, whole-mount in situ hybridization.

nteractions between mesenchymal and epithelial cells are guiding principles of tooth crown formation and lead to the final differentiation into the extracellular matrixsecreting cell types, odontoblasts and ameloblasts. The respective matrices, dentin and enamel, are different both in structure and in the mechanism of mineralization. Dentin matrix has a distinct developmental stage of non-mineralized fibrous predentin, where the predominant protein is collagen type I. The mineral is deposited later. On the other hand, the major constituent of enamel matrix is a mixture of amelogenin peptides (Eisenmann, 1994). Amelogenin protein is synthesized from a group of differentially spliced RNAs and also degraded to a variable extent during the maturation of the enamel matrix (Gibson et al., 1991; Lau et ai, 1992). Both effects render amelogenin highly heterogeneous. Newly deposited enamel already contains about 15% hydroxyapatite by weight. During the course of enamel maturation, the amount of enamel proteins and water reduces considerably, whereas the mineral content increases (Robinson et al., 1988). So far, no structural enamel matrix protein with cellanchoring properties has been identified. In the search for new tooth proteins, we have been studying gene expression in matrix-forming cells by in situ hybridization. We constructed a cDNA library from developing rat teeth and screened for frequently occurring sequences by hybridization of plaque lifts with cDNA from molars of four-day-old rats. The screening resulted in a finding of several novel sequences not represented in databases (GenBank and EMBL). One of those was highly expressed in ameloblasts. Its encoded protein, which we named amelin, had the characteristic properties for extracellular localization (Cerny et al., 1996). In the present paper, we compare the amelin and amelogenin expression patterns in growing rat molars and incisors by in situ hybridization. Also, we introduce the whole-mount in situ hybridization technique into dental research. By this method, the hybridization signal of the whole explant can be inspected at once. Reactions in groups of cells can be observed without the need for serial sections. MATERIALS AND METHODS

Presented at the Third Carolina Conference on Tooth Enamel Formation, held October 18-20, 1995, at the University of North Carolina, Chapel Hill.

Synthesis of probes DIG-labeled RNA probes complementary to the RNA sequences of amelin and amelogenin were synthesized in vitro. A probe with the same sequence as amelogenin RNA served as control. The DNA templates were obtained by restriction enzyme cleavage of Bluescript SK plasmids (Stratagene, La Jolla, CA, USA). The synthesis of probes has been described in detail (Cerny et al., 1996).


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Fig. I—An illustration of a rat skull to outline the areas examined in Figs. 2-6. (A) This area was used in the molar sections for comparison of the expression patterns ofRNAs during different developing stages in Figs. 2-4. (B) This area was used in the incisor section in Fig. 5. (C) The apical end of the mandibular incisor in area C was dissected for wholemount in situ hybridization. Regular in situ hybridization

Preparation of sections. To examine different development stages of the tooth, we included Sprague-Dawley rats (B & K Universal, Sollentuna, Sweden) aged 2, 5, 10, and 15 days. The upper jaws of the rats were isolated and fixed with newly prepared 4% paraformaldehyde in PBS (phosphate-buffered saline, 137 mM NaCl, 2.7 mM KC1, 4.3 mM Na 2 HPO 4 , 1.4 mM KH2PO4) for 24 hrs at 4°C. For the rats aged 10 days and above, the teeth were decalcifed in EDTA (144 g EDTA dissolved in 1 liter of de-ionized sterilized water, adjusted with NaOH to pH 7.0). The effect of decalcification was monitored by radiographic examination. Specimens were then dehydrated and embedded in paraffin. Longitudinal sections were cut through the first molars or through the midline of the incisor and mounted on Vectabond-coated (Vector, Burlingame, CA, USA) slides. The section thickness was 7 jam. The slides were stored at 4°C.

ADV DENT RES NOVEMBER 1996

placed on top of the liquid. The slides were placed in a humid chamber overnight at 42°C. Post-hybridization stringency washes were performed with decreasing salt concentrations at room temperature. It started with the rinsing with 4 x SSC, followed by washes with 2 x SSC four times for 10 min, then 0.1 x SSC four times for 10 min. The hybridized probe was detected by means of the DIG nucleic acid detection kit (Boehringer Mannheim). An anti-digoxigenin antibodyalkaline phosphatase complex was bound to the hybridized probe and revealed by the phosphatase reaction in the presence of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt, to form a blue precipitate over the hybridized RNA. Whole-mount in situ hybridization

Tissue preparation. The most apical thirds of the mandibular incisors of two 25day-old female Sprague-Dawley rats were carefully dissected. The apical ends of the teeth were kept untouched. The explants were fixed with newly prepared 4% paraformaldehyde in PBS for 24 hrs at 4°C. The fixative was removed by immersion in PBS for 1 hr. The PBS was then replaced with PBT (PBS with 0.1% Triton X-100, SIGMA) for 2 hrs. The explants were dehydrated with ascending proportions of ethanol in PBT and stored at -20°C.

Pre-hybridization treatments. The tooth explants were rehydrated with descending concentrations of ethanol in PBT, incubated with proteinase K (20 ug/mL) at 37°C for 30 min, transferred to glycin (0.2%), and washed 5x for 5 min in PBT. Post-fixation was performed with 4% paraformaldehyde in PBT for 20 min. The fixative was washed away 3 x 10 min with PBT. The PBT was then replaced with hybridization mixture (50% deionized formamide, 5 x SSC, 0.2 mg/mL of boiled sonicated salmon testis DNA, 0.1 mg/mL tRNA, adjusted to pH 5.0 by the addition of HC1) at 42°C overnight.

Hybridization. Pre-hybridization treatments. The paraffin was removed with xylene. The sections were rehydrated with descending concentrations of ethanol, rinsed in PBS, treated with proteinase K (20 jig/mL, SIGMA, St. Louis, MO, USA) at 37°C for 30 min, and then rinsed in 0.2% glycin. Sections were post-fixed with 4% paraformaldehyde for 5 min, submerged in triethanolamine and acetic anhydride, rinsed with PBS, and pre-hybridized in 2 x SSC (150 mM NaCl, 15 mM sodium citrate as 1 x SSC) with 50% de-ionized formamide at 42°C for one hr. Hybridization. Pre-hybridization solution was replaced by 20 uL of hybridization buffer [0.3 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, lx Denhardt's reagent (Watkins, 1994), 0.1 g/L dextrane sulphate (SIGMA, St. Louis, MO, USA), 50% formamide] containing 0.5 ng/pL RNA probe. Coverslips treated with aminopropyl triethoxysilane (SIGMA) were

Digoxigenin-labeled amelin and amelogenin RNA probes were added to a fresh hybridization mixture at a final concentration of 1 ng/uL. Hybridization was performed at 42°C overnight. For post-hybridization washes, 5 x SSC, containing 0.1% Triton-X 100, 50% formamide was mixed with 2 x SSC containing 0.1% Triton-X 100. Subsequent washes contained descending concentrations of the first component and increasing concentrations of the second. The explants were immersed in 2 x SSC and then in 0.2 x SSC, containing 0.1% Triton-X 100, 3x for 20 min each. Subsequently, the explants were transferred to 100 mM TrisHCl, pH 7.5, 150 mM NaCl, and 0.1% Triton-X 100 for 10 min. The explants were incubated for 3 hrs at room temperature with blocking solution (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton-X 100, 1% blocking powder; Boehringer Mannheim). An anti-digoxigenin alkaline phosphatase antibody was used to detect the hybridized probe as described for the regular in situ hybridizations.


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Fig. 2—Neighboring sagittal sections through the first and second molars of a five-day-old rat showing the expression of RNA for amelogenin (a) and amelin (b). The blue staining shows the location of the hybridized probe. At this age, all ameloblasts in the first and second molars are at the secretory stage, and both RNAs are expressed in these cells. M 1 = first molar; M2 second molar; scale bar - 200 urn. Sections. Serial longitudinal sections through the midline of the incisors and serial cross-sections were made. The section thickness was 7 |jm.

RESULTS Fig. 1 illustrates the parts of the rat skull which were analyzed by in situ hybridization. Sections through the molars were performed from an area of the upper jaw (A). The incisor sections used for conventional in situ hybridization were taken from area (B). The explants for whole-mount in situ hybridization were dissected from area (C). The RNAs for both amelogenin and amelin were expressed in the ameloblasts. The highest signal of amelogenin RNA was observed in the secretory ameloblasts and then gradually decreased in the post-secretory ameloblasts to a level no longer demonstrable prior to the eruption of the tooth. On the other hand, the RNA for amelin was expressed at about the same level in the secretory and post-secretory ameloblasts up to the time of tooth eruption. The results of the whole-mount in situ hybridization were essentially the same as those obtained with regular in situ hybridization. In the molars of two- (not shown) and five-day-old rats, the signal for both amelin and amelogenin RNAs appeared in the ameloblasts at about the time of the start of enamel matrix formation (Fig. 2). In the molars of 10-day-old rats, the signal for the amelogenin RNAs was highly expressed in the secretory ameloblasts and to a lower degree in the post-secretory stage. There was no dramatic change of expression at the end of the secretory stage, but there was a gradual decrease. Also, the RNA for amelin was highly expressed in the secretory ameloblasts. In contrast to amelogenin, this level of expression also remained essentially unchanged in the postsecretory ameloblasts (Fig. 3). In the molars of 15-day-old rats, the amelogenin RNA was

highly expressed in the secretory ameloblasts which, in Fig. 4, are confined in the cervical third of the second molar. The expression gradually decreased to a non-demonstrable level in the ameloblasts covering the cusps of the first molar. The expression of RNA for amelin remained unchanged up to the fusion between the oral and dental epithelia at the time of eruption (Fig. 4). The patterns of RNA expression in the incisors for amelogenin and amelin, respectively, were the same as in the molars at all ages studied. Thus, the RNA for amelogenin was highly expressed in secretory ameloblasts. The expression then gradually decreased after the cells had reached the post-secretory stage. No RNA for amelogenin was demonstrable in the late post-secretory stage. The RNA for amelin was highly expressed in both secretory and postsecretory ameloblasts. The expression remained high in all cells until the dental epithelium fused with the oral epithelium (Fig. 5). The whole-mount studies showed a rather homogeneous distribution of the RNAs of amelogenin (Fig. 6) and amelin (not shown) in the secretory ameloblasts, with an intense staining at the labial side of the incisor. The examination of the sections made from the explant confirmed the results of the conventional in situ hybridization.

DISCUSSION The differentiation of the cells of the inner enamel epithelium is guided at the outset by the interaction with mesenchymal cells. Later, the inner epithelial cells become secretory ameloblasts and express RNA for amelogenin. The peripheral mesenchymal cells facing the ameloblastic layer give rise to odontoblast-synthesizing collagen. During the maturation stage of the enamel matrix, the ameloblasts reduce their content of amelogenin RNA substantially. Amelin RNA appears in in situ hybridization experiments concomitantly with amelogenin RNA, i.e., during the


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Fig. 3—Neighboring sagittal sections through the first and second molars of a 10-day-old rat showing the expression of RNA for amelogenin (a) and amelin (b). At this age, the ameloblasts in the cervical third of the first molar and cervical two-thirds of the second molar are at the secretory stage. In the secretory ameloblasts, both the RNAs are expressed. In the post-secretory ameloblasts, the expression of the RNAfor amelogenin is gradually decreasing with increasing distance from the secretory stage, while the signal for amelin RNA remains equally intense in all stages of ameloblasts. Arrows (—>) indicate the end of the secretory stage as accessed by the height of the ameloblasts. Mag. as in Fig. 2. elongation of the ameloblasts at the beginning of the secretory stage. In later stages, amelogenin and amelin RNA exhibit profoundly different hybridization patterns. Amelogenin RNA disappears to a great extent in the maturation stage, with only small amounts remaining at a later stage of mature ameloblasts, in agreement with the findings of Wurtz et al. (1996). The signal intensity obtained with the amelin probe, however, was virtually the same during the maturation stage of the ameloblasts. Functionally, the two stages are different in that no additional enamel matrix is deposited during the maturation phase. However, mineral seems to be deposited in both

phases. The newly deposited enamel already contains mineral, and the mineral content is increasing during the maturation phase. When these events are correlated with the appearance of the respective RNAs, it is possible that amelin is involved in the mineralization process. The amelin cDNA sequence was recently determined. It codes for an extracellular-like protein with putative cell-binding domains (Cerny et al., 1996). Thus, amelin might mediate interactions between ameloblasts and enamel matrix. The technique of whole-mount in situ hybridization was developed for studies of the early development of Drosophila embryo (Tautz and Preifle, 1989). It facilitated the detection

Fig. 4—Neighboring sagittal sections through the first and second molars of a 15-day-old rat showing the expression of RNA for amelogenin (a) and amelin (b). At this age, secretory ameloblasts can be found only in the cervical third of the second molar. The expression of RNA for amelogenin gradually decreases from the level seen in the early post-secretory ameloblasts in the cervical area of the first molar to a level almost below detection in the most occlusal area. In the second molar, the RNAfor amelogenin is highly expressed in the secretory ameloblasts and, to a lesser degree, in the post-secretory cells. The RNAfor amelin is expressed at the same level in secretory as well as post-secretory ameloblasts. Mag. as in Fig. 2. Downloaded from adr.sagepub.com by guest on July 12, 2011 For personal use only. No other uses without permission.

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Fig. 5—Neighboring sagittal sections through one of the maxillary incisors of a 15-day-old rat showing the expression of RNA for amelogenin (a) and amelin (b). The RNA for amelogenin is expressed in the secretory and early post-secretory ameloblasts but not in the late post-secretory ameloblasts. The RNA for amelin is expressed in secretory and in all post-secretory ameloblasts. Scale bar — 400 /jm. of RNA expression when it was limited to a small number of cells, and it also gave the three-dimensional distribution of these cells. Furthermore, it was possible to select the area of interest for sectioning and further examination. The wholemount in situ hybridization was later modified and has been used in other tissues (Hemmati-Brivanlou et al., 1990; Herrmann, 1991; Lepage et al., 1991; Wilkinson, 1992; Rosen and Beddington, 1993). This paper seems to be the first description of this technique for studies of developing teeth. It allowed us to study the distribution of amelogenin and amelin in the apical end of the rat incisor. The results were in agreement with those obtained by means of in situ hybridization of tissue sections. In addition, it revealed that the enamel epithelial layers prior to hard tissue formation were often markedly folded. The technique may be useful in future studies of the three-dimensional distribution of cells expressing specific RNAs in the developing teeth. It may also be advantageous for the in situ hybridization to be performed before the preparatory procedures that are necessary for

paraffin or ground sections of dental hard tissues to be obtained. However, possible limitations of the technique relate to the property and thickness of the specimen and to the penetration of the probe as well as the substances required for the visualization of the hybridized RNA (Hafen and Levine, 1986; Tautz et al., 1992).

ACKNOWLEDGMENT This investigation was supported by the Stockholm County Council and Swedish Medical Research Council Grant #06001.

REFERENCES Cerny R, Slaby I, Hammarstrom L, Wurtz T (1996). A novel gene expressed in rat ameloblasts codes for proteins with cell binding domains. J Bone Min Res 11:883-891. Eisenmann DR (1994). Amelogenesis and enamel structure.

6b Fig. 6—The explants from the apical ends of mandibular incisors in the experiment of whole-mount in situ hybridization with DIG-labeled amelogenin probe, (a) The explant after hybridization but before the enzyme-histochemical visualization of the signal, (b) The explant after the enzyme-histochemical visualization of the signal, (c) A sagittal section of the explant after enzyme-histochemical incubation showing that the RNAfor amelogenin is expressed in the ameloblastic layer. Scale bar = 1 mm. Downloaded from adr.sagepub.com by guest on July 12, 2011 For personal use only. No other uses without permission.

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In: Oral histology. 4th ed. Ten Cate AR, editor. St. Louis: Mosby, pp. 218-256. Gibson CW, Golub E, Ding W, Shimokawa H, Young M, Termine J, et al. (1991). Identification of the leucine-rich amelogenin peptide (LRAP) as the translation product of an alternatively spliced transcript. Biochem Biophys Res Commun 174:1306-1312. Hafen E, Levine M (1986). In: Drosophila—A practical approach. Roberts DB, editor. Oxford: IRL Press, pp. 139174. Hemmati-Brivanlou A, Frank D, Bolce ME, Brown BD, Sive HL, Harland RM (1990). Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization. Development 110:325-330. Herrmann BG (1991). Expression pattern of the Brachyury gene in whole-mount Twis/Twis mutant embryos. Development \ 13:913-917. Lau EC, Simmer JP, Bringas P Jr, Hsu DD-J, Hu C-C, Zeichner-David M, et al. (1992). Alternative splicing of the mouse amelogenin primary RNA transcript contributes to amelogenin heterogeneity. Biochem Biophys Res Commun 188:1253-1260. Lepage T, Sardet C, Gache C (1991). Spatial expression of the hatching enzyme gene in the sea urchin embryo. Dev Biol 150:23-32.


Robinson C, Kirkham J, Hallsworth AS (1988). Volume distribution and concentration of protein, mineral and water in developing bovine teeth. Arch Oral Biol 33:159-162. Rosen B, Beddington RSP (1993). Whole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions. Technical Focus 9:162-167. Tautz D, Preifle C (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98:81-85. Tautz D, Hulskamp M, Sommer RJ (1992). Whole-mount in situ hybridization in Drosophila. In: In situ hybridization, a practical approach. Wilkinson DD editor. Oxford: IRL Press, pp. 61-73. Watkins S (1994). In situ hybridization and immunohistochemistry. In: Current protocols in molecular biology. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JD, Smith JA, et al., editors. New York: John Wiley. Wilkinson DD (1992). Whole-mount in situ hybridization of vertebrate embryo. In: In situ hybridization, a practical approach. Wilkinson DD editor. Oxford: IRL Press, pp. 75-83. Wurtz T, Lundmark C, Christersson C, Bawden JW, Slaby I, Hammarstrom L (1996). Expression of amelogenin RNA sequences during development of rat molars. J Bone Min Res 11:125-131.

DISCUSSION Simmer: We were searching the data bank for a clone we had for porcine enamel and discovered that the Karolinska group and a group at NIDR (Yamada) have independently cloned the same sequence that you refer to as amelin. We now have it as well. Deutsch: How much of the enamel protein is this? Fong: Looking at the RNA level in situ hybridization, we estimate that expression of amelin is about 7% that of amelogenin. Deutsch: Are there no similarities between amelin and amelogenin? Slaby: We have it at the cDNA level but not at the protein level. Based on computer generation of the protein from the cDNA, there is about a 21% similarity to amelogenin and 2 1 % similarity to collagen. Prostak: In the whole-mount preparation, can the probe get to the odonto-blasts? Fong: It is more difficult. Wurtz: We used a collagen probe, and the odontoblasts stained. But in general, the deeper the tissue, the more difficult it is. O'Connor: What stage of development were you looking at when estimating relative amount? Fong: Secretory stage. Slaby: These values are only estimates because you cannot get quantitative values using this method. McKee: I am suspicious when the staining extends all the way through maturation and the reduced enamel epithelium. What evidence do you have that this is a secreted extracellular protein? Slaby: There is a signal peptide for export, the structure of the protein is hydrophobic, and it has a cell-binding sequence. So it looks like an extra-cellular

protein. Zaki: I notice that you have amelin staining over the enamel-free areas. We and others have noted that something is secreted and removed prior to tooth eruption. Fong: We got staining for amelogenin at two days but not at five days, but got amelin staining into those areas at all stages. Warshawsky: It's interesting that amelin has similarities to Type IV collagen because ameloblasts generate a basement membrane. Nanci: No one has succeeded in localizing Type IV collagen to that structure so far. Warshawsky: They have at the beginning. Wurtz: The lysine-x-y repeats that are a requirement for triple helix formation are not present in the amelin sequence. Bonass: Did you get any signal when you did your controls with sense probe? Fong: No. Wright: Do you know where the amelin gene is localized in the genome? Fong: No. Young: The Yamada group at NIDR is in the process of doing that. I think that they do have antibodies to the secreted protein. We will find out in Wisconsin. They call it ameloblastin. McKee: I still have a problem with this being a bona fide extracellular matrix product. The expression pattern does not show the variability one would expect with the disappearance and re-constitution of the basement membrane. And there is no evidence of matrix secretion in the late development stages that you show. Is that correct, Charlie (Smith)? Smith: Localization requires the use of mineralized sections, and that work has not actually been done.
