I Int. .I. Un. iii..!. ~n: 11~1-1150 (1996) 1141 I Origillal Arlid,' I I Ameloblastin expression in rat incisors and I human tooth germs I SUK KEUN LEE', PAUL H. KREBSBACH', YUTAKA MATSUKI", ANTONIO NANCI2, I KENNETH M. YAMADA' and YOSHIHIKO YAMADA" I 'Laboratory of Developmental Biology, Nations/Institute of Dental Research, National Institutes of Health. Bethesda, MD. USA and lDepartment of Stomatology, Facultr of Dentistry, Universite de Montreal. Montreal, Canada I ABSTRACT We recently identified ameloblastin as an ameloblast~specific gene product from a rat incisor cDNA library (Krebsbach et a/., J. B;o/. Chern. 271: 4431-4435, 1996). Here we report the I developmental pattern of expression of ameloblastin in rat incisors and human tooth germs as visualized by in situ hybridization and immunochemistry. Compared to the expression of amelogenin, I the major ameloblast product, ameloblastin mRNA was more widely expressed in ameloblasts from the presecretory to the late maturation stage of development, Ameloblastin mRNA was first observed in the juxtanuclear cytoplasm of pre secretory stage ameloblasts, gradually increased in the distal I cytoplasm of secretory stage ameloblasts and was found throughout the cytoplasm of early to late maturation stage ameloblasts. The immunostaining of ameloblastin, using a monospecific antibody I raised against a recombinant protein, showed intense reactivity in Tomes' processes of secretory stage ameloblasts and surrounding enamel. The immunoreaction was concentrated in the juxtanuclear I cytoplasm of late maturation stage ameloblasts, High-resolution colloidal gold immunocytochemistry established the presence of ameloblastin antigenicity in the Golgi apparatus. secretory granules in Tomes' process and enamel. Humantooth germs in early to late bell stage also expressed ameloblastin I mRNA and ameloblastin antigenicity in the ameloblasts, Western blot analysis of protein extracts from rat incisor tissues indicated that ameloblastin can be found in the enamel epithelial tissue and in I mineralized enamel, as well as in the EDTA decalcification solution. These data indicate that ameloblastin is an ameloblast secretory product which is sequentially expressed from the presecretory I to the late maturation stage in rat and human teeth. This unique developmental pattern suggests that ameloblastin may have a broader role in amelogenesis than amelogenin and tuttelin. I KEY WORDS: flU/flob/flMil/. fl"'f'logt'l/t'.~i\, f'lIflmt'i Inottin. tooth I I Introduction maturalion stage, and 4) late maturation stage (Warshawsky and Smith, 1974) (Fig. 1). I Tooth organogenesis results from reciprocal interactions be- To date, two major classes of enamel proteins called amelogenins tween neural crest.derived ectomesenchyme and oral epithelium and non.amelogenins have been identified and characterized (Lumsden, 1988; Kollar and Mina, 1991; Ruch etal., 1995; Zeichner- (Termine et al., 1980a; Ogata et a/., 1988; Inage et a/.. 1989; I David et a/., 1995). Specialized cells called odontoblasts and Deutsch et a/.. 1991, 1995a). Amelogenins are relatively small (22 ameloblasts produce the dentin extracellular matrix (denfnogen- to 30 kDa), proline-rich, hydrophobic proteins, which are prone to I esis) and the enamel extracellular matrix (amelogenesis), respec- aggregate. Among the non-amelogenins, enamel ins are relatively large (48 to 70 kDa), glycine-rich, hydrophilic proleins, which are tively. Amelogenesis is characterized by the stage- and spatial- I specific regulation of different enamel proteins (Slavkin et a/., 1988; acidic and are posttranslalionally modified by sugar and phosphate Snead et a/., 1988; Inage et al.. 1989; Ruch et al., 1995). The rat moieties (Limeback and Simic, 1990; Menanleau et al.. 1988; I incisor is a useful system 10 study the molecular mechanism Ogata et al., 1988; Fincham et at., 1991 ).It is broadly accepted that underlying amelogenesis since the continuously erupting incisor contains a complete series of developmental stages in a single I tooth, i.e., 1) presecretory stage; 2) secretory stage; 3) early I ----- -Address for reprints: Laboratory of Developmental Biology, National Institute of Dental Research, Building 30, Room 413, 30 Convent Dr. MSC 4370, Bethesda, MD 20892.4370, USA. FAX: 301.402-0897. e-mail: [email protected] I 'Present address: Department of Periodontology, Niigata University School of Dentistry, Niigala 951, Japan. I 021~-62R2/96/$OJ.OO C) l'BC P..'u PlIn,cd,nSp.,n I I I I - --- j I 11*2 5.1\.Lee,'1"I. I the amelogenins comprise the largest fraction of enamel matrix removed during tooth germ extraction. the cervical enamel epithe- (Termine et al.. 1980b: Deutsch. 1989) in developing teeth and is lium which remained closely attached to the forming dentin and I lost during the maturation stage when the total mineral content of enamel matrix expressed ameloblastin mRNA (Fig. 2-1 D). In situ this tissue increases. In addition, several uncharacterized enamel hybridization in microsection of human bell stage tooth germs proteins that do not belong to the amelogenin or enamelin protein showed that cuspal ameloblasts also expressed ameloblastin I families have been identified (Graham, 1985: Strawich et al.. mRNA (Fig. 2-1 E). 1993). We recently cloned and sequenced ameloblastin, a novel amel- In situ hybridization I oblast-specific protein whose gene maps to mouse chromosome In situ hybridization sections of paraffin embedded rat incisors 5 (Krebsbach et al., 1996), Ameloblastin mRNA encodes an open showed that ameloblastin expression is limited to the ameloblast reading frame of 422 amino acids corresponding to a protein of 45 cell layer . Presecretory stage ameloblasts first showed expression I kDa, and it is predicted as an acidic protein (pI: 5,54), rich in proline of ameloblastin mRNA as a diffuse granular pattern throughout (15.2%), glycine (9.9%) an'd leucine (9.9%). From these data, it is their cytoplasm (Fig. 3A-area b, Fig. 3B). Ameloblastin mRNA likely that ameloblastin does not belong to the amelogenin or increased substantially in secretory stage ameloblasts (Fig. 3A- I enamel in families. In this report, we have studied the expression of area c, Fig. 3C) and was particularly prominent in early maturation ameloblastin in developing rat and human teeth, and compared its stage ameloblasts (Fig. 3A-area d, Fig. 3D). The expression of expression pattern to amelogenin. amelcblastin persisted at reduced levels in late maturation stage I ameloblasts (Fig. 3A-area e, Fig. 3E) where little amelogenin Results mRNA is expressed (Snead et al., 1988: Lee, unpublished). The in situ hybridization signal was characteristically concentrated in the I Whole-mount in situ hybridization juxtanuclear cytoplasm of secretory, early and late maturation Whole-mount preparations of 3-4 week old rat mandibles re- stage ameloblasts. In comparison to the pattern of expression of vealed a linear pattern of expression of ameloblastin mANA that amelogenin mRNA, ameloblastin mRNA appeared to be expressed I was restricted to the inner aspect of the enamel epithelium (Fig. 2- earlier during the presecretory stage of development (Fig. 3B). 1B and Fig. 2-2A, schematic diagram). This expression pattern was similar to that exhibited by amelogenin, but ameloblastin Immunohistochemistry mANA was distributed more apically and more incisally than the Immunolabeling with an antibody to arneloblastin showed in- I amelogenin mRNA (Fig. 2-1A,B). tense reaction in ameloblasts and linear staining at the dentino. Ameloblastin mRNA was also consistently expressed in human enamel junction (composite pictureof immunostaining in Fig. 4A). tooth germs. Whole-mount in situ hybridization revealed that No reactivity was seen in the negative control using the antibody I particularly intense expression of ameloblastin mRNA was found preabsorbed with recombinant ameloblastin (Fig. 4J). Ameloblastln in the cuspal regions of early bell stage tooth germs (Fig. 2-1 C and was first immunodetecled in the distal cytoplasm of preameloblasts Fig. 2-28. schematic diagram). The secretory ameloblast zone (Fig. 4A-area b, Fig. 4B). The labeling was intense in the Tomes' I showed strong staining while the immature enamel epithelial zone processes of secretory ameloblasts (Fig. 4A-areas c,d,e: Fig. was negative (Fig. 2-1C). Similar staining was also found in the 4C,D,E) and then appeared localized to the distal cytoplasm of cervical region of late bell stage tooth germs (Fig. 2-10), where early maturation stage ameloblasts. At a later developmental I early stage secretory ameloblasts begin to produce enamel matrix. stage, ameloblastin was also weakly detected in the papillary iayer Although the cuspal enamel epithelium in Figure 2-' D was partly of the enamel organ (Fig. 4A-areas g,l, Fig. 4G,F). In late matura- I Pre-secretory Secretory Early maturation Late maturation I stage stage stage stage I Outer enamel epithelium Capillary Stratum intermedium cell / I Inner enamel epithelium '" I I Fig. 1. Schematic represen- Tomes' process Odontoblast Dentine Enamel tation of the sequential I stages of ameloblast cell dif- Dentino-enamel junction ferentiation. I n_ __n__ Aml!lohlaSfill t'xpressioll in dt>\'l!/oping ft'('fll 1143 I .' / immunostaining Fig. 2.1. Expression of ameloblastin in rat incisor and human tooth germ. IA,BI Whole-moum in SItu hvbridlzation of 3 week-old rat mandibles. IAI Ame/ogenln mRNA eApression. (8) Ame/oblastin mRNA expression, arne/ablastin mRNA Is more widely expressed towards the apical end and the inCisal area rhan amelogenin (extent of e~preSSlon demarcated by arrows) IC,D) Whole-mount in situ hybridization of human tooth germs. ICI Early bell stage; enameJ.-producing zone In rhe cuspal area IS strongly poSl(Jve (brown) and shows a clear-cut border (arrows) with the immature ameloblast zone. IDI Late bell stage; ca/cdlcation IS advanced, and remarning enamel epithelium at the cervical margin (arrows) shows strong reaction (blue). IE.F) Microsections of human tooth germ {x200}; lEI In situ hybridization of arneloblastln mRNA In m;crosecrion of earty bell stage of human tooth germs; secretory amelobfasts of the cusp area (arrow' are positive. IFI fmmunostainlng with anti-amefobfasM antibody.