Sequential expression and differential function of multiple enamel proteins during fetal, neonatal, and early postnatal stages of mouse molar organogenesis

Differentiation

Differentiation (1988) 37:26-39

0 Springer-Verlag 1988

Sequential expression and differential function of multiple enamel proteins during fetal, neonatal, and early postnatal stages of mouse molar organogenesis Harold C. Slavkin’ *, Conny Bessem’, Pablo Bringas, Jr.’, Margarita Zeichner-David’, Antonio Nanci’ and Malcolm L. Snead’ Laboratory for Developmental Biology, Department of Basic Science, School of Dentistry, University of Southern California, Los Angeles, California 90089-0191 Department de Stomatologie, Faculte de Medecine Dentaire, Universite de Montreal, Canada Montreal, Quebec

Abstract. We have established the time and position of expression for multiple enamel proteins during the development of the mouse molar tooth organ. Using high-resolution two-dimensional gel electrophoresis coupled with immunoblotting and immunocytochemistry, a 46-kDa enamel protein (PI, 5.5) was detected during late cap stage (28-days gestation, E l 8d) within differentiation-zone-I1 inner enamel epithelia associated with an intact basal lamina. At E19d a second enamel polypeptide of 72 kDa (PI, 5.8) was identified at the time and position of initial biomineralization in differentiation zone V. At 20 days, differentiation-zoneVI ameloblasts without basal lamina (late bell stage) expressed 46- and 72-kDa enamel proteins and, in addition, expressed a relatively more basic 26-kDa enamel protein (PI, 6.5-6.7) ; detected after initial formation of calcium hydroxyapatite crystals. Antibodies raised against chemically synthesized enamel peptides cross-reacted with both the 72-kDa and 26-kDa polypeptides, but did not cross-react with the 46-kDa enamel polypeptide. The sequential expression of multiple enamel proteins suggests several functions : (a) the anionic enamel proteins may provide an instructive template for calcium hydroxyapatite crystal formation ; (bj the more neutral proteins possibly serve to regulate size, shape and rates of enamel crystal formation. We suggest that initial expression of enamel gene products during mouse tooth development possibly recapitulates ancestral features of amelogenesis documented in prereptilian vertebrates. These results imply that multiple instructive signals may be responsible for mammalian enamel protein induction and that the sequential expression of a family of enamel proteins reflects the evolutionary acquisition of a more complex genetic program for amelogenesis.

Introduction

A consequence of molecular differentiation is the tissuespecific expression of unique genes. The developmental events that determine cell-lineage-dependent patterns of transcription, translation and posttranslational modifications have not yet been defined. Part of the difficulty in elucidating these events during epidermal organogenesis (e.g., skin, scale, feather, teeth, mammary gland), stems from the limited state of our knowledge concerning when, where and how epithelial-specific structural gene products

* To whom offprint requests should be sent

are activated and expressed. To approach the problem of regional specification of epithelial determination and differentiation during epidermal organogenesis, it is necessary to first determine when and where ectodermally-derived epithelial-specific gene products are expressed during development. Enamel protein gene expression during mouse molar tooth organogenesis is a model system that is potentially very useful for investigating these issues, because it provides several experimental advantages. First, the morphological and cytological specialization of the mouse mandibular first molar tooth organ (MI) is well-documented [S, 16, 53, 561. Detailed ultrastructural descriptions of differentiation in inner enamel epithelia (“differentiation zones I-VI ”) provide excellent positional orientation [25, 441. Second, cranial neural-crest-derived ectomesenchyme induction of epithelia to become determined to differentiate into functional ameloblasts that produce enamel extracellular matrix (ECM) constituents has been described at several levels of biological organization [27, 28, 29, 34, 47, 69, 73, 791. Third, several enamel-specific gene products have been identified (i.e., amelogenins and enamelinsj, and these products can serve as molecular markers of ameloblast differentiation [57, 72, SO]. Fourth, a cDNA clone encoding the major mouse ameloblast gene product has been isolated and characterized [65, 671. Finally, embryonic and fetal M I can be readily dissected and cultured in serumless, chemically-defined medium for extended periods of time, resulting in morphogenesis, cytodifferentiation, production of tissue-specific ECM macromolecules, and tissue-specific patterns of biomineralization [5, 121. Previous studies showed that ameloblast-specific messenger RNAs encode a minimum of one enamelin and three amelogenin polypeptides when immunoprecipitated from a cell-free translation system 157, 651. Reports from this laboratory have also provided evidence that the major amelogenin mRNA as well as its nascent corresponding polypeptide (26 kDa) are first expressed at birth during M I development [66]. The precise timing of enamelin biosynthesis remains in question [lo, 45, 54, 721. The subfamily of enamel proteins termed amelogenins are relatively low-molecular-weight (approx. 30-20 ), hydrophobic, proline-rich (more than 220 proline amino acid residues per thousand) proteins. They appear as peptides of increasingly lower molecular weight during the process of biomineralization and enamel maturation [ l l , 15, 45, 691. The increased number of progressively lower-molecu-

27

lar-weight amelogenins is assumed to be the result of enzyme-mediated posttranslational processing [70]. In contrast, enamelins are of relatively high molecular weight (approx. 7240) and are hydrophilic glycoproteins, which appear to be retained in association with calcium hydroxyapatite crystals during the process of enamel maturation [lo, 13, 21, 721. It has been assumed that both amelogenins and enamelins are expressed in tandem during the process of enamel ECM production [lo, 45, 721. Polyclonal antibodies raised against mammalian enamel proteins cross-react with enamel polypeptides from a number of disparate vertebrate species, including hagfish, shark, fish, alligator, mouse, hamster, rabbit, pig, cow, and human enamel matrix constituents [22, 57, 58, 60, 621. Our experimental protocol was designed to define the time and position of enamel-protein synthesis and secretion during fetal, neonatal and early postnatal M, development. We report that an anionic enamel protein of 46 kDa (PI, 5.5) was synthesized and secreted from differentiation-zone111 inner enamel epithelia at 18 days of gestation (El8d). At E19d a second enamel protein of 72 kDa (PI, 5.8) was synthesized. These enamel proteins were secreted into the ECM by inner enamel epithelia of differentiation zones IIIV prior to removal of the basal lamina, and in concert with initial biomineralization. Both anionic enamel proteins persisted during subsequent neonatal and early postnatal stages. Synthesis and secretion of a lower molecular weight (26 kDa), more neutral (PI, 6.5-6.7), and hydrophobic protein was detected at 20 days. This was correlated with the disruption and removal of basal lamina associated with the differentiation-zone-VI ameloblast phenotype and progressive biomineralization. This pattern of synthesis and secretion of multiple enamel gene products, orchestrated in a precise temporal and spatial pattern, is consistent with a model in which several members of a multigene family are individually regulated by epigenetic signal(s). These results provide a basis for further attempts to define ectomesenchyme factors that regulate ameloblast-specific gene expression. Methods

Gel electrophoresis and immunodetection. Ampholites and kits for isoelectric focusing and electrophoretic calibration were obtained from Pharmacia Inc. (Pistcataway, New Jersey). Prestained protein-molecular-weight standards were obtained from Bethesda Research Laboratories (Bethesda, Maryland). All other electrophoresis and immunostaining chemicals were from Bio-Rad Laboratories (Richmond, California). Other reagents, unless otherwise indicated, were obtained from Sigma Chemical Co. (St. Louis, Missouri). Tissue preparation and protein extraction. M, were dissected from timed-pregnant Swiss-Webster mice (a vaginal plug indicating day 0), staged according to Theiler [73], and sampled each day (& 12 h) from cap (El 5d) through crown stages of molar tooth formation (2 days postnatal development). The molars were rinsed in cold PBS (phosphatebuffered saline, pH 7.4) and frozen in dry ice until sufficient material was accumulated. For each developmental timepoint at least 20 MI were pooled. In addition, isolated M from each developmental stage were labeled by incubation at 37" C for 3 h with 100 pCi/ml [35S]methionine (New England Nuclear, Boston Mass.

800 Ci/mmole) in BGJb medium (GIBCO, Staten Island, New York) and subsequently chased for 30 min with nonlabeled excess L-methionine in culture medium. MI were rinsed three times with PBS, homogenized and extracted in homogenization buffer. The protein content was then determined as previously described [57]. Radiolabeled samples were analyzed by two-dimensional gel electrophoresis followed by fluorography [3, 321. All experiments were performed in triplicate. Antibodies directed against amelogenin and enamel. The sensitivity and specificity for the rabbit antibody to mouse amelogenin has been previously reported [57-601. Rabbit antibody to human enamelin was prepared and characterized as described by Zeichner-David and colleagues [81]. Synthetic enamel peptide. The oligopeptide corresponding to the amino termini (amino acid residues 3-13) of human [14,15], bovine [71] and mouse [67] amelogenin was synthesized by Applied Protein Technology (Cambridge, Massachusetts) and was shown by amino acid sequence determination to be LPPHPGHPGYI. The synthetic peptide was coupled to keyhole limpet hemocyanin (KLH) [23] and used to immunize young adult female New Zealand White rabbits by multisite subcutaneous injections. Boosting was performed every 7 days, and immune sera collected after three boosts. The IgG fraction was prepared by DEAE-cellulose chromatography and used in enzyme-linked immunoabsorbent assays (ELISA) to determine the titer and specificity of the antibody (manuscript in preparation). Histochemical and ultrastructural procedures f o r biomineralization. The crown of the mouse mandibular first molar tooth organ consists of five cusps with an enamel-free area at the occlusal surface of each cusp. To optimize comparisons between different stages of morphogenesis, observations were confined to the mesial-buccal cusp region at each stage of MI development. The labial surface of young postnatal continuously forming mandibular incisor tooth organs was used to confirm Kallenback's stages of cytodifferentiation in the mouse incisor and molar specimens [24]. Nondemineralized tooth organs from El 5d through 2 days of postnatal development were processed for von Kossa staining to identify the time and position of initial calcium-salt deposition [75].In replicate studies, tooth organs were fixed in ethylene glycol and then processed for ultrastructural analysis using the anhydrous preparation method [31, 781. Immunogold cytochemistry. The protein-A-gold technique was used for detecting enamel protein antigenic sites in thin sections from either Lowicryl- (LKB, Piscataway, New Jersey) or epon-embedded rodent incisor and molar tooth organs [37,38]. Thin sections prepared from osmicated incisor and molar tissues embedded in epon were treated with sodium metaperiodate for 1 h at room temeprature, washed with distilled water, and then incubated with rabbit antibodies to amelogenin (1/ I 0 dilution) as recently described [37, 381. The sections were washed, and the antigen-antibody complex was detected by washing the section with protein-A complexed to gold. Sections of tissues embedded in Lowicryl were incubated with the primary antibody without prior treatment with sodium metaperiodate. The sections were then stained with uranyl acetate and lead citrate

28

and examined with a Phillips 300 or JEOL 1200 transmission electron microscope. Controls consisted of incubating sections with: (a) antibody that had been exposed to an excess of amelogenin antigen, (b) preimmune IgG antibody, or (c) protein-A-gold [38]. High-resolution two-dimensional gel electrophoresis. Isoelectric focusing-polyacrylamide gel electrophoresis (IEFPAGE) was conducted as described by O’Farrell [39]. The first-dimension gel used a mixture of ampholites (1.6% pH 5-8 and 0.4% pH 3-10) in a 10.5-cm-long tube gel with a diameter of 2 mm. The sample buffer contained 9.5 A4 urea, 5% (v/v) 2-mercaptoethanol, 2% (v/v) Nonidet P-40, and 2% (v/v) ampholites. Extracted proteins (1 00-200 pg) from mouse teeth were mixed with an equal volume of lysis buffer after the addition of solid urea. Isoelectric focusing was conducted for 16 h at 400 V and 1 h at 800 V. The second dimension was run according to the method of O’Farrell [39], employing the Laemmli PAGE-system [30]. Briefly, the IEF gels were incubated in SDS equilibration buffer for 1-2 h and anchored into place with 1OO/ agarose solution in the same buffer. After electrophoresis through the stacking gel (4% acrylamide), at 10 mA per gel, gels were run overnight through the resolving gel (loo/, acrylamide) at 3 mA per gel. It was observed that slow runs eliminated streaking and improved separation of proteins. Samples were silver-stained after electrophoresis [81]. Additional studies processed the metabolically-labeled extracted proteins for two-dimensional gel electrophoresis and fluorography. Electroblotting and immunostaining. The electrophoretic transfer of proteins was based on Burnette’s modifications [6] of Towbin’s method [76]. The transfers were conducted at 4” C for 75 min at 0.6 A (60-90 V) after equilibration of the gels in transfer buffer (0.192 M glycine; 0.025 M Tris, pH 8.3; 20% v/v methanol). It was necessary to use at least 200 pg protein per sample for Western blotting and immunodetection following protein separation by IEFPAGE. Resolution of either subfamily was complicated, since the relatively hydrophobic amelogenins represent over 90% of the total enamel matrix proteins, whereas anionic enamelins represent only 1%-3% of total enamel matrix proteins [45, 61, 721. Proteins of interest were determined to transfer only toward the anode. After transfer, the membranes were removed and washed for 10 min in TBS (20 m M Tris, pH 7.5; 0.5 M NaC1) and then immersed in TBS containing 3% gelatin for 30 min. The membranes were then incubated in a solution of TBS with 1% gelatin [19] containing rabbit IgG antibodies to either mouse amelogenins or human enamelins, in 1 :2000 dilution for 16 h at 30” C with gently agitation. The incubated membranes were rinsed once with distilled water and twice for 10 min with TBS, and then incubated with goat antirabbit horseradish-peroxidase-conjugated (GAR/HRP) IgG at a 1 :2000 dilution in 1 % gelatin/TBS for 1 h. The filters were washed with water and TBS as described above, and the peroxidase color reaction was completed by placing membranes in freshly prepared TBS solution containing 16% methanol, 0.05% 4-chloro-lnaphthol, and 0.05% hydrogen peroxide for 15-30 min. The colorimetric reaction was terminated by washing the membranes twice for 10 min in distilled water.

Results Developmental features ef inner differentiation and biomineralization

enamel

epithelial

A survey of selected histological features of inner enamel epithelial differentiation into ameloblasts, with production of enamel ECM, and ectomesenchyme differentiation into odontoblasts, with production of dentine ECM, from the cap through the crown stages of M I are shown in Fig. 1. During the cap stage (E1&17d), cuboidal inner enamel epithelia differentiated into columnar epithelia (differentiation zones 1-111) and retained a continuous metachromatic basement membrane. The histological characteristics of mouse mandibular first molar tooth organogenesis in situ have been extensively described (see comprehensive descriptions in [8, 16, 20, 34, 57, 611). The data reported in the present communication use the terminology inner enamel epithelial differentiation into ameloblasts (i.e., “differentiation zones I-VI”) as defined by Kallenbach [25]. Biomineralization of mouse dentine ECM has been reported to precede that of enamel ECM [12]. In the present studies, biomineralization was monitored using the von Kossa histochemical assay for calcium phosphate deposition. By that criteria, biomineralization during MI formation was identified at E19d (Fig. 2). Two-dimensional immunoblot analysis of stage-specific enamel protein expression

Two-dimensional immunoblot analysis of proteins detected the expression of an anionic (PI, 5.5) 46-kDa enamel protein initially at the EI8d stage of development (Fig. 3B, arrow). Prior to ElSd, no enamel antigen was detected using either antiamelogenin or antienamelin (Figs. 3A, and 4A, respectively). A 72-kDa enamel protein (PI, 5.8) was detected at E19d using antimouse amelogenin IgG or antihuman enamelin IgG at 1 :2000 dilution (Figs. 3 C and 4D, respectively). At 20 days, the 26-kDa amelogenin was identified (PI, 6.5-6.7) using the rabbit IgG antibodies to amelogenin (Fig. 3 D, arrows). This confirmed previous documentation using Western transfer from one-dimensional gel electrophoretograms, as well as cytoplasmic RNA dot-blot hybridization analysis with cDNA probe pM5-5 for mouse amelogenin [66]. However, under these experimental conditions, the antibody to human enamelin did not react with the amelogenin antigen (Fig. 4E, F). The 46-kDa and 72-kDa enamel proteins continued to be detected through neonatal and early postnatal stages of development (Figs. 3D, E, F and 4E, F). The antibody to mouse amelogenin recognized the 46-kDa, but not the 72-kDa, enamel protein under these experimental conditions following birth (Fig. 3 E, F). The increase in polypeptide number and the alteration in their charge and mass were possibly the result of posttranslational modifications such as glycosylation and enzymatic cleavage (Fig. 3 D-F). Antibodies raised against synthetic enamel oligopeptides (LPPHPGHPCYI) cross-reacted with only the 72- kDa protein at E19d (Fig. 5B), and with both the 72-kDa and 26-kDa enamel proteins at the neonatal stages of development. The antipeptide antibody did not crossreact with the 46-kDa enamel protein (Fig. 5). The order of enamel protein expression was further analyzed by comparison of the immunodetected enamel protein

29

Fig. 1A-E. Histological survey of epithelial and ectomesenchymal cell differentiation during late embryonic, fetal, neonatal and postnatal stages of M, organogenesis. A E16d (16days of embryonic life). B E17d. C Birth. D One-day after birth. E Two-days after birth. x 700

Fig. 2A-C. Dentine and enamel biomineralization as demonstrated by von Kossa histochemical staining of calcium salts at selected stages of development. A E18d, a progenitor dentine extracellular matrix (ECM; arrows) is evident at the interface between the inner enamel epithelia (iee) and adjacent preodontoblasts (pod). B Birth; odontoblast cells (04are associated with predentine (pD),a mineralized dentine ECM (D),and ameloblasts ( A m ) are producing the enamel ECM with initial biomineralization (arrows). C Two days after birth, enamel biomineralization is evident as aggregations of spherical calcium salt deposits. preenamel ($E) is evident. x 700

30

MW KDa

7.4 I

6.5 I

5.5 I

7.4 I

PH 6.5

68-

I

5.5 I

7.4 I

6.5 I

I)I 5.5 I

EF

$. S O S

43-

2 6-

6843-

26Fig. 3A-F. Analysis of enamel proteins expressed during late embryonic, fetal, neonatal and postnatal stages of development using high-resolution, two-dimensional gel electrophoresis and immunoblotting with antibody to amelogenin. A El 7d. B E18d. C E19d. D Birth. E One day after birth. F Two days after birth. Immunoblotted proteins (200 pg protein per gel) were assayed with IgG antibody against amelogenin at 1 :2000 dilution, and the immune complex detected with GAR (goat antirabbit)/HRP (horse raddish peroxidase) at 1 : 2000 dilution. Arrows indicate proteins immunodetected during fetal and neonatal molar development

with the order of expression for [35S]methioninemetabolically-labeled proteins (Fig. 6A-F). A major change in synthetic activity was observed at E18d, correlated to the synthesis of a 46-kDa protein (Fig. 6C, arrow). The 72-kDa protein was also synthesized at E19d (Fig. 6D, arrows). The 46- and 72-kDa anionic proteins and the 26-kDa enamel protein were identified at 20 days (Fig. 6F, arrows). Immunocytochernical localization of multiple enamel proteins during inner enamel epithelial dgferentiation into ameloblasts Continuously erupting rodent incisor model. T o define the anatomic position of enamel protein expression during tooth organ development, experiments were designed to take advantage of the well-defined sequential representation of inner enamel epithelial differentiation (differentiation zones I-VI) found along the labial surface of the continuously erupting rodent incisor tooth organ [24, 56, 64, 771. The regions sampled (Fig. 7, regions labeled A-D) represented Kallenbach’s differentiation zones 111 (region A), V (region B); VI (region C); and secretory ameloblast with Tonics’ process (region D). Along this gradient of differentiation, immunolocalization of enamel proteins was observed within inner enamel epithelial at differentiation zone I11 (Fig. SA, region A in Fig. 7). Enamel protein was identified within preameloblast (approx. 50 Krn in length) secretory granules. It was also identified within the ECM, in association with coarse gran-

ular material. Invaginations of the preameloblast apical cell membranes were evident in juxtaposition to the continuous basal lamina. Fine filaments, presumably the type VII collagen anchoring fibrils recently described by Keene nd colleagues [26], were associated with the undersurface of the basal lamina. Odontoblast cell processes extended towards the undersurface of the basal lamina. These were associated with electron-dense coarse granular material containing immunogold particles. No calcium hydroxyapatite crystals were detected. Differentiation zone V (region B in Fig. 7) consisted of preameloblasts (approx. 60 pm in length), devoid of basal lamina, and producing enamel proteins (Fig. 8 B). Immunogold localization was apparent within coarse-textured material and in droplets of fine granular electron-dense ECM materials. Odontoblast processes closely approached the apical surfaces of preameloblasts. The first mineral deposits in the ECM were evident at this position. In differentiation zone VI the ameloblasts were 65-70 pm in length; enamel ECM formation was evident along a 1- to 2.5-pm-thick mineralized dentine ECM (Fig. 8 C). The coarse-textured material was no longer evident. Secretory ameloblasts with Tomes’ processes continued to produce fine electron-dense material consisting of enamel proteins (Fig. 7, region D, and Fig. 9D). Fetal and neonatal M I development. The cap stage consisted of an enamel organ of inner (preameloblasts) and outer enamel epithelia, and a continuous basal lamina, which sep-

31

MW 7-4 I KDa 68-

6.5

5.5

I

I

7.4

6.5

5.5

7.4

6.5

I

I

I

I

I

S D S

43-

26-

68-

43-

26Fig. 4A-F. Analysis of enamel proteins expressed during late embryonic, fetal, neonatal and postnatal stages of development using high resolution, two-dimensional gel electrophoresis and immunoblotting with antienamelin. A E16d. B E17d. C E18d. D E19d. E Birth. F One day after birth. Irnmunoblotted proteins (200 pg protein per gel) were assayed with IgG antibody against enamelin at 1 :2000 dilution and the immune complex detected with GAR/HRP as the secondary antibody at 1 :2000 dilution. Arrows indicate proteins which were immunodetected during fetal and neonatal molar development

+IEF

PH

M W 7.4 I KDa 68-

6.5 I

5.5 I

7.4 I

6.5 I

5.5 I

7.4 I

6.5 I

5i5

$. S

D

S

43-

26-

Fig. 5A-C. Analysis of enamel proteins expressed during late embryonic, neonatal and postnatal stages of development using highresolution, two-dimensional gel electrophoresis and immunoblotting with antipeptide. A E18d. B Birth. C Four days after birth. Immunoblotted proteins (200 pg protein per gel) were assayed with IgG antibody against peptide at 1 :2000 dilution, and the immune complex was detected with GAR/HRP as secondary antibody at 1:2000 dilution

arated the ectodermally-derived epithelia from the adjacent dental papilla ectomesenchyme. The epithelial-mesenchyma1 interface was folded into a cap form with a definitive pattern of five major cusp areas and fossae. At the major cusp site, ectomesenchyme-derived preodontoblast cells ceased dividing, formed a sheet of cells coupled through gap junctions, polarized, and then elongated into mero-

crine-type secretory cells, which produced a progenitor ECM consisting primarily of type I collagen and glycosaminoglycans. The inner enamel epithelia in juxtaposition to these odontoblasts illustrated differentiation zones I-IV [25].The enamel protein (Figs. 3 C and 4B) was localized within differentiation zone 111 intracellular secretory vesicles as well as within the ECM, appearing as coarse-textured

32

PH MW 7.4 I KDa

6.5 I

5.5 I

7.4 I

6.5 I

5.5 I

7.4 I

6.5 I

+I

EF

5.5 I

$. S

68-

D S

43-

2 6-

6843-

26-

6843-

26Fig. 6A-G. Developmental order for expression of metabolically labeled proteins from MI tooth organs. Molars were labeled for 3 h with [35S]-methioninefollowed by a 30-min chase with excess nonlabeled methionine in culture medium. Extracted radioactive proteins were analyzed by two-dimensional gel electrophoresis followed by fluorogrdphy. A E16d. B E17d. C E18d. D E19d. E Birth. F One day after birth. G Two days after birth. Radiolabeled protein samples of 50000 cpm were analyzed for each stage of development

electron-dense granular stippled material (Fig. 9A). The continuous basal lamina was associated with fine filamentous fibrils and matrix vesicles. At E19d, the transition from differentiation zone 1V to V resulted in the removal of the basal lamina and an increase in the enamel protein detected within the ECM (Fig. 9B). This stage was characterized by the expression of 46-kDa and 72-kDa enamel proteins (Figs. 3d and 4C). Calcium hydroxyapatite crystal formation associated with matrix vesicles was found in differentiation zones V and VI in forming dentine. Odontoblast cell processes extended toward the differentiation-zone-V preameloblast apical surfaces (Fig. 9C) and were associated with granular stippled material and enamel proteins (Fig. 9C, arrows). Presumably, this material was the multiple-anionic polypeptides that were identified within secretory vesicles throughout the apical regions of the preameloblasts and as coarse-textured electron-dense material in the ECM (Fig. 9D, arrow). Large

deposits of this material were associated with extended odontoblast cell processes (Figs. 9D, arrow; and 9E, arrows). The immunolocalization patterns were similar for the biosynthesis of mouse enamel matrix proteins, as visualized by high-resolution, light-microscopic autoradiography following the administration of [3H]-tryptophan [56].During neonatal development, differentiation zone VI and the terminally differentiated secretory ameloblast phenotype were well-delineated. Discussion The present studies provide new information indicating that several anionic enamel proteins are synthesized and secreted into the extracellular matrix (ECM) prior to the initiation of biomineralization. High-resolution two-dimensional gel electrophoresis coupled with metabolic labeling, immunoblotting techniques, and immunocytochemistry indicated

33

the sequential appearance and localization of enamel proteins during late embryonic and fetal stages of morphogenesis. At E18d, a 46-kDa (PI, 5.5) protein, which crossreacted with antibodies to both amelogenin and enamelin, but did not crossreact with antipeptide, was synthesized and secreted into the ECM by inner enamel epithelial cells associated with an intact basal lamina (differentiation zones IIIIV), a developmental stage prior to overt ameloblast cytodifferentiation and prior to dentine ECM biomineralization (Figs. 1-5 and 8A). At E19d, a second enamel protein of 72-kDa (PI, 5.8) was detected within differentiation zones 111-V. Ultrastructural evidence suggested that initial biomineralization, as determined by the appearance of calcium hydroxyapatite crystal electron-diffraction patterns and positive von Kossa staining for calcium-salt deposition, was evident at E19d. At 20 days, a 26-kDa amelogenin polypeptide was identified in association with overt ameloblast differentiation (zone VI and secretory ameloblasts with Tomes’ processes) and with dentine biomineralization. The studies described in this report lead to a number of general conclusions related to the ameloblast biochemical phenotype. First, the inner enamel epithelia become determined and initiate the process of enamel protein gene expression significantly earlier than previously considered. Second, the sequence of enamel protein synthesis and secretion is protracted over a 3-day period in the developing M I ; a 46-kDa enamel protein is expressed at E18d, the 46-kDa and a 72-kDa proteins are expressed at E19d, and these two proteins plus a 26-kDa amelogenin are expressed in the newborn. This order of expression suggests the possibility that multiple epigenetic signals are required to regulate and coordinate this sequential pattern of enamel gene expression [68]. Since only one amelogenin gene has been identified in mouse, human and/or bovine genomic DNA [50, 51, 651, we assume that: (a) different gene(s) comprise the enamel gene family, being minimally represented by one enamelin and one amelogenin gene [65], and/or (b) one structural gene with alternative splicing pathways gives rise to multiple polypeptides [4]. Third, the temporal and spatial associations between the 46- and 72-kDa anionic enamel proteins, the initiation of calcium hydroxyapatite crystal formation, and attendant dentine/enamel junction formation, suggest that these anionic molecules probably serve as templates for initiation of crystal formation. Subsequent expression and secretion of amelogenin probably regulates the size, orientation, and rate of crystal growth associated with enamel formation [15, 45, 67, 72, 791. Classification of enamel polypeptides according to temporal sequence of expression and isoelectric points

The timing and sequence of enamel protein expression during fetal, neonatal and postnatal molar tooth organogenesis in mouse is summarized in Table 1 . We have adopted a classification scheme based on the chronology of expression, relative molecular weight and isoelectric point determinations, and crossreactivity with enamel antibodies. Fig. 7. Low-magnification light-photomicrograph showing regions of postnatal incisor labial surface (A-D) in rodent young, used to evaluate the immunocytochemical localization of enamel protein during differentiation of inner enamel epithelial into secretory ameloblast. x 200

34

Fig. 8A-D. Ultrastructural localization of enamel proteins during inner enamel epithelial differentiation into ameloblasts using IgG antibody against amelogenin and the protein Aimmunogold technique. A Inner enamel epithelia from region A in Fig. 7 with associated basal lamina (bl), intracellular secretory granules containing enamel proteins (sg), and extended preodontoblast cell processes (cp) in proximity to the undersurface of the basal lamina. Note the distribution of antigens related to aggregates of electron-dense granular stippled material within the ECM. B Preameloblasts from region B of Fig. 7 without a basal lamina engaged in the synthesis and secretion of enamel proteins into the ECM. C Ameloblast extension of Tomes’ process containing secretory granules associated with the enamel ECM formation from region C of Fig. 7 ; d, dentine ECM ; e, enamel. D Secretory ameloblast from region D of Fig. 7 with secretory granules (sg); bar, 1 pm (original magnification, x 30000)

35

Fig. 9A-F. Immunocytochemical localization of enamel proteins during mouse molar tooth organ organogenesis. Lowicryl embedding molars were utilized to provide enhancement of protein A-immunogold localization within epithelial cells. A Immunolocalization of enamel proteins at 18 days of gestation during M, development; su, secretory vesicles and adjacent progenitor dentine ECM; Bf,basal lamina. B Basal lamina has been disrupted and removed, with immunogold labeling observed in association with progenitor ECM prior to dentine biomineralization. C Odontoblast cell processes (rncp) extend toward the undersurface of preameloblasts and are often associated with electron-dense granular materials labeled with immunogold staining (urrows). At this time and position biomineralization of dentine ECM was evident (see also Fig. 1C, D). Note the intracellular secretory vesicles (su) containing enamel proteins. D Electrondense granular stippled material (arrow) stained with the immunogold particles in proximity to the extended odontoblast cell process (mcp). E Survey of the interface between preameloblasts (prior to the formation of Tomes processes) showing the secretion of enamel proteins into the dentine ECM. F Control section using immunoabsorbed IgG directed against mouse amelogenin (immunoabsorbed with the mouse immunogen proteins), which did not detect tissue localized epitopes; bar 0.5 pm

36 Table 1. Sequential enamel protein expression during mouse molar

tooth organogenesis Days of development

Rclatively acidic

Relatively basic

MW”

PI

MW

a-->46

5.5 5.8 5.5

PI

16 11 18

19

b-->12 a-->46

20

b-

-

>I2

a-->46

5.5 C-

21

b-->l2 a-->46

-

>26 6.5-6.1

5.8 5.5 - >28 6.5-6.1 ~ - - > 2 6 6.1

d-

a

MW, molecular weight

The first enamel polypeptide detected was the 46-kDa protein with PI 5.5. This crossreacted with antibodies to enamelin and amelogenin, but did not crossreact with the antibody to peptide (Fig. 5). At E19d, a 72-kDa enamel protein with PI 5.8 was detected. This protein crossreacted with both antienamelin and antiamelogenin, as well as with antipeptide. These two anionic enamel polypeptides were expressed at E19d in tandem, at the time of initial ECM biomineralization. They continued to be expressed through neonatal and early postnatal stages of M I . At the newborn stage of development, a 26-kDa amelogenin polypeptide with PI 6.5-6.7 was identified. Thereafter, we observed an increase both in the number of amelogenin polypeptides and in their respective isoelectric PI values (PI, 6.5-7.4). The data presented corroborate evidence for enamel biogenesis provided by a number of studies in disparate vertebrate species that have suggested the anionic enamel proteins are expressed before amelogenins. Levine et al. [33] showed that protein(s) of shark enameloid have an amino acid composition similar to that of enamelins, but do not contain amelogenins. Enamelins appear to be expressed prior to amelogenins in fetal bovine teeth [72]. More recently, shark enameloid was found to consist of two major anionic proteins of 72 kDa and 46 kDa; amelogenins were not detected [49]. Deutsch et al. [lo] showed that relatively high-molecular-weight anionic human fetal enamelins are expressed prior to amelogenins. Zeichner-David et al. [82] recently showed that polyclonal antibodies against the synthetic oligopeptide “ LPPHPGHPGYI” crossreact with high-molecular-weight anionic fetal rabbit enamelins. Enamelins have been shown to be directly associated with calcium hydroxyapatite enamel crystals, whereas amelogenins are detected between enamel crystals [21]. Taking this data cumulatively, we suggest that the anionic enamel proteins are the ancestral enamel proteins derived from primitive vertebrates and are used in mammals to initiate crystal nucleation. Belcourt has reported that a high-molecular-weight, amelogenin-like protein (approx. 40 kDa) appears in fetal bovine enamel matrix 11, 21. However, the hallmark amelogenin amino acid sequence LPPHPGHPGYI, which is found within a number of different amelogenin polypep-

tides [14, 15, 51, 671, was not detected in the 46-kDa enamel protein in the present study (see Fig. 5). This finding suggests that either: (a) the 46-kDa enamel protein does not contain this sequence; (b) the molecule was posttranslationally processed, eliminating the amino terminal domain containing the epitope(s); (c) the epitope was masked by the tertiary structure of the molecule; or (d) the protein is not an amelogenin. Studies are in progress to determine the amino acid sequence fo the 46-kDa fetal mouse enamel protein. Epithelial-mesenchymal intercellular communication prior to amelogenin gene expression

Whereas direct cell-cell contacts have been observed between inner enamel epithelia and adjacent ectomesenchyme cells during late-cap and early-bell stages of tooth development [24, 25, 551, little information is available pertaining to the putative function(s) associated with this suggested transmission of inductive signals between heterologous tissues by these contacts. It is apparent that odontoblast cell processes make contact with the undersurface of the basal lamina in apposition to inner enamel epithelial, and that direct cell-cell contacts between these dissimilar cell types are observed following the removal of the basal lamina [47, 55, 56, 741. Previous reports indicate that odontoblast cell processes are positioned at sites where electron-dense, tryptophan-labeled, granular stippled materials released from preameloblasts are secreted, which precedes biomineralization of the dentine ECM [56]. In the present study, this electron-dense stippled material was identified as representing enamel proteins by immunocytochemical assays (see Figs. 7 A and 8 C, D, E). Here, we have confirmed that relatively low-molecularweight, hydrophobic enamel proteins first appear at birth during development. Previous analysis indicated that the amelogenin mRNA and polypeptide are first detected at birth [66]. In the present study we note that direct cell-cell contact between extended odontoblast cell processes and adjacent inner enamel epithelial were observed in the M, tooth organs in vivo prior to amelogenin expression. Further, odontoblast cell processes were routinely identified in association with enamel protein antigens released from the inner enamel epithelial cells (Fig. 8C, D, E). We interpret these findings to suggest that ectomesenchyme cell processes may transmit the signals required to activate the induction and/or secretion of amelogenins. Evolutionary importance of heterochrony in enamel epithelial cytodifferen tiation

Vertebrate tooth organogenesis spans nearly 450 million years of evolution (see critical discussions in [13, 42, 431). During enameloid synthesis and secretion from inner enamel epithelia in chondrichthyes, the basal lamina is not degraded [25, 37, 381. The enameloid proteins which crossreact with antibodies to mouse or bovine enamel proteins are secreted into the ECM by epithelia prior to odontoblast differentiation and denticle formation [22, 58, 59, 631. Enameloid consists of relatively high-molecular-weight proteins that have amino acid compositions resembling mammalian enamel anionic glycoproteins (enamelin) and distinct from the amino acid composition of mammalian amelogenins [33, 49, 59, 621. Moreover, It is now well established that

37

epitopes in enameloid of chondrichthyes and osteichthyes and epitopes of enamel proteins of amphibian, reptilian, and mammalian species are shared [22, 49, 58, 591. These observations suggest that one of the conserved features of inner enamel epithelial production of ECM associated with biomineralization is the synthesis and secretion of anionic protein molecules. We conclude that the two initially expressed mouse anionic enamel proteins identified in this study may represent conserved protein domains that are also identified as epithelial-derived enameloid gene products localized during tooth formation in chondrichthyes [49, 59, 621. We suggest that changes in the relative timing and rates of development during the process of amelogenesis are dominant variables in the genesis of vertebrate enamel evolutionary biology. Early ancestral events associated with cytodifferentiation of inner enamel epithelium (i.e., Kallenbach’s proliferation zone and differentiation zones I-IV), including the synthesis and secretion of anionic proteins that proposedly nucleate calcium hydroxyapatite crystal formation, are conserved during vertebrate odontogenesis but are altered in their timing and rates of expression within extant postreptilian vertebrate species [I 71. The substantial odontological literature [34-36, 40-43, 491 can be interpreted to indicate that ancestral stages of cytodifferentiation initially persisted within inner enamel epithelia during mammalian tooth development [25, 37, 38, 601. The present study and substantive literature argue that the evolutionary importance of heterochrony (e.g., changes in the relative time of appearance and rate of development for characters already present in ancestors ; see discussion in [17]) is operant and accessible for experimental testing during vertebrate enamel formation of tooth development. We further suggest that the shark enameloid proteins derived from inner enamel epithelium function as anionic macromolecular templates to nucleate calcium hydroxyapatite prior to dentine ECM biomineralization, whereas the more hydrophobic amelogenins identified for terrestrial vertebrates possibly regulate rates of enamel crystal growth and provide orientation for species-specific prismatic enamel [13, 18, 61, 63, 671. In summary, our results demonstrate that the 72-kDa and 46-kDa enamel proteins are synthesized and secreted by preameloblasts into the adjacent ECM prior to basal lamina removal and prior to overt ameloblast cytodifferentiation. These observations support the hypothesis that several enamel proteins are expressed in tandem with the initiation of ECM biomineralization and the proteins are synthesized prior to the expression of the 26-kDa amelogenin enamel protein. These results recapitulate the situation for enameloid formation in osteichthyes and chondrichthyes and suggest that multiple epigenetic signals from ectomesenchyme are responsible for the coordinated expression of the enamel gene family [68]. We are presently pursuing the experimental opportunities related to temporal and spatial coordinated enamel-gene expression during tooth morphogenesis. Acknowledgements. We wish to thank Dr. Alan Fincham for reading the manuscript and providing valuable discussion. We also thank Mr. Valentino Santos for photography and Mr. Dwain Lewis for assistance in the preparation of the manuscript. These studies were supported in part by research funds granted by the NIH, USPHS (DE-06425, DE-06988 and DE-02848). MLS is supported by a Research Cdreer Development Award from the NIH, NIDR.

References 1. Belcourt AB (1984) High molecular weight protein in developing enamel: their probable interaction. In: Fearnhead RW, Suga S (eds) Tooth Enamel IV. Elsevier, Amsterdam pp. 54C542 2. Belcourt AB, Fincham AG, Termine J D (1982) Acid-soluble bovine fetal enamelins. J Dent Res 61 : 1031-1032 3. Bonner WH, Laskey RA (1974) A film detection method for tritium-labeled proteins and nucleic acid in polyacrylamide gels. Eur J Biochem 46:83-88 4. Breitbart RE, Andreadis A, Nadal-Ginard B (1987) Alternative splicing: a ubiquitous mechanism for the generation of multiple protein isoforms from a single gene. Annu Rev Biochem 56:467495 5. Bringas P, Nakamura M, Nakamura E, Evans J, Slavkin HC (1987) Ultrastructural analysis of enamel formation during in vitro development using chemically defined medium. Scanning Microscopy International 1 : 1103-1 108 6. Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein-A. Anal Biochem 112:195-203 8. Cohn SA (1957) Development of the molar teeth in the albino mouse. Am J Anat 101 :295-320 10. Deutsch D, Shapira L, Abayoff A, Leviel D, Yoeli Z, Arad A (1984) Protein and mineral changes during prenatal and postnatal development and mineralization of human deciduous enamel. In: Fearnhead RW, Suga S (eds) Tooth Enamel IV. Elsevier, Amsterdam, pp. 234-239 11. Eastoe J (1979) Enamel protein chemistry - past, present and future. J Dent Res 58(B):753-764 12. Evans J, Bringas P, Nakamura M, Nakamura E, Santos V, Slavkin HC (1988) Metabolic expression of intrinsic developmental programs for dentine and enamel biomineralization in serumless, chemically-defined, organotypic culture. Calcif TissueInt42:l-12 13. Fearnhead RW (1979) Matrix-mineral relationships in enamel tissues. J Dent Res 588 :909-916 14. Fincham AG, Belcourt AB, Termine JD, Cothran WC, Butler WT (1981) Dental enamel matrix: sequence of two amelogenin polypeptides. Biosci Rep 1 :771-778 15. Fincham AG, Aptel JD, Belcourt AB (1984) The protein matrix of developing dental enamel: structure and function. I n : Bclcourt AB, Ruch JV (eds) Tooth Morphogenesis and Differentiation, Inserm, Paris, pp. 315-332 16. Gaunt WA (1955) The development of the molar pattern of the mouse (Mus Musculus). Acta Anat (Basel) 24:249-268 17. Gould SJ (1977) Ontogeny and pylogeny. The Belknap Press of Harvard University Press, Cambridge, Massachusetts 18. Greenberg G, Bringas P, Slavkin HC (1983) The epithelial genotype controls the pattern of extracellular enamel prism formation. Differentiation 25: 3 2 4 3 19. Hawkes R, Niday E, Gordon J (1982) A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem 119:142-1 47 20. Hay M F (1961) The development in vivo and in vitro of the lower incisor and molars of the mouse. Arch Oral Biol 3:8&109 21. Hayashi Y , Bianco P, Shimokawa H, Termine JD, Bonucci E (1986) Organic-inorganic relationships and immunohistochemical localization of amelogenin and enamelins in developing enamel. Basic Appl Histochem 30:291-299 22. Herold R, Graver H, Cristner PJ (1980) Immuno-histochemical localization of amelogenin in enameloid of lower vertebrate teeth. Science 207 :1357-1 358 23. Kagan A, Click M (1979) In: Taffc BM, Behram HA (eds) Methods of human radioimmunoassay. Academic Press, New York, pp. 328-329 24. Kallenbach E (1971) Electron microscopy of the differentiating rat incisor ameloblast. J Ultrastruct Res 35 :508-531

38 25. Kallenbach E, Piesco N P (1979) The changing morphology of the epithelium-mesenchyme interface in the differentiation zone of the growing teeth of selected vertebrates and its relationship to possible mechanisms of differentiation. In: Ruch JV (ed) Tooth morphogenesis I. Inserm, Paris, pp. 315-332 26. Keene DF, Sakai LY, Lunsprum GP, Morris NP, Burgeson RE (1987) TypeVII collagen forms an extended network of anchoring fibrils. J Cell Biol 104:611-621 27. Koch WE (1967) I n vitro differentiation of tooth rudiments of embryonic mice. Transfilter interactions of embryonic incisor tissues. J Exp Zool 165:155-170 28. Kollar EJ (1983) Epithelial-mesenchymal interaction in the mammalian integument. In: Sawyer RH, Fallon J F (eds) Epithelial-mesenchymal interaction in development. Praeger Publishers, New York, pp. 27-50 29. Kollar EJ, Baird G (1970) Tissue interaction in embryonic mouse tooth germs. 11. The inductive role of dental papilla. J Embryo1 Exp Morphol24: 173-1 86 30. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 68G695 31. Landis WJ (1983) Special techniques for problem tissues. In: Jones BR (ed) Electron Microscopy, Academic Press, New York, pp. 459476 32. Laskey RA, Mills aD (1975) Quantitative film detection of 3H and I4C i n polyacrylamide gels by fluorography. Eur J Biochem 56:335-341 33. Levine PT, Glimcher MJ, Seyer JM, Huddleston JI, Hein JV (1966) Noncollagenous nature of the proteins of shark enamel. Science 154: 1192-1 194 34. Lumsden AGS (1979) Pattern formation in the molar dentition of the mouse. J Biol Buccale 7 :77-103 35. Lumsden AGS (1987) The neural crest contribution to tooth development. In: Madcrson PFA (cd) Developmental and evolutionary biology of the neural crest. John Wiley and Sons, New York, pp. 3-43 36. Moss ML (1977) Skeletal tissues in sharks. Am Zool 17: 335-342 37. Nanci A, Bendayan M, Slavkin H C (1985) Enamel protein biosynthesis and secretion in mouse incisor secretory ameloblasts as revealed by high resolution immunocytochemistry. J Histochem Cytochem 33: 1153-1160 38. Nanci A, Slavkin HC, Smith CE (1987) Immunocytochemical and radioautographic evidence for secretion and intracellular degradation of enamel proteins by ameloblasts during the maturation stage of amelogenesis in rat incisors. Anat Rec 217(2): 107-123 39. O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007a021 40. Orvig T (1967) Phylogeny of tooth tissues: Evolution of some calcified tissues in early vertebrates. In: Miles AEW (ed), Structure and chemical organization of teeth. Academic Press, New York, Vol 1. pp. 45-110 41. Osbourne JW (1978) Morphogenetic gradients: Fields versus clones. In: Butler PW, Joysey KA (eds) Development, function and evolution of teeth. Academic Press, New York, pp. 171-201 42. Peyer B (1968) Comparative odontology, University of Chicago Press, Chicago, pp. 89-99 43. Poole DFG (1967) Endmeloid and enamel in recent vertebrates. In: Miles AEW (ed) Structure and chemical organization o f teeth. Academic Press, New York, Vol 1. pp. 111-149 44. Reith EJ (1970) The stages o f amelogenesis as observed in molar teeth of young rats. J Ultrastruct Res 30: 133-151 45. Robinson C, Kirkham J (1985) Dynamics o f amelogenesis as revcdled by protein amino acid composition studies. In: Butler W (ed) The chemistry and biology of mineralized connective tissues. Ebsco Media, Inc., Birmingham, Alabama, pp. 248-263 47. Ruch JV (1985) Epithelial-mesenchymal interactions. I n : Butler W (ed) The chemistry and biology o f mineralized connective tissues. Ebsco Media, Inc., Birmingham, Alabama, pp. 5 4 6 2 49. Samuel NB, Bessem C, Bringas P, Slavkin H C (1987) Immunochemical homology between elasmobranch scale and tooth ex-

tracellular matrix protein in Cephalscyllium ventriosum. J Craniofac Genet Dev Biol4:371-386 50. Shimokawa H, Wassmer P, Sobel ME, Termine J D (1984) Characterization of cell-free translation products of mRNA from bovine ameloblasts by monoclonal and polyclonal antibodies. In: Fearnhead RW, Suga S (eds) Enamel IV. Elsevier, Amsterdam pp. 163-166 51. Shimokawa H, Sobel ME, Sasaki M, Termine JD, Young M F (in press) Heterogeneity of amelogenin mRNA in the bovine tooth germ. J Biol Chem 52. Slavkin HC (1974) Embryonic tooth formation: A tool for developmental biology. In: Melcher AH, Zarh GA (eds) Oral sciences reviews, Vol. 4. Munksgaard, Copenhagen, pp. 1-1 36 53. Slavkin HC (1985) Regional specification of cell-specific gene expression during craniofacial development. J Craniofac Genet Dev Biol [Suppl] 1 : 57-66 54. Slavkin HC (1985) Current perspectives on enamel proteins. In: Butler W (ed), The chemistry and biology of mineralized connective tissues. Ebsco Media, Inc., Birmingham, Alabama, pp. 237-239 55. Slavkin HC, Bringas P (1976) Epithelial-mesenchymal interactions during odontogenesis 1V : Morphological evidence for direct heterotypic cell-cell contacts. Dev Biol 50:428-442 56. Slavkin HC, Mino W, Bringas P (1976) The biosynthesis and secretion o f precursor enamel protein by ameloblasts as visualized by autoradiography after tryptophan administration. Anat Rec 185:289-312 57. Slavkin HC, Zeichner-David M, MacDougall M, Bringas P, Bessem C, Honig L (1982) Antibodies to murine amelogenins: localization of enamel proteins during tooth organ development in vitro. Differentiation 23: 73-82 58. Slavkin HC, Zeichner-David M, Ferguson MWJ, Termine JD, Graham EE, MacDougall M, Bringas P, Bessem C, Grodin M (1982) Phylogenetic and immunogenetic aspects of enamel proteins. In: Rivere GR, Hildemann WH (eds) Oral immunogenetics and tissue transplantation. Elsevier, Amsterdam, pp, 241-251 59. Slavkin HC, Graham E, Zeichner-David M, Hildemann W (1983) Enamel-like antigens in hagfish : possible evolutionary significance. Evolution 37: 404412 60. Slavkin HC, Samuel N, Bringas P, Nanci A, Santos V (1983) Selachian tooth development: 11. Immunolocalization of amelogenin polypeptides in epithelium during secretory amelogenesis in Squalus acanthius. J Craniofac Genet Dev Biol 3 :43-52 62. Slavkin HC, Snead ML, Zeichner-David M, Bringas P, Greenberg GL (1984) Amelogenin gene expression during epithelialmesenchymal interactions. In: Trelstad R (ed) The role of extracellular matrix in development. Alan Liss, Inc., New York, pp. 221-253 62. Slavkin HC, Zeichner-David M, Snead ML, Graham EE, Samuel N, Ferguson MWJ (1984) Amelogenesis in reptilia: evolutionary aspects of enamel gene products. In: Ferguson MWJ (ed) The structure, development and evolution of Reptiles. Academic Press, New York, pp. 275-304 63. Slavkin HC, Zeichner-David M, Snead ML, Samuel N, Bessem C, Bringas P Jr. (1984) Genetic and phylogenetic aspects of enamel. In: Ruch JV, Belcourt A (eds) Tooth morphogenesis and differentiation. INSERM, Paris, pp. 341-354 64. Smith CE (1979) Ameloblasts, secretory and resorptive functions. J Dent Res 58 :695-706 65. Snead ML, Zeichner-David M, Chandra T, Robson KJH, Woo SLC, Slavkin HC (1983) Construction and identification of mouse amelogenin cDNA clones. Proc Natl Acad Sci USA 80:72547258 66. Snead ML, Bringas P, Bessem C, Slavkin HC (1984) De novo gene expression detected by amelogenin gene transcript analysis. Dev Biol 104:255-258 67. Snead ML, Lau E, Zeichner-David M, Fincham A, Woo SLC, Slavkin HC (1985) DNA sequence for cloned cDNA for murine amelogenin reveals the amino acid sequence for enamel-specific protein. Biochem Biophys Res Commun 12:812-818

39 68. Snead ML, Lau E, Zeichner-David M, Nanci T, Bendayan M, Bringas P, Bessem C, Slavkin HC (1987) Relationship of the ameloblast biochemical phenotype to morpho- and cytodifferentiation. In: Firtel A, Davidson E (eds) Molecular approaches to developmental biology. Alan Liss, New York, pp. 641-652 69. Strawich E, Glimcher MJ (1985) Synthesis and degradation in vivo of a phosphoprotein from rat dental enamel. Biochem J 230:423433 70. Suga s (1970) Histochemical observations of proteolytic enzyme activity in the developing dental hard tissues of the rat. Arch Oral Biol 15 : 555-558 71. Takagi T, Suzuki M, Baba T, Minegishi K, Sasaki S (1984) Complete amino acid sequence in developing bovine enamel. Biochem Biophys Res Commun 121 : 592-597 72. Termine JD, Belcourt AB, Christner PJ, Conn WM, Nylen MU (1980) Properties of dissociatively extracted fetal tooth matrix proteins. I. Principal molecular species in developing bovine enamel. J Biol Chem 255:9760-9765 73. Theiler K (1972) The house mouse. Springer-Verlag, Berlin, pp. 109-143 74. Thesleff I, Hurmerinta K (1981) Tissue interactions in tooth development. Differentiation 18: 75-88 75. Thompson SW, Hunt RD (1966) Histochemical procedures: Von Kossa staining for calcium. In: Selected histochemical and histopathological methods. Thomas Publishers, Springfield, 11linois, pp. 581-584

76. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitro-cellulose sheets: procedures and some applications. Proc Natl Acad Sci USA 76:43504354 77. Warshawsky H (1968) The fine structure of secretory ameloblasts in rat incisors. Anat Rec 161:211-230 78. Watt IM (1985) The principles and practice of electron microscopy. Cambridge University Press, England, pp. 117-195 79. Weinstock A, Leblond CP (1971) Elaboration of the matrix glycoprotein of enamel by the secretory ameloblasts of the rat incisor as revealed by radioautography after g a l a ~ t o s e - ~injecH tion. J Cell Biol51: 26-43 80. Zeichner-David M, MacDougall M, Slavkin HC (1983) Enamelin gene expression during fetal and neonatal rabbit tooth organogenesis. Differentiation 25: 148-1 53 81. Zeichner-David M, MacDougall M, Vides J, Snead ML, Slavkin HC, turkel SB, Pavlova A (1987) Immunochemical and biochemical studies of human enamel proteins during neonatal development. J Dent Res 66: 5&56 82. Zeichner-David M, Vides J, MacDougall M, Fincham A, Snead ML, Bessem C, Slavkin HC (1988) Biosynthesis and characterization of rabbit tooth enamel extracellular matrix proteins. Biochem J (in press) Received October 1987 / Accepted in revised form December 31, 1987