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American Journal of Medical Genetics Supplement 47-22 (1988)

Molecular Determinants of Cranial Neural Crest-Derived Odontogenic Ectomesenchyme During Dentinogenesis

Harold C. Slavkin, Mary MacDougall, Margarita Zeichner-David, Peter Oliver, Masanori Nakamura, and Malcolm L. Snead

Laboratory for Developmental Biology, Department of Basic Sciences, School of Dentistry, University of Southern California, L 0s Angeles, California

Positional information on tooth morphogenesis is investigated by the identification of when and where phenotypic markers are expressed during odontogenesis. This temporal and positional information is correlated with the instructive and permissive signaling required for both dentinogenesis and amelogenesis. Of particular interest is the establishment of a map for the cranial neural crest-derived dental papilla ectomesenchyme and the odontoblast cell lineages. The expression of ectomesenchyme- derived cytotactin, dentin phosphoprotein, and epithelial-derived enamel proteins was studied in mice using embryonic, fetal, and postnatal mandibular first molar tooth organ development. This review summarizes the observations in the context of instructive epithelial-mesenchymal interactions and suggests that amelogenesis imperfecta and dentinogenesis imperfecta may in part be explained by alterations in these differentiation markers. Recombinant DNA methods should facilitate future investi- gations of these inherited dental disorders.

Key words: cytotactin, dentin phosphoprotein, amelogenin, enamelins, inner enamel epithelia, odontobhts, tooth morphogenesis, epithelial-mesenchyml interactions, extracellular matrix

INTRODUCTION

One of the most cogent problems in developmental biology concerns how tissue- specific gene expression is regionally specified during embryonic, fetal, and postnatal morphogenesis. Cranial neural ixest cells produce embryonic progenitor lineages for a number of different neuronal and nonneuronal cell types, including chondroblasts, fibroblasts, osteoblasts, and odontoblasts during craniofacial morphogenesis [40,41,48,73]. During embryogenesis, caudal mesencephalic and rostra1 rhombencephalic cranial neural crest migrate through pathways along the epithelial basal lamina, the extracellular matrix spaces, and other cues derived from other embryonic cell types en route to the forming maxillary and mandibular processes. How are cranial neural

Received for publication June 2,1987; revision received June 26,1987.

Address reprint requests to Dr. Harold C. Slavkin, Laboratory for Developmental Biology, Gerontology Center, Rm 314, University of Southern California, Los Angeles, CA 90089-0191.

0 1988 Alan R. Liss, Inc.

8 Slavkinetal.

crest-derived dental ectomesenchymal cells instructed to express substrate adhesion molecules (e.g., cytotactin) according to spatiotemporal coordinates, to influence the morphogenetic patterning of tooth organs, to provide inductive signals for enamel organ epithelial cytodifferentiation, and subsequently to express tissue-specific dentin phosphoprotein and other dentin extracellular matrix constituents within odontoblasts?

Premigratory cranial neural crest cells do not appear to be preprogrammed craniofacial cell lineages. Rather, crest cells appear to be instructed according to the temporal and microenvironmental conditions within which they migrate [2,21,40,41,48,73]. This general type of structural gene regulation by epigenetic signals has previously been described by Gurdon [22] as “cell-type specificity of gene control.” Considerable progress has been made in the last few years on several experimental strategies designed to understand when, where, and how cranial crest cells become unique regional ectomesenchyme that in turn provide signals for tissue-specific epithelial cytodifferentiation during the process of mammalian tooth development. This review highlights recent progress related to the problems of 1) molecular determinants of cranial neural crest-derived odontogenic ectomesenchyme during dentinogenesis and 2) molecular processes suggested for instructive extracellular matrix influences on gene expression during mammalian tooth morphogenesis and cytodifferentiation. The recent and rapid advances in recombinant DNA methodology applied to problem-solving in experimental embryology, molecular biology, and human medical genetics offers substantial promise toward understanding the molecular determinants of craniofacial development.

EPITHELIAL-MESENCHYMAL INTERACTIONS DURING TOOTH MORPHOGENESIS

Epithelial-mesenchymal interactions are defined as reciprocal interdependent tissue interactions that result in profound changes in one or both of the tissue interactants (e.g., thyroid, mammary gland, lung, kidney, tooth, hair, scales, feather, and skin) [ I9,20,3& 35,40,41,55,63]. Induction of epithelial development by region-specific mesenchyme- derived instructive signals may be mediated by 1) close cell-cell contact between heterotypic tissues, 2) autocrine and/or paracrine factors produced by mesenchyme and/or epithelia, and/or 3) by extracellular matrix constituents accumulating between the two tissue interactants [2,8,11,12,14,15,19,20,23,3 1-35,49,55,59,63,69].

MANDIBULAR FIRST MOLAR TOOTH MORPHOGENESIS IN THE MOUSE ANIMAL MODEL SYSTEM

During late neurulation of the mouse embryo, as the trigeminal ganglion is forming between 8.5 and 9.5 days gestation (Theiler stage 15), caudal mesencephalic and rostra1 rhombencephalic levels of neural crest cells migrate into the developing maxillary and mandibular processes and differentiate into ectomesenchyme cell lineages that subsequently become chondroblasts, osteoblasts, dental papilla ectomesenchyme, odontoblasts, and fibroblasts [40,41,73]. By Theiler stage 16, mandibular processes merge at the midline to form the mandibular arch. Discrete populations of ectcdermally derived epithelial cells proliferate, resulting in the formation of a four to five cell thick oral ectodermal region corresponding to the bilateral positions for molariform teeth at Theiler stage 18. At Theiler stage 20 (12 days gestation), the oral ectoderm invaginates into the ectomesenchyme to form dental laminae, which delineate incisor and molar positions. At Theiler stages 22-23 (1 3 days gestation) M, bud stage morphogenesis is apparent, and by 14 days of gestation the enamel organ divides into inner and outer enamel epithelia

Neural Crest-Derived Odontogenic Ectomesenchyme 9

observed along the anterior or medial surfaces of the forming MI. At 15-16 days gestation the enamel organ becomes concave at the interface between the inner enamel epithelia and adjacent presumptive odontoblasts (initial odontoblast differentiation is evident by immunolocalization of dentin phosphoprotein late on day 16 in MI), resulting in cap stage morphogenesis [43]. Theiler stages 24-25 represent late cap stage and indicate the positions for five forming cusps (three buccal and two lingual) that characterize the molariform of MI. The bell and subsequent crown stages further define the major cusps and intercuspal regions during the neonatal and early postnatal phases of development. M, crown dentinogenesis extends from 16 days gestation (initial odontoblast cytodifferentiation) through 12-1 5 days postnatal development; biomineralization is first detected at 19 days gestation along the occlusal region of the major midbuccal cusp. Amelogenesis, as defined by the first expression of enamel proteins, begins at 18 days gestation and continues through postnatal development [57]. The position of the dentin-enamel junction (DEJ) is evident at 20 days postfertilization [57]. Root formation extends from 5 through 28 days postnatal development, when the mandibular MI tooth surface occludes with the opposing maxillary M, tooth surface [ 18,28,32-35,4O,4l,49,57,6O761,63,69].

During the initial determination of mouse tooth morphogenetic positions, branchial arch-derived epithelia appear to provide positional information for subsequent tooth development. Recent evidence demonstrated that heterotypic tissue recombinations between first branchial arch epithelia and second branchial arch ectomesenchyme showed that early mandibular arch epithelia, before 12 days of gestation, possess odontogenic potential and can elicit the formation of a dental papilla in nonodontogenic neural crest-derived ectomesenchyme of the second branchial arch [4&42,46]. Mandibular ectomesenchyme appears to be required to interact with mandibular epithelia to possess competence to induce nonodontogenic epithelia to produce tooth organs [4O-42,46]. Subsequent determination for tooth morphogenesis (e.g., incisiform and molariform), and epithelial cytodifferentiation to become ameloblasts and to produce the enamel extracellular matrix, are regulated by regional mesenchyme-specific instructions [ 32-35,4O- 42,49,55,69]. However, the significant developmental problem remains: When, where, and how do epithelia and/or ectomesenchyme signal during instructive and permissive interactions (Fig. l)?

i n s t r u c t i v e in f luences

p e r m i s s i v e i n f l u e n c e s and m a i n t e n a n c e

PROGRESSIVE DIFFERENTIATION OF

> EPITHELIA AND/OR ECTOMESENCHYME

Fig. 1. Reciprocal and interdependent epithelial-mesenchymal interactions are requisite for morphogenesis and cytcdifferentiation in a number of developing epidermal organ systems. Two general types of interaction have been identified: 1) instructive interactions, in which either epithelia or mesenchyme induce the other tissue to express a unique biochemical and cytological phenotype (e.g., dental papilla ectomesenchyme induce inner enamel epithelia to express enamel proteins and differentiate into secretory ameloblasts); and 2) permissive interactions, in which tissues provide maintenance and stabilization of already expressed phenotypes.

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COUPLED EXPRESSION AND COLOCALIZATION OF CELL ADHESION AND SUBSTRATE ADHESION MOLECULES DURING TOOTH MORPHOGENESIS AND ODONTOBLASTS AND AMELOBLAST CYTODIFFERENTIATION

It is well-documented that cell-cell and cell-extracellular matrix interactions participate in morphogenetic patterning during embryogenesis [2,6,8,10,14,15, 19,20,24,27,3&35,41,49,60,63]. The regulation of coordinating the timing and position of epithelia and mesenchyme may reside in the sequential expression of cell transmembrane linkage molecules (e.g., integrin), cell surface adhesion molecules (CAMs), and substrate or extracellular matrix (ECM) adhesion molecules [2,11,14,15,20,23,26,31,49, 55,60, 63,691. Such a regulatory scheme assumes that cell surface adhesion molecules (e.g., L-CAM, N-CAM) and/or substrate adhesion molecules (e.g., fibronectin, laminin, cytotactin, and tenascin) are expressed at defined times and positions during development [8-12,14,15,21-27,30,50,5 1,54,68-711. A substantial literature now provides a number of examples suggesting that the extracellular matrix and the cytoskeleton of cells are intimately related [2,8-12,21,23-27,30,38,50,5 1,57,63,66,69]. For example, the fibronectin (FN) cell attachment domain binds to a 140 kDa FN receptor complex that codistributes with intracellular microfilament bundles at the cell-substrate adhesion sites; a sequence of Arg-Gly-Asp-Ser within the FN cell attachment domain appears to provide the attachment biological activity [9,54]. A biophysical connection between extracellular matrix constituents (e.g., fibronectin) and intracellular cytoskeletal constituents (e.g., actin) has been established [2,9,23-27,30,5 1,54,66]. Crossin et al. [ 111 and Edelman [ 151 have recently shown that the N-CAM glycoprotein is present in neural crest cells before migration from the neuroepithelium, disappears during migration, and reappears once neural crest cells reach their final destination and further differentiate. A major interpretation of this scheme is that the sequential expression and distribution of transmembrane linkage molecules, CAMs, and ECM molecules implies developmental regulation, presumably associated with regional specification of morphogenetic patterns and tissue-specific cytodifferentiation.

Site-Restricted Expression of Cytotactin During Cranial Neural Crest-Derived Ectornesenchyme Differentiation

An extracellular matrix glycoprotein, cytotactin, has recently been isolated and partially characterized [2 11. Cytotactin appears to be a cell-substrate adhesion molecule found in both neural and nonneural tissues, synthesized and secreted from cells and consisting of related polypeptides of 220, 200, and 190 kDa associated through disulfide linkages to form a complex polymer [ 12,211. Cytotactin, like laminin and fibronectin, are disulfide-linked, high-molecular-weight ECM molecules that all bind to proteoglycans. Immunolocalization of cytotactin during development has identified distribution along cell surfaces, within the ECM, and within basement membranes [21]. Whereas cytotactin was originally purified from 95,000 g supernatants of homogenates of 14-day embryonic chick brains, it appears to be identical to the mouse J1 glycoprotein, which has been found to mediate neuron-glial interactions, and to tenascin isolated from the conditioned medium of primary chick embryo fibroblast cultures [ 101.

The tissue distribution of cytotactin during mouse M, morphogenesis finds cytotactin localized along discrete positions of the basement membrane associated with dental lamina epithelium during 12 days gestation. At this stage of M, morphogenesis, the thickened epithelium is surrounded by a condensing cranial neural crest-derived ectomesenchyme. By 13 days gestation the tissue distribution of cytotactin includes a localized


Fig. 2. Immunocytochemical localization of a substrate adhesion molecule, cytotactin, during selected stages of mouse M, morphogenesis and cytdifferentiation. A Cranial neural crest-derived ectomesenchyme cells adjacent to the thickened 12-day mouse embryonic presumptive molar region. Arrows indicate localization in basement membrane. dl, dental laminus B Initial cytotactin distribution along the basement membrane and adjacent presumptive dental papilla ectomesenchymal cells shown in A. epi, epithelia C The distal surface of the bud stage tooth organ shows cytotactin localization in discrete patches along the basement membrane and adjacent ectomesenchyme. eo, enamel organ D Higher magnification showing punctate localization (arrows). E Bell stage tooth organs (19 days postcoitus) show no cytotactin localization within odontoblasts and dental papilla ectomesenchyme, except in regions of enhanced vascularization (arrows). iee, inner enamel epithelia; dpm, dental papilla ectomesenchyme. F Region in E (asterisk) showing vascular elements with cytotactin. Arrows indicate location of mesenchyme. od, odontoblasts; iee, inner enamel epithelia.

subpopulation of ectomesenchyme and a discrete region of the basement membrane (Fig. 2). At 14 days gestation (bud stage), additional dental papilla ectomesenchyme expresses cytotactin in areas suggested with expansion of the M, tooth form. The basement membrane is not uniformly labeled, but rather shows immunostaining that is site- restricted to the center of the tooth bud and the extreme perimeter surfaces adjacent to the

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outer enamel epithelia. This distribution pattern continues through the cap stage, with cytotactin presumably becoming site-restricted to the nonexpanding regions of the enamel organ epithelial basement membrane and adjacent ectomesenchyme cells, similar to the patterns described for type I11 collagen and fibronectin [38,68-701.

Major changes during enamel organ epithelial morphogenesis occur during the cap and bell stages of tooth formation. Cytotactin immunostaining was inversely related to odontoblast differentiation; cranial neural crest-derived dental ectomesenchyme cells that were stained for cytotactin lost this staining as they differentiated into the odontoblast lineage.

During early bell stages (1 8 days gestation), immunostaining was observed primarily in regions similar to distribution patterns previously reported for type I11 collagen and fibronectin [70]. During late bell and early crown stages of tooth development (i.e., 19 days gestation), cytotactin immunostaining is restricted to regions of increased vascularization. The present evidence suggests that major qualitative changes in the expression of cytotactin take place during embryonic and fetal stages of M, development. Detailed aspects of these findings will be published elsewhere [Slavkin et al., in preparation]. Comparable observations have been reported for tenascin during fetal rat tooth development [ lo].

Basal Lamina Constituents Laminin, Type IV Collagen, Basement Membrane Heparan Sulfate Proteoglycan, and Fibronectin Expression During Embryonic and Fetal Mouse Molar Tooth Development

Adhesive interactions of cells with their ECM appear to mediate morphogenetic movements and cytodifferentiation during embryonic and fetal development [2,8- 12,14,15,19,20,23-25,30,49,50,54,59,66,69]. These intricate processes could be guided by timing and positional changes in the composition and localization of cell adhesion molecules within the plasma membrane surface that interacts with ECM molecules such as laminin, fibronectin, cytotactin, types I, 11, 111, IV, and VII collagens, proteoglycans, and glycosaminoglycans. Of course, it becomes readily apparent that changes in the basal lamina, ECM, and/or CAMS could guide or direct developmental events such as epithelial morphogenesis in the forming tooth organ, odontoblast cytodifferentiation, ameloblast cytodifferentiation, and the formation of the dentin and enamel ECM [47,49,55,58,60,63,69]. Regulation of instructive and/or permissive cell-matrix interactions could be accomplished by transmembrane molecules (e.g., talin, vinculin, and integrin) facilitating cell adhesion molecule/ECM interactions outside of the cell and cytoskeletal constituents inside the cell [2,9,20,24-27,30,50,5 1,54,66].

The distribution of fibronectin, basement membrane proteoglycans, laminin, and types I, 111, and IV collagens have been investigated by indirect immunofluorescence microscopy during embryonic, fetal, and neonatal stages of mouse molar tooth organogenesis [6,38,57,6&70]. During bud stage morphogenesis (14 days gestation), fibronectin and type I11 collagen were colocalized throughout the condensing dental papilla ectomesenchyme and within the basement membrane [70]. Laminin, fibronectin, proteoglycan, and type IV collagen were colocalized in the dental basement membrane throughout early bud and cap stage morphogenesis 138,681. During initial odontoblast differentiation (cap stage, 16 days gestation) and thereafter, fibronectin and type I11 collagen localization was no longer detected; type I collagen was detected in association with odontoblasts and the production of the dentin ECM [38,68-701. Regions of dentin biomineralization were associated with a loss of basement membrane along the undersurface of the inner enamel epithelia, and they increased positive immunostaining for type I collagen [69].


Three functions of the basement membrane are suggested during tooth morphogenesis: 1) to regulate nutrient and regulatory growth factor diffusion between ectomesenchyme and epithelia; 2) to facilitate major morphogenetic patterns within the enamel organ epithelia through transmembrane linkage molecules, cell adhesion molecules, and substrate or ECM adhesion molecules [48]; and 3) to serve as a required substratum for ectomesenchyme cell processes and odontoblast cytodifferentiation [49,69]. Relatively anionic enamel proteins, other than amelogenins, are expressed by inner enamel epithelia associated with an intact basal lamina [57]. Initial extracellular matrix biomineralization appears before the removal of the basement membrane [57]. Following basal lamina removal from the Kallenbach terminology of differentiation zones V-VI inner enamel epithelia [28], ameloblasts then synthesize and secrete the 26 kDa amelogenin protein associated with enamel extracellular matrix formation and subsequent biomineralization [55,56,57,60,6 1,631.

Dentin Phosphoprotein Expression by Odontoblasts

Dentin phosphoprotein (DPP) is a tissue-specific biochemical marker that characterizes the odontoblast cell lineage. During tooth development odontoblasts express types I and V and type I trimer collagens, several proteoglycans, glycoproteins, glycosaminoglycans, gamma-carboxyglutamate-containing proteins (GLA proteins or dentin osteocalcin), and dentin phosphoprotein, which form the dentin extracellular matrix [39,72].

DPP is a highly phosphorylated (P-serine) acidic protein (isoelectric point approxi- mating 1 ,O), with serine and aspartic acid amino acid residues representing approximately 70-8895 of the total amino acids [39,72]. At the moment only limited primary structure data are available; however, it has been suggested that bovine DPP contains repetitive blocks of the type (Asp)n, (P-Ser)n, and (Asp-Y)n, where Y is serine or phosphoserine [36,37]. A partial amino terminus sequence for rat DPP is Asp-Asp-Asp-Asn and Asp-Asp-Pro-Asn [7]. Polyclonal antibodies have been produced against mouse DPP [44], and these have been used to establish an exquisite site-restricted expression of DPP within odontoblasts [43]. Preliminary characterization indicates that mouse DPP mRNA appears to code for a 43 kDa polypeptide in contrast to the 72 kDa archetypal mouse dentin phosphoprotein that characterizes the mineralizing dentin ECM [45]. We assume this apparent discrepancy is due to the fact that DPP is a highly posttranslationally modified dentin ECM constituent.

A biological function for DPP has been suggested based on its strong affinity for calcium ions [39,72]. This physicochemical property can be used in the isolation, purification, and partial characterization schemes for calcium-binding proteins, including DPP [39,72].

Additional evidence for DPP serving a putative function in the regulation of dentin calcium hydroxyapatite crystal formation comes from recent immunolocalization studies that report the distribution of mouse DPP within forming odontoblasts during early cap stage M, morphogenesis (16 days gestation), well in advance of the initiation of extracellular matrix mineralization [43]. Thereafter, DPP is localized only within the odontoblast cytoplasm enriched in the Golgi region, the odontoblast cell processes, and subsequently secreted at the mineralizing front of the dentin ECM at 19 days gestation; no immunostaining is observed within the adjacent dental papilla ectomesenchyme cells or in the predentin ECM [43] (Fig. 3). This site-restricted expression pattern is also reported for osteocalcin or dentin GLA protein during rodent tooth development [5,13,17]. However, osteocalcin is not tissue specific for odontoblasts in that it is also found in forming bone appearing after the initiation of osteogenic mineralization [ 131.

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Fig. 3. lmmunocytochemical localization of dentin phosphoprotein (DPP) during the process of ectomesenchyme differentiation into odontoblasts. The initial dontoblast phenotype shows positive immunostaining with antimouse DPP plyclonal antibody in differentiation zones 11-111 [28]. Localization of DPP is restricted to the odontoblasts and their cell processes until dentin extracellular biomineralization first appears; DPP is then identified at the mineralization front (arrows) but is not detected within the predentin regions of the forming mouse molar. iee, inner enamel epithelia; dpm, dental papilla ectomesenchyme.

Enamel Protein Expression by Inner Enamel Epithelia

Simplistically, the biochemical phenotype of the mammalian ameloblast is repre- sented by two classes of enamel proteins-amelogenins and enamelins [67]. The amelogenins are relatively hydrophobic proteins of lower molecular weight (5-30 kDa), and they consist of enriched levels of proline, glutamic acid, leucine, and histidine. During enamel matrix formation amelogenins represent at least 90% of the total enamel protein [%I. These neutral (PI 6.5-7.5 polypeptides appear to regulate the size and rates of enamel calcium hydroxyapatite crystal growth. Recently, several mammalian amelogenin cDNAs have been produced, sequenced, and used to predict the complete primary structure of bovine and mouse amelogenin [53,62,64]. Both amelogenin cDNA probes hybridize with human genomic DNA [53,64].

The enamelins, in contrast, are relatively hydrophilic proteins of higher molecular weight (circa 68-72 kDa), represent perhaps 1-3% of the total enamel matrix protein, and are characterized by a high content of glycine, aspartic acid, serine, and glutamic acid [67]. These relatively anionic molecules appear to be synthesized and secreted before amelogenins and may serve to initiate calcium hydroxyapatite crystal formation [ 57,671. Polyclonal antibodies have recently been reported for human enamelin [75]. The de novo expression of rabbit enamelin mRNA during late cap and early bell stages of molar tooth morphogenesis has been described [74]. Comparisons between ameloblast poly(A)- enriched RNA fractions expressed in a cell-free translation system show a high degree of conservation among enamel translation products in most mammals [76].


TABLE I. Sequential Enamel Protein Expression During Mouse Tooth Ogranogenesis*

Enamel proteins

Days of development (postcoitus) M W (kDa) PI - - 16 .-

I /

18 19

20 (birth)

- - 46 5.5 12 5.8 46 5.5 12 5.8 46 5.5 26 6.5-6.1

*11:08 AM.

Fig. 4. Immunocytochemical localization of enamel proteins within inner enamel epithelia (iee), differentiation zones IV-VI, and ameloblasts (am) during mouse molar tooth development. Antimouse amelogenin polyclonal antibodies were used to identify intracellular and forming enamel extracellular matrix distribution of enamel proteins. Dental papilla ectomesenchyme (dpm), odontoblasts (od), and dentin matrix (d) were not stained.

The biochemical phenotype of the inner enamel epithelia has recently been defined according to the coordinated sequence of enamel proteins synthesized and secreted during ameloblast cytodifferentiation in the embryonic, fetal, and postnatal stages of mouse MI [57]. Table I summarizes the sequence of enamel protein expression during M, morphogenesis. According to Kallenbach’s description [28] of inner enamel epithelial “differentiation zones” I-VI, a 46 kDa enamel protein is first expressed by differentiation zones Ill-IV at 18 days gestation; the basement membrane is continuous at this stage of tooth development [57] (Fig. 4). At 19 days gestation the 46 kDa enamel protein and another 72 kDa enamel protein are expressed by differentiation zones 111-VI; the basement membrane is discontinuous in differentiation zone V and completely removed in differen-

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tiation zone VI [50]. At 20 days postfertilization, these relatively acidic (PI 5.5) enamel proteins continue to be produced, and a third amelogenin polypeptide of 26 kDa is synthesized and secreted into the enamel matrix by functional secretory ameloblasts [57,61]. Thereafter, increased numbers of amelogenins, ranging from 5 to 30 kDa and presumably derived from posttranslational processing, become the major constituent of the enamel ECM [56]. With advanced enamel mineralization, termed enamel maturation, water and protein are removed from the enamel ECM coincident with increased calcium hydroxyapatite crystal formation, eventually forming a 99.9% inorganic enamel tooth covering.

EPIGENETIC REGULATION OF ODONTOGENIC EPITHELIAL-MESENCHYMAL INTERACTIONS

When, where, and how do epithelia and/or ectomesenchyme signal during instructive interactions related to tooth morphogenesis and cytodifferentiation?

Epigenetic Regulation

Epigenetic regulation operates during tooth development; heterotypic tissue recombinations between dental and nondental epithelia and/or ectomesenchyme provide results that indicate that presumptive dental epithelia initiate dental morphogenetic fields of ectomesenchyme [40-42,46] and that region-specific dental ectomesenchyme induces tooth form and epithelial differentiation into ameloblasts and the production of enamel [ 32-35]. The dental extracellular matrix appears to regulate epithelial and ectomesenchy- ma1 cytodifferentiation [47,49,55,5840,63,65,68-701.

Cell-Cell and Cell-Matrix Interactions

Cell-cell and cell-matrix interactions regulate morphogenesis and cytodifferentiation during development [2,15,20,24,33,41,49,55,63]. Migrating neural crest cell surface galactosyltransferase activity recognizing and binding to terminal N-acetylglucosamine residues on glycoconjugates within the extracellular matrix [50] may provide temporal and positional cues for crest cell pathways during embryogenesis. Cysteine-rich repeats within the laminin B1 chain bind to cell adhesion molecules and promote cell-cell aggregation and cell growth [51]. Integrin is one of the complex of cell membrane glycoproteins that form part of the transmembrane connection between the ECM and the cytoskeleton [26]. One paradigm for cell-cell and cell-matrix interactions is the suggested functions of transmembrane receptor molecules (e.g., talin, integrin) to bind specifically to domains of a number of ECM molecules (e.g., fibronectin, laminin, vitronectin, cytotactin, various collagens, and heparin sulfate-containing proteoglycans) [6,9,15,21,25,27,30,54]. Epithelial polarity is influenced by collagen components within the extracellular matrix [2,6,8,30]. Epithelial cell surface heparin sulfate proteoglycans, consisting of a core protein bearing polyanionic heparin sulfate chains and localized within the apical basal lamina, may facilitate cell-matrix interactions via specific binding to type I collagen or to other ECM constituents [30]. Another dimension of this model is the interactions between transmembrane receptors, CAMS, and ECM or substrate adhesion molecules (SAMs) [ 1 1,15,26]. Each of these models predicts coupled expression and colocalization of specific molecules during the time and position within complementary epithelial and ectomesenchymal cell lineages and their extracellular matrix boundary (Fig. 5).

Tooth Morphogenesis and Cytodifferentiation

Several opportunities to pursue these questions experimentally have recently been established. First, branchial arch-derived mandibular epithelia appear to instruct ecto-

Neural Crest-Derived Odontogenic Ectomesenchyme

I I I Ill

17

I V V V I

Fig. 5 . Summary of sequential inner enamel epithelial and ectomesenchymal cytodifferentiation according to the scheme of Kallenbach [28] (“differentiation zones I-VI”) indicating molecular determinants expressed according to restricted temporal and positional regulation. I: Cytotactin is localized to mesenchymal cells (mes) and basal lamina (bl) at the epithelial-mesenchymal interface. Microfilaments (mf) may be type VII collagen fibrils associated with anchoring filaments. iee, inner enamel epithelia. Ik Cytotactin is no longer evident, and dentin phmphoprotein is detected in preodontoblast cell lineage. af, anchoring filament; mv, matrix vesicles. Ilk preameloblasts produce a 46 kDa enamel protein, and apical cell membrane undulates along basal lamina (arrows). Odontoblast cell processes (cp) are evident, and cells are DPP positive. rV: Preameloblast basal lamina becomes discontinuous and is eventually removed. DPP is localized within odontoblasts and cell processes. mv, matrix vesicles. V: Initial mineralization is observed in matrix vesicles, and 46 and 72 kDa enamel proteins are expressed and secreted by epithelial cells. VI: Preameloblasts produce 46 and 72 kDa enamel proteins into the forming enamel matrix (e), and DPP is localized within odontoblasts, cell processes, and along the forming dentin mineralizing front (d).

mesenchyme (prior to 12 days gestation during mouse embryogenesis) to aggregate into a dental morphogenetic pattern [40,41]. The initial immunolccalization of cytotactin along the presumptive dental lamina epithelial basement membrane suggests a possible partici- pation during instructive epithelial-mesenchymal interactions associated with dental pattern formations. The molecular determinant cytotactin serves to identify specific subpopulations of cranial neural crest-derived dental ectomesenchyme prior to the formation of a dental papilla and prior to odontoblast cytodifferentiation.

Second, the loss of cytotactin immunostaining coincident with odontoblast cytodifferentiation may also serve as an important factor in determining when and where basement membrane constituents mediate the putative induction of DPP.

Third, the transient colocalization of fibronectin and type I11 collagen during major morphogenetic changes in bud and cap stages provide additional markers for when and where ectomesenchyme mediates epithelial morphogenesis [70].

Fourth, a number of ectomesenchyme-derived odontoblast phenotypic changes appear to precede and accompany major differential gene expression within the adjacent inner enamel epithelial (proliferation and differentiation zones I-VI) [5,13,1& 18,28,38,39,43,47,49,58,69]. Inner enamel epithelia produce both types I and IV collagens [71] and may also produce type VII collagen, which serves to form anchoring fibrils along the undersurface of the basement membrane [26]; anchoring filaments may facilitate odontoblast cytodifferentiation [29,49,58].

Fifth, during the late cap or early bell stages inner enamel epithelia have become determined and initiate a sequential pattern for enamel protein synthesis and secretion [57] (Table I).

18 Slavkin et al.

CHROMOSOMES 1

proteins t ransmembrane

o y t o s k e l e t o n

/ r e c e p t o r s plasma membrane \ , I 1 1 , I

I

extra? e I I u ,,or rn at * I1 r i x I “ I ? A

ECTOMESENCHYME INSTRUCTIONS

Fig. 6. General scheme for ectomesenchyme-derived instructions for epithelial cytodifferentiation. Putative signals (e.g., epidermal growth factor, laminin, heparin sulfate protmglycan, entactin, cytotactin, and fibronectin) serve as developmental information providing regional specification for epithelial cytodifferentiation. The transmission mode enlists cell-cell and/or cell-matrix communication. Fxtomesenchymederived signals may be received by epithelial integral plasma membrane glycoproteins, termed transmembrane receptors ( eg , talin, integrin), which extend from the interior of the epithelial cell, through the plasma membrane, and into the basal lamina and adjacent extracellular matrix. Epithelial reception of the signal(s) affects a series of steps that provide biophysical linkages between the extracellular matrix, transmembrane receptors, the cytoskeleton matrix, and the nuclear matrix, resulting in differential gene expression.

Finally, cap stage molar tooth organs cultured organotypically have recently been shown to express morphogenesis through crown formation, odontoblast and ameloblast cytodifferentiation, dentin and enamel extracellular matrix production, and dentin and enamel tissue-specific patterns of biomineralization in serumless, chemically defined medium using in vitro culture conditions [4,16]. We interpret these studies to suggest that intrinsic autocrine and paracrine regulatory factors (e.g., fibroblast growth factor, epidermal growth factor, transferrin, insulin-like factor, and so forth) produced by ectomesenchyme and/or epithelia during in vitro morphogenesis mediate instructive and/or permissive morphogenesis and cytodifferentiation in the absence of serum or exogenous steroid or polypeptide hormones (Fig. 6 ) .

Human Genetic Studies

Amelogenesis imperfecta (AI) and dentinogenesis imperfecta (DGI) are inherited dental disorders that reflect alterations in regulatory and/or structural genes associated with enamel (e.g., types I, IV, and VII collagens, amelogenins, enamelins, and enamel peptidases) and dentin (eg, heparin sulfate proteoglycans, fibronectin, cytotactin, types I and 111 collagens, dentin phosphoproteins, dentin GLA-protein, and vitamin D-binding protein) formation. DGT is an inherited defect affecting both primary and secondary dentitions. DGI has been classified into three subtypes: type I (DGI-I), which is always associated with osteogenesis imperfecta; type I1 (DGI-IT), being the more common or “classical hereditary DGI”; and type I11 (DGI-III), originally found in a large inbred population known as the “Brandywine isolate” located in southern Maryland [52]. Based on a vitamin D-binding protein groupspecific component (Gc) localized to bands ql1-13 of human chromosome 4 by deletion mapping and on DGI-I1 linkage to Gc, DGI-I1 has been assigned to chromosome 4 [l]. More recently, an autosomal dominant form of juvenile periodontitis has been localized to chromosome 4 with linkage to DGI-111 [3]. The


recent advances in biochemistry, immunochemistry, developmental biology, and recombinant DNA technology applied to human genetic diseases offer a number of approaches toward understanding the molecular genetics of A1 and DGI. Antibodies now available that are directed against molecular determinants expressed during normal amelogenesis and dentinogenesis (e.g., cytotactin, N-CAM, L-CAM, integrin, laminin, fibronectin, type VII collagen, vitamin D-binding proteins, calcium-binding glycoproteins, dentin phosphoprotein, and enamel proteins) can be used to screen cDNA libraries. Tissue-specific and position-specific cDNA probes, which hybridize with human genomic DNA, for example, enable restriction fragment length polymorphism (RFLP) analysis of multiple-generation kindred, containing individuals affected with A1 or DGI. The intellectual and collabora- tive opportunities, therefore, between experimental embryology, molecular biology, and human medical genetics may provide for rapid advances in understanding the molecular genetics of inherited dental tissue diseases.

ACKNOWLEDGMENTS

The authors dedicate this paper to the pioneering efforts of Professor Clifford Grobstein (University of California at San Diego) for his untiring efforts toward understanding epithelial-mesenchymal interactions. The authors thank Professor Gerald Edelman and Dr. Kathy Crossin (Rockefeller University, New York) for their generous advice. The excellent technical assistance by Pablo Bringas, Jr., and Valentino Santos is gratefully acknowledged. This work was supported in part by National Institutes of Health research grants DE02848 and DE06425. M.L.S. is a recipient of a National Institutes of Health Research Career Development Award.

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Edited by John M. Opitz, James F. Reynolds, and Robert J. Gorlin