Molecular aspects of tooth morphogenesis and differentiation

Molec. AspectsMed. ©Pergamon

V o l . 4, pp. 125 - 188.

0098 - 2 9 9 7 / 8 1 / 0 7 0 1 - 0125 $ 0 5 . 0 0 / 0

Press Ltd, 1981. Printed in Great Britain.

MOLECULAR ASPECTS OF TOOTH MORPHOGENESIS A N D DIFFERENTIATION Harold C. Slavkin and Margarita Zeichner-David

Laboratory for Developmental Biology, School of Dentistry, University of Southern California, Los Angeles, Ca 90007, USA and M. A. Q. Siddiqui Roche Molecular Biology Institute, Nutley, New Jersey 07110, USA

Contents PART

i

I:

2. 3. 4.

I •

2. 2.1 2.2 33.1 3.2

FEATURES

OF TOOTH M O R P H O G E N E S I S 127

Tooth Development: A Model System in D e v e l o p m e n t a l Biology M o r p h o l o g i c a l C h a r a c t e r i s t i c s of Human Tooth Development Biochemical C h a r a c t e r i s t i c s of Tooth Development Genetic Disorders of Human Tooth M o r p h o g e n e s i s and Differentiation

°

PART

GENERAL D E V E L O P M E N T A L AND D I F F E R E N T I A T I O N

II.

CELLULAR AND MOLECULAR ISSUES EPITHELIAL DIFFERENTIATION

REGARDING

128 I33 134

TOOTH

General C o n s i d e r a t i o n s Instructive E p i t h e l i a l - M e s e n c h y m a l Interactions Structure and Function of the Basal Lamina Mesenchy~al S p e c i f i c i t y Possible Epigenetic Signals During E p i t h e l i a l Hesenchymal Interactions Differentiation Alloantigens Regulatory Levels of Mesenchyme Induction

125

127

~36 136 138 141 143 145 146 147

126 PART

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

~o 5.

5.1 5.2 5.3 5.4 5.5 5.6 6. 7.

I. 2. 3. 4. 5. 6. 7.

PHENOTYPE OF A M E L O B L A S T

148

CELLS

General Characteristics o f Enamel P r o t e i n s Current Biochemical Definition o f Enamel P r o t e i n s F u n c t i o n s o f Fnamel P r o t e i n s Possible E x p l a n a t i o n for Enamel Protein Heteroqeneity Enamel Protein R i o s y n t h e s i s Kinetics and L o c a l i z a t i o n of Fnamel Protein Biosynthesis Enamel Gene E x p r e s s i o n T r a n s l a t i o n of Enamel mR~IAs P o s t - T r a n s l a t i o n a l Processing of Enamel Proteins Enamel B i o s y n t h e s i s Amelogenin Biosynthesis Partial Amin~, Acid S e q u e n c e s of Enamel Proteins S i g n i f i c a n t Research O p p o r t u n i t i e s

I• 2°

PART

BIOCHEMICAL

III.

IV.

P H Y L O G E N E T I C AND I M M U N O G E N E T I C ENAMEL PROTEINS

CHARACTERISTICS

OF

Introduction to Vertebrate Enamel E v o l u t i o n Phylogeny of Secretory A m e l o g e n e s i s : E n a m e l o i d and Enamel Comparative Biochemical Features of Selected Vertebrate Enamel P r o t e i n s I m m u n o g e n e t i c A s p e c t s o f Enamel P r o t e i n s A Phylogenetic Hypothesis Summary: Prospectus Acknowledgements

REFERENCES

148 [49 150 150

150 150 153 155 L58 159 159 166 168

169 169 169 172 175 ]77 177 179 180

Part I

General Developmental Features of Tooth Morphogenesis and Differentiation

1. Tooth Development: A Model System in Developmental Biology A crucial problem in d e v e l o p m e n t a l b i o l o g y is to d e t e r m i n e the nature of m e c h a n i s m s that control a c t i v a t i o n and e x p r e s s i o n of structural genes. In order for an o r g a n i s m or an i n d i v i d u a l organ system to serve as a p a r a d i g m for studying cell d e t e r m i n a t i o n and d i f f e r e n t i a t i o n , the selected example should be a s s e s s i b l e to e x p e r i m e n t a l m a n i p u l a t i o n and also be a m e n a b l e to b i o c h e m i c a l , i m m u n o l o g i c a l and genetic analyses. To f a c i l i t a t e these purposes, the entire d e v e l o p mental sequence should be d i v i s i b l e into d i s c r e t e units (with repect to t e m p o r a l and spatial issues) during which the e m e r g e n c e of only a limited number of i n t e r a c t i o n s and unique p h e n o t y p e s need to be a c c o u n t e d for.

The d e v e l o p i n ~ m a m m a l i a n tooth organ is an e x c e p t i o n a l l y s u i t a b l e organ system for q u a n t i t a t i v e studies of the i n t e r a c t i o n s o c c u r r i n g during d e t e r m i n a t i o n , d i f f e r e n t i a t i o n and ~ c r o h o g e n e s i s . In addition, this u n i q u e e p i d e r m a l organ system can be used to study epit h e l i a l - m e s e n c h y m a l i n t e r a c t i o n s , b i o c h e m i s t r y and cell b i o l o g y of e x t r a c e l l u l a r m a t r i x f o r m a t i o n and s u b s e q u e n t m i n e r a l i z a t i o n . The d e v e l o p m e n t a l sequence a s s o c i a t e d with m e s e n c h y m e d i f f e r e n t i a t i o n into t e r m i n a l l y - d i f f e r e n t i a t e d o d o n t o b l a s t s and a s s o c i a t e d d i f f e r e n t i a t i o n of inner enamel e p i t h e l i a l cells into t e r m i n a l l y - d i f f e r entiated s e c r e t o r y a m e l o b l a s t cells, is e x p r e s s e d in several distinct sta~es, each being p r e c i s e l y separated in time and space. These d e v e l o p m e n t a l events appear in s y n c h r o n y : the m o r p h o g e n e t i c phase of d e v e l o p m e n t is p r e c i s e l y known, growth and d i f f e r e n t i a t i o n occur at r e l a t i v e l y d i f f e r e n t times in the cycle of o d o n t o g e n e s i s , the a b s o l u t e cell number related to e p i t h e l i a l and m e s e n c h y m a l cell d i f f e r e n t i a t i o n can be c o n t r o l l e d in the absence of serum or e x o g e n ous e m b r y o n i c extracts, and a number of m o l e c u l a r m a r k e r s are now available for studying d e f e c t i v e m u t a n t s appearing in selected m a m m a l i a n species.

127

128

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

The m e c h a n i s m s by which ~ene e x p r e s s i o n is r e g u l a t e d can be studied most p r o f i t a b l y in a system that e x h i b i t s dramatic program alterations in the s y n t h e s i s of but a few structural ~ene products. We assume that one of the best of these e x a m p l e s for b i o c h e m i c a l analysis is enamel protein s v n t h e s i s during embryunic fetal, neonatal and postnatal s e c r e t o r y amelo~enesis. Enamal matrix proteins are produced e x c l u s i v e l y by a sin~]e tissue, the inner enamel e p i t h e l i a l cells which become d e t e r m i n e d and d i f f e r e n t i a t e into a sinKle sheet of terminally-differentiated, s e c r e t o r y a m e l o b l a s t cells. Their s y n t h e s i s of enamel matrix proteins is d e t e r m i n e d , turned-on, maintained for a c o n s i d e r a b l e period of time, and then t u r n e d - o f f during the d e v e l o p m e n t a l process termed a m e l o g e n e s i s . During the form a t i v e phases of these active d e v e l o p m e n t a l periods, enamel matrix proteins d o m i n a t e the synthetic a c t i v i t y of the ameloblasts which results in the accumulation of the e x t r a c e l l u l a r enamel matrix. Enamel protein s y n t h e s i s cannot be detected in any other tissue in the body. This c l e a r l y defined pattern of gone e x p r e s s i o n is archtypical of a t e r m i n a l l y - d i f f e r e n t i a t e d e p i t h e l i a l tissue.

2. Morphological Characteristics of Human Tooth Development Human tooth d e v e l o p m e n t is initiated during the Embryonic Period of e m b r y o g e n e s i s (circa sixth week). F o l l o w i n g the m i g r a t i o n ef the cranial neural crest cells into the forming m a x i l l a r y and m a n d i b u lar segments, derived from the first and second b r a n c h i a l arches, a series of i n t e r a c t i o n s between oral e c t o d e r m and adjacent cranial neural c r e s t - d e r i v e d e c t o m e s e n c h y m e initiates the formation of the dental lamina and tooth m o r p h o g e n e s i s (see reviews by Gaunt and Hiles, 1967; Kollar, 1972; Koch, I~72; Slavkin, 1974; 1979; Thesleff, 1977). INITIATION

~

STAGE

"-~

,I~, ~. Ep th t

'

Mesenchyme (ecto)

BUD

STAGE

pap,il~ De{}t al Papilla CROWN FORMATION

cA~ ST~

Molecular Aspects of Tooth Morphogenesis and Differentiation Fig. 1.

129

The d e v e l o p m e n t of the human d e c i d u o u s incisor tooth organ (i.e., initiation, bud, bell and cap stages of m o r p h o z e n esis, h i s t o g e n e s i s and d i f f e r e n t i a t i o n t h r o u g h crown formation). Note the det e r m i n a t i o n of the p e r m a n e n t incisor tooth analagen is c l e a r l y evident by the bell stage (after d i a g r a m s provided by Dr. Charles Smith, NcGill University, Montreal, Canada). F-r6m-~lavkin, et.al., 1980.

In Table I. " h i g h l i g h t s " of human tooth morgno~,enesis, h i s t o g e n e s i s c y t o d i f f e r e n t i a t i o n and e x t r a c e l l u l a r matrix f o r m a t i o n are presented in a c o m p a r i s o n of e m b r y o n i c and fetal a~e, c r o w n - r u m p length and ~eneral features of tooth d e v e l o p m e n t . TABLE

I. H i g h l i g h t s

Embryo

or Fetal

ef Human

Age

Embryonic

Crown-Rump

. . . . . . . . . . . . . . . . . . . . . . . . . .

.

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Fetal

Length

~m-) .

and

Developmental

. . . . . . . . . . .

.

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Tooth

.

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Development Character-

i-~ ~ ~Z%- ..... .

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.

.

.

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.

.

40-44

days

%-14

42-48

days

12-15

Dental lamina forms for deciduc)us incisor, canine and molar tooth anala~en.

50-56 days

17-22

Tooth "bud stage" of deciduous incisor, canine and molar tc~oth anala~en.

70 days

40-45

Tooth "cap sta~e" is expressed by d e c i d u o u s tooth anala[en with dental papilla m e s e n c h y m e ceil c o n d e n s a t i o n a d j a c e n t to the basal lamina of the enamel organ inner enamel epithelium.

14 weeks

116

Tooth "bell stage" is expressed by d e c i d u o u s tooth organs with the d i f f e r e n t i a t i o n of o d o n t o b l a s t s , p r e d e n t i n e and d e n t i n e ext r a c e l l a r matrix f o r m a t i o n

Oral e c t o d e r m a l p r o l i f e r ation in p r o s p e c t i v e regi<)ns of the m a x i l l a r y and m a n d i b u l a r d e c i d u o u s Ist and 2rid incisor, canine and molar tooth organs.

130

H . C . Slavkin, M. Zeichner-David and M. A, Q. Siddiqui ,and c a ] . c i f i c a t i o n ; manent tooth bud now e x D r e s s e d .

From

the perstage" is

18 weeks

164

Dentine calcification cont i n u e s in d e c i d u o u s t o o t h t o o t h o r g a n s and e n a m e l extracellular matrix formation b e g i n s w i t h s e c r e t o r y a m e l o b l a s t cell d i f f e r e n t i a t i o n in i n c i s o r , canine, and m o l a r teeth; 9 e r m a n e n t "cap s t a g e " is e x p r e s s e d .

32 w e e k s

302

All d e c i d u o u s t o o t h c r o w n s are c o m p l e t e d w i t h d e n t i n e and e n a m e l e x t r a c e l l u l a r m a t r i x f o r m a t i o n and c a l c i f i c a t i o n ; c u s p s of t e e t h are fused; o e r m a n e n t molar t o o t h o r g a n s now s h o w d e n tine c a l c i f i c a t i o n .

36 w e e k s

341

Slavkin,

1079;

glav!
D e n t i n e and e n a m e l e x t r a cellular matrix formation begins in p e r m a n e n t Ist molar t o o t h o r g a n s . 1980.

!

Molecular Aspects of Tooth Morphogenesis and Differentiation

Fig.2.

Light photomicrograph of early cap stage during human inciscor tooth development. Note the condensation of cranial neural crest-derived dental papilla ectomesenchyme cells adjacent to the inner enamel epithelia of the enamel <~r~an. From Slavkin, 1979.

Fig.3.

Inner enamel epithelial cells differentiate into secretory ameloblast cells approximately 48 hours following dentine formation. From Slavkin 1979.

131

132

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqb

Fig.4.

Incisor crown formation. Each tooth is a d i s c r e t e e p i d e r m a l organ system. From Slavkin, 1979.

Molecular Aspects of Tooth Morphogenesis and Differentiation

133

3. Biochemical Characteristics of Tooth Development Regulatory processes operating during epithelial-mesenchymal interactions associated with tooth morphogenesis are crucial determinants in normal development (see reviews by Slavkin, 1974; 1979; Slavkin and Zeichner-David, (in press). Each tooth organ represents a high degree of developmental programming for sequential synthesis and secretion of unique extracellular matrix macromolecules derived from either cranial neural crest-derived ectomesenchyme (e.g. preodontoblast and odontoblast cells) or from the inner enamel epithelial cells differentiating into secretory ameloblast cells (Slavkin et.al, 1977). During differentiation a changing program of macromolecular synthesis and secretion characterize tooth morphogenesis. For example, the odontoblast cells synthesize preprecollagen, subsequently process this gene product to procollagen and then secrete type I collagen and ~(1) trimer collagen coincident with dentine proteoglycans (Butler, Finch and Desteno, 1972; Dimuzio and Veis, 1978; Munksgaard, et al.. 1978). Subsequently, odontobl~st cells synthesize and secrete dentine phosphoproteins after the initiation of calcium hydroxyapatite crystals within matrix vesicles (Slavkin, Iq75). Since the dental papilla mesenchy~e, including the odontoblast cells, synthesize and secrete type I collagen various types of proteoglycans, ¢lycosaminoglycans and dentine phosphoDroteins (constituents of the forming dentine organic extracellular matrix), one would predict that genetic diseases such as Ehlers-Danlos syndrome, osteogenesis imperfecta, achondroplasia, and mucopolysaccharidosis IV (or Morquio Syndrome) will have associated tooth malformations especially related to the connective tissue elements involved in dentine formation (Bailey, 1975; Dorfman, 1975; Fessler and Fessler, 1978; Martin, Byer and Piez, 1975; McKusick, 1966; Slavkin, 1979; Slavkin, et al., 1980). Adjacent inner enamel epithelial cells synthesize tonofilaments and also synthesize and secrete a basal lamina, allegedly formed with type IV collagens (Trelstad, Hayashi and Toole, 1974). Thereafter, these preameloblast epithelial cells demonstrate a cessation in cell division, become highly elongated as tall columnar epithelial cells, acquire extreme nuclear polarity, no longer possess a discernible basal lamina, and synthesize and secrete enamel matrix proteins (see reviews by Leblond and Warshawsky, 1979; Weinstock, 1972). Secretory ameloblast cells also regulate mineralization of calcium hydroxyapatite crystals in the enamel matrix (see Fearnhead, 1979; Frank, 1979). Subtle genetic and/or epigenetic factors which perturbate the flow of information within and between these heterotypic cell types are reflected in ma,~er and minor malformations of the deciduous and/or permanent dentitions.

134

TABLE

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

2.

Phenotype

Harkers

Structure/Cell-Type Epithelial

Basal

Lamina

For

OdontoKenic Rioche~ical Type

Basement

Differentiation Constituent

IV Collaqen, ~embrane

Laminin,

Proteoglycans,

and

Fibronectin

O d o n t o b l a s t ...... Dentine

Type I Collamen Type I Trimer Collagen, Dentine Proteoglycans, G l y c o p r o t e i n s , and Dentine P h o s p h o p r o t e i n s

Ameloblast

Enamelins and Amelogenins

Enamel

4. Genetic Disorders of Human Tooth Morphogenesis and Differentiation In all tyoes of epidermal o r g a n o g e n e s i s (for example, hair, salivary gland, skin and tooth m o r p h o ~ e n e s i s ) , both embryonic and maternal g e n o t y p e s are e x t r e m e l y relevant to the structural and functional integrity of the resulting developing organ system. Each tooth organ in the deciduous or primary dentition, as well as in the p e r m a n e n t dentition, is an individual organ system; each organ has independent n~rve innervation, vascularization and lymphatic drainage (Kraus and Jordan, 1965; Nery, Kraus and Croup,1970; van der Linder and Duterl(~o, 1976). £ince tooth m o r p h e ~ e n e s i s , histogenesis, cytodifferentiation, dentine and ena~]el e x t r a c e l l u l a r matrix formation and c a l c i f i c a t i o n spans a d e v e l o p m e n t a l period extending from the Embryonic Period to well beyond birth, numerous genetic as well as envirc~nmental factors are implicated in congenial m a l f o r m a t i o n s involving dental tissues (Slavkin, 1979). Obvious defects of tooth d e v e l o p m e n t include adontia, malformed teeth, s u p e r n u m e r a r y teeth, d e n t i n o g e n e s i s imperfecta (reflecting the extracellular organic matrix and/or subsequent mineralization and calcification) and a m e l o g e n e s i s imperfecta.

Molecular Aspects of Tooth Morphogenesis and Differentiation TABLE

3.

Genetic

Examples of Genetic Disorders

135

in Human Tooth M o r p h o g e n e s i s ,

Pattern(s)

Disorder .

.

.

.

.

.

.

.

.

.

.

.

of .

.

.

.

.

Inheritance .

.

.

.

.

.

N o n s y n d r o m e a b e r r a t i o n s in number, size, and shape of teeth x-linked r e c e s s i v e no central incisors autosomal dominant absent or "pegged" lateral incisors autosomal dominant or m u l t i f a c t o r i a l dens invaginatus autosomal dominant or m u l t i f a c t o r i a l hypodontia autosomal duminant or m u l t i f a c t o r i a l microdontia autosomal doi~inant, X-linked recess u p e r n u m e r a r y teeth sive and/or m u l t i f a c t o r i a l Syndromes with h y p o d o n t i a El]is-van Creva]d syndrome Goltz syndrome h y p o h i d r o t i c ectodermal dysDlasia Syndromes with s u n e r n u m e r a r y c l e i d o c r a n i a l dysplasia Gardner syndrome

autosomal r e c e s s i v e X-linked dominant X-linked or autosomal

teeth autosomal autosomal

dominant dominant

N o n s y n d r o m e enamel matrix d y s p l a s i a s hypoplasia autosomal Jcminant hypocalcification autosomal dominant hypomaturation X-linked r e c e s s i v e h y p o m a t u r a t i o n with pigautosomal recessive mentation Syndromes with enamel matrix dysplasias Aarskog syndrome X-linked r e c e s s i v e Goltz syndrome X-linked dominant trichodentoosseous syndrome autosomal recessive N o n s y n d r o m e dentin matrix dysplasias dentin d y s p l a s i a - - I autosomal dentin d y s p l a s i a - - I I autosomal Brandywine o p a l e s c e n t dentin autosomal

dominant dominant dominant

Syndrome~ with dentin matrix hypophosphatasia o s t e o ~ e n e s i s imperfecta

dominant ,Jominant

dysplas ias autosomal auto somal

N o n s y n d r o m e c e m e n t u m matrix dysplasias multiple c e m e n t o m a s autosomal dominant idiopathic juvenile periodontitis (periodontosis) X-linked recessive From

Slavkin,

et al,

1980.

recessive

Part// Cellular and Molecular Issues Regarding Tooth Epithelial Differentiation

1. General Considerations It has become a x i o m a t i c that d e v e l o p m e n t a l i n s t r u c t i o n s for diff e r e n t i a t i o n are related to d i f f e r e n t i a l ~ene e x p r e s s i o n leadinK to unique Kene products, and that all somatic cells within an o r g a n i s m c(,ntain identical c o n c e n t r a t i o n s of d e o x y r i b o n u c l e i c acid (DN&) (see Davidson, 1976). The timing and sequence of genetic e x p r e s s i o n r e s i d e within inherited s t r u c t u r a l genes contained within c h r o m a t i n . W h e r e a s so much i n f o r m a t i o n is encoded within DNA [circa 80,000 d i f f e r e n t unique structural genes in addition to the e n o r m o u s number of r e p e t i t i v e genes for transfer ribonucleic acid (tRNA) and ribosomal r i b o n u c l e i c acid (rRNA)]. it is also evident that subtle p e r t u r b a t i o n s o u t s i d e of chromatin, so-called "extrachromosomal" or " e p i g e n e t i c , " serve to regulate timing, sequence and d u r a t i o n of d i f f e r e n t i a l gene expression. This t h e m e is f u n d a m e n t a l towards understanding epithelial cell differentiation, h i s t o g e n e s i s and m o r p h o g e n e s i s during tooth dev e l o p m e n t (see Slavkin, 1972; 1974; 1979; Slavkin and Z e i c h n e r David, in press). One very i n t e r e s t i n g question r e g a r d i n g the nature of e p i t h e l i a l m e s e n c h y m a l i n t e r a c t i o n s during tooth m o r p h o g e n e s i s is the issue of d e t e r m i n a t i o n (Slavkin, et al, 1977). When are these heterotypic interactions determined? When have scecific RNA polymerases initiated the t r a n s c r i p t i o n of unique sequences of DNA leadin5 to b i o c h e m i c a l m a r k e r s for d i f f e r e n t i a t i o n ? When do inner enamel e p i t h e l i a l cells initiate the t r a n s c r i p t i o n of cell surface-specific glycoproteins, orenrocollamens, g l y c o s y l a t i n K enzymes, p h o s p h o k i n a s e s , preproenamel proteins, or alkaline phosphatases? When 4o p r e d o n t o b l a s t cells initiate p r e p r o c o l l a g e n synthesis? When does the induction for dentine p h o s p h o p r o t e i n , glycosyl t r a n s f e r a s e , or p r e t e o g l y c a n s occur? How are mc~lecu]ar determinants reKulated?

136

Molecular Aspects of Tooth Morphogenesis and Differentiation

instruc ive

influences

l

permissive influences and maintenance

PROGRESSIVE D I F F E R E N T I A T I O N OF EPITHELIA

Fig.5.

During embryogenesis both instructive and permissive or maintenance influences are required for e--D~helial differentiation. Instructive influences include (I) direct cell-cell contacts between mesenchyme and adjacent epithelial cells, (2) specific interactions between epithelial cells and constituents of the forming extra-cellular matrix (e.g colla~ens, Droteoglycans, glycosamino~lycans, anions, cations), (3) diffusion of "chemical signals" from adjacent cells (so-called "shortrange" intercellular communications), or (4) diffusion from other parts of the organism (e.g. long-range diffusion of growth-promoting humoral factors such as insulin, thyroxin, glucocorticosteriods, somatomedin, epidermal growth factor) suggests those nutritional requirements essential to maintain already determined epithelial phenotype. From Slavkin, et.al., 1980.

137

138

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

These q u e s t i o n s survey various m o l e c u l a r r e s p o n s e s that individual cells make as a c o n s e q u e n c e of both e p i t h e l i a l - m e s e n c h y m a l i n t e r a c t i o n s and pattern i n f o r m a t i o n d u r i n g tooth m o r p h o g e n e s i s . How are these r e s p o n s e s i n i t i a t e d ? How do e p i g e n e t i c processes m e d i a t e the a m p l i f i c a t i o n or s u p p r e s s i o n of gene e x p r e s s i o n ? That h e t e r o t y p i c tissue i n t e r a c t i o n s are required for these various types of gene e x p r e s s i o n appears today to be unequivocal. What is not clear is whether these e p i t h e l i a l - m e s e n c h y m a l interactions are c a u s a l l y related to the d e t e r m i n a t i o n of d i f f e r e n t i a l gone e x p r e s s i o n a s s o c i a t e d with d i f f e r e n t i a t i o n , or whether these same i n t e r c e l l u l a r interactions serve to regulate predetermined events; events d e t e r m i n e d prior to the h i s t o l o g i c a l identification of inner enamel e p i t h e l i u m within the d e v e l o p i n g tooth organ system. D e t e r m i n a t i o n of e p i t h e l i a might be e n v i s i o n e d to begin with ind u c t i o n of unique mRNAs. The popular p a r a d i g m for induction has been to consider "on" ( d e r e p r e s s i o n ) and "off" (repression) controis of Kene a c t i v a t i o n and e x p r e s s i o n . Unique s t r u c t u r a l ~enes, such as collagen, myosin, actin, keratin, and enamel protein, are either "on" or "off." Epithelial-mesenchymal i n t e r a c t i o n s could provide a m e c h a n i s m to s e l e c t i v e l y a c t i v a t e previously inactive and unique s t r u c t u r a l ~.enes in the inner enamel epithelium. An alternative concept is to consider that all unique structural ~enes are e s s e n t i a l l y activated in the e p i t h e l i u m and a p p r o p r i a t e hnRNAs (heterogeneous nuclear RNAs) are s y n t h e s i z e d earlier in embryronic development. In this concept p o t e n t i a l mRNA (messenKer RNA) t r a n s c r i p t s are p h y s i c a l l y available for t r a n s l a t i o n prior to e p i t h e l i a l - m e s e n c h y m a l interactions, yet require "activation" as a direct c o n s e q u e n c e of heterolc~gous t i s s u e - t i s u e interactions. D e t e r m i n a t i o n at the gene level of biologic o r g a n i z a t i o n has already o c c u r r e d . S u b s e q u e n t l y , d i f f e r e n t i a t i n g inner enamel e p i t h e l i a l cells b e c o m e increas-J[ng]--y-Testr-ic-ted as a c o n s e q u e n c e of a net loss in their capacity to express gene products and c e s s a t i o n of cell division. Differentiation is a n o n - r e v e r s i b l e process. N o n - d i v i d i n g cells, such as a m e l o b l a s t s , s y n t h e s i z e p h e n o t y p i c a l l y c h a r a c t e r i s t i c enamel protein gone products as their terminal expression of diversity. Aberrations in either rates of p r o d u c t i o n and/or absolute c o n c e n t r a t i o n of gene products can produce various forms of amel(~genesis imperfecta, including hypoplasia, hypomineralization, and h y p o c a l c i f i c a t i o n .

2. Instructive EpitheliaI-Mesenchymal Interactions Despite a p l e t h o r a of p u b l i c a t i o n s spanning nearly .six decades by innumerable experimental embryologists, it remains unclear as to whether epithelial-mesenchymal interactions are inductive (i.e. in the m o l e c u l a r d e f i n i t i o n of d_ere_p_ression of uniqu@ s C r u c tural Kene products) , or are p e r m i s s i v e for the e x p r e s s i o n of p r e v i o u s l y d e t e r m i n e d d e v e l o p m e n t a l pru~ramming,. Moreover, ohenom e n o l o g y observed in h i s t o l o g i c a l p r e p a r a t i o n s has been r e p e a t e d ly advanced by i n v e s t i g a t o r s as direct evidence for "embryonic i n d u c t i o n . " Critical d i s c u s s i o n of this l i t e r a t u r e have been carefully analyzed in c o n t r i b u t i o n s by Spemann, 1996; Weiss and Taylor, 1960; Grobstein, 1967, 1975; Holtfreter, 1968; Rutter et al, 1968; Saxen, et al, 1976; Caplan and Ordahl, 1978; and Slavkin, 1979.

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D e v e l o p m e n t a l c o m m u n i c a t i o n b e t w e e n tissues and cells is an outstanding feature of the genesis of m u l t i c e l l u l a r organisms. Cells and tissues t r a n s m i t to each other i n f o r m a t i o n about their presence, identity and f u n c t i o n s by contact, through cell surface m o l e c u l e s , across i n t e r f a c e s or by c l o s e - r a n g e d i f f u s i o n processes. The result of such c o m m u n i c a t i o n is the r e g u l a t i o n of individual cellular activities and i n t e r c e l l u l a r activities related toward the a d v a n c e m e n t of cellular d i v e r s i t y and s p e c i a l i z a t i o n s . As yet unknown aspects of these intricate processes are termed 'intercellular c o m m u n i c a t i o n s , " which e x p r e s s e s the complex developmental problem of how i n d i v i d u a l cells of tissues affect adjacent and often d i s s i m i l a r cells and tissues. Perhaps the heart of the p r o b l e m resides in whether (I) cells or a tissue type c o n t a i n s and stores intrinsic i n f o r m a t i o n which is e x p r e s s e d in a p e r m i s s i v e e n v i r o n m e n t , or whether (2) d e v e l o p m e n tal i n f o r m a t i o n is produced de novo and e x p r e s s e d as i n t e r c e l l u l a r c o m m u n i c a t i o n ; the direct c o n s e q u e n c e of h e t e r o t y p i c i n t e r a c t i o n s .

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One e x p e r i m e n t a l s t r a t e g y used in epithelial-mesenchymal interactions has been to design e x p e r i m e n t s using either h o m o t y p i c or h e t e r o t y p i c cell or tissue r e c o m b i n a t i o n s in vitro. For example, v a r i o u s sources of m e s e n c h y m e r e c o m b i n e d with a specific e p i t h e l i a . Such studies have c o n c l u d e d that in m o s t e p i d e r m a l organ systems the m e s e n c h y m e i n s t r u c t s the e p i t h e l i a to differenti~e--i-n-t-o a p h e n o t y p e c o m p l e m e n t a r y to that of the mesenchyme.

140

H.C. Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

25 day embryonic incisor

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Summary of experimental studies designed to evaluate epithelial-mesenchy~al interactions durin~ embryonic rabbit incisor tooth organ development using ×enografts explanted to the chick chorioallantoic membrane (CAM). Both cell types were quired for either mesenchymal or epithelial differentiation (see Slavkin, 1972; 1974).

re

Molecular Aspects of Tooth Morphogenesis and Differentiation

141

One of the most useful levelopmental systems with which to address these important issues of intercellular communication during epithelial-mesenchymal interactions is the embryonic mammalian tooth organ (see discussions by Koch, 1972; Kollar, 1972; Slavkin, 1972, 1979; Thesleff, 1979). In 1967 Koch demonstrated the first successful transfilter experimentation using embryonic mouse odonto~enic epithelial and mesenchymal tissues; inner enamel epithelial cells differentiated into ameloblasts and secreted enamel matrix in juxtaposition to odontoblasts which secreted dentine matrix cultured in juxtaposition on MilliDore cellulose acetate tilters of 0.45 pm pore size and 25 pm thickness. It was then assumed that "diffusible morpho~enetic factors," perhaps mesenchyme-produced collagen, mediated the transfilter epithelial-mesenchymal interactions (Grobstein, 1967; Koch, 1967). The advent of Nucleopore filters and the approaches of Saxen and colleagues prc~vided a novel interpretation to the classical "Grobstein Experiments" (see discussions by Saxon, 1975; Saxen, et.al, 1976). Saxen postulated that direct cell-to-cell contact between mesenchymal cell processes and adjacent epithelia represented a possible mechanism for mesenchymal specificity upon subsequent epithelial cytodifferentiation and histogenesis (e.g. kidney, salivary, mammary and tooth morphogenesis). Nucleopore filters of 0.1 pm pore size prevented mesenchymalepithelial interactions during kidney and tooth development in vitro (Lethtonen, et al, 1975; Saxen, et al, 1976; Thesleff~ 1977, 1979; Thesleff, et al, 1977). Pore sizes of 0.2 pm or greater were permissive to epithelial-mesenchymal interactions. Increased Nucleopore filter thicknesses (greater than 50 ~ ) retarded morphogenesis (Wartiovaara, et al, 1972). The duration of alleged direct mesenchyme-epithelial cell-to-cell contact was suggested to be 12-24 hours (Saxon, 1975; Saxon, et al, 1976; Thesleff, 1977, 1979). The hypothesis of direct cell-to-cell cc~ntact durin~ instructive phases of epithelial-mesenchymal interactions was further supported from descriptions <~f predontoblast cell contact with adjacent inner enamel epithelial cells in vivo (Pannesse, 1962; Kallenbach, Iq71, 1976; Kallenbach and Piesco, 197~; Silva and Kailis, 1972; Slavkin and Bringas, 1976 These direct c~ntacts occurred in precise positions and for transitory periods of time (Slavkin and Bringas, 1976). 2.1

Structure and Function of the Basal Lamina

Another critical issue is related to the composition, structure and function of the basal lamina associated with the undersurface of the epithelia in juxtaposition to the adjacent mesenchyme (see discussions by Bernfield and Banerjee, 1978; Thesleff, 1979). Does the basal lamina have a major function related to mesenchymal instruction of epithelial differentiation? Thesleff (1979) states that the basal lamina (i.e. basement membrane) may regulate mesenchymal differentiation into odontoblasts during tooth development. Recently, Osman and Ruch (in press) supported this assertion by demonstrating that EDTA- (ethylenedinitro-tetracetic acid) dissociated cap stage dental papilla mesenchyme (circa 18-days gestation) is determined and will differentiate into odontoblast cells and produce predentine--within ~8 hours without adjacent inner enamel epithelia. Trypsin-dissociated mesenchyme did not differentiate into odontoblasts under comparable experimental conditions. Unique to the EDTA-dissoc-

142

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

iated mesenchyme tissues tact b a s a l lamina.

Fig.

8.

was

the

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and

in-

A sequential series of electron photom i c r o g r a p h s of comparable m a g n i f i c a t i o n (bar line = 0.5 pm) illustrating the d i f f e r e n t i a t i v e changes at the interface during embryonic tooth e p i t h e l i a l - m e s e n chymal interactions. (a) Inner enamel epithelial cells secrete a basal lamina (b) The e c t o m e s e n c h y m e cells have differentiated into p r e o d u n t o b l a s t cells e x t e n d i n g long cell processes towards the u n d e r s u r f a c e of the inner enamel epithelial cells. (c) The preodontoblast cells have d i f f e r e n t i a t e d into nondividJn~, elongated, and polarized o d o n t o b l a s t cells which synthesize and secrete type I collaKen, proteo~lycans, and dentine D h o s D h o D r c t e i n s at least 48 hours before epithelial cells differentiate into secretory ameloblasts. From Slavkin, et al, 1975, 1980.

Our l a b o r a t o r y suggested that basal lamina degradation in vivo and in vitro may be regulated by matrix vesicle and/or predontoblast cell p r o c e s s e s - d e r i v e d enzyme activity, and that basal lamina degradation precedes direct cell c<~ntact between mesenchymal cell processes and epithelial cell surfaces (Slavkin and Bringas, 1976;

Molecular Aspects of Tooth Morphogenesis and Differentiation

143

Slavkin, et al, 1977; Sorgente, et al, 1977; Slavkin, 1979). A similar argument has been postulated by Bernfield and colleagues for salivary and mammary gland morpho~enesis (Bernfield and Banerjoe, 197£; David and Bernfield, 1979). They suggested that mesenchyme-derived enzymatic activity selectively de~rades the epithelial basal lamina substrate composed of proteoglycans during active phases of morphoCenesis (Cohn, et al, 1977; ~ernfield, et al, 1972). Our laboratory also considered this suggestion and detected mammalian collagenase activity derived fr~m preodontoblast cell processes and matrix vesicles (Sorgente, et al, 1977; Slavkin, et al, 1977). Several recent studies support these suggestions. Juhnson-Wint (1980) has recently demonstrated that epithelial cells may provide several regulatory controls for mesenchymal cell collagenase production; epithelial cells do not appear to synthesize or secrete mammalian collagenases yet provide both stimulatory as well as inhibitory regulation of connective tissue stromal cell collagenase synthesis and/or secretion in vitro. Cytochalasin B was required for both secretion of stimulators by epithelial cells and production of collagenase by mesenchymal connective tissue stromal cells (Johnson-Wint, 1980). In addition, the neutral protease responsible for triple-helical basement membrane collagen degradation is different from those collagenases active against interstitial collagen types I, II and III (Uitto, Schwartz and Veis, 1980). More recently it has been shown that the embryonic mouse mandibular first molar tooth organ (e.g. "cap stage" from Theiler sta~e 25 embryos) has an intact basement membrane which contains type IV collagen, laminin, basement membrane proteo~lycan and fihronectin (Thesleff, et al, 1979; Thesleff, et al, in press; Osman, et al, in Dress). Type IV collagen appears to be located in most basal lamina (Timpl et al, 1979). Laminin is considered to be ubiquitous in all basal lamina (Timpl et al, 1979; Foidart et al, in press). Fibrenectin is not secreted from epithelia yet has been localized within various basal lamina and is not ubiquitous (Mayer and Hay, in press; Mayer et al 1979). Fibronectin is cross-reactive with all verte~ brate species (Kuusela et al, 1976). Basement membrane proteoglycans have been localized in basal lamina using immunochemical and transmission electron microscopic methods (Foidart et al, in press; Foidart et al, 1978). 2.2.

Mesenchymal

Specificity

Another important issue is the specificity of mesenchymal "instruction" for epithelial differentiation (see discussions by Holtfreter, 1968; Grobstein, 1975; Kollar, 1973; Slavkin, 1979; Garber et al, 1968; Slavkin et al, 1977). Specifically, embryonic mouse dental papilla mesenchyme has been demonstrated to be highly "inductive" by providing developmental instructions for mammalian non-oral epithelia to differentiate into secretory ameloblasts within heterotypic and heterochronic tissue recombinants (see examples from Kollar and Baird, 1969, 1970; Hertier, 1971; Ruch et al, 1973).

144

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

vi~.

Q.

Indirect immunofluorescence localization of fibronectin in Theiler S t a g e 25 ( 1 7 - 1 8 d a y s ~ e s t~tion) mouse m a n d i b u l a r f i r s t molar tooth orK a n s . (A) S t a i n i n g shows r e g i o n a l localization of f l u o r e s c e n c e associated with dental papilla m e s e n c h y m e (M) and adjacent oral m e s e n c h y m a l tissues, and the lack of f l u o r e s c e n c e with oral e c t o d e r m (E) and enamel organ e p i t h e l i a l tissues (E) bar equals i00 ~m. (B) Higher m a g n i f i c a tion i l l u s t r a t e s the intense staining of basement m e m b r a n e between m e s e n c h y m e and inner enamel epithelia; bar equals 450 pm. (C) Control showing no f l u o r e s c e n c e with normal rabbit serum; bar equals 100mm. From Brownell, Bessem and Slavkin, in Dress.

Molecular Aspects of Tooth Morphogenesis and Differentiation

145

A number of f a s c i n a t i n g studies have d e m o n s t r a t e d that e p i t h e l i a l m e s e n c h y m a l i n t e r a c t i o n s can be d e m o n s t r a t e d using iso- and heterochronic h e t e r o l o g o u s tissue r e c o m b i n a t i o n s within and between higher v e r t e b r a t e species (e.g. reptile vs. avian, reptile vs. mammal, avian vs. mammal, etc.) (Sengel and Dhouailly, 1977). Despite these positive d e m o n s t r a t i o n s that regional dermal or m e s e n c h y m a l specificity will influence adjacent h e t e r o l o g o u s e p i t h e l i a to d i f f e r e n t i a t e with c o m p l e m e n t a r i t y to that of the m e s e n c h y m e (e.g. chick feather dermis plus ~ouse epidermis results in feather-like m o r p h o g e n e s i s ) , a number of attempts to d e m o n s t r a t e this usinz cap stage dental papilla mesenchyme from mice r e c o m b i n e d with embryonic chick epithelia did not resuit in tooth m o r p h o g e n e s i s , no dentine was produced, nor was enamel detected (Ruch et al, 1973]. In contrast to these negative results by Ruch et al (1973), Kollar and Fisher (1980) showed that dental m e s e n c h y m e isolated by trypsin from 16-18-davs ~estation CD-1 mouse molars (cap stage of tooth development) recombined with 5-day embryonic chick first and second pharyngeal arch e p i t h e l i u m formed discrete tooth organs and expressed both d e n t i n o g e n e s i s and a m e l o g e n e s i s as intraocular xenografts in the eyes of adult nude athymic mice. It must also be emphasized that these i n v e s t i g a t o r s stated that their positive results were from xenografts after 30 days "in culture." The long period of time and rather low yields of positive heterologous r e c o m b i n a n t s raises a number of questions including the remote possibility of mouse tooth e p i t h e l i a l cell c o n t a m i n a t i o n during these procedures. To address this c r i t i c i s m and in hopes of c o n f i r m i n g the report by Kollar and Fisher (1980), our laboratory has recently been able to d e m o n s t r a t e that H a m b u r g e r - H a m i l t o n equivalent stages 22-25 quail m a n d i b u l a r e p i t h e l i u m in r e c o m b i n a t i o n with trypsin-isolated S w i s s - W e b s t e r mouse dental papilla m e s e n c h y m e (Theiler stage 25, cap stage molar tooth organ) results in the d i f f e r e n tiation of o d o n t o b l a s t s and the production of dentine within 3-days using a c h e m i c a l l y - d e f i n e d , serumless m e d i u m (Cummings, Bringas and Slavkin, 1981). Subsequent o b s e r v a t i o n s at 5,7 and 10 days in vitro did not indicate the d i f f e r e n t i a t i o n of quail epithelia to become s e c r e t o r y ameloblast cells (preliminary and unpublished observations). In all of these h e t e r o l o g o u s tissue recombinants, light microscopic o b s e r v a t i o n s have been used to assav for amelo~enesis.

3. Possible Epigenetic Signals During EpitheliaI-Mesenchymal Interactions Several advances in cell surface i m m u n o g e n e t i c s are critical towards this discussion. Differentiative signals can be introduced through direct c e l l - t o - c e l l or c e l l - s u b s t r a t u m contacts. For example, Rutter and his colleagues have shown a "mesenchymal factor" (e.g. 25,000 MW glycoprotein) which can be conjugated to Sepharose beads (Levine et al, 1973), which can enhance p r o t o d i f f e r entiated pancreatic epithelium to d i f f e r e n t i a t e (Pictet et al, 1975; Pictet and Rutter, 1977). McMahon and West (1976) showed that conjugated plasma fragments of D i c t y o s t e l i u m to polyacrylamide beads facilitated d i f f e r e n t i a t i v e events in p r o t o d i f f e r e n t i ated slime mold cells. Exogenous p o l y p e p t i d e hormones (e.g. insulin or somatomadin) have been d e m o n s t r a t e d to effect epithelial lens cell e l o n g a t i o n ( P i a t i g o r s k y et al, 1972). An i n c r e a s i n g l y

146

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

c o n v i n c i n g body of evidence s~]pports the concept that d e v e l o p m e n tal instructive siKnals impinge d i r e c t l y upon the outer cell surfaces of the r e s p o n d i n g cell (McMahon, 1974, 1976; Edelman, 1976; Nicholson, 1976, 1979; Mocona, 1977; Sa×en et al 1976).

3.).

P~rf_er_en_tLat_i£n A_Lt_£ant!ff~_n_s

One model for epigenetic signals binding to integral plasama membrane g l y c o p r o t e i n s and then translated into intracellular information is the H-2 system (Edelman, 1976; Edidin, 1976; Boyse and Cantor, 1971, White and Trump, 1972; Bennett, 1975; Bennett et al, 1972). The H-2 antigen is an integral plasma m e m b r a n e protein found in all somatic mouse cells (Klein, 1975). The H-2 antigen consists of a heavy chain (46,000 MW) which contains the antigenic d e t e r m i n a n t s and a c a r b o h y d r a t e prosthetic group, and also contains a light chain termed B2-microglobulin having a molecular weight of 12,O00 daltons. The H-2 m o l e c u l e amino terminus projects from the cell surface into the e x t r a c e l l u l a r milieu, whereas the carbo×yl terminus is located on the inner surface of the plasma m e m b r a n e facing the i n t r a c y t o p l a s m i c microfilaments and m i c r o t u b u l e s (see reviews by Edelman, 1976; Klein, 1975; Nicolson, 1979). By virtue of this structural c h a r a c t e r i s t i c , the H-2 molecule physically spans from the e x t r a c e l l u l a r e n v i r o n m e n t to the i n t r a c y t o p l a s m i c milieu. The H-2 antigens h~ve been identified and localized on the surfaces of Theiler stage 25 edonto~enic epithelium and mesenchymal cells using, i m m u n o c h e m i c a ! and transmission electron microscopic methods (Slavkin et al, 1974). H-2 antigens are located on p r e d o n t o b l a s t cell processes and on the s~rfaces of matrix vesicles (Slavkin, 1974; Slavkin et al, 1976). An additional feature which enhances the oossible d e v e l o p m e n t a l functions of H-2 or related cell surface integral proteins is that epigenetic signals, such as those possibly derived from dental mesenchyme, can form ligand-receptor binding (Mervelo and Edidin, 1980). Such bindin~ can be translated into phenotypic alteratic>ns within the responding epithelial cell via an e n e r g y - d e p e n d e n t assembly of m i c r o f i l a m e n t s and m i c r o t u b u l e s (Edidin, 1976; Nicolson, 1979; Poste et al, 1975; Slavkin, 1979).

Molecular Aspects of Tooth Morphogenesis and Differentiation

~.~.

Regulatory Level_~s o£ Mesen_chyme Ind_uctign

J CHROMOSOME (CHROMATIN)I )m RNA

l$0roi0o"0e0''°' plasma "-,,,. membrane ::-:

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Mesenchymal tissue, cells, subcellular fractions, or mesenchymal-derived factors appear to be critically required for specific epithelial cytodifferentiation in vivo and in vitro. Mesenchyme instruction" may (I) directly regulate differential gene expression within epithelial cells. (2) serve as a "signal" and engage epiithelial cell surface receptors; (3) regulate transcription of unique mRNAs (4) regulate translation; and/or (5) regulate post-translational modifications of unique gene products (e.g. glycosylation, phosphorylation, etc). From Slavkin et al, 1980.

147

Part III

Biochemical Phenotype of Ameloblast Cells

1. General Characteristics of Enamel Proteins The v e r t e b r a t e tooth enamel is a useful system for the study of a number of fundamental d e v e l o o m e n t a l processes on a molecular level. Vertebrate tooth enamel system can be used for the elucidation of a number of significant developmemtal problems including: (I) determination, (?) d i f f e r e n t i a t i o n , (3) protein processin~ including_ t r a n s l a t i o n and s p p r e c i a b l e p o s t - t r a n s l a t i o n a l covalent m o d i f i c a tions, (4) e x t r a c e l ] u l a r matrix formation, and (~) m i n e r a l i z a t i o n associated with the larKest known biolomical apatite crystals. In this review our attention is directed to the hiKhly specific enamel ~ene products. Their fate after synthesis on polysomes can be followed by a number of techniques through p o s t - t r a n s l a t i o n a l modifications includinE glycosylation, phosphorylation possible sulphation and subsequent proteolytic cleavages resulting in a large number of p o l y p e p t i d e s associated with the extracellular enamel matrix. Enamel proteins, assumed to be a major c o n s t i t u e n t of most lower and higher v e r t e b r a t e s (e.g. fish, amphibians, reptiles, mammals), can be extracted from various d e v e l o p m e n t a l stages of enamel matrix production, can be purified and are suitable for comparative studies of a,nino acid sequences (see Peyer 1968; Slavkin et al, in press). This d i s c u s s i o n has several objectives: (I) to review the general problem area of enamel protein b i o s y n t h e s i s ; and (2) to discuss critical problems related to gene regulation, t r a n s l a t i o n and postt r a n s l a t i o n a l processing of one or more unique enamel gene products. Since space l i m i t a t i o n s prevent an e x h a u s t i v e r e v i e w and citation of scientific p u b l i c a t i o n s , we shall indicate the most pertinent literature related to a biochemical approach of enamel biosynthesis. Our purpose is to provide the reader with a key to an expanding scientific literature. Our survey is complemented by several previous c o m p r e h e n s i v e reviews (see Fincham, in press; Nylon and Termine, 1970; glavkin et al, Iq7~; Weinstock, 1972).

148

Molecular Aspects of Tooth Morphogenesis and Differentiation

149

The concept of enamel proteins as an heterogeneous family of proteins is rapidly changing. The notion of a multi-aggregating assembly of numerous polypeptides, varying in molecular weight from 3,000 to 18,000 daltons, is being challenged. More recent evidence now suggests that secretory amelogenesis, the production of enamel proteins for a developing extracellular matrix, consists of the biosynthesis of two major enamel pr~,teins: enamelins and amelogenins (Termine, Torchia and Corm, 1979). We now define, operationally, two different enamel proteins; one consisting of a high molecular weight enamelin ( ~72,000 daltons) and the other a lower molecular weight amelogenin of approximately 30,000 daltons (Termine et al, 1980)-.---It-is likely that the initial translation gene products for each of these different secretory enamel proteins will have still longer amino acid sequences and will also have additional "signal sequences." A number of studies indicate that the in vitro translation products of the mRNAs for various secretory proteins (e.g. collagens, insulin, ovalbumin) carry a 20-30-residue NH -termin~I extension rich in hy~rophobic amino acids (Blobel, 1977; Steiner et al, Iq80). This extension region is thought to provide a "signal sequence" which functions in the initiation of the polysome-membrane junction leading to segregation of nascent polyDeptides (Blobel, 1977). The archtype of secretory proteins found in developinK extracellular matrices associated with mineralization is preprocolla~en (Fessler and Fessler, 1978; Martin, Byer and Piez, 1975). In view of the uncertainties concerning the actual number of structural genes for enamel, specific details regarding transcriptional, post-transcriptional processing, translation, and post-translational processing within the ameloblast cells as well as in the extracellular matrix, our discussion attempts to bring into focus biochemical studies and future opportunities of enamel biosynthesis.

2. Current Biochemical Definition of Enamel Proteins Recent studies have now provided important data to suggest that there are possibly two primary classes of enamel proteins: (I) enamelins of approximately 72,000 daltons, and (2) amelogenins of approximately 25-30,000 daltons (Termine, Torchia an-d- Cc-~nn, 1979; Termine et al, 1980). The enamelins appear to be tightly associated with enamel apatite crystals (they contain 0.32% organic phosphate) (Termine et al, 1980). The amelo~enins are also rich in the amino acids proline, ~lutamic acid, leucine and histidine and contain O. 14% organic phosphate. Enamel proteins do not contain hydroxproline. Several experimental strategies have been emplt,yed reKardin~ the extraction and isolation of newly secreted extracellular matrix enamel proteins. These studies have assumed that these polypeptides are insoluble under physiological conditions, they may form aKgregates using disulphide bonding, appear to be globular rather than fibrous proteins, and may be associated with several proteases related to "enamel maturation." Therefore, extraction methods using acetic acid at low temperature, urea-Tris borate solutions, protease inhibitors, guanidine hydrochloride, denaturing and reducing conditions, all appear relevant to this complex chemical problem (also see Eastoe, 1979; Eggert, Allen and Burgess, 1973; Mechanic, Katz and Glimcher, 1967; Robinson et al, 1978; and Seyer and Glimcher, 1971).

150

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

3. Functions of Enamel Proteins The function(s) of enamel matrix proteins appears to be: (I) to provide a protein o r i e n t a t i o n for ~rowing enamel apatite crystals, (P) serve as initial nucleation sites for enamel apatite crystal formation, (3) serve as c a l c i u m - b i n d i n g ~lycoproteins and serve to regulate and transport calcium within the secretory ameloblast cell and to facilitate c~llular r e g u l a t i o n for e x t r a c e l l u l a r mineralization, and (4) to provide a thixotropic gel-like structure which facilitates the formation of d e n s e l y - p a c k e d and remarkably large enamel apatite crystals.

4. Possible Explanation for Enamel Protein Heterogeneity One r e c e n t l y proposed explanation for the r e m a r k a b l e degree of hete r o g e n e i t y associated with enamel matrix proteins is that there is a significant amount of: (I) hydrogen bonding which require conditions e q u i v a l e n t to 8M urea for d i s s o c i a t i o n , (2) a significant amount of h y d r o p h o b i c bonding as a c o n s e q u e n c e of lipophilic proline and leucine amino acid side chains, and (3) e l e c t r o s t a t i c forces derived from the very high content of glutamic acid and the high h i s t i d i n e content which c o n t r i b u t e s p o s i t i v e - c h a r g e d groups at pH c o n d i t i o n s below 7.5 (Eastoe, 1979).

5. Enamel Protein Biosynthesis Early i n v e s t i g a t i o n s of developing enamel matrix proteins indicated a r e v e r s i b l e a ~ g r e g a t i n ~ system of h e t e r o g e n e o u s and r e l a t i v e l y low molecular weight p o l y p e p t i d e s (Eastoe, 1960). More recent investigations have been able to d i s c r i m i n a t e amongst enamel matrix constituents by c o n s i d e r i n g hydrogen bonding, hydrophobic bonding and electrostatic forces between diverse enamel polypeptides (Termine, Torchia and Corm, 1979; Termine et al, 1980; Eastoe, 1960; 1979; Fukae and Shimizu, 1974; Guenther et al, 1977). Advances in extraction methods using d e n a t u r i n g conditions in the presence of protease inhibitors, p o l y a c r y l a m i d e [el e l e c t r o p h o r e s i s in the presence of sodium d o d e c y l s u l p h a t e (SDS) , amino acid analysis including c o m p o s i t i o n and partial sequence data, gel and ion exchange c h r o m o t o g r a p h y and pulse/chase isotopic precursor labeling e x p e r i m e n t s have enabled additional penetration into this complex problem. 5.1.

.

.

.

.

.

Kinetics .

.

.

.

.

and .

.

.

Localization .

.

.

.

.

.

.

.

of .

.

Enamel .

.

.

.

.

Protein .

.

Biosynthesis

Light m i c r o s c o p i c and high resolution electron m i c r o s c o p i c autoradiographic i n v e s t i g a t i o n s of enamel protein b i o s y n t h e s i s in vivo and in vitro have provided kinetic and localization data describing the b i o s y n t h e s i s of enamel proteins in a number of different mammalian species (Belanger, 1956; Frank, 1970; Greulich and Slavkin, 1965; Warshawsky, 1966; 1979; W e i n s t o c k and Leblcnd, 1971). Following the a d m i n i s t r a t i o n of various isotopic amino acid precursors in situ, label in the form of silver grains is first detected over the rough endoplasmic reticulum within 5 minutes (Slavkin. Mino and Bringas, 1976; Warshawsky, 1966; Weinstock, 1972). Using isotopic m o n s a c c h a r i d e precursors, it has been demonstrated that enamel proteins are glycosylated in the Golgi Apparatus, packaged in condensing vacuoles derived from the inner

Molecular Aspects of Tooth Morphogenesis and Differentiation

151

saccules, t r a n s p o r t e d in secretory vesicles to the s e c r e t o r y regions of the ameloblast and then released into the forming enamel matrix within 30 minutes from the initiation of t r a n s l a t i o n (see review by Weinstock, 1972). A number of obvious t r a n s l a t i o n a l and post-translational controls are apparent for the regulation *of enamel b i o s y n t h e s i s .

90. /X /

80.

Enamel Matrix

//

0

,,jr

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:,,

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o

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tRouoh endoplasmic ~reticulurn

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a0.

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.

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.

.

~..~'Gronular

.

.

Material

C ID

o

I0

O 0.

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60

120

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24 hrs

Minutes Incubation

Fi~.

11

Light m i c r o s c o p i c and t r a n s m i s s i o n electron m i c r o s c o p i c a u t o r a d i o g r a p h i c studies have provided substantial resolution towards the problem of kinetics and l o c a l i z a t i o n of enamel protein b i o s y n t h e s i s and secretion in viva. The data shown was derived f r o m [3H]-tryptophan incorporation into neonatal mouse inciscor secretory amelogenesis. From Slavkin, Mino and Bringas, 1976.

152

H.C. Slavkin, M. Zeichner-Davidand M. A. Q. Siddiqui

MITOCHONDRIA

t

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Durin~ the e l o n g a t i o n of the s e c r e t o r y a m e l o b l a s t , enamel proteins are s y n t h e sized on p o l y s o m e s a s s o c i a t e d with ER and then t r a n s f e r r e d to the Golgi where these p o l y p e p t i d e s are p o s t - t r a n s l a t i o n ally m o d i f i e d including g l y c o s y l a t i o n . S y n t h e s i s and secretion r e q u i r e s a p p r o x i m a t e l y 30 minutes. F o l l o w i n g the initiation of enamel matrix production, the d u r a t i o n of secretion is protacted in numerous v e r t e b r a t e species a l l o w i n g for the d e t e r m i n e d t h i c k n e s s of enamel matrix to be formed. During s e c r e t i o n of enamel matrix proteins, changes occur in the subunit c o m p o s i t i o n and in the physical chara c t e r i s t i c s of enamel p o l y p e p t i d e s termed "enamel m a t u r a t i o n . "

Molecular Aspects of Tooth Morphogenesis and Differentiation

5.2.

153

Enam_melGene Expressi__on

The number of structural genes for enamel is not as yet known. Current information sugy~ests that most secretory proteins (e.~. ~lobin, colla~.en, insulin, ovalbumin, immunoglobulins) are translated from unique p o l y [ A ] f m R N A s . It is w e l l - d o c u m e n t e d that in most eucmryotic cell types, formation of t r a n s l a t a b l e mRNA is a complex process. Primary DNA transcripts, pre-mRNAs or HnRNAs, are synthesized in the nucleus and undergo post-transcriptional processing including cleavage of these e x t r e m e l y large RNA precursors, 3'-end p o l y a d e n y l a t i o n . 5'-end capping and methylation, internal m e t h y l a t i o n , and finally splicing together of d i f f e r e n t segments of pre-mRNA into the final and functional mRNA m o l e c u l e which is transported to the polysomes for subsequent translation into the unique gone products (Revel and Groner, 1978). Transcription and p o s t - t r a n s c r i p t i o n a l processing of enamel gene products is not as yet defined. Data is available, however, which indicates that t r a n s c r i p t i o n a l inhibitors such as actinomycin D (30 ~g/ml) or proflavine (60 ~M) reduce enamel protein synthesis by 60-72% in s e c r e t o r y ameloblast cells in vitro (Slavkin et al, 1 979) • TABLE

4.

Effects of T r a n s c r i p t i o n a l and T r a n s l a t i o n a l Inhibitors on E x t r a c e l l u l a r Matrix Protein Synthesis and Secretion

~ulture-Conditions 0° C

Inhibition

of

Control

97 %

Transcriptional Inhibitor Actinomycin D {?0 ug/ml} Actinomycin D {30 pK/ml} Proflavine {6 pM} Proflavine {12 uM}

62 76 _PO 45 50

% % % %

T r a n s l a t i o n a l Inhibitor Puromycin {~5 ~K/ml) ....... P_~ro~_r_~n {S_O ~ / _ ~ l ) . . . . . . . . . . . . . . . . . . . .

87 % 93

Groups of twenty 26-day fetal rabbit molar tooth organs were preincubated for 2 hours in the culture c o n d i t i o n s listed. The evaluation of inhibition began with the addition of 50 ~Ci/ml of [3,~,53H{N}]-leucine, (specific activity 110 Ci/mM), in the presence or absence of inhibitors. The pulse incubation was 20 hours in MEM supplemented with ascorbic acid, L-glutamine and antibiotics. Tooth organs were then washed, e x t r a c e l l u l a r matrices were d i s s e c t ed free of adherent tissue, sonicated and d e m i n e r a l i z e d , and then e x t r a c e l l u l a r matrix proteins were extracted. Data r e p r e s e n t means of triplicate e x p e r i m e n t s . From slavkin et al, 1979.

154

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Slavkin,

M.

Zeichner-David

and

M.

A. Q. Siddiqui

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The e f f e c t s of 12 pM p r o f l a v i n e ( c l o s e d c i r c l e s ) on the s y n t h e s i s and s e c r e t i o n o f e n a m e l m a t r i x prot e i n s d u r i n g fetal New Z e a l a n d W h i t e r a b b i t m o l a r d e v e l o p m e n t in v i t r o is s h o w n f o l l o w i n g 24 hrs. in o r g a n culture. Note that p r e c o l l a ~ e n b i o s y n t h e s i s was not r e d u c e d by t h i s transcriptional inhibitor. From S l a v k i n et al, 1979.

Molecular Aspects of Tooth Morphogenesis and Differentiation

5.3.

Translation

of

Enamel

155

mRNAs

The i s o l a t i o n and c h a r a c t e r i z a t i o n of nascent enamel p o l y o e p t i d e s on p o l y s o m e s has not been reported. Information is not as yet a v a i l a b l e r e K a r d i n g the d e t a i l s of nascent t r a n s l a t i o n of enamel mRNAs. Neither the number of mRNAs or the actual number of nascent p o l y p e o t i d e s is as yet a v a i l a b l e . Recently, our l a b o r a t o r y reported on the isolation and p r e l i m i n a r y c h a r a c t e r i z a t i o n of a m e l c b l a s t - d e r i v e d mRN&s and their r e s p e c t i v e gene products (Lee-Own et al, Iq77; Z e i c h n e r - D a v i d , W e l i k y and Slavkin, Iq,%0). A PolF[A]+mRNA fraction of 16-0_6S was isolated from d e v e l o p i n g fetal New Zealand White rabbit molar ameloblast cells by u l t r a c e n t r i f u ~ a t i o n en sucrose ~radients. When t r a n s l a t e d in a r e t i c u l o c y t e - l y s a t e , cell-free system, the mRNA r e s u l t e d in the synthesis of three proteins of m o l e c u l a r weights 65,000, 58,000 and 43,000 d a l t o n s ( Z e i c h n e r - D a v i d , Weliky and Slavkin, 1980). The higher m o l e c u l a r weight proteins were assumed to be enamel polypeptides. Further attempts to identify t h e s e t r a n s l a t i o n gene products using immunoprepi,oitation assays indicate that they are a n t i g e n i c a l l y c r o s s - r e a c t i v e with antisera which is produced against enamel matrix proteins. Attempts to produce a cDNA for enamel mRNAs are now in progress. T%BLE

5.

Enrichment

Translation

Variables

of mRNA

Translational

Activity

Intact Molars ~-S.A. Enrichment

Dental M e s e n c h y m e S.A. Enrichment

Total RNA 19521 I 10732 P o l y ( A ) - c o n t a i n i n ~ RNA 130166 6.6 13,%353 16-26S mRNA 244037 13 278011 ~ S p ec T f ~ - - A - c t - i v--ft-y-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I 12.8 25.8

The specific a c t i v i t y w a s d e t e r m i n e d a s c.p.m, of [ 3 5 S l m e t h i o n i n e i n c o r p o r a t e d into t r i c h l o r o a c e t i c a c i d - p r e c i p i t a b l e p r o t e i n / u ~ of RNA added to the r e a c t i o n mixture. Fr-om Z e i c h n e r - D a v i d , W e l i k y and Slavkin,-]q~-qT. . . . . . . . . . . . . . . . . . . . .

156

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

I

55,000--

Fiz.

2

5

4

Rill

~65,000 --58,000

12. G e l - e l e c t r o p h o r e t i c analysis of products o [ ~ t r a n s l a t i o n in the c e l l - f r e e system. ~-i-uorographs of 10{--polyacrylamide slab ~els c o n t a i n i n g the r e t i c u l o c y t e - l y s a t e , c e l l - f r e e system products when: no e x o g e n o u s mRNA was added (2), P~, of dental m e n s e n c h y me 16-26S mRNA was added (3). 14Clabelled amylase (55,000 mol.wt.) was used as m o l e c u l a r - w e i g h t marker (I). From Z e i c h n e r - D a v i d , Weliky and Slavkin, 1980.

Molecular Aspects of Tooth Morphogenesis and Differentiation

I

?-

3

4

55,000

Fig.

15. Relationship between mRnA species ~n--d~p-r-6-t-@zns p.res_~ent ~n the rabbTt tooth organ Compari-~o-E-of the cell-free-system translational products obtained with the 16-26 S mRNA (3) and the newly synthesized proteins (4) extracted fro~ intact molars shows the correlation between these gene products. The characterization of the proteins is facilitated by including the ~olecular-wei~ht marker (I) and the reticulocyte-iysate endogenous cellfree product (2~. Fro~ Zeichner-David, Weliky and Slavkin, 1980.

157

158 5.4.

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui Post-Translational

Processin<

of Enamel

Proteins

The available evidence now suggests that several major gene products, enamelin and amelogenin, characterize the ameloblast biochemical phenotyoe (Termine et al, 1080). These proteins appear to have significant p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s durin~ the course of i n t r a c e l l u l a r transport, secretion, e x t r a c e l l u l a r matrix formation and m i n e r a l i z a t i o n (Fukae, Tanabe and Shimizu, 1977; Fukae et al, 1980; Slavkin et al 1979). Our l a b o r a t o r y p r e v i o u s l y suglested the paradigm of a " p r e p r o e n a m e l ÷ ÷ + + p r o e n a m e l ÷ + + + e n a m e l " model (Chrispens et al, 1979) after the precedent discovered for a number cf secretory proteins including the various types of collagen (Fessler and Fessler, 1978; Steiner et al, 1980). We further suggest that the kinetics and relative processing for enamelin will be found to s i g n i f i c a n t l y differ- with those found for amelogenin (ZeichnerDavid et al, in press).

TABLE

6.

Effects of T r a n s c r i p t i o n a l , T r a n s l a t i o n a l and Post-Translational Inhibitors on Intracellular Protein Synthesis in vitro

................C . . .u.l. t. u. .r.e. . . . . .C. .o. n. .d. .i .t .i o n s

%- I n h T b T t - X - o n - - o f - - C 6 n t ~ o ]

0°C

Transcriptional Actinomycin Translational Puromycin

97 %

Inhibitor D {30 ~g/ml}

76.75

Inhibitor {2 mM}

Post-Translational Inhibitor PhenylmethyIsulDhonyl Fluoride

~£,6

{I aM)

Z

%

47

Groups of 4-12 molar tot)th organs were preincubated for 1 hour in c u l t u r e medium. The pulse period (5-15 minutes) be~a3n with the addition of either [ 35S]-methionine (50 ~Ci/mL), [ H]-leucine (50 pCi/m]) , or [ 3H]-proline (50 uCi/mL) in the presence or absence of inhibitors in 50 ul MEM in HEPES buffer (20 mM) omitting the amino acid used to label with. The chase medium consisted of MEM in HEPES buffer with I mM excess amino acid used. Molar tooth organs were washed and then boiled for 5 minutes in 50 pl of 125 mM TrisHCI, pH 6.8/2% SDS/5 % 2 - m e r c a p t o e t h a n o l / 2 5 0 mM sucrose. From Slavkin et al., 1979. Evidence for a " p r e c u r s o r - p r o d u c t " r e l a t i ~ n s h i p for enamel proteins has been suggested by a number of d i f f e r e n t laboratories. Porcine, iogamorph, rodent and bovine enamel protein b i o s y n t h e t i c studies have all suggested proteolytic processing of nascent enamel proteins during secretion and during subsequent enamel matrix maturation (Fukae and Shimizu, 1974; Fukae et al, 1980; Hoe and Birkedal-Hansen, 1979). Available information suggests that amelogenins are processed from a 2 5 , 0 0 0 - 3 0 , 0 0 0 d a l t o n s to a 21,000 daltons p o l y p e p t i d e during secretion (Fukae et al, 1980; Slavkin et al, 1979; Zeichner-

Molecular Aspects of Tooth Morphogenesis and Differentiation

159

David et al, in press). Of course, the nascent amelogenin polypeptide should prove to be of higher molecular weight. In contrast, enamelin polypeptides appear to be more stable, have different kinetic properties and probably possess very different functions.

5.5.

Enamel

Biosynthesis

A sequential dissociative extraction scheme has been reported in which fetal enamel matrix proteins are extracted first in 4 M guanidine HCL, DH 7.4, and subsequently in 4 M guanidine HCI, 0.5 M EDTA, pH 7.4, in the presence of protease inhibitors (Termine et al, 1980).

The latter procedure has been shown to be effective for demineralization and the extraction of those enamel proteins specifically associated with enamel calcium hydroxyapatite crystals. The initial dissociative step extracts proline-rich amelogenins, whereas the subsequent extraction with guanidine HCL/EDT~ removes enamefins (approximately 72,000 daltons), which stain positively for sialic acid, phosphate and carbohydrates, and have an amino acid composition distinct from that reported for amelogenins (Lyaruu et al, in press; Slavkin et al, in press). In addition to amino acid composition differences, enamelins also differ from amelogenins in their very high affinity for apatite crystals. Biosynthetic data indicate that amelogenins are secreted before enamelins, and that amelogenins are rapidly converted to increasingly smaller polypeptides by enamel proteases (Zeichner-David et al, in press). Enamelins appear to be highly stable during enamel maturation. Immunoprecipitation data indicates that both enamelins and amelogenins are antigenically cross-reactive (Herold, Graver and Christner, 1980; Slavkin et al, in press; Termine et al, 1980). These features and many others now provide opportunities for future studies of enamelin biosynthesis. 5.6.

Amelogenin

Biosynthesis

The biosynthesis of amelogenins in ameloblast cells include translation on polysomes, glycosylation of the polypeptides in the Golgi, phosphorylation during condensation within secretory vesicles, and subsequent proteolytic post-translational processing of secreted amelogenins into increasingly smaller polypeptides (Fincham, 1979; Fukae et al, 1980; Leblond and Warshawsky, 1979; Sasaki and Shimokawa, 1979; Seyer and Glimcher, 1977; Slavkin, Mino and Bringas, 1976; Slavkin et al, 1979. During fetal amelogenesis, newly secreted amelo~enins are approximately 25,000 to 30,000 daltons, which is considered the archtype of amelogenins (Fukae et al, 1980; Termine et al, 1980). Although controversy has pursued for many years regarding the actual number of different enamel gene products, current d a t a now provides evidence to indicate that the larger molecular weight amelogenin is the precursor for the numerous small polypeptides (Fukae et al, 1980). Pulse/chase isotopic labeling experiments, polyacrylamide gel electrophoresis and gel filtration chromatography have been used to establish the amelogenin precursor-product relationship; amelogenins of approximately 30,000 daltons are proteolytically-cleaved to produce 21,

160

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

000 da]_ton a m e l o ~ , e n i n s ~enin polypeptides.

which,

in

turn,

produce

6,000

dalton

amelo-

Amino a c i d c o m p o s i t i o n and p a r t i a l amino a c i d s e q u e n c e d a t a f u r ther indicate that amelogenins of anomalous molecular weights were a l l d e r i v e d f r o m t h e same p r i m a r y ~ene p r o d u c t (Fukae et al, 1980). Finally, partial characterization o f enamel p r o t e a s e s which mediate post-translational processing of amelcgenin suggest a pH o p t i m a l activity of 6, i n h i b i t i o n by p h e n y l m e t h y l s u l f o n y l fluoride (PMSF) and disopropyl p h o s p h o f l u o r i d a t e , and also indicate that these enzymes are not affected by EDTA (Fukae, Tanabe and Shimizu, 1977; Hoe and Birkedal-Hansen, 1979; Shimizu, Tanabe and Fukae, 1979).

Molecular Aspects of Tooth Morphogenesis and Differentiation

161

~ .~a]-(B~~=' ASE}F'["IN- FRANUCLEAR o

3

~- ..-~ IsTR,TuM' TERMEO'U~ ZONE

0 I

i

NUCLEARZONE

I

SUPRANUCLEAR ZONE

_I

I.-

IXIMAL

r INTERDIGITATING

Fig.

16.

ZONE OF TOMES' PROCESS

Diagram of secretory ameloblast cells. From Slavkin, Mino and Bringas, 1976.

162

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

Fig.

17.

Scanning electron m i c r ~ s c o p i c desc r i p t i o n of the t o p o g r a p h i c a l features during fetal rabbit s e c r e t o r y amelogenesis. Arrows indicate red blood cells (circa 7 m in diameter). The a m e l o b l a s t cells secrete enamelin and a m e l o g e n i n proteins from the lateral surfaces of To~es' processes.

Molecular Aspects of Tooth Morphogenesis and Differentiation

Fig.

18.

Secretory granules release enamel proteins as granular material (arrows) along the periphery of Tomes' processes. From Slavkin, Mino and Bringas, 1976.

163

164

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

Fig.

19.

T o p o g r a p h i c a l features of the enamel matrix surface following low-power s o n i c a t i o n to r e m o v e a d h e r e n t ameloblast cells. The enamel prism pattern is i n d i c a t i v e of the v e r t e b r a t e species Note the t r a n s i t i o n from no enamel matrix to o v e r t enamel matrix.

Molecular Aspects of Tooth Morphogenesis and Differentiation

G e n e (S)

HnRNA

mRNA

Intracellular

(s)

(s)

Polypeptides I I

Post-- translational modifications

,l

B

m

Proteins

Enamel

I I I I

Extracellular

Degradation and t mineralization

Enamel

Fig.

20.

Summary of enamel protein synthesis and secretion.

bio-

Posttranslational modifications

165

166

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

6. Partial Amino Acid Sequences of Enamel Proteins Another e x t r e m e l y i n t e r e s t i n { but as yet incomplete set of investig a t i o n s has dealt with the amino acid sequence of the p o l y p e p t i d e s p h y s i c a l l y isolated and c h a r a c t e r i z e d from fetal and/or m a t u r e enamel. Glimcher and his c o l l e a g u e s were able to identify four homogenous p h o s p h o r y l a t e d p o l y p e p t i d e s (El, E2, E3 and E4) which have been isolated by p r e p a r a t i v e acrylamide gel e l e c t r o p h o r e s i s and e v e n t u a l l y isolated in q u a n t i t i e s a p p r o p r i a t e for study using a c o m b i n a t i o n of Bio-Gel P-IO f i l t r a t i o n and DE-52 ion exhange chrom o t o g r a p h y using 6M urea (Glimcher, 1979; Papas, Seyer and Glimcher, 1977). Analysis of peptides derived from partial acid h y d r o l y s i s and e n z y m a t i c d e g r a d a t i o n of h o m o K e n e o u s samples of E3 and E4 provided data s u g g e s t i n g that each of these p o l y p e p t i d e s c o n t a i n e d serine r e s i d u e s which were p h o s p h o r y l a t e d . In extended studies, Glimcher (1979) reported that the amino acid seq~ence in E4 is idential to that found in E3 except for the sixth amino acid which is t y r o s i n e in ELI. E3:

~IH2HET-PRO-LEU-GLU-SER(P)-LEU-

Ed:

N H 2 H E T - P R O - L E U - G L U - S E R ( P ) -TYR-

Usin~ a c o m b i n a t i o n of t e c h n i q u e s the amino acid sequence of E4 p h o s p h o r y l a t e d p o l y p e p t i d e is suggested to be as follows:

the

NH2-MET-PRO-LEU-GLU-SER-TYR-VAL-LEU, [ ( T Y P - P R O - L E U ) , (ASPILE-PHE-TRY)], [PRO2 G L Y 2 H I S 2 L Y S ] - G L U - S E R - T Y R , (PRO 2 TYR,GLY, HIS)-MET-GLY-TRP-COOH

In c o n t r a s t , Fukae, ljiri, Tanabe and Shimizu (1979) described the partial amino acid s e q u e n c e s of two p r o t e i n s isolated from d e v e l o p ing porcine enamel. In their a p p r o a c h they wanted to d e t e r m i n e if the amino acid s e q u e n c e s of two d i f f e r e n t proteins p h y s i c a l l y isolated from d e v e l o p i n g porcine enamel were identical o r d i f f e r e n t . Their s t r a t e g y was to obtain r e s u l t s of sequence analyses of these p r o t e i n s by a s e q u e n t i a l Edman d e g r a d a t i o n and by h y d r o l y s i s with c a r b o x y p e p t i d a s e Y which could indicate whether or not the partial amino acid s e q u e n c e s of these proteins were identical or different. One of the p o l y p e p t i d e s was termed Fr.2 which had a m o l e c u l a r weight of 20,300 daltons, w h e r e a s another major c o m p o n e n t was termed Ft.4 which had a m o l e c u l a r weight of 5,700. Both of these proteins were soluble usin~ g u a n i d i n e h y d r o c h l o r i d e . The isolated Ft.2 and Ft.4 p r e p a r a t i o n s were f r a c t i o n a t e d into d i s c r e t e polypeptides. The amino acid sequences of the first thirteen amino acids and the c a r b o x y l - t e r m i n u s regions of Fr.2 and Fr.4 were found to be identical:

Molecular Aspects of Tooth Morphogenesis and Differentiation

167

Fr.2-1MET-PRO-LEU-PRO-PRO-X-PRO-GLY-X-PRO-GLY-X-ILE---MET-PHE-SER 3 MET-PRO-LEU-PRO-PRO-X-PRO-GLY-X-PRO-GLY-X-ILE---MET-PHE-SER 4 ~ET-PRO-LEU-PRO-PRO-X-PRO-GLY-~-PRO-GLY-X-ILE---MET-PHE-SER 5 MET-PRO-LEU-PRO-PRO-X-PRO-GIY-X-PRO-GLY-X-ILE---MET-PHE-S~R Ft.4-1MET-PRO-LEU-PRO-PRO-X-PRO-GLY-X-PRO-GLY-X-ILE---PRO-SER-HIS 2

MET-PRO-LEU-PRO-PRO-X-PRO-GLY-X-PRO-GLY-X-ILE---TYR-GLY-TRP(?)

Finally, Fukae and his c o l l e a g u e s (1980) have r e c e n t l y published the amino acid sequence of two stages of amelogenin polypeptide post-translational processing; 26,000 and a 21,000 dalton amel~genins (termed "2a protein," and "2b protein," respectively). The amino acid sequences of the first 54 residues of 2a and 2b proteins is as follows:

I 2a 2b

Protein Protein

2

3

4

5

6

7

8

9

10

11

12

13

MET-PRO-LEU-PRO-PRO-HIS-PRO-GLY-HIS-PRO-GLY-TYR-ILEMEY-PRO-LEU-PRO-PRO-HIS-PRO-GLY-HIS-PRO-GLY-TYR-ILE14

15

16

17

18

19

20

21

22

23

24

ASP-PHE-SER-TYR-GLU-VAL-LEU-THR-PRO-LEU-LYSASP-PHE-SER-TYR-GLU-VAL-LEU-THR-PRO-LEUX 27

28

P9

30

GLU-ASP-MET-ILEGLU-ABP-MET-ILE40

41

42

43

31

33

34

35

36

~7

X -TYRX -TYR38

44

45

46

47

48

49

50

51

54

21.

39

52

X -LEU-HIS-HIS-GLU-ILE-ILE-PRO X -LEU-HIS-HIS-GLU-ILE-ILE-PRO

Val-Val Val-Val

Fi~.

26

X -HIS-PRO-TYR-THR-SER-TYR-GLY-THRX -HIS-PRO-TYR-THR-SER-TYR-GLY-THR-

GLU-PRO-MET-GLY-GLYGLU-PRO-MET-GLY-GLY53

~?

25

The amino acid sequences of the first 54 r e s i d u e s of an alleged "precursor" amelo~enin of 26,000 and that of a 21,000 d a l t o n s amelogenin. From Fukae et al, 1980.

168

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

The recent findings of Zalut and c o l l e a g u e s (1980) d e s c r i b i n g the N-terminal sequence for the bovine E4 p o l y p e p t i d e is in close a g r e e m e n t with the data reported by the Japanese i n v e s t i g a t o r s for the porcine a m e l o g e n i n s .

7. Significant Research Opportunities Recently, an e x t r e m e l y s e n s i t i v e assay has been d e s c r i b e d to detect and c h a r a c t e r i z e a specific mRNA c o r r e s p o n d i n ~ to a specific protein of known amino acid sequence. The t e c h n i c a l a p p r o a c h is based upon the a r t i f i c i a l production of a d e o x y o l i g o n u c l e o t i d e probe specific for a p a r t i c u l a r mRNA; the n u c l e o t i d e sequence of the probe can be c a l c u l a t e d from the known amino acid sequence of the protein under e x a m i n a t i o n (Agarwal and Noyes, 19£0). For studyin~ gastrin mRNA, w h i c h has served as a model system for d e v e l o p m e n t s of this s c i e n t i f i c a p p r o a c h , the unique amino acid sequence TRPH E T - G L U - G L U was selecte4 for the d e d u c t i o n of the n u c l e o t i d e sequence of the s y n t h e t i c cDNA probe. Thereafter, an o l i ~ o n u c l e c tide sequence was s y n t h e s i z e d by a rapid triester method, artifically p h o s p h o r y l a t e d and then used in a p r i m e r - t e m p l a t e system for the synthesis of ho~ ~astrin eDNA which was subsequently analyzed by a number of t e c h n i q u e s including n u c l e o t i d e sequence analysis and a number of h y b r i d i z a t i o n studies. These m e t h o d s suggest a feasible approach to studies of enamel gene products.

Part IV

Phylogenetic and Immunogenetic Characteristics of Enamel Proteins

1. Introduction to Vertebrate Enamel Evolution All v e r t e b r a t e teeth form as the result of c o m p a r a b l e d e v e l o p m e n t a l p r o c e s s e s ; r e c i p r o c a l and i n d u c t i v e e p i t h e l i a l - m e s e n c h y m a l interactions b e t w e e n and o v e r l y i n g oral e c t o d e r m and an u n d e r l y i n g core of cranial neural c r e s t - d e r i v e d e c t o m e s e n c h y m a l cells (see extensive r e v i e w s by Moss, 1977; Peyer, 1968; Poole 1967; Slavkin, 1974). During the 500 m i l l i o n years of v e r t e b r a t e e v o l u t i o n from shark to Man, enamel has provided a r e m a r k a b l y r e l i a b l e record. A number of f a s c i n a t i n g q u e s t i o n s become r e a d i l y apparent. Of p a r t i c u l a r interest to our l a b o r a t o r y is the q u e s t i o n of how many enamel gene products c h a r a c t e r i z e each v e r t e b r a t e species? What is the degree of v a r i a t i o n among and within v e r t e b r a t e species? Might d i f f e r e n t v e r t e b r a t e species, such as shark and Man, share enamel protein antigenic d e t e r m i n a n t s ? Recent data now supports the h y p o t h e s i s that the e n a m e l o i d c o v e r i n g the o u t e r m o s t surfaces of C h o n d r i c h thyes, T e l e o s t e i , and Reptilia contain similar antigens to those characteristic o f mammalian enamel p r o t e i n s .

2. Phylogeny of Secretory Amelogenesis: Enameloid and Enamel It was suggested by F e a r n h e a d (1979) that "true enamel" has a number of d i s t i n g u i s h i n g c h a r a c t e r i s t i c s : (I) an e x t r a c e l l u l a r matrix product secreted by e c t o d e r m a l l y - d e r i v e d epithelial cells; (2) an e x t r a c e l l u l a r matrix c o n t a i n i n g apatite crystals which are much larger than those found in me s o d e r m a l l y - d e r i v e d mineralized e x t r a c e l l u l a r m a t r i c e s (e.g. dentine, cementum, bone, c a r t i l a g e ) ; (3) an e x t r a c e l l u l a r matrix which does not contain c o l l a g e n m o l e c u l e s ; and (4) when m i n e r a l i z e d the e x t r a c e l l u l a r enamel contains only trace amounts of organic matrix c o m p o n e n t s , the protein is degraded and lost from the matrix during "enamel m a t u r a t i o n " . These d i s t i n g u i s h i n g features of "true enamel" are c r i t e r i a with which to identify and c h a r a c t e r i z e enamel from other e x t r a c e l l u l a r m a t r i c e s such as c a r t i l a g e , bone, d e n t i n e and cementum. 169

170

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

Enameloid is the term used to describe the m i n e r a l i z e d extracellular matrix c o v e r i n ~ the incisal cusp of the d e n t i c l e s in many living and extinct E l a s m o b r a n c h s and Teleost fishes (Fosse et al, 1974; Garant~-- f970; 3~,ss, 1977-;[email protected], 196~; Poole, 1967; Shellis, 1975). Convincing, evidence suggests the e n a m e l o i d represents the ancestral e x t r a c e l l u l a r matrix to the hi~her v e r t e b r a t e e n a m e l s (Gaunt and Hiles, I~67). Several i n t e r e s t i n g approaches have been taken. C o m p a r i s o n s amc,ngst eiKht shark species, representing three E l a s m o b r a n c h families within C h o n d r i c h t h y e s , found that min-@r-a-l---~l-i-s-t-ributi(~n and patterns in---enamelc~id were h o m o l o g o u s to that for human enamel (Fosse et al, 1974; Gaunt and Miles, 1967). The E l a s m o b r a n c h dentitions have c o n t i n u o u s replacement of teeth; tn--.sh-a-rk]---£or example, the tooth form is flattened, triangular and r e p r e s e n t s a functional row of teeth r e s e m b l i n g "the cutting edge of a saw". Hature shark enamel proteins were found to be similar in amino acid c o m p o s i t i o n to adult bovine and human enamel proteins, rather than to embryonic mr d e v e l o p i n g m a m m a l i a n enamel proteins (Levine et al, 1966). These findings are c o n s i s t e n t with the i n t e r p r e t a t i o n that lower vertebrate enamel d e v e l o p m e n t a l p r o c e s s e s are a n a l o g o u s to those described during m a m m a l i a n a m e l o g e n e s i s (Kemp, 1980). The d e v e l o p m e n t a l p r o c e s s e s a s s o c i a t e d with inner enamel e p i t h e l i a l d i f f e r e n t i a t i o n into a m e l e b l a s t s and s u b s e q u e n t a m e l o g e n e s i s , appear to be h o m o l o g o u s between C h o n d r i c h t h y e s (e.g. shark), Teleostei (e.g. ballan wrasse, common e-e-i-,--pik-e3, and Eutheria ~ T g . mous-e, rat, rabbit, hamster and man) (Moss, 1977; Po(~l-@, 19-6-7). By a number of d i f f e r e n t criteria, including autoradioEraphy, light m i c r o s c o p y , t r a n s m i s s i o n and scanninK electron m i c r o s c o p y , the developinK patterns of synthesis and secretion ef enameloid matrices in Teleest fishes are c o m p a r a b l e to that observed in numerous m a m m a l i a n species. The c r o c o d i l i a n s a p p e a r t o be one o f t h e most i n t e r e s t i n ~ and atypical order within t h e Re p _ t i l i a n _ C l a s s ; phylo~entically they belonK to the Archosauria S u b c l a s s and a r e b e l i e v e d t e h a v e e m e r K ed d u r i n K t h e T r i a s s i c Era f r o m t h e T h e c o d e n t a i n s t o c k s<)me 230 million y e a r s age ( s e e e x t e n s i v e r e v i e w by F-e-r-K-0s<[n, 1 9 8 0 ) . It must also be e m p h a s i z e d that the Thecodontains also were t h e ancestors of Saurishian Dinosaurs, Fly~ng Reptiles and B i r d s (Walker, 1972). I t s h o u l d be n o t e d t h a t t h e c r o c o d i l ~ - a n - d e n i t i tion (e.g. Alligator m i s s i s s i p p i e n s i s ) is not of a c l a s s i c a l r e p t i l i a n type, but rather similar to that in mammals. The presence of teeth in the fossils of certain Jurassic and C r e t a c e o u s birds has led to a search for dental vestiges in m{,dern birds, all of which have been found t o o t h l e s s (Kerr, 1919).

Molecular Aspects of Tooth Morphogenesis and Differentiation

A r~

"'

Z UJ L9

ELASMOBRANCHS

'"

>-

Z UJ

O o O

-.I

(CHONDRICHTHYES)

o

~

0

,,,

~

~

~

u. 0

>-

~. I--

o o

m

W

u. A 0

~

>"

Z

__L

_o

1

~

TELEOST

1

0

o

n-

_

o

~

I1:

0

! ~

> <

>-

~"

-,._1

w I-

_1

n",

,"r

r,n

AMPHIBIA

REPTILIA !

/

09 (3

z

AVES

(?)

rr 121

T

Fi~.

22.

MAMMALIA

P h y l o g e n y of the m a j o r p a t h w a y s for the d i f f e r e n t i a t i o n of a m e l o b l a s t s and the p r o d u c t i o n of e n a of enamel extracellular matrix proteins.

171

172

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

This is an e x t r e m e l y significant set of o b s e r v a t i o n s , especially in light of the recent results published by Kollar and Fisher (1980) who showed that intraocular ~rafts of chick pharyngeal e p i t h e l i u m combined with mouse molar mesenchyme produced molariform tooth crowns with d i f f e r e n t i a t e d secretory ameloblasts depositing enamel matrix. They interpreted their results to suggest that the loss of teeth in modern birds was not the result of a loss in genetic coding for enamel biosynthesis, but an abberation in e p i t h e l i a - m e s e n c h y m a l interactions essential for" odontogenesis. What is not clear at this point is whether the Gallus d o m e s t i c a genome contains structural enamel genes, and if the "enamel matrix" observed by Kollar and Fisher in their heterologous tissue r e c o m b i n a n t s contained enamel proteins or keratin. This suggestion is offered since it has been r e p e a t e d l y shown that specific sources of m e s e n c h y m e (e.g. dermis) will determine the a r c h i t e c t u r e of a r e s p o n d i n g epithelia (i.e. scale, feather, hair, non-keratinized epidermis, keratinized epidermis), but will not effect the s p e c i f i c i t y of the e p i t h e l i a l - d e r i v e d gene product (Sengel and Dhouailly, 1977),

3. Comparative Biochemical Features of Selected Vertebrate Enamel Proteins In Figure 27 we present a comparison of selected vertebrate matrix proteins (e.~. shark, alligator, rabbit, mouse and hamster) as compared with fetal bovine enamelin and amelogenin (Termine et al, 1980). It is readily apparent that shark samples contain no det e c t a b l e amelogenin, whereas the other" vertebrates contain significant amounts of a m e l o g e n i n s and enamelins. Note the degree of h e t e r o g e n e i t y associated with enamel matrix proteins (see extensive d i s c u s s i o n s in the recent volume edited by Nylen and Termine, 1979), p r o b a b l y due to at least three critical issues (I) hydrogen bonding which might require conditions equivalent to 8 M urea for dissociation, (2) endogenous proteolytic activities within enamel e x t r a c e l l u l a r matrices, and (3) h y d r o p h o b i c bonding due to proline and leucine side chains, as well as e l e c t r o s t a t i c forces derived from the high content of glutamic acid and histidine. These issues and others have recently been considered by using extraction methods such as acetic acid at low temperature, urea-Tris borate solutions, the addition of protease inhibitors, guanidine h y d r o c h l o r i d e , d e n a t u r i n g and reducing conditions (Slavkin et al, 1979; Termine et al, 1979).

Molecular Aspects of Tooth Morphogenesis and Differentiation

COMPARISON

OF D I F F E R E N T

VERTEBRATES

/ / # / /.~'/.//,,/~7 / j Ioo/~ Zl. o,* !<.:°L,/~o-/o-/® ~ Z l y l / / E l ~.:/ / / 94,000

- - ~ -- ~-72,000

67,000--~ 43,000

- - i

20, Joo - - ~ P

® Fifo

23.

Comparison between proteins from selected vertebrates. Protein samples were applied to polyacrylagels containing 0,1% SDS and IM and then stained with Coomassie Blue. Bovine amelogenin and enamelin m o l e c ular weight markers were obtained by selective extraction with guanidine h y d r o c h l o r i d e (Termine et al, 1980) From Slavkin et al, in press.

173

174

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

TABLE

7. Amino teins

Acid

................... Amino

Acid

Proline

of

Selected

Vetebrate

Rabbit................ Bo~ne

"Enamelins"

Aspartic Acid Threonine

Serine

Composition

94 42

71

"Amelogenins"

~

~2 38

Enamelins 98 55

~

Enamel

Pro-

Shark Amelogenins 38 29

4~

Total 96 47

84

139

Alanine 126 29 72 Cystine trace 4 2 I 2 Valine 37 39 39 46 45 Methionine 27 11 23 51 17 Isoleucine 18 15 28 34 40 Leucine 63 ~ 53 ~ 73 Tyrosine 23 33 21 33 25 Phenylalanine 27 27 49 26 26 Histidine 20 ~ 27 I~ 14 Lysine 50 32 48 Arginine 46 30 40 19 43 Hydroxyproline . . . . traces traces 4 Hydroxylysine . . . . . . . . 8 Data expressed as residues/1000. a : 26-day-old NZW rabbit embryonic molars. Slavkin et al. J. Biol. Buccale 6:309-326 (1979). b = ~--mo~ths old Bovine fetuses. Termine et al. J. Biol. Chem. 2 5 5 : 9 7 6 0 - 9 7 6 8 (1980). c = Mature Hammerhead S h a r k Levine et al Science 154:1192-1194

(196~).

Molecular Aspects of Tooth Morphogenesis and Differentiation

175

In Table 7 we present a c o m p a r i s o n of the amino acid c o m p o s i t i o n s of selected v e r t e b r a t e enamel proteins (e.g. "enamelins" and '~melogenins"). Note that glutamic acid and glycine amino acid residues are high in enamelin, whereas increased leucine and histidine residues indicate amelogenins. The amino acid composition of the selected m a m m a l i a n species are comparable for enamelins and amelogenins. We assume that the amino acid c o m p o s i t i o n of the shark specimens r e s e m b l e s that for enamelins (see Kawasaki et al, 1980; and Levine et al, 1965).

4. Immunogenetic Aspects of Enamel Proteins The i m m u n o g e n i c i t y of enamel proteins has recently been reviewed by Schonfeld (1979) who clearly indicated that soluble enamel proteins are immunogenic across species barriers (e.g. rabbit a n t i - b o v i n e amelogenins) and solid-phase enamel proteins are immunogeneic within a species (e.g. mouse anti-mouse enamel matrix or rabbit a n t i - r a b b i t enamel matrix) (Guenther et al, 1977; Nikiforuk, and Gruca, 1971; Graver et al, 1978; Elwood and Apostolopoulos, 1975). Several months ago, Termine and his c o l l e a g u e s (1980) reported that rabbit anti-fetal bovine amelogenin antisera is crossreactive with enamelin. The same antisera had previously been described to be c r o s s - r e a c t i v e with d e v e l o p i n g porcine enamel matrix antigens as visualized by indirect immunofluorescent microscopy (Graver et al, 1978). Additional studies indicated that rabbit a n t i - b o v i n e amelogenin antisera is also cross-reactive with mouse enamel matrix antigens (Herold et al, 1979). Our l a b o r a t o r y has produced a rabbit anti-mouse enamel matrix antisera (Hyatt-Fischer et al, 1979) which is c r o s s - r e a c t i v e with enamel matrix antigens found in shark, alligator, hamster, rabbit and ~ouse. Our indirect immunohistochemcal localization of enamel protein antigens in the enameloid of various v e r t e b r a t e s and in enamel per se of several mammalian species confirms recent data reported by HerolJ and his colleagues (1980); enamel protein antigens are present in the enameloid of Chondrichthyes,_ Teleostei and Amphibia as well as in the enamel of Repitilia Nonm a m m a [ i a n enamel proteins share a n t i g e n i ~ - d e t e r { i n a n t s with mammalian e n a m e l i n s and ameloKenins.

176

H . C . Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

B Fi~

,

24,

Immunological cross-reactivity of e n a m e l p r o t e i n s in d i f f e r e n t vertebrates. Teeth were dissected, fixed in 4% b u f f e r e d - f o r m a l i n and f r o z e n s e c t i o n s (4-6 ~m) were preDated. S e c t i o n s w e r e i n c u b a t e d with r a b b i t a n t i - m o u s e e n a m e l organ epithelia I~G f r a c t i o n , washed with phosphate buffered feted - s a l i n e , and i n c u b a t e d w i t h flu<)rescein - ccn.]u~ated ~.oat anti r a b b i t IgG (A) shark, and (B) m o u s e toc,th o r g a n s .

Molecular Aspects of Tooth Morphogenesis and Differentiation

177

5. A Phylogenetic Hypothesis Evidence that antigens within either enameloid or enamel are immunologically cross-reactive is now available (Graver et a~, 1978; Herold et al, 1979; Herold et al, 1980; Hyatt-Fischer, et al, 1979; Schonfeld, 1979; Termine et al, 1980). Both enamelin and amelogenin are a n t i g e n i c a l l y cross-reactive (Termine et al, 1980), Albeit limited, available information also suggests that m a m m a l i a n and Reptilian enamel proteins consist of both enamelin and amelogenin. Several Fears ago we proposed a h y p o t h e s i s stating that s e c r e t o r y amelogenesis in m a m m a l i a n tooth d e v e l o p m e n t consisted primarily of a " p r e p r o e n a m e l polypeptide" which is synthesized on polysomes and, thereafter, experiences a number of e n z y m e - r e g u l a t e d , postt r a n s l a t i o n a l m o d i f i c a t i o n s r e s u l t i n g in the formation of a large number of h e t e r o g e n e o u s , low m o l e c u l a r weight, p o l y p e p t i d e s (Chrispens et al, 1979; Slavkin et al, 1976; Slavkin et al, 1977; Slavkin et al, 1979; Z e i c h n e r - D a v i d et al, 19~0). We now suggest, in attempting to interpret the structure and function of precursor forms, that secretory proteins, such as enamel polypeptides, r e f l e c t the e v o l u t i o n a r y h i s t o r y of specific functional c o n f o r m a t i o n s . Enamel protein's unique pathway of evolution imposes structural and functional d e t e r m i n a n t s . It seems likely that as evolution progressed from E l a s m o b r a n c h s to Mammals, new enamel p o l y p e p t i d e s were elaborated -fro~m ~pre-@xisting m a c r o m o l e c u l e s . If these notions are valid, one could predict that m o n o - s p e c i f i c a n t i b o d i e s directed against shark enamelin could provide an i m m u n o l o g i c a l probe with which to isolate the alleged " p r e p r o e n a m e l protein" during initial m a m m a l i a n secretory amelogenesis. Such studies are now in progress.

6. Summary: Prospectus During the past three years a number of technical advances have enabled more intensive biochemical i n v e s t i g a t i o n s into tooth morphogenesis and d i f f e r e n t i a t i o n . Combined with d e v e l o p m e n t a l and experimental embryological experimental designs, these scientific approaches have been quite productive towards ~aining an understandin~ of the m o l e c u l a r aspects of tooth m o r p h o g e n e s i s and differentiation. Extraction techniques designed to minimize potential protein d e g r a d a t i o n and aggregation of enamel polypeptides during isolation and subsequent c h a r a c t e r i z a t i o n , advances in twodimensional slab ~el p o l y a c r y l a m i d e e l e c t r o D h o r e s i s under d e n a t u r ing and reducing conditions, f l u o r o g r a p h y for high resolution detection and q u a n t i t a t i o n of very small amounts of i s o t o p i c a l l y labeled proteins, the d e v e l o p m e n t of i m m u n o p r e c i p i t a t i o n assays for enamel proteins and recent advances in DNA Recombinant t e c h n o l o g y all provide promise for future e l u c i d a t i o n of e p i t h e l i a l d e t e r m i n a tion and d i f f e r e n t i a t i o n into secretory ameloblast cells.

178

H.C. Slavkin, M. Zeichner-David and M. A. Q. Siddiqui

HnRNIA (s) ~

J

Intracellular

Polypeptides

'} ,1 I

Enamel

Post-translational

modifications

proteins

I

Degradation mineralization

Extracellular-

and

translational modifications

Enamel

Fig.

25.

Experimental design and for future research.

scheme

Molecular Aspects of Tooth Morphogenesis and Differentiation

179

Of particular promise are the technical advances which enable detection, isolation and c h a r a c t e r i z a t i o n of various v e r e b r a t e en~mel structural ~enes. Recently. Lee-Own and his colleagues (1977) and Zeichner-David, Weliky and Slavkin (1980) in our l a b o r a t o r y have provided p r e l i m i n a r y reports r e ~ a r d i n ~ the isolation of enamel mRNAs. More recently, stu4ies have been initiated in collaboration with Dr. M.~.Q. Siddiqui to produce complementary DNA molecules (cDNA) to the enamel mRNAs and to s u b s e q u e n t l y clone these cDNA m o l e c u l e s in an appropriate bacterial plasmid. The prospectus for these i n v e s t i g a t i o n s suzgest at least three objectives: (I) to isolate a desired sequence r e p r e s e n t i n g enamel structural gene(s) from a complex mixture of DNA m o l e c u l e s and the ability to replicate m i l l i g r a m quantities of enamel structural gene(s) for biochemical investigations; (2) to employ the isolated enamel genes as probes to investigate the c o n s e r v a t i o n and/or variability to enamel gene evolution from shark to Man; and (3) to d e m o n s t r a t e that b a c t e r i a can synthesize in large amounts enamel p o l y p e p t i d e s which represent the nascent polypeptides formed on the p o l y s o m e s before p o s t - t r a n s l a t i o n a l processing. Finally, a number of biomedical problems can also be approached including further b i o c h e m i c a l i n v e s t i g a t i o n s of normal and abnormal tooth m o r p h o g e n e s i s , amelogenesis and d e n t i n o g e n e s i s imperfecta, and, possibly, the d e v e l o p m e n t of biological enamel matrices to serve as m a t e r i a l s in the repair of dental caries.

7. Acknowledgements We wish to thank Drs. Alain Belcourt, Allan Boyde, Gerry Mechanic, and John Termine for their many stimulatin~ and p r o v o c a t i v e discussions and critical c o n t r i b u t i o n s toward our intellectual growth in this complex and intriguing problem area. We also want to thank the many c o n t r i b u t i o n s from our collea~ues Drs. Richard Croissant, Edward Graham, Harold Guenter and Lawrence Honi~ as well as Connv Bessem, Pablo Bringas, Jr., Elaine Cummings, Martin Grodin, Mary HacDou~all, Julia Vides, and Berta Weliky We want to e s p e c i a l l y thank Ms. Gwen Airkens for her untiring efforts in preparin~ this manuscript. The efforts of Ms. June Sayles and Ms. Olga Lewis are very much appreciated. Aspects of this research effort has been supported in part from research grants from the National Institute For Dental Research, U.S. Public Health Service (DE-02848, DE-03569 and DE-03513) and training grants DE-O0094 and DE-00134.

180

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