Enamelysin (MMP-20) Deficient Mice Display an Amelogenesis Imperfecta Phenotype.

John J. Caterina1, Zeidonis Skobe2, Joanne Shi1, Yanli Ding3, James P. Simmer4, Henning

Birkedal-Hansen1, and John D. Bartlett3,5,6

From the 1Matrix Metalloproteinase Unit, NIDCR, National Institutes of Health, Bethesda,

Maryland 20892, the 2Biostructure Core Facility, and the 3Department of Cytokine Biology,

Forsyth Institute, Boston, Massachusetts 02115, 4Department of Biologic and Material Sciences,

University of Michigan School of Dentistry, Ann Arbor Michigan 48108, and the 5Department

of Oral and Developmental Biology, Harvard Medical School, Boston, Massachusetts 02115

6To whom correspondence may be addressed: Dept. of Cytokine Biology, Forsyth Institute,

Boston MA 02115. Tel.: 617-262-5200 x388; Fax 617-456-7732; E-mail

jbartlett@forsyth.org.

Running Title: Enamelysin (MMP-20) knockout mouse

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Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on October 21, 2002 as Manuscript M209100200

SUMMARY

Enamelysin is a tooth-specific matrix metalloproteinase (MMP) that is expressed during the

early through middle stages of enamel development. The enamel matrix proteins amelogenin,

ameloblastin, and enamelin are also expressed during this same approximate developmental time

period suggesting that enamelysin may play a role in their hydrolysis. In support of this

interpretation, recombinant enamelysin was previously demonstrated to cleave recombinant

amelogenin at virtually all of the precise sites known to occur in vivo. Thus, enamelysin is likely

an important amelogenin-processing enzyme. To characterize the in vivo biological role of

enamelysin during tooth development, we generated an enamelysin-deficient mouse by gene

targeting. Although mice heterozygous for the mutation have no apparent phenotype, the

enamelysin null mouse has a severe and profound tooth phenotype. Specifically, the null mouse

does not process amelogenin properly, possesses an altered enamel matrix and rod pattern, has

hypoplastic enamel that delaminates from the dentin, and has a deteriorating enamel organ

morphology as development progresses. Our findings demonstrate that enamelysin activity is

essential for proper enamel development.

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7The abbreviations used are: AI, amelogenesis imperfecta; bp, base pairs; CBB, Coomassie

Brilliant Blue; ES, embryonic stem; HAT, hypoxanthine/thymine/aminopterin; HPRT,

hypoxanthine phosphoribosyl transferase; HSV, herpes simplex virus, kb, kilobase pairs; MMP,

matrix metalloproteinase; KLK-4, Kallikrein-4; PCR, polymerase chain reaction; Pgk,

phosphoglycerate kinase; RT, reverse transcriptase; SEM, scanning electron microscopy; tk,

thymidine kinase.

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INTRODUCTION

Dental enamel covers the crown of the tooth and is unique among mineralized tissues due to its

high mineral content, large crystals, and organized prism pattern. Other mineralized tissues such

as bone, dentin, and cementum are composed of approximately 20% organic material. In

contrast, mature enamel has less than 1% organic matter by weight (1,2). Moreover, enamel

crystallites possess a volume that is 100 times greater than the volume of crystallites found in

other mineralizing tissues. These enamel crystallites form enamel rods which, in turn, form a

unique interlacing (decussating) prism pattern. As a result, dental enamel is the hardest substance

in the body. Its hardness is intermediate between that of iron and carbon steel, yet it also has a

high elasticity (3).

Although mature enamel is a very hard protein-free tissue, it does not start this way. Enamel

development (amelogenesis) consists of several stages that include the secretory, transition, and

maturation stages. During the secretory stage, enamel crystallites elongate into long thin ribbons

that are only a few apatitic unit cells in thickness (about 10 nm) with a width of approximately

30 nm (4,5). The ribbons are evenly spaced, are oriented parallel to each other, and grow in

length but very little in width and thickness. Ultimately, enamel crystal length determines the

final thickness of the enamel layer as a whole [Reviewed in (6)]. It is during the secretory stage

that the columnar-shaped ameloblast cells, located adjacent to the forming enamel, secrete

specialized enamel proteins into the enamel matrix. These proteins include amelogenin (7),

ameloblastin (8), and enamelin (9). Amelogenin is the predominant component and comprises

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approximately 90% of total enamel matrix protein (10). Interestingly, the full-length enamel

proteins are found only at the mineralizing front suggesting that they participate in crystal

elongation (11-19). In contrast, the protein cleavage products are found throughout the enamel

layer suggesting that they prevent crystallite growth in width and thickness (16).

Enamelysin is a member of the matrix metalloproteinase family and its mRNA has been cloned

from pig (20), human (21), cow (22) and mouse (23). Enamelysin is secreted into the enamel

matrix during the secretory stage through transition stage of enamel development (24-27). Since

enamelysin is present in the mineralizing front, it is thought to participate in the early cleavage

events that allow the crystals to grow in length but not in width or thickness (25). Previously,

recombinant enamelysin was demonstrated to cleave recombinant amelogenin at virtually all of

the precise cleavage sites that were demonstrated to occur in vivo (28). Thus, enamelysin was

identified as a predominant amelogenin-processing enzyme.

As the secretory stage ends and the transition stage begins, the ameloblasts shrink in size and

down-regulate protein release into the enamel matrix. These changes are associated with an end

of the elongation of enamel crystals. The transition stage is followed by the maturation stage

where enamelysin expression is eliminated and the crystallites grow in width and thickness but

no longer in length. The remaining proteins within the enamel matrix are degraded by an enamel

matrix serine proteinase (KLK-4)7 prior to their export out of the enamel (25,29-32). Enamel

attains its final hardened form at the completion of the maturation stage. These general features

of amelogenesis are remarkably consistent among different species (33).

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Enamelysin is unique among the MMP family members because of its highly restricted pattern of

expression. One study assessed 51 different cell lines for enamelysin expression, but none were

positive (34). Conversely, enamelysin expression was observed in pathologic tissues such as in

ghost cells of calcifying odontogenic cysts (35), odontogenic tumors (36), and human tongue

carcinoma cells (37). Recently, enamelysin expression was also observed in bradykinin treated

granulosa cells isolated from the follicles of porcine ovaries (38). However, with the exception

of the ameloblasts of the enamel organ and the odontoblasts of the dental papilla (20-24,26,27),

no other intact physiologically normal tissue has been demonstrated to express enamelysin.

Therefore, to date, enamelysin is considered a tooth-specific MMP.

To characterize the in vivo role of enamelysin during amelogenesis, we have generated a mouse

with a null mutation that eliminates enamelysin activity. This mouse has a severe and profound

phenotype that includes altered amelogenin processing, enamel that delaminates from the dentin,

hypoplastic enamel, a disorganized prism pattern, and a deteriorating tooth morphology as

enamel development progresses. These results demonstrate that enamelysin plays a critical

protein-processing role during enamel development.

EXPERIMENTAL PROCEDURES

All animals used in this work were housed in AAALAC-approved facilities and all operations

were performed in accord with protocols approved by each Institute’s IACUC.

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Construction of the Targeting Vector and Generation of mutant mice--The enamelysin catalytic

domain targeting vector was constructed using gene sequences cloned from a 129 strain mouse

genomic library in the Lambda Fix II vector (Stratagene, La Jolla CA). The targeting vector was

pBluescript SK+ including a 4.6 kb 5’ homology spanning sequence from a PstI site in the 3’

region of intron 2 to the PstI site at the 5’ end of intron 4. The 1.1 kb 3’ homology arm

encompassed sequence from the BamHI site at the end of exon 5 to the XbaI site near the 3’ end

of intron 5. Sequence from the PstI site in intron 4 through the EcoRI site at the 5’ end of intron

5 were replaced with a phosphoglycerate kinase (Pgk) promoter-driven HPRT minigene EcoRI

cassette (39). The targeting vector was completed by addition of an HSV-tk minigene cloned

into the XbaI site in the 3’ terminus of the short homology arm and the SalI site of the plasmid

polylinker.

HM-1 mouse ES cells (40) were transfected with the targeting vector by electroporation and

cultured in selective growth medium containing 0.1 mM hypoxanthine, 16 ¼M thymidine, 0.4

¼M aminopterin (HAT supplement, GIBCO-BRL, Gaithersburg, MD), and 2 ¼M ganciclovir

(Roche Laboratories, Nutley, NJ). HAT-resistant clones were expanded and screened for the

legitimate targeting event using a primer complementary to the HPRT minigene 5’p01: 5’ ACC

CTC TGG TAG ATT GTA GCT TAT C 3’; and a primer complimentary to sequences not

included in the targeting vector 3’p02: 5’ CCT TTC CCA ACA TTG TCA CTG C 3’.

Cell clones containing the targeted allele were further characterized by Southern blot analysis.

An exon 6-specific probe was hybridized to EcoRI digested mouse genomic DNA. As a result of

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gene targeting, the endogenous EcoRI site present in the 5’ end of intron 4 was deleted and

replaced by an EcoRI site at the 3’ end of the HPRT cassette. This results in a band of

approximately 6.5 kb in cells with the targeted construct and a band of approximately 7.3 kb for

the native allele (Fig. 1A,B).

To generate chimeric mice, targeted ES cells were injected into 72 hr-old blastocysts from

C57BL/6 mice and implanted into pseudopregnant B6D2 or C57BL/6 x DBA females (NCI-

Frederick, Frederick, MD). Offspring were mated to C57bl/6 wild-type mice (NCI-Frederick,

Frederick, MD) to generate heterozygous animals for the targeted gene. These were subsequently

interbred to generate homozygous mutant progeny.

Genotyping of animals was performed by PCR amplification of DNA obtained from tail biopsies

with primers 5’p03: 5’ CTG CGT CCC CAG ACT TTT GAT TT 3’ and 3’p04: 5’ GCT TTT

CAT GGC CAG AAT GCT CT 3’ to detect the targeted allele; and primers 5’ p05: 5’ AAG

TAG ACT GAA GTC AGG AGA GCC 3’ and 3’ p06: 5’ CTG TAG TGG TGA CCC TAG

TCA TCT T 3’ to detect the wild-type allele.

Preparation of RNA--Total RNA was prepared by flash freezing tissue in liquid N2 followed by

extraction in Trizol (Invitrogen, Carlesbad, CA). Twenty µg samples of total RNA were size

fractionated on formaldehyde agarose gels, immobilized on nylon membranes, and hybridized to

an exon 5-specific radiolabeled probe.

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Protein Gels and Casein Zymography--First mandibular molars were extracted from 4.0-4.5

day-old pups, all non-mineralized tissues were removed, and proteins were extracted from

mineralized tooth caps by placing them in gel loading buffer (Zymograms: 62.5 mM Tris-HCl

[pH 6.8], 1.0% SDS, 0.3% glycerol, and 0.005% Bromophenol blue. In addition protein gel

loading buffers had 0.1% dithiothreitol). Casein zymography gels were prepared (12%

acrylamide, 375 mM Tris-HCl [pH 8.8], 0.1% casein, 0.0005% TEMED, and 0.05% ammonium

persulfate) and electrophoresis was performed at a constant current of 20 mA per gel for

approximately 2 hrs. After electrophoresis, protein gels were silver stained (Amersham

Biosciences, Piscataway, NJ) and zymography gels were washed twice for ten minutes in 50 ml

of 2.5% Triton X-100 solution (2.5% Triton X-100 in 100 mM Tris-HCl buffer [pH 8.0]). The

gels were incubated for one to two days at 37oC in 50 mM Tris-HCl buffer (pH 7.2) containing

10 mM CaCl2 and were stained with Coomassie Brilliant Blue (CBB) R-250 solution (0.23%

CBB R-250, 5.8% acetic acid, and 30% methanol) for 20 min and destained with 10% methanol

and 10% acetic acid until clear bands of substrate lysis were observed (41).

Histology and Scanning Electron microscopy (SEM)--Incisors obtained from three euthanized

wild type, three heterozygous, and six enamelysin null mice were fixed in 5% neutral

formalin/saline overnight, incubated in PBS containing 0.1% Triton X-100 for 8 hr, rinsed

overnight with running water and decalcified in 20% sodium citrate/45% formic acid for 2

weeks. This and all subsequent incubations were performed at ambient temperature. The jaws

were dehydrated in a graded series of ethanol washes and embedded in paraffin for sectioning.

Deparaffinized and rehydrated sections were stained with haemotoxylin/eosin. For SEM, erupted

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molar and incisor teeth were either examined whole or were fractured transversely, air-dried,

fastened to stubs, sputter coated, and examined using a JEOL 6400 scanning electron

microscope.

RESULTS

Targeted disruption of the enamelysin locus--The mouse enamelysin gene includes 10 exons

and is located within the MMP cluster at the centromeric end of chromosome 9 (42). To disrupt

the functional expression of the enamelysin gene, a 10.6 kb segment containing sequence starting

at the 3’ end of intron 2 and extending through most of intron 5 was modified such that the

majority of intron 4 and exon 5 was replaced by a PGK promoter controlled HPRT minigene

(Fig. 1A) (39,43). Exon 5 encodes the highly conserved zinc-binding site (HEXGHXXGXXH)

present in the catalytic domains of the MMP family. This deletion renders any polypeptide

expressed from this mutant gene catalytically inactive. The targeting construct was transfected by

electroporation into HM-1 (HPRT-deficient mouse embryonic stem) cells (40) and HAT

resistant clones were selected for further characterization. Targeted alleles were identified by

PCR and confirmed by Southern blot analysis. Chimeric offspring derived from two individual

cell clones were mated to C57bl/6 mice and germline transmission was obtained with chimeras

from both clones. Interbreeding of heterozygous mice yielded the expected Mendelian

distribution of homozygous mutant (enamelysin-/-), heterozygous (enamelysin+/-), and wild-

type (enamelysin+/+) mice (Fig. 1B). Total RNA prepared from enamelysin-/- homogenized

incisors probed with an exon 5-specific probe demonstrated the absence of transcripts containing

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this exon (Fig. 1C). Zymography of proteins extracted from 4.0-4.5 day-old first molars verified

the absence of enamelysin activity in the enamelysin deficient mice (Fig. 1C). Note that two

enamelysin bands are present on the zymogram. A study in which native enamelysin was

purified from porcine enamel suggests that the two bands represent active intact enamelysin and

active enamelysin with at least one cut site present within its hemopexin domain8.

Characterization of null mouse enamel--Maxillas from wild type and enamelysin null mice were

removed, the periradicular bone was dissected away, and the exposed molars were prepared for

SEM. The first maxillary molars from a wild type (Fig. 2A) and enamelysin null mouse are

shown (Fig. 2B). The dashed lines in figure 2 encompass the enamel-free areas of each molar.

As shown in the wild type, enamel free areas are normally present in the mouse molar at the

marsal plateau of the cusps. These areas provide troughs that are necessary for the efficient side-

to-side grinding of ingested food. In the enamelysin null mouse, however, the enamel that

surrounds the cusps is virtually absent. Only the cervical margin of the tooth had an enamel

covering that remained (Fig. 2). Thus, enamel from the null mouse delaminates from the dentin

surface.

To determine if the characteristic decussating rod pattern was altered in enamel from enamelysin

null mice, incisors were fractured and prepared for scanning electron microscopy. The fracture

plane of the wild type and heterozygous tooth extended through the enamel and dentin (Fig.

________________________8Y. Yamada, Y. Yamakoshi, R. F. Gerlach, J. C-C. Hu, K. Matsumoto, M. Fukae, S. Oida, J. D. Bartlett, and J. P. Simmer, manuscript submitted.

11

3A,B) whereas in the null mouse, fracture planes of enamel and dentin were separate (Fig. 3C).

This suggests that the null mouse enamel does not adhere properly to the dentin surface.

Littermate wild type and heterozygous mice had an inner enamel layer (100 µm) consisting of

alternating rows of enamel rods decussating at about 90o, an outer enamel layer of parallel rods

(15 µm) slightly inclined to the tooth surface, and a surface layer without rods (6 µm). Inspection

of the littermate null mouse inner enamel rods revealed the complete absence of the typical

decussating rod pattern and enamel rod diameters were notably uneven (Fig. 3C). Fractures in

the sagittal plane of null mouse incisors (not shown) did reveal the three distinct enamel layers:

The inner enamel layer (50 µm) with parallel rods inclined at about 45o to the dentin surface, an

outer enamel layer (15 µm) with rods nearly parallel to the tooth surface, and a surface layer

without rods (5 µm). In addition to the abnormal rod pattern present in the null mice, a

comparison of the enamel thickness from the dentin/enamel junction to the enamel surface

revealed that the null mice had a significantly thinner (hypoplastic) layer of enamel (70 µm) than

did the wild type (120 µm) mice (Fig. 3). Thus, the enamelysin null mouse incisor had enamel

that fractured independently of the dentin, abnormal enamel rod pattern, uneven enamel rod

diameter, and hypoplastic enamel.

The enamelysin null mouse tooth morphology--An advantage of observing tooth development

in rodents is that rodent incisors are continuously erupting and, therefore, all the stages of tooth

development are present along each forming incisor throughout adult life. A morphological

comparison of enamel development present in a demineralized incisor from littermate wild type

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mice (Fig. 4A-C), heterozygous mice (Fig. 4D-F), and enamelysin null mice (Fig. 4G-I) is

shown. Since the tissues were demineralized, only protein is observed. Thus, for the wild type

and heterozygous mouse incisors, the staining pattern beneath the ameloblasts becomes lighter

with each successive panel until, at the mid-late maturation stage (Fig. 4C,F), the most mature

enamel is clear. This clear area represents demineralized mature enamel that is almost protein-

free. Conversely, in the null mouse, with each successive panel (Fig 4G-I) the ameloblasts

become progressively more disorganized and the protein in the enamel matrix is not properly

resorbed. A comparison of the enamel proteins in the secretory and early maturation panels

between the wild type and null animals reveals that the normal protein pattern present in the wild

type is consistent with proteins that had surrounded mineralized prism structures (Fig. 4A,B).

This organized enamel protein pattern is absent in the enamelysin null mouse (Fig. 4G,H). Thus,

in comparison to wild type and heterozygous animals, the morphology of the enamelysin null

mouse incisor displays hypoplastic enamel, ineffective removal of proteins from the enamel

matrix, a disorganized protein pattern, and an increasingly disorganized ameloblast morphology

as development progresses.

Enamelysin null mice display altered amelogenin processing--To directly demonstrate that

enamelysin cleaves amelogenin in vivo, amelogenins were extracted from 4.0-4.5 day-old

mouse molars and size-separated by SDS-PAGE. A clearly different pattern of amelogenin

degradation was evident between the wild type and null mice (Fig. 5). Amelogenin proteins from

the null mice displayed a prominent band at approximately 27 kDa that was only faintly

detectable in the amelogenins from the wild type controls. Only one amelogenin band of less

13

than approximately 23 kDa was present in the enamel from the null mice whereas, in the

controls, at least 5 bands were present below this molecular mass. Thus, enamelysin activity is

responsible for generating at least 4 different amelogenin isoforms that are present in naturally

maturing dental enamel.

DISCUSSION

In summary, the enamelysin null mouse does not process amelogenin properly, possesses an

altered enamel protein and associated rod pattern, has hypoplastic enamel, has enamel that

delaminates from the dentin, and has a deteriorating tooth morphology as enamel development

progresses. Previously, several studies have shown that recombinant enamelysin cleaves

recombinant amelogenin (21,24,26,44) including a study demonstrating that recombinant

amelogenin was cleaved at virtually all of the precise cleavage sites that had previously been

observed in vivo (28). However, until now (Fig. 5), no study has presented direct evidence

demonstrating that enamelysin is responsible for these cleavages in vivo. Since enamelysin is

expressed primarily during the secretory stage of amelogenesis when the crystallites grow in

length, it appears that enamelysin functions to initiate hydrolysis of the structural enamel matrix

proteins so that the enamel crystals may grow in length. Prevention of this process by the

elimination of enamelysin activity results in thin, brittle enamel that does not mature properly.

Enamelysin activity is therefore essential for proper enamel development.

In addition, we have observed (not shown) that the first molar of the null mouse possesses less

14

enamel than the second molar, which in turn, has less enamel than the third molar. The mouse

molars erupt in this very sequence, from first to third. The same phenomenon was observed in

incisor teeth. Intact enamel covered the labial surface of the recently erupted incisor portion near

the gingival margin, but at the incisal tip the enamel was missing. This enamel wear pattern

suggests that the teeth erupt with a complete covering of enamel, but that over time the

malformed enamel wears or chips away presumably due to normal stresses encountered during

mastication.

Amelogenin comprises approximately 90% of the organic component of developing enamel.

Thus, the lack of amelogenin processing in the enamelysin null mouse is likely an important

aspect of the null mouse phenotype. Previously it was demonstrated that a solitary point mutation

(proline to threonine) in exon 6 of the amelogenin gene caused AI (45). This missense mutation

was positioned at P5 relative to a Trp/Leu enamelysin cleavage site and was demonstrated to

reduce the efficiency of hydrolysis by 25-fold compared to hydrolysis of the non-mutated

peptide (46). So, a small change in amelogenin structure can have a profound effect on enamel

development. Also, the hydrolysis of enamel proteins can alter their functional properties.

Proteolysis of amelogenin reduces both its crystal binding affinity and its solubility (47-51).

Thus, the lack of amelogenin processing in the enamelysin null mouse likely eliminated

necessary changes in the physical properties of amelogenin that are essential for proper enamel

development.

Interestingly, during the late maturation stage, the ameloblasts of the null mouse sometimes

15

surrounded abnormal nodule structures. In addition, an abnormally thick layer of protein

appeared to separate the ameloblasts from the enamel surface (Fig. 4 I). This result was difficult

to interpret given that both enamelysin and amelogenin are not normally expressed during this

late stage of enamel development. Perhaps, pre-processing of enamel proteins by enamelysin is

necessary for their proper removal from the enamel matrix and/or their subsequent degradation

by the ameloblasts.

Since, the dental enamel disease amelogenesis imperfecta (AI) affects only dental enamel, the

phenotype/genotype of the enamelysin null mice suggest that one form of human AI may be

caused by the recessively inherited inactivation of the enamelysin locus. The human amelogenin

gene in the p21.1-p22.3 region of the X chromosome and the human enamelin gene at 4q11-q21

are loci in known cases of AI (52,53). The human enamelysin gene locates to 11q22.3 which has

not yet been identified as an AI locus. However, in contrast to the phenotype observed for

mutations in the amelogenin gene (X-linked) or the enamelin gene (autosomal-dominant), an

enamelysin defect would likely be autosomal-recessive and therefore less prevalent within the

population. Thus, the likelihood of identifying an enamelysin deficient AI patient is greatly

reduced compared to the known genes that cause AI.

An intriguing aspect of the enamelysin null mouse is that because it displays a severe and

profound phenotype and survives to breed, it may be useful for transgenic studies to assess the

functional significance of MMP domain structure. MMPs are characterized by a domain structure

that consists of a signal peptide of approximately 20 amino acid residues that is removed after it

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has directed secretion of the enzyme from the cell; a propeptide composed of approximately 80

amino acids that folds back to mask and inhibit the catalytic pocket; a catalytic domain

composed of approximately 160 amino acids; and except for matrilysin and matrilysin-2, a

hemopexin-like domain comprised of approximately 200 amino acids [Reviewed in (54)]. In

general, MMP hemopexin domain function is poorly characterized. We are therefore currently

elucidating the function of the enamelysin hemopexin domain by inserting an enamelysin

transgene that encodes all but the hemopexin domain into the null mouse background. Thus, the

enamelysin null mouse may allow us an opportunity to identify functional aspects of specific

MMP domains as the tooth develops.

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Acknowledgments-We thank Glenn Longenecker and Ashok Kulkarni for gene targeting

expertise, Justine M. Dobeck, Nancy Marinos and Victor Morgan Jr. for histology expertise,

Susan Yamada for technical assistance, Jeffrey A. Engler and the University of Alabama at

Birmingham Cancer Center oligonucleotide core facility for intellectual input and

oligonucleotides, Charles E. Smith for sharing his zymography expertise, David Melton for his

generous gift of the HM-1 cells, and Conan Young and Daniel H. Lee for critical review of the

manuscript. This work was supported in part by a grant (DE14084) for J.D.B. from the National

Institute of Dental and Craniofacial Research.

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Fig. 1. Generation of enamelysin knockout mouse. A, a map of the targeted Pgk-HPRT mini

gene demonstrating the loss of most of intron 4 and exon 5. Exons are depicted as dark boxes.

Indicated below is the change in EcoRI restriction pattern between the wild type and targeted

enamelysin gene. B, PCR and Southern analysis of the F2 generation mice. Primers p03-p06

were used for PCR analysis where the 5’ primers were specific for intron 4 (wild type, wt) or the

HPRT minigene (null). Southern analysis was performed with an exon 6-specific probe after an

EcoRI restriction digest of genomic DNA. A 7.3 kb band demonstrated presence of the wild type

allele and a 6.5 kb band demonstrated presence of the knockout allele. C, total RNA from

incisors was probed with an exon 5-specific probe to confirm the loss of exon 5 in the

homozygous null mice. Proteins from immature mineralizing molars were subjected to

zymography to demonstrate the absence of enamelysin activity in the null mice. Note the doublet

present at approximately 42-46 kDa is missing in the null molars. This doublet represents zones

of casein degradation by enamelysin proteins (26) that differ in the size of their hemopexin

domains (M, marker; wt, wild type; null, enamelysin knockout).

Figure 2: Examination of wild type and null mouse molars. Scanning electron micrograph of a

first maxillary molar from a wild type (A) and an enamelysin null mouse (B). Dashed lines

encircle the enamel-free areas present on each molar. Note the pattern of enamel-free areas that

are typical of rodent molars at the marsal plateau of the cusps (A). In contrast, the first molar

from the enamelysin knockout mouse contains very little enamel (B). The only enamel that

remains is the enamel that surrounds the crown near the gingival margin. Most of the enamel has

delaminated from the dentin.

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Fig. 3. Examination of littermate enamel prism patterns. Enamel prism pattern of fractured

incisors from a wild type (A), heterozygous (B), and enamelysin null (C) mouse. The enamel

thickness is approximately 120 µm in the wild type and heterozygous, but is approximately only

70 µm in the null animal. The typical decussating inner enamel rod pattern can be observed in the

wild type (A) and heterozygous (B), but is absent in the enamel from the enamelysin null mouse

(C). Note the enamel from the null mouse did not fracture in the same plane as the dentin

indicating a faulty dentin/enamel junction.

Fig. 4. Examination of littermate incisor enamel organ morphology.. Demineralized sections of

wild type (A-C), heterozygous (D-F) and enamelysin null (G-I) mice showing ameloblasts

(Am), enamel space (En) and dentin (De). The wild type and heterozygous sections show tall

secretory-stage (A, D) ameloblasts with Tome’s processes penetrating into stained proteins of

the enamel layer. The secretory-stage ameloblasts from null mice (G) show ameloblasts that do

not have discernible Tome’s processes within the enamel protein layer. In the early maturation

stage (B, E, H) ameloblast length was reduced for all incisors examined. For the wild type and

heterozygous mice (B, E), the matrix was sparse and lightly stained indicating an increase in

mineralization and the loss of protein. In contrast, the enamel matrix protein from the null

mouse (H) persisted. In the late maturation stage the enamel matrix of wild type (C) and

heterozygous (F) mice was mostly removed. Conversely, enamel matrix in null mice persisted,

an abnormally thick layer of protein separated the ameloblasts from the enamel surface(I), and

nodule-like formations surrounded by ameloblasts were observed (I, arrows).

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Fig. 5. Examination of null mouse tooth proteins. Immature molars from 4-day-old mice were

dissected free of tissue, extracted for proteins, and subjected to SDS-PAGE. Note that the null

mouse has a strong amelogenin band of approximately 27 kDa whereas the wild type (Wt) has a

very weak band at this position. Also note that several lower MW amelogenin bands are missing

in the null lane compared to the bands present in the wild type lane.

25