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
Running Title: Enamelysin (MMP-20) knockout mouse
1
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.
2
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.
3
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
4
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).
5
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.
6
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
7
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.
8
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
9
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
10
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
12
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
16
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.
17
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.
18
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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