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Cytomegalovirus induced amelogenesis imperfecta

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Cytomegalovirus induced amelogenesis imperfecta

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CYTOMEGALOVIRUS INDUCED AMELOGENESIS IMPERFECTA

by

George Abichaker

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CRANIOFACIAL BIOLOGY)

May 2012

Copyright 2012 George Abichaker

ii
DEDICATION

To my family and all those that helped and worked with me in the lab throughout all
these years. To all my friends who supported me. It was fun. To my beloved
grandmother, my inspiration.

iii
ACKNOWLEDGMENTS

Drs. Michael Melnick and Tina Jaskoll
Thank you for everything.

iv
TABLE OF CONTENTS

DEDICATION ii
ACKNOWLEDGMENTS iii
LIST OF FIGURES v
ABSTRACT vi
INTRODUCTION 1
MATERIALS AND METHODS 9
RESULTS 18
DISCUSSION 42
CONCLUSIONS 55
REFERENCES 56
APPENDICES
APPENDIX A: FIGURES 68
APPENDIX B: TABLE 1 89

v
LIST OF FIGURES

FIGURE 1: Tooth Development 68
FIGURE 2: Cytomegalovirus 69
FIGURE 3: Clinical Picture of CMV-Induced Amelogenesis Imperfecta 70
FIGURE 4: mCMV-‐Induced
Histopathology
and
Viral
Distribution

71
FIGURE 5: Characterization
of
mCMV-‐Induced
Cellular
Changes

73
FIGURE 6: Acyclovir
Treatment
Ameliorates
mCMV-‐Induced
Pathology

75
FIGURE 7: mCMV
Dose
Response

76
FIGURE 8: mCMV-‐Induced
Stage-‐Dependent
Differences
in
Tooth

Pathology
and
Enamel
Defects
on
Day
17
of
Culture

77

FIGURE 9: mCMV-‐Induces
Stage
Dependent
Tooth
Defects
on

Day
19
of
Culture

79

FIGURE 10: Comparative
Amelx,
Enam,
Dspp
and
β-‐catenin
Expression

in
Control,
Cap
Stage-‐Infected
and
Bell
Stage-‐Infected
Molars

81

FIGURE 11: mCMV
Induces
Stage-‐Dependent
Changes
in
the

Distribution
of
DSP,
enamelin,

and
amelogenin
proteins

83

FIGURE 12: mCMV
Induces
Stage-‐Dependent
Changes
in
the

Distribution
of
C/EBPα
Protein

85

FIGURE 13: mCMV
Induced
Stage-‐Dependent
Qualitative
and

Quantitative
Spatial
Changes
in
PCNA
Localization

87

vi
ABSTRACT

BACKGROUND: Cytomegalovirus (CMV) is one of the most common causes of major
birth defects in humans. Of the approximately 8400 children born each year in the U.S.
with CMV-induced birth defects, more than 1/3 of these children exhibit hypoplasia and
hypocalcification of tooth enamel.

OBJECTIVE: Our objective was to initiate the investigation of the pathogenesis of
CMV-induced tooth defects and examine the effects of CMV infection on progressive
tooth differentiation and amelogenesis.

METHODS: Mouse Cap and Bell stage mandibular first molars were infected with
mouse CMV (mCMV) in vitro in a chemically-defined organ culture system and
analyzed utilizing histological and immunolocalization methodologies

RESULTS: CMV infection of embryonic mouse mandibular first molars in vitro induces
tooth dysmorphogenesis and enamel defects in a developmental stage- and duration-
dependent manner. Initial protein localization studies suggest that the pathogenesis is
mediated through NF-κB signaling and that there appears to be an unusual interaction
between abnormal mesenchymal cells and surrounding matrix. Rescue with acyclovir
indicates that mCMV replication is necessary to initiate and sustain progressive tooth

vii
dysmorphogenesis. Cap stage- and Early Bell stage-infected molars exhibit enamel
agenesis and Bell stage-infected molars exhibit enamel hypoplasia.

CONCLUSIONS: Our results indicate that mCMV-induced changes in signaling
pathways severely delays, but does not completely interrupt, tooth morphogenesis. This
viral-induced pathology is coincident with stage-dependent changes in Amelx, Enam and
Dspp gene expression, distribution of amelogenin, enamelin, C/EBPα and DSP proteins,
cell proliferation localization and dedifferentiation of secretory ameloblasts. Our data
indicate that specific levels of Amelx and Dspp gene expression define whether mouse
CMV induces enamel agenesis or hypoplasia. Importantly, our results demonstrate that
this well-defined embryonic mouse organ culture system can be utilized to delineate the
molecular mechanism underlying the CMV-induced tooth defects that characterize the
amelogenesis imperfecta phenocopy seen in many CMV-infected children.

1
INTRODUCTION

During the fifth week of embryonic life, teeth begin to form and this process continues
until the roots of the permanent third molars are completed around the adult age of 20
years [127]. Tooth development is the same for permanent and primary teeth, although
they develop at different times (Fig. 1). The process starts when the tooth germ develops
from the dental lamina, a sheet of epithelial cells that develop from the primary epithelial
band. The primary epithelial band then organizes into two discrete epithelial ingrowths:
the vestibular lamina and the dental lamina. The dental lamina then forms a series of
epithelial buds that grow out into the surrounding connective tissue [127]. These buds
represent the first stage in the development of the tooth-germs of the primary dentition.
The epithelial buds then continue to grow and become associated with a condensation of
mesenchymal cells to form a tooth germ at what is called the cap stage of development
[127]. The epithelial bud develops into the enamel organ and the condensation of
mesenchymal cells constitutes the dental papilla and comes to extend around the enamel
organ to form the dental follicle. The cells at the edge of the eptithelial bud continue to
proliferate and grow to enfold the mesenchymal cells of the dental follicle, producing a
tooth germ [127]. This stage of development is known as the bell stage. During the
transition from cap stage to bell stage, a process of histodifferentitation produces the
structures of the enamel organ, with the external and internal enamel epithelia, stratum
intermedium and stellate reticulum.

2
The cells of the inner enamel epithelium become longer to a columnar shape, with the
cell nuclei occupying the portion of the cell beside the stratum intermedium, away from
the dental papilla [127]. The cells of the dental papilla adjacent to the internal enamel
epithelium, will then also become longer to a columnar form, with their nuclei aligned
away from the enamel organ and towards the center of the dental papilla. These columnar
cells differentiate into odontoblasts which are cells that eventually form dentin, and
dentin always precedes enamel formation [127]. Even though dentin is formed by
odontoblasts of the dental papilla, the process is initiated by signaling from the epithelial
cells of the enamel organ [127]. As dentin formation begins, the cells of the internal
enamel epithelium change into ameloblasts and begin to form enamel enamel. Dentin
and enamel formation begin in the area around the incisal edges and cusp tips and
continues toward the cervical margins of the crowns [127]. A process similar to that of
the differentiation of odontoblasts and dentin formation occurs to form the roots of teeth.
The inner enamel epithelium and the outer enamel epithelium come together in the neck
region of the tooth where they form a fold, the epithelial root sheath (or Hertwig Root
sheath). This sheath grows into the mesenchyme and initiates root formation. This
process takes about 3-5 years after the eruption of the tooth crown to complete.
Odontoblasts adjacent to the epithelial root sheath form dentin that is continuous with
that of the crown [127]. As the dentin increases, it reduces the pulp cavity to a narrow
root canal through which vessels and nerves pass. And then the inner cells of the dental
sac differentiate into cementoblasts, which produce cementum that is restricted to the root.

3
Cementum is deposited over the dentin of the root and meets the enamel at the neck of
the tooth known as the cementoenamel junction (CEJ) [75].

Site and time specific interruptions of tooth development process results in defects such
as amelogenesis imperfecta and dentinogenesis imperfecta. Of particular concern in this
research report is cytomegalovirus (CMV) –induced amelogensis imperfecta in children
infected during prenatal and perinatal life.

Cytomegalovirus (CMV) is an enveloped, double-stranded DNA betaherpesvirus (Fig. 2).
Like all other herpes viruses, CMV shares a characteristic ability to remain latent within
the body for extended periods; and in fact, establishes a long-lasting persistence in
salivary glands [90]. Human CMV (hCMV) is found throughout all geographic locations
and socioeconomic groups, and infects between 50% and 80% of adults in the United
States as indicated by the presence of antibodies in much of the general population [98].
Although most healthy individuals who are infected with hCMV after birth show no
symptoms, those who are immunocompromised have been shown to be adversely
affected by the virus, as well as devastating systemic infections being seen in neonates.
hCMV is also the virus most frequently transmitted to a developing fetus. In infected
newborns, the virus is shed in saliva for months to years before termination of productive
infection and establishment of latency.

4
As the name implies, cytomegalovirus produces enlargement of infected cells. Infected
cells will exhibit gigantism of both the entire cell and its nucleus, and within the nucleus
is a large inclusion body surrounded by a clear halo.

Transmission of CMV can occur by several mechanisms depending on the age group
affected. These include:
• Congenital CMV. Transplacental transmission from a newly acquired or primary
infection in a mother who does not have protective antibodies.
• Perinatal CMV. Transmission of the virus through cervical or vaginal secretions
at birth or later through the breast milk of a mother who has an active infection.
• Transmission through saliva. Many toddlers are infected this way in preschool
and then pass on the infection to their parents.
• Transmission by the venereal route. This method is most common after 15 years
of age.
• Respiratory secretions and the fecal-oral route.
• Iatrogeneic transmission. This can occur at any age through organ transplants or
blood transfusions.

CMV can induce transient but severe immunosuppression. Mouse CMV (mCMV) can
infect dendritic cells and impair their function and maturation and their ability to
stimulate T-cell responses, and it appears that hCMV can also infect dendritic cells and
alter their function. Similar to other herpes viruses, CMV can elude immune responses

5
by down-modulating MHC class I and II molecules and producing homologues of TNF
receptor, IL-10, and MHC class I receptors. CMV can both activate and evade natural
killer cells by inducing ligands for activating receptors and class I-like proteins that
engage inhibitory receptors. Thus, CMV can both hide from immune defenses and
actively suppress immune responses.

Since the primary topic of this paper discusses congenital effects of CMV, a closer
understanding of congenital infection and CMV is critical. When infection takes place in
utero, it may take on many forms. In a vast majority of cases, it is asymptomatic,
however sometimes when the virus is acquired from a mother with primary infection
(who does not have protective immunoglobulins), a classic form of cytomegalic inclusion
disease (CID) develops. CID is similar to erythorblastosis fetalis and affected infants
may suffer intrauterine growth retardation, be severely ill, manifest jaundice,
hepatosplenomegaly, anemia, bleeding due to thrombocytopenia, and encephalitis. In
cases which are fatal, the brain is often smaller than normal exhibiting microcephaly.

Infants who survive often exhibit mental retardation, hearing loss, blindness and other
neurologic impairments. That said, congenital infection does not always equate with
devastating results. Most infants with milder CID recover with a small minority
developing mental retardation later. Uncommonly, a totally asymptomatic infection may
be followed by neurologic sequelae months to years later. This includes delayed-onset
mental retardation and hearing deficits [58].

6

It is established that about 2% of live born infants are congenitally infected with active
CMV. About 10% of this group has newborn symptoms and most of these infants will
exhibit subsequent abnormalities of the central nervous system [20, 94]. Thus, at least 1
in every 500 newborns will exhibit major CMV-induced congential pathology, making
CMV one of the most common causes of major birth defects in humans [11, 20, 94].

It has also been established that about 36% of children with CMV-induced birth defects
and 5% of CMV-infected asymptomatic infants also exhibit enamel hypoplasia and
hypocalcification of teeth [106-108, 110]. In these cases of amelogenesis imperfecta (Fig.
3), the absence of enamel significantly increases the likelihood of serious tooth wear and
fracture. These effects are primarily seen in primary teeth, but given that active CMV
infection can persist for 6-18 months postnatal, enamel defects may be expected in the
permanent dentition as well. It can be estimated that each year in the United States there
are an additional 3000 children with CMV-induced amelogenesis imperfecta (AI), and
that the prevalence to age 12 is more than 30,000. This is a significant problem in that
children have incisal and cuspal attrition, as well as rampant dental caries [108]. Further,
these children may require orthodontic therapy due to caries-induced loss of primary
teeth, as well as abnormalities of growth and development of the oral-facial complex
secondary to microcephaly and growth retardation [106].

7
Since the mouse CMV has many features in common with hCMV, the mouse model has
been widely employed for studying the pathogenesis associated with acute, latent and
recurrent infections [57]. Previous studies by Baskar et al. [1-3] have investigated the
effects of CMV on embryonic development in mice and have observed consistent fetal
growth retardation and fetal dysmorphogenesis particularly of the craniofacial complex.
Other studies by Tsustui [120] have reported viral-antigen positive cells were in
abundance in the mesenchyme of the oral and nasal complexes and he postulated that
mesenchymal infection is the critical step in disrupting organogenesis. If this is true, then
oral-facial organogenesis, which is highly dependent on mesenchymal integrity and
epithelial-mesenchymal interactions, would be particularly vulnerable to CMV infection.
Previous studies in our lab have shown that first brachial arch derivatives such as the
submandibular gland (SMG) [70] and mandible [46], are vulnerable to CMV infection
and that CMV has a particular tropism for neural-crest-derived ectomesenchyme (EM).

Currently, little is know about the mechanism underlying hCMV-induced AI. The strict
species specificity of hCMV and the inability of mouse CMV (mCMV) to cross the
placenta have hindered the study of this virus (see reviews [52, 88, 89]). Since mCMV
has many features in common with hCMV and has been widely employed for studying
the postnatal pathogenesis associated with acute, latent and recurrent infections [57], we
employed the well-established embryonic mouse tooth organ culture system to study the
effects of mCMV infection on early tooth morphogenesis [45].

8
In order to have a better understanding of the long term effects on enamel formation, the
effects of mCMV infection on progressive tooth differentiation and amelogenesis in
cultured embryonic mandibular mouse first molars were examined. Since initial CMV
infection in human fetuses can occur at different developmental times (for example, early
fetal period, late fetal period, perinatal period), we varied the stage of intial viral infection
(Cap stage, Early Bell stage, Bell stage) as well as the duration of infection. We
postulated that the later the tooth-developmental stage of initial mCMV infection, as well
as the shorter the duration, the less abnormal the anatomic phenotypes, and that these
difference will be correlated with notable stage-dependent differences in transcript and
protein expression. We delineated progressive tooth development from Cap stage to
crown formation and demonstrate that mCMV infection in-vitro induces tooth
dymorphogenesis and enamel defects in a developmental stage- and duration-dependent
manner, and models the pathology seen in children. This viral-induced pathology is
coincident with stage-dependent changes in amelogenin (Amelx), enamelin (Enam) and
dentin sialophosphoprotein (Dspp) transcript expression, dentin sialoprotein, enamelin,
amelogenin and C/EBPα protein distribution, localization of cell proliferation, and
dedifferentiation of secretory ameloblasts.

9
MATERIALS AND METHODS

Embryonic Culture System and mCMV Infection

Female C57BL6 mice (Charles River, Wilmington, Mass., USA) were mated overnight
as previously described [45, 70]; plug day = day 0 of gestation. Timed-pregnant females
were sacrificed on gestation day 15 (E15) by carbon dioxide narcosis and cervical
dislocation. All procedures were performed in accordance with the Institutional Animal
Care and Use Committee of USC in accordance with the Panel on Euthanasia of the
American Veterinary Medical Association. Embryos were dissected in cold phosphate-
buffered saline, and staged according to Theiler [112]. Mandibular first molar regions
were dissected and cultured for up to 19 days using a modified Trowell method
essentially as previously described [9, 45]. The medium was BGJb (Gibco Invitrogen,
Carlsbad, Calif., USA) supplemented with 50 mM ascorbic acid, 50 units/ml
streptomycin and penicillin and 10% fetal calf serum. Fetal calf serum was added to the
medium since it has been shown to promote epithelial differentiation and amelogenesis
[118, 131]. Cultures were maintained at 37 ° C in atmospheric conditions of 95% air and
5% CO 2 . The medium was adjusted to pH 7.4 and changed daily.

For mCMV infection, we cultured E15 Cap stage mandibular first molars and incubated
them for 24 h with 250,000 plaqueforming units/ml of lacZ -tagged mCMV RM427 +
[99] on culture day 0 (Cap stage), day 5 (Early Bell stage) or day 9 (Bell stage) and then

10
in virus-free control medium for a total culture period of 15 (E15 + 15), 17 (E15 + 17) or
19 (E15 + 19) days. Controls consisted of E15 molars cultured in virus-free control
medium for 12, 15, 17 or 19 days. We have previously demonstrated that after the initial
24-hour mCMV infection, the virus continues to replicate in the dental papilla
mesenchyme for the entire culture period and that active mCMV infection is necessary to
initiate and sustain progressive tooth pathogenesis [45]. We employed this experimental
design to analyze variation in both initial stage and duration of infection in cultured E15
molars rather than in molars of different gestational ages (E15, E16 and E17) in order to
reduce variation. Explants were collected and processed for whole-mount morphology,
histology or immunohistochemistry as previously described [45]. For each experimental
protocol, 3–10 tooth organs/treatment/day were analyzed for each assay.

mCMV infection

On day 0, E15 tooth organs were incubated with 50,000 or 100,000 plaque-forming units
(PFU)/ml of lacZ-tagged mCMV RM427
+,
[99] for 24 h and then cultured in virus-free
BGJb-defined media for a total of 12 (E15 + 12) and 15 (E15 + 15) days; controls
consisted of E15 mandibular molar organs cultured in BGJb-defined media for the entire
culture period. Explants were collected and processed for whole mount morphology,
routine histology, viral expression, and immunolocalisation as previously described [70].
For each experimental protocol, 3–20 tooth organs/treatment/day were analysed for each
assay. Since no marked difference was seen in tooth organs infected with 50,000 or

11
100,000 PFU mCMV, tooth organs were routinely cultured in 100,000 PFU except where
noted. For histological analyses, tooth organs were fixed for 4 h in Carnoy's fixative at
4 °C or overnight in 10% neutral buffered formalin at room temperature, embedded in
paraffin, serially-sectioned at 8 µm and stained with haematoxylin and eosin as
previously described [70].

mCMV analysis

We assayed β-galactosidase (lacZ) activity, and localisation of viral immediate early
(IE1) proteins as described in Melnick et al [70].

β-Galactosidase (β-gal) staining

Briefly, mCMV-infected E15 + 12 and E15 + 15 tooth organs were processed, stained for
4–6 h and photographed. Whole mounts were then dehydrated through graded alcohols,
embedded in paraffin, serially-sectioned at 8 µm and counterstained with eosin. A
minimum of three explants per day were analysed.

IE1 distribution

mCMV-infected E15 + 12 and E15 + 15 tooth organs were fixed in Carnoy's fixative,
serially-sectioned at 8 µm, and incubated overnight with anti-IE; controls consisted of

12
sections incubated with mouse IgG alone. A minimum of three explants per day were
analysed.

Cell Proliferation Assay

Cell proliferation in control molars cultured for 12 or 15 days and mCMV-infected
molars cultured for 15 days was determined by the cell-specific localization of
proliferating cell nuclear antigen (PCNA) using the Zymed mouse PCNA kit (Invitrogen
Corp., Carlsbad, Calif., USA) and counterstained with hematoxylin as previously
described [45]. In this experiment, the cytoplasm appears purple and PCNA-positive
nuclei appear dark brown. We compared the cell-specific distribution of PCNA-positive
nuclei in Cap stage-infected and Bell stage-infected molars to control E15 + 12 and/or
E15 + 15 molars. For quantitation, we determined the cell proliferation indexes for 3
regions: (1) the dental papilla mesenchyme (DPM) (PCNA-positive DPM cells/total
DPM cells/mm
2
); (2) stellate reticulum (SR) (PCNA-positive SR cells/total SR
cells/mm
2
); (3) cervical loop (CL) (PCNA-positive CL epithelial cells/total CL epithelial
cells mm
2
). The DPM, SR and/or CL cell proliferation indexes were determined in 3
areas per section, 3 sections per tooth and 3 teeth per group. For each region, we then
calculated the mean ratios per tooth and mean ratios per group. Since percent data is not
normally distributed, the data was arcsin transformed before the mean ratios of mCMV-
infected and controls were compared by t test. The cell proliferation indexes are
presented as the fold change in mCMV-infected/controls.

13
Antibodies and immunostaining

Immunolocalization was conducted using the following monoclonal (Mab) and
polyclonal (Pab) antibodies: Mab ATP-synthetase (Mitochondria marker) (#MAB3494,
Chemicon International, Temecula, CA); Mab α5-integrin (# 103801, Biolegend, San
Diego, CA); Mab β1-integrin (# 102201, BioLegend); Pab β-catenin (# AB19022,
Chemicon International); Pab cytokeratin (# ab9377, Abcam Inc., Cambridge, MA); Pab
COX-2 (# 160106, Cayman Chemical Company, Ann Arbor, MI); Mab E-cadherin (#
610181, BD Biosciences, San Jose, CA); Pab FN (# F3648, Sigma-Aldrich Corp., St.
Louis, MO); Pab IL-6 (# sc-1265, Santa Cruz Biotechnology, Inc., Santa Cruz,
California); Pab p120 (# sc-1101, Santa Cruz Biotechnology, Inc); Pab mucin [48]. For
immunofluorescent analyses, Pab's were incubated with biotin-labeled rabbit IgG or anti-
goat IgG (MP Biomedical, Aurora, OH) and then with Alexa-Fluor-labeled streptavidin
(Invitrogen Corporation). Mab's were incubated with biotin-labeled anti-mouse IgG or
anti-rat IgG (Jackson Laboratories, West grove, PA) and then with Alexa-Fluor-labeled
streptavidin. Hamster antibodies (β-1 integrin) were incubated in FITC-labeled hamster
IgG (Biolegend). Nuclei were counterstained with DAPI (Invitrogen Corporation).
Immunohistochemistry was conducted essentially as previously described, using the
Chemicon Tissue Staining Kit. Sections were viewed on a Zeiss Axioplan Microscope
and photographed using 10×, 20× and 40× objectives. Confocal images were obtained
using a Zeiss LSM-510 laser scanning confocal/multiphoton microscope (Carl Zeiss Inc.
Thornwood NY) and the accompanying LSM version 3.2 image acquisition and analysis

14
software. Cells were imaged using a plan-neofluor 1.3 numerical aperture, 40× objective
lens. Alexa-Fluor- and FITC-labeled images were captured using a 488 nm Argon laser
for excitation and a 505–530 nm band-pass filter to detect emission. DAPI images were
captured using a 800 nm Mira titanium sapphire laser for excitation and a 390–465 nm
band-pass filter to detect emission.

Protein Immunolocalization

NBF- or Carnoy’s-fixed, paraffin-embedded mCMV-infected molars cultured for 15 days
and control molars cultured for 12 or 15 days were serially sectioned at 8 µm and
immunolocalization performed using polyclonal anti-amelogenin (FL-191), anti-dentin
sialoprotein (M300; Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and anti-
enamelin (courtesy of Jan Hu, University of Michigan) antibodies and the STAT-Q
Rabbit/Mouse Peroxidase-DAB staining kit (Innovex Biosciences, Pinole, Calif., USA).
Controls consisted of sections incubated in phosphate-buffered saline alone. Three to five
teeth per treatment group per antibody were analyzed. The data were analyzed using a
Zeiss Axioplan microscope and a digital camera.

CMV replication and pathology

Acyclovir, a synthetic purine nucleoside analogue, is a highly selective agent for CMV
with low toxicity to the host cell [10]. Acyclovir sodium (100 mg/20 ml) was purchased

15
from American Pharmaceutical Partners, Inc. (Schaumberg, Il). We employed 10 µg/ml
acyclovir, a dose shown in our laboratory to be nonteratogenic to embryonic salivary
glands [70] and tooth organs in vitro (unpublished). E15 Cap stage mandibular molar
regions were infected with 50,000 or 100,000 PFU mCMV for 24 h and then cultured in
control medium with or without 10 µg/ml acyclovir (CMV vs. CMV + Acy) for a total of
12 or 15 days in culture; controls consisted of E15 mandibular molar regions cultured in
control medium for 24 h and then with or without 10 µg/ml acyclovir (CONT vs. Acy)
for a total of 12 or 15 days. Tooth organs were collected and analysed for whole mount
morphology, mCMV infection (β-gal staining), and histopathology. For each assay, 3–5
tooth organs/group/day were analyzed.

Histological Analysis

Tooth organs were fixed for 4 h in Carnoy’s fixative at 4°C or overnight in 10% neutral-
buffered formalin (NBF) at room temperature, embedded in paraffin, serially sectioned
on the mesial-distal axis of the tooth germ at 8 µm and stained with hematoxylin and
eosin as previously described [45]. For semi-thin sections, control and mCMV-infected
molars cultured for 19 days were fixed overnight in 10% NBF, dehydrated through
graded alcohols, and embedded in JB-4 Embedding Medium (Polysciences Inc.,
Warrington, Pa., USA) as previously described [16]. Serial sections were cut at 6 µm
with a Sorvall JB-4 microtome and stained with 0.03% toluidine blue. All sections were
analyzed using a Zeiss Axioplan microscope and a digital camera.

16
Quantitative RT-PCR

For analysis of gene expression, quantitative RT-PCR (qRT-PCR) was conducted as
previously described [71]. Control E15 + 15 and mCMV-infected E15 molars infected on
days 0 and 9 of culture were pooled (3–4 molars per independent sample). We performed
qRT-PCR on 3 independent samples per treatment group. RNA was extracted and 1 µg
RNA was reverse transcribed into first-strand cDNA using ReactionReady™ First Strand
cDNA Synthesis Kit: C-01 for reverse transcription (Superarray Biosciences, Frederick,
Md., USA). The primer sets used were prevalidated to give single amplicons and
purchased from Superarray Biosciences: Amelx (No. PPM29897A); β-catenin (No.
PPM03384A); Cebpa (No. PPM04674A); Dspp (No. PPM40292A); Egfr (No.
PPM03714A); Enam (No. PPM25461A); Fn1 (No. PPM04224A); Nfkb1 (No.
PPM02930A); Rela (No. PPM04224E); Relb (No. PPM03202A). Primers were used at
concentration of 0.4 µM. The cycling parameters were: 95°C, 15 min; 40 cycles of 95°C,
15 s; 55°C, 30–40 s, and 72°C, 30 s. Specificity of the reactions was determined by
subsequent melting curve analysis. RT-PCR of RNA (not reverse transcribed) were used
as negative controls. GAPDH was used to control for equal cDNA inputs and the levels
of PCR product were expressed as a function of GAPDH. The relative fold changes of
gene expression between the gene of interest and GAPDH, or between the controls and
mCMV infected, were calculated by the 2
–ΔΔCT
method. Significant differences between
mCMV-infected and control teeth were determined by Student’s t test, with α = 0.01 and

17
the null hypothesis of R = 1. The calculated expression ratios (R) were log or arcsin
transformed prior to analysis.

Probabilistic Neural Network Analysis

We used probabilistic neural network (PNN) analyses to determine the contribution of
each individual gene to the discrimination between experimental groups with 100%
sensitivity and specificity. As such, PNN analyses identify the relative importance (0–1,
with 0 being of no relative importance and 1 being relatively most important) of specific
gene expression changes that discriminate between phenotypes. It is the contextual
change in expression, not the direction of change that is important in defining the
molecular phenotype. The foundational algorithm we used is based upon the work of
Specht and colleagues [13, 103, 104]. The proprietary software designed by Ward
Systems Group (Frederick, Md., USA) formulates Specht’s procedure around a genetic
algorithm [28]. A genetic algorithm is a computational method modeled on biologic
evolutionary processes that can be used to find the optimum solution to a problem that
may have many solutions [35]. These algorithms have been found to be very powerful in
solving optimization problems that appear to be difficult or unsolvable by traditional
methods. They use a minimum of information about the problem and they only require a
quantitative estimation of the quality of a possible solution. This makes genetic
algorithms easy to use and applicable to most optimization problems.

18
RESULTS

To determine the affect of mCMV infection on embryonic mouse tooth development, we
cultured embryonic Cap stage (E15) mandibular first molars (Fig. 4A, insert) with LacZ-
labeled mCMV [70] or control medium for up to 15 days in vitro using a chemically-
defined organ culture system [9, 47, 70, 102]. Exposure of embryonic mouse teeth to
mCMV in vitro substantially disrupted tooth morphogenesis and differentiation.

Histopathology and Viral Expression

After 12 days in culture, the uninfected (control) E15 mandibular first molars have
undergone cuspal morphogenesis and reached the Bell stage. At this stage, the
odontoblasts (dentin forming cells) have differentiated from the neural-crest-derived
ectomesenchyme (EM) and have secreted dentin matrix and the ameloblasts (enamel
forming cells) have differentiated from the inner dental epithelium (Fig. 4A, D). With
mCMV infection, E15 + 12 mandibular first molars are severely dysmorphic, smaller and
developmentally-delayed compared to controls, having only reached the Early Bell stage
(compare Fig. 4B to 4A). The mCMV-infected molars are characterized by shallow,
broad and misshapen cusps, abnormally-short dental epithelia, undifferentiated
odontoblasts, and the absence of dentin matrix. (Fig. 4B, E). The major cytological
differences between mCMV-infected and control molars are seen in the dental papilla,
with disorganized odontoblasts, stromal hypercellularity and altered mesenchymal

19
cytology being seen (compare Fig. 4B, E to A, D). Clusters of large basophilic,
pleiomorphic cells (some with inclusion bodies) are found in the dental papilla, as well as
in the peripherally-localized EM surrounding the tooth germ (i.e., the presumptive dental
sac).

After 15 days in culture, developmental delay and cuspal dysmorphogenesis persist in
mCMV-infected mandibular first molars (Fig. 4C, F). Although an additional 3 days in
culture is not sufficient to reach the Bell stage phenotype seen in control E15 + 12 molars
(compare Fig. 4C, F to A, D), morphogenetic advances are seen between mCMV-infected
E15 + 12 and E15 + 15 molars (compare Figs. 4C, F to B, E). Notably, E15 + 15 molars
exhibit preodontoblast alignment at the epithelial junction, predentin matrix, and
epithelial cells having elongated to become preameloblasts. This more developed
phenotype is similar to that seen in Cap stage mandibular first molars cultured for 9 days
in control medium. Since predentin induces ameloblast differentiation, the presence of
predentin matrix likely accounts for improved epithelial morphology.

To determine which cells are infected by mCMV, we analyzed the distribution of mCMV
in E15 + 12 Cap stage mandibular first molars by the cell-specific localization of viral
inclusion body (Fig. 4E, F), lacZ (β-galactosidase) expression (Fig. 4G) and viral
immediate early protein 1 (IE1) (Fig.4H). mCMV is found in dental papilla
ectomesenchymal cells and is absent from inner dental epithelia.

20
mCMV Infection Alters Cell Proliferation

To determine whether mCMV infection of Cap stage tooth organs alters cell proliferation,
we compared the spatial distribution of PCNA-positive cells (compare Figs. 5A and B),
as well as quantitative differences in proliferation index (PCNA-positive cells/total
cells/mm
2
), in mCMV infected and uninfected (cont) teeth. In controls, PCNA-positive
nuclei are detected primarily in the ameloblasts and, to a far lesser extent, in some dental
papilla EM (Fig. 5A). Since withdrawal from the cell cycle is a prerequisite for
odontoblast terminal differentiation, and differentiation progresses from the tip of the
cusp downward [96], the absence of PCNA-positive nuclei in odontoblasts clearly
indicates that they are terminally differentiated. In contrast, mCMV-infected molars
exhibit a very different proliferation pattern. PCNA-positive nuclei are found in infected
and affected EM throughout the dental papilla and, to a lesser extent, in the short dental
epithelial cells (Fig. 5B). mCMV infection induced a significant 161% increase
(P<0.003) in mesenchymal cell proliferation (PCNA-positive mesenchymal cells/total
mesenchymal cells/ mm
2
: CMV 81 ± 13 v. CONT 31 ± 8.3), as well as a significant 50%
decrease (P<0.0065) in epithelial cell proliferation (PCNA-positive epithelial cells/total
epithelial cells/ mm
2
: CMV 41 ± 3.2 v. CONT 83 ± 4.7). The increase in EM
proliferation is correlated with stromal hypercellularity.

21
Cell Characterization: Fibronectin and β-Catenin

To begin to delineate the molecular mechanisms underlying mCMV-induced tooth
dysmorphology, we analyzed spatial differences in the distribution of components of
several cell signaling pathways important for tooth morphogenesis and shown to be
altered by mCMV infection of embryonic SMGs [70]. mCMV infection of embryonic
mouse SMGs induced marked differences in the localization of fibronectin (FN) [66], a
basement membrane component important for odontogenesis [25, 95]. Thus, we
postulated that changes in FN distribution would also be seen in mCMV-infected molars.
This is exactly what we found: FN is seen surrounding individual cytomegalic dental
papilla cells and is relatively absent from the basement membrane (Fig. 5D). This FN
distribution pattern markedly differs from that seen in control Bell stage molars: FN was
most prominent in the basement membrane at the epithelial-mesenchymal interface and,
to a lesser extent, throughout dental papilla extracellular matrix (compare Fig. 5C to D).
Given that (1) FN may be involved in odontoblast polarization [95, 97]

and (2)
preameloblasts adhere to FN [25], this substantial change in FN localization in mCMV-
infected tooth organs may account for the disrupted odontoblast and ameloblast
alignment and differentiation.

Moreover, given the relationship between FN and β-catenin expression [31] and our
observation that mCMV infection induced β-catenin translocation in embryonic SMGs
[70], we analyzed the spatial distribution of β-catenin in control and mCMV-infected E15

22
+ 12 mandibular first molars. In Bell stage controls, β-catenin (both an adherens junction
constituent and a transcription factor) is primarily localized in the apical and basal
regions of ameloblasts and in aligned odontoblasts, and barely in centrally-localized
dental papilla mesenchyme (Fig. 5E); this distribution pattern is consistent with previous
reports [21, 81]. Importantly, mCMV infection induces the accumulation of β-catenin in
the cytoplasm of cytomegalic mesenchymal cells found throughout the dental papilla and
in abnormal epithelia, as well as in cell membranes (Fig. 5F). Cytoplasmic accumulation
of β-catenin is indicative of its potential function as a transcription factor that binds to the
DNA and regulates the expression of effector genes.

mCMV and NF-κB Expression

NF-κB signaling plays an important role during morphogenesis, including tooth
development [15, 82, 100]. Given that CMV infection has been shown to induce the
canonical [NF-κB1/RelA; NF-κB1/RelB] and noncanonical (NF-κB2/RelB) NF-κB
pathways [5, 17, 18, 49, 70, 125, 133, 134], we analyzed the spatial distribution of
RelA(p65) and RelB (components of both the canonical and noncanonical pathways) in
control and mCMV-infected molars. In Bell stage control molars, RelA(p65) is nuclear
localized in polarized odontoblasts (Fig. 5G) whereas RelB is primarily detected in the
apical and basal regions of polarized ameloblasts (Figs. 5I). Since activation of the NF-
κB/Rel complex requires translocation into the nucleus, our observation of nuclear-
localized RelA and cytoplasmic-localized RelB indicates that, at this stage, RelA (but not

23
RelB) has been activated. mCMV infection induces marked differences in RelA and RelB
protein expression. Specifically, there is de novo cytoplasmic localization of both RelA
and RelB proteins in infected and affected centrally-localized cytomegalic dental papilla
mesenchymal cells (Fig. 5H, J); RelB also exhibits an epithelial distribution similar to
that seen in controls (compare Fig. 5J to I). Since the noncanonical NF-κB2/RelB
pathway had appeared to be important for mCMV-induced SMG embryopathology [70],
we also determined the distribution of NF-κB2 in control and mCMV-infected molars.
The absence of immunodetectable NF-κB2 protein in both control and mCMV-infected
mandibular first molars (data not shown) suggests that the noncanonical NF-κB2/RelB
pathway is not involved in normal and abnormal odontogenesis.

Active mCMV Infection Required for Tooth Pathology

To determine if active mCMV infection is necessary to initiate and sustain progressive
tooth pathogenesis, we utilized acyclovir, an antiherpesviral nucleoside active against
CMV [10], to inhibit mCMV replication. Since acyclovir has been shown at high doses to
be teratogenic to rat embryos [12, 109], we use a dose shown in our laboratory to be
nonteratogenic to embryonic mouse SMGs [70] and tooth organs (data not shown) in
vitro. E15 (Cap stage) mandibular first molars were infected for 24 hrs with mCMV and
then cultured in the presence or absence of 10 µg/ml acyclovir sodium for up to 15 days
in vitro (Fig. 6); controls consisted of tooth organs cultured in chemically-defined
medium alone. Acyclovir treatment suppresses mCMV replication and spread, as

24
indicated by the absence of lacZ (mCMV) expression (compare Fig. 6 C, D to A, B), and
promoted differentiation (Fig. 6). Acyclovir-treated, mCMV-infected molars are
characterized by improved cuspal formation compared to mCMV-infected organs, with
differentiated, polarized odontoblasts and ameloblasts, as well as secreted dentin matrix,
being seen (compare Fig. 6D to B, G to F). The acyclovir-treated tooth phenotype
resembles that seen in control (compare Fig. 6G to E). Since the mCMV replication cycle
is incomplete after only 24 hrs of infection, when our antiviral treatment is initiated, it
appears that completion of the viral replication cycle beyond DNA replication is critical
to the initiation of tooth pathogenesis.

These results demonstrate that mCMV dysregulation of key signaling pathways disrupted
early stages of tooth morphogenesis and histodifferentiation in vitro [45]. However, what
remained unclear was whether mCMV infection of embryonic mouse mandibular first
molars in vitro results in enamel defects similar to those seen in congenitally-infected
children.

CMV and Progressive Tooth Development

The aim of the second half of this study was to examine the effects of mCMV infection
on progressive tooth differentiation and amelogenesis in cultured embryonic mandibular
mouse first molars. Since initial CMV infection in human fetuses can occur at different
developmental times (e.g. early fetal period, late fetal period, perinatal period), we varied

25
the stage of initial viral infection (i.e. Cap stage, Early Bell stage, Bell stage), as well as
the duration of infection. We postulated that the later the tooth developmental stage of
initial mCMV infection, as well as the shorter the duration, the less abnormal the
anatomic phenotypes, and that these differences will be correlated with notable stage-
dependent differences in transcript and protein expression.

mCMV dose response: The severity of tooth defects is correlated to mCMV dosage

To determine the relationship between mCMV dosage and tooth dysmorphogenesis, we
cultured E15 Cap stage molars for 15 days in 10,000, 25,000 or 100,000 PFU/ml mCMV
(Fig 7. B, C, D). There is a marked increase in cusp morphogenesis, aligned ameloblast
and dentin (d) expression in molars cultured in 10,000 PFU/ml compared to 25,000
PFU/ml (compare Fig. 7 B to C). The most severely abnormal and developmentally-
delayed phenotype is seen with 100,000 PFU/ml mCMV (compare Fig. 7D to B, C).
These teeth are smaller, with shallow cusps and exhibit a small amount of predentin (pd).
The cytomegalic infected and affected cells (
*
) are more widespread the higher the
mCMV dosage, with the greatest extent being seen with 100,000 PFU/ml (Fig. 7 D) and
the least with 10,000 PFU/ml (Fig. 7 B).

26
mCMV-induced stage-dependent differences in tooth pathology and enamel defects
on day 17 and day 19 of culture

To determine if mCMV-induced enamel defects is stage-dependent, we infected E15 Cap
stage mandibular first molars with mCMV at the Cap stage (day 0 of culture), Early Bell
stage (day 5 of culture) or Bell stage (day 9 of culture) (Fig. 8B-D, inserts), and cultured
them for a total of 17 (Fig. 8) or 19 (Fig. 9) days. Exposure of embryonic mouse molars
to mCMV infection disrupted tooth morphogenesis, dentinogenesis and amelogenesis in a
stage and duration-dependent manner; the earlier the initial stage and the longer the
duration of infection, the more severely abnormal the phenotype and enamel defect.
On day 17 of culture, uninfected (control) E15 Cap stage mandibular first molars have
undergone normal morphogenesis and differentiation (Fig. 8A, E). Dentin mineralization
and formation of enamel has proceeded apically from the mesial cusp tip along the mesial
side of the cusp and the dentino-enamel junction (DEJ) is readily visualized. Lobular
cuspal morphology is evident coronally and root formation is seen apically, with the
cervical loop extension of the inner and outer enamel epithelia bilayer forming Hertwig’s
Epithelial Root Sheath (HERS).

By contrast, mCMV infection disrupts tooth morphogenesis, dentinogenesis and
amelogenesis in a stage-dependent manner (compare Fig. 8B-D, F-H to 8A, E). Cap
stage-infected molars are the most severely dysmorphic (compare Fig. 8B, F to C-D, G-
H) and Bell stage-infected molars are the best developed (compare Fig. 8D, H to B-C, F-

27
G). All mCMV-infected molars are substantially smaller compared to controls, as
evidenced by the marked decrease in volume of dental papilla mesenchyme (DPM),
reduced cusp tip to cervical loop length, and HERS absence or hypoplasia (compare Fig.
8B-D to A). Cap and Early Bell stage-infected molars appear more developmentally-
delayed and dysmorphic compared to controls than Bell stage-infected molars, as
revealed by the lack of dentin mineralization and the absence of enamel matrix in these
molars (compare Fig. 8B-C, F-G to A, E). Although the Bell stage-infected molars are the
best developed, their phenotype is abnormal compared to controls (compare Fig. 8D, H to
A, E). The infected Bell stage molars are characterized by marked decreases in
mineralized dentin matrix, enamel formation, cusp size and root (HERS) formation
(compare Fig. 8D, H to A, E). Although enamel matrix is seen in Bell stage-infected
molars, there is limited, discontinuous enamel formation, a substantial decrease in enamel
thickness, and uneven enamel matrix mineralization (compare Fig. 8H to E). In addition,
the DPM in all mCMV-infected molars, regardless of stage of initial infection, appear as
two distinct cell populations rather than one: round and ovoid cells within a fibromyxoid
stroma, and basophilic, cytomegalic infected (some with inclusion bodies) and affected
cells (Fig. 8B-C, F-G). These cytomegalic infected and affected cells are more
widespread in Cap stage-infected molars than in Early Bell stage molars (compare Fig.
8B to C) and in Early Bell stage than in Bell stage-infected molars (compare Fig. 8C to
D). Note that in Cap stage-infected molars, the striking degree of infection obliterates the
normal morphology and cellular architecture of the parenchyma and stroma (Fig. 8B, F).

28
On day 19 of culture, tooth development continues to exhibit stage-dependent differences
in mCMV-induced pathology (Fig. 9). In control molars (Fig. 9A-D), polarized
odontoblasts with associated predentin and mineralized dentin are seen, and the elongated
ameloblasts are polarized. Tome’s processes can be seen at the secretory ends of
ameloblasts inserting into the newly deposited enamel (Fig. 9D) and tubules traversing
through the dentin are also found (data not shown). Dental papilla cells reside in a
fibromyxoid stroma and appear as round to ovoid with scant nucleoli and granular
cytoplasm. The stratum intermedium is composed of 1-2 layers of cuboidal cells (Fig.
9B), stellate reticulum cells reside in a myxoid stroma and appear as angulated or stellate
cells with scant clear cytoplasm (Fig. 9A, B), and a well-formed, elongated HERS is
found (data not shown).

With mCMV infection, phenotypic changes, alterations in the integrity of epithelial and
mesenchymal structures, and the presence of viral infection (viral inclusion bodies) are
more profound on day 19 than on day 17 of culture (compare Fig. 9E-P to 8B-D, F-H).
As noted above, stage-dependent differences in pathology are seen: molars infected at
earlier stages are more dysmorphic than those infected at later stages (i.e. Cap stage v.
Early Bell or Bell stage; Early Bell stage v. Bell stage) (compare Fig. 9E-P to A-D, E-H
to I-P, I-L to M-P). Coronal morphology is disorganized and dentin mineralization is
either absent (Fig. 9E-H) or appears attenuated, incongruent and irregularly scalloped in
many areas (Fig. 9I-P); HERS is either absent (Fig. 9E, G, I, K) or severely dysmorphic
(Fig. 9M, O). On higher magnification, it is evident that in all virally-infected molars,

29
ameloblasts, odontoblasts, and DPM have lost their characteristic morphology, with
odontoblasts and DPM being more severely affected (compare Fig. 9F-H, J-L, N-P to B-
D). In Cap stage and Early Bell stage-infected molars (Fig. 9F-H, J-L), disorganized
ameloblasts appear as blunted (often multilayered) cuboidal/low columnar cells and no
enamel matrix is seen. In contrast, in Bell stage-infected molars (Fig. 9N-P), ameloblasts
are composed of low cuboidal or short, elongated (but not polarized) cells and appear to
have a wavy structure, with enamel matrix being present. Although enamel formation is
seen in infected Bell stage molars, notable enamel defects are seen compared to control
(compare Fig. 9M-P to A-D). There is a marked decrease in amount of enamel matrix and
length of DEJ, and Tomes’ processes cannot be visualized as in controls. The enamel
depth dramatically differs from control teeth, being attenuated, irregular, and less
abundant. In addition, the stratum intermedium and stellate reticulum are abnormal in all
mCMV-infected molars, being composed of multilayers of basophilic and cytomegalic
infected and affected cells (Fig. 9F-H, J-L, and N-P). Taken together, these findings
indicate that mCMV infection results in abnormal dentinogenesis, amelogenesis and root
formation, the severity of which is dependent on the developmental stage of initial
infection, as well as the duration of infection.

mCMV induces significant stage-dependent changes in transcript expression

Having noted the variant pathology of Cap stage-infected and Bell stage-infected molars
after 17 and 19 days in culture (Figs. 8, 9), it is critical to study the natural history of the

30
progressive viral-induced pathogenesis, particularly that of Cap stage-infected molars. To
wit, the ameloblasts of infected Cap stage molars cultured for 15 days are elongated and
mostly polarized (Fig. 10B). By 17 days in culture, as noted above (Fig. 8B, F), the
ameloblasts are disorganized and nonpolarized, and the odontoblasts appear
undifferentiated. Thus, we chose to investigate the molecular pathology in mCMV-
infected molars cultured for 15 days (E15 + 15) (Fig. 10B, C), that is, just prior to the
histopathologic changes 48-96 hours hence (compare Fig. 10B, C to 8F, H and 9H, P).
We chose infected Cap and Bell stage molars since the most abnormal tooth phenotype is
evident in Cap stage-infected molars and the least abnormal phenotype in Bell stage-
infected molars on days 17 and 19 of culture (Figs 8-9).

To delineate the molecular basis for progressive mCMV-induced tooth pathology, we
determined if mCMV induced stage-dependent changes in the expression of 10 molecules
known to be important for tooth development, dentinogenesis and amelogenesis [26, 38-
40, 61, 113, 121, 128, 129, 137] and/or shown to be involved in mCMV-induced
pathology of oral structures [45, 46, 70]. We compared transcript expression in control
(Fig. 10A), Cap stage-infected (Fig. 10B), and Bell stage-infected (Fig. 10C) molars
cultured for 15 days using quantitative RT-PCR (qRT-PCR).

The results of these qRT-PCR-derived measurements are shown in Table 1. For each
transcript, the relative expression ratio (R) was calculated as the mean increase or
decrease in gene expression in mCMV-infected teeth compared to uninfected control

31
teeth. The variation of R is calculated as gene expression noise (η); η is statistically
equivalent to the coefficient of variation and ranges from 0 to 1 [η = gene expression
noise = s
R
/R (where s
R
= standard deviation of R)]. The value of η reflects fluctuations in
promoter-binding efficiency, specific transcription factor abundance, variation in post-
transcriptional modifications, and a host of other stochastic events that comprise intrinsic
and extrinsic noise [92]. In the absence of prohibitively large sample sizes, as η
approaches 1 it becomes extremely difficult to detect small, but potentially important,
true expression differences, should they exist. Nevertheless, it is highly unlikely that
substantial differences that characterize mCMV-infected tooth organs will be masked by
noise. In this regard, tests of significance were determined by student t, with α = 0.01 and
the null hypothesis of R = 1. The calculated expression ratios (Rs) were log or arcsin
transformed prior to analysis.

mCMV induces many highly significant stage-dependent changes in the expression of
genes essential for dentinogenesis and amelogenesis [amelogenin (Amelx), enamelin
(Enam), dentin sialophosphoprotein (Dspp)], as well as in genes important for tooth
development and altered in mCMV-infected embryonic oral structures. As shown in
Table 1, there are similarities, as well as differences, in transcript expression changes
between infected Cap stage and Bell stage molars. Compared to controls, Cap stage-
infected molars exhibit significant differences in 8 of the 10 genes whereas Bell stage-
infected molars show significant differences in 5 genes. Of genes essential for
dentinogenesis and amelogenesis, both Cap stage and Bell stage-infected molars show

32
significant decreases in the expression of Enam [Cap stage- 50%↓ (P <0.001); Bell stage-
47%↓ (P <0.001)] and Dspp [Cap stage-72%↓ (P <0.001); Bell stage- 38%↓ (P <0.01)].
Interestingly, only Cap stage-infected molars have a significant 25% (P<0.001) decrease
in Amelx expression, with normal levels of Amelx transcripts being found in Bell stage-
infected molars.

In addition, stage-dependent differences in β-catenin transcript expression are evident.
Since the canonical Wnt/β-catenin signaling is important for cusp morphogenesis, tooth
shape and root formation [61, 80, 129] and mCMV-infected Cap stage molars are
characterized by more severely abnormal crown and root phenotypes than seen in
infected Bell stage molars (Fig. 8), it was reasonable to postulate that mCMV would
induce greater changes in β-catenin transcript expression in Cap stage-infected molars
than in Bell stage-infected molars. Our observation of a significant ~37% decrease
(P<0.001) in β-catenin expression in Cap stage-infected molars but normal levels in Bell
stage-infected molars supports this hypothesis.

Further, we also found common significant changes in the levels of specific transcripts at
both stages of initial viral infection. Cap stage and Bell stage-infected molars exhibit
similar significant ~ 35% declines (P<0.001) in Fn1 and Egfr gene expression. Given that
fibronectin (FN) mediates odontoblast polarization and ameloblast differentiation [25],
EGFR is important for tooth morphogenesis and root formation [24, 37], and FN
activation of signaling pathways is mediated via EGFR [66], the absence of stage-

33
dependent changes in Fn1 and Egfr expression suggests that the FN and EGFR pathways
participate during early and later stages of mCMV-induced tooth pathology.

Transcript changes and stage-dependent abnormal phenotypes

The stage-dependent phenotypic differences in tooth morphology and enamel pathology
in Cap stage and Bell stage-infected molars are very distinct (Figs. 8 and 9), with enamel
agenesis and hypoplasia being seen in infected Cap stage and Bell stage molars,
respectively. Given the above, we predicted sharp mathematical discrimination of the
expression of genes important for enamel formation and tooth shape (Amelx, Enam, Dspp,
and β-catenin) among the 2 different initial stages of viral exposure. Comparisons
between infected Cap stage and Bell stage molars demonstrate that Bell stage-infected
molars are 1.3-fold higher (P<0.001) in Amelx expression, 2.2-fold higher (P<0.01) in
Dspp expression and 1.4-fold higher (P<0.001) in β-catenin expression, but not
significantly different in Enam expression, as compared to Cap stage-infected molars (Fig.
10D). These results suggest that higher levels of Amelx, Dspp and β-catenin transcripts
account for the better tooth morphology and enamel formation in Bell stage-infected
molars as compared to Cap stage-infected molars.

We then used Probabilistic Neural Network (PNN) analysis to characterize the molecular
pathology with respect to the relationship of individual gene expression changes to stage
of initial infection. An optimized (neural network) gene expression model was derived,

34
resulting in a molecular signature that is able to blindly distinguish (i.e. without bias)
between Cap stage and Bell stage-infected phenotypes with 100% sensitivity and
specificity. As shown in Figure 10E, PNN analysis revealed that changes in Amelx and
Dspp transcript expression levels are relatively most important in correctly classifying the
gene expression signature as infected Cap stage molars or infected Bell stage molars.
That is, our observation of enamel hypoplasia or enamel agenesis in infected Bell stage or
Cap stage molars, respectively, is clearly defined by specific differences in Amelx and
Dspp gene expression. As discussed later, this has important implications for
understanding the stage-dependent differences in histopathology observed above (Figs. 8,
9) and below (Fig. 13).

mCMV induces stage-dependent changes in dentin sialoprotein, amelogenin, and
enamelin protein localization

Since mCMV infection clearly compromises amelogenesis and amelogenesis is
dependent on sequential interactions between odontoblasts and ameloblasts, we
investigated whether mCMV altered the cell-specific distribution of amelogenin and
enamelin proteins, the predominant proteins found in developing enamel matrix [22], as
well as dentin sialoprotein (DSP), one of the two principal dentin proteins cleaved from
DSPP [65], in a stage-dependent manner. These three proteins have been shown to be
important for enamel formation [26, 38, 39, 42, 111]. Given that our data indicates that
mCMV-infected molars are developmentally-delayed compared to controls, we compared

35
the distribution of DSP, enamelin, and amelogenin proteins in Cap stage and Bell stage-
infected molars cultured for 15 days to control molars cultured for 12 (E15 + 12) and 15
(E15 + 15) days (Fig. 11).

Analysis of DSP protein distribution in control E15 + 12 and E15 + 15 molars
demonstrates DSP localization predominantly in the polarized, secretory odontoblasts and
adjacent dental papilla mesenchyme (DPM), as well as in the polarized ameloblasts (Fig.
11A, B), with increased immunostaining seen on day 15 (compare Fig. 11B to A). In E15
+ 15 controls, DSP is also seen in dentinal tubules, mineralized matrix, and at the DEJ
(Fig. 11B). These results are similar to those previously reported (e.g. [29, 33, 64, 132]).
mCMV-infected molars exhibit a clear reduction in DSP staining intensity, as well as
notable stage-dependent changes in cell-specific localization (compare Fig. 11C to A, B,
D and D to A, B). In Cap stage-infected molars (Fig. 11C), DSP is weakly localized in
the polarized ameloblasts, in adjacent stratum intermedium and stellate reticulum, in
polarized odontoblasts and throughout DPM. Since Bell stage-infected molars exhibit
mineralized dentin matrix and enamel formation, we expected a staining pattern
somewhere between E15 + 12 and E15 + 15 controls. This is exactly what we found:
DSP immunostaining is primarily in elongated odontoblasts, dentinal tubules and
mineralized matrix, and more weakly in polarized ameloblasts (compare Fig. 11D to A,
B). Our observation of greater differences in DSP immunostaining in Cap stage-infected
molars than in Bell stage-infected molars is consistent with the more severely dysmorphic
DPM morphology and cellular architecture seen in Cap stage-infected molars (Figs. 8-9).

36
Enamelin, the largest enamel protein, is strongly localized in secretory ameloblasts
(mostly in the secretory end) and forming enamel in E15 + 12 and E15 + 15 controls (Fig.
11E, F); it is also weakly seen in odontoblasts and dentinal tubules in the DEJ region (Fig.
11E, F). These results are consistent with previous reports (e.g. [30, 41, 77]). Importantly,
mCMV induced changes in enamelin localization in a stage-dependent manner (Fig.11G,
H). Cap stage and Bell stage-infected molars exhibit notable decreases in
immunolocalized enamelin in secretory ameloblasts and in odontoblasts, as compared to
E15 + 12 and E15 + 15 controls (compare Fig. 11G, H to E, F). Enamelin is also seen in
dentinal tubules in infected Bell stage (but not Cap stage) molars (compare Fig. 11H to
G). Further, although the pattern of enamelin immunostaining in odontoblasts and
dentinal tubules in Bell stage-infected molars is the similar to controls, Bell stage-
infected molars show a substantial reduction in immunostaining (compare 11H to E, F).
Amelogenin, the primary protein component of enamel, is strongly localized in polarized
ameloblasts and young, polarized odontoblasts, and more weakly in adjacent
mesenchyme and predentin in control E15 + 12 molars (Fig. 11I). In E15 + 15 control
molars, strong staining is detected in polarized ameloblasts, associated with granular-like
structures in the distal parts of the secretory ameloblasts and on the DEJ; weak staining is
detected in enamel and dentin matrixes; and no staining is seen in mature odontoblasts
(Fig. 11J). These results are similar to those previously reported (e.g. [78, 85, 86, 136]).
mCMV induces stage-dependent changes in amelogenin protein localization (compare
Figs. 11K, L to A, I, J). Compared to controls, infected Cap stage molars show reduced
amelogenin immunostaining in polarized ameloblasts (compare Fig. 11K to I, J). Of

37
particular note is the misexpression of amelogenin proteins, with amelogenins being
localized in odontoblasts, DPM in the crown region and stellate reticulum, as well as in
the extracellular matrix surrounding apically-located cytomegalic DPM cells (Fig. 11K).
In infected Bell stage molars, amelogenin immunostaining is seen in elongated
ameloblasts, and more weakly, in odontoblasts and adjacent DPM (Fig. 11L), a
distribution pattern somewhere between E15 + 12 and E15 + 15 controls (compare Fig.
11L to I, J).

mCMV induces stage-dependent changes in C/EBPα protein localization

C/EBPα is a potent transactivator of the mouse X-chromosomal amelogenin gene [137].
Our studies indicate stage specific differences in the localization of amelogenin protein
(Fig. 11), thus it is reasonable to postulate that there will be differences in C/EBPα
localization due to its affiliation with amelogenin. In table 1 we see that transcript levels
of C/EBPα are not significantly different from the control in both the Cap stage and Bell
stage infected. However, just because the transcript did not change in the Cap and Bell
stage infected teeth relative to the controls, does not mean that you will not see changes
at the protein level [137]. The issue at hand is whether or not the transcript is always
reflected in the protein. Even though the transcripts did not change relative to the control,
we investigated to see if there would be changes in the spatial distribution of C/EBPα in
the mCMV infected teeth because we see that there are changes in the localization of
amelogenin in the Cap and Bell stage infected relative to the controls.

38

As shown in figure 11, we can see where the amelogenin protein is localized, and in the
control 15+12 and 15+15, you can clearly see that the amelogenin protein is normally
localized to the ameloblasts (Fig. 11I,J). Additionally, when you look at the Cap stage
infected tissue (Fig. 11K), you can that amelogenin is located in the mesenchyme, while
in Bell stage (Fig. 11L) you see it in the ameloblasts and weakly in the dental papilla
mesenchyme. Due to the changes in the distribution of amelogenin, and since C/EBPα
plays an important role in activating the amelogenin gene, we investigated whether there
is a correlation in the changes of C/EBPα and the abnormal distribution of the
amelogenin protein. What we found was that there was in fact a corresponding abnormal
distribution of C/EBPα in the mesenchyme in the Cap and Bell stage infected tissue (Fig.
12) relative to the controls. In the CMV Cap infect E 15+15 (Fig. 12C) C/EBPα is clearly
seen in the DPM further away from the cuspal region and more toward where the root
would form (*) and not on the odontoblasts, which is what we would expect in the
control (compare Fig. 12A to C). Additionally, in the Bell E15+15 infected tissue,
C/EBPα is primarily in the DPM around the cuspal region (black arrowheads) and
slightly dispersed throughout other regions of the DPM with some found on the
odontoblasts toward the CL (black arrow) (Fig.12 G). While this may look more
developmentally advanced compared to the Cap infected tissue, it does not look normal
compared to the Cont E15+15 (compare Fig. 12 E to G), rather it looks more like the
earlier developmental stage of Cont E15+12 (compare Fig. 12 G to A). This is also
evident in the location of the cells marked with DAPI. (Fig. 12) Thus it is clear C/EBPα

39
protein is abnormally distributed in both the Cap and Bell stage infected tissue relative to
the controls, even though the transcript levels may indicate otherwise.

mCMV infection induces stage-dependent changes in cell proliferation
Since previous studies have demonstrated that mCMV infection of embryonic oral organs
altered the spatial localization of proliferating cells [45, 70], we investigated whether
mCMV induces stage-dependent changes in the cell-specific localization of proliferating
cells in embryonic mandibular first molars in vitro. We compared the cell-specific
distribution of proliferating cell nuclear antigen (PCNA), a marker of cells in early G1
and S phases of the cell cycle, in mCMV-infected Cap stage and Bell stage molars
cultured for 15 days to control molars cultured for 12 (E15 + 12) and 15 (E15 + 15) days
(Fig. 13A-D). In E15 + 12 controls, PCNA-positive nuclei are primarily seen in the
cervical loop extension of the inner and outer enamel epithelia which forms HERS and,
to a lesser extent, in surrounding DPM and in stellate reticulum (SR) cells (Fig. 10A). In
E15 + 15 controls, PCNA-positive nuclei are found in the well-formed HERS, in
surrounding DPM and in SR cells (Fig. 13B).

Since viral-induced spatial differences in cell proliferation are predicted, we compared
the cell-specific distribution of PCNA-positive nuclei and the cell proliferation indexes in
3 different regions [(1) DPM (PCNA-positive DPM cells/total DPM cells/ mm
2
); (2) SR
(PCNA-positive SR cells/total SR cells/ mm
2
); and (3) CL (PCNA-positive CL epithelial
cells/total CL epithelial cells/ mm
2
]. Notable stage-dependent qualitative (compare Fig.

40
13C, D to A, B, D to C) and quantitative (Fig. 13E) changes are seen. Cap stage-infected
molars exhibit highly significant ~6-fold increase in PCNA-positive nuclei in DPM
[mCMV v. CONT E15 + 12 (P<0.0001); mCMV v. CONT E15 + 15 (P<0.005)]
(compare Fig. 13C to A, B; 13E); no significant differences in DPM cell proliferation are
found between Bell stage-infected molars and controls (compare Fig 13D to A, B; Fig.
13E). For SR, mCMV infection induced similar significant ~1.5-2-fold increases
(P<0.05) in cell proliferation in Cap stage and Bell stage-infected molars (Fig. 13C-E).
Given that the SR of mCMV-infected Cap and Bell stage molars consists of multilayers
of basophilic, cytomegalic infected and affected cells (Figs. 8-9), our data suggests that
significantly increased cell proliferation on day 15 results in increased populations of
abnormal SR cells seen on days 17 (Fig. 8B, D) and 19 (Fig. 9E-H, M-P) of culture.
In mCMV-infected molars, root development is markedly delayed and dysmorphic on
day 15 of culture as compared to controls in a stage-dependent manner (compare Fig.
13C, D to A, B). Normally, after completion of crown formation, the root (HERS)
initially develops as an extension of cervical loop inner and outer enamel epithelia (Fig.
13A), with depletion of the core of SR cells within the cervical loop being required for
HERS formation (Fig. 13B) [117, 122, 123]. However, HERS formation is not seen in
mCMV-infected molars. Rather, infected Cap stage molars exhibit severely hypoplastic
cervical loops (CL) (Fig. 13C); infected Bell stage molar CLs are abnormal, composed of
multilayered inner and outer enamel epithelia separated by an expanded population of
disorganized SR cells (Fig. 13D). Since a more developmentally-advanced, elongated
HERS is seen in E15 + 15 than in E15 + 12 controls (compare Fig. 13B to A), we

41
compared the distribution of PCNA-positive nuclei and proliferation indexes in the CL
region of mCMV-infected molars to the younger day 12 controls. With mCMV infection,
notable stage-dependent differences in the CL region are seen (compare 13C, D to A; E).
In Cap stage-infected molars, few PCNA-positive nuclei are found in its severely
hypoplastic CL (Fig. 13C). This absence of cell proliferation in Cap stage-infected molar
indicates that, even with additional days in culture, CL elongation to form HERS will not
occur; thus, this viral induced abnormality is not merely due to developmental delay but
rather to mCMV-induced histopathology. In contrast, Bell stage-infected CLs exhibit
PCNA-positive nuclei in the multilayered inner and outer enamel epithelia, as well as in
the disorganized population of stellate reticulum cells which persist between the inner
and outer epithelial layers (Fig. 13D). We found a significant 1.7-fold increase (P<0.02)
in the CL cell proliferation index in Bell stage-infected molars as compared to controls
(Fig. 13E). Since cessation of cell proliferation and disappearance of SR cells in the CL
is a key event regulating the timing and onset of HERS formation [24], our data suggests
that active cell proliferation in stellate reticulum cells in Bell stage-infected molars likely
delays HERS formation.

42
DISCUSSION

It is well established that reciprocal interactions between epithelium and mesenchyme are
critical for the development of many tissues and organs, tooth being a classic example
[113, 116]. Tooth development is a continuous process during which the oral epithelium,
a derivative of surface ectoderm, thickens and buds into the underlying ectomesenchyme
(EM), and then grows and folds to form several crown shapes of varying complexity.
This progressive differentiation is regulated by sequential and reciprocal epithelial-
mesenchymal interactions [73, 113, 116]. These communications are effected by
paracrine signaling molecules from the TGFβ, FGF, Hedgehog, Wnt, and TNF families
and their cognate receptors and transcription factors. Typically, during Mid to Late Bell
stage, the odontoblasts and ameloblasts differentiate at the interface of the epithelium and
mesenchyme and deposit dentin and enamel matrixes, respectively. Recombination
experiments have shown that the dental epithelium governs tooth development prior to
the Bud stage and odontoblast differentiation [54, 63, 74, 115] and the dental papilla
regulates tooth shape and ameloblast differentiation in the Cap and Bell stages [55, 56].

In this study, mCMV infection of Cap stage mouse mandibular first molars induced
smaller, developmentally-delayed and severely dysmorphic tooth organs. After 12 days in
culture, the mCMV-infected molars exhibit shallow, misshapen cusps, abnormal EM
cellularity, undifferentiated odontoblasts and ameloblasts, and the absence of dentin
matrix. Analysis of viral distribution indicates that mCMV exclusively infects dental
papilla EM. These results are consistent with our prior observations in first branchial arch

43
derivatives (SMG, mandible) that mCMV has a particular tropism for neural-crest-
derived EM ([70]; unpublished). The absence of mCMV infection in affected abnormal
dental epithelia suggests that, as in mCMV-infected SMGs, paracrine factors likely
mediate the viral effect on dental epithelia. The same may reasonably be concluded for
affected, but not infected, dental papilla EM.

Terminal differentiation of odontoblasts involves withdrawal from the cell cycle,
polarization and secretion of predentin/dentin matrix [59]. Since the dental epithelium
induces odontoblast differentiation [54, 115], the presence of infected and affected
mesenchyme and absence of differentiated odontoblasts in mCMV-infected teeth suggest
that mCMV infection interrupts the reciprocal epithelial-mesenchymal interactions
critical for their differentiation. Our observation of cell proliferation in unaligned,
nonpolarized EM adjacent to the inner dental epithelium provides additional evidence
suggesting that mCMV infection interferes with differentiation. Moreover, since
odontoblast secretion of predentin, the first layer of the dentin matrix, induces ameloblast
differentiation [72], the absence of polarized odontoblasts and the marked delay in
predentin secretion in mCMV-infected tooth organs likely results in the substantial
abnormality of ameloblast differentiation. Taken together, our results indicate that defects
in the dental epithelium are secondary to defects in dental papilla mesenchyme.

Further, since mCMV-infected tooth organs exhibit morphogenetic advancement between
day 12 and 15 of culture, with the mCMV-infected E15 + 15 molar phenotype resembling

44
that seen in control E15 + 9 molars, it is reasonable to expect that odontoblast and
ameloblast differentiation would progress with additional days in culture. It appears then
that mCMV infection severely delays, but does not completely interrupt, tooth
morphogenesis. This is consistent with the AI phenocopy seen in children who exhibit
active perinatal and postnatal CMV infection [106-108, 110].

Over the last decades, it has become increasingly clear that basement membrane
components are important for tooth development [59, 60, 95, 96, 114]. The functional
network of matrix molecules, including FN, mediates the complex series of cell-cell
interactions between the epithelium and mesenchyme. We demonstrate that FN is
intensely localized to the basement membrane (Fig. 5C); this observation is similar to
previous reports [60, 95, 114]. Importantly, viral infection of Cap stage molars induces a
marked decrease in basement membrane localization; it also induces FN localization
surrounding individual infected and affected dental papilla EM (Fig. 5D). Given that FN
may be involved in odontoblast polarization [95, 97] and preameloblasts must adhere to
FN prior to differentiation

[25], the relative absence of FN in the basement membrane in
mCMV-infected molars may account for the disrupted odontoblast and ameloblast
alignment and differentiation.

β-catenin has dual roles as both a cell adhesion molecule and a transcription factor and
appears to be an important molecule linking cell adhesion and cell signaling during
organogenesis [6, 62, 79]. β-catenin stabilizes cell-cell adhesions by anchoring cadherins

45
to the actin cytoskeleton and transduces the Wnt signal by interacting with transcription
factor TCF/Lef . In the absence of Wnt signaling, cytoplasmic levels of β-catenin are kept
low through continuous ubiquitination and proteosome-mediated degradation. The
presence of Wnt signaling inhibits the degradation pathway, resulting in β-catenin
accumulation in the cytoplasm and translocation into the nucleus where it binds to the
transcription factor TCF/LEF to regulate gene expression. The canonical Wnt/β-catenin
pathway has been shown to be essential for normal odontogenesis [44, 72] and may
possibly link the differentiation of odontoblasts and cusp morphogenesis [129].

Herein, we demonstrate that mCMV infection of Cap stage tooth organs induces a
substantial increase in cytoplasmic accumulation of β-catenin in cytomegalic
mesenchyme and abnormal epithelia (Fig. 5F). This cytoplasmic localization is indicative
of its potential function as a transcription factor. Given that (1) β-catenin can induce Fn
gene transcription in fibroblasts [31] and (2) FN can activate tyrosine kinase receptors
and activated tyrosine kinases can increase β-catenin signaling [66, 79], the concomitant
increase of β-catenin and FN in dental papilla cells is likely correlated to tooth pathology.
NF-κB/Rel signaling is important to normal odontogenesis [15, 69, 82, 100]. Abnormal
molar phenotypes were seen in IKBαΔN and Ikkα mutant mice with reduced NF-κB/Rel
signaling [82, 100]. Sharpe and coworkers [15] found similar abnormal molar phenotypes
in Eda, Edar, Traf6 and NEMO (IKKγ) mutant mice with decreased NF-κB/Rel function.
NF-κB is composed of homo- and hetero-dimeric complexes of Rel family polypeptides.
Mammals express five NF-κB subunits: NF-κB1(p50), NF-κB2(p52), RelA(p65), RelB
and c-Rel [4, 8, 32]. Since NF-κB1(p50) and NF-κB2(p52) lack a transactivation domain,

46
they can only promote transcription when heterodimerized with transactivating Rel units.
The IκB family of inhibitory proteins keeps inactive NF-κB/Rel dimers in the cytoplasm.
Activation of the NF-κB/Rel complex requires degradation of IκB which allows nuclear
translocation, followed by binding to NF-κB and/or Rel recognition sites to regulate gene
transcription. Two NF-κB pathways, the canonical NF-κB1(p50)/Rel pathway and
noncanonical NF-κB2(p52)/Rel pathways, have been identified and their functions
investigated [4, 8, 32]. Since nuclear localization indicates NF-κB/Rel activation, our
observation in uninfected (control) Bell stage molars of nuclear-localized RelA (but not
RelB) suggests that the canonical NF-κB1/RelA pathway mediates cuspal morphogenesis.
In contrast, the absence of NF-κB2 protein in developing teeth suggests that the
noncanonical NF-κB2(p52)/Rel pathway does not play a role during normal
odontogenesis.

It is well established that CMV infection induces the canonical and noncanonical NF-κB
pathways [5, 17, 18, 49, 76, 125, 133]. mCMV and hCMV activation of the canonical
NF-κB pathway during infection of fibroblasts and other cell types facilitates viral
replication [5, 17, 18, 133, 134].

In addition, viral IE1 induces nuclear translocation
(activation) of NF-κB and upregulates RelB transcription [5, 49, 76, 125]. IE1 is also a
NF-κB response gene, with NF-κB binding sites being identified in the promoter and
enhancer regions of ie genes (He and Weber 2004). Moreover, we have previously
demonstrated that mCMV-induced SMG embryopathology was centered around both the
canonical and the noncanonical NF-κB pathways [70].

47

In this study, we report viral IE1 expression in dental papilla mesenchymal cells (Fig.
4G), with a concomitant upregulation of RelA(p65) and RelB protein expression being
seen in these infected and affected dental papilla EM cells (Fig. 5G-J). Our observation
that mCMV infection induced a shift in RelA localization from odontoblast nuclei to the
cytoplasm of undifferentiated dental papilla mesenchyme suggests that viral-induced
pathology is mediated by reduction of canonical NF-κB1(p50)/RelA signaling. Our
results are consistent with the previous findings that (1) reduced NF-κB/Rel signaling is
associated with abnormal odontogenesis [15, 82, 100] and (2) activation of the canonical
NF-κB1(p50)/RelA pathway and binding of p50/RelA to the major ie gene promoter is
not required for CMV replication in cultured fibroblasts [5].

Finally, the absence of NF-
κB2 protein (a required component of the noncanonical pathway) in mCMV-infected
tooth organs indicates that the noncanonical NF-κB2/RelB pathway is not involved in
mCMV-induced tooth pathogenesis.

It is well established that the phenotype of tooth pathology is dependent on the
teratogen’s time of initial exposure or duration of exposure (e.g. [87, 91]). Thus we
postulated that the later the tooth developmental stage at initial mCMV infection, as well
as the shorter the duration of the infection, the less abnormal will be the tooth phenotype.
Our results clearly demonstrate the time (stage and duration)-dependency of mCMV-
induced tooth pathogenesis (Figs. 9-13; Table 1). Only mCMV-infected Bell stage molars,
and not Cap stage or Early Bell stage-infected molars, have enamel matrix formation,

48
albeit hypoplastic and dysmorphic (Figs. 8, 9). This mCMV-induced AI is coincident
with stage-dependent differences in Amelx, Enam, and Dspp transcript expression,
localization of DSP, enamelin and amelogenin proteins, localization of cell proliferation,
and dedifferentiation of polarized ameloblasts, as one observes the time-series from 15
(Fig. 10-13) to 17 (Fig. 8) to 19 (Fig. 9) days in culture. Interestingly, although the
ameloblast morphology seen in mCMV-infected Cap and Bell stage molars on day 15
(Fig. 10) would not predict the substantial phenotypic differences seen between Cap stage
and Bell stage-infected molars on days 17 (Fig. 8) and 19 (Fig. 9) of culture, the major
differences detected in molecular profiles (Table 1, Fig. 10D) foreshadows the
differences seen 2-4 days later.

Dedifferentiation is the progression of cells from a more differentiated state to a less
differentiated state, i.e. a loss of specialization [14, 51]. After 15 days in culture, E15
tooth explants that were infected at Cap or Bell stages all exhibit ameloblasts with
apically polarized nuclei and basally-localized amelogenin and enamelin proteins (Figs.
10, 11). Infected Cap stage ameloblasts somewhat resemble those seen in controls
cultured for only 12 days, and infected Bell stage ameloblasts appear similar to controls
somewhere between 12 and 15 days in culture. The presence of enamel matrix in Bell
stage-infected molars suggests that the secretory ameloblasts have been thus for a longer
period of time. By 19 days in culture, the Cap stage and Early Bell stage-infected molars
display ameloblasts with a cuboidal morphology characteristic of their prior
undifferentiated state (Fig. 9E-L); the Bell stage-infected molars display ameloblasts still

49
elongated, but no longer polarized (Fig. 9M-P). It would appear, then, that the extent of
phenotype reversal is proportional to the length of time the ameloblasts were functionally
differentiated. This is consistent with what has been previously well-documented for
dedifferentiation [51].

Dedifferentiation is very much a regulated process involving the down-regulation of
developmental genes and the degradation of their extant mRNAs [51]. Beyond this
reverse of differentiation, there are gene and protein expression changes that are specific
for dedifferentiation, including genes that may regulate both processes. It has been
proposed that development harbors checkpoints that insure a return path to the
undifferentiated state in the context of perturbation (e.g. mCMV infection); the
checkpoint conditions developmental progression on the accumulation of a protein that is
essential for dedifferentiation [51]. Given the significant stage-dependent differences in
levels of Dspp, Enam, Amelx transcripts in mCMV-infected molars (Table 1, Fig. 10), as
well as marked stage-dependent changes in DSP, enamelin and amelogenin protein
distribution (Fig. 11), it is reasonable to speculate that DSP, enamelin and amelogenin
play important roles in both differentiation and dedifferentiation of ameloblasts.
Since mCMV infection clearly compromised tooth morphogenesis and amelogenesis in a
stage-dependent manner, we focused on the expression of putative ameloblast (Enam,
Amelx) and odontoblast (Dspp) markers known to play key roles during enamel formation
and mineralization [26, 39, 40, 84, 128, 137], as well as genes important for
odontogenesis and previously shown to be involved in mCMV-induced pathogenesis [45,

50
46, 61, 70, 121, 129]. Since common signaling pathways are utilized at different stages of
tooth development (see reviews, [113, 121]), we postulated that specific pathways altered
at early stages would also be affected at later stages. Moreover, since the stage-dependent
phenotypic differences are distinct, we also postulated sharp mathematical discrimination
of gene expression among the 2 different stages of initial viral infection.

Enamelin and amelogenin, the major protein components of enamel matrix [22], were
chosen because (1) they play essential roles in enamel formation; (2) mutations in
AMELX and ENAM cause X-linked AI and autosomal dominant AI, respectively; and (3)
amelogenin and enamelin null mice have abnormal teeth with disorganized, hypoplastic
enamel (see reviews, [26, 38, 39]). We also chose dentin sialophosphoprotein (Dspp), the
major noncollagenous protein in dentin matrix, since initiation of enamel matrix
formation at the DEJ is associated with DSPP expression [42], mutations in DSPP cause
various types of dental disorders [dentinogenesis imperfecta (DGI) type II, DGI type III,
and dentin dysplasia type II] (see reviews [53, 68]) and Dspp null mice are characterized
by abnormal teeth similar to human DGI type III [105]. DSPP, one of the most abundant
proteins in dentin matrix, is immediately processed into two dentin matrix proteins,
dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) [65]. Both DSP and DPP
have been shown to play important roles during dentin and enamel formation [84, 111,
128, 132]. In this study, we demonstrate that mCMV infection in vitro significantly
changed Dspp, Enam, and Amelx gene expression in a stage-dependent manner (Table 1;
Fig. 10) and our protein localization studies (Fig. 11) confirmed these results. Importantly,

51
our observation that mCMV altered the cell-specific localization of DSP, enamelin and
amelogenin proteins clearly shows the location of these molecules as they participate in
normal v. abnormal enamel formation. Thus, these protein localization results provide
further evidence of their pathophysiologic relevance for CMV-induced AI.

C/EBPα is a sequence-specific DNA binding protein that regulates gene expression in
certain mammalian cells [137]. C/EBPα is implicated to regulate mouse amelogenin gene
expression during tooth enamel formation in vitro [137]. It is has been shown to be a
strong transactivator for amelogenin. Thus, eventhough the transcript levels showed no
significant difference from the controls and both Cap and Bell stage infected, protein
changes seen with amelogenin suggested that similar changes would be seen with
C/EBPα at the protein level. Our hypothesis was proven correct as our results clearly
showed that C/EBPα is abnormally distributed in both the Cap and Bell stages of
infection relative to controls, an indication of C/EBPα’s relationship with amelogenin
(Fig. 12).

Amelogenins are the most abundant secreted proteins in developing enamel matrix,
comprising up to 90% of the proteins present [22]. Since amelogenin transcripts were
initially found only in ameloblasts by in situ hybridization [7, 41, 50, 78], and enamel
hypoplasia and abnormal mineralization are seen in Amelx null mice [27], it was
reasonably thought that amelogenins solely regulate enamel matrix formation and
mineralization. However, amelogenin transcripts and proteins have also been found in

52
odontoblasts, pulp cells, and cementoblasts in developing teeth [83, 85, 86, 124], as well
as in tissues not related to odontogenesis (e.g. brain cells, hematopoietic cells,
chondrogenic/osteogenic cells) (see review, [19]). In vitro and in vivo studies have clearly
demonstrated that amelogenins have signaling properties (e.g. [34, 67, 118, 119, 124, 126,
130, 135]).

Alternative splicing of the primary mRNA transcript results in several long- and short-
spliced isoforms of amelogenin [36, 101]. Iacob and Veis [43] demonstrated that
ameloblasts, odontoblasts, and stratum intermedium cells each have distinct spatial and
temporal patterns of amelogenin isoforms. Of particular note, is the M59/[A-4] (LRAP)
isoform, for which there is a putative receptor [118, 119]. The [A-4] isoform is secreted
by maturing mouse odontoblasts before the onset of dentin mineralization and inhibits
ameloblast maturation; it is proposed that this permits a sufficiently thick layer of dentin
to be produced, blocking any further epithelial-mesenchymal signaling [118]. When
cultured E16 tooth germs are exposed to exogenous [A-4] protein, there is normal dentin
matrix formation but disrupted ameloblast maturation, ranging from multiple cuboidal
cells to elongated cells with indeterminate polarization [118]. It is proposed that the
diffusion of [A-4] into the epithelial cell layer may provide a gradient opposite to that in
control cultures.

Eventhough dedifferentiation of polarized ameloblasts as one observes the time-series of
Cap stage-infected molars from 15 (Figs. 10, 11) to 17 (Fig. 8) to 19 (Fig. 9) days in

53
culture is associated with a reduction of Amelx transcript expression (Table 1) and
misexpression of amelogenin protein in DPM (Fig. 11), it remains reasonable to
hypothesize that upregulation of [A-4] in the context of overall amelogenin protein
decline may be driving the dedifferentiation in a manner similar to that seen in the study
by Tompkins et al., [118]. It is a recognized principle of concentration-dependent
signaling that if the amount or duration of the signal is too great, development is diverted
from its normal course [23]. Even a mere three-fold increase in ligand concentration or
time of exposure will dramatically alter cell fate. Furthermore, the decrease in Dspp and
Enam transcript expression, as well as in immunolocalized DSP and enamelin proteins, in
both infected Cap stage and Bell stage molars suggest that their downregulation also
participates in dedifferentiation of ameloblasts and enamel pathogenesis.

The least abnormal tooth phenotype is seen in Bell stage-infected molars, with enamel
hypoplasia (and not agenesis) being seen. This less severe pathology is associated with
Amelx and β-catenin transcript expression levels similar to those seen in controls, as well
as the significantly higher Dspp expression compared to Cap stage-infected molars.
Given that canonical Wnt/β-catenin signaling regulates tooth shape and root formation
[61, 80, 129], the significantly higher β-catenin expression in Bell stage-infected molars
as compared to Cap stage-infected molars likely mediates the improved tooth
morphology and root formation. Furthermore, since amelogenins activate the canonical
Wnt/β-catenin pathway [67, 126] and Wnt/β-catenin signaling regulates Dspp transcript
expression [129], the significantly higher Dspp expression in Bell stage-infected molars

54
may be due, in part, to the significantly ~25-30% higher (P<0.001) Amelx and β-catenin
transcript expression. Moreover, PNN analysis revealed that specific differences in Amelx
and Dspp gene expression is relatively most important for distinguishing between the
infected Cap stage and Bell stage enamel defect (i.e. enamel agenesis v. hypoplasia). Our
results further indicate that the significant upregulation of Amelx gene expression to
normal levels, as well as the significantly higher Dspp expression, in Bell stage-infected
molars likely accounts for enamel formation (albeit abnormal).

Finally, our qRT-PCR-derived results also indicate that common key pathways are
utilized at different stages of mCMV-induced pathogenesis. Given that FN and EGFR are
important for normal tooth morphogenesis [25, 36], FN activation of signaling pathways
is mediated through EGFR [66], and mCMV-induced downregulation of Egfr and Fn1
expression is not stage-dependent (Table 1), the similar significant reductions in Fn1 and
Egfr expression in Cap stage and Bell stage-infected molars suggest that the FN and
EGFR pathways are involved during early and later stages of viral-induced tooth
pathology. Also, given that EGFR signaling is important for the onset and timing of
HERS formation [24] and Wnt/β-catenin signaling is important for root formation [80],
the significant decrease in Egfr transcripts, but stage-dependent reduction in β-catenin
expression, likely accounts for HERS agenesis or hypoplasia in infected Cap stage or Bell
stage molars, respectively.

55
CONCLUSIONS

In conclusion, we have demonstrated for the first time that mCMV induces tooth
pathology and enamel defects in a stage- and duration-dependent manner: the earlier the
initial stage and the longer the duration of infection, the more severely abnormal the
phenotype. This viral-induced pathology is coincidental with stage-dependent changes in
Amelx , Enam and Dspp expression, amelogenin, enamelin, DSP and C/EBPα protein
distribution, cell proliferation localization, and dedifferentiation of secretory ameloblasts.
Our observation of disorganized and blunted cuboidal ameloblasts in Cap stage- and
Early Bell stage-infected molars indicates that the absence of enamel formation is not
merely due to developmental delay, but rather to mCMV-induced ameloblast
histopathology. Finally, our data indicate that whether mCMV induces enamel agenesis
or hypoplasia is defined by the specific levels of Enam, β-catenin and Dspp gene
expression. Further studies are needed to delineate the proteomic changes related to
mCMV infection of mouse tooth organs. Together, these studies will reveal drug targets
that will ameliorate enamel defects in the permanent dentitions of about 3,000 children
born each year with CMV-induced amelogenesis imperfecta.

56
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APPENDIX A: FIGURES
FIGURE 1: Tooth Development [75]

69
FIGURE 2: Cytomegalovirus [93]

70
FIGURE 3: Clinical Picture of CMV-Induced Amelogenesis Imperfecta [108]

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FIGURE 4: mCMV-Induced Histopathology and Viral Distribution
mCMV-induced histopathology and viral distribution. A-F. Histological analysis of
control (A, D) and mCMV-infected (B-C, E-F) E15 Cap stage first mandibular molars
cultured in serumless, chemically-defined medium. A, D. After 12 days in culture,
control molars have undergone cuspal morphogenesis and reached the Bell stage, with
polarized odontoblast (o) and ameloblasts (a), as well as dentin matrix (d), being seen.
dp-dental papilla. Insert A: starting E15 Cap stage mandibular first molar. B, E. After 12
days in culture, the mCMV-infected molars are markedly smaller, developmentally-
delayed, and severely abnormal, having only achieving the Early Bell stage. mCMV
infection results in shallow, broad and misshapen cusps composed of abnormally short,
undifferentiated dental epithelium (arrow), disorganized undifferentiated odontoblasts
(double arrowheads), and no dentin matrix. The dental papilla is composed of clusters of
large basophilic, pleiomorphic infected [with viral inclusion bodies (insert)] and affected
cells being seen (double arrows). C, F. After 15 days of infection, the smaller,
underdeveloped and abnormal tooth phenotype persists. The presence of aligned (but not
polarized) preodontoblasts (arrowhead), secreted predentin matrix (white arrow), and
elongated (but not polarized) epithelia (white arrowhead) indicates developmental
advancement. insert F: viral inclusion bodies. G-H. mCMV distribution in E15 + 12 tooth
organs. Expression of β-galactosidase staining of lacZ (mCMV) (G) and viral IE1
protein (H) demonstrates the presence of viral infection throughout dental papilla EM
cells but not in dental epithelia (e). Bar, A-C: 30 µm; A insert: 60 µm; D-F, H: 20 µm; E,
F insert: 12 µm; G: 50 µm.

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FIGURE 4: Continued

73
FIGURE 5: Characterization of mCMV-Induced Cellular Changes.
Characterization of mCMV-induced cellular changes. A, B. Cell proliferation. Cell
proliferation was determined by the distribution of PCNA-positive nuclei (brown color).
In control molars (A), PCNA-positive nuclei are seen in ameloblasts (a) and, to a lesser
extent, in dental papilla (dp) mesenchyme. Note the absence of PCNA-positive nuclei in
polarized odontoblasts (o) in the cuspal tip, indicating differentiation. With mCMV
infection (B), PCNA-positive nuclei are seen throughout the cytomegalic mesenchymal
cell population (*) and, to a lesser extent, in short, nonpolarized epithelial cells (double
arrows). C, D. FN distribution. In control molars (C), FN is strongly immunolocalized
localized in basement membrane (white arrowhead) and weakly throughout the dental
papilla extracellular matrix. With mCMV infection (D), FN intensely surrounds
individual cytomegalic dental papilla cells (white asterisks) and is relatively absent from
the basement membrane. E, F. β-catenin distribution. In control molars (E), β-catenin is
seen in the apical and basal regions (arrows) of polarized ameloblasts and more weakly in
aligned odontoblasts. In mCMV-infected molars (F), β-catenin is strongly accumulated in
the cytoplasm of dental papilla mesenchymal (insert) and short, nonpolarized epithelial
(e) cells (white arrows); β-catenin is also detected in cell membranes. G, H. RelA
distribution. In control molars (G), polarized odontoblasts exhibit nuclear-localized RelA
(insert). With mCMV infection (H), RelA is seen in the cytoplasm of centrally-localized
cytomegalic dental papilla cells (insert) and is absent from presumptive odontoblasts. I, J.
RelB distribution. In control molars (I), RelB is seen in the apical and basal regions of
polarized ameloblasts (black arrowheads). A similar cytoplasmic distribution pattern
(black arrowheads) is seen in the mCMV-infected epithelia (D) which consists of
nonpolarized cuboidal epithelial cells (white arrowheads). Note that mCMV infection
induced a marked increase in RelB immunostain in the cytoplasm of centrally-localized
dental papilla mesenchyme (insert D) compared to control (insert C). Bar, A-B, E-J: 20
µm; C-D: 10 µm. F-J inserts: 13 µm.

74
FIGURE 5: Continued

75
FIGURE 6: Acyclovir Treatment Ameliorates mCMV-Induced Pathology.

Acyclovir treatment ameliorates mCMV-induced pathology. A-D. β-galactosidase
(LacZ) expression. LacZ is detected in EM dental papilla cells in mCMV-infected tooth
organs (A, B) and was absent from acyclovir-treated, mCMV-infected (CMV + Acy)
tooth organs (C, D). E-G. Histological sections of control (E), mCMV-infected (F) and
mCMV+ Acy (G) molars. mCMV infection induces abnormal tooth development (F),
characterized by unaligned, disorganized odontoblasts (arrow), undifferentiated
ameloblasts, and abnormal mesenchymal cellularity. In contrast, acyclovir treated,
mCMV-infected molar phenotypes (G) are similar to that seen in control (E). Note the
presence of aligned, polarized odontoblasts (double arrows) and ameloblasts (double
arrowheads), increased dentin matrix deposition (arrowhead), and dental papilla cells
exhibiting a normal, fibroblastic appearance. Bar, A, C: 30 µm; B, D-G: 20 µm.

76
FIGURE 7: mCMV Dose Response

mCMV dose response: The severity of tooth defects is correlated to mCMV dosage.
E15 Cap stage molars are cultured for 15 days in 10,000, 25,000 or 100,000 PFU/ml
mCMV(Fig 4. B, C, D). There is a marked increase in cusp morphogenesis, aligned
ameloblast and dentin (d) expression in molars cultured in 10,000 PFU/ml compared to
25,000 PFU/ml. The most severely abnormal and developmentally-delayed phenotype is
seen with 100,000 PFU/ml mCMV. These teeth are smaller, with shallow cusps and
exhibit a small amount of predentin (pd). The cytomegalic infected and affected cells (
*
)
are more widespread the higher the mCMV dosage, with the greatest extent being seen
with 100,000 PFU/ml (D) and the least with 10,000 PFU/ml (A).

77
FIGURE 8: mCMV-Induced Stage-Dependent Differences in Tooth Pathology and
Enamel Defects on Day 17 of Culture.
mCMV-induced stage-dependent differences in tooth pathology and enamel defects
on day 17 of culture. A, E. CONT: E15 mandibular first molars cultured in control
medium. B, F. CMV CAP: E15 mandibular first molars infected with mCMV at the Cap
stage. B, upper insert-Cap stage molar. C, G. CMV E. BELL: E15 mandibular first
molars infected with mCMV at the Early Bell Stage. C, insert-Early Bell stage molar. D,
H. CMV BELL: E15 molars infected with mCMV at the Bell stage. D, insert-Bell stage
molar. E-H. Representative higher magnifications of control (E), Cap stage-infected (F),
Early Bell stage-infected (G), and Bell stage-infected (H) molars. A, E. Control molars
exhibit normal morphogenesis and cusp formation, characterized by polarized
ameloblasts (a) and odontoblasts (o), the presence of predentin (pd), dentin (d) and
enamel (e) matrixes, and the formation of HERS (A, double black arrows). DPM: dental
papilla mesenchyme. B-D, F-H. mCMV infection induces smaller, developmentally-
delayed and dysmorphic molars compared to controls. Cap stage-infected molars (B, F)
are the most severely dysmorphic and developmentally-delayed. They are characterized
by short, shallow cusps, disorganized, nonpolarized ameloblasts (F, red arrowheads),
undifferentiated odontoblasts, a marked decrease in predentin matrix, the absence of
dentin and enamel matrixes, a short, abnormal cervical loop (B, black arrowhead), and
disorganized, multilayered stellate reticulum (s). Note the widespread distribution of
basophilic, cytomegalic cells (*), some with inclusion bodies (B, lower insert),
throughout dental papilla mesenchyme. Early Bell stage-infected molars (C, G) are
smaller and severely dysmorphic as compared to controls but are better developed than
Cap stage-infected molars. Compared to Cap stage-infected molars, they exhibit
improved cusp formation, elongated ameloblasts, polarized odontoblasts, more predentin
matrix, a better-developed cervical loop (C, blue arrowhead) and a more limited
distribution of abnormal infected and affected DPM cells (C, double black arrowheads).
Bell stage-infected molars (D, H) are the least dysmorphic. Compared to Cap stage-
infected and Early Bell stage-infected molars, they exhibit greater cusp formation,
mineralized dentin and enamel matrixes, and HERS formation. Compared to controls,
Bell stage-infected molars have shorter cusps, shorter polarized ameloblasts and
odontoblasts, a marked decrease in mineralized dentin matrix, and reduced HERS
formation (D, black arrow). Although enamel is present, there is limited, discontinuous
formation, reduced thickness, and uneven matrix mineralization. Note the marked
decrease in abnormal affected and infected DPM cells (D, double black arrowheads)
compared to infected Cap stage (B) and Early Bell stage (C) molars. Bar, A-D: 40 µm;
top insert B:130 µm; bottom insert B: 20 µm; top inserts C-D: 160 µm; E-H: 25 µm.

78
FIGURE 8: Continued

79
FIGURE 9: mCMV-Induces Stage Dependent Tooth Defects on Day 19 of Culture.
mCMV induces stage-dependent tooth defects on day 19 of culture. Semi-thin plastic
sections of control and mCMV-infected molars. A-D. Control molars (representative
higher magnifications shown in B-D). E-H: CMV CAP: Cap stage-infected molars
(representative higher magnifications shown in F-H). I-L. CMV E. BELL: Early Bell
stage-infected molars (representative higher magnifications shown in J-L). M-P. CMV
BELL: Bell stage-infected molars (representative higher magnifications shown in N-P).
On day 19, control molars (A-D) exhibit polarized odontoblasts (o) with associated
predentin (pd) and mineralized dentin (d) matrixes, elongated, polarized ameloblasts (a)
and associated enamel (e) matrix, and a well-developed HERS. At the secretory ends of
differentiated ameloblasts, Tome’s processes (D, red arrowheads) insert into the newly
deposited enamel and tubules traverse through the dentin (data not shown). DPM cells are
round to ovoid with scant nucleoli and granular cytoplasm. The stellate reticulum (s)
consists of angulated or stellate cells with scant clear cytoplasm. Cap stage-infected
molars (E-H) are the most severely abnormal. They have a multilayer of primarily
cuboidal ameloblasts (white arrowhead), pleiomorphic, undifferentiated odontoblasts,
predentin (but not dentin) matrix, abnormal CL/absent HERS (E, black arrowhead), and
abnormal stellate reticulum cytology. Basophilic, cytomegalic cells are seen in
throughout dental papilla (E, red star) and stellate reticulum (F, *), with many of the cells
exhibiting viral inclusion bodies (F-H, blue arrowhead). Early Bell stage-infected molars
(I-L) are slightly better developed than Cap stage-infected molars, with taller cusps and
the presence of predentin and mineralized dentin matrixes being seen. Ameloblasts are
composed of a multilayer of cuboidal epithelia (white arrowhead) and HERS remains
absent (black arrowhead). Basophilic, cytomegalic cells are seen in dental papilla (I, red
star) and stellate reticulum (L, double red arrows), many exhibiting viral inclusion bodies
(J-K, blue arrowhead). Bell stage-infected molars (M-P) are the best developed but are
abnormal compared to control. Compared to controls, Bell stage-infected molars exhibit
abnormal DPM (M, red star), shorter elongated ameloblasts (blue arrows), less
mineralized dentin and predentin matrixes, enamel hypoplasia and a shorter CL/HERS
(M, double black arrowheads). Fewer viral inclusion bodies (blue arrowhead) are seen in
DPM and stellate reticulum than in Cap stage and Early Bell stage-infected molars. Bar,
A, E, I, M: 50 µm; B-C, F-G, J-K, N-O: 40 µm; D, H, L, P: 27 µm.

80
FIGURE 9: Continued

81
FIGURE 10: Comparative Amelx, Enam, Dspp and β-catenin Expression in Control,
Cap Stage-Infected and Bell Stage-Infected Molars.
Comparative Amelx, Enam, Dspp and β-catenin expression in control, Cap stage-
infected and Bell stage-infected molars. A-C. Ameloblast morphology in control
molars (A), Cap stage-infected (B) and Bell stage-infected (C) molars cultured for 15
days. In control (A) and Bell stage-infected (C) molars, elongated, polarized ameloblasts
(arrowhead) are found. In contrast, Cap stage-infected molars (B) exhibit elongated
polarized (arrowhead) and non polarized (arrows) ameloblasts. (a), ameloblasts; (d),
dentin; (e), enamel; (pd), predentin. Bar, 20 µm. D. Quantitative RT-PCR-derived mean
relative expression ratios in Cap stage or Bell stage-infected molars compared to control
molars. Differences in relative expression levels between infected Cap and Bell stage
molars were calculated by t-test. Brackets indicate comparisons. **P<0.001; *P<0.01. †
not significant. E. Probabilistic Neural Network (PNN) analysis was used to determine
the contribution of each gene to the blind classification of molars as either Cap stage-
infected or Bell stage-infected. PNN analysis identifies the relative importance (0-1, with
0 being of no relative importance and 1 being relatively most important) of specific gene
expression changes that distinguish between Cap stage and Bell stage-infected molars
with 100% specificity and sensitivity. Amelx and Dspp transcript levels are relatively
most important in correctly classifying molars as either Cap stage-infected or Bell stage-
infected; Enam and β-cat transcript levels are relatively unimportant.

82
FIGURE 10: Continued

83
FIGURE 11: mCMV Induces Stage-Dependent Changes in the Distribution of DSP,
enamelin, and amelogenin proteins.
mCMV induces stage-dependent changes in the distribution of DSP, enamelin, and
amelogenin proteins. A-D. DSP localization. E-H. Enamelin localization. I-L.
Amelogenin localization. A-D. In E15 + 12 (A) and E15 + 15 (B) controls, DSP is
strongly localized in polarized, secretory odontoblasts (double arrows) and adjacent DPM
(*), and in polarized ameloblasts (a). In E15 + 15 controls, DSP is also found in dentinal
tubules (B, arrowhead), mineralized matrix, and at the DEJ. (e), enamel. In Cap stage-
infected molars (C), weak DSP immunostaining is seen in the polarized ameloblasts,
stratum intermedium and stellate reticulum, as well as in abnormal odontoblasts and
throughout DPM. In Bell stage-infected molars (D), DSP immunostaining is found
primarily in odontoblasts, adjacent DPM (*), dentinal tubules and mineralized matrix,
and to a lesser extent, in polarized ameloblasts. E-H. In E15 + 12 (E) and E15 + 15 (F)
controls, enamelin is seen primarily in secretory ameloblasts and forming enamel; weak
immunostaining is also seen in polarized odontoblasts and dentinal tubules in the DEJ
region (double arrowheads). Infected Cap stage (G) and Bell stage (H) molars are
characterized by markedly decreased enamelin immunostaining in polarized ameloblasts,
odontoblasts and dentinal tubules as compared to both E15 + 12 and E15 + 15 controls. I-
L. In E15 + 12 controls (I), strong amelogenin immunostaining is seen in secretory
ameloblasts and polarized odontoblasts (double arrows), and weakly in adjacent
predentin and DPM. In E15 + 15 controls (J), strong amelogenin immunostaining is seen
in polarized, secretory ameloblasts and on the DEJ; weak staining is seen in predentin,
dentin and enamel matrixes. In Cap stage-infected molars (K), mCMV induces a marked
decrease in amelogenin immunostaining in ameloblasts and misexpression of amelogenin
protein in odontoblasts, DPM cells in the crown area (white *), the extracellular matrix
surrounding individual cytomegalic stromal cells (arrowhead), and stellate reticulum. In
Bell stage-infected molars (L), immunostaining is localized in ameloblasts, particularly in
granular-like structures in the distal parts of secretory ameloblasts, and diffusely in DPM
in crown region (white *). Bar, A-D, G-L: 30 µm, E, F: 40 µm.

84
FIGURE 11: Continued

85
FIGURE 12: mCMV Induces Stage-Dependent Changes in the Distribution of
C/EBPα Protein.
mCMV induces stage-dependent changes in the distribution of C/EBPα protein. (a)
Ameloblasts. (e) Enamel. C/EBPα localization is seen in E15+12 (A) controls with
C/EBPα localized to the odontoblasts from the cusp down to the cervical loop (black
arrow) and spread evenly throughout the dental papilla mesenchyme (dpm). E15+15 (E)
controls show the developed enamel (e) and the C/EBPα localized throughout the
epithelium. mCMV infected Cap stage E15+15 molars (C) shows a different distribution,
with C/EBPα protein not present around the odontoblast, but rather in the dental papilla
mesenchyme that is more apical (*) and not present at all in the more cuspal dental
papilla mesenchyme. In the mCMV Bell stage-infected E15+15 (G) you can see that the
C/EBPα protein is present in the odontoblasts toward the CL (black arrowheads) and in
the more cuspal region of the dental papilla mesenchyme (black arrow). It is also found
sparsely throughout the dental papilla mesenchyme. DAPI staining of E15 + 12 and
E15+15 controls and mCMV infected Cap and Bell stage-infected tooth (B,D,F,H).

86
FIGURE 12: Continued

87
FIGURE 13: mCMV Induced Stage-Dependent Qualitative and Quantitative
Spatial Changes in PCNA Localization.
mCMV induced stage-dependent qualitative and quantitative spatial changes in
PCNA localization. A-D. The cell-specific localization of PCNA-positive nuclei in E15
+ 12 control molars (A), E15 + 15 control molars (B), Cap stage-infected molars (C) and
Bell stage-infected molars (D). In E15 + 12 controls (A), extension of the CL inner
enamel epithelium (IEE) and outer enamel epithelium (OEE) forms the early HERS.
PCNA-positive nuclei are seen primarily in IEE and OEE and, to a lesser extent, in
surrounding mesenchymal cells and stellate reticulum (s). Red bracket indicates the
apical end of CL/HERS. In E15 + 15 molars (B), PCNA-positive nuclei are seen in the
fused IEE and OEE bilayer of HERS (double red arrows), in surrounding mesenchyme
and stellate reticulum. (e), enamel. In Cap stage-infected molars cultured for 15 days (C),
there is a substantial difference in the spatial localization of PCNA-positive nuclei; they
are found in the cytomegalic infected and affected DPM (dpm) cells but are relatively
absent in the severely abnormal, hypoplastic CL (black bracket). In Bell stage-infected
molars cultured for 15 days (D), PCNA-positive nuclei are found in the multilayered IEE
and OEE of the CL (blue bracket), as well as in the expanded population of remaining
stellate reticulum cells persisting between IEE and OEE (*); very few PCNA-positive
nuclei are seen in DPM. In both infected Cap stage and Bell stage molars, a marked
increase in PCNA-positive nuclei in the disorganized, multilayered stellate reticulum
cells is seen. Bar, 30 µm. E. mCMV induced stage-dependent differences in cell
proliferation indexes. Cell proliferation indexes in DPM and stellate reticulum of infected
Cap stage or Bell stage molars were compared to E15 + 12 and E15 + 15 controls; cell
proliferation indexes in CL regions of infected Cap stage and Bell stage molars were only
compared to E15 + 12 controls. *Significant differences in cell proliferation indexes: (1)
DPM: Cap stage-infected v. Cont E15 + 12: 5.8-fold↑ (P<0.0001); Cap stage-infected v.
CONT E15 + 15: 6.2-fold↑ (P<0.005). (2) Stellate reticulum: Cap stage-infected v.
CONT E15 + 12: 1.6-fold↑ (P<0.0001); Cap stage-infected v. CONT E15 + 15: 2-fold↑
(P<0.01); Bell stage-infected v. CONT E15 + 12: 1.5-fold↑ (P<0.0005); Bell stage-
infected v. CONT E15 + 15: 1.9-fold↑ (P<0.05). (3) CL: Bell stage-infected v. CONT
E15 + 12: 1.7-fold↑ (P < 0.02).

88
FIGURE 13: Continued

89
APPENDIX B: TABLE 1

Abstract (if available)

Abstract BACKGROUND: Cytomegalovirus (CMV) is one of the most common causes of major birth defects in humans. Of the approximately 8400 children born each year in the U.S. with CMV-induced birth defects, more than 1/3 of these children exhibit hypoplasia and hypocalcification of tooth enamel. ❧ OBJECTIVE: Our objective was to initiate the investigation of the pathogenesis of CMV-induced tooth defects and examine the effects of CMV infection on progressive tooth differentiation and amelogenesis. ❧ METHODS: Mouse Cap and Bell stage mandibular first molars were infected with mouse CMV (mCMV) in vitro in a chemically-defined organ culture system and analyzed utilizing histological and immunolocalization methodologies. ❧ RESULTS: CMV infection of embryonic mouse mandibular first molars in vitro induces tooth dysmorphogenesis and enamel defects in a developmental stage- and duration-dependent manner. Initial protein localization studies suggest that the pathogenesis is mediated through NF-κB signaling and that there appears to be an unusual interaction between abnormal mesenchymal cells and surrounding matrix. Rescue with acyclovir indicates that mCMV replication is necessary to initiate and sustain progressive tooth dysmorphogenesis. Cap stage- and Early Bell stage-infected molars exhibit enamel agenesis and Bell stage-infected molars exhibit enamel hypoplasia. ❧ CONCLUSIONS: Our results indicate that mCMV-induced changes in signaling pathways severely delays, but does not completely interrupt, tooth morphogenesis. This viral-induced pathology is coincident with stage-dependent changes in Amelx, Enam and Dspp gene expression, distribution of amelogenin, enamelin, C/EBPα and DSP proteins, cell proliferation localization and dedifferentiation of secretory ameloblasts. Our data indicate that specific levels of Amelx and Dspp gene expression define whether mouse CMV induces enamel agenesis or hypoplasia. Importantly, our results demonstrate that this well-defined embryonic mouse organ culture system can be utilized to delineate the molecular mechanism underlying the CMV-induced tooth defects that characterize the amelogenesis imperfecta phenocopy seen in many CMV-infected children.

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Asset Metadata

Creator Abichaker, George (author)

Core Title Cytomegalovirus induced amelogenesis imperfecta

School School of Dentistry

Degree Master of Science

Degree Program Craniofacial Biology

Publication Date 05/04/2012

Defense Date 02/21/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag amelogenesis imperfecta,cmv,Cytomegalovirus,OAI-PMH Harvest,tooth

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Melnick, Michael (committee chair), Jaskoll, Tina T. (committee member), Sameshima, Glenn (committee member)

Creator Email abichake@usc.edu,georgeabichaker.usc@gamil.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-30882

Unique identifier UC11289537

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Legacy Identifier etd-AbichakerG-771.pdf

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Document Type Thesis

Rights Abichaker, George

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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