expression and characterization of c-myb in prenatal odontogenesis

Develop. Growth Differ. (2011) 53, 793–803 doi: 10.1111/j.1440-169X.2011.01287.x

The Japanese Society of Developmental Biologists

Original Article

Expression and characterization of c-Myb in prenatalodontogenesis

Eva Matalova,1,2* Marcela Buchtova,1,2 Abigail S. Tucker,3 Timothy P. Bender,4

Eva Janeckova,1,5 Vlasta Lungova,2 Simona Balkova1 and Jan Smarda5

1Institute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Czech Republic;2Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic;3Department of Craniofacial Development and Orthodontics, KCL, London, UK; 4Department of Microbiology, University

of Virginia, Charlottesville, Virginia, USA; and 5Department of Experimental Biology, Faculty of Science, MasarykUniversity, Brno, Czech Republic

*AuthorEmail: mReceive

acceptedª 2011Develop

Society of

The transcription factor c-Myb is involved in the control of cell proliferation, survival and differentiation. As theseprocesses accompany the morphogenesis of developing teeth, this work investigates the possible role of c-Mybduring odontogenesis. Analysis of the expression of c-Myb in the monophyodont mouse was followed by similaranalysis in a diphyodont species, the pig, which has a dentition more closely resembling that of the human. Thedistribution of c-Myb was correlated with the pattern of proliferation and apoptosis and the tooth phenotype ofc-Myb mutant mice was also assessed. In the mouse, c-Myb expression was detected throughout prenataldevelopment of the first molar tooth. Negative temporospatial correlation was found between c-Myb expressionand apoptosis, while c-Myb expression positively correlated with proliferation. c-Myb-positive cells, however,were more abundant than the proliferating cell nuclear antigen positive cells, suggesting other roles of c-Myb inodontogenesis. In the minipig, in contrast to the mouse, there was an asymmetrical arrangement of c-Myb posi-tive cells, with a higher presence on the labial side of the tooth germ and dental lamina. A cluster of negativecells was situated in the mesenchyme close to the tooth bud. At later stages, the number of positive cellsdecreased and these cells were situated in the upper part of the dental papilla in the areas of future cusp for-mation. The expression of c-Myb in both species was strong in the odontoblasts and ameloblasts at the stageof dentin and enamel production suggesting a possible novel role of c-Myb during tooth mineralization.

Key words: morphogenesis, mouse, Myb, pig, tooth.

Introduction

Tooth development involves a series of sequential and

reciprocal homo- and heterotypical molecular cas-

cades between the stomodeal epithelium that lines the

inside of the oral cavity, and the cranial neural crest-

derived mesenchyme (Tucker & Sharpe 2004).

Regardless of shape or type, mammalian teeth pass

through the same developmental stages and are

formed by the same differentiated tissue (Stock et al.

1997). The first morphological sign of tooth develop-

ment is a thickening of the oral epithelium–dental

placode. Subsequently, tooth buds are formed by

to whom all correspondence should be [email protected]

d 24 November 2010; revised 15 April 2011;2 May 2011.The Authorsment, Growth & Differentiation ª 2011 JapaneseDevelopmental Biologists

coordinated interactions between epithelial and mesen-

chymal tissues. Cells of the epithelial thickening proli-

ferate and invaginate further into the mesenchyme that

reciprocally condenses around the epithelium. Later,

the epithelium expands deeper and wraps around thecondensing mesenchyme, forming a tooth cap and

subsequently a bell (Luckett 1993). The process of

tooth cap morphogenesis is controlled by a signaling

center, the primary enamel knot (Jernvall et al. 1994).

Non-dividing cells of the enamel knot produce signaling

molecules essential for the proliferation of surrounding

cells and the formation of the dental papilla enclosed

by a cervical loop. In multicuspid teeth, after loss of theprimary enamel knot, secondary enamel knots develop

at the sites of future cusps. Signals from the secondary

enamel knots are responsible for further folding of the

inner enamel epithelium, resulting in several epithelial

invaginations and multiple cusps (Coin et al. 1999).

Differentiation of cells producing dental hard tissue

proceeds once the basic cusp pattern is formed.

794 E. Matalova et al.

Mesenchymal cells facing the basement membranedifferentiate into dentin-producing odontoblasts and

start to secrete organic dentin matrix that serves as a

scaffold for deposition of hydroxyapatite crystals.

Immediately after initial predentin deposition, the adja-

cent layer of epithelial cells differentiates into amelo-

blasts. Ameloblasts produce organic enamel matrix

forming the three dimensional organization of enamel

and subsequently mediate its maturation into a highlymineralized tissue (Smith 1998). During development,

controlled proliferation, differentiation and apoptotic

elimination of particular cell populations are considered

to contribute to the final tooth shape, size and position

in the jaw (Cobourne & Sharpe 2003).

The Myb locus encodes the c-Myb (Myb, myeloblas-

tosis oncogene) transcription factor involved in control

of cell proliferation, differentiation, survival, and celldeath (Oh & Reddy 1999; Ramsay & Gonda 2008).

c-Myb was originally described as an essential regula-

tor of hematopoiesis being expressed predominantly

in undifferentiated precursors (Akashi et al. 2000).

Absence of proper c-Myb function is lethal; c-Myb defi-

cient mice die in utero from failure of fetal hematopoie-

sis around day 14 (Mucenski et al. 1991). Expression of

the Mybl1 locus has been reported in mammary glandductal epithelium, testis, central nervous system and in

the germinal centre of B-lymphocytes (Sleeman 1993;

Mettus et al. 1994; Trauth et al. 1994). c-Myb expres-

sion has been documented in a number of embryonic

and adult tissues (Sitzmann et al. 1995; Ess et al. 1999)

and there are multiple data suggesting a role for c-Myb

in the control of developmental processes (Zorbas et al.

1999; Hoffman et al. 2006; Greene et al. 2007).c-Myb is expressed in mouse dental tissue at

embryonic day 14, indicating a possible involvement in

tooth development (Ess et al. 1999). However, the role

of c-Myb in this process has not been investigated.

This work therefore aims to follow c-Myb expression

during the course of prenatal odontogenesis and cor-

relate it with cell proliferation and apoptosis. Further-

more, c-Myb expression was compared in an animalwith a single generation of teeth (the monophyodont

mouse) and an animal with two generations of teeth

(the diphyodont minipig), to assess the potential role in

formation of a replacement dentition. Finally, these

processes, along with the phenotype of the tooth bud,

were investigated in myb null embryos.

Materials and methods

Animals

Mice. Mouse strain CD1 was used and samples

were obtained at embryonic day 12.5, 15.0, 17.5

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Development, Growth & Differentiation ª 2011 Japanese Society of De

and perinatally (P0). After fixation in 4% bufferedformaldehyde, heads were dehydrated in a gradient

series of ethanol, and wax embedded after xylene

treatment. Frontal serial sections were split over four

slides to simultaneously follow production of the

c-Myb protein as well as cell proliferation ⁄ apoptosis

and to correlate expression of myb encoded mRNA

and protein levels.

Minipigs. Embryos and fetuses of minipigs wereobtained from Libechov animal facility (Czech Repub-

lic, strain LiM). They were collected between embry-

onic day (E) 20 and 67 and fixed in 4% buffered

formaldehyde. After paraffin processing, 5 lm serial

tissue sections were prepared and stained with hema-

toxylin-eosin (HE). Alternative slides were left unstained

for immunohistochemical analyses of selected

embryos and fetuses (E20, E36, E56, E67).All procedures were conducted following protocols

approved by the Animal Science Committee of the

Institute of Animal Physiology and Genetics, v.v.i.,

Academy of Sciences of the Czech Republic.

c-myb null embryos

A floxed myb allele was generated. Cre mediated dele-

tion of the loxP-flanked exon II generated the c-myb

deletion (d) allele (Bender et al. 2004). This deletedallele was a true null as no c-Myb protein (antibody

against the C-terminal portion) was detected in myb

d ⁄ d embryonic livers. Embryos die at E14–E15 with

extreme anemia similar to conventional c-myb deficient

mice. Embryonic heads of the mutants and wild type

littermates at E14 were processed in the same way as

described above for the CD1 mouse samples.

Cloning of the mouse c-myb cDNA for in situ

hybridization

Mouse tissues were collected from stage E13.5 and

total RNA was isolated using the Midi RNeasy kit (Qia-

gen). Complementary DNA (cDNA) was synthesized

using SuperScript III (Invitrogen) and primers were

designed based on mouse gene reference sequence

NM_010848.3 (Fig. S1). The primer set flanked the

mouse c-myb cDNA from 497 to 1342 bp (forward5¢-GTGCCAACACCGGTGGCAGA-3¢, reverse 5¢-GCC-

ACCCCATCTCTGCCTGC-3¢). Transcripts were then

amplified using Taq polymerases. Polymerase chain

reaction (PCR) product was subcloned into TOPO-II

vector (Invitrogen) and sequenced using the endoge-

nous M13F primer site. These fragments were used as

templates to generate RNA probes for in situ hybri-

dization (ISH).

velopmental Biologists

c-Myb and tooth morphogenesis 795

c-Myb in situ hybridization

Radioactive in situ hybridization of 35S-UTP-labelled

c-myb probe was performed as described (Tucker

et al. 1999). c-myb was linearized with HindIII and

transcribed with T7. Sections were counterstained withmethyl green and photographed under darkfield and

brightfield.

Immunohistochemistry

After deparaffinization and rehydration of sectioned

mouse heads, endogenous peroxidase was inhibited by

3% hydrogen peroxide in phosphate-buffered saline

(PBS) ⁄ RT ⁄ 5 min and non-specific secondary antibody

binding by incubation in goat serum for 20 min ⁄ RT.Primary antibody (anti-c-Myb, Abcam, ab59233; anti-

proliferating cell nuclear antigen (PCNA), Santa Cruz,

sc-7907; anti-B-Myb, Abcam, ab76009; osteocalcin,

Abcam, ab93876, respectively) was applied in the

concentration of 20 lg ⁄ mL (anti-Myb) or 4 lg ⁄ mL (anti-

PCNA-osteocalcin) for 1 h. In the case of B-Myb and

osteocalcin, citrate pretreatment (10 min ⁄ 97�C) was

applied prior to the primary antibody. The biotinylatedsecondary antibody and the streptavidin-peroxidase

complex (1:500, ABC kit, Vectastain) conjugations were

performed for 30 min ⁄ RT each. Final color reaction was

achieved using 3,3¢-diaminobenzidine tetrachloride

(DAB) chromogenic substrate (Dako) and samples were

counterstained by hematoxylin.

Tartrate resistant acid phosphatase assay

Tartrate resistant acid phosphatase (TRAP) substratereaction was used to detect osteoclastic activity along

with morphological confirmation of the cell type in HE

sections. After rehydration, slides were immersed into

the reaction mixture prepared according to the manu-

facturer’s directions (Sigma-Aldrich, 387A-1KT) and

kept at 37�C ⁄ 2 h to achieve the color reaction with

Fast Red substrate. Slides were counterstained by

hematoxylin.

Detection of apoptosis by TUNEL assay

After deparaffinization and rehydration, samples were

processed according to the manufacturer’s protocol

(Millipore, S7100). Briefly, samples were pretreated

with proteinase K (Millipore) 20 lg ⁄ mL, RT, 15 min,

and endogenous peroxidase was inhibited by 3%

hydrogen peroxide in PBS ⁄ RT ⁄ 5 min. Equlibration

buffer was applied for 15 min ⁄ RT, reaction mixturefor 60 min ⁄ 37�C. The digoxigenin-peroxidase com-

plex was conjugated for 30 min ⁄ RT. Final color

Development, Growth & Dif

reaction was achieved using DAB chromogenic sub-strate (Dako) and samples were counterstained by

hematoxylin.

To express relative quantity of TUNEL (terminal de-

oxynucleotidyl transferase-mediated dUTP nick end

labeling) positive cells in the mutant versus wild type

primary enamel knot (PEK), cells were counted in serial

sections using the IMAGE J plugin Cell Counter pro-

gram (Research Services Branch, Bethesda, MD,USA). Statistical significance was based on results of

the following t-test and F-test (P > 0.05).

Control reactions

As c-myb expression is well known to occur in the

duodenal crypts, mouse adult duodenum was used to

confirm the primary antibody reaction (Abcam,

ab59233) and the ISH probe specificity (Figs S1–3).

The negative control for IHC was achieved by omittingthe primary antibody from the reaction mixture.

Results

c-Myb expression in a monophyodont dentition: the

mouse

Initiation of odontogenesis at embryonic (E) day 12.5,

the bud stage at E13.5, the early bell at E15.5, the late

bell stage at E17.5 and the mineralized stage in the

perinatal period (P0) were investigated in the mouse

lower first molar by immunohistochemistry. At E12.5,

when the budding tooth germ becomes morphologi-

cally obvious, c-Myb positive cells were scattered in

the epithelium as well as in the surrounding mesen-chyme (Fig. 1A). Later on, at E13.5, c-Myb protein

concentrated particularly in the epithelial part of the

tooth germ. Nevertheless, scattered positive cells were

found also in the mesenchyme (Fig. 1B). At the early

bell stage, E15.5, the c-Myb protein production was

strong in the dental lamina and in the areas of the

growing cervical loop (Fig. 1C). Some c-Myb-positive

cells were detected also in the epithelial layer of thefuture ameloblasts and in the mesenchyme, particularly

in the region adjacent to the epithelium (Fig. 1C). Dif-

fused c-myb expression in and around the primary

enamel knot (as detected by Shh and Fgf-4 expres-

sion) was confirmed by in situ hybridization (ISH)

(Fig. 2A–C). At E17.5, the c-Myb-positivity shifted from

the epithelium to the mesenchyme. c-Myb in the epi-

thelium remained in the dental lamina and some scat-tered cells were present also in other epithelial regions

of the tooth germ (Fig. 1D). Mesenchyme continued to

be positive for c-Myb, particularly in the region facing

the oral epithelium. Restricted c-myb expression in the

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ferentiation ª 2011 Japanese Society of Developmental Biologists

(A) (B)

(C) (D)

(E) (E′) (E′′)

Fig. 1. c-Myb expression in mouse

monophyodont dentition. c-Myb

positive cells scattered in the

epithelium and the surrounding

mesenchyme at E12.5 (A). c-Myb

protein concentrated particularly in

the epithelial part of the tooth germ at

13.5 (B). c-Myb protein in the dental

lamina, in the growing cervical loop

and the epithelial layer of the future

ameloblasts at E15.5 (C). c-Myb-

positivity at E17.5 found in the

mesenchyme and in the epithelium

facing the mesenchyme – particularly

in the dental lamina (D). c-Myb-

positive cells in the dental lamina

epithelium, the oral part of the

mesenchymal dental papilla and the

border line facing the surrounding

mesenchymal tissue at P0 (E) with

detail of odontoblast and ameloblast

populations (E¢) and growing areas

of future tooth roots (E¢¢), am,

ameloblasts; cl, cervical loop; d,

dentin; od, odontoblasts. Scale bar =

100 lm.

(A) (B) (C)

(D) (E)

Fig. 2. Restricted c-myb expression

at the enamel knot stages (E15.5,

E17.5) – in situ hybridization. No

distinct patches of c-myb expressing

cells were found at the primary enamel

knot stage (E15.5) (A) as confirmed by

the primary enamel knot markers Shh

(C) and Fgf-4 (B). However, distinct

patches of c-myb expression were

found in the mesenchyme adjacent to

the secondary enamel knots of the bell

stage tooth germ (D), as confirmed by

the secondary enamel knot marker

Fgf-4 (E). Arrows point to the epithelial

secondary enamel knots facing the

c-myb positive mesenchyme. Scale

bar = 100 lm, sek, secondary enamel

knot.

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Development, Growth & Differentiation ª 2011 Japanese Society of Developmental Biologists



mesenchyme adjacent to the secondary enamel knots(as detected by Fgf-4 expression) was clearly demon-

strated at the mRNA level by ISH (Fig. 2E, F).

Perinatally, the c-Myb-positive cells in the epithelium

were still found in the dental lamina. The mesenchymal

dental papilla contained c-Myb protein particularly in

the oral part and also in the border facing the sur-

rounding mesenchymal tissue (Fig. 1E). Abundant c-

Myb expression corresponded to the odontoblast andameloblast populations (Fig. 1E¢) and the growing

areas of the future tooth roots as they invaded into the

mesenchyme (Fig. 1E¢¢).

c-Myb in the bone tissue surrounding the tooth

In addition to expression in the tooth itself, c-Myb pro-

tein was also observed in the surrounding alveolar

bone. To clearly distinguish osteoblasts and osteo-

clasts in this tissue, c-Myb protein distribution wascorrelated in serial sections with osteocalcin as a mar-

ker of osteocytes and activity of tartrate resistant acid

phosphatase (TRAP) characteristic for osteoclasts

(Fig. 3A–D). From such analysis, c-Myb appeared to

be expressed in osteoblasts and osteoclasts.

c-myb expression versus B-myb expression

As B-Myb exhibits the same growth and cell depen-

dence as c-Myb, the relationship between c-Myb andB-Myb in the tooth was shown by localization of B-

Myb protein performed at P0 and correlated with c-

Myb in serial sections (Fig. 4A–F). B-Myb as c-Myb,

(A)

(C)

Fig. 3. c-Myb in the tooth surroun-

ding bone. Detail of the bone

surrounding the dental papilla (A) and

c-Myb protein distribution (B) are

shown along with osteocalcin as the

osteoblast marker (C) and tartrate

resistant acid phosphatase (TRAP) as

the osteoclast marker (D) in the serial

section of the mouse lower molar at

P0. Scale bar = 100 lm, cl, cervical

loop; dp, dental papilla; arrows point

to positive cells.


was present in both, epithelial and mesenchymal partsof the tooth germs (Fig. 4A, D). However, B-Myb unlike

c-Myb was not detected in odontoblasts (Fig. 4E,

compare 4E) and the distribution pattern in the dental

papilla and surrounding bone did not overlap (Fig. 4C,

compare 4F).

c-Myb expression in a diphyodont dentition:

the minipig

There are morphological differences in formation of themouse monophyodont dentition and the pig diphy-

odont dentition. In the mouse, teeth are initiated in

close proximity to the oral epithelium while in the pig

the dental lamina first initiates and grows deep into the

mesenchyme before the teeth bud off. To compare

differences or similarities in c-Myb expression to the

mouse, immunohistochemical analysis of minipig early

odontogenesis was performed. The third and forthpremolars of the minipig were chosen for this study as

they are of a similar shape to mouse molars.

At E20, c-Myb positive cells were evenly distributed

through the mesenchyme and oral epithelium without

obvious clustering into restricted areas (Fig. 5A). At the

time when the dental lamina grows deep into the me-

senchyme (E36), positive cells were concentrated on

the labial side of the dental lamina and in the dentallamina connecting the tooth germ to the oral epithe-

lium (Fig. 5B). Clusters of negative cells were located

in the mesenchyme in close proximity to the bud or

cap anlagen (Figs. 5C, D). The enamel knot area

contained a few dispersed c-Myb positive cells at the

(B)

(D)

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(A) (B) (C)

(D) (E) (F)Fig. 4. c-Myb expression versus B-

Myb expression at P0. As c-Myb

protein (A), B-Myb was found in both

tooth germ epithelium and mesen-

chyme (D). However, compared to

c-Myb (B), B-Myb was not present in

the odontoblast (E). Occurrence of

c-Myb (C) and B-Myb differed also in

the mesenchymal papilla and sur-

rounding bone (F). am, ameloblast;

od, odontoblast. Scale bar = 100 lm.


cap stage; however, apoptotic cells were negative.

The enamel organ at the cap stage contained regularly

dispersed positive cells (Fig. 5D).

At a later stage (E56), only a few positive cells were

situated in the enamel organ and inner enamel epithe-

lium of the early bell stage (Fig. 5E) with the highest

concentration in the cervical loop areas (Fig. 5F). The

teeth at secretion stage (E67) contained c-Myb positiveodontoblasts that were regularly arranged along the

dentin producing region (Fig. 5G, H). In the mesen-

chyme close to the cervical loop where predentin pro-

duction has not started yet, most odontoblasts were

negative (Fig. 5H). The apical area of the dental papilla

contained more positive cells than the basal part of

the mesenchyme (Fig. 5G). Osteoclasts and osteo-

blasts located on the bone lamellae facing the teethwere c-Myb-positive (Fig. 3B, 5F, H).

c-Myb expression versus proliferation and apoptosis

The morphogenetic events of proliferation and apopto-

sis were correlated with the localization of c-Myb

protein using serial sections in the mouse at E15.5,

and the pig at E56. This is the stage when clear popu-

lations of proliferating cells (PCNA-positive) at the

cervical loops, and apoptotic cells (TUNEL-positive)can be distinguished in both species. Negative tempo-

rospatial correlation was found between c-Myb protein

localization and apoptosis in the mouse (Fig. 6A, C),

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and the pig (Fig. 6D, F, in detail Fig. 6G, I). Positive cor-

relation appeared between c-Myb and proliferation in

the mouse (Fig. 6A, B), and the pig (Fig. 6D, E, in

detail Fig. 6G, H). In general, apoptotic cells were c-

Myb negative, while c-Myb positive cells were more

abundant than the PCNA positive cells. This trend was

also observed at later stages (data not shown).

Tooth morphology in c-myb null mice

c-myb null embryos were investigated at E14, which

represents the latest stage available before embryonic

lethality. By this time-point, the first molar tooth germ

had reached the late bud stage in both mutants and

wild type (WT) littermates. The morphology of the first

molar did not significantly differ in shape or size from

the WT littermates (Fig. 7A, compare 7D). As in WT lit-

termates, proliferation in the mutant was found particu-larly in the bud epithelium, and excluded from the tip

of the bud. Scattered mesenchymal cells were also

PCNA-positive (Fig. 7B, E). Apoptotic (TUNEL-positive)

cells were concentrated at the tip of the bud and some

were present also in the middle axis of the bud, similar

to the WT (Fig. 7C, F). Despite the slightly different

number of TUNEL positive cells in comparable indivi-

dual sections, counting of positive cells per entire toothgerm did not show any significant differences (data not

shown). Therefore, along with no obvious alterations in

morphology, localization and quantity of proliferation


(A) (B)

(C) (D)

(E) (F)

(G) (H)

Fig. 5. c-Myb expression in pig

diphyodont dentition. c-Myb positive

cells evenly distributed in the oral

epithelial region as well as in the

mesenchyme at E20 (A). c-Myb

positive cells concentrated on the

labial side of the lamina at dental

lamina stage (B). Clusters of negative

cells (arrow) situated in the

mesenchyme surrounding bud (C)

and cap tooth anlagen (D). Positive

cells in the mesenchyme at early bell

stage (E), with higher number in the

inner enamel epithelium and in the

cervical loops (F). Ameloblasts (am)

and odontoblasts (od) at secretion

stage (G), c-Myb positive cells

concentrated to the apical tip of

dental papilla. Border of odontoblast

positivity (H) with beginning of

predentin production (arrow) in the

cervical loop, am, ameloblasts; d,

dentin; od, odontoblasts. Scale

bar = 100 lm.


and apoptosis were found to be similar in the mutant

compared to the wild type. These results would sug-

gest that c-Myb is not essential for the early develop-

ment of the first molar tooth and corresponding

proliferation-apoptosis dynamics.

Discussion

The first mouse molar, which has been widely exa-

mined in odontogenic studies, was used as a model

system to follow the expression of c-Myb at RNA and

protein levels. During early tooth development, when


the dental epithelium starts to invaginate and extend

into the underlying mesenchyme, c-Myb protein was

found to be concentrated in the epithelium with scat-

tered positive cells in the mesenchyme. The finding

that only a few c-Myb-positive, and PCNA positive,cells are found in the mesenchyme corresponds with

the fact that the major dynamics and increased density

of the mesenchyme at this stage is achieved by

condensation rather than by proliferation (Lesot &

Brook 2009). Interestingly, in the minipig at the bud

stage, an asymmetrical c-Myb expression in the dental

lamina was noticed and correlated with asymmetrical

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(A) (B) (C)

(D) (E) (F)

(G) (H) (I)

Fig. 6. c-Myb expression versus

proliferation and apoptosis. In both,

the mouse (A–C) at E15.5 and the

pig at E56 (D–I), c-Myb expression

(A, D, G) positively correlates with

proliferating cells, proliferating cell

nuclear antigen (PCNA)-positive

(B, E, H), cells at the cervical loops,

and negatively with apoptotic,

terminal deoxynucleotidyl transferase-

mediated dUTP nick end labeling

(TUNEL)-positive (C, F, I), cells in

the primary enamel knot. Scale

bar = 100 lm, cl, cervical loop; pek,

primary enamel knot. Arrows point to

the positive cells, sections in line are

parallel.


arrangement of PCNA-positive cells (Stembirek et al.

2010). Furthermore, asymmetrically dispersed c-Myb-positive cells were located in the facial mesenchyme

and around the dental lamina. The primary enamel

knot formed by terminally differentiated cells appeared

almost c-Myb-negative in both species. In general

throughout early tooth development, proliferating

regions were found to be c-Myb-positive, whereas

apoptotic populations were c-Myb-negative, agreeing

with published work that describes c-Myb as a posi-tive regulator of proliferation and PCNA transcription

(Travali et al. 1991; Malaterre et al. 2007; Lieu & Red-

dy 2009).

A greater number of c-Myb-positive cells in the mes-

enchyme was detected at the early bell stage (E15.5)

suggesting synchrony with proliferation of the future

dental pulp. At the later bell stage (E17.5), a strong c-

Myb immunohistochemical signal was detected in thedental lamina and some cells were scattered also in

other epithelial parts of the tooth germ. Expression

was detected at a high level in the mesenchyme. The

mesenchymal expression was particularly clear in the

in situ data, with two distinct patches of c-myb mRNA

in the mesenchyme facing the secondary enamel

knots. There are a number of signaling molecules

active in the primary and secondary enamel knots,

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such as Shh and Fgf4, which have been reported to

co-ordinate proliferation in surrounding cells (Jernvallet al. 1994; Dassule and McMahon, 1998). It is possi-

ble that these signaling molecules may functionally

interact with c-Myb and induce its expression in the

mesenchyme underlying the enamel knots. Expression

of c-myb, however, was not highly associated with the

mesenchyme under the primary enamel knot, indicat-

ing perhaps a different role for c-Myb at primary and

secondary enamel knot stages.As the tooth developed, epithelial cells in the dental

lamina remained positive and high c-Myb protein levels

were revealed in the growing molar roots as well as in

the mesenchymal border between the dental pulp and

surrounding tissues. The mesenchymal border of the

dental pulp and alveolar bone appear to be estab-

lished by cells invading from the dental mesenchyme

close to the dental lamina at the bud stage (Diep et al.

2009). Strong c-myb expression would support exclu-

sivity of these cells, which move downwards as the

tooth grows and later differentiates into the alveolar

osteoblasts, thereby contributing to a functional tooth-

bone connection (Diep et al. 2009).

Interestingly, in the minipig a clear border of c-Myb

expression was detected in the dental papilla where

predentin production just started with the condensa-


(A) (B) (C)

(D) (E) (F)

Fig. 7. Wild type versus c-myb null tooth phenotype. Morphology (D) and proliferation (E) of the first molar tooth germ of the mutant

with no obvious difference compared to the wild type (A, B) at E14 (survival limit). Detail of apoptotic cells in the wild type (C) and the

mutant bud (F). Scale bar = 100 lm.


tion of positive cells in the area with a thick layer ofdentin. Similarly, there was a clear border between the

strong c-Myb expression in the respiratory epithelium

of E15.5 mouse, and the olfactory epithelium (Fig. S3),

resembling the strong expression reported in the

developing trachea and proximal bronchial epithelial

cells at E14 (Ess et al. 1999). c-Myb expression there-

fore appears to be linked to the whole respiratory

tract. Only scattered c-myb positive cells were visiblein the olfactory epithelium of the nasal cavity (Fig. S3).

Epithelial cells in the respiratory part of the nasal cavity

at this stage do not show morphological signs of dif-

ferentiation and goblet cells are not recognizable. The

strong expression in this area therefore corresponds to

cells in an undifferentiated state. The lack of c-Myb in

the olfactory epithelium, which has been shown to act

as an important signaling center during early develop-ment in the craniofacial area, may indicate the more

differentiated nature of these cells (Szabo-Rogers et al.

2009).

In most tissues, abundant c-Myb expression is asso-

ciated with immature stages and decreases during

development and differentiation (Ess et al. 1999). How-

ever, the link does not appear to be so straightforward

in odontogenesis as c-Myb positive cells were alsofound in differentiated cells of the tooth germ (amelo-

blasts, odontoblasts). A similar observation was

reported in the brain, where c-myb transcript and

c-Myb protein levels significantly increased with the

age of mice (Hwang et al. 2007). One possible func-

tion supported by our findings could be a role for

c-Myb in calcium metabolism of odontoblasts as

Myb-dependent regulation of intracellular calcium level


was previously described in embryonic fibroblasts(Bein et al. 1997). Therefore, c-Myb might regulate the

calcium level in odontoblasts or ameloblasts and con-

tribute to the mineralization of dentin or enamel during

its production. Similarly, osteoblasts showed c-Myb

expression in both mouse and minipig samples. There

is only limited information published about the role of

c-Myb in osteogenesis (Falany et al. 2001). To date,

the c-Myb activity in bone cells has been consideredto be mediated by glucocorticoids and mutation in a

binding site for c-Myb abrogated the inhibitory effect of

cortisol on activity of the IGFBP-5 promoter (Gabbitas

et al. 1996), which contains functional cis regulatory

elements for Myb (Perez-Casellas et al. 2009). c-Myb

expression was also observed in osteoclasts.

Despite abundant c-Myb in odontogenesis, loss of

c-Myb in the myb null embryos did not cause anyapparent changes in tooth morphogenesis, with the

bud stage moving towards the cap stage with forma-

tion of a flattened epithelial base. As c-Myb has been

directly linked to PCNA transcription, loss of c-Myb

would have been predicted to lead to a loss of PCNA

positive cells in the tooth germ. However, PCNA levels

appeared normal. In addition, no significant alteration

in the number of TUNEL positive cells was observed inthe mutant. It is possible that c-Myb does not play a

role in co-coordinating these processes of proliferation

and death in the tooth, as it does during hemato-

poiesis (Oh & Reddy 1999; Peng et al. 2007; Lieu &

Reddy 2009). For example, in the intestine c-Myb

correlates with suppression of differentiation rather

than proliferation (Rosenthal et al. 1996; Zorbas et al.

1999). Alternatively, another member of the myb family

ª 2011 The Authors



may compensate for the loss of c-myb in the tooth(Bein et al. 1997; Toscani et al. 1997). The vertebrate

genome contains three myb genes (A-myb, B-myb

and c-myb) that are located on different chromosomes

(Davidson et al. 2005). There is a highly conserved

transcriptional activation domain present in A-Myb and

c-Myb but not in B-Myb (Davidson et al. 2005).

Expression of the mybl1 locus has been reported in

mammary gland ductal epithelium, testis, central ner-vous system and in the germinal center of B-lympho-

cytes (Sleeman 1993; Mettus et al. 1994; Trauth et al.

1994). Mice with a disruption of the A-myb locus are

viable but with the failure of spermatogenesis and

mammary gland proliferation (Toscani et al. 1997).

B-myb is ubiquitously present throughout mouse

development (Reiss et al. 1991; Sitzmann et al. 1996).

B-Myb exhibits the same growth and cell dependenceof expression as c-Myb (Lyon et al. 1994). Based on

these observations together with our results it is possi-

ble that other Myb family members can functionally

replace c-Myb during odontogenesis (Bein et al.

1997). Given the overlapping expression pattern of

c-Myb and B-Myb in some dental tissues, B-Myb

may compensate for loss of c-Myb during tooth deve-

lopment.Although a defect was not observed in the mutant

mice around E14 this does not rule out a role for

c-myb in tooth development at later stages of embryo-

genesis. Conditional knockouts will be needed to test

this hypothesis. At day 14 the only defect so far

reported in these mice involves the cardiovascular and

hematopoietic system (Mucenski et al. 1991; Bender

et al. 2004) although a potential defect in skin develop-ment has been suggested in myb null embryos

(Mucenski et al. 1991). Interestingly, the addition of

ectopic c-Myb to tissues did not lead to a significant

effect on mouse development. However, the overex-

pression of c-Myb in the thymus caused degenerative

abnormalities in skeletal and cardiac muscles accom-

panied by vacuolar degeneration of muscle fibers

(Furuta et al. 1993), supporting the idea that c-Mybmay have multiple roles in skeletal and muscular sys-

tems during postnatal stages.

In conclusion this study demonstrates the dynamic

expression pattern of c-Myb during prenatal odonto-

genesis and its correlation with proliferation in the

mouse and minipig. The expression patterns open up

several questions related to the role of c-Myb in hard

tissue development and differentiation, as well asthe functional relevance of the asymmetrical expres-

sion of c-Myb in our diphyodont species. We hope

to be able to address these questions in further

investigations.

ª 2011 The Authors


Acknowledgments

Mouse work was supported by GAAV (grantKJB500450802) and GACR (grant P304/11/1418),

minipig work by GACR (grant 304 ⁄ 08 ⁄ P289) and the

international cooperation by the Royal Society (grant

JP080875) and GACR (524/08/J032). The IAPG CAS

v.v.i. labs run under IRP IPAG No. AVOZ 5045015,

the FS MU under the Ministry of Education

(MSM0021622415).

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Supporting Information

Additional Supporting information may be found in the

online version of this article:

Fig. S1. Sequence of mouse c-Myb mRNA with

labeled primers and probe.

Fig. S2. Alignment of mouse c-Myb, A-Myb and

B-Myb with labeled sequence for antibody binding.

Fig. S3. c-Myb antibody and probe used for detection.

Please note: Wiley-Blackwell are not responsible for

the content or functionality of any supporting materials

supplied by the authors. Any queries (other than miss-

ing material) should be directed to the corresponding

author for the article.

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expression and characterization of c-myb in prenatal odontogenesis

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