postnatal histomorphogenesis of the mandible in the house mouse

Postnatal histomorphogenesis of the mandible in thehouse mouseCayetana Martinez-Maza,1,2,3 Laetitia Montes,1,2 Hayat Lamrous,1,2 Jacint Ventura4 andJorge Cubo1,2

1UPMC Univ Paris 06, UMR 7193, ISTEP, Paris, France2CNRS, UMR 7193, ISTEP, Paris, France3Department of Paleobiology, Museo Nacional de Ciencias Naturales – CSIC, Madrid, Spain4Departament de Biologia Animal, de Biologia Vegetal i d’Ecologia, Facultat de Biociencies, Universitat Autonoma de

Barcelona, Barcelona, Spain

Abstract

The mandible of the house mouse, Mus musculus, is a model structure for the study of the development and

evolution of complex morphological systems. This research describes the histomorphogenesis of the house

mouse mandible and analyses its biological significance from the first to the eighth postnatal weeks. Histologi-

cal data allowed us to test a hypothesis concerning modularity in this structure. We measured the bone growth

rates by fluorescent labelling and identified the bone tissue types through microscopic analysis of histological

cross-sections of the mandible during its postnatal development. The results provide evidence for a modular

structure of the mouse mandible, as the alveolar region and the ascending ramus show histological differences

throughout ontogeny. The alveolar region increases in length during the first two postnatal weeks by bone

growth in the posterior region, while horizontally positioned incisors preclude bone growth in the anterior

region. In the fourth postnatal week, growth dynamics shows a critical change. The alveolar region drifts later-

ally and the ramus becomes more vertical due to the medial growth direction of the coronoid region and the

lateral growth of the ventral region of the ramus. Diet changes after weaning are probably involved in these

morphological changes. In this way, the development of the masticatory muscles that insert on the ascending

ramus may be particularly related to this shape modeling of the house mouse mandible.

Key words: bone histology; development; mandible; modularity; morphology; Mus musculus; postnatal growth.

Introduction

The house mouse, Mus musculus, represents a well estab-

lished model in mammalian biological research. In particu-

lar, the mandible has been the subject of genetic,

embryological, and functional anatomical studies providing

an extensive background on mechanisms explaining pheno-

typic variation (Atchley & Hall, 1991). Many works have

used the house mouse mandible as a model system to study

the development and evolution of complex morphological

structures (Atchley & Hall, 1991; Atchley, 1993; Klingenberg

& Leamy, 2001; Klingenberg, 2002; Klingenberg et al. 2004;

Monteiro et al. 2005). As well, functional approaches have

shown the influence of the masticatory and paramasticato-

ry activities on the morphology of the mammalian mandi-

ble (Kesner, 1980; Satoh, 1997; Ravosa et al. 2007; Enomoto

et al. 2010; Renaud et al. 2010; Ventura & Casado-Cruz,

2011). However, the histomorphogenesis and the bone

growth dynamics associated with the morphology of the

mouse mandible have never been analyzed.

The mandible originates from neural crest cells that

migrate to form mesenchymal condensations that differen-

tiate into the six major morphogenetic units (the ramus, the

molar and incisor alveolar components, and the coronoid,

condylar and angular processes) and Meckel’s cartilage

(Atchley & Hall, 1991; Ramaesh & Bard, 2003). The early

alveolar region forms by intramembranous ossification

surrounding Meckel’s cartilage and the processes are

formed by secondary cartilages located at the proximal end

that are replaced later on by bone through endochondral

Correspondence

Cayetana Martinez-Maza, Department of Paleobiology, Museo

Nacional de Ciencias Naturales – CSIC, Jose Gutierrez Abascal 2,

28006 Madrid, Spain. T: + 34 91 5668981; E: martinezmaza.

[email protected] and [email protected]

Jorge Cubo, Universite Pierre et Marie Curie, Equipe Biomineralisa-

tions et Environnements Sedimentaires – UMR7193. 4, Pl. Jussieu BC

19, 75005 Paris, France. T: + 33 14 4273124; E: jorge.cubo_garcia@

upmc.fr

Accepted for publication 1 February 2012

Article published online 28 February 2012

ªª 2012 The AuthorsJournal of Anatomy ªª 2012 Anatomical Society

J. Anat. (2012) 220, pp472–483 doi: 10.1111/j.1469-7580.2012.01488.x

Journal of Anatomy

ossification (Hall, 2003; Ramaesh & Bard, 2003). Later in the

intrauterine development, the mandible grows and two pri-

mary functional units or modules are identified: the alveo-

lar region, which is the anterior part bearing the teeth, and

the ascending ramus, which articulates with the cranium

and provides surfaces for muscle attachment (Atchley &

Hall, 1991). Geometric morphometric analyses have sup-

ported the division of the mandible into these two develop-

mental modules ‘that are separate from each other but

they are not completely independent’ (Klingenberg et al.

2003; see also Zelditch et al. 2008; Klingenberg, 2009).

After birth, the mouse mandible undergoes morphologi-

cal changes involving size, shape, and its relative position

into the craniofacial system to fulfill the functional require-

ments (Moss, 1960; Enlow & Hans, 1996). These morphologi-

cal changes are often mediated by the action of soft tissues

(e.g. muscular insertions) and the external (e.g. biomechani-

cal) and internal stimuli (e.g. hormonal factors; Moss, 1960;

Enlow & Hans, 1996). Bone growth occurs through the

modeling mechanism involving the coordinated activity of

osteoblasts (bone-producing cells) and osteoclasts (bone-

removing cells) (e.g. Bloom & Fawcett, 1994; Enlow & Hans,

1996). Osteoblasts or bone-forming cells secrete an organic

matrix mainly composed by collagen fibers and then bio-

mineralize it with calcium phosphate. After mineralization

of the matrix, osteoblasts lose their ability to undergo mito-

sis and become osteocytes, which communicate with each

other through a network of canaliculi. Osteoclasts are mul-

tinucleated cells that remove the bone mineral and degrade

the organic bone matrix. These cellular activities are

recorded in the bone tissue as structural features containing

information about morphogenetic changes (bone tissue

types reflecting bone growth rates; Amprino, 1947). There-

fore, histological organization of bone tissue provides infor-

mation about the growth directions and rates of the

functional regions and developmental modules underlying

the genesis of the mandibular shape. Previous works have

approached the analysis of the postnatal development from

a histological perspective in the mandible of the mink

(Buffrenil & Pascal, 1984), rabbit (Bang & Enlow, 1967), and

several species of primates (Enlow, 1963; Enlow & Hans,

1996). These studies have provided information on both the

bone tissue structure and the bone growth rates of differ-

ent mandible regions (Buffrenil & Pascal, 1984) as well as

the bone growth directions (Bang & Enlow, 1967; Enlow &

Hans, 1996). Histological data obtained for each species

have pointed out the particular growth dynamics explain-

ing the morphological changes of the mandible during the

postnatal development.

The aim of this study is to characterize the histomorpho-

genesis of the house mouse mandible from birth to the

eighth postnatal week analyzing the bone histology, the

growth directions and the bone growth rates. These results

are discussed in the light of similar studies to infer specific

bone growth dynamics and to establish a bone growth

model for the normal postnatal development of the

M. musculus mandible. In addition, we will use these histo-

logical data to test the division of the mouse mandible into

the alveolar and the ascending ramus modules as hypothe-

sized in prior studies (e.g. Atchley & Hall, 1991; Klingenberg

et al. 2003). According to this hypothesis and considering

the developmental and functional differences proposed for

these two modules (Atchley & Hall, 1991), we predict that

both modules will differ in their histological organization

of bone tissue and bone growth rates. Understanding how

these modules grow and interrelate during development

will provide useful information about the biological pro-

cesses underlying the development and evolution of mor-

phological structures in the house mouse mandible.

Materials and methods

Thirty-eight mice of the strain C57BL ⁄ 6J (Janvier Inc., France)

from the first to eight postnatal weeks were used in this study.

Mice were housed in standard cages in an animal room under

controlled environmental conditions and provided with food

and water ad libitum. Animals were handled in accordance with

the European Communities Council Directive (86 ⁄ 609 ⁄ EEC). In

this study, only female mice were employed. According to most

literature, no sexual dimorphism is observed in mice, although

Renaud et al. (2010) have suggested that strain C57BL ⁄ 10 pre-

sents some degree of sexual dimorphism that may affect model-

ing processes. However, a detailed analysis of strain C57BL ⁄ 6J

has yielded inconclusive results (Boell et al. 2011). Thus, we

assume that sexual dimorphism will be very limited, particularly

in young animals.

To study the periosteal growth rates, mice received an intra-

peritoneal injection of dicarboxymethyl aminomethyl fluores-

cein (DCAF) 25 mg kg)1 of body weight. These fluorescent dyes

specifically color the mineralizing zone of growing bone tissue.

A week after the intraperitoneal injection of DCAF, mice were

sacrificed by cervical dislocation. Mandibles were dissected and

cleaned by hand. They were dehydrated in graded ethanol, de-

fatted in acetone and trichloroethylene and finally dried at 38–

40 �C in a stove. Right mandibles were embedded in a polyester

resin. Histological cross-sections 100 ± 10 lm thick were made

using a diamond-tipped circular saw in four representative man-

dibular regions: diastema, first molar, second molar, and ascend-

ing ramus at the level of the coronoid process. Each thin section

was ground and polished before being mounted on a slide.

They were observed under ultraviolet and natural light micro-

scope (Zeiss Axiovert 35; Jena, Germany) and digitalized

through a camera (Olympus, Japan). To determine the perio-

steal growth rates (lm day)1), pictures taken under ultraviolet

light were analyzed through the image processing program IMA-

GEJ. The bone growth rate was obtained using the distance

between the DCAF-label (fluorescein) and the bone surface

divided by the time elapsed (7 days). Figure 1 shows the points

in the histological cross-sections used to calculate this rate.

Results

Histological cross-sections (diastema, first molar, second

molar, and ascending ramus) from the first to the eighth


Postnatal growth of the mouse mandible, C. Martinez-Maza et al. 473

postnatal week are shown in Figs 2–5. To provide a detailed

description, we first describe the bone histology of the four

mandibular regions and then analyze the bone growth

dynamics (growth directions and rates). Finally, we use the

spatial and temporal distribution of bone tissue types and

modeling activities in the mandible to test the modular

structure proposed in previous studies (Atchley & Hall, 1991;

Cheverud et al. 1997; Mezey et al. 2000; Klingenberg et al.

2003).

Histological description

Diastema region

In the first and second postnatal weeks, this mandibular

region showed woven bone tissue (Fig. 6A) with low

ordered spatial arrangement of the collagen fiber bundles,

randomly distributed rounded osteocytic lacunae, and high

vascularity (Fig. 2: diastema, weeks 1 and 2; woven bone:

label w). The third week was characterized by a histological

change consistent with a more parallel-fibered arrange-

ment of collagen fibers including rounded osteocytic lacu-

nae in the dorsal half of the labial side (Fig. 2: diastema

week 3; parallel-fibered bone: label pf) and with flattened

osteocytic lacunae in an aligned distribution in the lingual

side (Fig. 2: diastema week 3, label pf). The diastema

showed woven bone tissue with high vascularity and ran-

domly distributed osteocytic lacunae (Fig. 2: diastema, week

3, label w) dorsally and in the ventral half of the labial side.

In the fourth week, the dorsal region and ventral half of

the labial side showed woven bone tissue (Fig. 2: diastema,

week 4, label w), while the dorsal half of the labial side dis-

played parallel-fibered bone tissue (Fig. 2: diastema, week

4, label pf; Fig. 6b). The lingual side displayed an internal

region (endosteal) of woven bone tissue (Fig. 2: diastema,

week 4, label w), whereas the outer region (periosteal)

showed parallel-fibered bone tissue with flattened osteocy-

tic lacunae in an ordered disposition (Fig. 2: diastema, week

4, label pf). In addition, the bone histology structure and

the DCAF label of the vascular cavity located in the dorsal

internal ridge suggest a centripetal bone growth associated

with the formation of the primary osteon (Fig. 2: diastema,

week 4; primary osteon: label po). The diastema region in

the fifth postnatal week showed an increase of the woven

bone tissue in the lingual and the labial sides (Fig. 2: dia-

stema week 5, w), whereas the parallel-fibered bone tissue

was located in the dorsal area of this region (Fig. 2: dia-

stema week 5, pf). The vascular cavity of the dorsal internal

ridge showed concentric bone lamellae related to the cen-

tripetal bone growth that could be involved in the forma-

tion of a primary osteon (Fig. 2: diastema, week 5, po). In

the sixth and seventh postnatal weeks, the diastema region

showed parallel-fibered bone tissue with flattened osteocy-

tic lacunae in the outer region (periosteal) of the labial and

lingual sides (Fig. 2: diastema, week 6 and diastema, week

7, pf), whereas woven bone tissue was observed in the

internal region (endosteal) of labial sides (Fig. 2: diastema,

week 6 and 7, label w) and in the ventral region (Fig. 2: dia-

stema, week 7, label w). The distribution of the bone tissues

and the concentric lamellae observed around some vascular

cavities or primary osteons (Fig. 2A: diastema, week 6 and

7, po) suggest a decrease of the bone growth in this postna-

tal week. In the eighth postnatal week, the labial and lin-

gual sides of the diastema region showed parallel-fibered

bone tissue with flattened osteocytic lacunae (Fig. 2: D8,

Fig. 1 Histological cross-sections from four regions of the Mus musculus mandible (from left to right): diastema, first molar region, second molar

region, and ascending ramus. Pictures taken under ultraviolet microscope show the green label (DCAF). Numbers on the DCAF line denote the

points used to measure the bone growth rates in this study. Scale bar: 1 mm.


Postnatal growth of the mouse mandible, C. Martinez-Maza et al.474

label pf), whereas the ventral and dorsal regions displayed

woven bone tissue with rounded osteocytic lacunae

randomly distributed (Fig. 2: diastema, week 8, label w).

The first and second molar regions

Both regions showed, in the first and second postnatal

weeks, woven bone tissue with high vascularity (Fig. 2: first

molar, weeks 1 and 2 and Fig. 3: second molar, weeks 1

and 2, label w), which is related to high osteogenesis. In

the third week, mandibles showed a histological change in

the first molar region but not in the second one. The first

molar region displayed woven bone tissue in the ventral

half of the labial side (Fig. 2: first molar, week 3, label w),

whereas the dorsal half of this side and the lingual side

Fig. 2 Histological cross-sections under natural light of the diastema region (bottom) and the first molar region (top) obtained in the Mus

musculus mandible from the first (right) to the eighth postnatal week (left). Labels indicate the bone tissue type observed in different areas of

each mandibular region, w: woven bone tissue; pf: parallel-fibered bone tissue; po: primary osteon. Scale bar: 1 mm.

Fig. 3 Histological cross-sections under natural light of the second molar region (bottom) and the ascending ramus region (top) obtained in the

Mus musculus mandible from the first (right) to the eighth postnatal week (left). Labels indicate the bone tissue type observed in different areas of

each mandibular region, w: woven bone tissue; pf: parallel-fibered bone tissue; po: primary osteon. Scale bar: 1 mm.



displayed parallel-fibered bone tissue with rounded osteo-

cytic lacunae in an ordered disposition (Fig. 2: first molar,

week 3, label pf). However, the second molar region was

characterized by woven bone tissue with a random distri-

bution of the osteocytic lacunae (Fig. 3: second molar,

week 3, label w). In the fourth and the fifth weeks, the

region of the first and second molars was characterized by

an increase of parallel-fibered bone tissue with rounded

osteocytic lacunae in an ordered disposition in a small area

of the dorsal half of the labial side and in the lingual side

(Fig. 2: first molar, week 4 and Fig. 3: second molar, week

4, pf), whereas the labial side displayed woven bone tissue

(Fig. 2: first molar, week 4 and Fig. 3: second molar week

4, w). In addition, both labial and lingual sides showed

concentric lamellae in some vascular cavities related to the

formation of primary osteons (Figs 2 and 3, po). The bone

histology of the molar region suggests a high bone growth

rate in the ventral half and slow bone growth in the lin-

gual and the dorsal half of the labial side. In the sixth and

seventh postnatal weeks, the first and second molar

regions showed parallel-fibered bone tissue with flattened

osteocytic lacunae in an ordered disposition on the lingual

side, the ventral and the outer regions (periosteal) of the

dorsal half of the labial side (Fig. 2: first molar, weeks 6

and 7, Fig. 3: second molar, weeks 6 and 7, pf). Woven

bone tissue was identified in two areas of the labial side,

the internal area (endosteal) of the dorsal half, around the

molar root, and close to the mandibular crest in the ven-

tral half (Fig. 2: first molar, weeks 6 and 7, Fig. 3: second

molar, weeks 6 and 7, label w). In the eighth postnatal

week, the molar region showed parallel-fibered bone tis-

sue with flattened osteocytic lacunae on the lingual side,

the ventral region, and in the dorsal half of the labial side

(Fig. 2: first molar, week 8, pf). The ventral half of the

labial side displayed woven bone tissue with randomly dis-

tributed rounded osteocytic lacunae (Fig. 2: first molar,

week 8, w). Also, the second molar region was character-

ized by parallel-fibered bone tissue with flattened osteocy-

tic lacunae in an ordered disposition (Fig. 3: second molar,

week 8, pf) and primary osteons close to the mandibular

crest and in the dorsal area of the lingual side (Fig. 3:

second molar, week 8, label po).

Ascending ramus region

From the first to the fifth postnatal week, the ascending

ramus was characterized by woven bone tissue with

rounded osteocytic lacunae indicating fast bone formation

(Fig. 3: ramus, weeks 1–5, label w). In the third and fifth

weeks, centripetal bone growth was observed in the vascu-

lar cavities, associated with the formation of a primary

osteon (Fig. 3: ramus, weeks 3 and 5, label po). In the sixth

week, the ascending ramus was characterized by woven

bone tissue, except in the labial side of the area surround-

ing the mandibular foramen, which showed parallel-

fibered bone tissue with flattened osteocytic lacunae

(Fig. 3: ramus, week 6, pf). There was a histological change

in the seventh week; the ascending ramus displayed paral-

lel-fibered bone tissue with flattened osteocytic lacunae on

the labial and lingual sides of the area surrounding the

mandibular foramen and in the labial side of the ventral

half of the ramus (Fig. 3: ramus, week 7, label pf), whereas

the coronoid and the lingual side of the ventral half of the

ramus displayed woven bone tissue (Fig. 3: ramus, week 7,

label w). The ascending ramus in the eighth week showed

parallel-fibered bone tissue with flattened osteocytic

lacunae on the labial and lingual side of the area

Fig. 4 Histological cross-sections under ultraviolet light of the diastema region (bottom) and the first molar region (top) obtained in the Mus

musculus mandible, from the first (right) to the eighth postnatal week (left). Labels indicate the bone modeling activity observed in different areas

of each mandibular region, bf: bone formation activity; BR: bone resorption activity. Scale bar: 1mm.



surrounding mandibular foramen and on the labial side of

the ventral half of the ramus (Fig. 3: ramus, week 8, pf),

whereas other areas were characterized by a transition

between woven bone and parallel-fibered bone tissue with

randomly distributed, rounded osteocytic lacunae.

Bone growth dynamics

Fluorescence-DCAF labeling provided information about

bone growth directions and rates of different mandibular

regions during postnatal development. On the one hand,

presence or absence of the DCAF-label was associated with

bone formation or resorption activities, respectively. As the

lack of fluorescence label could result from bone resorption

activity in the periosteal or endosteal surface, we analyzed

the profile of the bone surface to identify the region (perio-

steal or endosteal surface) where bone is resorbed (Enlow &

Hans, 1996). Bone growth rates were measured at different

points of each mandibular region (Fig. 1) to determine the

growth changes during development.

Diastema region

The histological data and the DCAF label observed in the

first 3 weeks showed periosteal bone formation surfaces

that indicated a general growth in all directions. The bone

resorption activity in the endosteal surface of the ventral

and lingual sides (Fig. 4: diastema, weeks 1–3, label br)

reveals bone growth in a ventral direction. From the fourth

to the eighth week, the labial side and the ventral area

were characterized by bone formation, but the lingual side

showed a field of bone resorption of variable size (Fig. 4:

diastema, weeks 4–8, label br). This bone modeling field dis-

tribution was associated with a lateral growth of the dia-

stema.

The bone growth rate data showed a high rate of osteo-

genesis during the first three postnatal weeks, followed

by a marked decrease of the rates in the fourth week and,

from the fifth week on, growth slowed gradually until the

eighth postnatal week. A thorough analysis of the bone

growth rates throughout development allows us to distin-

guish three areas in the diastema. The first area, which

includes the dorsal region and the dorsal half of the labial

side (from point 1 to 5, Fig. 1), was characterized by low

growth rate (4–5 lm day)1) in the first and second weeks, a

slight increase in the third and fourth weeks, mainly occur-

ring on the labial side (from point 4 to 5), and a gradual

decrease of the bone growth in the last weeks

(< 1 lm day)1). The second (the ventral half of the labial

side from point 6 to 11, labial side), showed a high bone

growth rate during the first four postnatal weeks and then

a gradual decrease of the rate with age (< 2 lm day)1). Part

of the ventral border of the diastema region (from point 9

to 11) displayed a high growth rate (> 20 lm day)1) during

the first 3 weeks. Growth of this area decreased abruptly in

the fourth week (� 9 lm day)1) and subsequently showed

a gradual decrease, with growth rates similar to those of

other points of this region. The third area corresponds to

the lingual side (from point 12 to 18) and is characterized

by lower ventral bone growth rates (< 3 lm day)1) than the

growth registered on the labial side during the first 3 weeks

of development. From the fourth week to the eighth, this

lingual side showed bone resorption.

First and second molar regions

These mandibular regions showed similar growth directions

throughout postnatal development. The DCAF label indi-

cated periosteal bone formation surfaces (see label bf in

Fig. 4: first molar and Fig. 5: second molar) but in the first

3 weeks, the lack of the DCAF label associated with bone

resorption activity in the ventro-lingual area of the endos-

teal surface indicated a dorso-ventral growth (Fig. 4: first

molar, weeks 1–3 and Fig. 5: second molar, weeks 1–3, label

br). From the fourth to the sixth weeks, the molar regions

showed bone resorption on the lingual side of their alveoli.

During the seventh and eighth weeks, mandibles showed

bone formation surfaces in both molar regions but bone

resorption activity occurred in the lingual side, as revealed

by the lack of the DCAF label and the histological data (see

label br in Fig. 4: first molar and Fig. 5: second molar, weeks

7–8).

The analysis of bone growth rates revealed differences

between the first and second molar regions. The first molar

region showed an increase of bone growth rates from the

first to the third postnatal weeks but subsequently the

growth rates decrease gradually until the eighth week. In

this mandibular region, four areas characterized by a partic-

ular variation of the bone growth rates during develop-

ment were identified. The first area corresponds with the

dorsal half of the labial side (from point 1 to 5) and showed

a gradual decrease of the bone growth (from 4–5 to

< 1 lm day)1). The second area (the mandibular crest of

the labial side from point 6 to 8) showed high growth rates

during the first 2 weeks (20–16 lm day)1), a decrease in

growth rates in the fourth week (6 lm day)1) and a marked

new decrease in the sixth week (2 lm day)1). The third area

included the ventral border (from point 9 to 11) and

showed high bone growth rates during the first two post-

natal weeks and in the fifth week (11–15 lm day)1). Both

in the third and fourth weeks and from the sixth postnatal

week onwards, the bone growth rates decreased in this

mandibular region (< 4 lm day)1). The fourth area was

associated with the lingual side (from point 12 to 17) and

showed lower ventral growth rates than in the labial side.

This side of the first molar displayed a gradual decrease in

bone growth rates during development (< 2 lm day)1).

Only the area associated with the incisor alveoli (points 12

and 13) showed a high growth rate (4–6 lm day)1) from

the first to the fifth week and then abruptly decreased

growth rates (� 1 lm day)1) until the eighth postnatal

week.



In the second molar region, three areas with similar varia-

tions in bone growth rate during development were distin-

guished. The first area corresponds with the labial side

(from point 1 to 5) and showed low growth rates in the first

2 weeks (3–4 lm day)1), except in the area of the mandibu-

lar crest (points 4 and 5), which showed high growth rates

(� 16 lm day)1). In this region, all individuals showed a

constant rate (2–3 lm day)1) from the third to the eighth

weeks. The second area, which covers the area from the

labial crest to the ventral border (points 6–10), showed the

highest bone growth rates (10–30 lm day)1) during the first

three postnatal weeks; growth rates gradually decreased

from the fourth to the eighth weeks (� 5 lm day)1). The

third area (points 11–14) was associated with the lingual

side and was characterized by low growth rates

(� 3 lm day)1) that gradually decreased during the postna-

tal development (1 lm day)1).

Ascending ramus region

This region was characterized by the lack of the DCAF label

due to the periosteal and endosteal resorption activity. The

fluorescence label and the histomorphometric data indicate

complex growth dynamics in this region (Fig. 5: ramus

weeks, 1 and 2, label bf). The first and the second weeks

showed the DCAF label only in small areas of the labial side

of the coronoid tip and in the ventral area of the ramus,

which is associated with endosteal bone formation. The lack

of fluorescence was due to high endosteal resorption activ-

ity and suggests growth in all directions by periosteal bone

formation. In the third week, the ramus was entirely deposi-

tory, revealing general growth, but in the labial side of the

coronoid region there was a small area of bone resorption

activity (Fig. 5: ramus week 3, label br). From the fourth to

the eighth postnatal weeks, there was a lack of the DCAF-

label in the labial side of the coronoid tip and in the lingual

side of the ventral half of the ramus (Fig. 5: ramus, weeks

from 4 to 8, label br).

The analysis of bone growth rates was limited by the lack

of the DCAF label. Nevertheless, histomorphometric data

provided information indicating ranges of osteogenesis. In

the first and second postnatal weeks, the ascending ramus

showed regions without fluorescence label that revealed

high bone growth rates with endosteal resorption. These

data were associated with the increase in size of the ramus.

From the third postnatal week on, the ramus showed a

change in bone growth dynamics. In the third week, the

ramus showed bone formation surfaces except on the labial

side of the coronoid (points 1–3), as well as bone resorption

activity. Bone growth rates indicated a generally high

osteogenesis (3–8 lm day)1), particularly on the lingual

side, close to the mandibular foramen (points 15), which

showed the highest rate (> 10 lm day)1) in that week.

From the fourth to the eighth postnatal weeks, bone mod-

eling fields allowed us to distinguish two areas character-

Fig. 5 Cross-sections under ultraviolet light of the second molar region (bottom) and the mandibular ramus region (top) obtained in the Mus

musculus mandible from the first (right) to the eighth postnatal week (left). Labels indicate the bone modeling activity observed in different areas

of each mandibular region, bf: bone formation activity; BR: bone resorption activity. Scale bar: 1 mm.



ized by bone resorption fields: the labial side of the coro-

noid (points 1–3) and the lingual side of the ventral area of

the ramus (points 12–14). Likewise, two zones showed bone

formation surfaces: the lingual side of the coronoid (points

18–20) and the labial side of the ventral half of the ramus

(points 6–9). During the last four postnatal weeks, bone

growth rates revealed a general growth with a slight

decrease in rates in the areas with bone formation surfaces,

the lingual side of the coronoid, the mandibular foramen

area (labial and lingual sides), and the labial side of the ven-

tral-half of the ramus.

Modularity in the mouse mandible

The histological data provide new evidence to test the

hypothesis about modularity in the mouse mandible pro-

posed in previous works (e.g. Atchley & Hall, 1991; Klingen-

berg et al. 2003). As illustrated in Table 1 and Fig. 7A,

histological features observed in the diastema, the first and

second molar regions (from here on, we consider these

three regions the alveolar region) are similar to each other

but different from the ascending ramus at different times

of postnatal ontogeny. Although both modules (alveolar

region and ascending ramus) are characterized by woven

bone in the first 2 weeks after birth, the alveolar region

seems to mature earlier and presents fibrolamellar bone,

whereas the ascending ramus retains the woven bone until

the seventh week. Parallel ontogenetic changes are

observed in the modeling patterns (Fig. 7B). Both modules

show bone deposition in their external surfaces during the

first 3 weeks. Later on, the alveolar region shows a consis-

tent pattern characterized by resorption on the lingual side.

On the contrary, the ramus shows a complex pattern char-

acterized by the occurrence of resorption in the labial side

of the coronoid and the lingual side of the ventral area.

Together, bone histological data agree with the existence

of two natural modules showing different developmental

patterns. Differences between both modules become

apparent in the third postnatal week.

Discussion

The mouse mandible is a model structure for the study of

the development and evolution of complex morphological

systems (Atchley & Hall, 1991). In this study, our results on

the microstructure of bone tissue and the bone growth

rates allowed us to determine the histomorphogenesis of

the M. musculus mandible during postnatal development.

In addition, our histological data suggest that the house

mouse mandible shows a modular structure consisting of

the alveolar region and the ascending ramus. The growth

pattern obtained provides clues to the dynamics and the

biological significance of the histological changes that occur

after birth in the house mouse mandible. Patterns of bone

growth in the house mouse mandible were compared with

available data obtained from the growth models reported

for other species, such as the rabbit Oryctolagus cuniculus

(Bang & Enlow, 1967), the American mink Mustela vison

(Buffrenil & Pascal, 1984), the rhesus monkey Macaca mulat-

ta, and modern human Homo sapiens (e.g. Kurihara et al.

1980; Enlow & Hans, 1996). Of these studies, only Buffrenil

& Pascal (1984) and Kurihara et al. (1980) documented the

histological changes during the postnatal growth in

Mustela vison and Homo sapiens mandibles, respectively.

In the first two postnatal weeks, the M. musculus mandi-

ble (both the alveolar region and the ascending ramus)

grows in all directions, increasing its size, as suggested by

the presence of woven bone tissue and confirmed by mea-

sured growth rates. Furthermore, bone growth rate data

from labial and lingual sides of the diastema and molar

regions, and the resorption activity on the endosteal surface

of their ventral area indicate a main lateral and dorsoven-

tral growth while the mandible increases its size. In the

ascending ramus, the highest growth rates registered reveal

a dorsoventral and lateral growth. A similar dynamics has

A

B

Fig. 6 Bone tissue types observed in the Mus musculus mandible. (A)

Woven bone tissue (first postnatal week, buccal side of the first molar

section), and (B) parallel-fibered bone tissue (eighth postnatal week,

buccal side of the first molar section). Scale bar: 50 lm.



been also described in minks at the age of 2 months and in

humans in the first postnatal year, both characterized by

bone formation surfaces (Buffrenil & Pascal, 1984; Enlow &

Hans, 1996). The inferred bone growth dynamics in mice

are in agreement with Buffrenil & Pascal’s (1984) interpreta-

tion of the increase in height of the alveolar region of the

mink, mainly in a ventral direction. In addition, and consid-

ering the interpretations of the growth in length in other

mammals (Bang & Enlow, 1967; Buffrenil & Pascal, 1984; En-

low & Hans, 1996), we hypothesize that the increase in

length of the mouse mandible occurs by bone formation in

the posterior region of the alveolar region, while it grows

mainly laterally and the ramus is relocated in a posterior

position. Nevertheless, Buffrenil & Pascal (1984) suggested

that the increase in length of the mink alveolar region was

associated with bone growth in both posterior and anterior

regions. This anterior and posterior growth is similar to that

observed in M. mulatta and H. sapiens (Enlow & Hans,

1996) but differ from the growth in length of the mouse

and rabbit mandibles, which show only posterior bone

growth (Bang & Enlow, 1967). Differences among these spe-

cies are related to the position of the incisors with respect

to the alveolar region. Mink and primate mandibles show a

vertical position of the incisors, whereas in the rabbit and

mouse mandible, the incisors show a horizontal position,

preventing bone growth in the anterior region of the man-

dible (Buffrenil & Pascal, 1984). Therefore, the posterior

growth of the alveolar region proposed in this study for the

mouse mandible may be associated with the horizontal

position of the incisor.

In the third postnatal week, the mouse mandible shows

growth dynamics similar to those in previous weeks,

although differences in the histological organization and in

bone growth rates were observed between the alveolar

region and the ascending ramus. In the alveolar module,

parallel-fibered bone tissue is related to low osteogenesis,

whereas bone resorption fields identified in the diastema

and the first molar regions suggest an increase in height of

the alveolar region in a mainly ventral direction. The

ascending ramus is characterized by woven bone tissue and

depository surfaces, indicating a general growth in all direc-

tions. Interestingly, the second molar region shows similari-

ties with the ascending ramus (both characterized by

woven bone). This result supports the statement of Klingen-

berg et al. (2003) arguing that ‘modularity in the mandible

appears to be a question of degrees’. Differential labial ⁄lingual growth rates indicate a lateral growth of the alveo-

lar region and ramus. Growth dynamics inferred for the

alveolar region in M. musculus are in agreement with the

increase in height of the alveolar region and the lateral

(labial) drift of the mandible suggested in other species pre-

viously analyzed (Bang & Enlow, 1967; Buffrenil & Pascal,

1984; Kurihara et al. 1980; Enlow & Hans, 1996).

In the fourth postnatal week, the growth dynamics of

the mouse mandible shows a noticeable change compared

with previous weeks. The particular bone modeling field

distribution observed in the fourth week in the alveolar

region and in the ascending ramus was also observed in the

next postnatal weeks, suggesting similar growth directions.

From the fourth to eighth postnatal weeks, the bone mod-

eling data indicate a lateral drift of the alveolar region,

whereas the ascending ramus has a vertical arrangement

due to medial growth of the coronoid region and lateral

growth of the ventral part of the ramus. Although these

growth dynamics are similar from the fourth to the eighth

postnatal weeks, bone tissue type distribution and bone

growth rates show slight differences that suggest similar

growth dynamics but different intensity of bone growth.

On the one hand, the alveolar region module shows a slight

growth in the ventral half of the labial side and in the

ventral area which is simultaneous with the lateral drift.

Such dynamics indicate a main dorsoventral direction of

Table 1 Distribution of bone tissue types observed in the four regions of the Mus musculus mandible in each postnatal week.

Region area

Age

Diastema First molar Second molar Ascending ramus

dor lab ven lin lab ven lin lab ven lin C lab C lin V lab V lin

Week 1 w w w w w w w w w w w w w w

Week 2 w w w w w w w w w w w w w w

Week 3 w pf ⁄ w w pf pf ⁄ w w pf w w w w w w w

Week 4 w pf ⁄ w w pf ⁄ w pf ⁄ w w pf pf ⁄ w w pf w w w w

Week 5 pf w w w pf ⁄ w w pf pf ⁄ w w pf w w w w

Week 6 w pf ⁄ w w pf pf ⁄ w w pf pf ⁄ w w pf w w w w ⁄ pf

Week 7 w pf ⁄ w w pf pf ⁄ w w pf pf ⁄ w w pf w w w ⁄ pf w ⁄ pf

Week 8 w pf w pf pf ⁄ w pf pf pf ⁄ w pf pf w ⁄ pf w ⁄ pf pf w ⁄ pf

w, woven bone tissue; pf, parallel-fibered bone tissue; pf ⁄ w, woven and parallel-fibered bone tissues observed in the same region;

dor, dorsal; lab, labial; ven, ventral; lin, lingual; C lab, labial side of the coronoid area; C lin, lingual side of the coronoid area; Ven

lab, labial side of the ventral half; V lin, lingual side of the ventral half.



growth increasing the height of the alveolar region and a

lateral growth increasing the width of the ventral part of

the alveolar region. This is in agreement with the results

obtained for minks by Buffrenil & Pascal (1984) that sug-

gested (from the resorption of the alveolar area) an

increase in height of the mandible, particularly in the

molar region. The growth rate and the gradual variation

from woven bone (high osteogenesis) to fibrolamellar

bone tissue (low osteogenesis) indicate lateral and dorso-

ventral growths that gradually decrease from the fourth

to the sixth postnatal week. The growth model obtained

in this work for the mouse alveolar region is highly similar

to that reported for the rabbit (Bang & Enlow, 1967).

These growth dynamics suggest an increase in size of the

alveolar region in proportion to the overall size of the

entire growing mandible, whereas the lingual resorption

of the diastema region may indicate a downward develop-

ment of the genial tuberosity area (Bang & Enlow, 1967).

These similarities may be related to the presence of the

diastema in both species.

On the other hand, the ascending ramus shows specific

growth dynamics in the mouse mandible different from

those observed in the alveolar region. These bone growth

dynamics indicate a vertical arrangement of this region that

may be related to the proximity of the condyles to maintain

the contact with the cranial base through the temporoman-

dibular joint during development. The particular growth of

the ascending ramus is influenced by the large complex of

muscles that insert in this mandibular region (Atchley &

Hall, 1991). As reported in other rodent species, diet

changes after weaning are involved in the shape modeling

of the mandible during growth (Cardini & Tongiorgi, 2003;

Ventura & Casado-Cruz, 2011). In M. musculus the weaning

occurs around 21 days after birth and at the age of the

fourth week there is an increase of activities related to

drinking and feeding (Marques & Olson, 2007). The mandi-

ble responds to physiological loads resulting from the new

dietary requirements during the postweaning period

through the modeling mechanism. As a consequence, dif-

ferences in the bone mineral density have been reported

also in the mouse mandible (Ravosa et al. 2007). Therefore,

we hypothesize that changes here reported in the growth

dynamics in the fourth week are related to the diet change.

From the seventh to eighth postnatal weeks, the mouse

mandible shows similar growth dynamics to that in previous

weeks but bone growth rates decrease notably, and both

the alveolar region and the ascending ramus show bone tis-

sue types associated with low osteogenesis.

Considering the mandible as a whole, the growth pattern

of this structure in the house mouse shows a characteristic

growth based on the V principle established by Enlow

(1963), which has also been reported in the rabbit (Bang &

Enlow, 1967) and the macaque (Enlow & Hans, 1996).

During the postnatal development, the alveolar region is

relocated laterally and increases its length posteriorly, while

the ascending ramus is placed in a posterior and medial

position. Furthermore, histological data have shown a

change from immature bone tissue type (woven bone with

high vascularity) to mature bone tissue (fibrolamellar bone

tissue with flat osteocytic lacunae without vascularization

B

A

Fig. 7 Schematic maps show the main postnatal changes in structural organization of bone tissues (A) and in the distribution of bone modeling

fields (B) in the Mus musculus mandible. (A) A series of three mandibles from the first (left) to the eight postnatal weeks (right). On the left, the

mandible is characterized by woven bone (light gray) in the alveolar region and the ascending ramus (the first and second postnatal weeks). In the

middle, the mandible displays parallel-fibered bone tissue (dark gray) in the alveolar region and woven bone in the ascending ramus (from the

third to the sixth postnatal week). On the right, the mandible shows predominantly parallel-fibered bone tissue (the seventh and eighth postnatal

week). The broken line in the second mandible delimited the natural modules established in previous works (Atchley & Hall, 1991; Klingenberg

et al., 2003). (B) Two generalized bone modeling patterns observed throughout ontogeny. On the left, the mandible is characterized by bone

depository surfaces (light gray; from the first to the third postnatal week). On the right, mandible displays the distribution of the bone depository

and bone resorption (dark gray) fields (from the fourth to eighth postnatal week).



or with primary osteons). This histological change occurred

anteroposteriorly, that is, changes were first observed in

the diastema and later in the molar and the ascending

ramus. Our findings provide new evidence supporting the

hypothesis that the mouse mandible is divided into two

developmental modules (alveolar region and the ascending

ramus), which agrees with results obtained in previous work

(Atchley et al. 1985; Leamy, 1993; Cheverud et al. 1997;

Mezey et al. 2000; Klingenberg et al. 2003). Results

obtained here reveal differences in the timing of bone tis-

sue development and the growth dynamics between the

alveolar region and the ascending ramus modules during

the postnatal development. Histological analyses allowed

us to establish the bone growth mechanism of the mouse

mandible, providing useful information to understand the

normal histomorphogenesis of the mandible. This growth

model can be useful for future studies focused on the deter-

mination of the growth changes associated with pheno-

typic variability due to genetic or epigenetic factors.

Acknowledgements

We thank two anonymous referees for their valuable comments

and their constructive suggestions that have contributed to

improve this article. We also thank Michel Laurin (Museum

National d’Histoire Naturelle, Paris, France) for checking the

English of this manuscript. Support for this study was provided

by a grant from the Spanish government, Ministerio de Ciencia

e Innovacion (CGL2010-15243). C.M.M. is funded by the

‘National Programme of Mobility and Humans Resources from

the I-D+I 2008-2011 National Plan’ of the Spanish Ministry of Sci-

ence and Innovation (MICINN). We thank the ‘Plateforme Ani-

malerie Rongeurs’ de l’IFR 83 de l’Universite Pierre et Marie

Curie.

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