Postnatal histomorphogenesis of the mandible in the house mouse

Authors


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.cayetana@gmail.com and cayetana@mncn.csic.es
Jorge Cubo, Université Pierre et Marie Curie, Equipe Biominéralisations et Environnements Sédimentaires – UMR7193. 4, Pl. Jussieu BC 19, 75005 Paris, France. T: + 33 14 4273124; E: jorge.cubo_garcia@upmc.fr

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. Histological 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 laterally 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.

Introduction

The house mouse, Mus musculus, represents a well established model in mammalian biological research. In particular, the mandible has been the subject of genetic, embryological, and functional anatomical studies providing an extensive background on mechanisms explaining phenotypic 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 paramasticatory activities on the morphology of the mammalian mandible (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 differentiate 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 ossification (Hall, 2003; Ramaesh & Bard, 2003). Later in the intrauterine development, the mandible grows and two primary functional units or modules are identified: the alveolar 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 supported the division of the mandible into these two developmental 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 morphological changes involving size, shape, and its relative position into the craniofacial system to fulfill the functional requirements (Moss, 1960; Enlow & Hans, 1996). These morphological changes are often mediated by the action of soft tissues (e.g. muscular insertions) and the external (e.g. biomechanical) 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 biomineralize it with calcium phosphate. After mineralization of the matrix, osteoblasts lose their ability to undergo mitosis and become osteocytes, which communicate with each other through a network of canaliculi. Osteoclasts are multinucleated 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). Therefore, histological organization of bone tissue provides information 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 (Buffrénil & 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 different mandible regions (Buffrénil & 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 explaining the morphological changes of the mandible during the postnatal development.

The aim of this study is to characterize the histomorphogenesis 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 histological data to test the division of the mouse mandible into the alveolar and the ascending ramus modules as hypothesized 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 processes underlying the development and evolution of morphological 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 presents some degree of sexual dimorphism that may affect modeling 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 intraperitoneal injection of dicarboxymethyl aminomethyl fluorescein (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, defatted 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 μm thick were made using a diamond-tipped circular saw in four representative mandibular regions: diastema, first molar, second molar, and ascending 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 microscope (Zeiss Axiovert 35; Jena, Germany) and digitalized through a camera (Olympus, Japan). To determine the periosteal growth rates (μm day−1), pictures taken under ultraviolet light were analyzed through the image processing program imagej. 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.

Figure 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.

Results

Histological cross-sections (diastema, first molar, second molar, and ascending ramus) from the first to the eighth 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).

Figure 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.

Figure 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.

Figure 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.

Figure 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.

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 arrangement of collagen fibers including rounded osteocytic lacunae 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 randomly 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 displayed 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 osteocytic 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: diastema week 5, w), whereas the parallel-fibered bone tissue was located in the dorsal area of this region (Fig. 2: diastema week 5, pf). The vascular cavity of the dorsal internal ridge showed concentric bone lamellae related to the centripetal bone growth that could be involved in the formation 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 osteocytic 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: diastema, 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 postnatal week. In the eighth postnatal week, the labial and lingual sides of the diastema region showed parallel-fibered bone tissue with flattened osteocytic lacunae (Fig. 2: D8, 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).

Figure 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 μm.

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 displayed parallel-fibered bone tissue with rounded osteocytic 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 distribution 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 lingual 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 ventral 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 tissue 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 distributed rounded osteocytic lacunae (Fig. 2: first molar, week 8, w). Also, the second molar region was characterized by parallel-fibered bone tissue with flattened osteocytic 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 vascular 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 surrounding 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 parallel-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 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 (periosteal 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 distribution was associated with a lateral growth of the diastema.

The bone growth rate data showed a high rate of osteogenesis 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 distinguish 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 μm day−1) in the first and second weeks, a slight increase in the third and fourth weeks, mainly occurring on the labial side (from point 4 to 5), and a gradual decrease of the bone growth in the last weeks (< 1 μm 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 μm day−1). Part of the ventral border of the diastema region (from point 9 to 11) displayed a high growth rate (> 20 μm day−1) during the first 3 weeks. Growth of this area decreased abruptly in the fourth week (∼ 9 μm 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 μm 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 indicated 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 endosteal 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 particular variation of the bone growth rates during development 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 μm 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 μm day−1), a decrease in growth rates in the fourth week (6 μm day−1) and a marked new decrease in the sixth week (2 μm day−1). The third area included the ventral border (from point 9 to 11) and showed high bone growth rates during the first two postnatal weeks and in the fifth week (11–15 μm 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 μm 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 μm day−1). Only the area associated with the incisor alveoli (points 12 and 13) showed a high growth rate (4–6 μm day−1) from the first to the fifth week and then abruptly decreased growth rates (∼ 1 μm day−1) until the eighth postnatal week.

In the second molar region, three areas with similar variations in bone growth rate during development were distinguished. 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 μm day−1), except in the area of the mandibular crest (points 4 and 5), which showed high growth rates (∼ 16 μm day−1). In this region, all individuals showed a constant rate (2–3 μm 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 μm day−1) during the first three postnatal weeks; growth rates gradually decreased from the fourth to the eighth weeks (∼ 5 μm day−1). The third area (points 11–14) was associated with the lingual side and was characterized by low growth rates (∼ 3 μm day−1) that gradually decreased during the postnatal development (1 μm 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 activity and suggests growth in all directions by periosteal bone formation. In the third week, the ramus was entirely depository, 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 μm day−1), particularly on the lingual side, close to the mandibular foramen (points 15), which showed the highest rate (> 10 μm day−1) in that week. From the fourth to the eighth postnatal weeks, bone modeling fields allowed us to distinguish two areas characterized by bone resorption fields: the labial side of the coronoid (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 ventral-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 proposed in previous works (e.g. Atchley & Hall, 1991; Klingenberg 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 consistent pattern characterized by resorption on the lingual side. On the contrary, the ramus shows a complex pattern characterized 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.

Table 1.   Distribution of bone tissue types observed in the four regions of the Mus musculus mandible in each postnatal week.
 Region area
AgeDiastemaFirst molarSecond molarAscending ramus
dorlabvenlinlabvenlinlabvenlinC labC linV labV lin
  1. 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.

Week 1wwwwwwwwwwwwww
Week 2wwwwwwwwwwwwww
Week 3wpf/wwpfpf/wwpfwwwwwww
Week 4wpf/wwpf/wpf/wwpfpf/wwpfwwww
Week 5pfwwwpf/wwpfpf/wwpfwwww
Week 6wpf/wwpfpf/wwpfpf/wwpfwwww/pf
Week 7wpf/wwpfpf/wwpfpf/wwpfwww/pfw/pf
Week 8wpfwpfpf/wpfpfpf/wpfpfw/pfw/pfpfw/pf
Figure 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).

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 (Buffrénil & Pascal, 1984), the rhesus monkey Macaca mulatta, and modern human Homo sapiens (e.g. Kurihara et al. 1980; Enlow & Hans, 1996). Of these studies, only Buffrénil & 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 mandible (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 measured 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 dorsoventral 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 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 (Buffrénil & Pascal, 1984; Enlow & Hans, 1996). The inferred bone growth dynamics in mice are in agreement with Buffrénil & Pascal’s (1984) interpretation of the increase in height of the alveolar region of the mink, mainly in a ventral direction. In addition, and considering the interpretations of the growth in length in other mammals (Bang & Enlow, 1967; Buffrénil & Pascal, 1984; Enlow & 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, Buffrénil & 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 species 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 mandible (Buffrénil & 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 directions. Interestingly, the second molar region shows similarities with the ascending ramus (both characterized by woven bone). This result supports the statement of Klingenberg 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 alveolar 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 previously analyzed (Bang & Enlow, 1967; Buffrénil & 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 modeling 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 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 Buffrénil & Pascal (1984) that suggested (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 dorsoventral 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 development 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 temporomandibular 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 mandible responds to physiological loads resulting from the new dietary requirements during the postweaning period through the modeling mechanism. As a consequence, differences 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 tissue 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 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 tissue 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 determination of the growth changes associated with phenotypic 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 Innovación (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 Science and Innovation (MICINN). We thank the ‘Plateforme Animalerie Rongeurs’ de l’IFR 83 de l’Université Pierre et Marie Curie.

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