Localization and function of the anion exchanger Ae2 in developing teeth and orofacial bone in rodents

Abstract

To explore the functions of the anion exchanger 2 (Ae2) in the development of bones and teeth we examined the distribution of Ae2 in cells involved in the formation of teeth and surrounding bone in young hamsters, mice and rats. In all three species strongest immunostaining for Ae2 was obtained in basolateral membranes of maturation ameloblasts and in osteoclasts resorbing bone. In hamsters a weaker staining was also seen in the Golgi apparatus of secretory ameloblasts, young osteoblasts and osteocytes, odontoblasts and fibroblasts of the forming periodontal ligament. In adult Ae2(a,b) (-/-) mice, in which Ae2-targeted disruption precluded the expression of Ae2a, Ae2b1 and Ae2b2 isoforms, the immunostaining for Ae2 in ameloblasts and osteoclasts was totally abolished. The enamel formation was abnormal but teeth erupted, osteoclasts in jaw bone were functional and structure of dentin and bone was normal. In another mouse model, Ae2(-/-) mice in which the expression of all five Ae2 isoforms was disrupted, teeth failed to erupt and the alveolar bone proved poorly formed with giant but apparently functional osteoclasts. Our data indicate that basolaterally located Ae2a, Ae2b1 or Ae2b2 (or a combination of these) is present in maturation ameloblasts critical for the cells' normal functioning. Although isoforms of Ae2 were also present in basolateral membranes of osteoclasts, they proved to be not critical to osteoclast resorption of orofacial bone. Poorly formed bone and the failure of teeth to erupt seen in the Ae2(-/-) mice with gene disruption affecting all isoforms may result from secondary (systemic) changes that are different from Ae2(a,b) (-/-) mice.

Fig. 1. Ae2 localization in rodent incisors

(a) Major part of an adult mouse mandibular incisor, presented in two parts. Left top represents apical end with secretory stage ameloblasts (SA) that develop downwards into maturation ameloblasts (MA). The part at the bottom (asterisk) continues in the second part at the top (asterisk) towards incisal end (bottom). Note that secretory ameloblast are immunonegative, that transitional ameloblast (TA) gradually become positive, and that maturation ameloblast are intensely stained, particularly near the incisal end. The immunopositive maturation zone is abruptly interrupted three times by small groups of ameloblasts that are virtually negative (sharp gaps; brackets), whereas other groups of ameloblasts stain variably (fuzzy gaps, arrows). Intense staining is seen in erythrocytes in the lumen of blood vessels (BV). Some erythrocytes are detached and spread over the enamel. E, enamel matrix (blue). ES, enamel space; D, dentin (pink); B, Bone. (b) A high power detail of mouse incisor secretory ameloblasts (SA), negative for Ae2, whereas the outer enamel epithelium of the enamel organ (EO) is weakly positive. (c, d). A longitudinal (c, not decalcified, E enamel) and cross section (d) through mouse maturation stage ameloblasts showing the membrane associated staining for Ae2. (e) Detail of an abrupt change in Ae2 staining in maturation stage ameloblast (MA) of an adult mouse incisor. PL: papillary layer (epithelial tissue) (f) Abrupt change in Ae2 staining in maturation stage in neonatal hamster incisor. Weak staining is seen in the papillary layer (PL). (g) Absence of staining of maturation stage ameloblasts (MA) in an adult Ae2_a,b^-/- mouse incisor. Note disorganization of ameloblasts and papillary layer (PL). Strong staining is seen in erythrocytes in blood vessel (BV) which are stained for Ae1. Original magnifications: Fig.1a, 50x; Fig 1b-d & 1f, 400x; Figs 1e&g, 250x;. Figs 1c&1d, counterstaining methyl green, Figs 1a,b, e &g, hematoxylin, Fig 1f hematoxylin and eosin. Scale bars represent indicated length in micrometer

Fig. 2. Localization of Ae2 in developing hamster molar tooth germs

(a) A weak immunostaining (brown) is seen in the Golgi area of secretory ameloblasts (SA) depositing the first layer of enamel (E, blue). The odontoblasts (o) contain a well delineated staining in the Golgi area; pulp cells do not stain. D, dentin. Asterisks indicate shrinkage artefacts. (b) Low power image of a developing molar indicating the different intensities of staining in the various cell types. Secretory ameloblasts (SA) and enamel organ cells (EO) stain weakly, maturation stage ameloblast (MA) near the tip of the cusp and erythrocytes in dental pulp (DP) intensely. Odontoblast staining is weak and pulp cells do not stain. (c) Detail of molar cusp. Maturation stage ameloblast (MA) are intensely stained and staining is continuous, but at some locations (arrows) staining in distal part of a group of cells is weak or lacking, reminiscent of the fuzzy gap in incisors. Note the elongated Golgi apparatus in the odontoblasts (O). EO, enamel organ; OE, oral epithelium. Asterisk indicates shrinkage artefact (d) Staining in cytoplasm of fibroblasts (arrows) of developing periodontal ligament (PDL) along the root dentine (RD) formed by the root odontoblasts (O). BV, blood vessel (e) Staining along the forming root dentin (RD) near the cemento-enamel junction (CEJ). B, bone with positive osteocytes; PDL, periodontal ligament. Strong Golgi staining is seen in both young root and crown odontoblasts (O), not seen in dental pulp cells (DP). BV, blood vessel. Original magnifications Figs 2a&2c, 250x; 2b, 50x; Figs 2d&2e, 400x; Fig 2d methyl green; Figs 2a-c& Fig 2e haematoxylin. Scale bars represent indicated length in micrometer.

Fig. 3. Ae2 localization in osteoclasts and bone cells

(a) Low power view of tip of incisor of lower hamster jaw containing strongly positive maturation stage ameloblasts (MA) with cytoplasmic and membrane staining. No counterstaining. ES, enamel space; D, dentin. The enamel organ (EO) is weakly stained and separated from the alveolar bone (B) by soft connective tissue (SCT) with blood vessels (BV). The inner side of the bone is being resorbed by osteoclasts whose plasma membranes are stained (arrows) to make way for tooth eruption. M, epithelial cells of Malassez in the developing root analogue (lingual portion) stain weakly. (b) Large multinucleated cell at the bone (B) surface with basolateral staining for Ae2; the arrow indicates the membrane portion containing the alleged ruffled border that is immunonegative (7 days old hamster; nuclei counterstained blue with hematoxylin). (c) Double staining for Ae2 (brown) and TRACP (pink), counterstained with methyl green (green blue, nuclei) identifying the large multinucleated cells as osteoclasts (wild type mouse, 9 days old). (d) Osteoclast (OCL) in a resorption lacunae at the alveolar bone (B) surface of the periodontal gap of an adult Ae2_a,b^-/- mouse immunonegative for Ae2. Note that the red blood cells in the vessels of the periodontal ligament are immunopositive, likely staining for Ae1. (e) Control section of osteoclast (OCL) against alveolar bone (B) of periodontium of wild type adult littermate mouse positive for Ae2. (f) Osteoblasts (OB) lining embryonic woven bone, and osteocytes (OC) immunostain in Golgi area (9 days hamster). Note that periosteal cells (upper part) at some distance from the bone surface are immunonegative. (g) Osteoblasts (OB) forming bone (B) trabeculae in the jaw as well as osteocytes (OC) embedded in bone are immunopositive (9 days hamster). Original magnifications: Fig. 3a, 250x; Figs 3b, 3c, 3f & 3g, 1000x; Figs 3d&3e, 400x; hematoxylin counterstaining. Scale bars represent indicated length in micrometer.

Fig. 4. Effect of disruption of all Ae2 isoforms in the Ae2_a,b^-/- mice on tooth eruption and jaw bone structure

(a) Low power of two wild type molars that are erupting into the oral cavity (ORC). Roots are well developing. Root dentin (RD) is surrounded by bone (B) present at some distance from the roots. G, gingiva. (b) Low power of two knockout molars. The molars are smaller than wild type and irregularly formed. Bone (B) is still covering the tooth crowns. Enamel space (ES) on top of the dark blue crown dentin contains no enamel matrix indicating maturation stage has progressed. Roots are distorted and short. Arrow indicates globular dentin matrix in the predentin (pd). Predentin is wider than usual. RD is root dentin, C is a cyst in the enamel organ (c) Detail of 5b. The root is poorly developed and bone (B) is very close by. Odontoblasts are well structured whereas predentin (PD) is wider than normal. D, dentin. (d) Wild type incisor with maturation stage ameloblasts (MA) near enamel space (ES). Double arrow indicates distance between ameloblasts and bone (B) surface. (e) Ae2^-/- incisor with maturation stage ameloblasts (MA), shorter and more irregular than in wild type. Note that bone (B) is closer to the ameloblast layer than in Fig 5d. Arrow in dentin (D) indicates a defect (not stained) in blue stained dentin. (f, g) Alveolar bone covering incisors in Ae2^-/- (f) and wild type (g) mice. In Ae2^-/-mice bone trabeculae are very thin and irregular with widely spaced osteocyte lacunae. There is relatively much osteoid. OC, osteocytes; OCL, osteoclasts; OB, osteoblasts. (h) Alveolar bone of an Ae2^-/- mouse with giant multinuclear osteoclasts (OCL). Arrow indicates a dark blue stained reversal line at the surface of a resorption pit indicating bone remodelling. (i) A giant multinuclear osteoclast at the surface of irregularly formed bone with large osteocyte lacunae in an Ae2^-/- mouse. Plastic sections, Richardson staining. Original magnifications: Fig. 4a-b, 50x; Fig 4c, 250x; Figs 4d-g, 400x. Scale bars represent indicated length in micrometer.