PTHrP receptor in odontoblasts alters odontoblast and ameloblast function and maturation

Mechanisms of Development 121 (2004) 397–408 www.elsevier.com/locate/modo

Constitutively active PTH/PTHrP receptor in odontoblasts alters odontoblast and ameloblast function and maturation L.M. Calvia, H.I. Shinb, M.C. Knightc, J.M. Webera, M.F. Youngd, A. Giovannettic, E. Schipanic,* a

Endocrine Unit, Department of Medicine, University of Rochester School of Medicine, Rochester, NY, USA Department of Oral Pathology, School of Dentistry, Kyungpook National University, Daegu, South Korea c Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, 501 Wellman Building, Boston, MA, USA d CranioFacial and Skeletal Diseases Branch NIDCR, NIH, Bethesda, MD, USA b

Received 14 October 2003; received in revised form 28 January 2004; accepted 2 February 2004

Abstract Parathyroid hormone (PTH)-related protein (PTH-rP) is an important autocrine/paracrine attenuator of programmed cell differentiation whose expression is restricted to the epithelial layer in tooth development. The PTH/PTHrP receptor (PPR) mRNA in contrast is detected in the dental papilla, suggesting that PTHrP and the PPR may modulate epithelial– mesenchymal interactions. To explore the possible interactions, we studied the previously described transgenic mice in which a constitutively active PPR is targeted to osteoblastic cells. These transgenic mice have a vivid postnatal bone and tooth phenotype, with normal tooth eruption but abnormal, widened crowns. Transgene mRNA expression was first detected at birth in the dental papilla and, at 1 week postnatally, in odontoblasts. There was no transgene expression in ameloblasts or in other epithelial structures. Prenatally, transgenic molars and incisors revealed no remarkable change. By the age of 1 week, the dental papilla was widened, with disorganization of the odontoblastic layer and decreased dentin matrix. In addition, the number of cusps was abnormally increased, the ameloblastic layer disorganized, and enamel matrix decreased. Odontoblastic and, surprisingly, ameloblastic cytodifferentiation was impaired, as shown by in situ hybridization and electron microscopy. Interestingly, ameloblastic expression of Sonic Hedgehog, a major determinant of ameloblastic cytodifferentiation, was dramatically altered in the transgenic molars. These data suggest that odontoblastic activation of the PPR may play an important role in terminal odontoblastic and, indirectly, ameloblastic cytodifferentiation, and describe a useful model to study how this novel action of the PPR may modulate mesenchymal/epithelial interactions at later stages of tooth morphogenesis and development. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Parathyroid hormone; PTH-related protein; PTH/PTHrP receptor; Odontoblast; Ameloblast; Cytodifferentiation; Sonic Hedgehog

1. Introduction Mammalian tooth formation illustrates fundamental aspects of organogenesis and pattern formation (Peters and Balling, 1999; Weiss et al., 1998). Teeth develop as discrete organs, and the development of individual teeth involves epithelial –mesenchymal interactions that are essential for tooth initiation, morphogenesis and cytodifferentiation (Jernvall and Thesleff, 2000). A number of factors involved in these interactions have been identified (Jernvall and Thesleff, 2000; Miletich and Sharpe, 2003; Zhao et al., 2000). * Corresponding author. Tel.: þ 1-617-726-3966; fax: þ1-617-726-7543. E-mail address: [email protected] (E. Schipani). 0925-4773/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mod.2004.02.004

Parathyroid hormone-related protein (PTHrP), an epithelial factor mediating epithelial – mesenchymal interactions resulting in the development of structures such as the mammary gland (Wysolmerski et al., 1998) and the hair follicle (Wysolmerski et al., 1994) is also likely to play an important role in tooth development. This peptide was first discovered by the cause of the syndrome of humoral hypercalcemia of malignancy (Suva et al., 1987), and is produced in a large variety of normal adult and fetal tissues (Broadus and Steward, 1994). PTHrP and parathyroid hormone (PTH), a major regulator of calcium homeostasis, are phylogenetically related, but they share only eight amino acids in their first 13 residues (Broadus and Steward, 1994). Despite their limited structural homology, they bind to

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and activate the same PTH/PTHrP receptor (PPR) with almost indistinguishable high affinity (Abou-Samra et al., 1992; Juppner et al., 1991; Schipani et al., 1993). The broad tissue distribution pattern of PTHrP strongly suggests that this peptide plays a role as an auto/paracrine factor important in organ development. Consistent with this hypothesis is the occurrence of dramatic developmental abnormalities in each of the several different transgenic mouse models in which PTHrP overexpression was targeted to different tissues, or in which the gene encoding PTHrP was disrupted through homologous recombination (Karaplis et al., 1994; Vasavada et al., 1996; Weir et al., 1996). Studies analyzing mice lacking the PPR (Lanske et al., 1999, 1996) or expressing a constitutively active PPR (caPPR) (Schipani et al., 1997) have shown that these actions of PTHrP are likely mediated by the PPR. In the tooth, PTHrP is expressed in the inner and outer enamel epithelia of the enamel organ (Beck et al., 1995; Lee et al., 1995; Liu et al., 1998), while the PPR is expressed in the dental papilla (DP) and in the dental follicle, the mesenchymal components of the developing tooth (Lee et al., 1995; Liu et al., 1998). Analysis of the role of PTHrP in the developing tooth has been difficult since the deletion of the PTHrP gene impairs tooth eruption, resulting in distortion of the anatomy of the developing tooth (Philbrick et al., 1998). Indeed, PTHrP appears to be absolutely required for tooth eruption (Kitahara et al., 2002; Philbrick et al., 1998; Schipani et al., 1997). Similarly, analysis of the role of the PPR in the later stages of tooth development has been limited, since mice lacking the PPR die at birth (Lanske et al., 1996). We recently described transgenic mice in which a caPPR is expressed in osteoblasts, with expression driven by the 2.3 kb fragment of the a1(I) collagen promoter (col1-caPPR mice) (Calvi et al., 2001). In these mice, expression of this activated receptor is also present in the developing odontoblasts and DP, allowing us to evaluate the role of activation of this receptor at later stages of tooth development as well as in odontoblastic biology. We show here that expression of the activated PPR in odontoblastic cells alters their differentiation, and disrupts the differentiation of ameloblastic cells. These data therefore, suggests that activation of the PPR, likely by PTHrP, may be important for normal odontoblastic and ameloblastic differentiation and tooth morphogenesis.

2. Results 2.1. Expression of the activated PPR in the developing tooth Mice expressing a constitutively active human PPR have a vivid postnatal bone and tooth phenotype (Calvi et al., 2001). Consistent with the bone phenotype described previously (Calvi et al., 2001), both mandibular and maxillary

bones were widened compared to the ones from sex-matched littermates (Fig. 1A,B). While tooth eruption was normal, transgenic teeth had friable and abnormally yellowed crowns (Fig. 1A,B). Both molars and incisors in the transgenic mice were abnormally wide, and transgenic molars had irregular and poorly defined cusps (Fig. 1A,B). The cusps of the molars were crenellated and difficult to enumerate even after eruption. To understand the impact of activation of the receptor in the developing tooth prior to the confounding presence of cavities, the developing molars were histologically analyzed prior to eruption. The normal pattern of expression of the PPR was confirmed in wild-type newborn molars by in situ hybridization. As shown by others previously (Lee et al., 1995; Philbrick et al., 1998), faint expression of the PPR was present in the DP and odontoblasts of molars from 1 week old wild-type animals (Fig. 1C,E). The increased expression of the PPR that was detected in odontoblastic cells as well as DP (Fig. 1D,F) of transgenic littermates at 1 week of age was very likely secondary to the cross-reactivity of the cRNA for the mouse PPR with the transgene. As noted previously, increased PPR expression was also noted in osteoblastic cells lining the alveolar maxillary and mandibular bone of the transgenic mice (Fig. 1D,F). A cRNA capable of specifically identifying expression of the transgenic construct was generated previously (Calvi et al., 2001). No expression of the transgene could be detected at E15.5 in the developing tooth (data not shown). Transgene mRNA expression was seen at birth in the DP (Fig. 1G,H) and, at 1 week postnatally, in odontoblasts (Fig. 1I,J). The pattern of expression of the transgene in transgenic animals overlapped with that of the native PPR described by us (Fig. 1C,E) and others (Lee et al., 1995; Liu et al., 1998) in wild-type animals. No transgene expression in the epithelial enamel organs or ameloblasts could be detected in the developing teeth of transgenic mice (Fig. 1G – J). 2.2. Abnormal morphology in molars from the transgenic mice At the cap stage, E 15.5, molars from the transgenic mice (Fig. 2A) were indistinguishable from those of normal littermates (Fig. 2B). At birth, the alveolar bone/mesenchyme surrounding the molars, as well as the long bones (Calvi et al., 2001) appeared normal in both wild-type (Fig. 2C) and transgenic littermates (Fig. 2D). At this stage, the DP, stellate reticulum (SR) and the developing cusps are clearly seen in the inferior second molar of a wild-type newborn animal (Fig. 2C). In contrast, in the corresponding molar from a transgenic littermate, there was widening of the DP, and the number of developing cusps was increased compared to the wild-type (Fig. 2D). At 1 week of age, in normal tooth development of the second molar, the DP was maturing into dental pulp, and cytodifferentiation had occurred, as was evident histologically (Fig. 2E). Analysis by electron microscopy from

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Fig. 1. Col1-mutPPR mice have abnormal incisors and molars. Dissected maxillae (A) and mandibles (B) from wild-type (wt) and transgenic (tg) littermates at 12 weeks of age. The large incisors and molars have erupted, but are abnormally wide and yellowed, with multiple poorly formed cusps. In situ hybridization with the cRNA for the PTH/PTHrP receptor in histologic sections of molars from 1 week old wild-type (C,E) and transgenic littermates (D,F), ameloblasts (ab); odontoblasts (ob); dental papilla (dp). Faint expression is seen in the dental papilla and odontoblasts as well as in osteoblastic cells. Darkfield (C,D) and brightfield (E,F) images are shown. Slides are counterstained with hematoxylin and eosin. In situ hybridization with the cRNA for the transgene in histologic sections of molars from a representative transgenic mice at birth (G,H) and 1 week of age (I,J). No transgene expression was present in wild-type littermates. Darkfield (G,I) and brightfield (H,J) images are shown. Slides are counterstained with hematoxylin and eosin. Scale bars: 150 mm (E, also applies to C,D,F); 100 mm (G, also H); 200 mm (I, also J).

identical portions in the respective wild-type and transgenic tooth germs confirmed that in images from the wild-type, molar odontoblasts were terminally differentiated, elongated, and polarized, and they produced dentin matrix (Fig. 2G). Ameloblasts had also undergone differentiation, and become highly polarized cells with an elongated cytoplasm, which secreted an enamel matrix (Fig. 2E). The SR was invaded by blood vessels and fibroblasts, derived from the dental sac (Fig. 2E and data not shown). In contrast, in the second molar from a transgenic littermate, as was seen at an earlier stage, the DP/dental pulp was widened compared to that of the normal littermates (Fig. 2F) and the DP cells were increased in number, as it could be clearly appreciated by electron microscopy (Fig. 2J), compared to cells in the DP of a normal littermate (Fig. 2I). Instead of distinct cusps, as were seen in the wild-type molar (Fig. 2E), the transgenic molar had multiple, poorly formed cusps (Fig. 2F). Morphological cytodifferentiation of odontoblasts was incomplete and the odontoblastic layer was disorganized, without cellular elongation and polarization (Fig. 2F,H).

Dentin matrix was substantially decreased (Fig. 2K) and had a disorganized, rather than lamellar pattern (Fig. 2H) compared to the dentin of the wild-type molars (Fig. 2G,L). Differentiation of the ameloblasts was also impaired, particularly in the ameloblasts adjacent to the odontoblastic layer (Fig. 2F), with disorganization of the ameloblastic layer, and a dramatic decrease of the enamel matrix (Fig. 2K) compared to the wild-type enamel matrix (Fig. 2L). In addition, the SR persisted abnormally (Fig. 2E,F). Of note, just as in the wild-type molar (Fig. 2E), an eruption pathway was clearly patent above the transgenic molar, suggesting normal osteoclastic activity in the alveolar bone of the transgenic mice (Fig. 2F). 2.3. Transgenic molars have abnormal odontoblastic cytodifferentiation To confirm the abnormal cytodifferentiation in the transgenic mice, markers of odontoblastic differentiation were analyzed by in situ hybridization in the mandible

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Fig. 2. Morphologic analysis of Col1-mutPPR molars. Histologic sections from molars from E15.5 (A,B), newborn (C,D) and 1 week old (E,F,K,L) wild-type (A,C,E,L) and transgenic littermates (B,D,F,K).ameloblasts (ab); odontoblasts (od); dental papilla (dp); stellate reticulum (sr); dentin matrix (d); enamel matrix (e); alveolar bone (AlvB). Slides are counterstained with hematoxylin and eosin. Electron microscopic images from identical portions of molars of wild-type (G,I) and transgenic (H,J) littermates at 1 week of age, focusing on the odontoblasts and dentin matrix (G,H) and the dental papilla (I,J). Note disorganization of dentin matrix and osontoblastic layer in H. Scale bars: 75 mm (A, also applies to B); 100 mm (C, also D); 150 mm (E, also F); 5 mm (G, also H, I,J); 50 mm (K, also J).

and maxilla of wild-type and transgenic littermates at 1 week of age. Alkaline phosphatase was expressed by osteoblastic cells as well as odontoblasts and immature odontoblasts in the wild-type upper and lower molars (Fig. 3A,E). In the transgenic mandibular and maxillary bones, there was an increase in alkaline phosphatase (Fig. 3B,F), consistent with the increased number and function of osteoblastic cells seen throughout the endosteal bone of the transgenic mice (Calvi et al., 2001). In the transgenic

molars, alkaline phosphatase was clearly up-regulated and expressed throughout the odontoblastic layer, as well as in mesenchymal cells from the DP proximal to the odontoblastic layer. In spite of the increase in alkaline phosphatase expression, there was an overall decrease in the dentin matrix in the transgenic mice (Fig. 2K) compared to wild-type littermates (Fig. 2L), secondary perhaps to the abnormal maturation of the odontoblastic layer.

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Fig. 3. Abnormal odontoblastic differentiation in teeth from Col1-mutPPR transgenic mice. In situ hybridization with the cRNA for Alkaline Phosphatase (A,B,E,F) and Osteocalcin (C,D,G,H) in histologic sections of molars from 1 week old wild-type (wt) (A,E,C,G) and transgenic (tg) littermates (B,F,D,H). molar (m); incisor (i). Darkfield (A –D) and brightfield (E–H) images are shown. Slides are counterstained with hematoxylin and eosin. Scale bar: 200 mm (E, also applies to all other panels).

Next, we examined the osteocalcin expression. Although osteocalcin is a well established marker of osteoblastic differentiation (Malaval et al., 1999, 1994), its expression during odontoblastic differentiation is more controversial, even though recent studies do suggest expression of osteocalcin by terminally differentiated odontoblasts (Bidder et al., 1998). In wild-type animals of 1 week of age, osteocalcin expression was limited to mature osteoblasts (Fig. 3C,G) and to the mature odontoblastic layer, while no expression was seen in the mesenchymal cells proximal to the odontoblastic layer (Fig. 3C,G). These data suggest that osteocalcin is expressed only by the mature odontoblasts in the developing molar. In the transgenic mice, osteocalcin was also expressed by the mature osteoblasts in both maxillary and mandibular bones (Fig. 3D,H). Interestingly, particularly in the upper molar, cells of the odontoblastic layer, which clearly had expressed alkaline phosphatase (Fig. 3B,F), expressed osteocalcin heterogeneously (Fig. 3D,H), with some odontoblasts having low level of expression, while others had levels of expression higher than that of the wildtype counterpart. Interestingly, no expression of osteocalcin could be detected in the transgenic incisors at this stage of development (Fig. 3D,H). Taken together, these data suggest

that differentiation of odontoblasts was impaired in the transgenic molars. 2.4. Transgenic ameloblasts have delayed differentiation Markers of the ameloblastic differentiation were analyzed next by in situ hybridization. In wild-type animals at 1 week of age, amelogenin (Fig. 4A,E), an early marker of developing ameloblasts (Sommer et al., 1996) and ameloblastin (Fig. 4C,G), were both expressed throughout the ameloblastic layer in the wild-type maxillary and mandibular bones. In contrast, amelogenin was expressed heterogeneously in the ameloblastic layer of transgenic molars (Fig. 4B,F), with some cells having very high levels of expression, while others showed expression that was barely detectable, suggesting incomplete differentiation of the ameloblastic layer. In addition, ameloblastin also had heterogeneous expression in the transgenic molars (Fig. 4D,H), with some amelogenin-expressing cells lacking ameloblastin message, again supporting a lack of full differentiation of the transgenic ameloblastic cells. Of note, the developing wild-type incisor had mature ameloblasts, which expressed both amelogenin (Fig. 4A,E) and at much

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Fig. 4. Abnormal ameloblastic differentiation in teeth from Col1-mutPPR transgenic mice. In situ hybridization with the cRNA for Amelogenin (A,B,E,F) and Ameloblastin (C,D,G,H) in histologic sections of molars from 1 week old wild-type (wt) (A,E,C,G) and transgenic (tg) littermates (B,F,D,H). molar (m); incisor (i). Darkfield (A –D) and brightfield (E–H) images are shown. Slides are counterstained with hematoxylin and eosin. Scale bar: 200 mm (E, also applies to all other panels).

lower levels ameloblastin (Fig. 4C,G), whereas the transgenic incisor lacked expression of both markers of ameloblastic differentiation (Fig. 4B,F,D,H). This finding supported once again the delay in differentiation of the ameloblastic cells in transgenic teeth in which the caPPR was expressed in the DP and developing odontoblasts. To address whether the delay in transgenic ameloblastic differentiation persisted as the tooth continued to develop, molar ameloblasts in wild-type and transgenic mice were studied at 2 weeks of age. At this stage of differentiation, the molars are about to erupt. Once again, the dental pulp was widened in the molars from transgenic mice (Fig. 5F,H), compared with wild-type littermates (Fig. 5E,G). In addition, multiple abnormal cusps were present in the transgenic molar (Fig. 5F,H) compared with the corresponding wild-type (Fig. 5E,G). While much dentin matrix was present in the wild-type teeth (Fig. 5G), only a thin layer of dentin matrix and enamel could be seen in the transgenic molars (Fig. 5F,H). By in situ hybridization, ameloblastin was expressed throughout the ameloblastic layer in both the wild-type (Fig. 5C,G) and the transgenic molars (Fig. 5D, H). As ameloblasts completed their differentiation, the expression of amelogenin decreased to a level that was

barely detected in the most mature normal ameloblasts (Fig. 5A,E), as shown by others (Sommer et al., 1996). In contrast, amelogenin expression persisted strongly in ameloblasts from transgenic mice (Fig. 5B,F), suggesting a persistent delay of the ameloblast differentiation at 2 weeks after birth in the transgenic animals. Interestingly, at this developmental stage there was also a drastic delay in root formation in the molars of the transgenic mice (Fig. 5B, F,D,H) compared to wild-type littermates (Fig. 5A,E,C,G). This phenomenon could be at least in part explained by the abnormal architecture of the alveolar bone in the transgenic mice. Fully differentiated ameloblasts are postmitotic and studies have suggested that maintenance of these cells in a proliferative stage may be one mechanism that controls the pace of differentiation in ameloblasts (Gritli-Linde et al., 2002). To assess ameloblastic proliferation, BrdU incorporation was studied in wild-type and transgenic ameloblasts at 1 week of age. In the wild-type molar, all ameloblasts were postmitotic, since none of their nuclei incorporated BrdU (Fig. 6A). In contrast, in the transgenic ameloblastic cells, a few nuclei incorporated BrdU (Fig. 6B), suggesting that these cells were still actively proliferating. These data again

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Fig. 5. Abnormal ameloblastic differentiation in teeth from Col1-mutPPR transgenic mice persists at 2 weeks of age. In situ hybridization with the cRNA for Amelogenin (A,B,E,F) and Ameloblastin (C,D,G,H) in histologic sections of molars from 2 weeks old wild-type (wt) (A,E,C,G) and transgenic (tg) littermates (B,F,D,H). molar (m,); incisor (i). Darkfield (A –D) and brightfield (E–H) images are shown. Slides are counterstained with hematoxylin and eosin. Scale bar: 200 mm (E, also applies to all other panels).

Fig. 6. Abnormal differentiation of epithelial structures in teeth from Col1-mutPPR transgenic mice. BrdU immunohistochemistry of histologic sections of molars from 1 week old wild-type (A) and transgenic littermates (B). Arrows point to positive (black) staining in the crown of a transgenic molar. In situ hybridization with the cRNA for PTHrP in histologic sections of molars from 1 week old wild-type (C,E) and transgenic littermates (D,F). Darkfield (C,D) and brightfield (E,F) images are shown. Slides are counterstained with hematoxylin and eosin. Scale bars: 50 mm (A, also applies to B); 100 mm (C, also D, E,F).

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confirmed that at least some of the ameloblasts from the transgenic mice were less differentiated than the corresponding ameloblasts in the wild-type mice. 2.5. Transgenic molars have a delay in differentiation of the SR, another dental structure of epithelial origin The SR persisted morphologically in 1-week-old molar of the transgenic animals (Fig. 2F). To confirm this finding, PTHrP expression was studied by in situ hybridization in 1week-old animals. PTHrP is expressed in the SR of developing teeth at the cap stage (Philbrick et al., 1998). While no expression of PTHrP was detected above the ameloblastic layer in the wild-type molars (Fig. 6C,E), expression of PTHrP was clearly detected in the area that morphologically was identifiable as the SR in the molars of the transgenic littermates (Fig. 6D,F). Since the SR is derived from the epithelial enamel organ, this finding suggests that, in the transgenic mice, expression of the caPPR in DP and odontoblastic cells leads to abnormal differentiation of multiple cell types of epithelial origin. 2.6. Transgenic molars have normal enamel knots, but abnormal ameloblastic expression of Sonic Hedgehog The enamel knot is constituted by a population of cells in the center of the cap producing several growth factors, and is thought to be an organizing center that patterns the tooth crown (Thesleff and Sharpe, 1997). In molars, which contain multiple cusps, secondary enamel knots develop at the tips of the future cusps (Jernvall et al., 1994; Jernvall and Thesleff, 2000). Sonic Hedgehog (Shh), a member of

the vertebrate Hedgehog family (Ingham and McMahon, 2001) encodes a secreted signaling peptide that is exclusively expressed in the epithelial portion of the murine tooth (Dassule et al., 2000). Expression of Shh at the cap stage is confined to the enamel knot (Hardcastle et al., 1998; Vaahtokari et al., 1996). While neither Shh expression (Dassule et al., 2000) nor signaling downstream of it (Gritli-Linde et al., 2002) are essential for enamel knot formation, expression of this gene constitute a useful marker for this structure (Gritli-Linde et al., 2002; Jernvall and Thesleff, 2000). Since the transgenic mice had abnormal crown morphology, we evaluated the enamel knot in developing molars from wild-type and transgenic littermates. Expression of Shh was found to be normal at E15.5 by in situ hybridization in cap stage developing molars of both transgenic and wild-type littermates (data not shown). This finding suggested that abnormal cusp patterning in the molars from the transgenic mice was not due to abnormalities of the primary enamel knot. Shh expression continues in differentiating ameloblasts (Dassule et al., 2000), in which it regulates ameloblastic differentiation and proliferation (Gritli-Linde et al., 2002). Since ameloblastic cytodifferentiation was significantly altered in molars from the transgenic mice, we studied the expression of Shh at later stages of dental differentiation as well. At the bell stage, the domain of Shh expression broadens from the primary enamel knot to other epithelial derived structures: the inner enamel epithelium (IEE), the stratum intermedium (SI) and the SR (Gritli-Linde et al., 2001). In molars from wild-type newborn mice (Fig. 7A,E), expression of Shh was indeed present in these epithelial structures, and was weakest at the apices of the developing

Fig. 7. Abnormal ameloblastic SHH expression in teeth from Col1-mutPPR transgenic mice. In situ hybridization with the cRNA for Sonic Hedgehog in histologic sections of lower molars (A,B,E,F) and upper molars (C,D,G,H) from newborn (A,B,E,F) and 1 week old (C,D,G,H)) wild-type (wt) (A,E,C,G) and transgenic (tg) littermates (B,F,D,H). Darkfield (A –D) and brightfield (E– H) images are shown. stellate reticulum (sr); stratum intermedium (si); inner enamel epithelium (iee). Slides are counterstained with hematoxylin and eosin. Scale bars: 150 mm (E, also applies to A,B,F); 200 mm (G, also C,D,H).

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cusps. In contrast, in newborn molars from transgenic littermates (Fig. 7B,F), expression of Shh was much diminished in the IEE, SI and SR. This pattern of expression in the IEE was the opposite of the wild-type expression, with highest expression at the apices of the developing cusps (Fig. 7B,F). Molars from 1-week-old wild-type mice had diffuse and homogeneous expression of Shh in the ameloblastic layer (Fig. 7C,G). In contrast, in the ameloblastic layer of molars from 1-week old transgenic littermates, expression of Shh was heterogeneous with some ameloblasts having low-level expression, while others had levels of expression higher than that of the wild-type counterpart (Fig. 7D,H). The lower levels of expression were seen in ameloblastic cells that produced enamel, while the strongest Shh expression was seen in ameloblastic cells that were in close proximity to the odontoblastic layer and in which almost no enamel nor dentin were present (Fig. 7D, H). Interestingly, the area of increased Shh expression in the transgenic ameloblasts (Fig. 7D,H) corresponded to the region in which abnormally proliferating ameloblastic cells were detected by BrdU staining in sections from the transgenic mice (Fig. 6B). These data again suggest abnormal differentiation in the transgenic ameloblastic cell, and are consistent with the important role of Shh in ameloblastic proliferation and differentiation.

3. Discussion The broad tissue distribution pattern of PTHrP strongly suggests that this peptide plays a role as an auto/paracrine factor (Broadus and Steward, 1994) with important developmental function. Several studies have shown PTHrP expression, as well as PPR expression in developing dental structures (Beck et al., 1995; Lee et al., 1995; Liu et al., 1998). Evaluation of the role of PPR signaling in tooth development has been limited by the perinatal mortality of the PPR null mice (Lanske et al., 1996), as well as by the severe tooth disruption due to lack of eruption in the PTHrP null mice (Philbrick et al., 1998; Schipani et al., 1997). In the col1-caPPR transgenic model, expression of the caPPR in the DP and odontoblasts was not surprising given the pattern of expression conferred by the particular fragment of the collagen I promoter, which was utilized in the design of the transgene. The 2.3 kb fragment of the mouse a1(I) collagen promoter has been shown previously to drive the expression specifically in cells of the osteoblast lineage and in odontoblasts (Rossert et al., 1995), with a dental pattern of expression consistent with the expression of the native PPR. The col1-caPPR transgenic model allows the study of the effects of PPR activation in the target organs of PTHrP signaling, the DP and the developing odontoblasts. The onset of transgene expression appears to be just prior to birth, as detection of the transgene is not present at E15.5, but is evident in the newborn DP. In a system in which morphogenesis and cytodifferentiation are governed by

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sequential, reciprocal and reiterative epithelial – mesenchymal interactions (Jernvall and Thesleff, 2000), the col1caPPR transgenic model has the advantage of leaving tooth development undisturbed until just prior to birth, so that the initial epithelial– mesenchymal interactions occur normally, after the odontoblastic and ameloblastic phenotypes have been determined. Therefore, our model focuses on the effects of PPR activation after the enamel organ and the DP are already established, and can address the question of whether PPR activation in the DP and in odontoblasts may have important effects on odontoblastic maturation, crown morphogenesis and ameloblastic differentiation and function. Since both incisors as well as molars in the adult transgenic mice were abnormally large and susceptible to cavities, we elected to study in detail teeth prior to eruption. While we observed changes in the incisors as well as in the molars of transgenic mice, we decided to focus our analysis on the second molar, since each tooth develops as a separate organ and with different timing (Jernvall and Thesleff, 2000). We were particularly interested in studying the molars since their morphology and development are similar to those of other mammals, and especially primates, while rodent incisors are characteristically continuously growing and asymmetric. Odontoblastic cells share many features of osteoblasts. The effect of the PPR activation on odontoblasts strikingly resembled the effect of PPR activation on osteoblasts. Just as PPR activation expands the osteoblastic population in trabecular bone, increasing the number of immature as well as mature osteoblasts (Calvi et al., 2001), DP cells were increased in molars from the transgenic mice with widening of the DP and dental structure that was evident histologically and grossly at all ages studied. In vitro studies have suggested that activation of the PPR by its ligands delays osteoblastic differentiation (Ishizuya et al., 1997). Likewise, odontoblastic differentiation was impaired in transgenic mice, as shown by heterogeneous expression of osteocalcin, a marker of odontoblastic differentiation, and by morphologic heterogeneity in the odontoblastic population. In addition, activation of the PPR affected osteoblastic function in transgenic mice, with differential effects depending on whether the osteoblasts were in the trabecular or the periosteal compartment (Calvi et al., 2001). Similarly, in the developing tooth, odontoblastic expression of the activated PPR resulted in decreased dentin in the molar crowns, whereas the incisors had large amounts of dentin. These data therefore, suggest that in odontoblast, activation of the PPR triggers responses similar to those in osteoblasts, with expansion of the odontoblastic pool and changes in odontoblastic maturation and function. Expression of the transgene was not detected in ameloblasts by in situ hybridization. While it is possible that very low levels of the transgene may be expressed in ameloblasts and not detected using the in situ hybridization technique, ameloblasts are cells of epithelial origin, and

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they are not known to express collagen I. Studies analyzing the pattern of expression conferred by this particular fragment of the collagen I promoter have never detected the expression of the transgene in ameloblasts or other tissues of epithelial origin, even while specifically focusing on the tooth (Rossert et al., 1995). Therefore, it is very unlikely that even low levels of transgene expression occurred in the enamel organs and ameloblasts. The specificity of transgene expression allowed us to study the action of the activated PPR in the mesenchymal portion of the developing tooth, and how this action may have an impact indirectly upon the epithelial portion. Surprisingly, ameloblastic differentiation was altered in the transgenic teeth, as shown by heterogeneous and delayed expression of amelogenin, persistence of ameloblastic proliferation at a stage in which the ameloblasts from wild-type littermates were postproliferative, and decreased the enamel matrix. In addition, PPR activation in odontoblasts had effects on other structures of epithelial origin, as was shown by persistence of the SR. Interestingly, since the primary enamel knot was not disrupted in the transgenic molars, the abnormalities in cusp morphology in the transgenic mice were likely due to the abnormal cytodifferentiation of odontoblastic and ameloblastic cells (Jernvall et al., 1994; Jernvall and Thesleff, 2000). Taken together, the data presented above suggest that the postnatal PPR activation in odontoblastic cells mediates further mesenchymal – epithelial interactions in tooth development, and results in alteration of normal ameloblastic cytodifferention. Although terminal odontoblastic differentiation is thought to trigger ameloblastic cytodifferentiation (Slavkin and Bringas, 1976), the factors mediating these late steps in tooth development are incompletely understood, partly because most knock-out experiments result in early developmental arrest (Jernvall and Thesleff, 2000; Miletich and Sharpe, 2003; Zeichner-David et al., 1997). Sonic Hedgehog (Shh), a member of the vertebrate Hedgehog family (Ingham and McMahon, 2001) encodes a secreted signaling peptide that is exclusively expressed in the epithelial portion of the murine tooth (Dassule et al., 2000). Shh expression continues in differentiating ameloblasts (Dassule et al., 2000), where it is known to regulate ameloblastic differentiation and proliferation (Gritli-Linde et al., 2002). In fact, absence of Shh signaling from the dental epithelium disrupts ameloblastic differentiation (Gritli-Linde et al., 2002). Given the disrupted ameloblastic cytodifferentaition in the col1-caPPR transgenic mice, the Shh expression was studied and found to be abnormal postnatally. These data confirm the abnormal differentiation of the transgenic ameloblasts. Notably, transgenic ameloblastic cells with higher level of expression of Shh had the most striking retardation of ameloblastic differentiation, with abnormal persistence of proliferation and the lowest amount of enamel. The indirect Shh effects of odontoblastic PPR activation are particularly intriguing given the interdependence of Hedgehog and PTHrP signaling in other

systems, such as in chondrogenesis and osteogenesis (Chung et al., 2001; Karp et al., 2000; Kobayashi et al., 2002; Lanske et al., 1996; Minina et al., 2001). Therefore, we speculate that Shh may act as one of the mediators of the arrest in ameloblastic differentiation induced by odontoblastic PPR activation. Further studies are needed to identify the signaling pathway(s) necessary to mediate this novel action of the PPR in the developing tooth. In conclusion, odontoblastic expression of a caPPR had cellular effects similar to those associated with PPR action in osteoblasts, including expansion of the odontoblastic population, delay in maturation and abnormal matrix formation. Ameloblastic differentiation was also unexpectedly and indirectly altered by odontoblastic caPPR expression, suggesting that PPR activation may play an important role in mediating the later mesenchymal – epithelial interactions that are necessary for terminal odontoblastic and ameloblastic cytodifferentiation. The col1-caPPR transgenic model may therefore, provide a useful tool to investigate the role of PPR activation at later stages of tooth development.

4. Experimental procedures 4.1. Identification of transgenic mice Mice expressing a caPPR under the control of the 2.3 kb fragment of the mouse a1(I) collagen promoter were generated previously (Calvi et al., 2001). Genotypying and confirmation of transgene expression were performed as described (Calvi et al., 2001). All studies performed were approved by the institutional animal care committee. 4.2. Sample preparation and histologic analysis Fresh tissues were obtained by cesarean section from fetuses at 15.5 days of gestation after timed matings. In addition, newborn, 1 and 2 week old transgenic mice and sex-matched wild-type littermates were sacrificed by cervical dislocation. For standard histology, tissues from transgenic and wild-type littermates were fixed as described (Calvi et al., 2001). After fixation, the maxilla and mandible were dissected. Tissue from 1 and 2 week old mice was decalcified in 20% EDTA, as described previously. Paraffin blocks were prepared by standard histological procedures and counterstained by hematoxylin and eosin. All slides were interpreted by at least two observers unaware of the genotypes of the particular mice being examined. 4.3. Preparation of adult tissue Mandibles and maxillae from 12 weeks old transgenic and wild-type littermates were dissected and boiled to remove any of the soft tissue. Maxillae and mandibles were photographed.

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4.4. Sample preparation for electron microscopic analysis

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Acknowledgements The authors thank H. M. Kronenberg and P. G. Robey for helpful discussion. This work was supported by Korea Health 21 R & D project, Ministry of Health and Welfare, Republic of Korea (01-PJ3-PG6-01GN11-0002) and by NIH Grants K08 DK64381 (to L.M.C.) and AR-44855 (to E.S).

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