See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/227836681
Differential responses to parathyroid hormone‐ related protein (PTHrP) deficiency in the various craniofacial cartilages Article in The Anatomical Record · August 1999 DOI: 10.1002/(SICI)1097-0185(19990801)255:43.0.CO;2-E
CITATIONS
READS
17
57
7 authors, including: Paul V Senior
Beck Felix
University of Melbourne
University of Leicester
47 PUBLICATIONS 1,605 CITATIONS
173 PUBLICATIONS 6,155 CITATIONS
SEE PROFILE
SEE PROFILE
All content following this page was uploaded by Paul V Senior on 06 October 2014.
The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately.
THE ANATOMICAL RECORD 255:452–457 (1999)
Differential Responses to Parathyroid Hormone-Related Protein (PTHrP) Deficiency in the Various Craniofacial Cartilages M. ISHII-SUZUKI,1 N. SUDA,1* K. YAMAZAKI,1 T. KURODA,1 P.V. SENIOR,2 F. BECK,3 AND V. E. HAMMOND3 1Second Department of Orthodontics, Faculty of Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8549, Japan 2Department of Anatomy and Cell Biology, University of Melbourne, Parkville 3052, Victoria, Australia 3Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville 3052, Victoria, Australia
ABSTRACT PTHrP null mutant mice exhibit skeletal abnormalities both in the craniofacial region and limbs. In the growth plate cartilage of the null mutant, a diminished number of proliferating chondrocytes and accelerated chondrocytic differentiation are observed. In order to examine the effect of PTHrP deficiency on the craniofacial morphology and highlight the differential feature of the composing cartilages, we examined the various cartilages in the craniofacial region of neonatal PTHrP deficient mice. The major part of the cartilaginous anterior cranial base appeared to be normal in the homozygous PTHrP deficient mice. However, acceleration of chondrocytic differentiation and endochondral bone formation was observed in the posterior part of the anterior cranial base and in the cranial base synchondroses. Ectopic bone formation was observed in the soft tissue-running midportion of the Meckel’s cartilage, where the cartilage degenerates and converts to ligament in the course of normal development. The zonal structure of the mandibular condylar cartilage was scarcely affected, but the whole condyle was reduced in size. These results suggest the effect of PTHrP deficiency varies widely between the craniofacial cartilages, according to the differential features of each cartilage. Anat Rec 255:452–457, 1999.
r 1999 Wiley-Liss, Inc. Key words: PTHrP; nasal septal cartilage; condylar cartilage; cranial base synchondrosis; Meckel’s cartilage
Parathyroid hormone-related peptide (PTHrP) was originally identified as the causative factor responsible for humoral hypercalcemia of malignancy (Rodan et al., 1983; Stewart et al., 1983). Amino-terminal PTHrP can mimic many of the effects of PTH and is known to share the same receptor, PTH/PTHrP receptor (Ju¨ppner et al., 1991). Mice homozygous for a disrupted PTHrP allele, generated by homologous recombination in embryonic stem cells, have been reported (Amizuka et al., 1994; Karaplis et al., 1994). The mutant shows widespread skeletal abnormalities and exhibits a chondrodysplastic phenotype characterized by a domed skull, short snout and mandible, short thorax and disproportionally short limbs. Histological examination of r 1999 WILEY-LISS, INC.
the growth plate cartilage of the null mutant has revealed a diminished number of proliferating chondrocytes and
Grant sponsor: Ministry of Education, Science, Sports, and Culture of Japan; Grant number: 5808; Grant sponsor: Japan Society for the Promotion of Science; Grant number: JSPS-RFTF 96I00205. *Correspondence to: Naoto Suda, 2nd Department of Orthodontics, Faculty of Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8549, Japan. E-mail: n⫽
[email protected] Received 5 January 1999; Accepted 3 May 1999
PTHrP DEFICIENCY AND CRANIOFACIAL CARTILAGES
accelerated chondrocytic differentiation. All these reports suggest that PTHrP is an essential factor regulating the pace of chondrocytic differentiation in an autocrine/ paracrine manner. It is known that the craniofacial morphology is greatly influenced by the growth and development of the cartilages (Diewert, 1982). There are several types of characteristic cartilages in the craniofacial region. Nasal septal cartilage, permanent cartilage that remains as cartilage throughout life (Koski, 1975); cranial synchondrosis, replaced by bone through endochondral bone formation (Roberts and Blackwood, 1983); Meckel’s cartilage, which appears transiently and has three different fates (Bhaskar, 1953); and condylar cartilage, classified as secondary cartilage (Ghafari et al., 1992). In order to examine the effect of PTHrP deficiency on craniofacial development and to highlight the differential character of each type of cartilage, we carried out histological observations in neonatal PTHrP deficient mice.
MATERIALS AND METHODS Animals Mice heterozygous for a disrupted PTHrP allele were generated using standard gene targeting methods in embryonic stem cells (Tucci et al., 1996). Heterozygous males and females, in a mixed C57BL/6J-129Sv genetic background, were mated, and the neonatal offspring [0 day, Theiler’s stage 27 (Theiler, 1989), 9 homozygous, 3 heterozygous and 4 wild type mice] were sacrificed under deep anesthesia of diethyl ether. Genotyping of the offspring was carried out according to the previous method (Tucci et al., 1996). In brief, extracted DNA was digested with EcoRI-KpnI. Following digestion, samples were electrophoresed on 0.8% agarose gels and transferred to membranes (Hybond N⫹, Amersham, Bucks, UK) by Southern blotting. A 1.0 kb fragment, encompassing mouse PTHrP exon V, was used as a cDNA probe. The unaltered PTHrP allele was seen as an 11 kb fragment. Heterozygous mice showed both normal and disrupted (6.5 kb) fragments, while PTHrP deficient mice displayed only the altered 6.5 kb fragment.
Tissue Processing The heads of neonatal mice were fixed in 2.0% paraformaldehyde–2.5% glutaraldehyde solution. After decalcification in 10% EDTA and dehydration in a graded series of ethanol, they were embedded in a water-soluble resin, cut in 2 µm serial sections in the sagittal, frontal, or coronal plane. All the sections were stained with toluidine blue (pH 7.0).
Skeletal Staining For visualization of the skeletal components in the craniofacial region, heads were dissected, fixed in 95% alcohol and acetone, stained with alizarin red S and alcian blue [0.3% alcian blue 8GX (EM Science) in 70% ethanol-1 volume, 0.1% alizarin red S (Wako chemical) in 95% ethanol-1 volume, acetic acid-1 volume, 70% ethanol-17 volumes] for 3 days, cleared first in 1% KOH for 3 days, then in 20% glycerin containing 1% KOH for 5 days and in 50% glycerin for 7 days, stored in 70% glycerin as previously described (McLeod, 1980).
453
RESULTS Anterior Cranial Base The midline structure of the anterior cranial base was cartilaginous in the new born wild type mice. The phenotype of the chondrocytes were spherical in the anterior part (Fig. 1A), flattened in the central part (Fig. 1C) and somewhat distended in the posterior part (Fig. 1E). In the homozygous PTHrP deficient mice, chondrocytes composing the anterior and central part showed no remarkable difference from those of the wild type mice (Fig. 1B,D). However, the chondrocytes in the posterior part had a marked hypertrophic phenotype (Fig. 1F). The structure of the anterior cranial base in the heterozygous PTHrP deficient mice did not show any difference from that of wild type mice (data not shown).
Posterior Cranial Base In the wild type mice, the basioccipital (bo) and basisphenoid bone (bs) had already ossified. The presphenoid bone (ps) was just beginning to form bone and remained mostly cartilaginous, consisting of hypertrophic chondrocytes (Fig. 2A). The intersphenoid synchondrosis (iss) and the sphenooccipital synchondrosis (sos) were already formed, and the former was longer than the latter (Fig. 2A,B,C). The histological appearance of the posterior cranial base in the homozygous mice was quite different from that of the wild type mice. While the basioccipital bone and basisphenoid bone had already ossified as in wild type, the presphenoid bone showed accelerated endochondral ossification (Fig. 2D). In addition, the intersphenoidal synchondrosis was ovoidal and all of the chondrocytes showed the hypertrophic phenotype (Fig. 2E). In the spheno-occipital synchondrosis, the process of endochondral bone formation was dramatically advanced on both sides. Hypertrophic chondrocytes remained only in the central part of the synchondrosis and thick layers of the bone-like matrix covered the spheno-occipital synchondrosis from the upper and lower sides (Fig. 2F). The structure of the posterior cranial base in the heterozygous mice did not show any difference from that of wild type mice (data not shown).
Meckel’s Cartilage The skeletal staining of the wild type mice showed that the soft tissue-running mid-portion of Meckel’s cartilage was observed as a cartilaginous rod structure stained by alcian blue (Fig. 3A,B). Histological examination of this portion showed that it was composed of hypertrophic chondrocytes and metachromatic stained matrix (Fig. 3C). This portion, which ultimately degenerates and converts to ligament in the normal developmental process (Mu¨hlhauser, 1986), was dramatically different in the homozygous mice. Alizarin red staining showed that calcification was well underway in the corresponding portion (Fig. 3D,E). Chondrocytic cells were scarcely observed and the cartilage matrix was scattered inside the rod, which was surrounded by a thin layer of bone-like matrix (Fig. 3F). There was no difference on histological observation between heterozygous and wild type mice (data not shown).
Condylar Cartilage The skeletal staining revealed the condylar cartilage at the distal edge of the mandibular ramus, above the
454
ISHII-SUZUKI ET AL.
Fig. 1. Cartilaginous anterior cranial base in wild type mice (A,C,E) and in homozygous mice (B,D,F). Anterior (A,B), central (C,D) and posterior regions (E,F). Note that the size and morphology of the chondrocytes located in the anterior and central part of homozygous mice were almost the same as those of the wild type mice. In the posterior region, the chondrocytes of the homozygous mice were much larger and more hypertrophic, compared with those of wild type mice. Scale bars ⫽ 50 µm.
Fig. 2. Sagittal section of the posterior cranial base in wild type mice (A,B,C) and in homozygous mice (D,E,F). Higher magnification of the intersphenoid synchondrosis (B,E) and sphenooccipital synchondrosis (C,F). Note the accelerated chondrocytic differentiation in the intersphenoid and spheno-occipital synchondrosis of the homozygous mice, the latter is more severely accelerated. ps ⫽ presphenoid bone, iss ⫽ intersphenoid synchondrosis, bs ⫽ basisphenoid bone, sos ⫽ spheno-occipital synchondrosis, bo ⫽ basioccipital bone. Scale bars ⫽ 500 µm in A,D and 200 µm in B,C,E,F.
angular cartilage (Fig. 4A). The cell layers of the cartilage were examined histologically in the condylar cartilage of wild type mice by toluidine blue staining. From the articular, an articular layer (a), flattened cell layer (f) and hypertrophic cell layer (h) were distinguishable (Fig. 4B). In the homozygous mice, the condylar cartilage showed a proportional reduction in size compared with that of wild type (Fig. 4C). However, the cell layers were scarcely affected and appeared to have similar architectures to those of the wild type (Fig. 4B,D).
The condylar cartilage of the heterozygous mice did not show any difference from that of wild type mice (data not shown).
DISCUSSION The role and function of PTHrP during the processes of chondrocytic differentiation and endochondral ossification has been highlighted by reports using the PTHrP deficient mouse (Amizuka et al., 1994; Karaplis et al., 1994).
PTHrP DEFICIENCY AND CRANIOFACIAL CARTILAGES Fig. 4. Lateral view of the skeletal staining showing the ramus region in wild type mice (A) and in homozygous mice (C). Note that the size of articular process is markedly diminished in the homozygous mice compared to that of wild type mice (stars). Coronal section of the condylar cartilage in homozygous mice (D) and wild type mice (B). Note that the cell layers of the mandibular condylar cartilage were scarcely affected in homozygous mice. a ⫽ articular layer, f ⫽ flattened cell layer, h ⫽ hypertrophic cell layer. Scale bars ⫽ 1 mm in A, C and 100 µm in B, D.
455
Fig. 3. Skeletal staining with alizarin red S and alcian blue in wild type mice (A,B) and in homozygous mice (D,E). Oblique ventral view of the mandibular bone (A,D) and higher magnification of the ramus region (B,E). Note the rod of Meckel’s cartilage (arrows) stained by alcian blue in the wild type littermate and the corresponding area stained by alizarin red S (arrowheads) in the homozygous mice. The histological appearance of the mid-portion of Meckel’s cartilage in the homozygous mouse (F) and the wild type mice (C), respectively. Note the ectopic bone formation in the homozygous mice (*). Scale bars ⫽ 1 mm in A,B,D,E and 50 µm in C,F.
456
ISHII-SUZUKI ET AL.
Detailed examinations of growth plate cartilage have indicated PTHrP as an essential factor for growth and maturation of growth plate cartilage (Amizuka et al., 1996; Weir et al., 1996), and for regulation of the pace of chondrocytic development and terminal differentiation (Lee et al., 1996; Vortkamp et al., 1996). The mRNA expression of PTHrP and PTH/PTHrP receptor suggests that PTHrP functions in an autocrine/paracrine manner during these processes (Amizuka et al., 1996). In addition to the abnormalities observed in the limbs, the null mutant mice have been reported to exhibit craniofacial abnormalities, such as a domed shaped skull, and short snout and mandible (Karaplis et al., 1994). We hardly recognized any difference in the cartilaginous anterior cranial base, except for the posterior part, in the mutant mice. The anterior part of the anterior cranial base remains cartilaginous throughout life and forms the nasal septum cartilage (Koski, 1975). Other permanent cartilages, such as the cartilage of trachea, larynx, and articular surfaces are also reported to be histologically normal in the homozygous mice (Karaplis et al., 1994). In contrast to this anterior part, chondrocytes showed marked hypertrophy in the posterior part of the anterior cranial base of homozygous mouse. This region is known to undergo endochondral bone formation soon after birth and forms the perpendicular plate of the ethmoid bone (Ruch, 1990). Our observations showed that PTHrP deficiency induces an acceleration in the process of ethmoid bone formation. The synchondroses can be described as double-faced growth plates. Chondrocytic differentiation progresses from the center of synchondrosis towards both edges, where endochondral bone formation is taking place (Koski, 1975; Ghafari, 1992). In the homozygous mouse, marked acceleration of chondrocytic differentiation was observed in the intersphenoid and spheno-occipital synchondrosis. Interestingly, the level of acceleration of endochondral bone formation was not equivalent between these synchondroses, but was further progressed in the spheno-occipital synchondrosis than in the intersphenoid synchondrosis. The caudal-rostral gradient is a feature of bone development of the midline cranial base (Roberts and Blackwood, 1983). At birth, the posterior located spheno-occipital synchondrosis was considerably shorter than the intersphenoid synchondrosis in the normal animal, hence considered it was in a more advanced state of endochondral bone formation. Since PTHrP deficiency affects the process of endochondral bone formation, the spheno-occipital synchondrosis was affected more severely in the homozygous mouse than the intersphenoid synchondrosis at this developmental stage. Compared with the cranial base cartilages, Meckel’s cartilage and condylar cartilage showed characteristic changes in homozygous mice. Meckel’s cartilage has been reported to play a supportive role for mandibular development (Hall, 1982; Yamazaki et al., 1997) and there are three distinct regions each having an apparently different fate (Bhaskar, 1953; Yamazaki et al., 1997). The anterior region of Meckel’s cartilage contributes to mandibular development and undergoes endochondral ossification. The most posterior region also undergoes endochondral ossification and gives rise to the malleus and incus. The mid region, distal to the ossification center of the mandibular anlage, degenerates and gives rise to the sphenomandibular ligament (Richany et al., 1956; Savostin-Asling et al., 1973; Frommer and Margolies, 1971; Goret-Nicaise
and Pilet, 1983). The most dramatic difference observed in Meckel’s cartilage of the PTHrP deficient mice was in this soft tissue-running mid-region. During normal development, the degeneration of this region has been reported to start at around day 18 of gestation in mouse (Frommer and Margolies, 1971) with a lack of type X collagen in the extracellular matrix (Chung et al., 1995). Mu¨hlhauser (1986) studied this process histologically in rat and described that this starts first at the perichondrium, where macrophage- and fibroblast-like cells appear to degrade the unmineralized cartilage matrix, and the chondrocytes are finally attacked by the giant cells. In the homozygous mice, the cartilage of this area was degenerating and was surrounded by the presumptive bone matrix. Similar ectopic bone formation has also been reported in the perichondrium of rib cartilage in the homozygous mice (Karaplis et al., 1994). These observations suggest that the PTHrP deficiency might alter the direction of normal cell differentiation. Compared to other cartilage, condylar cartilage has several distinguishing characteristics (Luder et al., 1988; Shibata et al., 1997). It appears late in fetal life after the formation of other cartilages, that is, condylar cartilage appears at around day 15 of gestation, with peculiar type I collagen containing extracellular matrix (Tengan, 1990; Ishii et al., 1998), while the cartilages of the limb buds, for example, appear at around day 13 of gestation (Ruch, 1990). Moreover, unlike other cartilages which start as mesenchymal cell condensation, condylar cartilage appears from the posterior end of the periosteum covering mandibular anlage and shares a common cellular origin which undergoes intramembranous bone formation (Miyake et al., 1997). This cartilage functions as a growth cartilage during the fetal and early postnatal periods, however, it serves only as an articular cartilage after growth (Copray et al., 1988). A difference is also indicated in the way of growth: interstitial in growth plate cartilage vs. appositional in condylar cartilage (Dibbets, 1990). The condylar cartilage of homozygous mice showed a proportional reduction in size, although the histological architecture and cell layers (articular layer, flattened cell layer and hypertrophic cell layer) were conserved. The response of condylar cartilage to PTHrP deficiency might be related to one of the above mentioned characteristic features, however, the exact mechanism remains unclear. In summary, we have examined the various cartilages in the craniofacial region of the PTHrP deficient mouse and found the effect of PTHrP deficiency had wide variations in these cartilages. The permanent cartilage forming the future nasal septum did not show any difference. However, the chondrocytic maturation and the process of endochondral bone formation were accelerated in the posterior part of the anterior cranial base and in the cranial base synchondroses. A proportional reduction in the size of the condylar cartilage and perichondrial ectopic bone formation in the mid-portion of Meckel’s cartilage were also observed. These variations in response to lack of PTHrP might be related to the characteristic features and fates of each cartilage.
ACKNOWLEDGMENTS The authors are grateful to Professor T. John Martin, St. Vincent’s Institute of Medical Research, for helpful discussion.
PTHrP DEFICIENCY AND CRANIOFACIAL CARTILAGES
LITERATURE CITED Amizuka N, Warshawskiy H, Henderson JE, Goltsman D, Karaplis AC. 1994. Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation. J Cell Biol 126:1611–1623. Amizuka N, Henderson JE, Hoshi K, Warshawskiy H, Ozawa H, Goltzman D, Karaplis AC. 1996. Programmed cell death of chondrocytes and aberrant chondrogenesis in mice homozygous for parathyroid hormone-related peptide gene deletion. Endocrinology 137:5055– 5067. Bhaskar SN, Weinmann JP, Schour I. 1953. Role of Meckel’s cartilage in the development and growth of the rat mandible. J Dent Res 32:398–410. Chung KS, Park HH, Ting K, Takita H, Apte SS, Kuboki Y, Nishimura I. 1995. Modulated expression of type X collagen in the Meckel’s cartilage with different developmental fates. Dev Biol 170:387–396. Copray JCVM, Dibbets JMH, Kantomaa T. 1988. The role of condylar cartilage in the development of the temporomandibular joint. Angle Orthod 58:369–380. Dibbets JMH. 1990. Introduction to the temporomandibular joint. In Enlow DH, editor. Facial growth, 3rd ed. Philadelphia: WB Saunders; p 149–163. Diewert VM. 1982. Contributions of differential growth of cartilages to changes in craniofacial morphology. In Dixon AD, Sarnat BG, editors. Factors and mechanisms influencing bone growth. Proceedings of the international conference held at Center for the Health Sciences, University of California, Jan. 5–7, 1982. New York: Alan R. Liss; p 229–242. Frommer J, Margolies MR. 1971. Contribution of Meckel’s cartilage to ossification of the mandible in mice. J Dent Res 50:1260–1267. Ghafari J, Heeley JD, Shapiro IM. 1992. Morphologic study of cartilages of the rat craniofacial skeleton: classification as osteogenic and/or supportive. In Davidovich Z, editor. The biological mechanisms of tooth movement and craniofacial adaptation. The Ohio State University: p 445–453. Goret-Nicaise M, Pilet D. 1983. A few observations about Meckel’s cartilage in the human. Anat Embryol 167:365–370. Hall BK. 1982. How is mandibular growth controlled during development and evolution? J Craniofac Genet Dev Biol 2:45–49. Ishii M, Suda N, Tengan T, Suzuki S, Kuroda T. 1998. Immunohistochemical findings of type I and type II collagen in prenatal mouse mandibular condylar cartilage compared to the tibial anlage. Arch Oral Biol 43:545–550. Ju¨ppner H, Abou-Samra AB, Freeman M, Kong XM, Schipani E, Richards J, Kolakowski LF Jr, Hook J, Potts JT Jr, Kronenberg HM, Serge GV. 1991. A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide. Science 254:1024– 1026. Karaplis AC, Luz A, Glowacki J, Bronson RT, Tybulewicz VLJ, Kronenberg HM, Mulligan RC. 1994. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev 8:277–289. Koski K. 1975. Cartilage in the face. Birth Defects: Original Article Series, Volume XI, 7:231–254. Lee K, Lanske B, Karaplis AC, Deeds JD, Kohno H, Nissenson RA. 1996. Parathyroid hormone-related peptide delays terminal differen-
View publication stats
457
tiation of chondrocytes during endochondral bone development. Endocrinology 137:5109–5118. Luder HU, Leblond CP, von der Mark K. 1988. Cellular stages in cartilage formation as revealed by morphometry, radioautography and type II collagen immunostaining of the mandibular condyle from weanling rats. Am J Anat 182:197–214. McLeod MJ. 1980. Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratology 22:299– 301. Miyake T, Cameron AM, Hall BK. 1997. Stage-specific expression patterns of alkaline phosphatase during development of the first arch skeleton in inbred C57BL/6 mouse embryos. J Anat 190:239– 260. Mu¨hlhauser J. 1986. Resorption of the unmineralized proximal part of Meckel’s cartilage in the rat: a light and electron microscopic study. J Submicrosc Cytol 18:717–724. Richany SF, Bast TH, Anson BJ. 1956. The development of the first branchial arch in man and the fate of Meckel’s cartilage. Q Bull Northwestern Univ Med School 30:331–355. Roberts GJ, Blackwood HJJ. 1983. Growth of the cartilages of the mid-line cranial base: a radiographic and histological study. J Anat 136:307–320. Rodan SB, Insogna KL, Vignery AMC, Stewart AF, Broadus AE, D’Souza SM, Bertolini DR, Mundy GR, Rodan GA. 1983. Factors associated with humoral hypercalcemia of malignancy stimulate adenylate cyclase in osteoblastic cells. J Clin Invest 72:1511–1515. Ruch R. 1990. Organogeny. In: The mouse: its reproduction and development. New York: Oxford University Press; p 208–295. Savostin-Asling I, Asling CW. 1973. Resorption of calcified cartilage as seen in Meckel’s cartilage of rats. Anat Rec 176:345–359. Shibata S, Fukada K, Suzuki S, Yamashita Y. 1997. Immunohistochemistry of collagen type II and X, and enzyme-histochemistry of alkaline phosphatase in the developing condylar cartilage of the fetal mouse mandible. J Anat 191:561–570. Stewart AF, Insogna KL, Goltzman D, Broadus AE. 1983. Identification of adenylate cyclase-stimulating activity and cytochemical glucose-6-phosphate dehydrogenase-stimulating activity in extracts of tumours from patients with humoral hypercalcemia of malignancy. Proc Natl Acad Sci USA 80:1454–1458. Tengan T. 1990. Histogenesis and three-dimensional observation on condylar cartilage in prenatal mice. J Oral Stomatol 57:17–42. Theiler K. 1989. The house mouse: atlas of embryonic development. New York: Springer-Verlag. Tucci J, Hammond VE, Senior PV, Gibson A, Beck F. 1996. The role of fetal parathyroid hormone-related protein in transplacental calcium transport. J Mol Endocrinol 17:159–164. Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. 1996. Regulation of rate of cartilage differentiation by indian hedgehog and PTH-related protein. Science 273:613–621. Weir EC, Philbrick WM, Amling M, Neff LA, Baron R, Broadus AE. 1996. Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation. Proc Natl Acad Sci USA 93:10240–10245. Yamazaki K, Suda N, Kuroda T. 1997. Immunohistochemical localization of parathyroid hormone-related protein (PTHrP) in developing mouse Meckel’s cartilage and mandible. Arch Oral Biol 42:787–794.
