Bone Morphogenetic Protein-7 (Osteogenic Protein-1, OP-1) and Tooth Development by Marilia Vidal Villar

Journal of Dental Research http://jdr.sagepub.com/

Bone Morphogenetic Protein-7 (Osteogenic Protein-1, OP-1) and Tooth Development M.N. Helder, H. Karg, T.J.M. Bervoets, S. Vukicevic, E.H. Burger, R.N. D'Souza, J.H.M. Wรถltgens, G. Karsenty and A.L.J.J. Bronckers J DENT RES 1998 77: 545 DOI: 10.1177/00220345980770040701 The online version of this article can be found at: http://jdr.sagepub.com/content/77/4/545

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On behalf of: International and American Associations for Dental Research

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J Dent Res 77(4): 545-554, April, 1998

Bone Morphogenetic Protein-7 (Osteogenic Protein-i, OP-i) and Tooth Development M.N. Helderla, H. Kargla, T.J.M. Bervoets1, S. Vukicevic3, E.H. Burgerl, R.N. D'Souza4, J.H.M. Wioltgens1, G. Karsenty2, and A.L.J.J. Bronckerslb 1Department of Oral Cell Biology, ACTA, Vrije Universiteit, van der Boechorststr 7, 1081 BT Amsterdam, The Netherlands; 2Department of Molecular Genetics, M.D. Anderson Cancer Center, Houston, Texas, USA; 3Bone Research Branch, National Institutes of Health, Bethesda, Maryland, USA; 4Department of Anatomical Sciences, Dental Branch, University of Texas, Houston, Texas, USA; aauthors who contributed equally to this work; bcorresponding author

Abstract. Bone morphogenetic proteins (BMPs) form a family of growth factors originally isolated from extracellular bone matrix that are capable of inducing bone formation ectopically. We studied the expression, tissue localization, and function of BMP-7 (OP-1) during tooth development in rodents. Patterns of BMP-7 gene expression and peptide distribution indicated that BMP-7 was present in dental epithelium during the dental lamina, bud, and cap stages. During the bell stage, BMP-7 mRNA expression and protein distribution shifted from dental epithelium toward the dental mesenchyme. With advancing differentiation of odontoblasts, BMP-7 protein staining in the dental papilla became restricted to the layer of fully functional odontoblasts in the process of depositing (pre)dentin. Secretorystage ameloblasts exhibited weak immunostaining for BMP7. A restricted pattern of staining in ameloblasts became apparent in post-secretory stages of amelogenesis. Also, cells of the forming periodontal ligament were immunopositive. Histological analysis of tooth development in neonatal BMP-7-deficient mice did not reveal obvious changes compared with wild-type mice. We conclude that, in developing dental tissues, BMP-7 has distribution and expression patterns similar to those of other BMP members but is not an essential growth factor for tooth development, possibly because of functional redundancy with other BMP members or related growth factors.

Key words: bone morphogenetic protein-7, tooth formation, in situ hybridization, immunolocalization, gene knockout.

Received December 29, 1996; Last Revision July 14, 1997; Accepted August 28,1997

Introduction Intramuscular implantation of demineralized bone matrix induces the ectopic formation of cartilage, bone, and bone marrow (Urist, 1965). The cascade of cellular events occurring in ectopic bone formation is reminiscent of embryonic endochondral bone differentiation in long bones and fracture healing (Rosen and Thies, 1992; Vukicevic et al., 1993). The factors responsible for the bone-inducing activity form a Bone Morphogenetic Protein (BMP) subfamily of the TGF3 superfamily (e.g., Wozney et al., 1988; Sampath et al., 1990; Ozkaynak et al., 1992). All BMP members-extracted from tissues as well as their recombinantly produced forms, e.g., recombinant human BMP-2 (BMP-2A), -4 (BMP-2B), -6 (Vgr-1) and -7 (OP-1)-are capable of ectopically inducing cartilage and bone formation in vivo (Wozney et al., 1988; Wang et al., 1990; Ogawa et al., 1992; Sampath et al., 1992). BMPs, including BMP-7 (osteogenic protein-i, OP-1), can also induce osteoblast and chondroblast differentiation in vitro and accelerate bone healing in vivo (Rosen and Thies, 1992; Asahina et al., 1993; Dieudonne et al., 1994; Maliakal et al., 1994). Consistent with their putative role during skeletogenesis, BMPs are highly expressed in skeletal tissues (Vukicevic et al., 1994; Helder et al., 1995a,b; Lyons et al., 1995). The absence of some of the BMPs (BMP-4, -5, -7) or of BMP-like factors (growth and differentiation factor-5, GDF5) in mutant mouse strains caused skeletal abnormalities which provide genetic evidence for their role in bone and cartilage formation (Kingsley, 1994; Storm et al., 1994; Dudley et al., 1995; Luo et al., 1995). There is accumulating evidence that BMPs exert important regulatory roles other than bone induction, in particular as signaling molecules during the formation of extraskeletal tissues (Vainio et al., 1993; Wall et al., 1993; Vukicevic et al., 1994; Helder et al., 1995a,b; Lyons et al., 1995). Based on functional studies using gene targeting methods to inactivate BMP members (e.g., Storm et al., 1994; Dudley et al., 1995; Luo et al., 1995), it becomes increasingly clear that the BMPs have multiple roles in the development 545

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Holder et al.

I Dciit Rcs 77(4) 1998 mice; 12 days embryonic 5 days post-natal hamsters; I to 3 days neonatal rats) were recovered and decapitated, and the heads were fixed overnight at 4 C in a freshly prepared solution of 4%/ paraformaldehyde in phosphate-buffered saline (PBS). Some specimens were decalcified for 3 to 7 days in a solution containing 2% of the disodium salt of ethyleniediaminetetraacetic acid (EDTA) and 4', paraformaldelhyde in PBS. Next, tissues were dehydrated in a graded series of ethaniol, embedded in paraffin, and sectioned sagittally, unless otherwise indicated. Experimental protocols concernlilng animal handling were approved by the ethical committee of the Free University in Amsterdam.

Figure 1. In situ hybridization for BMP-7 mRNA in the craniofacial area of the embryonic mouse (E18). High expression is noted in bell-stage first molar tooth germs (M1, M2), incisor (1), and the lessadvanced second molar (M), and in the surrounding osteogenic areas, whisker and hair follicles (small arrows), choroid plexes, meninges of the brain, and suprabasal levels of the skin. Darkfield (9x). Bar= I mm.

of skeletal as well as extraskeletal tissues. Like bone, dentin extracts contain BMP activity (Butler et al., 1977; Bessho et al., 1991; Nakashima, 1994), and studies have indicated that BMPs are locally produced during early embryonic tooth development (Vainio et al., 1993; Heikinheimo, 1994; Bennet et al., 1995; Helder et al., 1995a,b; Turekova ct al., 1995). Evidence that BMPs are capable of acting as signaling molecules during tooth development came from functional in vitro studies of embryonic teeth (Lesot et al., 1993; Vainio et al., 1993; Begue-Kirn et al., 1994) or dental pulp cells (Nakashima et al., 1994). In mature erupted teeth, BMP-3 stimulates repair of periodontal ligament (Ripamonti et al., 1994), and BMP-7 enhances the formation of reparative dentin (Rutherford et al., 1993; Nakashima, 1994) and dental cementum (Ripamonti et al., 1996). These findings support the idea that BMP members act as important signaling molecules in early tooth development and are also effective stimulators of repair processes in mature dental tissues. To study the function of BMP-7 in dental development, we examined the expression pattern and tissue localization of BMP-7 (osteogenic protein-1) in embryonic and neonatal stages of tooth development, using immunohistochemistry and in situ hybridization. We also examined tooth development in a BMP-7-deficient mouse strain (Luo et al., 1995).

Materials and methods Tissues Time-staged pregnant mice (CD-1 Swiss; gestation time, 20 days; vaginal plug 0.5 days p.c), golden hamsters (Mesocricetus auratis L; gestation time, 16 days), and rats (Sprague-Dawley) were purchased from local suppliers (Harlan, Zeist, The Netherlands). Embryos (17 days embryonic-3 days post-natal

Probe preparation For the detection of BMP-7 transcripts in the mouse, two different stretches of a full-length mouse BMIP-7 cDNA clone (Ozkaynak ft al., 1991) were selected: (1) a 676-base-pair (bp) BstXI/BglJ fragment covering amino acids 92 to 291 of the prepro region and the first 25 amino acids of the N-terminal part of the mature molecule; and (2) a 338-bp stretch of the 3' noncoding region, obtained by digestion of the full-length cDNA clone with Earl and Pstl. Both fragments were subcloned in a pBluescript II (SK)+ vector (Stratagene, La Jolla, CA), from which the Kpnl-Clal and Spel-Sacl Multiple Cloning Site (MCS) fragments were deleted to reduce the chance of crosshybridization with ribosomal RNA sequences (Witkiewicz et al., 1993). On the basis of computer analysis, all probe constructs were determined to be specific for BMP-7 and non-crossreactive with any of the other known BMPs. Ia-35SIUTP-labeled single-stranded sense and antisense RNA probes (specific activities, 0.3 to 1 x 109 dpm/pg) were prepared by in oitro transcription. The sizes of the long riboprobe fragments were reduced to an average length of 200 bp by limited alkaline hydrolysis, while the transcripts of the short constructs were used without hydrolysis. In situ hybridization For in situ hybridization, mouse sections were de-paraffinized, rehydrated, and post-fixed for 5 min in 4% paraformaldelhyde. Pre-treatments included: incubation in 0.2 N HCI (5 min), proteinase K-digestion (20 pg/mL; 7.5 min), additional postfixation (5 min); blocking with iodoacetamide (0.37 g/400 mL) and N-ethylmaleimide (0.25 g/400 mL), and treatment with acetic anhydride (0.5K7O in 0.1 M triethanolamine-HCI, pH 8.0; 2 x 10 min). After a short pre-hybridization, sections were hybridized under siliconized coverslips for 16 to 18 hrs at 50°C in a humid chamber in hybridization buffer (50'S formamide, 10% dextrane sulfate, 4 x SSC [SSC = 0.15 M NaCl, 0.015 M NaCitrate, pH 7.0], 10 mM dithiothreitol (DTT), I x Denhardt [0.02% each of bovine serum albumin, polyvinylpyrolidone, Ficoll 400], 500 pg/mL each of salmon sperm DNA and yeast tRNA, and 0.2 to 0.4 ng/pL "5S-labeled riboprobe). Washing steps included: 15 min in 2 x SSC at 50°C; 20 min in 500% formamide/2 x SSC/20 mM DTT at 65°C; 2 x 10 min in TEN buffer (TEN=10 mM Tris-HCI, pH 7.5; 5 mM EDTA; 0.5 M NaCI) at 37°C; 30 min in TEN buffer containing 20 pg/mL RNAse A at 37°C; again 10 min in TEN buffer at 37°C; twice for 15 min in 2 x SSC at 65°C; and finally twice for 15 min in 0.1 x

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BMP-7 and Tooth Development

j Dent Res 77(4) 1998

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SSC at 65째C. Sections were then dehydrated through an ascending series of ethanol containing 0.3 M NH4Ac, covered with NTB2 emulsion (Kodak), and exposed between I and 3 wks. After being developed, slides were counterstained for 10 sec with 0.1% toluidine blue, dehydrated, and mounted in Depex mounting medium.

Immunohistochemistry Polyclonal antibodies raised in rabbits and chicken and mouse monoclonal antiFigure 2. In situ/ hybridization of BMP-7 mRNA in early bell-stage first molar mouse tooth germ (E17), bodies were kindly proantisense (A, 100x) and sense (B, 100x). Signal in (A) is concentrated over the inner enamel epithelium of vided by Drs. J. Maliakal both first molars and second molar. Bar =100 pm. and K. Sampath (Creative Biomolecules, Hopkinton, sorbed antiserum. Furthermore, the immunostaining patterns of MA, USA). The rabbit anti-BMP-7 antibodies (batch #3331) were mouse embryos were verified on in sitiu data on BMP-7 mRNA raised against a mixture of the mature domain of human (Helder et al., 1995a; Luo et al., 1995; Lyons et al., 1995). For recombinant (rec h) BMP-7 (amino acids 293 through 431) and tissue sections from mice, rats, and hamsters immunostaining the prodomain of BMP-7 (amino acids 29 to 292; Jones et al., the antibodies, we used ABC-peroxidase Elite with polyclonal chromatography A affinity by protein 1994) and purified kits (Vector Labs, Burlingame, CA, USA). To avoid cross(Vukicevic et al., 1994). The chicken polyclonals (#881) and reactivity of the second anti-mouse IgG antibody with mouse monoclonals (12G3, Vukicevic et al., 1994)) were raised against the mature domain of rec hBMP-7. On b Western blots, these antibodies reacted with BMP. 7; the chicken polyclonals and mouse monoclonals reacted with the mature b domain only; the rabbit

polyclonals reacted with both

the

prodomain

(strongly) and the mature domain (weak, personal communication, Drs. W.K. Jones and Maliakal, Creative Biomolecules). The strongest one, the rabbit polyclonals, cross-reacted very slightly with BMP-2 (Dr. Maliakal, Creative Biomolecules, personal communication). Preliminary experiments in which these antibodies were cross-adsorbed to the mature portion rec hBMP2 (kindly donated by Dr. E.A. Wang, Genetics Institute, Cambridge, MA, USA) gave the same distribution pattern as unad-

3Figure 3. Inz situ hybridization for BMP-7 mRNA in late-bell and mineralization stages of a two-day-old mouse maxilla. Brightfield (a, 48x; bar = 200 pm) and darkfield (b, 55x; bar = 200 pm). In M2 tooth germs, transcripts are present in the lay of differentiating and young odontoblasts (arrowheads). In M1, is increased in functio: nal odontoblasts and also detectable in pre-ameloblasts and secretory expression inn er aspect of the surrounding alveolar bone tissues facing the tooth germs ameloblasts (large arrows). The (small arrows) contains signals for BMP-7, maybe associated with osteoclasts. a, ameloblasts; b, bone; df, dental follicle; dp, dental papilla; o, odontoblasts.

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J Dent Res 77(4) 1998

BMP-7 and Tooth Development

Figure 4. (left) Immunostaining for BMP-7 during dental lamina formation. (a) Mouse E12.5 first molar (440x, bar =50 pm); (b) hamster E12 first molar (272x, bar = 50 pm). Oral epithelium (oe) starts to form a dental lamina. Figure 5. (left) Immunostaining for BMP-7 in cap-stage first molar tooth, hamster E13 (110x, bar = 50 pm). The central portion of the enamel organ (eo) stains moderately strongly, and the inner enamel epithelium adjacent to condensing dental mesenchyme (dm) stains weakly.

Figure 6. (left) Immunostaining for BMP-7 in early bell-stage first molar tooth, hamster E15 (110x, bar = 100 pm). Most cells of the inner enamel epithelium (iee) and all cells of the stratum intermedium are positive for BMP-7; dental papilla (dp) of the left cusp stains weakly. Inner enamel epithelium between both arrows is negative. Not counterstained. Figure 7. (left) Immunostaining for BMP-7 in late bell-stage and mineralization stages in first molar tooth germs in the mouse on post-natal day 3 (61x, bar = 200 pm). Odontoblasts (o), some subodontoblastic cells, pre-ameloblasts, and early secretory ameloblasts (sa) in the first molar (Ml) are moderately to strongly positive. Staining in the ameloblast layer is variable and sometimes occurs in groups. Small arrows indicate strong staining in the basal (infranuclear) portions of ameloblasts. Staining is less pronounced in the less-advanced second molar (M2). po, pre-odontoblasts. Not counterstained. Figure 8. (left) Immunostaining for BMP-7 in the lower incisor, illustrating differences in staining patterns between cells of the developing enamel organ and those in the dental papilla (parasagittal section, hamster, post-natal day 5; 43x, bar = 200 Pm). Consecutive stages of differentiation, from bottom to top in the direction of the arrow: inner enamel epithelium (iee), preameloblast (pa), secretory ameloblasts (sa), and maturation phase ameloblasts (ma). Strongest staining is observed in all fully differentiated odontoblasts (o). In the enamel organ, most staining is detected in the inner enamel epithelium, in some preameloblasts, and in early secretory ameloblasts. Fully secretory ameloblasts stain only very weakly (not apparent at this magnification). Individual post-secretory (maturation phase) ameloblasts (arrowheads) near positive-staining maturation-phase enamel (asterisk) stain strongly. Strong staining is also seen in bone cells in or along alveolar bone (ab), particularly in osteoclasts (small arrows). e, enamel; d, crown dentin; rd, root dentin; p, pulp; pd, predentin; pl, forming periodontal ligament; po, preodontoblasts. Not counterstained.

endogenous IgG when using mouse monoclonals (particularly on mouse sections), we purchased a HistomouseTM SP kit (Zymed, San Francisco, CA, USA), especially designed for staining mouse tissues with (primary) mouse antibodies. Peroxidase was developed in a freshly prepared solution of 0.05% 3,3'-diaminobenzidinetetrahydrochloride (DAB), and 0.01 % H202 in 0.05 M Tris, pH 7.2. Unless otherwise stated, sections were counterstained with methylene green. In control sections, the primary antibodies were substituted by normal, non-immune IgGs from the same species (Vector Labs, Burlingame, CA).

Analysis of tooth development in BMP-7-deficient mice By embryonic stem cell technology, BMP-7-deficient C57BL mice were generated in which exons 5 and 6, coding for the mature BMP-domain, were deleted (Luo et al., 1995). Within 1

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hr after birth, pups were decapitated; the heads were fixed in 5% paraformaldehyde in phosphate buffer or in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer and embedded in Spurr's resin. Tail samples were used for Southern blotting for confirmation of genotype. In a prelimary analysis of two mutant heads, sections were examined in the approximate area of the developing dentition. In a more systematic analysis, 4 heads (2 wild-type, 2 mutants with apparent eye and patterning defects) were serially sectioned by means of a motor-driven Polycut S Reichert-Jung microtome. To examine the consecutive stages of differentiation of developing molars and incisors within the same section, we cut 1 wild-type and 1 mutant head in half along the median plane and sectioned all 4 halves sagittally (380 to 500 sections/block). Sections 5 pm thick were collected per block, mounted on glass slides, and stained with toluidine blue. To investigate bilateral symmetry of the developing dentition, we also sectioned 1 wild-type and 1 mutant head in a frontal plane, rostrally to caudally, and collected and stained 640 (-I-) and 995 (+/+) serial 5-pm-thick sections.

Results In situ hybridization of BMP-7 in developing tooth germs BMP-7 mRNA transcripts in 17- (E17) and 18- (E18) day mouse embryos revealed expression in a number of developing craniofacial tissues (Fig. 1), including the dental tissues, osteogenic and chondrogenic tissues, whisker and hair follicles, muscular tissues, and epithelia of the skin. In early bell-stage tooth germs, BMP-7 transcripts were mainly present in cells of the inner enamel epithelium and weakly in the adjacent cells of the dental papilla (Fig. 2). In more advanced bell-stage molar development (E18), the intensity of BMP-7 signal over the dental papilla increased compared with E17, with slightly higher levels in the pre-odontoblast layer (not shown). Labeling decreased (but did not disappear) in enamel epithelium and gradually increased in odontoblasts in the late bell stage (Fig. 3, second molar). In the odontoblast layer, the signal for BMP-7 mRNA became progressively stronger during odontoblast differentiation. In neonatal explants after the onset of dentin formation, high expression levels were apparent in functional odontoblasts and weak-to-moderate levels in differentiating and secretory ameloblasts (Figs. 3A, 3B; first molar). Essentially the same distribution pattern was seen in developing incisor tooth germs: At the apical aspect containing most embryonic parts, transcripts were detected in the inner enamel epithelium, and with ongoing development, signals decreased in the enamel epithelium but increased in (pre)odontoblasts (not shown).

Immunolocalization of BMP-7 in developing tooth germs Generally, the rabbit antibodies (#3331) reacted strongly at intracellular locations but very weakly extracellularly. The chicken polyclonal antibodies (#881) and the much weaker mouse monoclonal antibodies (12G3) reacted both intracellularly and extracellularly. The extracellular staining obtained with the latter two antibodies obscured the sites of synthesis. To demonstrate the co-localization of immuno-

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J Dent Res 77(4) 1998

BMP-7 and Tooth Development

Figure 10. (left) Immunostaining for BMP-7 in post-secretory (maturation phase) ameloblasts, hamster incisor, post-natal day 3 (170x, bar = 50 pm). Note that not all ameloblasts stain. ma, maturation ameloblasts; e, enamel; d, dentin. Not counterstained. Figure 11. (left) Immunostaining for BMP-7 during late secretory amelogenesis and surrounding bone; hamster incisor, post-natal day 3, decalcified (11Ox, bar = 100 pm). Distal and basal portions of late secretory ameloblasts (sa), stratum intermedium, and stellate reticulum (sr) stain positive. Note that enamel becomes positive throughout its thickness (left) as secretory ameloblasts become post-secretory, as indicated by a shortening of their long-body axis. Strong staining is seen in osteoclasts (ocl) actively resorbing alveolar bone (ab). A group of osteoblasts (ob) is positive as well, but other osteoblasts, more incisally (direction of arrow), and many osteocytes are not. Figure 12. (left) Immunostaining for BMP-7 of secretory-stage amelogenesis; hamster molar on post-natal day 3, decalcified (330x, bar = 50 pm). Staining in cusp portion of the pulp is confined to the odontoblast layer (o) and predentin (pd). In the enamel compartment, staining is seen in some secretory ameloblasts (sa), not in enamel (e). D, dentin; SR, stellate reticulum. Not counterstained. Figure 13. (left) Immunostaining for BMP-7 in developing periodontal ligament; hamster on post-natal day 5, decalcified (440x, bar = 50 pm). Odontoblasts (o) at the lingual portion of the incisor produce root dentin (rd). On top of this dentin, no enamel is deposited but rather (acellular) cementum, which attaches the collagenous fibers of the periodontal ligament (pl) to the root surface (see Fig. 8). Fibroblasts from the developing ligament adjacent to the root dentin are positive for BMP-7. v, blood vessel. Not counterstained. staining with the BMP-7 mRNA distribution, we will show the prominent intracellular immunostaining with the rabbit polyclonal antibodies, but basically all three antibodies gave the same intracellular distribution. With the rabbit antibodies, E12-E15 mouse embryos showed strong staining in developing kidney and heart tissue, developing muscular tissues, cartilaginous tissues of the spine, and epithelial tissues of the skin and bladder (data not shown). In the developing jaws, BMP-7 pro-tein was detected in the oral epithelium during thickening and invagination of the dental lamina into the underlying mesenchyme (Figs. 4A, 4B). During the cap stage, BMP-7 protein was still restricted to the enamel epithelium, was more intense in the central cells, and appeared only weakly in the forming layer of the inner enamel epithelium (Fig. 5). In the early bell stage, BMP-7 staining was moderate to strong in the inner enamel epithelium and adjacent stratum intermedium (Fig. 6). Occasionally, groups of cells in the inner enamel epithelium were negative. Staining of the dental papilla cells was weak to moderate. At the late bell stage (Fig. 7), staining in the dental papilla became restricted to the odontoblast layer and increased progressively in fully functional odontoblasts. Pre-ameloblasts and early secretory ameloblasts stained variably (Fig. 7). Essentially the same staining pattern as in molar tooth germs was noticed in the developing incisors (Fig. 8). Odontoblasts, once differentiated and functional, always exhibited very strong, unerupted staining for BMP-7 throughout the active stages of dentin formation. Odontoblastic processes and predentin were also immunopositive. Decalcified dentin matrix (and bone matrix) were negative (Figs. 9, 12) but stained weakly with

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the chicken and mouse monoclonals in undecalcified specimens. In the enamel organ, enamel epithelial cells near the apical end stained strongly, showing variable staining with the progression of differentiation and low staining in the secretory stage of amelogenesis. Staining in secretory ameloblasts was concentrated at either the supranuclear (near enamel) or infranuclear portion of the cell (Fig. 9A) or throughout the cell body, with some concentration infranuclearly (Fig. 9A). The other cells of the enamel organ occasionally stained as well (Fig. 9A), particularly the stratum intermedium and papillary layer. Staining in ameloblasts increased at late secretory stages (Fig. 11) but became variable in post-secretory stages: Either all post-secretory ameloblasts or only cells or s-mall groups of cells were positive (Figs. 8, 10). Secretory-stage enamel exhibited weak staining at the surface layer and in the deepest layers of enamel near the dentin in undecalcified sections (data not shown) but was negative in decalcified sections. However, in post-secretory stages, the complete layer of enamel became progressively more intensely stained (Figs. 8, 11). The same distribution pattern was observed in 3- to 5-dayold molar tooth germs (Fig. 12). In incisors, odontoblasts stained as intensely at the lingual side as at the labial side (Fig. 8), but in the pulp, most cells barely stained. Prospective periodontal ligament cells near the root dentin were also positive for BMP-7 (Figs. 8, 13). Undecalcified sections generally gave an overall background over the soft tissues, including blood vessels and blood vessel lumina, suggesting that BMP-7 was systemically present. Other forming tissues around the oral cavity stained positively for BMP-7 as well: groups of osteoblasts, osteocytes, osteoclasts (very strongly) in alveolar bones (Fig. 11), muscular tissues, hypertrophic cartilage cells in long bones, oral epithelium, and the epithelial portions of hair and whisker follicles (not shown). Sections stained with non-immune antibodies were negative (not shown). Tooth formation in BMP-7-deficient mice All mice lacking the BMP-7 gene died within 1 day after birth from kidney failure, a site which in the wild-type shows high expression for BMP-7. The mutant mice appeared to have patterning defects of skeletal tissues, and a major portion (70%) of them showed anophthalmia/ microphthalmia, sometimes only unilaterally (Luo et al., 1995). Immunohistochemistry of the kidney from E12-E14 mutant embryos with the mouse monoclonal 12G3 antibody failed to show positive immunostaining, in contrast to the wild-type (Luo et al., 1995). However, the rabbit polyclonal anti-BMP-7 showed positive intracellular staining in the mutant (data not shown). To verify whether expression of the prodomain of the BMP-7 might be responsible for this positive immunoreaction in the mutant, we probed Northern blots from mutant kidney tissues with a probe specific for the mature region of BMP-7 and another probe specific for the prodomain. The latter probe indeed gave a positive reaction on Northern blots, but not the former probe (data not shown). To examine tooth formation, we selected newly born mutant animals that exhibited bilateral eye defects. Serial

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sectioning through the heads of mutant mice revealed no qualitative differences in dental development. First and second molars as well as incisors were present in upper and lower jaws in the correct position. There was a small retardation in dental development of approximately 0.5 to 1 day that seemed related to a general growth retardation in these animals; mutant animals were smaller and weighed from 10 to 20% less than their wild-type littermates (Luo et al., 1995). Most advanced stages were seen in the developing lower incisors that had formed a thin layer of secretorystage enamel and dentin, both mineralized. Tooth eruption of the incisors had not yet occurred in either wild-type or mutant mice. Also, developing whisker follicles, hairs, and muscular tissues appeared to be normal. Surrounding bone and tissue structures, osteoblasts, osteocytes, and osteoclasts did not show obvious qualitative differences and seemed to function normally.

Discussion In this paper, we report the spatial and temporal expression and localization of BMP-7 protein in developing dental tissues in embryonic and neonatal rodents. Our data show that BMP-7 protein is present throughout all stages of embryonic and neonatal tooth development. We observed the earliest immunostaining for BMP-7 in the oral epithelium during formation of the dental lamina and in bud- and cap-stage dental epithelium, in agreement with the recently published location of mRNA for BMP-7 (Vaahtokari et al., 1996). Furthermore, we found co-localization between immunostaining and mRNA for BMP-7 in forming dental and non-dental tissues. During the course of dental development, BMP-7 distribution resembled the distribution patterns of other BMP members but with characteristic differences (Vainio et al., 1993; Begue-Kirn et al., 1994; Heikinheimo, 1994; Bennet et al., 1995; Tureckova et al., 1995; Vaahtokari et al., 1996). For instance, in stages of dental epithelium invagination and the bud stage, both BMP-2 and BMP-7 but not BMP-3, -5, and -6 (Helder et al., 1995b; Vaahtokari et al., 1996) are expressed in dental epithelium, but BMP-2 is expressed in a more restricted pattern than BMP-7 (Vaahtokari et al., 1996). At this stage, BMP-4 is expressed only in the dental mesenchyme (Vainio et al., 1993; Heikinheimo, 1994; Vaahtokari et al., 1996). BMP-7 protein and mRNA transcripts and BMP-2 and -4 mRNA (Vainio et al., 1993; Begue-Kirn et al., 1994; Heikinheimo, 1994) are all localized in the cap-stage inner dental epithelium, and expression then gradually shifts from dental epithelium toward the dental mesenchyme in the bell stage during odontoblast differentiation. In bell-stage dental papilla, BMP-7 and -4 mRNA transcripts are localized peripherally in the cuspal mesenchyme, and BMP-7 becomes restricted to the odontoblast layer; BMP-2, however, is located more in the central parts of the papilla (Vainio et al., 1993). In short, BMP-7 distributes similarly to other BMP members but with detailed differences. The failure of mature rec hBMP-7 to immunostain on Western blots as well as in tissue sections of null mutant mice in which the mature BMP-7 domain had been deleted suggests that the monoclonal 12G3 antibodies are specific

for BMP-7. In contrast, our collective data suggest that the rabbit polyclonal antibodies strongly reacted with the prodomain of BMP-7. This was suggested by the strong intracellular staining pattern that co-distributed with the BMP-7 mRNA pattern, and the fact that, on Western blots, the prodomain reacted strongly. The positive staining with the rabbit polyclonals in the BMP-7-deficient mice was attributed to the expression of the BMP-7 prodomain, still present and expressed in the null mutant. The rabbit antibody slightly cross-reacted with BMP-2, whose expression in a number of developing tissues co-localizes with BMP-7 (Lyons et al., 1995); cross-adsorption of the rabbit antibodies with BMP-2 gave the same tissue distribution. Although we cannot totally exclude the possibility that the rabbit polyclonal antibody weakly cross-reacts with other BMP members, the fact that it highly cross-reacts with the BMP-7 prodomain makes this possibility unlikely. All members of the BMP family share the 7-cystein C-terminal end of the mature protein, so that any cross-reactivity between different members is expected to be in this area; conservation of the prodomain between different members, however, seems unlikely. Little is known of the physiological functions of BMPs during tooth development. Our analysis of tooth development in BMP-7-deficient mice did not give evidence that tooth patterning, tooth morphogenesis, or cytodifferentiation of odontoblasts and ameloblasts were altered in these mice. This suggests that BMP-7 is not an essential factor for the formation of dental tissues. One explanation for the fact that tooth development is unaltered in the BMP-7 null mutants may be the maternal transfer of BMP-7 through the placenta, as reported for TGFI1 in TGFf1- null mutant mice (Letterio et al., 1994; cf. D'Souza and Litz, 1995). In that case, maternal transfer of BMP-7 might have rescued a putatively critical phase of development for which BMP-7 is required. Alternatively, other BMP members which are present at the same stage of development may substitute for BMP-7 in binding to BMP-7 receptor complexes. In this respect, the BMP-7 type I receptors (ALK-3 and ALK-6), in combination with a type II receptor, also have binding activity for BMP-2 (for only ALK-3) and BMP-4 (for both ALK-3 and ALK-6) (ten Dijke et al., 1994; DeWulf et al., 1995; Ikeda et al., 1996), and ALK-2, an activin type I receptor, can also bind BMP-7 (ten Dijke et al., 1994). BMP-2, -4 ,-5, and -6, TGF3 1-3 (Vainio et al., 1993; Begue-Kirn et al., 1994; D'Souza and Litz, 1995), and activin (Roberts and Barth, 1994) are all expressed in embryonic dental cells at some stage of development and thus may act as substitute ligands for BMP-7. In sum, BMP-7 is synthesized in dental tissues and surrounding bone tissues in all stages of development. Expression of BMP-7 may be developmentally regulated. Induction, morphogenesis, and differentiation of dental tissues are not affected in BMP-7-deficient mice. These data suggest that BMP-7 is not essential for dental development, possibly due to functional redundancy between different BMP members.

Acknowledgments The authors acknowledge Dr. G. Luo (M.D. Anderson,

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Houston, TX, USA) for technical assistance, Drs. K. Sampath, J. Maliakal, and W.K. Jones (Creative Biomolecules, Hopkinton, MA, USA) for providing antibodies to BMP-7 and rec hBMP-7, and Dr. E. Wang (Genetics Institute, Cambridge, MA, USA) for her gift of rec hBMP-2. We acknowledge the skillful assistance of Mrs. W. Goei. This study was made possible in part by a grant from the Royal Netherlands Academy of Arts and Sciences (A.B)., the Netherlands Institute for Dental Sciences (HK and MH), NIH grant AR-41059 (GK), and NIDR grant DE-10617 (RDS).

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