DEVELOPMENTAL DYNAMICS 213:500–511 (1998) PDGF-A and PDGFR-␣ Regulate Tooth Formation Via Autocrine Mechanism During Mandibular Morphogenesis In Vitro YANG CHAI,1* PABLO BRINGAS, JR.,1 ALI MOGHAREI,1 CHARLES F. SHULER,1 AND HAROLD C. SLAVKIN2 1Center For Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, California 2Craniofacial Development Section, National Institute of Arthritis and Musculoskeletal and Skin Disease, National Institute of Health, Bethesda, Maryland ABSTRACT Platelet-derived growth factor A (PDGF-A) binding to the PDGF receptor alpha (PDGFR-␣) mediates signal transduction processes related to DNA synthesis, cell migrations, cytodifferentiation, and wound healing. Recent studies indicate that PDGFR-␣ functions during cranial neural crest cell migrations and first branchial arch morphogenesis (Stephenson et al.  Proc. Natl. Acad. Sci. USA 88:6–10; MorrisonGraham et al.  Development 115:133–142; Hu et al.  Int. J. Dev. Biol. 39:939–945; Soriano  Development 124:2691–2700). The present studies were designed to test the hypothesis that PDGF-A, interacts with its cognate receptor PDGFR-␣ via an autocrine mechanism that regulates the timing, rates, and size of embryonic mouse tooth morphogenesis. Both PDGF-A and PDGFR-␣ transcripts were coordinately expressed in mandibular prominences prior to and during tooth formation using reverse transcriptase-polymerase chain reaction (RT-PCR). During the dental lamina stage, ligand and receptor were present in both enamel organ epithelium and adjacent mesenchymal cells. During the bud stage, ligand and receptor were localized mainly to the enamel organ epithelium. Exogenous PDGF-A at 20 ng/ml enhanced tooth development to reach the cap stage with increased tooth size (P F 0.05) using embryonic day (E)10 mandibular explants cultured in serumless, chemically defined medium. A significant increase in DNA synthesis was observed within enamel organ epithelium at E10⫹4 when the mandibular explants were treated with PDGF-A at 20 ng/ml. These data suggest that PDGF-A and its cognate receptor (PDGFR-␣) regulate the size and stage of tooth development via an autocrine mechanism during odontogenesis in vitro. Dev. Dyn. 1998;213:500–511. r 1998 Wiley-Liss, Inc. Key words: PDGF-A; PDGFR-␣; mouse tooth development; in vitro organ culture INTRODUCTION Platelet-derived growth factor (PDGF) is a disulfidebonded dimer of two subunit proteins; A-chain and r 1998 WILEY-LISS, INC. B-chain (Heldin and Westermark, 1990). Whereas all dimeric combinations (AA, AB, BB) have been identified in various biological tissues, unique dimers have been identified in specific tissues during amphibian and mammalian embryonic development and wound healing (Stroobant and Waterfield, 1984; Heldin et al., 1986; Hammacher et al., 1988a,b; Bowen-Pope et al., 1989; Nilsen-Hamilton, 1989; Rizzino, 1989; Hart et al., 1990; Orr-Urtreger et al., 1992, Schatteman, et al., 1996; Betsholtz and Raines, 1997). PDGF-A transcripts are expressed by mouse teratocarcinoma stem cell lines and a host of embryonic mouse tissues during development (Mercola et al., 1990). The presence of PDGF-A is required for normal murine cardiovascular and lung development (Schatteman et al., 1996; Bostrom et al., 1996; Lindahl et al., 1997; Betsholtz and Raines, 1997). PDGF receptor alpha (PDGFR-␣) is required for neural crest cell development and for normal patterning of the somites (Soriano, 1997). Multiple binding sites exist for the different isoforms of PDGF (Hart et al., 1988; Heldin et al., 1988). The biologically active high-affinity PDGF binding site is a dimer of two receptor subunit proteins designated PDGFR-␣ and PDGFR-␤ (Bishayee et al., 1989; Hammacher et al., 1989; Heldin et al., 1989; Seifert et al., 1989). The binding of PDGF isoforms to different cell types appears to be determined by the differential ability of the two PDGF subunit chains to bind to the two receptor subunits (Seifert et al., 1989); PDGF-A only binds to PDGFR-␣, whereas PDGF-B can bind to PDGFR-␣ and PDGFR-␤. PDGF isoforms and PDGFR-␣, ␤ are expressed at critical phases of mouse embryogenesis, and have been implicated in pre- and postimplantation embryogenesis including the regulation of first branchial arch morphogenesis and many mesodermal derivatives during embryonic and fetal development (Rappolee et al., 1988; Nilsen-Hamilton, 1989; Mercola et al., 1990; Schatteman et al., 1992; Orr-Urtreger et al., 1992; Morrison- Grant sponsor: NIDR Center; Grant number: DE-09165. *Correspondence to: Dr. Yang Chai, Center For Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Health Sciences Campus, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033. E-mail: firstname.lastname@example.org Received 11 November 1997; Accepted 14 September 1998 PDGF-A, PDGFR-␣ AND TOOTH FORMATION Graham et al., 1992, Hu et al., 1995; Bostrom et al., 1996; Soriano, 1997). The Patch mutation was identified as a deletion of the PDGFR-␣ gene that maps to chromosome 5 (Stephenson et al., 1991; Smith et al., 1991; Soriano, 1997). This mutation in homozygous Ph/Ph demonstrates pleiotropic effects associated with cranial and trunk neural crest cells and produces significant first branchial arch deformities including facial clefting, hemifacial microsomia, micrognathia, adontia, open neural tube, heart deformities, and large interstitial ‘‘blebs,’’ and is lethal by embryonic day (E)12 (Gruneburg and Truslove, 1960). One interpretation of these studies is that a depletion of cranial neural crest-derived ectomesenchymal cells, possibly resulting from a defect in PDGF-A ligand-mediated signal transduction using the PDGFR-␣, produces first arch deformities including hypodontia and adontia. However, the functions of PDGF-A or its cognate receptor during tooth morphogenesis are unknown. To investigate that PDGF-A signal transduction mediates instructive epithelial-mesenchymal interactions which determine regional and temporal patterns of early stages of tooth morphogenesis, (DNA synthesis, tooth size, and tooth shape), we designed experiments to test the hypothesis that both PDGF-A and PDGFR-␣ are coordinately expressed prior to and during initial tooth formation, and that gain of function of PDGF-A ligand up-regulates DNA synthesis in enamel organ epithelium and thereby controls the size and shape patterns for tooth morphogenesis. Tooth morphogenesis and cytodifferentiation are dependent upon instructive epithelial-mesenchymal interactions (Slavkin, 1974; Lumsden, 1988; Jowett et al., 1993; Vainio et al., 1993; Kratochwil et al., 1996). Tissue recombination experiments have clearly shown that specific regions within ectodermally derived oral epithelium initiate tooth formation (Lumsden, 1988; Kollar and Mina, 1991; Vainio et al., 1993; Thesleff et al., 1996), whereas the inductive role appears to shift from the oral epithelium to adjacent mesenchymal during the bud stage of mouse tooth morphogenesis (Kollar and Baird, 1969, 1970a,b; Ruch, 1984, Kratochwil et al., 1996). PDGF-A and PDGFR-␣ may play a critical role in the signaling required for tooth formation. The present study reports that both PDGF-A and the PDGFR-␣ transcripts are expressed during early mandibular morphogenesis. Both ligand and receptor are colocalized to the enamel oral epithelium and crestderived ectomesenchymal cells adjacent to the dental lamina at the initiation of tooth formation. As development advances into bud stage, however, both PDGF-A and its cognate receptor (PDGFR-␣) are associated with enamel organ epithelium, indicating an autocrine mechanism. Using a simple embryonic E10 mandibular explant model in serumless, chemically defined medium, we found that PDGF-A gain of function at 20 ng/ml significantly enhanced the stage of development and size of tooth organs (P ⬍ 0.05) in the mandibular explants. Three-dimensional reconstruction showed a 501 Fig. 1. Platelet-derived growth factor A (PDGF-A) and PDGF receptor alpha (PDGFR-␣) expression in mandibular prominences during different culture stages using reverse transcriptase-polymerase chain reaction (RT-PCR). A: The expression of PDGF-A transcripts. (M) marker X 174/Hae III. Lane 1: Late embryonic day (E)10 (42–44 somite pairs) mandibular processes. Lane 2: Late E10 mandibular explants plus 2 days in vitro. Lane 3: E10⫹4 days in vitro. Lane 4: E10⫹6 days in vitro. Lane 5: E10⫹9 days in vitro. Lane 6: E13 (Theiler stage 21) mandibular prominences. Lane 7: Reverse-transcription negative control. Lane 8: Polymerase chain reaction negative control. B: The expression of PDGFR-␣ transcripts. The marker and the RNA samples for RT-PCR are the same as in A. C: The expression of ␤-actin transcripts using the same RNA samples for both A and B, indicating that there is sufficient amount of mRMA from each sampling point. PCR products size: PDGF-A ⫽ 225 base pairs; PDGFR-␣ ⫽ 189 base pairs; ␤-actin ⫽ 209 base pairs. significant overall increase in tooth size. An increased DNA synthesis activity in the tooth organ was first seen within cranial neural crest-derived mesenchymal cells at E10⫹2 and then within the enamel organ epithelium during the bud stage of tooth formation. Collectively, this study demonstrated that PDGF-A and its cognate receptor (PDGFR-␣) regulated the size and stage of tooth development via an autocrine mechanism during early odontogenesis. RESULTS PDGF-A and PDGFR-␣ Transcripts Identified Before and During Tooth Formation PDGF-A and PDGFR-␣ transcripts were both detected within microdissected first branchial arch segments at E9 (data not shown) and at E10 (Fig. 1A,B). E10 mandibular explants cultured for 2, 4, 6, and 9 days in a serumless, chemically defined medium continued to express PDGF-A and PDGFR-␣ transcripts as detected by reverse transcriptase- polymerase chain reaction (RT-PCR; Fig. 1A,B). Meanwhile, both PDGF-A and PDGFR-␣ are present in vivo at E13 (Fig. 1 A,B, lane 6). As demonstrated in our previous study, tooth 502 CHAI ET AL. Fig. 2. PDGF-A and PDGFR-␣ immunolocalization on adjacent serial sections through the same tooth during early tooth morphogenesis in vitro. A: At E10⫹2, PDGF-A was present in the dental lamina, basement membrane, and adjacent mesenchymal cells. B: PDGFR-␣ was localized to identical locations comparing to its ligand at E10⫹2. After 4 days, PDGF-A (C) and PDGFR-␣ (D) were present in enamel organ epithelium and its basement membrane of the tooth (tb). By 6 days, PDGF-A (E) and PDGFR-␣ (F) remained in the enamel organ epithelium and basement membrane, while adjacent dental mesenchyme presented with faint positive staining. By the end of 9 days, PDGF-A (G) and PDGFR-␣ (H) were present in enamel organ epithelium, basement membrane, and adjacent dental mesenchyme as the tooth development getting ready to advance into cap stage. Scale bar ⫽ 100 µm. PDGF-A, PDGFR-␣ AND TOOTH FORMATION 503 Fig. 3. Dose response experiment showed that PDGF-A enhanced tooth morphogenesis and enlarged the size or volume of the tooth. A: E10⫹9 control group showed oral epithelium (oe) and bud stage tooth development (tb). B: PDGF-A at 20 ng/ml enhanced development to the cap stage and enlarged tooth size. Three-dimensional reconstruction from serially sectioned mandibular explants were analyzed to define the size of the tooth organ in E10 mandibles cultured for 9 days. C: Control. D: PDGF-A at 20 ng/ml treated mandibular explant. Tooth organs are indicated in red. Meckel’s cartilage is indicated in blue. The outline of mandibular explants are shown in green. Explants are made to be transparent and presented with increased intervals to show the tooth and Meckel’s cartilage positions and size. Scale bar ⫽ 100 µm. formation within E10 mandibular organ culture begins at 1–2 days after the initiation of tissue culture and is marked by the formation of dental lamina (Chai et al., 1994). However, the expression of PDGF-A and PDGFR-␣ are present before the initiation of tooth formation and continues throughout the culture process. This is further demonstrated by the results of immunolocalization of PDGF-A and PDGFR-␣ during mandibular morphogenesis in vitro. detected in the oral epithelium. Meanwhile, PDGFR-␣ was also localized to these mesenchymal cells and blood vessels (data not shown). At E10⫹2, PDGF-A was detected in enamel organ epithelium, basement membrane and adjacent mesenchymal cells (Fig. 2A) while PDGFR-␣ had a similar distribution associated with the dental lamina (Fig. 2B). Four days after the culture, PDGF-A was present in the enamel organ epithelium and the basement membrane (Fig. 2C). PDGFR-␣ was also found within the same tissue type (Fig. 2D). As tooth development continues to E10⫹6, PDGF-A and PDGFR-␣ were localized to the enamel organ epithelium and basement membrane (Fig. 2C,D). At the termination time of mandibular organ culture, PDGF-A was located within enamel organ epithelium and surrounding dental mesenchyme (Fig. 2G), while PDGFR-␣ was localized within the enamel organ epithelium with PDGF-A Colocalization With PDGFR-␣ During Tooth Formation In Vitro Immunohistochemistry on adjacent serial sections from the same tooth germ demonstrated that PDGF-A and PDGFR-␣ were associated with tooth formation (Fig. 2). At E10, PDGF-A was present in the cranial neural crest-derived mesenchymal cells, but it was not 504 CHAI ET AL. detectable expression in adjacent dental mesenchyme (Fig. 2H). PDGF-A Induces Precocious Tooth Morphogenesis A number of growth factors have been found to be pleiotropic, being potent mitogens at a particular concentration and target cell, or at a specific stage in development, or as regulators of cell differentiation by controlling gene expression and protein synthesis. Exogenous PDGF-A induced a dose-response effect on tooth morphogenesis added as a supplement to the serumless, chemically defined medium used to culture E10 mandibular explants for 9 days in vitro (Fig. 3A–D). In control nontreated explants as well as explants cultured with 2 ng/ml or 10 ng/ml PDGF-A (data not shown), tooth formation reached the bud stage within 4 days in vitro and remained at the bud stage up to 9 days in vitro (Fig. 3A). In contrast, PDGF-A at 20 ng/ml not only advanced the tooth development into the early cap stage but also increased the size of the tooth form (Fig. 3B). Statistical analyses indicated that tooth sizes were 50% larger in PDGF-A-treated cultures than the nontreated controls (P ⬍ 0.05). Three-dimensional reconstruction of serially sectioned mandibular explant further demonstrated the substantial increase of tooth size when mandibular explants were treated with PDGF-A at 20 ng/ml (Fig. 3D). This size increase was not limited to a particular section, but was consistent throughout the entire tooth organ when compared to controls (Fig. 3C). PDGF-A Stimulates DNA Synthesis of Enamel Oral Epithelium During Odontogenesis In Vitro DNA synthesis was monitored in E10 explants cultured for 9 days in chemically defined medium. Immunodetection of 5’-bromo-2’-deoxyuridine (BrdU) labeling of DNA was used to identify epithelial or mesenchymal cells engaged in DNA synthesis. Whereas a few epithelial cells were labeled with BrdU at the initial incisor and molar dental lamina stage of tooth morphogenesis (Fig. 4A,B), the majority of dividing cells in both incisor and molar tooth buds were identified within the condensed dental mesenchymal cells at 4 and 9 days of culture (Fig. 4C–F). Exogenous PDGF-A ligand at 20 ng/ml induced precocious cap stage tooth morphogenesis with increased number of cells showing positive BrdU labeling in incisor and molar tooth organs (Fig. 5A–F). At E10⫹2, PDGF-A increased DNA synthesis activity mainly in the cranial neural crest-derived mesenchyme (Fig. 5A,B). Interestingly, PDGF-A (20 ng/ml) significantly increased cell proliferation within molar enamel organ epithelium at E10⫹4 (Fig. 5D), which might have contributed to the advancement of tooth development from bud stage to cap stage at E10⫹9 (Fig. 5F). Furthermore, quantitation of percentage of BrdUlabeled cells showed that PDGF-A at 20 ng/ml significantly increased the DNA synthesis (fourfold) within enamel organ epithelium (Fig. 6 and Table 1). Meanwhile, the proliferation of cranial neural crest-derived dental mesenchymal cells remained relatively comparable to the control group during bud stage tooth morphogenesis. PDGF-A at 20 ng/ml also significantly increased the size (P ⬍ 0.05) of the tooth organ as compared to nontreated bud stage controls. However, the proliferation of enamel organ epithelium in PDGF-A-treated mandibular explants did not advance these epithelial cells to become ameloblasts which are marked by the expression of amelogenin (data not shown). In previous studies, dose responses of exogenous epidermal growth factor (EGF) and transforming growth factor-␤2 (TGF␤2) in this E10 mandibular explant in vitro model resulted in significantly smaller and less-developed tooth primordium (Shum et al., 1993; Chai et al., 1994). The presence of PDGF-A and other possible factors presumably act with EGF and TGF ␤ subtypes to regulate the size and shape of tooth form during morphogenesis of the mandibular prominence. DISCUSSION This study presents detailed analyses of the biological activity of PDGF-A during tooth morphogenesis in a serumless, chemically defined medium. It is also the first report describing the localization of both PDGF-A and PDGFR-␣ during initiation and subsequent morphogenesis of developing embryonic mouse tooth organs. An autocrine functional role for PDGF-A and its cognate receptor was demonstrated during the early inductive stage of tooth morphogenesis. Both ligand and its cognate receptor transcripts are expressed in the mandibular prominence prior to and during the initiation of embryonic mouse tooth formation, and both gene products were immunolocalized mainly to the enamel organ epithelium. Gain of function of PDGF-A in a serumless, chemically defined in vitro explant culture model resulted in precocious tooth development reaching the cap stage, whereas endogenous PDGF-A appears sufficient for the initiation and formation of small bud stage tooth organs. In light of the earlier identification of the Patch mutant mouse showing severe first branchial arch dysmorphogenesis, open neural tube, hypodontia, and adontia (Gruneberg and Truslove, 1960; MorrisonGraham et al., 1992), and the discovery that the Ph/Ph results from a deletion of the PDGFR-␣ (Stephenson et al., 1991), the present investigation provides additional evidence to support the hypothesis that PDGF-mediated signal transduction functions in early inductive epithelial-mesenchymal interactions that are required for the initiation and progression of tooth morphogenesis. PDGF-A Ligand Binds to PDGFR-␣ to Regulate Tooth Morphogenesis In Vitro The hypothesis that PDGF-A ligand-mediated signal transduction is involved in early developmental processes is based on the proposition that the timing and PDGF-A, PDGFR-␣ AND TOOTH FORMATION position of expression implies function, and that the major function of PDGF ligands is to regulate DNA synthesis of odontogenic epithelium and surrounding mesenchymal cells (Rizzino, 1989; Nilsen-Hamilton, 1989; Mercola et al., 1990; Orr-Urtreger and Lonai, 1992; Orr-Urtreger et al., 1992; Betsholtz and Raines, 1997; Soriano, 1997). The present report provides new evidence which indicates PDGF-A and PDGFR-␣ act mainly through an autocrine mechanism in regulating early tooth morphogenesis in vitro. In particular, PDGF-A and its cognate receptor are present in both odontogenic epithelium and condensed mesenchyme at E10⫹2 during the formation of the dental lamina. The cell/tissue distribution of the growth factor and its cognate receptor suggest a role in regulating epithelialmesenchymal interaction during the initiation stage of tooth morphogenesis (Fig. 2A,B). Four days after the culture, however, both PDGF-A and its cognate receptor PDGFR-␣ are mainly present within the ectodermally derived odontogenic epithelium (Fig. 2C,D) and continued to be present until E10⫹9. Collectively, our results indicate that PDGF-A and its cognate receptor are mainly responsible for regulating the proliferation of odontogenic epithelial cells during early tooth morphogenesis in vitro. Several lines of evidence are considered which support our hypothesis: (1) a number of endogenous growth factors including TGF-␣, EGF, TGF-␤1, TGF-␤2, or TGF-␤3 are expressed in odontogenic epithelium and/or mesenchyme prior to and during early mouse tooth formation which do not increase the size nor advance the stages of tooth morphogenesis. (Kronmiller et al., 1990; Heikinheimo et al., 1993; Shum et al., 1993; Chai et al., 1994); (2) underexpression of TGF-␤2 results in an increased size and precocious advancement in the stage of tooth morphogenesis, supporting the suggestion that endogenous growth factor(s) play critical roles in regulating tooth development, and that some of these function to inhibit DNA synthesis, growth and morphogenesis (Chai et al., 1994); (3) PDGF-A (this report), fibroblast growth factor (FGF), as well as insulin-like growth factor (IGF-II) all increase DNA synthesis, growth, and induce precocious tooth morphogenesis (Neubuser et al., 1997; Kettunen and Thesleff, 1998; unpublished results), resulting in late cap stage tooth organs with attendant epithelial and mesenchymal cell differentiation. Furthermore, the expression pattern of PDGF-A and PDGFR-␣ strongly suggests that PDGF-A is an important mediator for enamel organ epithelial cell proliferation during the process of odontogenesis. It is also noted, however, that PDGF-A or PDGFR-␣ immunolocalization need not correspond to the site of PDGF-A or PDGFR-␣ synthesis. This can only be determined by in situ hybridization. Although the possibility is remote, PDGF-A could conceivably diffuse and collect on cell surfaces of an adjacent PDGFR-␣ expressing tissue. In addition to an autocrine mechanism, a paracrine mechanism is not completely ruled out. 505 PDGF-A Induces DNA Synthesis in Odontogenic Epithelium During Early Tooth Formation In Vitro Overexpression of PDGF-A, achieved by supplementation of PDGF-A (20 ng/ml) to a serumless, chemically defined medium devoid of exogenous growth factors, produced a significant increase in DNA synthesis first seen within the mesenchyme at E10⫹2 (Fig. 5A,B) and then within the ectodermally derived enamel organ epithelium at E10⫹4 (Fig. 5D). This increase in proliferation of enamel organ epithelium advanced the tooth development into cap stage at E10⫹9 (Fig. 5F). Coexpression of PDGF-A and PDGFR-␣ in the enamel organ epithelium during the progression of early tooth development (Fig. 2) indicates an autocrine mechanism that PDGF-A is able to induce the proliferation of these epithelial cells. The number of BrdU-labeled mesenchymal cells remains high throughout the culture period in both control and exogenous PDGF-A-treated explants. It is, however, the significant increase in DNA synthesis within enamel organ epithelium which might be responsible for the advancement of both size and stage of tooth development following the addition of exogenous PDGF-A. These observations support the suggested functions for PDGF-A and PDGFR-␣ in postimplantation mouse embryogenesis (Mercola et al., 1990; Morrison-Graham et al., 1992; Orr-Urtreger and Lonai, 1992; Orr-Urtreger et al., 1992). Furthermore, our study indicates that PDGF-A and its cognate receptor PDGFR-␣ transcripts are expressed by cranial neural crest-derived mesenchymal cells within the forming first arch and early tooth primordium, subsequently expressed in the enamel organ epithelium, and are responsible for mediating the critical epithelial-mesenchymal interaction and subsequent proliferation of enamel organ epithelial cells in odontogenesis. These observations also support the interpretation that cranial neural crest-derived tooth mesenchymal cells are deficient in the Patch mutant, resulting in hypodontia and adontia due to the deletion of the PDGFR-␣. PDGF-A may control several downstream regulatory genes such as bone morphogenetic protein 4 (BMP-4) and several transcription factors (Lef1, Msx-1, and Msx-2). Several recent studies have elegantly demonstrated that BMP-4 and the transcription factors are initially expressed in oral epithelium prior to the initiation of tooth formation; moreover, these regulatory molecules also shift from initial expression within oral epithelium prior to tooth formation to expression within adjacent mesenchyme during the bud stage of tooth morphogenesis (Mackenzie et al., 1991, 1992; Vainio et al., 1993; Kratochwil et al., 1996; Jernvall et al., 1998). Homologous recombinations in transgenic mice have produced a null mutation for Msx-1 which results in tooth development arrest at the terminal bud stage in the homozygous animals (Satokata and Maas, 1994). Certain exogenous growth factor signals can substitute for the odontogenic placode as the initiation 506 CHAI ET AL. Fig. 4. Using 5-bromo-2’-deoxyuridine (BrdU) as cell proliferation marker to follow the DNA synthesizing activity in both enamel oral epithelium and condensed mesenchymal cells in the control group of E10⫹9 cultured mandibles. A, B: At 2 days, both incisor (i) and molar (m) enamel organ epithelium showed cell proliferation activity. The condensed mesenchymal cells surrounding both the incisor and molars also revealed DNA synthesizing activity. C, D: By 4 days, cell proliferation activity had increased in the mesenchymal cells surrounding both the incisor and molar enamel organ epithelium. There were few cells stained positive with Brdu within enamel organ epithelium of both incisor and molar tooth organs. E, F: At 9 days, there were still large numbers of condensed mesenchymal cells with positive BrdU labeling, while only a few cells were positive in both the incisor and molar enamel organ epithelium. Scale bar ⫽ 100 µm. center for tooth morphogenesis and induce transcription factors such as Lef1, Msx1, and Msx2 within the primitive odontogenic mesenchyme (Vainio et al., 1993; Kratochwil et al., 1996; Thesleff and Nieminen, 1996; Jernvall et al., 1998). Further study on the interaction of different growth factors/receptors, such as PDGF-A/ PDGFR-␣, and transcription factors, such as Lef1, Msx1, 2, can help us to understand the molecular mechanism that regulates the morphogenesis and cytodifferentiation of different phenotypes during embryogenesis. In summary, the present investigation provides evidence that PDGF-A and its cognate receptor PDGFR-␣ transcripts are expressed in the first branchial arch PDGF-A, PDGFR-␣ AND TOOTH FORMATION 507 Fig. 5. PDGF-A at 20 ng/ml increased the proliferation enamel organ epithelial cells and resulted in the advancement of tooth development. A, B: At 2 days, there was increased cell proliferation activity in both enamel organ epithelium and condensed mesenchymal cells in incisors (i) as well as molars (m) compared to the controls (Fig. 4). C, D: By 4 days, enamel organ epithelial cells showed increased cell proliferation activity, especially in the molar (m) as indicated by the intense Brdu staining. The condensed mesenchymal cells surrounding the enamel organ epithelium also revealed intense DNA synthesizing activity, but without a difference compared to the control groups. E, F: By 9 days, cell proliferation activity continued to be active in both incisor (i) and molars (m), especially in the enamel organ epithelium compared to the controls. PDGF-A at 20 ng/ml enhanced tooth development to reach the cap stage. Scale bar ⫽ 100 µm. prior to the initiation of tooth development. Immunostaining evidence for both ligand and its cognate receptor indicates localization within the cranial neural crest-derived tooth mesenchymal cells during the initial dental lamina stages of morphogenesis. During later bud stage, PDGF-A and its receptor (PDGFR-␣) are predominantly localized to the enamel organ epithelial cells which are highly responsive to PDGF-A- induced DNA synthesis. These observations support the hypothesis that PDGF-A and its cognate receptor PDGFR-␣ act through an autocrine mechanism to regulate tooth morphogenesis. As seen in the control group, a constantly active DNA synthesis activity and the expression of PDGFR-␣ are mainly associated with the cranial neural crest-derived mesenchymal cells which may in part reflect the well-documented influ- 508 CHAI ET AL. (Millipore, Bedford, MA), and supported on a stainless steel raft. All explants were positioned with the oral epithelium face upward, opposite to the filter surface. Approximately 6–8 explants per culture dish were cultured in Grobstein Falcon dishes under optimal humidity conditions in an atmosphere containing 5% CO2 and 95% air. The culture dishes were filled with BGJb medium (Gibco, Grand Island, New York) supplemented with 0.1 mg/ml ascorbic acid and 50 units penicillin/streptomycin (Flow Laboratories, McLean, VA). The medium was changed every other day and kept within a pH range of 7.2 to 7.4. All mandibular explants were cultured for 9 days in vitro. Reverse Transcription Polymerase Chain Reaction Fig. 6. PDGF-A at 20 ng/ml increased the percentage of BrdU-labeled odontogenic epithelial cells by almost fourfold. In the control group, only 8.6% of total counted cells within enamel organ epithelium were labeled with BrdU, while PDGF-A at 20 ng/ml increased BrdU-labeled odontogenic epithelial cells to 33.4%. ence of dental papilla mesenchyme on tooth shape and form. Addition of exogenous PDGF-A initially induced DNA synthesis activity within the cranial neural crestderived mesenchymal cells adjacent to the enamel oral epithelium and then dramatically increased cell proliferative activity within the enamel organ epithelium at E10⫹4, which may contribute to the advancement of tooth morphogenesis from bud to cap stage in this in vitro system. EXPERIMENTAL PROCEDURES Mouse Embryos Virgin female Swiss-Webster mice, obtained from Simonsen Labs (Gilroy, CA), were housed for at least 2 weeks in the breeding colony with other animals of the same age in a controlled light and temperature environment with ad libitum food and water. By mating these mice with adult male mice (11–14 weeks old) for 3 hours, timed pregnancies were obtained. The presence of a vaginal plug was used as an indication of pregnancy day 0. At selected developmental stages, the pregnant mice were killed by cervical dislocation. Mouse embryos were dissected from uterine decidum and staged by their external features according to Theiler’s method (1989). Mandibular processes including the tuberculum impar were removed from embryos by microdissection and used as explants for organ culture (Slavkin et al., 1989; Shum et al., 1993; Chai et al., 1994). Mandibular Organ Culture A modified Trowell method was used to culture mandibular processes as previously described (Slavkin et al., 1989). Dissected mandibular processes (E10, 42–44 somite pairs) were placed on type AA, 0.8 µm pore size Millipore filter paper 6 mm in diameter RT-PCR was performed according to Rappolee et al. (1988). E10 and E13 mandibular processes as well as E10 mandibular explants cultured for 2, 4, 6, and 9 days were isolated and placed on dry ice to ensure the stability of RNA within explants. Five mandibular explants or tissue isolates were pooled for each RNA extraction. The 5’ and 3’ amplimers used in the present studies were designed based on the cDNA sequences of PDGFR-␣ and PDGF-A (Orr-Urtreger and Lonai, 1992; Mercola et al, 1990). The primer set specific for PDGFR-␣ was for a 189-base pair product using primers 5’-AGG AAG CCA TTC CTG CA (17 mer), and 3’-CTT GAC ACT GCG GTG GTG (18 mer; Orr-Urteger and Lonai, 1992). The PDGF-A was amplified with a product size 435 using a pair of primers of 5’-CTC CAG CGA CTC TTG GAG ATA G (22 mer) and 3’-TTCAGG TTG GAG GTC GCACAT G (22 mer; Mercola et al., 1990). The internal positive control was ␤-actin, a 209-base-pair product using primers 5’-GAC ATC CGCAAAGAC C (16 mer) and 3’-CCACAT CTG CTG GAA G (16 mer). The amplification of ␤-actin confirmed that the amount of RNA in each sample was not degraded and differences in signal were not due to differences in the amount of RNA amplified. The PCR reaction mixture contained PCR buffer with 1.5–2.5 mM magnesium chloride, 1.0 mM dNTP, 0.25 unit Taq polymerase (Amplitaq, Perkin-Elmer Cetus, Norwalk, CT), 2 µl of each of the 5’ and 3’ amplimers, and 4 µl of the reverse transcription product. PCR was done in thermal cycles (ERICOMP, San Diego, CA). Subsequently, the PCR products were visualized on a 4% agarose gel (NuSeive [FMC, Rockland, ME]: BRL Agarose [BRL, Gaithersburg, MD]; 3:1; w/w) by staining with 1 µg/ml ethidium bromide. The size of the amplified product was evaluated using (⫻174 RF DNA/Hae III marker (BRL). The amplified products were verified by comparison with PDGFR-␣ and PDGF-A plasmid controls and product sequence analysis. In PCR control, diethylpolycarbonate (DEPC)-treated water was used to replace the reverse transcription product as a negative control. ␤-actin was used as an internal positive control. 509 PDGF-A, PDGFR-␣ AND TOOTH FORMATION TABLE 1. Number of BrdU-Labeled Cells in Odontogenic Epitheliuma Section 1 2 3 4 5 Mean ratio S.D. BK 1b 9 (102) 11 (91) 13 (109) 6 (81) 10 (96) Control BK 2 5 (79) 9 (98) 8 (97) 8 (93) 8 (95) 0.086 0.011 BK 3 7 (85) 8 (91) 5 (78) 5 (76) 6 (68) BK 1 36 (103) 43 (142) 38 (131) 41 (121) 34 (119) PDGF-A (20 ng/ml) BK 2 48 (136) 40 (125) 26 (72) 46 (129) 40 (116) 0.334 0.017 BK 3 43 (124) 42 (128) 44 (134) 47 (131) 41 (121) a58-bromo-28-deoxyuridine (BrdU) labeled cells and the total number of cells counted (in parentheses) within enamel organ epithelium. Three E10⫹9 cultured mandibular explants were randomly chosen from each group. All sections contained tooth organs. Two adjacent sections were chosen with at least 40 µm distance between them to avoid the same cells being counted twice. Platelet-derived growth factor-A (PDGF-A) at 20 ng/ml substantially (P ⬍ 0.05) increased the percentage of BrdU-labeled cells within enamel organ epithelium. bBK 1, 2, and 3 ⫽ E10⫹9 cultured mandibular explants 1, 2, and 3. PDGF-A and PDGFR-␣ PCR Products Sequence Analysis The PCR products were confirmed using PCR product nucleic acid sequence analysis (Tracy and Mulcahy, 1991). Ten microliters of PCR product observed on an agarose gel was eluted through a GLASSMAX (BRL) spin column. The resulting elucidate (40 µl) was found to be free of the primers, unincorporated dNTPs, enzymes, etc. The eluted PCR product (7.5 µl) along with 3.2 pMoles PDGF-A or PDGFR-␣ primers and water up to a total volume of 10.5 µl was provided to the Microchemical Core Facility of the USC Norris Cancer Center at this Institution. A fluorescent cycle sequencing reaction was performed and analyzed on an Applied Biosystems (ABI) automated sequencer (Model 373A). The resulting sequences were compared to the August, 1994, Genebank database and the results confirmed a 100% match of the observed PCR products with that of PDGFR-␣ and PDGF-A. Light Microscope Immunocytochemistry Mandibular explants were fixed in Carnoy’s fixative solution for 6 hr at 4°C, dehydrated in 100% ethanol overnight, cleared in xylene, and embedded in paraffin. Serial sections of 5 µm were cut and mounted onto slides coated with Histostick (Accurate Chemical & Scientific Co., Westbury, NY). The average cultured mandibular explant represented approximately 160 five-micron sections inclusive of the anterior (rostral), middle, and posterior extensions of Meckel’s cartilage which were used for orientation. The slides were heated in 60°C oven for 45 min and subsequently hydrated to water through a series of decreasing concentration of ethanol. Following the protocol of Zymed StreptavidinBiotin system (Zymed Laboratories Inc., San Francisco, CA), anti-PDGFR-␣ and anti-PDGF-A antibodies (UBI, Lake Placid, NY) developed in rabbits were used as the primary antibody at a dilution of 1:100 for antiPDGFR-␣, and 1:150 for anti-PDGF-A. The immunostaining was done on adjacent serial sections from the same tooth germ to localize both PDGF-A and its cognate receptor. Normal rabbit serum at a 1:100 dilution was used as negative control. The primary antibody was incubated overnight at room temperature in a moisture chamber prior to washing with PBS and reacting with goat-anti-rabbit secondary antibody. Adult mouse lung was used as positive control. PDGFR-␣ and PDGF-A antigens were indicated by orange-red coloration. The slides were counterstained with hematoxylin. To verify the distribution patterns of PDGF-A and PDGFR-␣, another set of primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Immunostainings using these antibodies confirmed our findings. PDGF-A Stimulation of DNA Synthesis The highly purified recombinant PDGF-A isoform was commercially obtained (UBI, Lake Placid, NY), and used in overexpression experiments at concentrations ranging from 0.25 ng to 40 ng/ml diluted in serumless, chemically defined medium. DNA synthesis activity was monitored in E10 mandibular explants using BrdU in chemically defined medium. Mandibular explants were incubated with BrdU at 100 µM in serumless chemically defined medium for 2 hours at 4°C. Then these mandibles were placed in the tissue culture dish with either control medium or medium plus PDGF-A at 20 ng/ml. The mandibular organ culture process was identical to the procedure described above. The mandibular explants were harvested and fixed for immunostaining at E10⫹2, 4 and 9 days of culture. Mandibular explants were fixed in Carnoy’s fixative solution for 6 hr at 4°C, dehydrated in 100% ethanol overnight, cleared in xylene, and embedded in paraffin. Serial sections of 5 µm were cut and mounted onto slides coated with Histostick (Accurate Chemical & Scientific Co., Westbury, NY). The slides were heated in 60°C oven for 45 min and subsequently hydrated to water through a series of decreasing concentration of ethanol. Then the slides were treated with 2 N HCl for 60 min at 37°C to 510 CHAI ET AL. denature DNA and followed with neutralization with Borate buffer (pH 8.5) for three times (10 min each). The slides were rinsed in PBS. Following the protocol of Zymed Streptavidin-Biotin system (Zymed Laboratories Inc.), immunohistochemistry was performed. Morphometric and Statistical Analysis In order to evaluate the morphological effect induced by PDGF-A treatment on tooth formation, the size of tooth bud was measured. Three mandibles were randomly selected from each treatment group (control, PDGF-A at 2 ng/ml, 10 ng/ml, and 20 ng/ml). Each section of tooth bud was traced throughout the serial sections of mandibular explant. The volume of the tooth bud was calculated. Using Epistat Statistical Package one-way, one-level analysis of variance (ANOVA) was applied to test for significant changes in the size of tooth bud among nontreated control, PDGF-A at 2 ng/ml, 10 ng/ml, and 20 ng/ml treated groups. A difference was considered statistically significant if the P value was less than 0.05 (P ⬍ 0.05). The null hypothesis for this test was that there was no difference in the size of tooth bud for the various groups in the PDGF-A treatment experiments. Computer Analysis With the same enlargement, the outlines on the adjacent serial sections of mandible were traced using a Jandel digitizing tablet into the PC3D software program (Jandel Scientific, Sausalito, CA). 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