THE ANATOMICAL RECORD PART A 286A:841– 847 (2005) Role of IGFBPs in the Morphogenesis of Lingual Papillae YUKO SUZUKI* Department of Oral Anatomy, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Japan ABSTRACT The expression of insulin-like growth factor binding proteins (IGFBPs) during the morphogenesis of lingual papillae of mice was examined by in situ hybridization. Among seven mouse IGFBPs, IGFBP-1, -6, and -7 mRNAs were not expressed in the tongue tissue. At E12, though no papillae have formed yet, IGFBP-2, -4, and -5 were expressed in the entire tongue epithelium. At E14, fungiform papillae appeared in the anterior region and circumvallate papillae were distinguished in the posterior region. Strong expression of IGFBP-5 was observed in the apical region of both fungiform and circumvallate papillae. At this stage, the epithelial elevation of ﬁliform papillae was not clear; but IGFBP-5 was expressed in the apex. At E15, foliate papillae were distinguished and IGFBP-5 was expressed in the dorsal epithelium of ridges. In ﬁliform papillae, IGFBP-3 was expressed in the core of the connective tissue. At E17, the expression of IGFBP-5 disappeared from the apical region of fungiform, ﬁliform, foliate, and circumvallate papillae, whereas that of IGFBP-2 remained. This ﬁnding suggests that IGFBP-5 and -2 function to cause evagination of the epithelium into a raised structure. In the epithelium of trenches of foliate and circumvallate papillae, strong expression of IGFBP-4 was observed at E15 and E17. As previously suggested from a study on postnatal mice (Suzuki et al. J Comp Neurol 2005;482:74 – 84), IGFBP-4 acts in the epithelial invagination to form the trenches, grooves, or furrows of lingual papillae during development. © 2005 Wiley-Liss, Inc. Key words: insulin-like growth factor binding protein; in situ hybridization; fungiform papillae; circumvallate papillae; ﬁliform papillae The dorsal surface of the mammalian tongue is covered with four kinds of papillae: fungiform, circumvallate, foliate, and ﬁliform. These papillae are distributed in a speciﬁc pattern over the tongue in mice: fungiform and ﬁliform papillae are located on the anterior two-thirds of the tongue. The fungiform papillae have a mushroom-shaped structure, composed of a multilayered epithelium and mesenchymal core (Farbman and Mbiene, 1991). Filiform papillae are small conical surface projections found in great numbers among the fungiform papillae (Baratz and Farbman, 1975). Foliate papillae are located on the posterior lateral margins of the tongue, and circumvallate papillae on the posterior midline. Foliate papillae consist of several ridges that alternate with deep grooves in the mucosa. In mice and rats, single circumvallate papilla is present in the middle part of the terminal sulcus and it is surrounded by a deep circular groove into which open the © 2005 WILEY-LISS, INC. von Ebner’s glands. Except for the ﬁliform papillae, these papillae contain taste buds (Nosrat et al., 1997). Grant sponsor: Academic Frontier Project for Private Universities; Grant sponsor: mathcing fund pubsidy from MEXT (Ministry of Education, Culture, Sports, Science, and Technology), 2002-2006. *Correspondence to: Yuko Suzuki, Department of Oral Anatomy, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu 061-0293, Japan. Fax: 81-1332-3-1236. E-mail: firstname.lastname@example.org Received 21 February 2005; Accepted 11 April 2005 DOI 10.1002/ar.a.20219 Published online 27 July 2005 in Wiley InterScience (www.interscience.wiley.com). 842 SUZUKI Several signaling molecules regulate the morphogenesis of the papillae. For example, sonic hedgehog (Shh) is expressed only in the dorsal surface of fungiform papillae during embryonic days 13–18 (Hall et al., 1999, 2003; Jung et al., 2004), and its signal disruption alters the number and location of fungiform papillae (Hall et al., 2003; Liu et al., 2004). In pax 9-deﬁcient mice, a corniﬁed layer is absent, thus ﬁliform papillae lack anterior-posterior polarity (Jonker et al., 2004). In mice knocked out for brain-derived neurotrophic factor (BDNF) and its receptor TrkB, fungiform, foliate, and circumvallate papillae are smaller or have an aberrant morphology or both (Nosrat et al., 1997; Oakley et al., 1998; Mistretta et al., 1999). In our previous study, insulin-like growth factor binding protein (IGFBP)-4, a member of the family of insulin-like growth factor (IGF) and related molecules, was expressed in the bottom of the trenches of circumvallate papillae of postnatal day 2 mice, suggesting that it regulates the down-growth of grooves (Suzuki et al., 2005). The IGF family comprises IGF-I, IGF-II, their receptors (IGF-IR, IGF-IIR), and seven high-afﬁnity IGFBPs. The actions of these IGFs appear to be regulated and coordinated by a family of IGFBPs. The IGFBPs are thought to have four major functions that are essential to the regulation and coordination of the biological activities of IGFs. That is, IGFBPs are considered to act as transport proteins in plasma and to control the efﬂux of IGFs from the vascular space; to prevent IGFs from being degraded and to prolong the half-lives of IGFs; to provide a means of tissue and cell type-speciﬁc localization; and to modulate directly the interaction of the IGFs with their receptors and thereby indirectly control their biological actions (Duan, 2002). Recent evidence suggests that the IGFBPs can also have direct IGF-independent actions on cellular functions (Zhou et al., 2003). All lingual papillae begin as epithelial thickenings and then evaginate to form raised papillae with a mesenchymal core during embryonic development (cf. Farbman and Mbiene, 1991). Because IGF-I and -II are known to be expressed in the embryonic tongue in rats (Ayer-LeLievre et al., 1991) and in mice (Ferguson et al., 1992; Yamane et al., 2000), the IGF family has been suggested to regulate the morphogenesis of the lingual papillae. The aim of this article was to examine the expression of IGFBPs during embryonic development of lingual papillae. MATERIALS AND METHODS Animals Timed pregnant ddY mice, were obtained from Sankyo Laboratories (Tokyo, Japan). They were maintained in a heat- and humidity-controlled vivarium with food and water provided ad libitum. Experimental protocols concerning animal handling were reviewed and approved by the Institutional Animal Care Committee of the Health Sciences University of Hokkaido. Tissue Preparation The day of appearance of vaginal plug was designated as E0. The study was conducted on 12 pregnant female mice. To obtain embryos, mice were killed by cervical dislocation and their uteri with fetuses (E12–17) carefully dissected out. For in situ hybridization, the tongues were ﬁxed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4°C. For immunohistochem- istry, the tongues were ﬁxed with periodate-lysine-paraformaldehyde (PLP) solution. Each specimen was washed in phosphate-buffered saline (PBS) solution, cryoprotected with 25% sucrose, and embedded in OCT compound (Oken, Tokyo, Japan). The tissues were sectioned coronally at a thickness of 8 –10 m. Sections were collected and placed on silane-coated slides. RNA Probes and In Situ Hybridization cDNA fragments of IGF-IR, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, and IGFBP-7 were generated by reverse transcription-polymerase chain reaction (RTPCR) using total RNA extracted from the tongue and then used for the synthesis of cRNA probes. The sequences of the primers were as follows: 5⬘-TCTCTCTCTGGCCGACGAGT-3⬘ and 5⬘-GAGCAGAAGTCACCGAATCG-3⬘ (977 bp; AF056187) for IGF-IR; 5⬘-GACGCTACGCTGCTATCCCA-3⬘ and 5⬘-GTCTCCTGCTGCTCGTTGTA-3⬘ (614 bp; NM008342) for IGFBP-2; 5⬘-GGAAACATCAGTGAGTCCGA-3⬘ and 5⬘-GCTGAGGCAATGTACGTCGT-3⬘ (458 bp; X8158.1) for IGFBP-3; 5⬘-GGAGAAGCCCCTGCGTACAT-3⬘ and 5⬘-ACCCCTGTCTTCCGATCCAC-3⬘ (434 bp; X76066) for IGFBP-4; 5⬘-AGTAACGTTGAGTGACGCGT-3⬘ and 5⬘-CAGTGTTGGGGGTGCGTACT-3⬘ (750 bp; L12447) for IGFBP-5; 5⬘-TAATGCTGTTGTTCGCTGCG-3⬘ and 5⬘-CACTGCTGCTTGCGGTAGAA-3⬘ (552 bp; NM008344) for IGFBP-6; and 5⬘-AAGGTCCTTCCATAGTGACG-3⬘ and 5⬘-CAGGGTTATAGCTGTCGGCT-3⬘ (439 bp; NM008048) for IGFBP-7. The PCR was carried out for 35 cycles. Each resulting fragment was cloned into HindIII/EcoRI sites of pT7/T3 DH5␣-FT (Invitrogen, Tokyo, Japan) and sequenced. Digoxigenin (DIG)-labeled antisense and sense probes were produced by use of an RNA transcription kit (Roche Diagnostics, Mannheim, Germany). Sections were washed in PBS and treated for 20 min with 0.2 N HCl and for 15–20 min with proteinase K (1 g/ml in PBS; Takara, Kyoto, Japan) at 37°C. Next, the sections were washed in PBS and reﬁxed with 4% paraformaldehyde in 0.1 M phosphate buffer for 20 min. After having been washed twice in PBS, the sections were air-dried and hybridized. Hybridization was performed at 47°C for 16 hr with an RNA probe in a hybridization solution containing 50% formamide, 0.3 M NaCl, 0.02 M Tris-HCl, 1 mM EDTA, 10% dextran sulfate, 1 ⫻ Denhardt’s solution, 1 mg/ml yeast tRNA, and 0.02% SDS. Hybridized sections were washed at 47°C in a solution containing 50% formamide and 2 ⫻ SSC for 1 hr, and thereafter twice in 2 ⫻ SSC for 5 min each time. Then they were treated with 20 g/ml of RNase (TypeII-A; Sigma) at 37°C for 30 min and washed at 47°C in 50% formamide/ 2XSSC followed by 50% formamide/1 ⫻ SSC for 1 hr for each. After having been washed three times in PBS, the sections were incubated with 1% blocking reagent (Boeringer Mannheim, Mannheim, Germany) in maleic acid buffer (pH 7.5) for 1 hr at room temperature. Subsequently, they were incubated overnight at 4°C with alkaline phosphatase-conjugated anti-DIG Fab fragments diluted 1:500 in PBS. After three washes in PBS, chromogenic reactions were carried out by using NBT/ BCIP (Boeringer). Immunohistochemistry The sections were incubated in a blocking solution (Dako Protein Block Serum-Free; Dako, Carpinteria, CA) 843 ROLE OF IGFBP for 10 min at room temperature. Then the sections were incubated overnight at 4°C with antirabbit PGP9.5 antibody (Ultraclone, Isle of Wight, U.K.), diluted 1:100 in PBS. After having been washed in PBS, the sections were treated with Alexa Fluor 594-conjugated donkey antirabbit IgG (Molecular Probes, Eugene, OR) for 2 hr at room temperature. Sections were viewed with a Leica confocal laser scanning microscope. RESULTS Among IGFBPs, it was reported that IGFBP-1 mRNA was detected only in adult liver tissue (Suzuki et al., 2005) and so it was not examined in the present study. IGFBP-6 and -7 mRNAs were not expressed in tongue tissue by in situ hybridization in the present study. Immunohistochemically IGFBP-6 was detected in the nerve ﬁbers innervating the taste buds of circumvallate papillae (Suzuki et al., 2005). IGFBP-7 was speciﬁcally expressed in developing lens (not shown). Therefore, IGFBP-2, -3, -4, and -5 mRNAs were detected in the developing tongue. At embryonic day (E) 12, the early fetal tongue has a relatively homogeneous epithelium with no obvious papillary structure. Among IGFBPs, IGFBP-2 was expressed in the entire lingual epithelium (Fig. 1A); IGFBP-5 was expressed in the lingual epithelium and in myoblast cells (Fig. 1B). IGFBP-4 was expressed in the entire lingual epithelium and mesenchyme (not shown). IGFBP-3 was not detected in the tongue epithelium but was found in some mesenchymal cells (Fig. 1C). Fungiform and Filiform Papillae At E14, in the anterior region of tongue, the developing fungiform papillae were small and slightly elevated into the oral cavity. They also contained a small amount of connective tissue in their medullary core. IGFBP-2 and -4 were expressed in the epithelium of both fungiform papillae and nonpapilla tongue tissue. IGFBP-4 was expressed also in the mesenchyme (Fig. 1D). IGF-IR expression was observed in the tongue epithelium throughout the embryonic stages (Fig. 1E). IGFBP-5 was intensely expressed in the fungiform papillae (Fig. 1F). Although the surface of the tongue remained ﬂat, IGFBP-5 was expressed also in the presumed ﬁliform papillae, which were interspersed with fungiform papillae (Fig. 1F). At E15, regularly spaced dermal papillae appeared in the presumptive ﬁliform papillae. Expression of IGFBP-5 was observed in the apical to middle region of the ﬁliform epithelium (Fig. 1G), and IGFBP-3 was expressed in mesenchymal core (Fig. 1H). At E17, the ﬁliform papillae start to form a corniﬁed layer in the epithelium, which exhibit anterior-posterior polarity. IGFBP-3 was expressed in the core of these papillae (Fig. 1I). In fungiform papillae, expression of IGFBP-3 was weak (Fig. 1I, inset). IGFBP-2 was expressed in the basal layer of the epithelium in the anterior region of each ﬁliform papilla (Fig. 1J) and in the apex of the fungiform papillae (Fig. 1K). The expression of IGFBP-4 and IGFBP-5 had mostly disappeared from the anterior tongue epithelium by E17 (Fig. 1L and M). Circumvallate Papillae At E14 in the posterior region of the tongue, circumvallate papillae were observed to have epithelial elevations and shallow grooves. IGFBP-2 was expressed in the entire epithelium of these papillae (Fig. 2A). IGFBP-5 was ex- pressed in the elevated epithelium of papillae but not in the grooves (Fig. 2B). At E15, IGFBP-3 was expressed in a few cells of mesenchymal core (Fig. 2C). IGFBP-4 was intensely expressed in the bottom of the grooves (Fig. 2D). IGF-IR expression was observed in the epithelium throughout the embryonic stages (Fig. 2E). At E17, the circumvallate papillae were more developed. The trench grooves had deepened further into the underlying mesenchyme, and IGFBP-2 expression had become restricted to the elevated epithelium of the papillae (Fig. 2F). Intense expression of IGFBP-4 was still observed in the epithelium of the bottom of the trenches, and IGFBP-4 was expressed also in minor salivary glands, von Ebner’s glands (Fig. 2G). IGFBP-5 expression had mostly disappeared from the epithelium of the circumvallate papillae, but it was expressed in presumptive taste buds and some epithelial cells at the boundary with the connective tissue (Fig. 2H). At this stage, numerous PGP9.5-positive nerve ﬁbers entered the circumvallate papillae, and a few taste buds were located at the top of the papillae (Fig. 2I). Foliate Papillae At E15, foliate papillae were observed in the margin of the posterior region of tongue. At this stage, IGFBP-4 was intensely expressed in the shallow grooves among the ridges (Fig. 3A). IGFBP-5 was expressed in the dorsal epithelium of papillae but not in the grooves (Fig. 3B). IGF-IR expression was observed in the epithelium throughout the embryonic stages (Fig. 3C). The expression of IGFBP-2 was observed in the entire epithelium of papillae at E15 and had restricted to the dorsal epithelium of the papillae at E17 (Fig. 3D). At E17, IGFBP-3 was weakly expressed in the mesenchyme (Fig. 3E). At this stage, intense expression of IGFBP-4 was still observed in the bottom of grooves (Fig. 3F). These results were summarized in Table 1. DISCUSSION In the embryonic tongue of mice, IGF-I and -II were earlier found by immunohistochemical means to be expressed in the dorsal epithelium and muscles (Yamane et al., 2000). By in situ hybridization, high levels of IGF-II mRNA were found in the mesenchyme, muscles, and connective tissue of the embryonic tongues of mice (Ferguson et al., 1992) and rats (Ayer-LeLievre et al., 1991). The expression of IGF-I mRNA in the embryonic rat tongue was weak, and thus its localization was not clear (AyerLeLievre et al., 1991). IGF-1 and -II bind to the receptor IGF-IR. IGF-IIR has no known growth-mediating effects and may simply act as a cell-surface depot for storage of ligand (cf. Zhou et al., 2003). In the present study, IGF-IR mRNA was expressed weakly in lingual epithelium, including the papillae. Therefore, IGFs may function to cause the morphogenesis of the lingual papillae through IGF-IR, acting in an autocrine or paracrine manner. However, the expression of these molecules was not restricted to the lingual papillae. The present study revealed speciﬁc expression patterns of IGFBP-2, -3, -4, and -5 in the lingual papillae at E14 –17. Morphogenesis of lingual papillae includes a series of evaginations and invaginations, which are coordinated interactions between the epithelium and underlying connective tissue. In fact, proliferating cells were observed in the apex of epithelium, trench grooves, and core of connective tissue of the developing 844 SUZUKI Fig. 1. Expression of IGFBP mRNAs in developing fungiform and ﬁliform papillae of the mouse tongue detected by in situ hybridization with RNA probes. A–C: E12 tongue showing IGFBP-2 (A), IGFBP-5 (B), and IGFBP-3 (C) expressions. D–F: E14 tongue showing IGFBP-4 (D), IGF-IR (E), and IGFBP-5 (F) expressions. Expressions of IGFBP-4 and IGF-IR are seen in the epithelium and mesenchyme including fungiform papillae (fu in D and E). Note the intense expression of IGFBP-5 in the apex of fungiform (fu) and ﬁliform (ﬁ) papillae (F). G and H: E15 tongue showing mRNA expression of IGFBP-5 (G) and IGFBP-3 (H). IGFBP-3 is expressed in the mesenchymal core of presumptive ﬁliform papillae (H). I–M: E17 tongue showing IGFBP-3 (I), IGFBP-2 (J, K), IGFBP-4 (L), and IGFBP-5 m RNA (M) expression. Corniﬁed surface of each ﬁliform papilla (ﬁ) is seen (I). IGFBP-3 is expressed in the mesenchymal core of ﬁliform papillae (I), but very weak in fungiform papillae (I, inset). IGFBP-2 is expressed in the basal layer of the epithelium of ﬁliform papillae (J) and at the apex of fungiform papillae (K). Expression of IGFBP-4 is absent in the epithelium (L). Expression of IGFBP-5 mostly disappears from ﬁliform and fungiform papillae (M). Scale bar ⫽ 20 m. ROLE OF IGFBP 845 Fig. 2. Expression of IGFBP mRNAs (A–H) and immunohistochemistry for PGP9.5 (I) in developing circumvallate papillae of mouse tongue. A and B: E14 circumvallate papillae showing mRNA expression of IGFBP-2 (A) and IGFBP-5 (B). Note intense expression of IGFBP-5 in the apex of circumvallate papillae (B). C: Weak expression of IGFBP-3 is seen in the core of mesenchyme. E15. D: Intense expression of IGFBP-4 is seen in the epithelium of trench grooves (G). E15. E: Expression of IGF-IR is seen in the epithelium. E15. F–H: E17. Expressions of IGFBP-2 (F), IGFBP-4 (G), IGFBP-5 (H), and PGP9.5 (I). IGFBP-2 is expressed in the apex of circumvallate papillae (F), and IGFBP-4 in the grooves of circumvallate papillae (G). IGFBP-5 expression remains in presumed taste buds (arrows in H), whose location is limited to the apex of circumvallate papillae (arrows in I). Scale bar ⫽ 20 m in A–G and I; 10 m in H. lingual papillae (cf. Mbiene and Roberts, 2003). IGFs may act in this series of evaginations and invaginations, and this action may be regulated by their interaction with IGFBPs. Based on the present results, we suggest IGFBP-5 and -2 to be molecules involved in the evagination to form the raised epithelial papillae. At E12, IGFBP-5 showed broad expression, and at E14 –16, this expression was observed in the apex of ﬁliform, fungiform, circumvallate, and foliate papillae. At a later embryonic stage (E17), IGFBP-5 expression remained only in the taste buds and some epithelial cells. A similar expression pattern of Shh was reported in the taste bud-bearing papillae of mice (fungiform and circumvallate) (Hall et al., 1999; Jung et al., 2004; Liu et al., 2004). Also, mature taste buds expressed Shh (Miura et al., 2001). In fact, in early chick embryogenesis, embryos cultured in the presence of cyclo- pamine, a potent inhibitor of Shh signaling (Liu et al., 2004), showed downregulation of IGFBP-5 expression. IGFBP-5 expression was suggested to be regulated by Shh (Allan et al., 2003). Moreover, IGFBP-2, which was expressed in the entire epithelium until E15, became restricted to the dorsal epithelium of ﬁliform, fungiform, foliate, and circumvallate papillae at E17. IGFBP-2 and IGFBP-5 expression often overlap in the same cells or tissue or are in adjacent tissues, e.g., taste buds (Suzuki et al., 2005), lung alveolae (Schuller et al., 1993), ectoderm of limb buds, astrocytes (Green et al., 1994). In contrast, IGFBP-4 is related to invagination of the epithelium to form grooves or furrows. In our previous study, IGFBP-4 was expressed in the bottom of trenches of circumvallate papillae at postnatal day 2 (Suzuki et al., 2005). In the present study, IGFBP-4 was expressed in the 846 SUZUKI Fig. 3. Expression of IGFBP mRNAs in developing foliate papillae of mouse tongue. A: Intense expression of IGFBP-4 is seen in the grooves (G). E15. B: Note the expression of IGFBP-5 in the ridges among the grooves (G). E15. C: Expression of IGF-IR is seen in the epithelium. E15. D: Expression of IGFBP-2 is seen in the ridge epithelium. E17. E: IGFBP-3 is weakly expressed in the core of mesenchyme. E17. F: Expression of IGFBP-4 is still seen in the grooves (arrows). ﬁ, ﬁliform papillae; fo, foliate papillae. Scale bar ⫽ 20 m in A–E; 10 m in F. TABLE 1. IGFBP mRNA expression patterns in ﬁliform, fungiform, foliate, and circumvallate papillae of E14-17 mice* pillae of ﬁliform papillae, but very weakly expressed in those of other papillae. IGFBP-3 is the major IGF carrier protein in adult serum (Clemmons, 1992) and is also expressed in peripheral embryonic tissue, such as dermal papillae of ﬁliform papillae and hair follicle (Batch et al., 1996). IGF-1R IGFBP-2 IGFBP-3 IGFBP-4 IGFBP-5 E14 E15 E17 ⫹ ⫹⫹ NE ⫹⫹ NE ⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫹ ⫹ (all four papillae) ⫹⫹ (all four papillae) ⫹⫹ (ﬁliform) ⫺ (ﬁliform, fungiform) ⫹⫹ (groove, foliate, circumvallate) ⫺ (all four papillae) *Relative levels of expression are based on hybridization intensities of sections. ⫺, signal low to undetectable; NE, not examined. entire epithelium until E14 and then became restricted to the groove of circumvallate and foliate papillae. Among IGFBPs, IGFBP-4 uniquely inhibits IGF action (Zhou et al., 2003), and so in circumvallate and foliate papillae, it may be involved in the down-growth of grooves, with its activity continuing during early postnatal development. In ﬁliform papillae, regularly spaced dermal papillae appeared at E15 in the rat fetus, although the surface of the tongue remained ﬂat. 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