EXPRESSions THE ANATOMICAL RECORD PART A 288A:695– 699 (2006) Runx3 Expression During Mouse Tongue and Palate Development HIROMITSU YAMAMOTO,1* KOSEI ITO,2 MARIKO KAWAI,3 YOTA MURAKAMI,4 KAZUHISA BESSHO,1 AND YOSHIAKI ITO2 1 Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan 2 Institute of Molecular and Cell Biology and Oncology Research Institute, Singapore 3 Department of Oral Morphology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama, Japan 4 Institute for Virus Research, Kyoto University, Kyoto, Japan ABSTRACT Studies of molecular mechanisms underlying the development of the mammalian oral mucosa have revealed a major involvement of transforming growth factor ␤ (TGF-␤) and bone morphologic protein (BMP) signaling pathways. Here, we examined the expression of a downstream target of TGF-␤ and BMPs, Runx3, in oral mucosa. Runx3 is a runt-related transcription factor that acts as a gastric tumor suppressor and regulator of growth and differentiation in mammalian gastric epithelial cells. Another member of the Runx family in C. elegans, run, is involved in the development of a functional hypodermis and gut. In this report, we examined Runx3 expression using reverse transcription-polymerase chain reaction, immnunohistochemistry, and in situ hybridization and found that Runx3 is expressed in the tongue and palate epithelium of mouse embryos from embryonic day 12.5 to 16.5. The functional relationship between Runx3 and TGF-␤/BMPs signaling in tongue and palate development is discussed. Anat Rec Part A, 288A:695– 699, 2006. © 2006 Wiley-Liss, Inc. Key words: Runx3; tongue epithelium; palate epithelium; TGF␤/BMP signaling Tongue development in mice is initiated during embryonic day 11 (E11) by the appearance of two lateral lingual swellings on branchial arch I. By E13, the general proportions of the tongue occupied by the body, root, and epiglottis are established (Paulson et al., 1985). On the other hand, the palatal shelves ﬁrst appear later on at approximately E12.5 and rapidly grow vertically to ﬂank the developing tongue. The shelves, consisting of proliferating mesenchymal cells, undergo a sudden elevation that brings them into a horizontal apposition above the ﬂattening tongue. Cleft palate, the most common birth defect in human, occurs when the palatal shelves fail to meet in the horizontal plane (Stanier and Moore, 2004). Previous studies have revealed that several developmental factors are expressed in the epithelial cells of the tongue and palate. Signaling pathways, including those comprising bone morphogenetic proteins (BMPs), transforming growth factor ␤ (TGF-␤), sonic hedgehog (Shh), and ﬁbrobrast growth factors (FGFs), are known to be involved in vertebrate facial development (Francis-West et al., 1998). Among these various factors, the expression © 2006 WILEY-LISS, INC. of ligands and speciﬁc receptors of TGF-␤/BMPs in tongue and palate epithelial cells have been particularly well demonstrated (see Results and Discussion). This is especially the case for TGF-␤3, which was shown to be essential for the fusion of palatal shelves through the study of TGF-␤3-deﬁcient mice (Kaartinen, 1995; Proetzel et al., 1995). TGF-␤3 is expressed by medial edge epithelium cells just prior to the fusion of palatal shelves and ceases to be expressed shortly after the formation of the midline epithelial seam (MES) (Fitzpatrick et al., 1990). There- *Correspondence to: Hiromitsu Yamamoto, Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, 54 Shogoin-kawaharacho, Sakyo, Kyoto 606-8507, Japan. Fax: 81-75-761-9732. E-mail: email@example.com Received 21 September 2005; Accepted 22 February 2006 DOI 10.1002/ar.a.20339 Published online 7 June 2006 in Wiley InterScience (www.interscience.wiley.com). 696 YAMAMOTO ET AL. fore, TGF-␤3 is predicted to be involved in the removal of the MES, a process essential for the conﬂuence of the palatal mesenchyme. Moreover, Msx, a factor acting downstream of BMPs, is frequently mutated in people with orofacial clefting, suggesting distinct functions for BMP signaling. This was further corroborated by studies using Msx-, type IA Bmp receptor (Alk3)-, and Bmp4deﬁcient mouse models (Satokata and Maas, 1994; van den Boogaard et al., 2000; Liu et al., 2005). Thus, TGF-␤/ BMPs signaling pathways are heavily involved in mammalian palatogenesis. The Runt-related (RUNX) gene family includes three members, which encode heterodimeric transcription factors (Ito, 2004). Runx1/AML1 plays a role in the regulation of hematopoiesis (Okuda et al., 1996), and its dysregulation leads to acute myeloid leukemia (Look, 1997). Runx2/Cbfa1 is essential for osteoblast differentiation, bone formation, and chondrocyte maturation (Komori et al., 1997; Otto et al., 1997; Inada et al., 1999; Enomoto et al., 2000), and haploinsufﬁciency of Runx2 causes cleidocranial dysplasia, a human skeletal disease (Mundlos et al., 1997). Runx3/PEBP2␣C (Bae et al., 1995) has multiple functions and was ﬁrst correlated with the genesis and progression of human gastric cancer as a tumor suppressor. The gastric mucosa of Runx3-null (Runx3⫺/⫺) mice exhibits hyperplasia as a result of stimulated proliferation and suppressed apoptosis of epithelial cells (Li et al., 2002), and RUNX3 is inactivated in more than 80% of human gastric cancer cells by gene silencing or protein mislocalization (Ito et al., 2005). Runx3 has an essential role in the development of dorsal root ganglia neurons (Inoue et al., 2002; Levanon et al., 2002) and functions in development of CD8-lineage T-lymphocytes (Taniuchi et al., 2002; Woolf et al., 2003). Furthermore, together with Runx2, Runx3 plays fundamental roles in skeletal development by regulating and coordinating chondrocyte maturation and proliferation to form limb bones of the appropriate size and shape (Yoshida et al., 2004). Runx3 is expressed in the gastrointestinal tract of mouse and human (Li et al., 2002), and run, a homolog of the Runx family in C. elegans, is involved in the development of a functional epidermis and gut (Nam et al., 2001). Furthermore, RUNX3 interacts with receptor-regulated Smads (R-Smads), Smad1, 2, 3, and 5, and a commonpartner Smad (Co-Smad), Smad4, to regulate target gene expression downstream of TGF-␤/BMPs (Hanai et al., 1999; Chi et al., 2005). Thus, Runx3 possibly functions downstream of TGF-␤/BMPs in tongue and palate development. In order to address the involvement of Runx3 in oral epithelial development, we examined the expression of Runx3 in mouse embryonic oral mucosa using reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and in situ hybridization techniques. Our ﬁndings demonstrate that Runx3 is expressed in the tongue and palate epithelium from E12.5 to E16.5, which corresponds to the developmental stage involving TGF-␤/ BMPs. MATERIALS AND METHODS Generation of Runx3 Mutant Mice Runx3⫹/⫺ and Runx3⫺/⫺ mice were generated as described previously (Li et al., 2002). The lac-Z reporter sequence was inserted in-frame into the Runx3 gene to detect the expression of Runx3. The Runx3 genotype of each mouse was determined by PCR using tail DNA and the following primers: 5⬘-GACTGTGCATGCACCTTTCACCAA-3⬘ and 5⬘TAGGGCTCAGTAGCACTTACGTCG-3⬘, or 5⬘-GACTGTGCATGCACCTTTCACCAA-3⬘ and 5⬘-ATGAAACGCCGAGTTAACGCCATCA-3⬘. All experiments in this study were approved by the Animal Research Committee, Graduate School of Medicine, Kyoto University. Tissue Dissection and RT-PCR Whole tongues were dissected from E12.5 to 18.5 and neonate wild-type mice. Five or six samples were collected at each stage except at E12.5 and subjected to RT-PCR independently. The amount of tissue from the E12.5 embryos was so small that two preparations were mixed for each assay. Stomachs and left hind limb cartilages were also dissected from E14.5 wild-type embryos. Total RNA was isolated from these tissues using Isogen (Nippon Gene, Tokyo, Japan). Total RNA (1 g) was reverse-transcribed with reverse transcriptase and oligo-(dT) primers at 42°C for 1 hr (Invitrogen, SuperScript kit). Runx3 cDNA was ampliﬁed by PCR with EX Taq DNA polymerase (TaKaRa) under the following conditions: 94°C for 3 min, followed by 28, 30, and 32 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min. The primers used for ampliﬁcation were 5⬘-ACCGCTTTGGAGACCTGCGCATG-3⬘ and 5⬘-CGCTGTAGGGGAAGGCGGCAGA-3⬘ for Runx3, and 5⬘-CGTATTGGGCGCCTGGTCAC-3⬘ and 5⬘CCAGTGAGCTTCCCGTTCAC-3⬘ for GAPDH (G. Huang et al., 2001). ␤-Galactosidase Staining and Immunohistochemistry ␤-galactosidase activity was measured as described previously (Li et al., 2002). For whole-mount ␤-galactosidase histochemical staining, Runx3⫹/⫺ E15.5 embryos were ﬁxed with 2% paraformaldehyde and 0.2% glutaraldehyde in PBS, then ␤-galactosidase activity was detected by incubating the samples in an X-gal staining cocktail, which contained nitroblue tetrazolium (NBT) and potassium ferrocyanide as described (Huang et al., 1998; Li et al., 2002). For histological analysis, the embryos were ﬁxed with 4% paraformaldehyde, then embedded in parafﬁn and cut into 7 m thick sections. To detect ␤-galactosidase, immunohistochemistry was performed using an anti-␤-galactosidase mouse monoclonal antibody (Promega) and the Dako EnVision⫹ mouse Kit (Dako). In Situ Hybridization For in situ hybridization, digoxigenin-11-UTP-labeled single-stranded RNA probes were prepared using a DIG RNA-labeling kit (Roche Biochemica). A 1.1 kb fragment of Runx3 cDNA was used to generate antisense and sense probes. Sections prepared as described above were also used for in situ hybridization as described previously (Komori et al., 1997; Inada et al., 1999; Yoshida et al., 2002). Hybridization was performed at 55°C overnight and washed at the same temperature. RESULTS AND DISCUSSION Expression of Runx3 in the developing tongue from E12.5 to newborn mice was detected by RT-PCR. Runx3 mRNA was expressed in mouse tongue at E12.5. Expression was strong between E13.5 and E15.5, slowly de- Runx3 EXPRESSION Fig. 1. Runx3 expression in mouse tongue revealed by RT-PCR. A: Runx3 expression in the tongue from E12.5 to E18.5 wild-type mouse embryos and newborn mice. The number of PCR cycles was 30. B: Runx3 expression in the tongue, stomach, and hind limb cartilage at E14.5 in the wild-type mouse embryo. PCR was performed for the indicated number of cycles to detect expression. GAPDH expression was used as a control. creased after E16.5, and was barely detectable in newborn mice (Fig. 1A). The level of Runx3 expression in the tongue was comparable to that in the stomach and hind limb cartilage at E14.5 (Fig. 1B), where Runx3 expression has been previously observed (Li et al., 2002; Yoshida et al., 2004). The expression pattern of Runx3 in the oral mucosa was examined using Runx3 heterozygous (Runx⫹/⫺) embryos in which the lac-Z gene was integrated into exon3 of one allele of Runx3 (Li et al., 2002). ␤-galactosidase activity was measured in whole-mount embryos at various stages. Expression of the lac-Z allele was detected in the developing tongue at E15.5 (Fig. 2A–C). Both the dorsal and the ventral (sublingual) surfaces of the tongue showed ␤-galactosidase activity in Runx3⫹/⫺ embryos (Fig. 2B and C). No staining was detected on either surface of wild-type tongue (Fig. 2D and E). To determine Runx3 expression at the cellular level, we analyzed embryonic tissues histologically. In the oral region of the E12.5 embryos, Runx3 expression was observed in the epithelial lining of the dorsal mucosal surface of the tongue and throughout the epithelium of the palate (Fig. 2F–H). ␤-galactosidase expression was detected predominantly in the epithelial cells of the tongue and palate, and it was stronger on the dorsal than on the ventral surface of the tongue (Fig. 2F–H). In E13.5 embryos, when the structure of the epithelial layer seemed complete, the Runx3 expression pattern (Fig. 2I–K) was 697 Fig. 2. Runx3 expression in mouse tongue as revealed by ␤-galactosidase activity. A–E: Runx3 expression in Runx3⫹/⫺ (A–C) and wildtype (D and E) mice was detected by ␤-galactosidase staining of wholemount preparations from E15.5 embryos. The dorsal (B and D) and the ventral (sublingual; C and E) surfaces of the tongues are shown. F–N: Runx3 expression in the Runx3⫹/⫺ mouse oral region at E12.5 (F–H), E13.5 (I–K), and E14.5 (L–N) was revealed by immunodetection of ␤-galactosidase in sagittal sections of embryos. P, palate; T, tongue; E, epithelium; M, mesenchyme. Scale bars ⫽ 1 mm (A–E); 500 m (F, I, and L); 200 m (G, J, and M); 100 m (H, K, and N). similar to that seen in the E12.5 embryos. In contrast, Runx3 expression was observed in both the dorsal and the ventral mucosal epithelial layers of the tongue and in the palate in E14.5 embryos (Fig. 2L–N). We conﬁrmed the Runx3 mRNA expression in the oral mucosa by in situ hybridization (Figs. 3 and 4). The expression pattern in the tongue and palate epithelium at E13.5 and E14.5 was similar to that observed in the immunohistochemical analysis of ␤-galactosidase activity (Fig. 3A–D). Runx3 was expressed in the mesenchyme as well as in the epithelium of tongue as detected by increased ␤-galactosidase activity (Figs. 2L–M and 3C and D). Additionally, a fall-off in expression was observed at E16.5 as shown by the RT-PCR results in Figure 1A (Fig. 3G and H). In frontal sections of E13.5 and E15.5 embryos, we also observed Runx3 expression in the tongue and palate epithelium (Fig. 4) and intense expression in the eyelid mesenchyme, Meckel’s cartilage, and the mandibular chondrocytes (data not shown; Yamashiro et al., 2002). Runx3 expression was also observed in the nasal epithelium at E15.5 (data not shown). We observed that Runx3 is expressed in mouse tongue and palate epithelium from E12.5 to E16.5, suggesting that it may play a role downstream of TGF-␤/BMP signaling in these stages of oral epithelial development. In fact, 698 YAMAMOTO ET AL. epithelium, which have been reported to express Tgf-␤3, Bmp-2 and -4, and Alks (Zhang et al., 2002; Dudas et al., 2004a, 2004b; Liu et al., 2005). This is the ﬁrst comprehensive report to demonstrate Runx3 expression during mouse tongue development and palatogenesis. The gastric mucosa of Runx3-null mice exhibit hyperplasia due to the continued proliferation and low apoptosis of epithelial cells (Fukamachi and Ito, 2004), and the loss of Runx3 alters gastric differentiation (Fukamachi et al., 2004). Therefore, we analyzed epithelial proliferation and apoptosis in the oral region of these mutants. However, there was no signiﬁcant difference between Runx3⫺/⫺ and wild-type mice in cell proliferation or apoptosis with no morphological difference (data not shown). All RUNX family genes are important targets of TGF-␤ superfamily signaling (Ito and Miyazono, 2003). Runx1 shows similar expression to TGF-␤3 in contact epithelium during palatal fusion (Yamashiro et al., 2002). Therefore, it will be crucial to study the involvement of the other Runx genes with or without Runx3 in tongue and palate development. In addition, the differentiation of Runx3⫺/⫺ oral epithelium using established markers remains to be studied. ACKNOWLEDGMENTS The authors thank T. Komori and C.A. Yoshida for their advice on in situ hybridization techniques. LITERATURE CITED Fig. 3. Runx3 expression in the developing oral region from E13.5 to E16.5 revealed by in situ hybridization. Runx3 expression was observed in the tongue and palate epithelium (arrowhead). Sagittal sections of embryos were stained antisense (AS) or sense (S) probes of Runx3. Scale bars ⫽ 200 m (A–D); 500 m (E–H). Fig. 4. Runx3 expression in tongue and palate development at E13.5 and E15.5 revealed by in situ hybridization. Frontal sections of embryos were stained with antisense (AS) or sense (S) probes of Runx3. PS, palatal shelf. Scale bars ⫽ 500 m. it was found that the location of Runx3 expression coincided with that of Bmp-4 and -7 expression in chick (R. Huang et al., 2001) and occurred in the same place and with the same timing as Bmp-2 and -4 in mouse (Jung et al., 1999; Kim et al., 2003). 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