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Jun 6, 2016 - unlikely that Ikkα or Irf6 are involved in Hoxc13 or Pax9 function in filiform papillae development, since mutants show no filiform papillae ...
DEVELOPMENTAL DYNAMICS 245:937–946, 2016 DOI: 10.1002/DVDY.24427

RESEARCH ARTICLE

Regional Regulation of Filiform Tongue Papillae Development by Ikka/Irf6 a

Maiko Kawasaki,1,2 Katsushige Kawasaki,1,2,3 Shelly Oommen,2 James Blackburn,2 Momoko Watanabe,1,4 Takahiro Nagai,1,4 Atsushi Kitamura,1,4 Takeyasu Maeda,1,3 Bigang Liu,5 Ruth Schmidt-Ullrich,6 Taishin Akiyama,7 Jun-Ichiro Inoue,7 Nigel L. Hammond,8 Paul T. Sharpe,2 and Atsushi Ohazama1,2* 1

Division of Oral Anatomy, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan Department of Craniofacial Development and Stem Cell Biology, Dental Institute, Kings College London, London, United Kingdom 3 Oral Life Science, Research Center for Advanced Oral Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 4 Division of Oral and Maxillofacial Surgery, Department of Health Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 5 Department of Molecular Carcinogenesis, UT MD Anderson Cancer Center, Smithville, Texas 6 Department of Signal Transduction in Tumor Cells, Max-Delbr€ uck-Center for Molecular Medicine, Berlin, Germany 7 Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan 8 Faculty of Life Sciences and School of Dentistry, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, United Kingdom

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Background: Non-gustatory filiform papillae play critical roles in helping to grip food, drawing food to the esophagus, cleaning the mouth, and spreading saliva. The molecular mechanisms of filiform tongue papillae development however are not fully understood. Results: We found Ikka and Irf6 expression in developing tongue epithelium, and describe here specific tongue abnormalities in mice with mutation of these genes, indicating a role for Ikka and Irf6 in filiform papillae development. Ikka and Irf6 mutant tongues showed ectopic vertical epithelium at the midline, while lateral sides of mutant tongues adhered to the oral mucosa. Both the ectopic median vertical epithelium and adhered epithelium exhibited the presence of filiform tongue papillae, whereas epithelium between the median vertical epithelium and adhered tongue showed a loss of filiform tongue papillae. Timing of filiform papillae development was found to be slightly different between the midline and lateral regions of the wild-type tongue. Conclusions: Filiform papillae thus develop through distinct molecular mechanisms between the regions of tongue dorsum in the medio-lateral axis, with some filiform papillae developing under the control of Ikka and C 2016 Wiley Periodicals, Inc. Irf6. Developmental Dynamics 245:937–946, 2016. V Key words: Filiform papillae; Ikka; Irf6 Submitted 19 April 2016; First Decision 6 June 2016; Accepted 8 June 2016; Published online 15 June 2016

Introduction Tongue papillae are ectodermal structures that are important in the gustatory system. In rodents, there are four different types of tongue papillae; fungiform, circumvallate, foliate, and filiform. The fungiform, circumvallate, and foliate papillae contain taste buds, and are referred to as taste papillae or gustatory papillae, whereas filiform papillae contain no taste buds (non-gustatory papillae). Although taste papillae are distributed in a specific pattern over the tongue (a single circumvallate papilla in the center of the terminal sulcus, fungiform papillae on the anterior tongue in a pattern of longitudinal rows, bracketing a median furrow), non-gustatory filiform papillae cover the entire dorsal surface of the tongue (Fig. 1A; Iwasaki et al., 1999a,b).

Grant sponsor: the Ministry of Health, Labour and Welfare of Japan; Grant number: 16592080. *Correspondence to: Atsushi Ohazama, Division of Oral Anatomy, Department of Oral Biological Science, Niigata University, Graduate School of Medical and Dental Sciences, 2-5274, Gakkocho-dori, Chuo-ku, Niigata 951-8514, Japan. E-mail: [email protected]

The filiform papillae function to help to grip food, drawing food to the esophagus, cleaning the mouth and spreading saliva (Wong et al., 2000; Wojcik et al., 2001). Filiform papillae are further classified into two morphological subtypes, depending on their location on the dorsum of the tongue. The filiform papillae have pointed tips in the intermolar eminence, whereas the filiform papillae show rounded tips in other (anterior) parts of the tongue surface (Hirao et al., 2007). It has been shown that the morphological differences between the filiform papillae in the intermolar eminence and those in the anterior tongue are dependent on Bmp signaling (Beites et al., 2009; Kawasaki et al., 2012). However, the molecular mechanisms of filiform tongue papillae development are not fully understood. The first significant morphological changes of filiform papillae development can be seen at mouse embryonic day (E) 16 and they develop rapidly during the last few days before birth (Jung et al., 2004; Iwasaki et al., 2006a). At birth, filiform papillae show Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy. 24427/abstract C 2016 Wiley Periodicals, Inc. V

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Fig. 1. Tongue papillae in Ikka mutant mice. A: Schematic diagram of mouse tongue papillae. B: In situ hybridization on sagittal sections showing Ikka expression at E16.5. C-I: Frontal sections showing wild-type tongue (C-E) and Ikka mutant tongue at the birth (F-I). D,E: Higher magnification views of the areas indicated by arrow and arrowhead in C, respectively. G,H,I: Higher magnification views of the areas indicated by arrow, arrowhead, and double arrowhead in F, respectively. F: MVE-AE (arrow), median vertical epithelium (double arrowhead), adhesion tongue epithelium with oral mucosa (single arrowhead). J-L: In situ hybridization on frontal sections showing Krt1-5 expression in wild-type (J,K) and Ikka mutant tongue (L). K: Arrow indicating taste papillae in non-Krt1-5 expressing cells. L: Arrows indicating the lack of Krt1-5 expression at MVE-AT. M,N: SEM image of dorsum tongue of wild-type (M) and Ikka mutant (N). O,P: In situ hybridization on frontal sections showing Shh expression inwild-type (O) and Ikka mutant tongue (P). P: Arrow indicating Shh expression at MVE-AE and median vertical epithelium, respectively. Q,R: Parasagittal section showing posterior wild-type tongue (Q) and Ikka mutant tongue (R). t; tongue

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a similar distribution to that observed in the adult (Iwasaki et al., 1999a,b). The NF-kB pathway plays a major role in many physiological and pathological processes including ectodermal organogenesis (Li and Verma, 2002; Chariot, 2009; Sanz et al., 2010; Hacker and Karin, 2006; Israel, 2010; Oeckinghaus et al., 2011). Three pathways—classical/canonical, alternative/non-canonical, and a hybrid of both—have been shown to activate NF-kB. Canonical NF-kB activation is usually a rapid and transient response to a wide range of stimuli. NF-kB activity is regulated by interaction with an inhibitor, Inhibitor of kappa B (IkB), which acts to retain NF-kB in the cytoplasm by masking the nuclear localization sequence in non-stimulated cells. Exposure to stimuli results in rapid phosphorylation of IkB leading to its degradation. The resulting free NF-kB dimers translocate to the nucleus and regulate target gene transcription. IkB kinase (IKK) is composed of two catalytic subunits, IKKa and IKKb, and a regulatory subunit, IKKg (NEMO). The canonical pathway is dependent on functional IKKb and NEMO subunits, whereas IKKa plays only a minor role. The noncanonical pathway leads to slower activation of NF-kB but results in prolonged activation of NF-kB target gene transcription. In this instance, NF-kB inducing kinase (NIK) only recruits the Ikka subunit, which subsequently phosphorylates specific NF-kB dimers (Oeckinghaus et al., 2011). The non-canonical pathway thus depends solely on Ikka. Mice with targeted mutations in Ikka show fusion of oral mucosa and evagination of incisor epithelium (Ohazama et al., 2004). Ifn regulatory factor (Irf) genes regulate the transcription of interferons and proteins produced in response to the presence of pathogens, and function as an integral part of the immune system (Platanias, 2005). In common with Ikka mutants, Irf6 mutant mice show evagination of incisor epithelium and adhesion of oral epithelium (Ingraham et al., 2006; Richardson et al., 2006; Blackburn et al., 2012). We show here that the filiform papillae develop through distinct molecular mechanisms between the regions of the tongue dorsum, with some filiform papillae developing under the control of Ikka and Irf6.

Results Ikka was found to be weakly expressed in E16.5 tongue epithelium (Fig. 1B). Filiform tongue papillae are characterized as epithelial protrusions into the oral cavity and epithelial ridge structures that are located only on the dorsum of the tongue (Fig. 1C,D). The lateral side of the tongue does not possess these epithelial structures (Fig. 1E). Filiform tongue papillae as epithelial protrusions and epithelial ridge structures are thus clearly identified by histology and scanning electron microscope (SEM) analysis on newborn mice (Fig. 1C-E, data not shown). Although the anterior part of the Ikka mutant tongue often showed fusion of the entire tongue epithelium with oral and nasal epithelia, other parts of the tongue (including the presumptive intermolar eminence) showed both fused and non-fused regions (Fig. 1F, data not shown). At the regions exhibiting a non-fused surface, Ikka mutant tongues showed ectopic vertical epithelium at the midline and adherence with oral mucosa at the lateral sides (Fig. 1F). Keratins and certain keratin-associated proteins are distinct markers of epithelial differentiation and tissue types, as their expression is organ and cell differentiation specific. Hard keratins, including

Krt1-5, are mainly observed in nail, hair cortex, and hair cuticle (Chu and Weiss, 2002; Homberg and Magin, 2014; Loschke et al., 2015). In addition, Krt1-5 is used as marker of filiform papillae, as it is known to be expressed only in these tissues and not in the gustatory papillae of the newborn wild-type tongue (Fig. 1J,K; Jonker et al., 2004). In Ikka mutants, Krt1-5 expression was observed in the upper part of adherent epithelium and median vertical epithelium at birth, suggesting that the epithelium from the upper part of the adhered epithelium at the lateral side to the median vertical epithelium was the dorsum surface of tongue (Fig. 1L). In fact, both the upper part of adherent epithelium and vertical epithelium showed an epithelial ridge structure in mutants (Fig. 1H,I). The epithelium between median vertical epithelium (MVE) and the adherent epithelium (AE) (MVE-AE) however exhibited no epithelial protrusions on the surface or obvious epithelial ridge structures (Fig. 1G). SEM analysis confirmed no papillae structures on the tongue surface at the ultrastructural level of the MVE-AE region compared to wild-type tongues (Fig. 1M,N). Krt1-5 expression could not be detected at the MVE-AE region (Fig. 1L). Filiform papillae thus failed to develop at the MVE-AE region. These tongue phenotypes were observed in all mutants we examined (n ¼ 40). Shh is a marker of the gustatory tongue papillae, and is expressed in wild-type embryonic tongue papillae containing taste buds (Fig. 1O; Kim et al., 2003; Liu et al., 2004). In E14.5 mutants, Shh expression was retained both in ectopic vertical epithelium at the midline and the MVE-AE region (Fig. 1P). These results suggested that gustatory papillae were maintained in the MVE-AE region, although any obvious morphological sign of gustatory papillae could not be detected by SEM or histological analysis. Para-sagittal sections of mutant tongues showed no epithelial protrusions on the surface or obvious epithelial ridge structures compared to wild-type tongues, suggesting that the loss of the filiform papillae formation occurred at the MVE-AE region along anterior-posterior axis (Fig. 1Q,R). Tongue papillae development is known to require reciprocal epithelial-mesenchymal interactions (Kim et al., 2003; Jung et al., 2004). To determine whether Ikka mutant tongue phenotypes are caused by disruption of epithelial and/or mesenchymal function, we crossed Ikka mutants with mice over-expressing Ikka under the Keratin 5 (K5) promoter (K5-Ikka; Lomada et al., 2007). K5 is expressed in oral and tongue epithelium from E11.5, and K5-Ikka mice yielded no obvious tongue papillae phenotypes (Fig. 2A,B; data not shown). Filiform tongue papillae were rescued completely in K5-Ikka;Ikka-/suggesting that epithelial Ikka is required to control filiform papillae development at the MVE-AE region (Fig. 2C,D). NF-kB is known to be involved in the development of ectodermal organs (Li and Verma, 2002; Hacker and Karin, 2006; Chariot, 2009; Sanz et al., 2010). To confirm whether the lack of filiform papillae in Ikka mutants is caused by the disruption of canonical NF-kB activation, we examined the tongue papillae in transgenic mice expressing a super-repressor of IkBa (cIjBaDN), which inhibits NF-kB release and results in downregulation of canonical NF-kB activation (Schmidt-Ullrich et al., 2001). Epithelial protrusions, epithelial ridge structures and Krt1-5 expression were observed on the entire dorsum surface of the tongue in cIjBaDN mice, suggesting that the loss of filiform papillae in Ikka mutants was not caused by disruption of canonical NF-kB activation (Fig. 2E-G). In order to investigate whether the lack of filiform papillae in Ikka mutants is caused by disruption of noncanonical NF-kB activation, we examined mice with mutation of

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Fig. 2. Tongue papillae in mice with alteration of NF-kB related molecule. A,B: In situ hybridization with frontal embryonic sections showing K5 expression in wild-type tongue at E11.5 (A) and E12.5 (B). t ¼ tongue. C–H: Frontal sections showing K5-Ikka;Ikka-/- mice (C,D), cIjBaDN mice (E– G), and Aly/Aly mice (H). D,F: Higher magnification views of tongue papillae in C and E, respectively. G: In situ hybridization on frontal section of cIjBaDN showing Krt1-5 expression.

the non-canonical NF-kB essential molecule, NIK (Aly/Aly). Aly/ Aly mice did not show any loss of epithelial protrusions or epithelial ridge structures, suggesting that the role of Ikka in filiform papillae development is independent of the NF-kB pathway (Fig. 2H). Irf6 mutant (Irf6R84C/R84C) mice are known to show evagination of incisor epithelium, palatal clefting, and fusion of oral epithelium, which are also seen in Ikka mutant mice (Richardson

et al., 2006, 2009, 2014; Ingraham et al., 2006; Thomason et al., 2010; Blackburn et al., 2012; Ferretti et al., 2011; Iwata et al., 2013). Irf6 has additionally been shown to be involved in tongue muscle development (Goudy et al., 2013). Irf6 was strongly expressed in tongue epithelium including the non-dorsum part of tongue (Fig. 3A; Knight et al., 2006). To investigate whether Irf6R84C/R84C mice also have tongue papillae phenotypes, we examined the Irf6 mutant tongue. In common with Ikka mutant

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Fig. 3. Tongue papillae in Irf6 mutant mice. A: In situ hybridization on frontal sections showing Irf6 expression at E16.5. B–E: Frontal sections showing Irf6 mutant tongue. B: MVE-AE (arrow), median vertical epithelium (double arrowhead), adhesion tongue epithelium with oral mucosa (single arrowhead). C,D,E: Higher magnification views of the areas indicated by arrow, arrowhead and double arrowhead in B, respectively. F,G: SEM image of dorsum tongue of wild-type (F) and Irf6 mutant (G). H: In situ hybridization on frontal sections showing Krt1-5 expression in Irf6 mutant tongue.

mice, the Irf6 mutant tongue showed ectopic vertical epithelium at the midline and adherent epithelium with oral mucosa at lateral sides of the tongue, which displayed epithelial ridges (Fig. 3BD; Richardson et al., 2006, 2009, 2014). Histological and SEM analysis showed that Irf6 mutant MVE-AE region exhibited no obvious tongue papillae structures (Fig. 3E,G). In common with

Ikka mutants, Krt1-5 showed no expression at the MVE-AE region of Irf6 R84C/R84C mice (Fig. 3H). Para-sagittal sections of Irf6 mutant tongues showed no epithelial protrusions on the surface or obvious epithelial ridge structures along anteriorposterior axis, suggesting that the Irf6 mutant tongue phenocopies that of the Ikka mutant (data not shown).

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Fig. 4. Tongue papillae in Ikka-/-;Irf6R84C/R84C mice. A: In situ hybridization on frontal sections showing Irf6 expression in Ikka mutant tongue. B– D: Frontal sections showing Ikka-/-;Irf6R84C/R84C mouse tongue at birth. C,D: Higher magnification views of adhesion tongue epithelium with oral mucosa (C) and the areas of MVE-AT (D) in B.

Filiform papillae were lost at the MVE-AE region, whereas they were retained in the ectopic vertical epithelium at the midline and upper part of adhered epithelium with oral mucosa at lateral sides of the tongue, in both Ikka and Irf6 mutants. Irf6 expression was retained in all tongue epithelium including MVE-AE region in Ikka mutant mice (Fig. 4A). In order to investigate whether there is redundancy between Ikka and Irf6 to maintain filiform papillae at the midline and lateral regions of mutant tongue, we generated mice with double mutation of Ikka and Irf6 (Ikka-/-;Irf6R84C/R84C). Ikka-/-;Irf6R84C/R84C mice, however, show no qualitative difference of vertical epithelium and adherent epithelium compared to those in Ikka or Irf6 mutant mice (Fig. 4B-D). Oral epithelial adhesion with tongue epithelium has also previously been reported in mice with mutation of Stratifin (14-3-3 isoform s), which is known to have a pivotal role in cell regulation (Wang et al., 2000; Hermeking and Benzinger, 2006). Stratifin mutants exhibit thickened skin epithelium, palatal cleft and adhesion of oral epithelium: identical phenotypes to those seen in Ikka and Irf6 mutant mice (Guenet et al., 1979). It is possible that Stratifin functions with Ikka and Irf6 during oral epithelium and tongue papillae development. In order to investigate the role of Stratifin in tongue papillae formation, we examined the Stratifin mutant tongue. The ectopic vertical epithelium, fusion between the oral epithelium and the lateral side of the tongue, and MVEAE region were observed in Stratifin mutants (Fig. 5A). Unlike Ikka and Irf6 mutants, an epithelial ridge structure was observed at MVE-AE region, which showed Krt1-5 expression (Fig. 5B,C). These results indicated that Stratifin is not essential for the formation of filiform papillae at the MVE-AE region. Stratifin was expressed throughout the tongue epithelium and molar tooth epithelium, and was retained in epithelium at MVE-AE region in Irf6 mutants (Fig. 5D,E). Stratifin expression was, however, reduced in fused tongue epithelium with oral mucosa and epithelium at MVE-AE region in Ikka mutant mice, while it was retained in Ikka mutant molar tooth epithelium (Fig. 5F). These results suggest that Stratifin is not essential, but related to filiform formation through Ikka. Oral epithelial adhesion with tongue epithelium has also been reported in mice with mutation of Jagged2, which is known to be involved with Irf6 during oral epithelial development (Casey et al., 2006; Richardson et al., 2009). Abnormal epithelial development on the lateral side of the tongue has been demonstrated previously in Jagged2 mutants (Casey et al., 2006). It is possible that Ikka and Irf6 are involved with Jagged2 in oral epithelium and tongue papillae development. Jagged2 expression was, however, retained in epithelium at the MVE-AE region of Ikka and Irf6 mutant mice (Fig. 5H,J; Richardson et al., 2009). p63 is

known to be a key regulator of squamous epithelial development, and shows expanded expression into the superficial cell layer in abnormal epithelial adhesions of Irf6 mutants (Richardson et al., 2009). It is conceivable that p63 is also associated with Ikka and Irf6 in tongue papillae development. Similar changes in p63 expression were observed in Ikka mutant tongue epithelium compared to wild-type (Fig. 5K,L). Bmp signaling is known to play a critical role in regulating filiform papillae development (Kawasaki et al., 2012; Beites et al., 2009). It is possible that Ikka and Irf6 regulate tongue papillae development through Bmp signaling. Phosphorylated-Smad 1/5/ 8 (marker of Bmp signaling)-positive cells were however observed at the MVE-AE region of Ikka mutant tongue (Fig. 6B). Changes in canonical Wnt signaling have been shown in incisor development of Ikka and Irf6 mutants, and Wnt signaling is also known to be involved in tongue papillae development (Liu et al., 2007; Iwatsuki et al., 2007; Blackburn et al., 2012). It is conceivable that Ikka and Irf6 are involved in Wnt signaling during tongue papillae development. Expression of the canonical Wnt target gene, Axin2, was slightly reduced, but retained at the MVE-AT region of mutant tongue (Fig. 6D). Ikka and Irf6 mutant tongue papillae phenotypes thus revealed distinct filiform papillae developmental mechanisms between the regions of the dorsum tongue. SEM analysis showed slight morphological differences of filiform papillae between the midline and lateral regions in wild-type embryos when filiform papillae initiate at E16.5, although no obvious morphological changes could be observed at birth (Fig. 6E,F, data not shown). These results suggest that the timing of filiform development is slightly different between regions of the dorsum tongue.

Discussion Non-gustatory filiform papillae are important for helping to grip food, drawing food to the esophagus, cleaning the mouth, and spreading saliva. Although filiform papillae cover the entire dorsal surface of the tongue, it has been shown that there are morphological differences between the filiform papillae in the intermolar eminence and those in anterior region, which are regulated by Bmp signaling (Beites et al., 2009; Kawasaki et al., 2012). In addition to the differences between the anterior tongue and the intermolar eminence, our results suggest that there is another distinct developmental mechanism for the mesio-lateral axis of the dorsum tongue, which is regulated by Ikka and Irf6. Our data indicated that epithelial fusion was observed both between dorsum tongue and oral epithelium, and the lateral side of tongue and oral epithelium at the birth. It has been shown that fusion starts between the lateral side of tongue and oral epithelium

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Fig. 5. Stratifin in tongue papillae development. A,B: Frontal sections showing Stratifin mutant tongue at the birth. MVE-AE (arrow), median vertical epithelium (double arrowhead), adhesion tongue epithelium with oral mucosa (single arrowhead). B: Higher magnification views of the areas of MVE-AE in A. C–F: In situ hybridization on frontal sections showing Krt1-5 expression in Stratifin mutants (C), and Stratifin expression in wild-type (D), Irf6 mutants (E), and Ikka mutants (F). G–L: Frontal (G,H,K,L) and sagittal (I,J) sections showing Jagged2 (G–J) and p63 (K,L) expression in wild-type (G,I,K), Irf6 mutants (H), and Ikka mutants (J,L) at the birth. I,J: Arrows indicating Jagged2 expression in wild-type filiform (I) and Ikka mutant filiform papillae at MVE-AE region (J).

at E12.5 in Irf6 mutants, suggesting the possibility that epithelial fusion expands into the dorsum tongue from the lateral side of tongue (Richardson et al., 2006, 2009, 2014). Our results showed that overexpression of Ikka in the epithelial tissues (K5-Ikka) rescued filiform papillae anomalies in Ikka mutants, suggesting that the lack of filiform papillae is caused by disruption of Ikka function in filiform epithelia which is

independent of the NF-kB pathway. Newborn Ikka mutants exhibit hyperplastic skin keratinocytes, which are also rescued by overexpression of Ikka in the epithelium (K5-Ikka; Hu et al., 1999; Liu et al., 2011). Both tongue papillae and epidermis are stratified epithelial structures. Filiform papillae at the MVE-AE region were observed as a thin epithelial layer in Ikka mutants, whereas Ikka mutant epidermis is markedly thicker (Hu et al.,

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Fig. 6. Molecular changes in Ikka mutant and wild-type tongue. A,B: Immunohistochemistry on frontal sections showing p-Smad1/5/8 in the developing tongue at E16.5 in wild-type (A) and Ikka mutant mice (B). C,D: In situ hybridization on frontal sections showing Axin2 expression at E16.5 in wild-type (C) and Ikka mutant mice (D). E,F: SEM image of wild-type dorsum tongue at midline (E) and lateral region (F). t, tongue

1999). The function of Ikka in epithelium is thus different between skin and filiform papillae. Changes of p63 expression were similar between fused epithelium and epithelium at the MVE-AT region, although filiform papillae existed in the upper part of the fused epithelium, but not the epithelium at the MVEAE region. Epithelial differentiation regulated by p63 is thus different between the regions of dorsal epithelium. Irf6 has been shown to be involved in formation of the periderm of the epidermis and oral mucosa during development (Richardson et al., 2014). p63 has been shown to be related to Irf6 in regulating periderm formation of oral epithelium (Thomason et al., 2010). Periderm is also observed in the dorsum tongue (Frabman and Mbiene, 1991; Iwasaki et al., 2006b). We could not exclude the possibility that periderm function is different between the regions of the dorsum tongue, resulting in the different tongue papillae phenotypes in the mutant mice.

Irf6 mutant mice showed identical filiform papillae phenotypes to Ikka mutants. Interconnection between Ikka and Irf6 in governing epidermal development has been assumed (Leslie et al., 2015). Although our findings could not establish a genetic interaction between Ikka and Irf6, the possibility of direct interaction between Ikka and Irf6 (e.g., they function in the same pathway) could not be excluded in filiform papillae development. It is also conceivable that both genes work completely independently, but have the same downstream targets and thereby generate the same phenotype. Both Ikka mutation and Ikkb overexpression have been shown to result in the increase of Wnt signaling in incisor development (Blackburn et al., 2012, 2015). Wnt signaling is known to be involved in tongue papillae development (Liu et al., 2006; Iwatsuki et al., 2007). Slight changes in Wnt signaling was also observed in Ikka mutant tongue, suggesting the possibility that fine-tuning of

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Wnt signaling is important for tongue papillae development, but is dependent on the region of the tongue dorsum. Hoxc13 is expressed in filiform papillae and is known to be essential for the formation of the tip of the filiform papillae (Godwin and Capecchi, 1998). Pax9 also regulates the anteriorposterior polarity of filiform papillae (Jonker et al., 2004). It is unlikely that Ikka or Irf6 are involved in Hoxc13 or Pax9 function in filiform papillae development, since mutants show no filiform papillae structures. Filiform papillae cover the entire dorsal surface of the tongue. Our findings reveal that timing of filiform development is slightly different in the mesio-lateral axis of the tongue dorsum. It is possible that distinct molecular mechanisms between the regions of tongue dorsum in the medio-lateral axis lead to different developmental timing, which is crucial for tongue papillae formation.

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Experimental Procedures All experimental procedures detailed below were performed in duplicate.

Production and Analysis of Transgenic Mice Ikka mutant, Irf6 mutant (Irf6R84C/R84C), Stratifin mutant (Sfn; SfnEr/Er) and cIjBaDN mice were produced as described previously (Guenet et al., 1979; Hu et al., 1999; Schmidt-Ullrich et al., 2001; Richardson et al., 2006). Mice overexpressing Ikka under the Keratin 5 promoter (K5-Ikka) have been described previously (Lomada et al., 2007). Aly/Aly mice were purchased from CLEA, Japan. Embryonic day 0 (E0) was taken to be midnight prior to finding a vaginal plug. To accurately assess the age of embryos, somite pairs were counted and the developmental stage confirmed using morphological criteria, such as relative size of maxillary and mandibular primordial and extent of nasal placode invagination. Mouse heads were fixed in 4% paraformaldehyde, embedded and serially sectioned at 8 mm. Sections were split over 4-10 slides and prepared for histology and radioactive in situ hybridization. Decalcification using 0.5M ethylenediaminetetraacetic acid was performed after fixation of newborn mouse tissues. For histological analysis, sections were stained with hematoxylin and eosin or Masson’s trichrome.

In Situ Hybridization Radioactive in situ hybridization with [35S]UTP-labeled riboprobes was carried out as described previously (Ohazama et al., 2008). In brief, hybridization was performed overnight at 55degC. The slides were then washed at 65degC and treated with RNase A at 37degC to remove any nonspecifically bound probe. The slides were dipped in Ilford K.5 photographic emulsion. Autoradiography was performed by exposing the sections in a light-tight box at 4degC for 10–14 days. Slides were developed, fixed, counterstained with hematoxylin, and mounted. In some of sections, the darkfield images were inverted, artificially stained red, and combined with the brightfield image using Adobe Photoshop software.

Immunohistochemistry After deparaffinization, tissue sections were treated with proteinase K and then incubated with an antibody to Phosphorylated-

Smad 1/5/8 (Cell signaling Technology). Tyramide signal amplification system was performed for primary antibody detection according to manufacturer’s instructions (Perkin Elmer Life Science).

SEM Analysis Tongues were fixed with 2% glutaraldehyde in 0.1 M Nacacodylate buffer and 0.1 M sucrose, followed by postfixation with 1% osmium tetroxide in 0.1 M Na-cacodylate buffer. After critical point drying, the samples were coated with gold and photographed using scanning electron microscopy.

Acknowledgments We thank Tony Brain for SEM analysis, Michael Dixon for Irf6R84C/R84C mice, Yinling Hu for Ikka-/- mice and Heiko Peters for Krt1-5 plasmids.

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