www.nature.com/scientificreports OPEN Fibroblast growth factor 12 is expressed in spiral and vestibular ganglia and necessary for auditory Received: 5 February 2018 Accepted: 26 June 2018 and equilibrium function Published: xx xx xxxx Yukiko Hanada1,2, Yukiko Nakamura1, Yoshiyuki Ozono2, Yusuke Ishida1,3, Yasumitsu Takimoto1,2,4, Manabu Taniguchi5, Kazuya Ohata1,2, Yoshihisa Koyama1, Takao Imai2, Tetsuo Morihana2,6, Makoto Kondo1, Takashi Sato2, Hidenori Inohara2 & Shoichi Shimada1 We investigated fbroblast growth factor 12 (FGF12) as a transcript enriched in the inner ear by searching published cDNA library databases. FGF12 is a fbroblast growth factor homologous factor, a subset of the FGF superfamily. To date, its localisation and function in the inner ear have not been determined. Here, we show that FGF12 mRNA is localised in spiral ganglion neurons (SGNs) and the vestibular ganglion. We also show that FGF12 protein is localised in SGNs, the vestibular ganglion, and nerve fbres extending beneath hair cells. Moreover, we investigated FGF12 function in auditory and vestibular systems using Fgf12-knockout (FGF12-KO) mice generated with CRISPR/Cas9 technology. Our results show that the inner ear morphology of FGF12-KO mice is not signifcantly diferent compared with wild-type mice. However, FGF12-KO mice exhibited an increased hearing threshold, as measured by the auditory brainstem response, as well as defcits in rotarod and balance beam performance tests. These results suggest that FGF12 is necessary for normal auditory and equilibrium function. Hearing loss is a common problem in people of all ages. Te World Health Organization reports that 360 million people worldwide have hearing loss, with 32 million being children1. Hearing loss is a major sensory defect, as prelingual hearing loss afects oral language acquisition. As over 50% of prelingual hearing loss is genetic2, it is therefore necessary to identify and determine the function of causative genes for hearing loss. Recent studies of transcripts enriched in the inner ear have provided novel insight into hereditary hearing loss and normal auditory function3,4. In this study, we focused on fbroblast growth factor 12 (FGF12) as an inner ear-enriched transcript and performed experiments to investigate its localisation and function. FGF12 is a member of the FGF superfamily, which has 23 FGF members identifed to date. FGF11–14 are intracellular non-secreted proteins known as intracellular FGFs or fbroblast growth factor homologous factors (FHFs)5. Whereas other FGFs work by binding to FGF receptors (FGFRs), FHFs do not bind to FGFRs6. Although reports are limited and much is unknown about FHF function, recent studies have revealed that FHFs bind to and regulate voltage-gated sodium channels (Nav channels)7,8. For example, Wittmack et al., reported colocalisation between FGF13 and Nav1.6 at nodes of Ranvier in dorsal root axons. Moreover, cell lines in which both proteins were coexpressed showed increases in peak current amplitude9. Further, Goldfarb et al. overexpressed FGF12 and reported colocalisation with Nav channels in cultured neurons10. Tey also reported that FGF12 single-knockout 1Department of Neuroscience and Cell Biology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan. 2Department of Otorhinolaryngology-Head and Neck Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan. 3Division of Anatomy and Cell Biology, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsushima, Aobaku, Sendai, Miyagi, 981-8558, Japan. 4Department of Otorhinolaryngology, Osaka Police Hospital, 10-31, Kitayamacho, Tennoujiku, Osaka, 543-0035, Japan. 5Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan. 6Department of Otorhinolaryngology, National Hospital Organization Osaka National Hospital, 2-1-14, Hoenzaka, Chuo, Osaka, Osaka, 540-0006, Japan. Correspondence and requests for materials should be addressed to Y.N. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:11491 | DOI:10.1038/s41598-018-28618-0 1 www.nature.com/scientificreports/ Figure 1. Expression of FGF12 mRNA in the inner ear. (a) Detection of FGF12 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) in the mouse cochlea and vestibular ganglion. β-Actin positive controls are shown. (−) indicates reaction products without cDNA. Tese gel images are cropped, and full-length images are presented in Supplementary Information 1. (b) Quantitative real-time PCR for FGF12 mRNA using cDNA from various tissues of adult mouse (n = 3). Expression levels were normalised to endogenous β-actin. Each expression level was standardised to the vestibular ganglion (VG) and given a value of 1. (KO) mice showed no phenotype, while FGF12/FGF14 double-KO mice exhibited severe ataxia and decreased excitability of cerebellar granule neurons, thereby concluding that FGF12 played an important role together with FGF14 in neuronal action potentials. However, there are no reports showing abnormalities in the auditory or equilibrium function of FGF12-KO mice, and FGF12 function in the inner ear remains to be determined. In this study, we investigated FGF12 expression and localisation in murine inner ear tissue. Additionally, audi- tory and vestibular function was examined in the FGF12-KO mice that we generated. Consequently, this is the frst report to show FGF12 function in the inner ear, and our fndings provide novel insight into the contribution of FHF to normal auditory and vestibular function. Results FGF12 mRNA is abundantly expressed in the inner ear. We hypothesised that transcripts enriched in the inner ear play a key role in normal hearing and vestibular function. Consequently, investigation of these transcripts will aid understanding of the causes of hereditary hearing loss. cDNA libraries encompassing diferent tissues contain expression sequence tag (EST) information that enables mRNA expression levels to be compared. Accordingly, we compared GenBank EST clones from inner ear cDNA libraries to those of other tissues. We iden- tifed 13 candidate genes specifcally expressed in the inner ear. Next, we performed reverse transcription-pol- ymerase chain reaction (RT-PCR) on murine inner ear to confrm that these 13 candidate genes are specifcally and abundantly expressed in the inner ear. Among these genes, we found that FGF12 mRNA was abundantly expressed in the cochlea and vestibular ganglion (Fig. 1a). β-Actin was used as an internal control. To examine the specifcity of FGF12 mRNA expression in the inner ear compared with other tissues, we per- formed quantitative real-time PCR (qPCR) (Fig. 1b). FGF12 mRNA levels were compared in each tissue relative to β-actin as an internal control. FGF12 mRNA levels were highest in the vestibular ganglion, and relatively high in the brain and cochlea. Weak expression was also detected in the eye and testis, with virtually no expression detected in the other tissues examined. FGF12 mRNA is localised in spiral and vestibular ganglia. To examine FGF12 mRNA localisation, we performed in situ hybridisation in the inner ear using an FGF12-specifc complementary RNA (cRNA) probe. FGF12 mRNA expression was abundantly detected in the spiral and vestibular ganglia (Fig. 2a–c). Further, FGF12 mRNA was expressed in both the superior and inferior regions of the vestibular ganglion. Staining intensity in the vestibular ganglion was stronger compared with the spiral ganglion, confrming our qPCR fnding showing that FGF12 mRNA expression is more abundant in the vestibular ganglion (Fig. 1b). No FGF12 mRNA signal was detected in other parts of the inner ear, i.e., hair cells, supporting cells, stria vascularis, or spiral ligaments (Fig. 2b). We also performed in situ hybridisation using the sense probe as a negative control, with no staining detected (Fig. 2d,e). FGF12 protein is localised in the spiral ganglion, vestibular ganglion, and peripheral nerve fbres of the cochlea and vestibular organ. To examine FGF12 protein localisation, we performed immunohistochemistry in inner ear tissue using an anti-FGF12 antibody. FGF12 protein was expressed in the spiral ganglion and superior and inferior regions of the vestibular ganglion (Fig. 2f,g). Tis expression pattern corresponded with FGF12 mRNA (Fig. 2a). In addition, higher magnifcation images of the cochlea showed that FGF12 protein was expressed in nerve fbres (Fig. 2h, white triangles) extending from the spiral ganglion to immediately beneath the inner and outer hair cells (Fig. 2h,i). FGF12 protein was also expressed in nerve fbres of the saccule, extending from the vestibular ganglion through the supporting cell layer to below the hair cells (Fig. 2j). To confrm the specifcity and immunoreactivity of the utilized anti-FGF12 antibody, we performed immunohistochemistry on inner ear tissue from FGF12-KO mice. Using the same exposure times, no fuorescent signal was detected (Fig. 2k [cf. Fig. 2f]). Generation of FGF12-KO mice using the CRISPR/Cas9 system. To examine the contribution of FGF12 function to auditory and vestibular function, we generated FGF12-KO mice using the C57BL/6 J strain SCIENTIFIC REPORTS | (2018) 8:11491 | DOI:10.1038/s41598-018-28618-0 2 www.nature.com/scientificreports/ Figure 2. Localisation of FGF12 mRNA and protein expression in the inner ear. (a–c) FGF12 mRNA localisation in the inner ear by in situ hybridisation. (b) Te cochlea. (c)