Development 127, 4551-4560 (2000) 4551 Printed in Great Britain © The Company of Biologists Limited 2000 DEV4430

Hes1 is a negative regulator of inner differentiation

J. Lisa Zheng1, Jianyong Shou2, Francois Guillemot3, Ryoichiro Kageyama4 and Wei-Qiang Gao1* 1Department of Neuroscience, Genentech, Inc., South San Francisco, CA 94080, USA 2Molecular Oncoclogy, Genentech, Inc., South San Francisco, CA 94080, USA 3Institut de Genetique et de, Biologie Moleculaire et Cellulaire, 67404 Illkirch, C.U. de Strasbourg, France 4Institute for Virus Research, Kyoto University, Shogoin-Kawahara, Sakyo-ku, Kyoto 606-8507, Japan *Author for correspondence (e-mail: [email protected])

Accepted 15 August; published on WWW 9 October 2000

SUMMARY

Hair cell fate determination in the has been sensory . In the , Hes1 is selectively shown to be controlled by specific genes. Recent loss- expressed in the greater epithelial ridge and lesser of-function and gain-of-function experiments have epithelial ridge regions which are adjacent to inner and demonstrated that Math1, a mouse homolog of the outer hair cells. Co-transfection experiments in postnatal Drosophila gene atonal, is essential for the production of rat explant cultures show that overexpression of Hes1 hair cells. To identify genes that may interact with Math1 prevents hair cell differentiation induced by Math1. and inhibit hair cell differentiation, we have focused on Therefore Hes1 can negatively regulate hair cell Hes1, a mammalian hairy and enhancer of split homolog, differentiation by antagonizing Math1. These results which is a negative regulator of neurogenesis. We report suggest that a balance between Math1 and negative here that targeted deletion of Hes1 leads to formation of regulators such as Hes1 is crucial for the production of an supernumerary hair cells in the cochlea and of the appropriate number of inner ear hair cells. inner ear. RT-PCR analysis shows that Hes1 is expressed in inner ear during hair cell differentiation and its expression is maintained in adulthood. In situ hybridization with late Key words: Cell fate determination, Notch signaling, bHLH embryonic inner ear tissue reveals that Hes1 is expressed transcription factor, Hes, Math1, Cochlea, Utricle, Hair cell in supporting cells, but not hair cells, of the vestibular development, Hair cell

INTRODUCTION cochlea are probably derived from the greater epithelial ridge (GER) and the lesser epithelial ridge (LER) cells, respectively, The of the inner ear develops from the otic during embryogenesis (see Lim and Rueda, 1992). vesicle (Van de Water, 1983; Fekete, 1996) and consists of the Hair cell differentiation in the inner ear is likely to be cochlea and vestibular end organs including the utricle, controlled by specific genes (for recent reviews see Fekete, and three . Each of these structures contains 1996, 1999). So far one of the most crucial genes for the a sensory epithelium in which hair cells, mechanosensory cells, control of inner ear hair cell differentiation appears to be the which convert sound or motion signals into electrochemical mouse basic -loop-helix (bHLH) transcription factor energy, and supporting cells are located. Currently, little is Math1 (Atoh1 – Mouse Genome Informatics), a mammalian known about the signaling events underlying inner ear homolog of Drosophila atonal. Math1 has been shown to be a development, specifically, differentiation of hair cells. positive regulator for the differentiation of cerebellar granule Classical tritiated thymidine incorporation studies have shown (Ben-Arie et al., 1997), dorsal commissural that hair cells become postmitotic between E11.5 and E17.5, (Helms and Johnson, 1998) and inner ear hair with a peak at E13.5 in rodents (Ruben, 1967; Sans and Chat, cells (Bermingham et al., 1999; Zheng and Gao, 2000). 1982). Hair cells in the mammalian vestibular end organs Targeted deletion of the Math1 gene leads to a failure of hair appear to be derived from the progenitor cells or supporting cell differentiation (Bermingham et al., 1999). Overexpression cells located within the sensory epithelium (Forge et al., 1993; of Math1 in postnatal rat cochlear explant cultures induces Warchol et al., 1993; Li and Forge, 1997; Zheng and Gao, production of extra hair cells in the GER (Zheng and Gao, 1997; Kuntz and Oesterle, 1998) in a similar manner as shown 2000). in and lower (Corwin and Cotanche, 1988; A few other bHLH transcription factors can influence cell Ryals and Rubel, 1988; Balak et al., 1990; Fekete et al., 1998). fate determination by acting as negative regulators (Kageyama However, the exact origin of mammalian cochlear hair cells is and Nakanishi, 1997). To identify a bHLH transcription factor still unclear because no cell lineage studies using lineage that may interact with Math1 to inhibit hair cell differentiation, tracers have been performed. Previous histological studies we turned our interest to Hes1, a mammalian hairy and suggest that the inner and outer hair cells in the mammalian enhancer of split homolog, which is a negative regulator of 4552 J. L. Zheng and others neurogenesis (Ishibashi et al., 1995; Nakamura et al., 2000). graded ethanol, and dried using Hexamethyldisilazane (HMDS). The Targeted disruption of the Hes1 gene results in a precocious samples were mounted on scanning electron microscopy stubs using neuronal differentiation in the (Ishibashi et al., 1995) and carbon double sticky tabs (Ted Pella, Redding, CA), sputter coated in the retina (Tomita et al., 1996). The Hes1 null mutant mice with 10 nm gold-palladium (Hummer XP, Anatech, Alexandria, VA) display severe neural tube defects and die before or and viewed in a Philips SEM 525M at an accelerating voltage of 10 immediately after birth (Ishibashi et al., 1995). More recently, kV. Digital images were captured using Semicaps Genie version 1.22 software. data collected from Hes1 and Hes5 double null mutant mice has shown that Hes1 and Hes5 act together as Notch effectors Plasmid DNA construction, cochlear explant cultures, for the control of mammalian neuronal differentiation (Ohtsuka electroporation and immunocytochemistry et al., 1999). The pRK5-Math1-EGFP plasmid was constructed as previously To determine whether Hes1 influences inner ear hair cell described (Zheng and Gao, 2000), by inserting the EcoRI Math1 differentiation, we examined the inner ear phenotypes of Hes1- fragment spliced from the pCMV-Math1 plasmid (Akazawa et al., deficient mice. We found supernumerary hair cells in the 1995) into the multiple cloning site of pRK5-EGFP plasmid (Murone cochlea and in the utricle, a vestibular end organ, of the Hes1- et al., 1999). The pSV2CMV-Hes1 plasmid (Akazawa et al., 1995) ′ deficient mice. In addition, to find out the spatiotemporal contains the Ssp1(171)-EcoRI (the 3 end) fragment of rat Hes1 expression patterns of Hes1 in the inner ear, we performed RNA cDNA. Middle turn cochlear explants (with the stria vascularis removed) were dissected from P0-P1 rats as previously described in situ hybridization and RT-PCR analysis. We discovered that (Zheng and Gao, 1996, 2000). The cultures were transfected with the Hes1 was expressed in the inner ear during hair cell pRK5-Math1-EGFP plasmid, a mixture of pSV2CMV-Hes1 and differentiation period and its expression became elevated pRK5-EGFP plasmids at a ratio of 5:1 or a mixture of equal amount around birth and persisted into adulthood. Within the vestibular of pSV2CMV-Hes1 and pRK5-Math1-EGFP plasmids using an sensory epithelium, Hes1 was expressed in supporting cells, but electroporator (Model CUY-21, BEX, Tokyo) with a train of eight not hair cells. In the cochlea, Hes1 was selectively expressed in pulses: 25 V, 50 mseconds duration and 100 mseconds interval. The GER and LER regions, which are adjacent to inner and outer tissue was placed in a groove freshly made of 1% agarose gel, hair cells, respectively. Moreover, we performed co-transfection containing 3 mg/ml plasmid DNA. The top surface of the explants was positioned to face to the cathode. After electroporation, the experiments with plasmids expressing Hes1 and Math1 and µ obtained evidence that Hes1 can block the induction of ectopic explants were plated on a collagen-coated (80 g/ml rat tail collagen I in 0.02 N HCl) eight-well LabTek slide in serum-free medium as hair cells in the presence of Math1. This study suggests that described (Zheng and Gao, 1999). The cultures were then fixed at 7 Hes1 is involved in hair cell differentiation as a negative days after transfection and processed for double immunocytochemistry regulator possibly by antagonizing Math1. with anti-myosin VIIa and anti-EGFP (Chemicon) antibodies, mediated by Texas Red- and FITC-conjugated secondary antibodies as described (Zheng and Gao, 1997, 2000). MATERIALS AND METHODS Cell counts and statistical analysis Hes1 mutant mice, and immunostaining of the To count total myosin VIIa-positive cells from the serial cryostat inner ear tissue utricular sections, we used an ocular grid in a Zeiss Axiophot Hes1 gene disruption and genotyping of the mice were performed as microscope with 20× and 40× lenses as previously described (Zheng previously reported (Ishibashi et al., 1995). The inner ear phenotypes et al., 1999b). Phalloidin-positive and myosin VIIa-positive cells were were compared between Hes1−/− embryos and their Hes1+/− and counted from cochlear surface preparations dissected from E17.5 Hes1+/+ littermates. Cochlear explants were prepared from E17.5 Hes1−/−, Hes1+/− and Hes1+/+ mice, essentially in the same way as mice (a close time point to birth, as Hes1−/− mice die at birth) and previously described (Zheng and Gao, 1996, 1999). At E17.5, the fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) cochlea has formed only two turns, the basal and apical turns, and hair before they were processed with FITC-conjugated phalloidin (Zheng cells in the apical turn are not yet well-developed. We therefore and Gao, 1999) or anti-myosin VIIa antibody labeling (Hasson et al., performed hair cell counts only in the basal turn. For the inner hair 1995; Zheng and Gao, 2000). Some of the cochlear tissue was fixed cells numbers, cell counts were performed along 1 mm length starting in 10% neutral buffered formalin. The preparations were then washed, from the basal end of the cochlea. For outer hair cell numbers, cell dehydrated in ascending graded alcohols, cleared in xylene and counts were obtained from a randomly selected 100 µm length in the embedded in paraffin (Zheng et al., 1999b). Three micrometer paraffin middle region of the basal turn of the cochlea. Data collected from sections of the preparations were cut with a microtome. The sections each experimental group are expressed as mean±s.e.m. Student’s t- were processed for Hematoxylin and Eosin staining. test was used for statistical analysis. For the co-transfection Utricular whole-mount tissue was also dissected out from E17.5 experiments, total numbers of EGFP-positive and myosin VIIa/EGFP- mice and fixed in 4% paraformaldehyde before they were double-positive GER cells were counted as previously described cryoprotected in 30% sucrose solution and embedded in OCT (Miles). (Zheng and Gao, 2000). Serial sections (20 µm) were cut from the utricular preparations and collected on microscopic slides for myosin VIIa RT-PCR analysis immunocytochemistry (Zheng et al., 1999a). Labeled preparations Quantitative RT-PCR analysis was performed with the 5′-exonuclease were washed in PBS, mounted in Fluoromount-G (Southern assay using fluorescent non-extendible oligonucleotide probes Biotechnology, AL) and viewed using a Zeiss Axiophot (TaqMan PCR detector, Perkin Elmer Applied Biosystems) as epifluorescent. Images were captured with Compix imaging systems described (Gibson et al., 1996). We used specific probes and primers using a cold RGB CCD camera for Hes1 (probe, CGGCTTCCAAGTGGTGCCGG; forward primer, GGAGAGGCTGCCAAGGTTTT; reverse primer, GCAAATTGG- Scanning electron microscopy CCGTCAGGA) and Math1 (probe, CTGAACCACGCCTTCGAC- After fixation in 10% neutral buffered formalin, some of the cochlear CAGCTG; forward primer, AACGGCGCAGGATGCA; reverse preparations were post-fixed in 1% aqueous osmium tetroxide for 2 primer TTGAAGGACGGGATAACGTTG). The specific probe and hours at room temperature, washed in distilled H2O, dehydrated in primers for the control housekeeping gene, Gapdh, was same as Role of Hes1 in hair cell differentiation 4553 reported (Zheng et al., 1999a). Data were collected from three to five length of the cochleae ranged from 3.6 to 4.1 mm for all pairs of cartilageous capsules containing the entire inner ear labyrinth genotypes. No apparent shortening of the cochlea was seen in tissue dissected at various developmental stages and are expressed as Hes1−/− mice. mean±s.e.m. Examination of paraffin sections of the cochlear tissue −/− +/+ In situ hybridization prepared from Hes1 and Hes1 mice showed that while there were three outer hair cells and only one inner hair cell in E17.5 Wistar rat were dissected and immediately fixed in 4% Hes1+/+ preparations (Fig. 3A), doublet inner hair cells were paraformaldehyde in 0.1M phosphate buffer, pH 7.4, for 3.5 hours, −/− cryoprotected in 20% sucrose in PBS and embedded in OCT (Miles, seen in Hes1 preparations (arrows in Fig. 3B). Similarly, Elkhart, IN) for cryostat sectioning. Sections (20 µm) were dried scanning electron microscopy revealed that instead of regular (50°C for 20 minutes) and processed for in situ hybridization with four rows of hair cells observed in Hes1+/+ mice (Fig. 3C), digoxigenin-labeled (Boehringer Mannheim, Indianapolis, IN) probes there were extra inner hair cells produced in the tissue prepared as described previously (Hynes et al., 1995). Rat Hes1 cDNA from Hes1−/− mice (arrows in Fig. 3D). These results therefore templates were generated by PCR using T3 or T7 RNA promoter confirmed our observations with phalloidin and anti-myosin sequence coupled primers. The primer sequences are as follows: VII antibody labeling (Fig. 1) in cochlear surface preparations. forward, CTTCGATAACAGCGGAATCCCC; reverse, TGTGCTCA- GAGGCTGTCTTTGG; T3-forward, TTATTAACCCTCACTAAAG- Hes1 also influences hair cell production in the GGAAGCTTCGATAACAGCGGAATCCCC; T7-reverse, TTGTAA- utricle TACGACTCACTATAGGGCGATGTGCTCAGAGGCTGTCTTTGG. For Hes5 in situ hybridization, a 992 bp rat Hes5 cDNA, including To determine whether hair cell production in the vestibular the whole coding region and 3′ UTR, was subcloned into the EcoRI organs is also regulated by Hes1, we made serial cryostat and Hind III sites of pBluescript-SK and used as the template for generating digoxigenin-labeled probes. and antisense digoxigenin-labeled RNA probes were made by in vitro transcription using T3, T7 RNA polymerase accordingly. Some of the sections were double labeled with anti-myosin VIIa antibody, as described above, following in situ hybridization procedures.

RESULTS

Targeted disruption of the Hes1 gene leads to formation of extra inner hair cells in the cochlea We performed phalloidin labeling to determine the number of hair cells in the cochlea as previously reported (Zheng and Gao, 1999). As shown in Fig. 1A, there were four rows of hair cells including one row of inner hair cells and three rows of outer hair cells in the cochlear surface preparations dissected from E17.5 wild-type mice. However, examination of cochlear surface preparations obtained from E17.5 Hes1−/− mice revealed the presence of supernumerary inner hair cells (arrows in Fig. 1C). There was a mild increase in the number of inner hair cells in the Hes1+/− mice, suggesting a gene-dose effect (arrows in Fig. 1B). Such a phenotypic change in the Hes1- deficient mice was also observed when a cytoplasmic hair cell- specific marker, anti-myosin VIIa antibody (Hasson et al., 1995; Xiang et al., 1998), was used (Fig. 1D-F). Cell counts performed from cochlear surface preparations of different genotypes are summarized in Fig. 2. There was a significant increase in the number of doublet inner hair cells along 1 mm length starting from the basal end of the cochlea in the Hes1−/− mice (Fig. 2A; 17.92 ±3.69 s.e.m., n=13) as compared to the Hes1+/+ (0.20±0.16, n=8, P<0.01) or Hes1+/− litter mates (5.00±0.55, n=15, P<0.01 between Hes1−/− and Hes1+/+ mice). There was also a statistical difference between Hes1+/− and Hes1+/+ mice (P<0.01). In contrast, no statistical difference was found in outer hair cell numbers among the three types of animals (Fig. 2B, Hes1−/−, 63.69±1.82, n=13, Hes1+/+, Fig. 1. Formation of extra cochlear inner hair cells in Hes1-deficient 58.87±2.18, n=15, Hes1+/+, 58.25±2.18, n=8, P=0.058 −/− +/+ −/− mice. Phalloidin labeling (A-C) and myosin VIIa immunostaining between Hes1 and Hes1 mice). In general, the Hes1 (D-F) of the cochlear surface preparations obtained from E17.5 mice still showed three evenly patterned rows of outer hair cells Hes1+/+ (A,D), Hes1+/− (B,E) and Hes1−/− mice (C,F). Arrows (Fig. 1C,F), although sometimes a few extra hair cells were indicate the presence of extra inner hair cells. Scale bar: 15 µm in A- seen in the third outer hair cell row (see Fig. 3D). The entire C; 12 µm in D-F. 4554 J. L. Zheng and others

A B 25 80 * p < 0.01 * m 20 60

Fig. 2. Quantitative analysis of inner 15 and outer hair cells in the cochleae of Hes1 mutant mice. (A) Doublet 40 inner hair cells were counted along 10 1 mm length starting from the basal end of the cochlea. (B) Outer hair * 20 cell numbers were obtained from 5 randomly selected 100 µm length in the middle region of the basal turn of the cochlea. Cochlear tissue was 0 100 hair cells / outer of Number 0 prepared from E17.5 mice. Number of doublet inner hair cells / 1 mm Hes1+/+ Hes1+/- Hes1-/- Hes1+/+ Hes1+/- Hes1-/- sections of the utricles prepared from the Hes1−/−, Hes1+/+, and performed with sections prepared from E17.5 rat inner ear Hes1+/− mice at E17.5, as previously described (Zheng and tissue. As shown in Fig. 5A1 and 5A2, specific labeling was Gao, 1997). We then immunostained these sections with anti- seen in the supporting cell layer but not hair cell layer of the myosin VIIa antibody (Fig. 4A,B) and performed total cell vestibular sensory epithelium. Double labeling the sections counts (Fig. 4C). We found a significant increase in the total with anti-myosin VIIa antibody, a hair cell-specific marker number of hair cells in the utricles of Hes1−/− mice (Hasson et al., 1995; Xiang et al., 1997), confirmed that hair (3193.50±143.94, s.e.m., n=4, P<0.05), as compared with cells were devoid of labeling (Fig. 5A2). Nonsensory epithelial those in the Hes1+/+ mice (2347.17±80.99, n=6) (Fig. 4C). cells in the transitional zone and in the roof showed no signals The number of hair cells in Hes1+/− was in-between (Fig. 5A1). In the cochlea, Hes1 signal was seen in the GER (2594.5±163.19, n=4), but the difference was not statistically and LER areas, which are adjacent to inner and outer hair cells, significant relative to that of the Hes1+/+ mice (P=0.17, Fig. respectively (Fig. 5A3, 5A4). The sensory epithelium in which 4C). Because there are no reliable supporting cell-specific hair cells and supporting cells such as Deiter’s cells and pillar markers available for the mammalian inner ear, the numbers of cells are located showed minimal labeling (5A4). Double supporting cells could not be determined with sufficient labeling the sections with anti-myosin VIIa antibody confirmed accuracy in these cryostat sections and we did not perform cell that hair cells were essentially devoid of Hes1 signal (Fig. counts for supporting cells. 5A4). Given that Hes1 is expressed in both LER and GER, but Hes1 and Hes5 are differentially expressed in the extra inner hair cells are only formed in the GER, we inner ear wondered that expression of other genes such as Hes5 may To find out the cellular expression patterns of the Hes1 gene also play a role in the control of hair cell differentiation. Hes5 in the inner ear, nonradioactive RNA in situ hybridization was has been shown to be another negative regulator of

Fig. 3. Confirmation of the presence of extra cochlear inner hair cells with paraffin sectioning and scanning electron microscopy of the Hes1−/− cochleae. (A,B) Paraffin sections of Hes1+/+ and Hes1−/− cochleae, respectively. Arrows in B show the presence of doublet inner hair cells. (C,D) Scanning electron micrographs of Hes1+/+ and Hes1−/− cochlear surface preparations, respectively. Owing to the initial fixation in 10% formalin, the ultrastructural preservation was not optimal. Arrows in D shows the presence of several extra inner hair cells. Scale bar: 20 µm in A,B; 15 µm in C,D. Role of Hes1 in hair cell differentiation 4555

supporting cells in the striola region. Minimal signals or much lower levels were seen in the non-striola region (Fig.5B1, 5B2). Owing to RNA degradation resulting from bone decalcification necessary for processing the mature ear for sectioning, we were unable to determine by in situ hybridization analysis the expression profile of Hes1 in the mature ear. To address whether Hes1 was developmentally regulated at late postnatal stages and in adult, we performed real-time quantitative RT-PCR analysis with RNA extracted from the entire inner ear labyrinth tissue prepared from E13.5, E15.5, E17.5, P0, P5, P15 and adult mice. As shown in Fig. 6A, Hes1 was expressed as early as E13.5. Its expression became elevated around birth and was maintained in the adult. To explore possible relationship between Hes1 and Math1 during hair cell differentiation, we also performed TaqMan analysis of Math1 expression in the inner ear at various developmental stages (Fig. 6B). In contrast to Hes1, Math1 was expressed mainly at embryonic and early postnatal stages with its expression being greatly downregulated and became minimal in the adult (Fig. 6B). Hes1 prevents hair cell differentiation induced by Math1 Our recent study showed that the GER cells that normally give rise to inner sulcus epithelial cells in postnatal rat cochlear explant cultures can be induced to become hair cells when forced to express Math1 (Zheng and Gao, 2000). These

−/− results are consistent with gene targeting experiments Fig. 4. Production of extra hair cells in Hes1 mouse utricles. (Bermingham et al., 1999), indicating that Math1 is crucially (A,B) Myosin VIIa immunostaining of cryostat sections of Hes1+/+ and Hes1−/− mouse utricles, respectively. (C) Total hair cell counts involved in the control of hair cell differentiation. To attempt from Hes1+/+, Hes1+/− and Hes1−/− utricles. Scale bar: 30 µm for to understand the mechanisms by which Hes1 influences hair A,B. cell differentiation and whether there is any functional interaction between Hes1 and Math1, we co-transfected postnatal rat cochlear explant cultures with an equal amount neurogenesis (Ohtsuka et al., 1999). We carried out Hes5 in of Hes1-expressing (pSV2CMV-Hes1) and Math1-expressing situ hybridization on E17.5 rat inner ear tissue and found an plasmids (pRK5-Math1-EGFP) and compared them to overlapping, but distinct expression pattern, as compared cultures transfected with pRK5-Math1-EGFP plasmid only or with that of Hes1. In the cochlea, Hes5 signal was observed co-transfected with a mixture of pSV2CMV-Hes1 and pRK5- in the LER and supporting cells, including Deiter’s cells, and EGFP plasmids. Because the pRK5-Math1-EGFP and pRK5- in pillar cells in the sensory epithelium, but not in the GER EGFP plasmids contain the EGFP reporter gene, cells (Fig. 5B3, 5B4). In the utricle, strong Hes5 signal was seen transfected with the pRK5-Math1-EGFP plasmid, a mixture in the supporting cells (Fig. 5B1). However, unlike Hes1 of pSV2CMV-Hes1 and pRK5-Math1-EGFP plasmids, or a which is expressed in supporting cells throughout the sensory mixture of pSV2CMV-Hes1 and pRK5-EGFP plasmids can epithelium, Hes5 was expressed at high levels in the be easily identified in the cultures. We found that Math1

Table 1. Expression of Hes1 prevents hair cell differentiation induced by Math1 Math1-EGFP transfected Hes1/Math1-EGFP co-transfected Hes1/EGFP co-transfected Number of Number of Number of Number of Number of Number of transfected induced Induction transfected induced Induction transfected induced Induction Culture number GER cells hair cells (%) GER cells hair cells (%) GER cells hair cells (%) 1 45 43 95.6 129 10 7.8 338 0 0 2 172 171 99.4 198 17 8.6 224 0 0 3 98 96 98.0 96 5 5.2 126 0 0 4 139 138 99.3 165 20 12.1 138 0 0 5 285 284 99.6 65 7 10.8 65 0 0 6 76 73 96.1 144 20 13.9 79 0 0 7 149 149 100.0 223 12 5.4 85 0 0 8 327 325 99.4 172 12 69.8 117 0 0 Mean±s.e.m. 161.4±34.9 160.0±35.0 98.6±0.6 149.0±18.4 12.9±2.0 8.9±1.1 146.5±32.5 0 0 Transfection and cell counts were performed as described in the Materials and Methods. 4556 J. L. Zheng and others overexpression led to robust production of ectopic hair cells prevented by co-expression of Hes1 (Fig. 7D-F). in the GER region (Fig. 7A-C) as shown previously (Zheng Overexpression of Hes1 alone, however, did not show any and Gao, 2000). However, the induction of hair cell apparent effects and none of the Hes1 transfected cells differentiation by Math1 expression was dramatically became myosin VIIa positive (Fig. 7G-I). Previously, we have shown that there is a morphological change of the GER cells in the presence of Math1: conversion from an elongated, process-bearing morphology to a pear-shaped morphology (Fig. 7C) (Zheng and Gao, 2000). Consistently, the majority of the Hes1/Math1 co-transfected GER cells showed a process-bearing morphology (in Fig. 7D-F) similar to those of normal GER cells (Fig. 7G-I), suggesting that the presence of Hes1 also prevents the GER cells from undergoing a morphological change. Quantitative analysis of the cultures transfected with various plasmids revealed that while 98.6%±0.6 of the Math1-transfected GER cells became hair cells, only about 8.9%±1.1 Hes1/Math1 co-transfected GER cells converted into hair cells (Table 1). All of the Hes1 transfected GER cells remained myosin VIIa negative and did not differentiate into hair cells (Table 1).

6 A Hes1

4

2 mRNA levels (arbitrary units) 0 P0 P5 P15 Adult E13.5 E15.5 E17.5

1.0 B Math1 Fig. 5. Nonradioactive Hes1 and Hes5 RNA in situ hybridization labeling in the inner ear. (A) Low- and high-magnification images of 0.8 Hes1 expression in E17.5 rat utricular (A1,A2) and cochlear (A3,A4) sections, respectively. (A2,A4) Myosin VIIa 0.6 immunocytochemical labeling (mediated by Texas-Red-conjugated secondary antibody) of the sections shown in A1,A3, respectively. Note that specific Hes1 signals are seen in supporting cell (SC) layer, 0.4 but not hair cell (HC) layer of the utricular sensory epithelium. In the cochlea, Hes1 signal is seen in the GER and LER cells, but minimal in the sensory epithelium (SE). Hair cells (red labeling in A2,A4) are 0.2 devoid of Hes1 signal. Hybridizing the sections with sense control

probes under the same experimental conditions did not show any mRNA levels (arbitrary units) staining (data not shown). (B) Hes5 expression in E17.5 rat utricular 0.0 P5 (B1,B2) and cochlear (B3,B4) sections, respectively. (B2,B4) P0 P15

Myosin VIIa immunocytochemical labeling (mediated by Texas-Red Adult E13.5 E15.5 secondary antibody) of the sections shown in B1,B3, respectively. E17.5 Note that specific Hes5 signals are seen in Deiter’s cells (DC), pillar Fig. 6. Temporal expression patterns of Hes1 and Math1 genes in the cells (PC) and the LER cells in the cochlea (B4), and in striola inner ear. Quantitative RT-PCR using RNA extracted from supporting cells (SC) in the utricle (B2). Hair cells (red labeling in cartilaginous capsules containing the entire inner ear labyrinth tissue B2 B4), the and non-sensory epithelial cells (not at different developmental stages (see Materials and Methods). Note shown) are devoid of Hes5 signal. Scale bar: 50 µm in A1,A3,B1- that Hes1 expression is maintained in the adult while Math1 B4; 25 µm in A2,A4. expression and becomes downregulated in the adult. Role of Hes1 in hair cell differentiation 4557

Fig. 7. Hes1 blocks hair cell differentiation induced by Math1. EGFP (green, A,D,G) and myosin VIIa (red, B,E,H) double immunocytochemistry (double exposure, C,F,I) of the cultures transfected with the pRK5-Math1- EGFP plasmid (A-C), a mixture of equal amount of pSVCMV-Hes1 and pRK5-Math1-EGFP plasmids (D-F), and a mixture of pSVCMV- Hes1 and pRK5-EGFP plasmid at a ratio of 5:1 (G-I). Note that while virtually all Math1 transfected GER cells become hair cells (A-C), only a very small number of GER cells in the cultures co-transfected with Hes1 and Math1 (arrows in D-F) are able to differentiate into hair cells. All Hes1 transfected GER cells remain myosin- VIIa negative (G-I). Scale bar: 25 µm.

DISCUSSION respectively, yet no or minimal signals were seen in supporting cells within the cochlear sensory epithelium of the cochlea. The present study shows direct experimental evidence that Hes5, however, is expressed in the LER and supporting cells, Hes1 is involved in hair cell differentiation. When the Hes1 including Deiter’s cells and pillar cells, but not in the GER. gene is deleted, there is formation of supernumerary hair cells Classical histological studies suggest that inner hair cells are in both the cochlea and the utricle of the inner ear. When Hes1 derived from the most distal GER cells and outer hair cells is overexpressed, it can block hair cell differentiation induced arise from the most proximal LER cells (Lim and Rueda, by a positive regulator, Math1. Therefore, Hes1 is a negative 1992). Our recent finding that the GER cells can directly regulator of hair cell differentiation, which is similar to its role convert into hair cells under experimental conditions (Zheng during neurogenesis (Kageyama and Nakanishi, 1997; and Gao, 2000) provides further supporting evidence for this Nakamura et al., 2000). A balance between negative regulators model. The expression of Hes1 in the GER and LER cells may such as Hes1 and positive regulators such as Math1 appears to serve as a mechanism to fine-tune the appropriate number of be crucial for production of an appropriate number of hair cells inner and outer hair cells. While Hes1 clearly contributes to a in the inner ear. mechanism that fine tunes the number of inner hair cells, its Our RT-PCR and in situ hybridization analyses show distinct role in regulating the number of outer hair cells is less clear expression patterns of Hes1, Hes5 and Math1 during normal and may be masked by the cooperative influence of Hes5, as hair cell development. Previous experiments have shown that Hes5 is expressed in the cochlear sensory epithelium and the Math1 is expressed in the presumptive sensory epithelium at LER (Fig. 5B4). early embryonic stage (E12.5), but its expression becomes It is important to note that overexpression of Math1 can restricted to hair cells, downregulated in supporting cells and convert either GER cells of the cochlea or supporting cells of absent in other non-sensory epithelial cells, including GER and the utricle into hair cells (Zheng and Gao, 2000), even though LER cells at late embryonic stages (Bermingham et al., 1999). the two populations of cells express Hes1. One possible Expression of Math1 becomes minimal in the adult inner ear interpretation for these results is that transfection-induced (Fig. 6B). Hes1, however, is expressed at low levels at early Math1 expression is much higher (see Zheng and Gao, 2000) embryonic stages, and its expression becomes elevated at late than the physiological levels of Hes1. These results suggest embryonic and early postnatal stages, and is maintained in the that Math1 plays a dominant role in driving the cells to become adult. Within the utricular sensory epithelium, Hes1 is hair cells. Hes1 only acts as a negative regulator to balance the expressed selectively in all supporting cells, but not hair cells. activity of Math1. It remains to be determined whether Hes5, however, appears to be expressed in striola supporting overexpression of Hes1 in mature hair cells in which Math1 is cells with much lower levels in non-striola supporting cells. downregulated or absent would influence the identity of hair The presence of high levels of Hes1 and downregulation of cells: converting hair cells back into supporting cells. Math1 in all supporting cells at late embryonic stages may be Our findings provide further supporting evidence that Notch one of the mechanisms used to prevent formation of signaling is involved in hair cell fate determination, because supernumerary vestibular hair cells; this may also explain why Hes1 is believed to be a downstream target gene of the Notch extra hair cells are produced in Hes1−/− utricles. signaling pathway based on previous findings that It is interesting that Hes1 was expressed in the GER and constitutively active form of Notch can activate Hes1 promoter LER cells, which are adjacent to inner and outer hair cells, and induce Hes1 expression (Jarriault et al., 1995; Hsieh et al., 4558 J. L. Zheng and others

1997; Nishimura et al., 1998). Activation of Notch by its heterodimer with another HLH factor, E2a, which then binds exogenous ligand, Delta, is reported to induce either Hes1 or to the E-box to activate transcription. Hes1 may prevent the Hes5 expression in neighboring cells (Jarriault et al., 1998; binding of Math1 to E2a (Sasai et al., 1992) by competition, Wang et al., 1998). Recent studies have provided supporting to suppress gene transcription that is necessary for cell evidence for the involvement of Notch signaling mediated differentiation to occur. Second, Hes1 might bind directly to lateral inhibition in the control of hair cell fate determination promoter DNA and restrain transcription (Ohsako et al., 1994; (Corwin and Warchol, 1991; Lewis, 1991). During embryonic Van Doren et al., 1994). In addition, Hes1 may bind to Groucho development, Notch1 and Notch ligands are expressed in the (McLarren et al., 2000), a transcription co-repressor that can presumptive sensory epithelium at the onset of hair cell prevent cells from differentiation (Paroush et al., 1994). differentiation (Lewis et al., 1998; Lanford et al., 1999, Considered together with our previous work (Zheng and Morrison et al., 1999; Zine et al., 2000). When the jagged2 Gao, 2000), we believe that the postnatal cochlear explant gene which encodes one of the Notch ligands, is deleted, cultures could serve as a convenient model system for hair cell supernumerary inner hair cells are produced in the cochlea differentiation studies. When the GER cells in the cultures are (Lanford et al., 1999). Similarly, interference in Notch forced to express Math1, they differentiate into hair cells. signaling by either Notch1 or jagged1 antisense Transfection of the same type of cells with other genes such as oligonucleotides results in an increase in the number of hair Hes1 (Fig. 7G-I) or Brn3c (Pou4fc – Mouse Genome cells in cultures of developing cochlear tissue (Zine et al., Informatics; Zheng and Gao, 2000) does not lead to production 2000). In addition, during hair cell regeneration in chicks, of extra hair cells. One can use this model system to determine Delta1, another Notch ligand, is upregulated in regenerating whether another specific gene is sufficient to induce hair cell hair cells but downregulated in cells that do not acquire a hair differentiation by using Math1, Hes1 and Brn3c as positive and cell fate (Stone and Rubel, 1999). Moreover, in the mind bomb negative controls. Moreover, when the cultures are co- zebrafish mutants, in which Notch signaling is disrupted, the transfected with Hes1- and Math1-expressing plasmids, the ear sensory patches consist solely of hair cells, which are induction of hair cell differentiation by Math1 is blocked. produced precociously in great excess, while supporting cells Theoretically, one can co-transfect the cultures with two or are absent (Haddon et al., 1998). These data considered more plasmids to dissect genetic pathways between genes that together demonstrate that Notch signaling may influence the might regulate hair cell differentiation. choice of a precursor cell to adopt a hair cell fate after it exits Finally, understanding the mechanisms of hair cell the cell cycle. Our RT-PCR analysis indicates that Hes1 is differentiation would be helpful for us to stimulate hair cell expressed during hair cell differentiation and maintained in the regeneration following injury and could eventually lead to a adult, which appears to follow the expression of Notch1. Hes1- therapeutic treatment of and balance impairments. deficient mice show a similar inner ear phenotype as compared Over the past several years, a lot of efforts have been mainly to the jagged 2 null mutant mice, but less severely in terms of made to identify mitogenic growth factors that can stimulate the production of total cochlear hair cells (Lanford et al., 1999). proliferation of supporting cells (Lambert, 1994; Yamashita This could be due to the compensation from Hes5 in the Hes1 and Oesterle, 1995; Corwin et al., 1996; Gu et al., 1996; mutant mice, because Hes5 is expressed in supporting cells of Oesterle et al., 1997; Zheng et al., 1997, 1999a; Kuntz and the cochlear sensory epithelium and in the LER (Fig. 5B3, Oesterle, 1998; Staecker and Van De Water, 1998) and to 5B4). It is also possible that expression of other genes that understand the mechanisms controlling cell proliferation modulate Notch signaling (Zhang et al., 2000) in the sensory (Navaratnam et al., 1996; Chen and Segil, 1999; Lowenheim epithelium and the boundary areas can play important roles in et al., 1999) in the inner ear sensory epithelium. On the one the control of hair cell differentiation. Interactions from hand, cell proliferation appears to be needed to compensate the multiple genes may contribute together to the complex number of lost hair cells and the supporting cells that are morphogenesis of the mammalian cochlea. capable of converting into new hair cells. On the other hand, Based on the present experiments and previous findings additional signals or strategies might be required for the mentioned above, we favor the following model for hair proliferative and non-proliferative supporting cells to cell differentiation: Notch → Hes1/Hes5 –| Math1 → hair cell successfully convert into hair cells. Our recent findings that differentiation (Kegeyama and Nakanishi, 1997, Shailam et al., overexpression of specific genes such as Math1 can induce hair 1999). Activation of Notch would lead to the expression of cell differentiation in the cochlea and facilitate the conversion Hes1/Hes5, which antagonizes Math1. Math1 acts as a of supporting cells into hair cells in the utricle (Zheng and Gao, dominant, positive regulator for hair cell differentiation. In the 2000) have suggested an additional approach for the absence of or at low levels of negative regulators such as Hes1 production of new hair cells. In this regard, it is conceivable and Hes5, Math1 would be sufficient to drive the epithelial that downregulation of Hes1 in addition to upregulation of cells within and nearby the sensory epithelium to differentiate Math1 in the inner ear could be another strategy by which to into hair cells. When Hes1 is expressed at high levels, Math1 stimulate hair cell regeneration in adults, given that Hes1 activity can be inhibited and hair cell differentiation is expression persists in the adult inner ear. A combination of prevented. Support for the interaction between Hes1 and Math1 mitogenic supporting cell growth factors and upregulation/ also comes from a previous biochemical study (Akazawa et al., downregulation of specific genes involved in the control of hair 1995), in which Hes1 is shown to inhibit the Math1/E47- cell differentiation could be a more effective way to stimulate induced transcriptional activation. Although the exact hair cell regeneration in the inner ear. mechanism by which Hes1 interacts with Math1 is still unknown, there could be several possibilities (Akazawa et al., We thank Arnon Rosenthal for his helpful discussions, Tama 1995). First, it is believed that Math1 functions through a Hesson for her gift of anti-myosin VIIa antibody, Linda Rangell and Role of Hes1 in hair cell differentiation 4559

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