Spatiotemporal expression of Ezh2 in the developing mouse cochlear sensory epithelium

Abstract

The enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) is a histone-lysine N-methyltransferase enzyme that participates in DNA methylation. Ezh2 has also been reported to play crucial roles in stem cell proliferation and differentiation. However, the detailed expression profile of Ezh2 during mouse cochlear development has not been investigated. Here, we examined the spatiotemporal expression of Ezh2 in the cochlea during embryonic and postnatal development. Ezh2 expression began to be observed in the whole otocyst nuclei at embryonic day 9.5 (E9.5). At E12.5, Ezh2 was expressed in the nuclei of the cochlear prosensory epithelium. At E13.5 and E15.5, Ezh2 was expressed from the apical to the basal turns in the nuclei of the differentiating cochlear epithelium. At postnatal day (P) 0 and 7, the Ezh2 expression was located in the nuclei of the cochlear epithelium in all three turns and could be clearly seen in outer and inner hair cells, supporting cells, the stria vascularis, and spiral ganglion cells. Ezh2 continued to be expressed in the cochlear epithelium of adult mice. Our results provide the basic Ezh2 expression pattern and might be useful for further investigating the detailed role of Ezh2 during cochlear development.

Keywords

polycomb repressive complex / Ezh2 / expression / inner ear / cochlea / development

Introduction

The cochlea is probably the most complex sensory organ. It originates from a thickening of the surface ectoderm next to the hindbrain (otic placode), which quickly deepens to form the otocyst and undergoes a series of morphogenetic events to give rise to a coiled cochlear duct that functions as a detector of sound waves. The complicated developmental process of the cochlea depends on well-orchestrated signaling cascades and precise temporal and spatial gene expression patterns within the otic vesicle as well as interactions with the surrounding tissue.

Epigenetic regulation plays an important role in the development of the inner ear [ 1– 5]. The epigenetic modifier DNA methyl transferase (DNMT) 3A, which is expressed in the neural plate border region, influences the development of the otic placode [ 4]. Lysine demethylase 4B (KDM4B) plays a critical role during inner ear invagination by modulating the Dlx3 histone methylation [ 2]. A recent report has shown that the epigenetic regulation of Atoh1 underlies hair cell differentiation and subsequent maturation [ 5]. Dynamic changes in histone modifications H3K4me3/H3K27me3, H3K9ac, and H3K9me3 correlate with the onset of the Atoh1 expression during hair cell differentiation.

The enhancer of zeste homolog 2 (Ezh2) is a histone-lysine N-methyltransferase enzyme encoded by the Ezh2 gene, which participates in DNA methylation and, ultimately, in transcriptional repression [ 6]. Ezh2 is the functional enzymatic component of the polycomb repressive complex 2 (PRC2) [ 7]. It plays crucial roles in stem cell proliferation and differentiation during the development of many organs [ 8– 17]. Ezh2 controls the basal cell fate determination during lung endoderm development [ 14, 15]. Ezh2 is also essential for the proliferation of neural progenitor cells during adult hippocampal neurogenesis [ 16]. Ezh2 orchestrates gene expression during the stepwise differentiation of tissue-specific stem cells [ 8]. The suppression of Ezh2 prevents the shift of the osteoporotic mesenchymal stem cell fate into adipocytes and enhances bone formation during osteoporosis [ 11]. The loss of Ezh2 results in abnormal mammary gland development and heterogeneous malignancies [ 12, 13].

Our recent findings show that the polycomb group gene Bmi1, which belongs to the same family as Ezh2, is expressed in the cochlea [ 18]. Bmi1 plays an important role in hair cell survival by controlling redox balance [ 18]. Bmi1 also regulates the proliferation of supporting cells and progenitors via Wnt signaling [ 19]. However, the detailed expression profiles of Ezh2 have not been investigated during the development of the mouse inner ear. In this study, we describe the spatiotemporal Ezh2 expression in the embryonic and postnatal mouse cochlear epithelium.

Materials and methods

Animals

The morning of plug identification was defined as embryonic day (E) 0.5. The day of birth was defined as postnatal day (P) 0. C56BL/6J mice were housed with free access to food and water at the Experimental Animal Center, Shanghai Medical College of Fudan University, China. All experiments were approved by the Shanghai Medical Experimental Animal Administrative Committee (permit number: 2009-0082). All efforts were made to minimize suffering and reduce the number of animals used.

Tissue and frozen section preparation

Pregnant mice were anesthetized, and their embryos were removed. The embryo heads containing the developing inner ear were dissected and fixed overnight in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS), with pH 7.2. P0 and P7 mice were sacrificed, and their cochleae were fixed overnight in 4% PFA. P0 and P7 cochleae were decalcified in 10% EDTA for 2 h. The specimens were then embedded in sucrose and cut into 10 mm sections.

Immunofluorescence

After fixation, the cochlear samples were blocked with 10% normal donkey serum in 10 mmol/L PBS (pH 7.4) with 1% Triton-X100 for 1 h at room temperature. The samples were then incubated with primary antibody overnight at 4 °C. The tissue samples were incubated the next day for 1 h at 37 °C with Alexa Fluor® 488- or 594-conjugated donkey secondary antibody (1:500 dilution, Invitrogen) and 4,6-diamidino-2-phenylindole (DAPI, 1:800 dilution, Sigma-Aldrich). The omission of primary antibody served as the negative control. The following primary antibodies were used: Anti-Ezh2 (1:200 dilution, Cell Signaling), anti-myosin VIIA (Myosin 7a) (1:500 dilution, Proteus BioSciences), anti-parvalbumin (1:1000, Sigma-Aldrich), and anti-Sox2 (1:500 dilution, Santa Cruz). Images were taken using a Leica SP5 fluorescence microscope (Leica, Germany) or Leica SP8 confocal fluorescence microscope (Leica, Germany).

Results

Ezh2 was expressed in the otocyst

We first investigated the expression pattern of Ezh2 in the otocyst using Sox2 as the marker for sensory progenitor cells. The otic placode has invaginated and closed up to form the otocyst at E10.5. The ventral part of the otocyst will give rise to the cochlea [ 20]. Our data showed that Ezh2 was expressed in the dorsal and ventral parts of the otocyst at E9.5 and E10.5 (Fig. 1), thereby indicating that Ezh2 might play important roles in the proliferation and specification of the prosensory domain.

Ezh2 was expressed in the embryonic cochlear duct

We further studied the Ezh2 expression at later developmental stages to confirm its expression in the sensory lineage of the inner ear. Sox2 was used as the marker for sensory progenitor cells. The cochlear duct has formed as an outpocketing from the ventral medial region of the otocyst by E12.5 [ 21]. At this stage, we observed that Ezh2 was expressed in the cochlear prosensory epithelium (Fig. 2A).

During the cochlear duct development, the cells in the primordial organ of Corti exit the cell cycle between E13.5 and E14.5 to establish a distinct zone of non-proliferating cells delimited by the expression of the cyclin-dependent kinase inhibitors p27^Kip1 and p19^Ink4d in the cochlea. Subsequently, hair and supporting cells differentiate within the sensory primordium to form a precise mosaic of hair and supporting cells [ 20]. Hair cell differentiation starts at around E14.5 in the cochlea as indicated by the Math1 expression [ 22]. We observed that Ezh2 was broadly expressed in the nuclei of the cochlear epithelium at E13.5 and E15.5 (Fig. 2B and 2C, respectively).

Ezh2 was expressed in the cochlea of newborn mice

We next explored the Ezh2 expression in the postnatal cochlear epithelium. Myosin 7a and Sox2 were used as markers of hair and supporting cells, respectively. Ezh2 was expressed in auditory hair cells, supporting cells, the Kölliker organ, and the stria vascularis in all three turns of the cochlea at P0 (Fig. 3A). The spiral ganglion neuron was chiefly located at the Rosenthal tube at P0. The spiral ganglion neuron developed from the base to the apex. Ezh2 was expressed in the spiral ganglion in a similar manner as in the organ of Corti and the stria vascularis (Fig. 3A). Similarly, Ezh2 was expressed in the nuclei of the auditory hair and supporting cells and the stria vascularis at P7 (Fig. 4).

Ezh2 was expressed in the cochleae of adult mice

We next explored the Ezh2 expression in the adult cochlear epithelium. Parvalbumin and Sox2 were used as markers of hair and supporting cells, respectively. Fig. 5 shows that Ezh2 was expressed in the cochlear hair and supporting cells at P35. However, the Ezh2 expression in the outer hair cells was obviously less than that in the inner hair cells (Fig. 5A).

Discussion

The mammalian cochlea, which converts sound waves into neural impulses, is regarded as the most complicated and specialized sensory organ. The organ of Corti, which is located along the full length of the basilar membrane and serves as the terminal receptor, contains two types of hair cells and at least four types of supporting cells, all of which are terminally differentiated cells and derived from the same precursor cells during embryonic development [ 23].

Ezh2 is a critical protein in the polycomb repressor complex. PcG proteins form large complexes, including PRC1 and PRC2. As a part of PRC2, Ezh2 catalyzes H3K27 (H3K27me3) trimethylation, then attracts PRC1, which maintains the silencing effect of H3K27me3 [ 24]. The mutation or overexpression of Ezh2 has been linked to many forms of cancer [ 25], including breast [ 26], prostate [ 27], melanoma [ 28], and bladder [ 29] cancer. Aside from its important function in cancer development, Ezh2 also plays critical roles in normal developmental processes in many organs [ 10– 17, 30]. Ezh2 is involved in the cell differentiation and cell fate determination of tissue-specific stem cells [ 8], natural killer cells [ 10], osteoporotic mesenchymal stem cells [ 11], lung basal cells [ 14, 15], skeletal muscle cells, and embryonic stem cells [ 17]. Furthermore, Ezh2 is essential for the proliferation of adult neural stem cells [ 16]. The deletion of Ezh2 in hippocampal NSCs/progenitor cells results in a reduction in progenitor cell proliferation. The conditional knockout of Ezh2 ultimately results in impairments in spatial learning and memory [ 16].

In this study, we show that the Ezh2 protein is expressed in the whole otocyst nuclei (Fig. 1), thereby indicating that Ezh2 might be involved in the otocyst development. Ezh2 is expressed in the cochlear duct from E12.5 to E15.5 (Fig. 2). The prosensory area proliferates and differentiates into hair and supporting cells from E12.5 to E15.5, which implies that Ezh2 might take part in the proliferation and differentiation of the cochlear progenitor cells. Ezh2 is located in the hair and supporting cells (Figs. 3 and 4) from P0 to P7, thereby suggesting that Ezh2 might also be involved in the maturation of the hair and supporting cells. These results suggest that Ezh2 plays important roles in the differentiation and maturation of the organ of Corti. Our results also show that Ezh2 is expressed in the cochlear spiral ganglion and the stria vascularis (Figs. 3 and 4), which shows that Ezh2 might take part in the development of these tissue types. Ezh2 continues to be expressed in the hair and supporting cells in the cochlea of adult mice. However, the Ezh2 expression decreases in the outer hair cells compared to the inner hair cells (Fig. 5). Our results for the expression pattern of Ezh2 in the neonatal mouse cochlea are consistent with the previous results [ 31].

In conclusion, our data show that Ezh2 is broadly expressed in various cell types during the development and maturation of the inner ear. This indicates that Ezh2 might cooperate with different genes to play different roles in different cell types during different developmental stages. Our results provide the basic Ezh2 expression pattern, which might be helpful for further investigation of the detailed role of Ezh2 during the development of the cochlea.

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