MicroRNA gene expression in the mouse inner ear

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that function through the RNA interference (RNAi) pathway and post-transcriptionally regulate gene expression in eukaryotic organisms. While miRNAs are known to affect cellular proliferation, differentiation, and morphological development, neither their expression nor roles in mammalian inner ear development have been characterized. We have investigated the extent of miRNA expression at various time points throughout maturation of the postnatal mouse inner ear by microarray analysis. Approximately one third of known miRNAs are detected in the inner ear, and their expression persists to adulthood. Expression of such miRNAs is validated by quantitative PCR and northern blot analysis. Further analysis by in situ hybridization demonstrates that certain miRNAs exhibit cell-specific expression patterns in the mouse inner ear. Notably, we demonstrate that miRNAs previously associated with mechanosensory cells in zebrafish are also expressed in hair cells of the auditory and vestibular endorgans. Our results demonstrate that miRNA expression is abundant in the mammalian inner ear and that certain miRNAs are evolutionarily associated with mechanosensory cell development and/or function. The data suggest that miRNAs contribute substantially to genetic programs intrinsic to development and function of the mammalian inner ear and that specific miRNAs might influence formation of sensory epithelia from the primitive otic neuroepithelium.

Introduction

The sensory endorgans of the vertebrate inner ear are mosaics of supporting cells and hair cells that transduce mechanical energy into electrical impulses destined for the central nervous system. Organization of terminally differentiated cell types in the sensory epithelia provides a model system for understanding the molecular processes that govern cell type specification and maturation from a simple epithelium to the complex organ of Corti. Much progress has been made in identifying the molecular constituents required for various aspects of inner ear morphogenesis, maturation, and homeostasis (Fritzsch and Beisel, 2003, Eatock and Hurley, 2003, Barald and Kelley, 2004). A number of transcription factors, morphogens, growth factors, receptors, ion channels, and cytoskeletal proteins have been identified that affect various aspects of inner ear development from primitive otic neuroepithelium to cell fate assignment and functional maturation of afferent neurons, hair cells, and supporting cells. Approximately 29,000 genes are expressed throughout ear development with about 4000 being differentially regulated (Chen Z.Y., personal communication). Yet to be considered in these processes is the probable influence of an extensive class of regulatory molecules termed microRNAs.

MicroRNAs (miRNAs) are processed from the transcripts of endogenous genes and function through the RNA interference (RNAi) pathway to mediate post-transcriptional silencing of target genes (Ambros, 2004, He and Hannon, 2004). Among multi-cellular eukaryotic organisms, miRNA genes are evolutionarily conserved and abundant, supporting the view that miRNAs are part of an ancient and crucial genetic regulatory program (Zamore and Haley, 2005). Indeed, certain miRNAs are known to be critical determinants of developmental timing and cell fate specification (Grishok et al., 2001, Chen et al., 2004, Esau et al., 2004, Abbott et al., 2005), morphogenesis (Giraldez et al., 2005, Leaman et al., 2005), cell proliferation (Lee et al., 2005), or differentiated cell function (Poy et al., 2004). Notable examples are miRNAs of the let-7 family that function as repressors of target genes involved in various aspects of biology (Banerjee and Slack, 2002, Abbott et al., 2005, Johnson et al., 2005). These include RAS-dependent cell signaling and proliferation, where let-7 miRNA family members repress lin-60/RAS expression in C. elegans and human (Johnson et al., 2005), thus illustrating an expected conservation of miRNA function among eukaryotic organisms.

There are at least 270 mouse miRNA genes, many of which are orthologous to those of other vertebrate and mammalian species (Griffiths-Jones, 2004). MicroRNA genes are expressed as capped and polyadenylated RNA polymerase II transcripts (Cai et al., 2004, Lee et al., 2004). Among human and mouse miRNA genes, approximately 25% reside within introns and are presumably co-expressed with known genes, and approximately 40% reside in tandem with another miRNA gene(s) suggesting coordinated miRNA expression and function (John et al., 2004). From primary miRNA transcripts (pri-miRNA), the formation of precursor miRNAs (pre-miRNAs) precedes that of functionally mature miRNAs through successive processing events catalyzed by RNase III family members, Drosha and Dicer (Murchison and Hannon, 2004). Mature (∼ 20 nucleotide) miRNAs associated with RNA-induced silencing complexes (RISC) primarily direct translational repression of partially complementary target mRNAs by a mechanism that is not yet well understood (Filipowicz, 2005). Additionally, miRNAs accelerate target mRNA deadenylation and degradation in a manner distinct from small inhibitory RNA (siRNA)-directed mRNA cleavage (Lim et al., 2005, Giraldez et al., 2006, Wu et al., 2006). Thus, miRNAs and their target genes have co-evolved such that their expression in specified tissues is mutually exclusive (Lewis et al., 2003, John et al., 2004). Analyses of miRNA expression and function in zebrafish have offered the first insights regarding the relevance of miRNAs to ear biology. The RNAi pathway can be disrupted by knockout of the Dicer gene, which encodes a ribonuclease required to produce functional microRNAs. Maternal zygotic Dicer knockout zebrafish have been shown to exhibit defects in organogenesis that include a lack of otoconia formation in the ear (Giraldez et al., 2005), demonstrating that an absence of functional miRNAs affects vertebrate ear development. Additionally, in situ hybridization analysis of miRNA expression in zebrafish shows that certain miRNAs are expressed in mechanosensory organs (Wienholds et al., 2005). In particular, a set of miRNAs (miR-96, miR-182, and miR-183) are expressed in the hair cells of zebrafish ears and neuromasts. While these studies suggest that miRNAs have cell-specific expression and may fulfill critical functions in vertebrate ear development, no analysis of miRNA expression in the mammalian ear has been performed.

We have examined the expression of miRNAs in inner ears from newborn to adult mice in order to identify relevant miRNAs. Microarray analyses that indicate either precursor or mature miRNA expression were performed to assess the extent of miRNA expression and processing in the inner ear. The expression of a subset of individual miRNAs is validated by Q-PCR and northern blot detection of mature miRNAs. Moreover, in situ hybridization demonstrates that the cell-specific distribution of miRNA expression can be assessed in whole-mounted sensory epithelia of the mouse inner ear.