Research ReportMicroRNA gene expression in the mouse inner ear
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.
Section snippets
Microarray analyses of miRNA expression
To assess the breadth of miRNA expression in the mouse inner ear, two microarray analyses were performed. The first microarray analysis utilized probes designed to discriminate 213 mouse precursor miRNAs from total RNA samples, enabling identification of precursor miRNAs derived from different genetic loci that yield the same mature miRNA sequence. The second microarray analysis utilized probes designed to assess the expression of 344 mature miRNA sequences present in human, mouse, and/or rat
Discussion
MicroRNAs represent approximately 1% of the genes in mammalian organisms and a substantial component of abundant non-coding RNAs that contribute to the complexity of genetic regulation (Riddihough, 2005). The potential of miRNAs to regulate thousands of target mRNAs containing conserved complementary sites suggests that they contribute substantially to regulation of gene expression (John et al., 2004, Lewis et al., 2005). Our analysis of the mouse inner ear demonstrates that miRNA expression is
Animals
Animal care and handling complied with protocols approved by the Creighton University Institutional Animal Care and Use Committee (IACUC #0730) and employed measures to minimize pain or discomfort. FVB mice were purchased from Charles River Laboratories.
RNA extraction
Tissues were isolated from FVB mice in cold phosphate-buffered saline (PBS; 10 mM Na2HPO4, 1.7 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4). Inner ear included all tissues of the otic capsule (membranous and bony labyrinths) and cochlear and
Acknowledgments
We thank Bernd Fritzsch for helpful discussions and James Eudy for mature miRNA microarray analysis. This work was supported by NIH grant P20 RR018788 from the Centers of Biomedical Research Excellence (CoBRE) Program of the National Center for Research Resources (NCRR) and conducted in a facility constructed with support from NIH grant C06 RR017417 from the Research Facilities Improvement Program of the NCRR.
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