Little but loud: small RNAs have a resounding affect on ear development

Abstract

The impact of small RNA function has resonated throughout nearly every aspect of eukaryotic biology and captured the varied interests of researchers, whether they are endeavoring to understand the basis of development and disease or seeking novel therapeutic targets and tools. The genetic regulatory roles of microRNAs (miRNAs) are particularly interesting given that these often highly conserved factors post-transcriptionally silence many complementary target genes by inhibiting messenger RNA translation. In this regard, miRNAs can be considered as counterparts to transcription factors, the ensemble of which establishes the set of expressed genes that define the characteristics of a specific cell type. In this review, evidence supporting a resounding role for small RNAs in development and maturation of sensory epithelia in the mouse inner ear will be considered with an emphasis on the contribution of one hair cell miRNA family (miR-183, miR-96, and miR-182). Although there is much yet to be explored in this fledgling aspect of ear biology, the breadth of miRNA expression and functional requirement for ear development are already sounding off.

Fig. 1

miRNA biogenesis and function. (A) The miRNA pathway. Primary miRNA (pri-miR) transcript processing in the nucleus requires Dgcr8 and the ribonuclease III family member, Drosha. The short hairpin precursor miRNA (pre-miR) is exported from the nucleus and subsequently processed by another ribonuclease III family member, Dicer, with Trbp. The mature miRNA strand is separated from the complementary strand (so-called “star” strand denoted by an asterisk) and is associated with an Argonaute (Ago) protein within the RNA induced silencing complex (RISC). RISC utilizes the miRNA as a guide sequence to discriminately bind target mRNAs and primarily effects translational repression. (B) miRNA-target mRNA interaction. Target mRNA is primarily discriminated through Watson-Crick base pairing to the miRNA “seed” sequence comprising nucleotides 2 through 7 (red) or 8 (orange). Other base-pairing interactions near the 3′ end of the miRNA can contribute further specificity and/or affinity.

Fig. 2

Neurosensory miR-183 family. (A) miR-183 family member sequence and homology. miR-183, miR-96, and miR-182 are clustered within 2-4 kb in vertebrate genomes and co-expressed from a single pri-miR transcript. The vertebrate miRNAs (black) share substantial sequence homology, particularly with the seed sequence (red/orange). Other members of the neurosensory miRNA family include homologous miR-263b and miR-228 (blue) respectively found in D. melanogaster and C. elegans. (B) miR-183 expression in newborn mouse cochlea. miR-183 is detected specifically in inner and outer hair cells (IHC and OHC, respectively) in the organ of Corti by in situ hybridization using a digoxigenin-labeled LNA probe.

Fig. 3

Dicer conditional knockout (CKO) and affect of miRNA depletion on inner ear development. (A) Tissue-specific Cre recombination in transgenic Pax2-Cre mouse embryo (∼8.5 days post coitus). Cre-mediated recombination supports conditional reporter gene expression (blue) in the midbrain (BM), anterior hindbrain (HB), and otic placode (OP). Also indicated are the mid-hindbrain boundary (dashed line) and forebrain (FB). (B) Overview of developmental defects in Pax2-Cre Dicer CKO inner ear (∼17.5 days post coitus). Schematically depicted is normal morphology, histology and innervation of the inner ear and brain (left) versus that of the Dicer CKO (right). Dicer CKO and loss of miRNAs results in a substantial loss of mid-hindbrain structure including the cerebellum (CB), auditory and vestibular neurons, and certain sensory epithelia of the inner ear (colored) are variously affected. Remaining fibers innervating the utricular macula fail to project properly to the brain. AC, anterior crista; HC, horizontal crista; PC, posterior crista; U, utricular macula; S, saccular macula; CO, cochlea.

Fig. 4

Residual miRNA expression correlates with extent of hair cell differentiation in Dicer CKO inner ear sensory epithelia (∼17.5 days post coitus). Absence or presence of apical specializations for presumptive hair cells in the cochlea (CO), utricular macula (U), and posterior crista (PC) correlate with observed loss or retention of residual miR-183. Hair cells in the utricular macula appear as bulbous microvillus cells whereas those in the posterior crista exhibit well-developed kinocilia (arrowhead).

Fig. 5

Mutually exclusive expression of miR-183 family members and predicted target genes. Precursor and/or supporting cells express predicted target genes (red) including Sox2, Notch1, Jag1, and Hes1, whereas hair cells expressing miR-183 family members (black) exclude such factors and otherwise express Dll1, Jag2, and Atoh1 (green). Genetic and/or biochemical interactions among these factors are key to establishing supporting versus hair cell fates, suggesting that miR-183 family members help tip the balance in favor of hair cell differentiation.