4.1. Introduction to the Role of MicroRNAs in the Inner Ear
MicroRNAs (miRNAs) are small (~22 nucleotides-long), non-coding RNAs that regulate gene expression either by translation repression or mRNA destabilization or both [69]. miRNAs can be transcribed from either the introns of genes or from independent transcription units. In mammals, the primary transcript of miRNA, called pri-miRNA, is first cleaved by Drosha RNAse III endonuclease giving rise to precursor miRNA (pre-miRNA), a ~60–70 nucleotides-long stem-loop structure. This pre-miRNA is then transported to the cytoplasm, where it is again cleaved by another enzyme Dicer, leaving an imperfect double-stranded RNA. This double-stranded RNA is then loaded into the RNA Induced Silencing Complex and cleaved into a single-stranded mature miRNA containing the seed region (2–7 nucleotides) to target the mRNA transcripts that have either perfect or imperfect complementary sequences in their 3′ UTR regions [70]. The seed regions of miRNAs are highly conserved between many organisms [71]. miRNAs can regulate many cellular functions, such as cell proliferation and apoptosis, owing to their binding potential to many target mRNAs [70,72]. Dicer knockout studies in mice [73] and zebrafish [74] showed reduced mature miRNAs, indicating that Dicer is crucial in processing pre-miRNAs to mature miRNAs. miRNAs can occur as clusters in the genome, and over half of the known miRNAs in the Drosophila genome are clustered, although this is not the case with the human and worm genome, where only a small fraction of miRNAs are clustered, i.e., many are found to be isolated [70,75]. The regulation of miRNAs is dynamic, and they show remarkable variability in abundances in cells. For example, in the adult worm, miR-2 is present at more than 50,000 copies, whereas miR-124 is present at ~800 copies per cell [76]. 
A large number of miRNAs have been shown to affect hair cell development in zebrafish [77]. Interest in understanding the role of miRNAs in regulating mechanosensory hair cells has increased ever since the first identification of expression of miRNAs in the sensory epithelia of zebrafish [74]. Following this, many groups have started to explore the developmental and functional role of miRNAs in the inner ear of model organisms, including zebrafish [73,76], mouse [71,73,78,79,80,81,82], rat [83], chicken [84,85] and salamanders [86]. 
Microarrays can be used to detect a large number of miRNA molecules at a time. During the early development of the inner ear, both at the embryonic and post-natal stages, a large number of miRNAs have been detected [74,78,79,86]. Within the inner ear, miRNAs show regulation at both spatial and temporal levels [70,73,74]. For example, in mouse, miR-99a is expressed in hair and supporting cells in the cochlea, but it is expressed only in hair cells in the vestibule [73]. In zebrafish, the majority of the miRNAs examined at the embryonic stage showed very little expression, but they were expressed after the completion of organogenesis, suggesting that miRNAs are required during development, but not during embryonic growth [74]. Differential expression of miRNAs within different inner ear structures have been documented [73,78]. Microarrays done in newborn mouse inner ear showed that 24 miRNAs were differentially expressed between cochlear and vestibular structures [73]. Another study found differential expression of over 100 miRNAs in five developmental time points [78]. Table 2 lists differentially expressed miRNAs that have been found in the inner ear of model organisms. In order to understand the importance of mature miRNAs in the inner ear of mammals, some studies have knocked out the enzyme, Dicer, which processes pre-miRNA to mature miRNA. A conditional Dicerfl°x/fl°x knockout mouse was generated by expressing cre recombinase under the Pou4f3 promoter to study its role in hair and supporting cells of the mouse inner ear [73].
microarrays-02-00186-t002_Table 2 Table 2  List of differentially expressed microRNAs found in different organisms and tissues detected using microarray analysis.   No phenotype was observed in the inner ear in the early post-natal day 0 (P0) stage, but at P38, after maturation of hair cells in the cochlea, severe malformations were observed in the hair cells in the base, while a less severe phenotype was observed in the apex. Scanning electron microscopy in the cochlea and vestibule also revealed the ultrastructural changes, including loss of stereocilia, hair cells becoming round in shape and disorganized and fused stereocilia [73]. Another study knocked out Dicer conditionally in mouse using the Pax2 promoter, and profound morphological and developmental defects were observed in the early embryonic stages [81]. The phenotypes included smaller otocysts, absence of horizontal and anterior cristae, reduced saccules and utricles, reduced inner ear innervation and disorganized hair cell stereocilia. A third study using Atoh1-cre mouse for knocking out Dicer found a marked reduction in the number of outer hair cells and, to some extent, in the inner hair cells [88]. These studies utilized different promoters for knocking out Dicer, but all found severe defects in the development of the inner ear, suggesting that mature miRNAs play an essential role in the morphogenesis, development and innervation of hair cells.