Supporting Information

Friedman et al. 10.1073/pnas.0812446106 SI Results and Discussion intronic miR in these -coding genes. Because in General Phenotype of -PCKO Mice. Dicer-PCKO mice had many many cases the exact borders of the protein-coding genes are defects in additional to inner ear defects. Many of them died unknown, we searched for miR genes up to 10 kb from the around birth, and although they were born at a similar size to hosting- ends. Out of the 488 mouse miR genes included in their littermate heterozygote siblings, after a few weeks the miRBase release 12.0, 192 mouse miR genes were found as surviving mutants were smaller than their heterozygote siblings located inside (distance 0) or in the vicinity of the protein-coding (see Fig. 1A) and exhibited typical defects, which enabled their genes that are expressed in the P2 cochlear and vestibular SE identification even before genotyping, including typical alopecia (Table S2). Some coding genes include huge clusters of miRNAs (in particular on the nape of the neck), partially closed eyelids (e.g., Sfmbt2). Other genes listed in Table S2 as coding genes are [supporting information (SI) Fig. S1 A and C], eye defects, and actually predicted, as their transcript was detected in cells, but weakness of the rear legs that were twisted backwards (data not the predicted encoded protein has not been identified yet, and shown). However, while all of the mutant mice tested exhibited some of them may be noncoding RNAs. Only a single protein- similar deafness and stereocilia malformation in inner ear HCs, coding gene that is differentially expressed in the cochlear and other defects were variable in their severity. These defects most vestibular SE includes a miR gene: this is Trpm1, that is probably resulted from a nearly ubiquitous Cre expression expressed only in vestibular SE (and not in the cochlear SE) and detected in many organs of the Pou4f3-Cre mice, including the includes Mirn211 in one of its introns. Indeed, qRT-PCR con- heart, muscle, placental regions, olfactory turbinates, and bone/ firmed that miR-211 in expressed only in P0 whole vestibule but cartilage. In many organs, Cre expression was mosaic (D. Vetter, not in P0 whole cochlea (data not shown). unpublished work). Our second approach encompassed miRNAs that may be The smaller size of the adult mutants may be a consequence hearing-related. While at least 44 chromosomal protein-coding of an anterior crossbite. While in heterozygote littermates the genes have been linked with nonsyndromic hereditary hearing lower teeth were behind the upper teeth, the upper jaw of the loss (NSHHL) in thus far, there are 62 additional Dicer-PCKO mice was shorter than normal, and their upper teeth chromosomal loci that have been linked with NSHHL, but the were behind the lower teeth (Fig. S1 B and D) (consistent with responsible gene has not yet been identified (Hereditary Hearing a Col2a1-Cre recombination of the Dicer1 allele in ref. 1). This Loss Homepage; http://webh01.ua.ac.be/hhh). In some of these defect may interfere with suckling and eating. Similarly, jaw cases the responsible gene may miRNAs. Even when the defects and small size were reported recently in mutant mice in genes responsible for hearing loss in some of these loci are which the miR-199a-2 and miR-214 cluster was knocked out by protein-coding, those not yet verified as having cochlear expres- replacement of its gene (Dnm30s) with a lacZ gene. These sion would have been excluded from the predictions derived homozygote mice exhibited several skeletal defects, including from the first approach. Out of the 695 miR genes jaw closure, but this defect did not include a short upper jaw, in included in miRBase release 12.0, 155 miR genes were found to contrast to our mice. However, in both cases, many mutant mice be located in 54 NSHHL loci (Table S3). The gene for our died a short time after birth and the surviving homozygotes were positive-control miRNA, miR-182, which is known to be specif- smaller than their control littermates, most probably because of ically expressed in inner ear HCs both in and mice feeding problems (2). (6, 7), is included in a human deafness-related , DFNA50. Intersections between the predicted lists and miRNAs that miRNA Microarray Results and Predictions of Mammalian miRNAs that were found to be expressed in newborn mouse whole cochleae may Have Roles in Inner Ear SE. Microarrays were used to screen and vestibules by miRNA microarrays are listed in Tables S4 and which miRNAs are expressed in the newborn-mouse whole S5. Venn diagrams are presented in Fig. 2C.InTable S4, the list cochlea and vestibule (Table S1). miRNAs are very short (on of miRNAs that are expressed in the newborn-mouse whole average, 22 nucleotides) and their probes on the arrays are cochlea, according to miRNA microarrays (see Table S1) was antisense sequences. As a result, different probes on our arrays intersected with a list of miRNAs that are predicted to be have different melting temperatures (Tm). Therefore, we do not expressed in the cochlear SE (see Table S2, excluding miR-211) present the average intensity for each probe, taking into account and with the list of miRNAs that may be related with deafness that differences may result from different Tm. Nonetheless, we (see Table S3). The 6 miRNAs that are included in all 3 consider these microarray results as true/false values, with intersections (mir-25, mir-93, mir-126, mir-185, mir-210, and possible false negatives, and spots with high intensities can still mir-335) may have a role in hearing. Table S5 presents miRNAs indicate that a particular miRNA is expressed. Because the that are expressed in the newborn-mouse whole vestibule, ac- RNAs for microarray hybridization were purified from whole cording to miRNA microarrays (see Table S1) and are also cochleae and vestibules, they included miRNAs expressed in all predicted to be expressed in the vestibular SE (see Table S2). of the inner ear tissues. Therefore, there may be a bias for From the miRNAs that were selected for further study, the microarray results that emphasize miRNAs expressed in non- Mirn15a gene is very close to a transcript that is expressed in the sensory cells, while miRNAs that are specifically expressed in the mouse inner ear SE and is predicted to be protein-coding SE may appear to have low expression. (2810055G20Rik). Mirn199a-2 (together with its cluster member, Two complementary approaches were used to pick candidate Mirn214) gene is included in the antisense strand of a conserved miRNAs that are predicted to be expressed in the inner ear SE. intron of the protein-coding gene Dnm3, which is expressed in First, miR genes in introns of protein-coding genes that are the mouse inner ear SE. Because the Mirn199a-2/214 gene is not expressed in the cochlear and vestibular SE may be transcribed located in the same strand as Dnm3, it is not included in Table together with the hosting protein-coding gene, using the same S2. However, the antisense strand of this intron is known to be promoter (3–5). mRNAs that are expressed in cochlear and transcribed in mice (8). The genes for miRNAs 15a and 199a-2 vestibular SE of P2 C3H mice were profiled by Affymetrix chips are also included in human deafness-related loci. MIRN15a is (data not shown), and a Perl algorithm was written to identify included in the AUNA1 auditory neuropathy locus (9).

Friedman et al. www.pnas.org/cgi/content/short/0812446106 1of28 MIRN199a-2/214 is included in the human locus 1q24 that embryos are shown in Fig. S6. Embryos treated with MMO- contains a dominant modifier (DFNM1), which suppresses the 15a-1 show a delay in the appearance of the sensory cristae and recessive deafness DFNB26 locus (10). Although there are 2 a reduction in the size of the sensory maculae and the stato- Mirn199a genes in the mouse, the gene that is expressed in the acoustic ganglion (see Fig. S6 CϪE). The magnitude of HC mouse inner ear seems to be Mirn199a-2, because this gene is reduction in the maculae is not strongly correlated with the clustered with Mirn214 and both are very highly and similarly degree of body truncation (see Table S6). MMO-18a morphants, expressed in P0 mouse whole inner ears, according to our when compared with injected control fish, have a more subtle miRNA microarrays (see Table S1). Unexpectedly, miR-199a phenotype: they have the correct number of sensory organs in was not detected in P0 inner ear SE by ISH (Fig. 3), although a their inner ears, although the maculae have fewer HCs. small level of miR-199a was detected by quantitative real time PCR (data not shown). Selection of 3 Putative miR-15a Targets for Experimental Validation. To identify biologically relevant targets for miR-15a in the Mechanosensory Phenotypes in Zebrafish Morphants. Fertilized ze- mouse, 3 putative targets expressed in the inner ear SE (as brafish eggs were treated with morpholinos (MO) against miR- mRNAs) and may have a role in its function or survival were 15a-1, miR-18a or their pre-miRNAs. A control ‘‘standard’’ selected. Two of them, Slc12a2/Nkcc1 (solute carrier family 12, morpholino (Stnd) with a sequence that is not expected to target member 2/sodium-potassium-chloride cotransporter) (11) and any known zebrafish RNA served as a negative control for Bdnf (-derived neurotrophic factor) (12), were already injection artifacts. At 24, 30, and 55 hpf, chorions were removed linked with hereditary hearing loss in mice, although HC survival and fish were examined for gross morphogenetic defects, such as was not considered as their primary role. Slc12a2/Nkcc1 is absence of otoliths or canal pillars, indicating the initiation of essential for maintenance of the endolymph, a solution with a semicircular canal formation. To address the issue of off-target unique ion composition that fills the scala media and to which effects, several different MOs were used against each miRNA or the HCs face (11). The protein translated from Slc12a2 mRNA its longer precursor (the MOs are described in Fig. S3). is not detected in the WT newborn-mouse inner ear (as exam- Treatment with 3 different MOs targeting miR-15a-1 yielded ined by immunohistochemistry; data not shown), although it is significant reductions of miRNA-15a ISH signal in the brain and known to be expressed in cells that are involved in endolymph otocysts at 30 hpf for the majority of embryos when compared production in inner ears from older mice (e.g., in the cochlear with uninjected embryos (Fig. S4, Left). At this time, otocyts stria vacularis and in the dark cells of the cristae and the roof of looked normal (Fig. S4, Middle). At 54 hpf, MMO-15a-1 and the saccule in the vestibule; these cells do not express miR-15a MO-15a-1-G showed similar penetrance of tail truncations and in newborns) (11, 13). The neurotrophin Bdnf is released from inner ear phenotypes that included absence of canal pillar hair cells, guides their afferent innervation (14), and is required outgrowth in the majority of injected fish (Fig. S4, Right). for survival of vestibular and spiral ganglion nerve cells (12). It miR-15a-1-S morphants generally had normal bodies and only a also has a protective role on spiral ganglion cells following small fraction of injected embryos (Ϸ8%) were missing the canal exposure to ototoxic antibiotics and acoustic trauma (15). Bdnf pillars; similarly, a relatively large fraction of miR-15a-1-S protein is known to be expressed in all vestibular hair cells (16) morphants had detectable miR-15a-1 signal by ISH (data not and in cochlear hair and supporting cells (17) in newborn shown). rodents. The third putative target, Cldn12 (claudin 12), belongs Morphants of miR-18a were examined by ISH at 30 hpf and to a family of tight-junction and is known to be the majority treated with MMO-18a or MO-18a-G had reduced expressed in cochlear and vestibular SE, as well as in other inner signal when probed for miRNA-18, as compared to uninjected ear cells in adult mice (18). Gene-targeted inactivation of controls (Fig. S5, Left). While a few embryos treated with another claudin, Cldn14, with a similar expression pattern in the MO-18a-Sdi had undetectable expression of miRNA-18a, many adult cochlear SE (18), led to hearing loss in mice because of others had a clear signal in the head (data not shown). miR-18a degeneration of cochlear HCs (19). Immunohistochemistry morphants were notable for their defective formation of the staining of claudin 12 protein in newborn-mouse inner ears is anterior otoliths that was already evident at 24 hpf (data not presented in Fig. 5A. shown). Anterior otoliths were either missing or significantly smaller than normal, which was documented photographically at SI Materials and Methods 30 hpf (Fig. S5, Middle). This defect persisted at 54 to 56 hpf (Fig. S5, Right). The phenotype was highly penetrant for embryos Generation of Dicer-PCKO Mice. All procedures involving mice met injected with MMO-miR-18a and MO-18a-G; whereas only 12 to the guidelines described in the National Institutes of Health 25% of embryos injected with 0.25 mM or 0.5 mM of MO-18a- Guide for the Care and Use of Laboratory Animals and had Sdi showed small otoliths at 30 hpf that correlated with a reduced been approved by the Animal Care and Use Committees of the ISH signal for miRNA-18a, and none were missing the anterior National Institutes of Health and Tel Aviv University (M-06–042). otolith. Most MO-18a-Sdi morphants had a normally sized 129/SvEv mice that carry the Cre recombinase gene under the anterior otolith by 54 hpf. Those that did not also failed to control of Pou4f3 promoter (Pou4f3-Cre) were generously pro- initiate canal pillar outgrowth, suggesting perhaps this specific vided by D. Vetter (Tufts University, Boston, MA, USA) and MO may induce off-target effects. Z.-Y. Chen (Harvard University, Boston, MA, USA) (20). For HC counts in the lateral-line and inner ear sensory organs, 24 of Dicer1 in mice encodes most of the second embryos treated with MMO-15a-1 and MMO-18a were har- RNaseIII domain, which is crucial for the ability of Dicer1 to vested at 54 to 56 hpf and stained with a monoclonal produce mature miRNAs from pre-miRNAs. Homozygous Dicerflox/flox (HCS-1) that marks mechanosensory HCs. Some embryos were mice, in which exon 24 of Dicer1 is surrounded by two loxP sites, double-stained with a neuronal marker, HuC. Individuals were were generously provided by C. Tabin (Harvard University, grouped according to the severity of body dysmorphogenesis for Boston, MA, USA) (21). miR-15a morphants, so that quantification and statistical com- To conditionally knock out Dicer1 only in cells that express parisons could be performed separately and combined. Silencing Cre under the control of the Pou4f3 promoter in mice, male of miR-15a and miR-18a using MMO MOs induced a significant Dicerflox/flox mice were mated with female Pou4f3-Cre mice to decrease in the number of lateral-line neuromasts and in the generate heterozygote Dicerflox/ϩ mice that carry a Pou4f3-Cre number of hair cells per neuromast or inner ear macula (Table allele (Pou4f3-Creϩ/Ϫ;Dicerflox/ϩ). Then, male and female Pou4f3- S6). Confocal images through the inner ears of some sample Creϩ/Ϫ;Dicerflox/ϩ were mated to generate Dicer-PCKO progenies

Friedman et al. www.pnas.org/cgi/content/short/0812446106 2of28 that are homozygous for Dicerflox and carry a Pou4f3-Cre allele for Immunohistochemistry in Mice. Primary antibodies (Pou4f3-Cre;Dicerflox/flox). Mating of a Pou4f3-Cre–/–;Dicerflox/flox for immunohistochemistry were as follows: rabbit polyclonal (either male or female) to a Pou4f3-Cre;Dicerflox/ϩ (homozygote anti-myosin VI antibody (1:200 dilution; Proteus BioScience); or heterozygote for Cre) did not yield any viable Dicer-PCKO goat polyclonal anti-myosin VI antibody (1:100 dilution; Santa progeny (progeny died before or around birth). Littermate Cruz Biotechnology); rabbit polyclonal anti-Slc12a2 antibody heterozygote Dicerflox/ϩ mice with or without Pou4f3-Cre served (1:150 dilution; Chemicon/Milipore). Chicken anti-mouse as controls. Cldn12 antibody (used at 1:100 dilution) was a generous gift from K. Turksen (Ottawa Health Research Institute Canada) Genotyping. For Cre genotyping, forward: (5Ј-aggcgttttctgagcat- (24). acctgga-3Ј) and reverse (5Ј-acgctagagcctgttttgcacgtt-3Ј) primers Secondary antibodies for immunohistochemistry were: Alexa- were used. PCR was performed by using 95 °C for 2 min followed Flour 488-conjugated goat anti-rabbit (1:400 dilution) or donkey by 95 °C for 30 s, 58 °C for 1 min, and 72 °C for 1 min for 30 anti-rabbit (1:500 dilution) IgG (Molecular Probes, Invitrogen); cycles. The final annealing was 72 °C for 10 min. A band of 300 cy3-conjugated donkey anti-goat (1:250 dilution; Jackson Im- bp indicated the presence of the Cre allele. munoResearch); FITC-conjugated rabbit anti-chicken (1:50 di- For Dicer-loxP genotyping, forward (5Ј-cctgacagtgacggtc- lution; Sigma-Aldrich). caaag-3Ј) and reverse (5Ј-catgactcttcaactcaaact-3Ј) primers Auditory Brainstem Response. Click-evoked auditory nerve and were used. PCR was performed as follows: 94 °C for 3 min, brainstem response recordings for each ear were obtained from (94 °C for 30 sec, 61 °C for 1 min, and 72 °C for 30 sec) ϫ 3 mice anesthetized with Avertin 11.25 mg/kg injected intraperi- cycles, (94 °C for 30 sec, 59 °C for 1 min, and 72 °C for 30 ϫ tonially. The stimuli were alternating polarity clicks of around sec) 3 cycles, (94 °C for 30 sec, 57 °C for 1 min, and 72 °C 4,000 Hz or 8,000 Hz tone bursts through an insert earphone, for 30 sec) ϫ 3 cycles, (94 °C for 30 sec, 55 °C for 1 min, and ϫ using a Biologic Navigator Pro evoked potential system (Bio- 72 °C for 30 sec) 3 cycles, (94 °C for 30 sec, 53 °C for 1 min, logic Systems Corp.). Recording subdermal needle electrodes ϫ and 72 °C for 30 sec) 35 cycles. The final annealing was 72 °C were inserted at the vertex referred to the chin, with a ground for 5 min. A band of 420 bp indicated the presence of the loxP electrode in the hindlimb. The stimuli were presented at a rate sites (351 bp in WT). of 20 per second from a maximal intensity of 125 dB below threshold in 5-dB steps. The responses were filtered (band pass Zebrafish Strains and Standard Practices. ET20 adult zebrafish were 300–3,000 Hz), amplified, and 128 to 256 responses were aver- created by Parinov et al. (22) and obtained from a colony in the aged and displayed vertex-positive up. Threshold was defined as laboratory of A.J. Hudspeth (the Rockefeller University). These the lowest stimulus intensity required to elicit repeatable com- enhancer-trap fish express GFP in supporting cells in neuromasts ponents (usually the first wave) of ABR in at least 2 recordings. and the inner ear. Breeders of the AB strain were obtained from ‘‘No response’’ was determined when recordings obtained from the Zebrafish International Resource Center, University of the same stimuli did not show repeating components. In case no Oregon, Eugene, OR. Breeders of a mixed AB/TL genotype repeating components were detected, another measure was were obtained from a colony in the laboratory of J. Dowling at performed with a blocked earphone to confirm that the response Harvard University. Zebrafish embryos were raised in E3 solu- is similar. tion according to standard procedures (23). All protocols were approved by the Purdue University Animal Care and Use Scanning Electron Microscopy of Inner Ears. Whole inner ears from Committee. Beginning at 24 hpf, some embryos were raised in P38 mice were isolated and fixated in 2.5% glutaraldehyde in 0.2 mM 1-phenyl-2-thiourea in E3 media to block pigmentation. PBS overnight at 4 °C. After 3 PBS washes, inner ears were Before fixation, all embryos were anesthetized by immersion in fine-dissected to expose the SE of the vestibular and cochlear 0.08 mg/ml Tricaine for Ϸ10 min. They were fixed overnight by systems and run through the osmium- thiocarbohydrazide- immersion in 4% paraformaldehyde (PFA) in PBS (PBS) at 4 °C. osmium procedure: samples were incubated for1hin1% osmium, then incubated for 20 min in thiocarbohydrazide and ϫ Immunohistochemistry Staining in Mice. Inner ears were fixed in 4% washed with DDW 3 5 min. The osmium–thiocarbohydrazide– PFA for 2 to 18 h or declassified in 4% PFA-EDTA for 72 h DDW washes incubations were repeated twice and a final 1-h (inner ears from postnatal day 38) at 4 °C, dehydrated in a incubation in 1% osmium was followed by a DDW wash and a graded series of ethanol, and incubated for2hinisopropanol gradient of ice-cold ethanol (30%, 50%, 70%, and 100%; 15 min incubation each step). After dehydration, the samples were and 2 ϫ 2 h in toluene. critical-point dried and coated with gold for examination under For paraffin sections, samples were embedded in paraffin the JEOL 5400 scanning electron microscope. using a Leica Histoembedder. Sections (10 ␮m) were dewaxed with xylene and rehydrated with a decreasing ethanol gradient. Skeleton Staining. P38 mice were killed, the fur, viscera, and most For immunohistochemistry, antigens were unmasked using a of the skin were removed, and the skeletons were stained with citric acid-based antigen unmasking solution (Vector). Follow- Alcian Blue and Alizarin Red solution (0.014% Alcian Blue; ing 2-h incubation in a PBSTG blocking solution (0.2% Tween 0.006% Alizarin Red; 57% ethanol; 13% acetic acid) for 14 days. 20 and 0.2% gelatin in PBS), sections or whole-mount inner ears After KOH incubations (1.8% KOH for 3 days; 0.3% KOH were incubated overnight with a primary antibody at 4 °C. After overnight), muscles and fat were removed, and the skeletons PBS washes, a secondary antibody was applied to samples for 1 h were transferred through glycerol gradients. at RT (see Antibodies for Immunohistochemistry in Mice for details). In some cases, Rhodamin-Phalloidin (1:250 dilution; RNA Isolation. Inner ears and were dissected from WT Molecular Probes, Invitrogen) was added together with the C57BL/6 mice at indicated ages. From inner ears, cochleae and secondary antibody to stain F-actin. Thereafter, 4Ј-6-Diamidino- vestibules were separated, with meticulous separation of saccules 2-phenylindole (DAPI dilactate; Sigma-Aldrich) was applied to from cochleae. The mirVana miRNA Isolation Kit (Ambion, stain nuclei. Applied Biosystems) was used for isolation of small (Ͻ200 nt) RNAs, following the manufacturer’s instructions. The quality H&E Staining in Mice. Paraffin sections were stained with Hema- and integrity of the small RNAs were evaluated by denaturing toxylin and Eosin (Fluka). polyacrylamide gel electrophoresis (PAGE: 15% polyacryl-

Friedman et al. www.pnas.org/cgi/content/short/0812446106 3of28 amide/8M urea gels in TBE buffer) and ethidium bromide Tm). Antisense 3Ј-DIG-labeled miRCURY LNA Detection staining of the gels. probes to specific mouse miRNAs were purchased from Exiqon. For microarray experiments, tiny (Ͻ40 nt) RNAs were iso- After prehybridization, inner ears were hybridized with hybrid- lated from the small (Ͻ200 nt) RNA fraction, using flashPAGE ization mix containing 1-␮g LNA probe/200 ␮l overnight at Fractionator (Ambion), according to the manufacturer’s instruc- Thyb. Each run included positive controls hybridized with tions. The Ͻ40 nt RNA fractions, which are expected to include miR-182 probes and negative controls hybridized with Scramble mature RNAs without pre-miRNAs, were precipitated overnight probes. at –20 °C with a linear acrylamide carrier (Ambion) in the Anti-DIG-AP (alkaline phosphatase conjugated) Fab frag- presence of 0.06 M sodium acetate and 80% ethanol. After this ments were purchased from Roche and preincubated at 1:1,000 stage, the RNA amount was about 1% of the Ͻ200 nt sample dilution with a batch of PFA-fixed P0 inner ears in a blocking amount. solution (PBS containing 0.1% Tween-20, 2% sheep serum and 2 mg/ml BSA) overnight at 4 °C. miRNA Microarrays and Their Analysis. Arrays were printed in- The next day, the inner ears were washed briefly with 100% house with the mirVana miRNA Probe Set version 1 (Ambion) hybridization mix (HM) at Thyb, and incubated for 15 min in according to the manufacturer’s instructions, including spike-in each of the following solutions at Thyb: (i) 75% HM with 25% control probes. These arrays contained DNA probes to 206 2ϫ SSC; (ii) 50% HM with 50% 2ϫ SSC; (iii) 25% HM with 75% mammal miRNAs. 2ϫ SSC; (iv)2ϫ SSC. The inner ears were washed twice with A positive-control RNA (Ambion) was added to tiny (Ͻ40 nt) 0.2ϫ SSC for 30 min at Thyb, and incubated for 10 min in each RNAs, pooled from at least 20 WT cochleae or vestibules from of the following solutions at room temperature: (i) 75% 0.2ϫ P0 C57BL/6 mice. Each RNA sample was labeled with Cy3 or SSC with 25% PBST; (ii) 50% 0.2ϫ SSC with 50% PBST; (iii) Cy5 fluorescent dyes (Post-Labeling Reactive CyDyes, GE 25% 0.2ϫ SSC with 755% PBST; (iv) PBST. Following incuba- Healthcare), using the mirVana miRNA Labeling Kit (Ambion), tion in blocking solution for2hatroom temperature, the inner according to the manufacturer’s protocol. Each spotted microar- ears were labeled with the preincubated antibody overnight at ray was hybridized with 2 RNA samples that were labeled with 4 °C (1:5 dilution of preincubated anti-DIG-AP Fab in blocking different dyes, using the buffers and instructions of the mirVana solution). Thereafter, the inner ears were intensively washed miRNA Bioarray Essentials Kit (Ambion). Three microarrays with PBST for 3 days (at least 5 ϫ 1h,12ϫ 2 h, and 2 overnight were hybridized with P0 cochleae and vestibules, one of them washes), washed 3ϫ for 5 min with staining buffer (100 mM with crossed dyes. The microarrays were scanned by an Af- Tris-HCl pH9.5; 50 mM MgCl2; 100 mM NaCl and 0.1% fymetrix 428 array scanner and quantified using the ImaGene Tween-20), and incubated in NBT/BCIP (Sigma-Aldrich) until a (version 5.1) software (BioDiscovery). purple color developed. The reaction was stopped with 1 mM Interarray variability was compensated by the global expres- EDTA pH 5.5. sion of each array. For each probe at each tissue, the average To freeze the inner ears in tissue-freezing medium (TBS, intensity of 8 to 12 spots from 2 to 3 microarrays (including dye Triangle Biomedical Sciences), the inner ears were incubated in swap) was calculated. For each microarray, the global back- the following solutions: (i) 10% sucrose in PBS for1hatroom ground was calculated by averaging the intensities of buffer and temperature; (ii) 20% sucrose in PBS for1hatroom temper- empty spots, and only miRNAs with an average expression ature; (iii) 30% sucrose in PBS overnight at 4 °C; (iv) 15% higher than twice the fold of the array global background at least sucrose and 50% TBS in PBS overnight at 4 °C; (v) 100% TBS in 1 of the tissues were considered as expressed. The linear for2hatroom temperature. Inner ears were frozen in TBS and regression of the average vestibular versus cochlear miRNA 8- to 10-␮m cryosections were prepared. expression was calculated, and miRNAs that their vestibular expression is different by 15% or more than their expected ISH of miRNAs in Whole Zebrafish Embryos. For ISH of miRNAs in expression according to the linear regression were considered as zebrafish embryos, whole embryos were fixed, dehydrated differently expressed in the 2 tissues. through graded methanols, and stored at –20 °C. Whole-mount ISH for miRNAs was performed similarly to ISH in mouse inner qRT-PCR of miRNAs. Small (Ͻ200 nt) RNAs were isolated from ears, using dre-miR miRCURY LNA Detection DNA probes cochleae, vestibules, and brains from WT C57BL/6 mice at (Exiqon), anti-DIG-AP immunostaining, and alkaline phospha- indicated ages, P38 Dicer-PCKO or littermate mice. Specific tase detection, according to Kloosterman et al. (25). Probes were miRNAs and U6B small nuclear RNA (as endogenous control) either purchased prelabeled with DIG or they were DIG-labeled were reverse transcribed, and their expression was measured with DIG Oligonucleotide 3Ј-end labeling kit (Roche Applied using the TaqMan MicroRNA kits and the ABI Prism 7000 or Science). Probes were hybridized at 20 to 25 °C below the probe 7900 PCR machine (Applied Biosystems). Cycle thresholds were melting temperatures. Each run included positive controls hy- normalized according to U6B, and relative quantities to P0 brain bridized with miR-183 probes and negative controls hybridized are presented. with 5Ј-DIG-labeled Scramble miR (Exiqon). After develop- ment to detect alkaline phosphatase, specimens were put ISH of miRNAs in Whole Mouse Inner Ears. Whole inner ears from through 10%, 20%, and 30% sucrose in PBS, embedded in 15% WT P0 C57BL/6 mice were fixed in 4% PFA overnight, the sucrose/7.5% gelatin in PBS, frozen, and sectioned at 30 ␮mon cartilage was punctured and the stapes removed from the oval a Leica CM1900 cryostat. window to enable fast penetration and circulation of the fixative. After short rinses with PBS, the samples were fine-dissected to Morpholino Injections to Zebrafish Embryos. MOs directed against expose the cochlear and vestibular labyrinth to enable probe miR-15a-1 and miR-18a were purchased from GeneTools, LLC. penetration. MOs were designed to target different parts of the pre-miRNA Following intensive PBST washings, the inner ears were or mature miRNA. Because these miR genes are members of treated with 2.5 ␮g/ml proteinase K in PBST for 20 min at 37 °C. larger families, we show sequence comparisons for the known The inner ears were then refixed in 4% PFA for 20 min, washed family members, along with the position the antisense MOs are with PBST (5 ϫ 5 min), and prehybridized in hybridization mix designed against for the targeted family member (see Fig. S3). (50% formamide; 5ϫ SSC; 0.1% Tween-20; titrated with 1M Although some of the MOs can potentially target other members Citric acid to pH 6.0; 50 ␮g/ml heparin and 500 ␮g/ml tRNA) for of the same miRNA family, we considered this to be an asset in 2 h at hybridization temperature (Thyb, 21 °C below the probe’s their design. For example, miR-18a is a member of a larger

Friedman et al. www.pnas.org/cgi/content/short/0812446106 4of28 family that includes miR-18b and miR-18c. Their sequences are GCTCTAGACCCTGCATAATGAGCCTTTT; Cldn12 reverse highly similar in the targeted regions, and therefore a single MO primer–GCTCTAGAATGAATCTTTGGCGCTGAC. might reduce the activity of all miRNAs of a family and thus For sequencing of the 3Ј-UTRs in pGL3, the forward primer avoid potential problems of rescue because of redundancy. The TCGACGCAAGAAAAATCAGA, which binds the pGL3 se- MOs used in this study have the following sequences: quence upstream of the XbaI site, or the reverse cloning primer MMO-15a-1 5Ј-TTATAACTCACAAACCATTCTGTGCT- were used. GCTAC-3Ј MO-15a-1-G 5Ј-TCACAAACCATTCTGTGCTGCTAC-3Ј Dual Luciferase Assay. An miRVec vector for expression of miR- MO-15a-1-S 5Ј-TGCCGCAGCACAGTACGGCCTGCA-3Ј 15a was generously provided by R. Agami (29). HEK-293T MMO-18a 5Ј-TACTTCACTATCTGCACTAGATGCAC- (human embryonic kidney) cells were seeded in 6-well plates. CTTAG–3Ј The cells in each well were transfected using JetPEI transfection MO-18a-G 5Ј-CTATCTGCACTAGATGCACCTTAG-3Ј reagent (PolyPlus), according to the manufacturer’s instructions, MO-18A-Sdi 5Ј-CACTTAGGGCAGTAGGTGCTAGTCT-3Ј. with 4 ␮g DNA: 0.8 ␮g pGL3 containing the desired 3Ј-UTR; 0.2 MOs were reconstituted with water to 1.0 mM and diluted ␮g pRL-TK (to induce a control renilla luciferase expression; further for injections into embryos. One-cell fertilized embryos Promega), with or without 1.4 ␮g miRVec-15a; a vector express- received injection boluses of 1 nl at doses of 0.10 to 0.5 mM. As ing GFP from a standard CMV promoter was cotransfected (to a negative control for injection artifacts, a ‘‘standard’’ MO (Gene compare transfection efficiency in different wells), and pUC18 Tools, LLC) with a sequence that is not expected to target any vector was added to 4 ␮g. Each transfection was performed in known zebrafish RNA was injected. Embryos injected with triplicate. In addition, each experiment included, as negative standard MOs were normal. We tested a range of doses for each controls, a triplicate of nontransfected wells (NTC) to measure miR MO and toxic levels were based on the dose at which half the background firefly luciferase luminescence, and a triplicate or more embryos failed to survive to 24 hpf. The final MO doses of wells that were transfected with empty pGL3 without pRL-TK per embryo were reduced to obtain at least 20% normal-looking to measure the background renilla luciferase luminescence. embryos in a typical injection batch. To document body and Forty-eight hours after transfection, the transfection efficiency inner ear phenotypes, zebrafish embryos were photographed was verified by GFP expression, and firefly and renilla luciferase with a SPOT flex color-mosaic camera (Diagnostic Instruments) activities were measured using the Dual-Luciferase Reporter mounted on a Nikon E800 fluorescence microscope equipped Assay System kit (Promega) and a Veritas microplate luminom- with Nomarski optics. eter, according to Promega’s instructions.

Immunostaining of Zebrafish Whole-Mount Embryos and Phenotypic Prediction of miRNAs that Participate in the Development or Function Evaluation. Whole zebrafish embryos were fixed and then per- of Inner Ear SE. Gene coordinates (, strand, and base meabilized in 1% triton in PBS for 24 h to dissolve the otoliths numbers at 3Ј and 5Ј ends) are as in National Center for (26). Antibodies were used to detect hair cells [HCS-1, IgG2a, Biotechnology Information (NCBI) build 36 and 37 assemblies 1:250 dilution, gift of J. Corwin (27)], human acetylated tubulin for human and mouse genes, respectively. Coordinates of mouse (IgG2b, 1:100 dilution, Sigma), or neurons (HuC, IgG2b, 1:75 and human miR genes were downloaded from the miRBase dilution, Molecular Probes, Invitrogen) by single- or double- database [http://microrna.sanger.ac.uk/sequences/; release 12.0, labeling with the appropriate goat anti-mouse-IgG2a or -IgG2b updated for September 2008 (30, 31)], and coordinates of mouse AlexaFluor-conjugated secondary antibodies (1:500 dilution, protein-coding genes were downloaded by Ensembl BioMArt Molecular Probes), as previously described (28). Specimens were (http://www.ensembl.org/biomart/martview/; as of November cleared in 70% glycerol, mounted with Vectashield (Vector 2008). Symbols of mouse protein-coding genes are as in Mouse Laboratories) onto glass slides and cover-slipped, after removing Genome Informatics (MGI) database release 4.13. the yolk and, for lateral orientations, also 1 eye. Two approaches were used to identify miRNAs that may have Following immunohistochemistry staining, the number of roles in mouse inner ear development and function: mechanosensory organs and the number of hair cells per organ (i) A Perl algorithm was written to identify miR genes in were imaged and counted using 20ϫ or 40ϫ objectives of the introns and up to 10 kb from known ends of protein-coding genes Nikon E800 fluorescence microscope. Some samples were (in the same strand) that are transcribed in cochlear or vestibular counted on the Nikon and then imaged with a Bio-Rad MRC sensory epithelia (based on microarrays screening of mRNAs 1024 confocal microscope using a 60ϫ objective. Results showed isolated from sensory epithelia dissected from P2 C3H mice; that the Nikon counts underestimated hair cell numbers by 0 to Affymetrix GeneChip Mouse Genome 430 2.0 Arrays, R. 2 hair cells per macula, with larger errors typical of larger organs. Hertzano, personal communcation). Confocal image stacks were imported into ImageJ 1.36b (Na- (ii) All of the human loci correlated to NSHHL, in which the tional Insitutes of Health) for generating Z-projections. These responsible gene has not yet been identified, were downloaded and all other digital images were imported into Adobe Photo- from the Hereditary Hearing Loss Homepage (http:// shop CS for figure preparation. webh01.ua.ac.be/hhh/; May 2008 release). Sixty-two loci have published locations. Chromosomal locations of loci that are Constructs for Dual Luciferase Assay. Fragments of about 1 kb from included in NCBI build 36.3 (homo sapiens) were updated from the 3Ј-UTRs of Slc12a2, Bdnf, and Cldn12 mRNAs were cloned the Online Mendlian Inheritance in Man database (http:// into the XbaI restriction site of the pGL3 Luciferase Reporter www.ncbi.nlm.nih.gov/omim/). miR gene locations were down- Vector (Promega) as 3ЈUTRs of the firefly luciferase cDNA. For loaded from Univerisity of California Santa Cruz Genome this purpose, the 3Ј UTR fragments were PCR-amplified from Browser (human March 2006 assembly). We searched for human P0 cochlear cDNA (reverse transcribed from Ͼ200 nt RNA miR genes in these loci. fraction), and XbaI restriction sites were added (italics), with the following cloning primers: Slc12a2 forward primer–TCTA- Target Prediction. Only miRNAs that are expressed in inner ear GACGGTCCCAGATTTTTGTCAT; Slc12a2 reverse primer– SE, according to ISH results, were selected to look for their TCTAGATTGTGATGACTCCACTTCCTTT; Bdnf forward targets. Results of 3 Web-available target-prediction algorithms primer–GCTCTAGACAGGGTAAATTATTCAGTTAA- were intersected to look for possible targets for these miRNAs: GAAAAA; Bdnf reverse primer–GCTCTAGACAAAA- TargetScan (32)–version 4.0, conserved targets (http:// CAAAAAGAAGGGACCA; Cldn12 forward primer– www.targetscan.org/); miRanda (33)–human targets (as of

Friedman et al. www.pnas.org/cgi/content/short/0812446106 5of28 November 2007; http://www.microrna.org/microrna/getDown- for each miRNA, with only a subset of targets common to all loads.do); and PicTar (34)–mouse miRNAs with conserved 3 predictions. Then, Affymetrix mRNA microarrays data of targets in mammals (http://pictar.mdc-berlin.de/cgi-bin/ WT P2 C3H cochlear and vestibular sensory epithelia mRNAs PicTar࿝vertebrate.cgi). These algorithms base their predictions (R. Hertzano, personal communication) were used to filter on target site-seed complementation, evolutionary conserva- prediction results. Only predicted target mRNAs that are tion of the target site, and thermodynamic calculations. Hun- expressed in the P2 inner ear SE were considered as possible dreds of mRNAs were predicted by each algorithm as targets targets.

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Friedman et al. www.pnas.org/cgi/content/short/0812446106 6of28 Fig. S1. Dicer-PCKO mice defects outside the inner ear. Compared with their littermate heterozygote siblings (A and B), Dicer-PCKO mice exhibit defects also outside the inner ear (C and D). (C) P38 Dicer-PCKO mice exhibited a typical balding pattern and their eyelids were partially closed. (D) The P38 Dicer-PCKO skeleton appeared normal except for a shorter upper jaw. As a result, the teeth closure of the Dicer-PCKO mice was abnormal, with an anterior crossbite. The skeletons (B and D) were stained with Alcian Blue and Alizarin Red.

Friedman et al. www.pnas.org/cgi/content/short/0812446106 7of28 Fig. S2. Temporal expression of miRNA 15a, 18a, 30b, 99a, and 199a in the mouse whole cochlea (blue) and vestibule (green) along inner ear development, as determined by qRT-PCR at 4 ages (E16.5, E19.5, P0, and P30). miR-182 was used as a positive control, and no template samples were used as negative controls. The results are presented as relative quantities compared to P0 brain (arbitrary fold-changes). Error bars represent the maximum and minimum fold-change, while the bar represents the average fold-change. The data shown were obtained from 2 separate experiments. Similar results were obtained in 2 additional experiments.

Friedman et al. www.pnas.org/cgi/content/short/0812446106 8of28 Fig. S3. Comparison of the sequences of zebrafish miR-15 and miR-18 pre-miRNAs and the morpholinos used in this study. In zebrafish, both miR-15a-1 and miR-18a are members of larger miRNA gene families that have significant sequence similarity in the mature miRNAs (underlined) and beyond. For miR-15a-1 and miR-18b, the antisense (Star) strands are also expressed as mature miRNAs (underlined). Blue denotes positions conserved among all family members. Pink highlights positions that are similar to the targeted pre-miRNA in all but one family member. Yellow highlights positions that are similar to the targeted pre-miRNA in all but 2 family members. Beneath each family, dashed lines indicate the positions targeted by the antisense morpholino listed. The multiblocking morpholinos (MMOs) are 31 nucleotides in length and overlap with the 5Ј cleavage site and the 3Ј Dicer cleavage site on the targeted pre-miRNAs. All other MOs are 24 nucleotides in length. MO-18a-G and MO-15a-1-G target the ‘‘guide’’ strand and are designed to extend only 1 nucleotide on each side of the mature miRNA. Other MOs are designed to target either the antisense ‘‘star’’ (S) strand alone (MO-15a-1-S) or in combination with the 3Ј loop that includes the Dicer (di) cleavage site (MO-18a-Sdi), as indicated.

Friedman et al. www.pnas.org/cgi/content/short/0812446106 9of28 Fig. S4. Morpholino knockdown of miR-15a-1 using different MOs. (Left) Thirty-hpf embryos probed with miR-15a locked nucleic acid (LNA) probe to detect mature miRNAs. (Scale bar, 200 ␮m.) (Middle) Nomarski images of embryos at 30 hpf show presence of 2 otoliths in the inner ear; MMO-15a-1 morphants are indistinguishable from uninjected embryos (data not shown). (Right) Nomarski images of embryos at 55 hpf showing that miR-15a-1 morphants fail to show proper initiation of semicircular canal formation (image of MMO-15a-1 morphant ear at this stage is shown in Fig. 4B and Fig. S6B). Many embryos treated with MO-15a-1-S look normal at this stage; the embryo shown is representative of fewer than 10% that show the canal phenotype.

Friedman et al. www.pnas.org/cgi/content/short/0812446106 10 of 28 Fig. S5. Morpholino knockdown of miR-18a using different MOs. (Left) Thirty-hpf embryos probed with miR-18a LNA probe to detect mature miRNA. (Scale bar, 200 ␮m.) (Middle) Nomarski images of embryos at 30 hpf show either absent or smaller anterior otoliths in 18a morphants. MO-18a-Sdi morphants are either normal (data not shown) or have smaller anterior otoliths; they typically do not have a specific loss of the anterior otoliths. (Right) Nomarski images of embryos at 55 hpf showing that the phenotype of the anterior otoliths persists, although in most morphants the canals form normally. An exception are the small fraction of MO-miR-18a-Sdi morphants that continue to show reductions in anterior otolith size, which is correlated with a more general loss of canal pillar outgrowth as shown.

Friedman et al. www.pnas.org/cgi/content/short/0812446106 11 of 28 Fig. S6. Defects in body and inner ear morphology in zebrafish morphant embryos. MMOs against specific miRNAs were injected to 1-celled embryos and the phenotype 55 h later is presented. A ‘‘standard’’ morpholino (MO Stnd) with a sequence that is not expected to target any known zebrafish RNA served as a negative control for unspecific toxicity. (A) Body phenotypes shown at low magnification. A range of body types was apparent in miR-15a-1 morphants, including complete tail truncation as the most severe outcome (panel MMO-15a-1). miR-18a morphants had normal bodies (panel MMo-18a), except when MOs were used at very high doses (data not shown). (Scale bar, 500 ␮m.) (B) Higher power views of the inner ears from the same embryos as those shown on column (A), photographed with Normarski optics. These are the same images shown in Fig. 4. (Scale bar, 100 ␮m.) (C–E) Defects in the sensory organs and ganglia of the inner ears from miR-morphant zebrafish. Confocal image slices were obtained from lateral views of the inner ear and were collapsed as Z-projections to view hair cells (labeled with HCS-1 antibody; column C) and neurons (labeled with HuC antibody; column D) associated with the inner ear. Column (E) presents merged photos. Embryos injected with standard MO showed the normal distribution of 2 maculae, 3 cristae, and a comma-shaped statoacoustic ganglion (SAG). Unlabeled hair cell clusters are cranial neuromasts. 15a-1 morphants had smaller maculae and were missing the 3 cristae. 18a morphants had smaller maculae (notice the utricular macula assumes a curved distribution of hair cells). Abbreviations: AC, anterior crista; LC, lateral crista; PC, posterior crista; SAG, statoacoustic ganglion; SM, saccular macula; UM, utricular macula. (Scale bar, 30 ␮m.)

Friedman et al. www.pnas.org/cgi/content/short/0812446106 12 of 28 Table S1. miRNAs expressed in whole newborn mouse cochleae miRNAs expressed miRNAs expressed and vestibules in whole P0 cochleae in whole P0 vestibules miRNAs expressed miRNAs expressed miR24 miR423 in whole P0 cochleae in whole P0 vestibules miR200b miR191 let7f let7f miR143 let7g miR181a miR214 miR148a let7d miR214 miR206 miR103 miR200a miR29b miR198 miR411 miR341 miR17–5p miR181a miR188 miR199a* miR19a miR202 miR292–3p miR376b miR374 miR19a let7f miR203 miR100 miR199a let7b miR30a-3p miR198 miR100 let7e miR380 miR125a miR291–5p miR147 miR201 miR151 miR30a-5p miR1 miR181b miR182 miR300 miR361 let7f miR206 miR320 let7d miR185 miR202 miR93 miR351 miR188 miR423 miR374 miR367 miR137 miR126 miR338 miR203 miR383 miR384 miR182 miR339 miR9 miR184 miR204 miR380 miR183 miR30a-5p miR24 miR201 miR382 miR199a miR29b miR204 miR411 miR93 miR184 miR144 miR190 miR124a miR125b miR195 miR220 miR31 miR17–5p miR142–5p let7a miR220 miR26a miR293 miR15a miR221 let7c miR200a miR301 miR338 let7b miR155 miR302b* miR9* miR23b miR298 let7i miR320 miR151 miR197 miR144 miR30b miR124a miR207 miR155 miR22 miR9* miR370 miR142–5p miR300 miR125a miR182* miR16 miR217 miR30b miR301 miR27a miR410 miR122a miR25 miR297 let7c miR143 miR376a miR370 miR291–5p miR195 miR382 miR367 miR122a miR210 miR15a miR95 miR422a miR384 miR373* miR207 let7a miR422a miR297 miR351 miR190 miR200b miR30a-3p miR145 miR149 miR410 miR342 miR331 miR376b miR146 miR205 miR361 miR210 miR221 miR106a miR383 miR126 miR298 miR101 miR147 miR292–5p miR26a miR372 miR205 let7g miR368 miR138 miR28 miR103 miR182* miR181b miR31 miR293 miR146 miR22 miR7 miR125b miR148a miR140 miR215 miR217 miRNAs listed from highest to lowest expressed. miRNAs selected for miR34c miR101 further study are labeled in bold. miR23b miR18a miR335 let7e miR137 miR149 miR372 miR335 miR191 miR292–3p miR368 miR20 miR341 miR28 miR20 miR376a miR18a miR1 miR185 miR34c

Friedman et al. www.pnas.org/cgi/content/short/0812446106 13 of 28 Table S2. miR genes in introns or in the region of protein-coding genes expressed in P2 inner ear sensory epithelia Symbol of miR gene miR gene Coding gene name Distance (bases)*

Mirnlet7c-1 mmu-let-7c-1 2810055G20Rik 1126 Mirnlet7f-2 mmu-let-7f-2 Huwe1 0 Mirnlet7g mmu-let-7g Wdr82 0 Mirnlet7i mmu-let-7i Mon2 9439 Mirn1–2-as mmu-mir-1–2-as Mib1 0 Mirn7a-1 mmu-mir-7a-1 2210016F16Rik 7554 Mirn7a-1 mmu-mir-7a-1 Hnrpk 0 Mirn7a-2 mmu-mir-7a-2 Isg20l1 7554 Mirn9–2 mmu-mir-9–2 C130071C03Rik 0 Mirn15b mmu-mir-15b Smc4 0 Mirn16–2 mmu-mir-16–2 Smc4 0 Mirn21 mmu-mir-21 Tmem49 538 Mirn22 mmu-mir-22 2010305C02Rik 4269 Mirn22 mmu-mir-22 2210403K04Rik 0 Mirn23b mmu-mir-23b 2010111I01Rik 0 Mirn24–1 mmu-mir-24–1 2010111I01Rik 0 Mirn25 mmu-mir-25 Mcm7 0 Mirn25 mmu-mir-25 Zfp113 9577 Mirn26a-1 mmu-mir-26a-1 Ctdspl 0 Mirn26a-2 mmu-mir-26a-2 Ctdsp2 0 Mirn26b mmu-mir-26b Ctdsp1 0 Mirn27b mmu-mir-27b 2010111I01Rik 0 Mirn30c-1 mmu-mir-30c-1 Nfyc 0 Mirn30e mmu-mir-30e Nfyc 0 Mirn32 mmu-mir-32 D730040F13Rik 0 Mirn33 mmu-mir-33 Srebf2 0 Mirn92b mmu-mir-92b Trim46 6979 Mirn93 mmu-mir-93 Mcm7 0 Mirn93 mmu-mir-93 Zfp113 9779 Mirn98 mmu-mir-98 Huwe1 0 Mirn99a mmu-mir-99a 2810055G20Rik 405 Mirn101b mmu-mir-101b Rcl1 0 Mirn103–1 mmu-mir-103–1 Pank3 0 Mirn103–2 mmu-mir-103–2 Pank2 0 Mirn106b mmu-mir-106b Mcm7 0 Mirn106b mmu-mir-106b Zfp113 9993 Mirn107 mmu-mir-107 Pank1 0 Mirn125b-1 mmu-mir-125b-1 2610203C20Rik 0 Mirn126 mmu-mir-126 Egfl7 0 Mirn128–1 mmu-mir-128–1 R3hdm1 0 Mirn128–2 mmu-mir-128–2 Arpp21 0 Mirn130b mmu-mir-130b Sdf2l1 5998 Mirn132 mmu-mir-132 Smg6 9238 Mirn135b mmu-mir-135b 6430514M23Rik 0 Mirn135b mmu-mir-135b Lemd1 0 Mirn138–2 mmu-mir-138–2 Nup93 9245 Mirn139 mmu-mir-139 Pde2a 0 Mirn140 mmu-mir-140 Wwp2 0 Mirn141 mmu-mir-141 Emg1 5787 Mirn141 mmu-mir-141 Ptpn6 2732 Mirn142 mmu-mir-142 Bzrap1 4475 Mirn148b mmu-mir-148b Copz1 0 Mirn149 mmu-mir-149 Gpc1 0 Mirn151 mmu-mir-151 Ptk2 0 Mirn152 mmu-mir-152 Copz2 0 Mirn185 mmu-mir-185 D16H22S680E 0 Mirn186 mmu-mir-186 Zranb2 0 Mirn188 mmu-mir-188 Clcn5 0 Mirn190 mmu-mir-190 Tln2 0 Mirn190b mmu-mir-190b 1700094D03Rik 4145 Mirn190b mmu-mir-190b 4933434E20Rik 7576 Mirn190b mmu-mir-190b Tpm3 2638 Mirn191 mmu-mir-191 Dalrd3 1519 Mirn191 mmu-mir-191 Impdh2 2755

Friedman et al. www.pnas.org/cgi/content/short/0812446106 14 of 28 Symbol of miR gene miR gene Coding gene name Distance (bases)*

Mirn191 mmu-mir-191 Qrich1 8159 Mirn192 mmu-mir-192 Atg2a 2540 Mirn194–2 mmu-mir-194–2 Atg2a 2339 Mirn200a mmu-mir-200a Ttll10 0 Mirn200b mmu-mir-200b Ttll10 0 Mirn200c mmu-mir-200c Emg1 6195 Mirn200c mmu-mir-200c Ptpn6 2327 Mirn204 mmu-mir-204 Trpm3 0 Mirn207 mmu-mir-207 Dnaja1 0 Mirn207 mmu-mir-207 OTTMUSG00000008561 0 Mirn210 mmu-mir-210 1600016N20Rik 7302 Mirn211† mmu-mir-211 Trpm1 0 Mirn212 mmu-mir-212 Smg6 8944 Mirn218–1 mmu-mir-218–1 Slit2 0 Mirn218–2 mmu-mir-218–2 Slit3 0 Mirn219–1 mmu-mir-219–1 H2-Ke6 942 Mirn219–1 mmu-mir-219–1 Ring1 328 Mirn219–1 mmu-mir-219–1 Slc39a7 3181 Mirn297a-3 mmu-mir-297a-3 Sfmbt2 0 Mirn297a-4 mmu-mir-297a-4 Sfmbt2 0 Mirn297a-5 mmu-mir-297a-5 Sfmbt2 0 Mirn297b mmu-mir-297b Sfmbt2 0 Mirn297c mmu-mir-297c Sfmbt2 0 Mirn301a mmu-mir-301a 1110001A07Rik 0 Mirn301b mmu-mir-301b Sdf2l1 5644 Mirn302a mmu-mir-302a 4930422G04Rik 7925 Mirn302b mmu-mir-302b 4930422G04Rik 8188 Mirn302c mmu-mir-302c 4930422G04Rik 8059 Mirn302d mmu-mir-302d 4930422G04Rik 7800 Mirn326 mmu-mir-326 Arrb1 0 Mirn330 mmu-mir-330 Eml2 0 Mirn331 mmu-mir-331 Vezt 4237 Mirn335 mmu-mir-335 Mest 0 Mirn338 mmu-mir-338 Aatk 0 Mirn339 mmu-mir-339 3110082I17Rik 0 Mirn340 mmu-mir-340 Rnf130 0 Mirn342 mmu-mir-342 Evl 0 Mirn343 mmu-mir-343 Ppp1r13l 8110 Mirn350 mmu-mir-350 Cep170 0 Mirn361 mmu-mir-361 Chm 0 Mirn362 mmu-mir-362 Clcn5 0 Mirn367 mmu-mir-367 4930422G04Rik 7682 Mirn374 mmu-mir-374 B230206F22Rik 0 Mirn421 mmu-mir-421 B230206F22Rik 0 Mirn423 mmu-mir-423 Ccdc55 0 Mirn425 mmu-mir-425 Dalrd3 1050 Mirn425 mmu-mir-425 Impdh2 3213 Mirn425 mmu-mir-425 Qrich1 8617 Mirn429 mmu-mir-429 Ttll10 0 Mirn455 mmu-mir-455 Col27a1 0 Mirn455 mmu-mir-455 OTTMUSG00000008561 0 Mirn466a mmu-mir-466a Sfmbt2 0 Mirn466b-1 mmu-mir-466b-1 Sfmbt2 0 Mirn466b-2 mmu-mir-466b-2 Sfmbt2 0 Mirn466b-3 mmu-mir-466b-3 Sfmbt2 0 Mirn466c mmu-mir-466c Sfmbt2 0 Mirn466d mmu-mir-466d Sfmbt2 0 Mirn466e mmu-mir-466e Sfmbt2 0 Mirn466f-1 mmu-mir-466f-1 Sfmbt2 0 Mirn466f-2 mmu-mir-466f-2 Sfmbt2 0 Mirn466f-3 mmu-mir-466f-3 Sfmbt2 0 Mirn466g mmu-mir-466g Sfmbt2 0 Mirn466h mmu-mir-466h Sfmbt2 0 Mirn466l mmu-mir-466l Sfmbt2 0 Mirn467a mmu-mir-467a Sfmbt2 0

Friedman et al. www.pnas.org/cgi/content/short/0812446106 15 of 28 Symbol of miR gene miR gene Coding gene name Distance (bases)*

Mirn467b mmu-mir-467b Sfmbt2 0 Mirn467c mmu-mir-467c Sfmbt2 0 Mirn467d mmu-mir-467d Sfmbt2 0 Mirn467e mmu-mir-467e Sfmbt2 0 Mirn483 mmu-mir-483 Igf2 0 Mirn484 mmu-mir-484 Nde1 3614 Mirn486 mmu-mir-486 Ank1 0 Mirn488 mmu-mir-488 Astn1 0 Mirn491 mmu-mir-491 BC057079 0 Mirn491 mmu-mir-491 OTTMUSG00000008561 0 Mirn493 mmu-mir-493 Meg3 8514 Mirn500 mmu-mir-500 Clcn5 0 Mirn501 mmu-mir-501 Clcn5 0 Mirn504 mmu-mir-504 Fgf13 0 Mirn505 mmu-mir-505 Atp11c 0 Mirn511 mmu-mir-511 Mrc1 0 Mirn532 mmu-mir-532 Clcn5 0 Mirn546 mmu-mir-546 Ctdsp2 0 Mirn574 mmu-mir-574 9130005N14Rik 0 Mirn652 mmu-mir-652 Tmem164 0 Mirn669a-1 mmu-mir-669a-1 Sfmbt2 0 Mirn669a-2 mmu-mir-669a-2 Sfmbt2 0 Mirn669a-3 mmu-mir-669a-3 Sfmbt2 0 Mirn669b mmu-mir-669b Sfmbt2 0 Mirn669c mmu-mir-669c Sfmbt2 0 Mirn669d mmu-mir-669d Sfmbt2 0 Mirn669e mmu-mir-669e Sfmbt2 0 Mirn669f mmu-mir-669f Sfmbt2 0 Mirn669g mmu-mir-669g Sfmbt2 0 Mirn669h mmu-mir-669h Sfmbt2 0 Mirn669i mmu-mir-669i Sfmbt2 0 Mirn669j mmu-mir-669j Sfmbt2 0 Mirn669k mmu-mir-669k Sfmbt2 0 Mirn671 mmu-mir-671 2010209O12Rik 0 Mirn673 mmu-mir-673 Meg3 271 Mirn674 mmu-mir-674 Spred1 4846 Mirn675 mmu-mir-675 H19 0 Mirn677 mmu-mir-677 Atp5b 0 Mirn677 mmu-mir-677 Baz2a 0 Mirn677 mmu-mir-677 Ptges3 8032 Mirn678 mmu-mir-678 Prmt2 0 Mirn684–2 mmu-mir-684–2 OTTMUSG00000008561 0 Mirn686 mmu-mir-686 Psmb5 0 Mirn687 mmu-mir-687 Rb1 0 Mirn688 mmu-mir-688 Atp5g2 745 Mirn692–2 mmu-mir-692–2 Ftl2 0 Mirn693 mmu-mir-693 Xpo5 0 Mirn697 mmu-mir-697 Sf3a3 0 Mirn698 mmu-mir-698 Inpp5b 0 Mirn700 mmu-mir-700 Rcan3 0 Mirn702 mmu-mir-702 Plod3 0 Mirn704 mmu-mir-704 Pdia4 0 Mirn705 mmu-mir-705 Rab11fip5 0 Mirn705 mmu-mir-705 Sfxn5 2870 Mirn706 mmu-mir-706 Wnk1 0 Mirn707 mmu-mir-707 Akt1s1 0 Mirn707 mmu-mir-707 Pnkp 7368 Mirn709 mmu-mir-709 Rfx1 0 Mirn711 mmu-mir-711 Col7a1 0 Mirn717 mmu-mir-717 Gpc3 0 Mirn718 mmu-mir-718 Irak1 0 Mirn718 mmu-mir-718 Mecp2 2892 Mirn719 mmu-mir-719 Nupl1 0 Mirn721 mmu-mir-721 Cux1 0 Mirn744 mmu-mir-744 Map2k4 0

Friedman et al. www.pnas.org/cgi/content/short/0812446106 16 of 28 Symbol of miR gene miR gene Coding gene name Distance (bases)*

Mirn761 mmu-mir-761 Nrd1 0 Mirn761 mmu-mir-761 OTTMUSG00000008561 0 Mirn762 mmu-mir-762 Ctf1 4182 Mirn763 mmu-mir-763 Hmga2 0 Mirn770 mmu-mir-770 Meg3 0 Mirn872 mmu-mir-872 Ift74 0 Mirn872 mmu-mir-872 OTTMUSG00000008561 0 Mirn877 mmu-mir-877 Abcf1 0 Mirn1190 mmu-mir-1190 Ccdc88c 0 Mirn1192 mmu-mir-1192 Klf9 0 Mirn1195 mmu-mir-1195 Tgif1 9268 Mirn1198 mmu-mir-1198 Gripap1 0 Mirn1224 mmu-mir-1224 Ece2 7065 Mirn1892 mmu-mir-1892 1110002B05Rik 0 Mirn1893 mmu-mir-1893 Epc1 0 Mirn1894 mmu-mir-1894 Ppp1r10 0 Mirn1898 mmu-mir-1898 Zfr 0 Mirn1899 mmu-mir-1899 1700128F08Rik 4799 Mirn1902 mmu-mir-1902 Hipk3 841 Mirn1903 mmu-mir-1903 Nrp1 0 Mirn1905 mmu-mir-1905 Rab25 5654 Mirn1906 mmu-mir-1906 Meg3 1675 Mirn1907 mmu-mir-1907 Trps1 0

miRNAs further studied are labeled in bold. Gene coordinates (chromosome, strand and base numbers at 3’ and 5’ ends) were downloaded from NCBI build 37 (mus musculus). *The distance of the miR gene from the protein coding gene known end (up to 10 kb). For miR genes located inside the known range of the protein-coding genes, the distance is 0. †miR-211, in an intron of Trpm1, is expressed only in vestibular SE.

Friedman et al. www.pnas.org/cgi/content/short/0812446106 17 of 28 Table S3. Candidate miRNAs for non-syndromic hereditary hearing loss (NSHHL) in humans miR gene miR gene chromosomal Human locus miR gene symbol location

AUNA1 hsa-mir-320d-1 MIRN320d-1 13q14.11 AUNA1 hsa-mir-621 MIRN621 13q14.11 AUNA1 hsa-mir-16–1 MIRN16–1 13q14.3 AUNA1 hsa-mir-15a MIRN15a 13q14.3 AUNA1 hsa-mir-1297 MIRN1297 13q21.1 DFN2/DFNX1 hsa-mir-652 MIRN652 Xq22.3 DFN6/DFNX4 hsa-mir-651 MIRN651 Xp22.31 DFN6/DFNX4 hsa-mir-1308 MIRN1308 Xp22.11 DFNA21, DFNA31 hsa-mir-877 MIRN877 6p21.33 DFNA21, DFNA31 hsa-mir-1236 MIRN1236 6p21.32 DFNA21, DFNA31 hsa-mir-219–1 MIRN219–1 6p21.32 DFNA21, DFNA31 hsa-mir-1275 MIRN1275 6p21.31 DFNA23 hsa-mir-548h-1 MIRN548h-1 14q23.2 DFNA23 hsa-mir-625 MIRN625 14q23.3 DFNA24 hsa-mir-1305 MIRN1305 4q35.1 DFNA25, DFNB50 hsa-mir-1251 MIRN1251 12q23.1 DFNA25, DFNB50 hsa-mir-135a-2 MIRN135a-2 12q23.1 DFNA25, DFNB50 hsa-mir-1827 MIRN1827 12q23.1 DFNA30 hsa-mir-1276 MIRN1276 15q25.3 DFNA30 hsa-mir-1179 MIRN1179 15q26.1 DFNA30 hsa-mir-7–2 MIRN7–2 15q26.1 DFNA30 hsa-mir-9–3 MIRN9–3 15q26.1 DFNA30 hsa-mir-1469 MIRN1469 15q26.2 DFNA30 hsa-mir-1302–2 MIRN1302–2 15q26.3 DFNA30, DFNB48 hsa-mir-184 MIRN184 15q25.1 DFNA30, DFNB48 hsa-mir-549 MIRN549 15q25.1 DFNA32 hsa-mir-210 MIRN210 11p15.5 DFNA32 hsa-mir-675 MIRN675 11p15.5 DFNA32 hsa-mir-483 MIRN483 11p15.5 DFNA32 hsa-mir-302e MIRN302e 11p15.4 DFNA37, DFNB32 hsa-mir-137 MIRN137 1p21.3 DFNA37, DFNB32 hsa-mir-553 MIRN553 1p21.2 DFNA40 hsa-mir-484 MIRN484 16p13.11 DFNA42, DFNA54, DFNB60 hsa-mir-1289–2 MIRN1289–2 5q31.1 DFNA42, DFNA54, DFNB60 hsa-mir-886 MIRN886 5q31.2 DFNA42, DFNA54, DFNB60 hsa-mir-874 MIRN874 5q31.2 DFNA47 hsa-mir-491 MIRN491 9p21.3 DFNA47 hsa-mir-31 MIRN31 9p21.3 DFNA47 hsa-mir-876 MIRN876 9p21.1 DFNA47 hsa-mir-873 MIRN873 9p21.1 DFNA50 hsa-mir-593 MIRN593 7q32.1 DFNA50 hsa-mir-129–1 MIRN129–1 7q32.1 DFNA50 hsa-mir-182 MIRN182 7q32.2 DFNA50 hsa-mir-96 MIRN96 7q32.2 DFNA50 hsa-mir-183 MIRN183 7q32.2 DFNA50 hsa-mir-335 MIRN335 7q32.2 DFNA50 hsa-mir-29a MIRN29a 7q32.3 DFNA50 hsa-mir-29b-1 MIRN29b-1 7q32.3 DFNA53 hsa-mir-1201 MIRN1201 14q11.2 DFNA53 hsa-mir-208a MIRN208a 14q11.2 DFNA53 hsa-mir-208b MIRN208b 14q11.2 DFNA53, DFNB5 hsa-mir-624 MIRN624 14q12 DFNA57, DFNB15, DFNB68 hsa-mir-1181 MIRN1181 19p13.2 DFNA57, DFNB15, DFNB68 hsa-mir-1238 MIRN1238 19p13.2 DFNA57, DFNB15, DFNB68 hsa-mir-638 MIRN638 19p13.2 DFNA57, DFNB15, DFNB68 hsa-mir-199a-1 MIRN199a-1 19p13.2 DFNA7, DFNA49 hsa-mir-554 MIRN554 1q21.3 DFNA7, DFNA49 hsa-mir-190b MIRN190b 1q21.3 DFNA7, DFNA49 hsa-mir-92b MIRN92b 1q22 DFNA7, DFNA49 hsa-mir-555 MIRN555 1q22 DFNA7, DFNA49 hsa-mir-9–1 MIRN9–1 1q22 DFNA7, DFNA49 hsa-mir-765 MIRN765 1q23.1

Friedman et al. www.pnas.org/cgi/content/short/0812446106 18 of 28 miR gene miR gene chromosomal Human locus miR gene symbol location

DFNA7, DFNA49 hsa-mir-556 MIRN556 1q23.3 DFNB13 hsa-mir-671 MIRN671 7q36.1 DFNB13 hsa-mir-153–2 MIRN153–2 7q36.3 DFNB13 hsa-mir-595 MIRN595 7q36.3 DFNB14, DFNB17 hsa-mir-592 MIRN592 7q31.33 DFNB15 hsa-mir-24–2 MIRN24–2 19p13.12 DFNB15 hsa-mir-27a MIRN27a 19p13.12 DFNB15 hsa-mir-23a MIRN23a 19p13.12 DFNB15 hsa-mir-181c MIRN181c 19p13.12 DFNB15 hsa-mir-181d MIRN181d 19p13.12 DFNB15 hsa-mir-639 MIRN639 19p13.12 DFNB15 hsa-mir-1470 MIRN1470 19p13.12 DFNB15 hsa-mir-640 MIRN640 19p13.11 DFNB15, DFNB42 hsa-mir-1280 MIRN1280 3q21.3 DFNB15, DFNB72 hsa-mir-1302–2 MIRN1302–2 19p13.3 DFNB15, DFNB72 hsa-mir-1909 MIRN1909 19p13.3 DFNB15, DFNB72 hsa-mir-1227 MIRN1227 19p13.3 DFNB15, DFNB72 hsa-mir-637 MIRN637 19p13.3 DFNB15, DFNB72 hsa-mir-7–3 MIRN7–3 19p13.3 DFNB15, DFNB72 hsa-mir-220b MIRN220b 19p13.3 DFNB25 hsa-mir-572 MIRN572 4p15.33 DFNB25 hsa-mir-218–1 MIRN218–1 4p15.31 DFNB25 hsa-mir-573 MIRN573 4p15.2 DFNB25 hsa-mir-1255b-1 MIRN1255b-1 4p14 DFNB25 hsa-mir-574 MIRN574 4p14 DFNB26 hsa-mir-548g MIRN548g 4q31.23 DFNB27 hsa-mir-933 MIRN933 2q31.1 DFNB27 hsa-mir-10b MIRN10b 2q31.1 DFNB27 hsa-mir-1246 MIRN1246 2q31.1 DFNB27 hsa-mir-1258 MIRN1258 2q31.3 DFNB32 hsa-mir-760 MIRN760 1p22.1 DFNB32 hsa-mir-197 MIRN197 1p13.3 DFNB33 hsa-mir-126 MIRN126 9q34.3 DFNB33 hsa-mir-602 MIRN602 9q34.3 DFNB38 hsa-mir-1913 MIRN1913 6q27 DFNB39 hsa-mir-590 MIRN590 7q11.23 DFNB39 hsa-mir-1285–1 MIRN1285–1 7q21.2 DFNB39 hsa-mir-653 MIRN653 7q21.3 DFNB39 hsa-mir-489 MIRN489 7q21.3 DFNB39 hsa-mir-591 MIRN591 7q21.3 DFNB39 hsa-mir-25 MIRN25 7q22.1 DFNB39 hsa-mir-93 MIRN93 7q22.1 DFNB39 hsa-mir-106b MIRN106b 7q22.1 DFNB39 hsa-mir-548o MIRN548o 7q22.1 DFNB40 hsa-mir-648 MIRN648 22q11.21 DFNB40 hsa-mir-185 MIRN185 22q11.21 DFNB40 hsa-mir-1306 MIRN1306 22q11.21 DFNB40 hsa-mir-1286 MIRN1286 22q11.21 DFNB40 hsa-mir-649 MIRN649 22q11.21 DFNB40 hsa-mir-301b MIRN301b 22q11.21 DFNB40 hsa-mir-130b MIRN130b 22q11.21 DFNB40 hsa-mir-650 MIRN650 22q11.22 DFNB40 hsa-mir-548j MIRN548j 22q12.1 DFNB42 hsa-mir-568 MIRN568 3q13.31 DFNB42 hsa-mir-198 MIRN198 3q13.33 DFNB42 hsa-mir-548i-1 MIRN548i-1 3q21.2 DFNB48 hsa-mir-629 MIRN629 15q23 DFNB48 hsa-mir-630 MIRN630 15q24.1 DFNB48 hsa-mir-631 MIRN631 15q24.2 DFNB55 hsa-mir-1269 MIRN1269 4q13.2 DFNB57 hsa-mir-346 MIRN346 10q23.2 DFNB57 hsa-mir-107 MIRN107 10q23.31 DFNB57 hsa-mir-607 MIRN607 10q24.1

Friedman et al. www.pnas.org/cgi/content/short/0812446106 19 of 28 miR gene miR gene chromosomal Human locus miR gene symbol location

DFNB57 hsa-mir-1287 MIRN1287 10q24.2 DFNB57 hsa-mir-608 MIRN608 10q24.31 DFNB57 hsa-mir-146b MIRN146b 10q24.32 DFNB57 hsa-mir-1307 MIRN1307 10q24.33 DFNB57 hsa-mir-936 MIRN936 10q25.1 DFNB57 hsa-mir-609 MIRN609 10q25.1 DFNB57 hsa-mir-548e MIRN548e 10q25.2 DFNB58 hsa-mir-1285–2 MIRN1285–2 2p14 DFNB58 hsa-mir-1302–3 MIRN1302–3 2q14.1 DFNB58 hsa-mir-663b MIRN663b 2q21.2 DFNB60 hsa-mir-548f-3 MIRN548f-3 5q22.1 DFNB60 hsa-mir-1244 MIRN1244 5q23.1 DFNB62 hsa-mir-1244 MIRN1244 12p13.2 DFNB62 hsa-mir-613 MIRN613 12p13.1 DFNB62 hsa-mir-614 MIRN614 12p13.1 DFNB62 hsa-mir-920 MIRN920 12p12.1 DFNB63 hsa-mir-548k MIRN548k 11q13.3 DFNB65 hsa-mir-296 MIRN296 20q13.32 DFNB65 hsa-mir-298 MIRN298 20q13.32 DFNM1 hsa-mir-921 MIRN921 1q24.1 DFNM1 hsa-mir-1255b-2 MIRN1255b-2 1q24.2 DFNM1 hsa-mir-557 MIRN557 1q24.2 DFNM1 hsa-mir-1295 MIRN1295 1q24.3 DFNM1 hsa-mir-214 MIRN214 1q24.3 DFNM1 hsa-mir-199a-2 MIRN199a-2 1q24.3 DFNM2 hsa-mir-596 MIRN596 8p23.3 DFNM2 hsa-mir-548i-3 MIRN548i-3 8p23.1 DFNM2 hsa-mir-597 MIRN597 8p23.1 DFNM2 hsa-mir-124–1 MIRN124–1 8p23.1 DFNM2 hsa-mir-1322 MIRN1322 8p23.1 DFNM2 hsa-mir-598 MIRN598 8p23.1

miRNAs selected for further study are labeled in bold.

Friedman et al. www.pnas.org/cgi/content/short/0812446106 20 of 28 Table S4. Intersection of miRNAs expressed in newborn mouse whole cochlea and predicted to play role in cochlear sensory epithelium miR genes within human miR genes within human NSHHL miR genes within introns of P2 NSHHL loci and introns loci expressed in newborn cochlear SE coding genes expressed of P2 cochlear SE coding genes mouse whole cochlea in newborn mouse whole cochlea miR-25 † miR-9 let-7c miR-92b miR-15a let-7f miR-93 † miR-24 let-7g miR-106b miR-25 † miR-1 miR-107 miR-29b miR-9* miR-126 † miR-31 miR-22 miR-130b miR-93 † miR-23b miR-185 † miR-124a miR-24 miR-190b miR-126 † miR-25 † miR-210 † miR-137 miR-26a miR-218–1 miR-182 miR-93 † miR-219–1 miR-184 miR-103 miR-301b miR-185 † miR-125b miR-335 † miR-197 miR-126 † miR-483 miR-198 miR-142–5p miR-484 miR-199a miR-149 miR-491 miR-210 † miR-151 miR-574 miR-214 miR-185 † miR-652 miR-298 miR-188 miR-671 miR-335 † miR-190 miR-675 miR-191 miR-877 miR-200a miR-200b miR-204 miR-207 miR-210 † miR-301 miR-335 † miR-338 miR-339 miR-342 miR-361 miR-367 miR-374 miR-423

†miRNAs included in 3 lists. miRNAs studied further in bold.

Friedman et al. www.pnas.org/cgi/content/short/0812446106 21 of 28 Table S5. Intersection of miRNAs expressed in newborn mouse whole vestibule and predicted to play role in vestibular sensory epithelia miR genes within introns of P2 vestibular SE coding genes expressed in newborn mouse whole vestibule let-7c let-7f let-7g let-7i miR-1 miR-103 miR-125b miR-126 miR-138 miR-140 miR-142–5p miR-149 miR-151 miR-16 miR-185 miR-188 miR-190 miR-191 miR-200a miR-200b miR-204 miR-207 miR-210 miR-22 miR-23b miR-24 miR-26a mir-297a mir-301a miR-302b* miR-331 miR-335 miR-338 miR-361 miR-367 miR-374 miR-423 mir-7a miR-9, miR-9* miR-93

Friedman et al. www.pnas.org/cgi/content/short/0812446106 22 of 28 Table S6. Numbers of lateral line neuromasts and hair cells per sensory organ in controls, miR-15a (MMO-15a-1) and miR-18a (MMO-18a) zebrafish morphants #LLNMs(n) # HCs per NM (n) # HCs per UM (n) # HCs per SM (n)

Uninjected 5.4 Ϯ 0.6 (15) 4.0 Ϯ 1.0 (15) 21.5 Ϯ 8.9 (13) 24.5 Ϯ 8.6 (13) Stnd MO 4.7 Ϯ 1.2 (23) 3.36 Ϯ 0.9 (23) 21.8 Ϯ 5.4 (20) 26.1 Ϯ 6.1 (21) (All controls 5.0 ؎ 1.1 (38) 3.6 ؎ 0.8 (38) 21.6 ؎ 6.9 (33) 25.5 ؎ 7.1 (34

15a MO normal 3.7 Ϯ 2.0 (22)* 3.0 Ϯ 1.2 (22) 16.9 Ϯ 7.4 (22)* 16.9 Ϯ 7.7 (19)** 15a MO short tail 1.9 Ϯ 1.9 (9)** 1.8 Ϯ 1.8 (9)* 14.4 Ϯ 5.4 (7)* 17.0 Ϯ 6.7 (8)** 15a MO no tail 2.4 Ϯ 1.3 (7)** 2.1 Ϯ 0.8 (7)** 13.7 Ϯ 6.7 (7)* 16.7 Ϯ 8.5 (7)* **(All 15a morphants 3.1 ؎ 2.0 (38)** 2.6 ؎ 1.4 (38)** 15.0 ؎ 6.8 (36)** 16.9 ؎ 7.5 (34

**(All 18a morphants 3.4 ؎ 1.9 (28)** 2.1 ؎ 1.0 (28)** 18.0 ؎ 6.6 (25)* 19.9 ؎ 8.7 (24

Abbreviations: HCs, hair cells; LL, lateral line; MO, morpholino; NM, neuromasts; SM, saccular macula; Stnd MO, standard negative control morpholino; UM, utricular macula. Numbers shown are means Ϯ standard deviations. Statistical comparisons were computed with t-tests: two-sample assuming unequal variances: * P Ͻ 0.05 or ** P Ͻ 0.01 when compared with all controls. AB and ET20 genotypes were pooled for these analyses.

Friedman et al. www.pnas.org/cgi/content/short/0812446106 23 of 28 Table S7. Predicted targets for miR-15a expressed in P2 mouse inner ear sensory epithelia Target gene Description

PAPPA pregnancy-associated plasma protein A, pappalysin 1 LUZP1 leucine zipper protein 1 PVRL1 poliovirus receptor-related 1 (herpesvirus entry mediator C; nectin) TGIF2 TGFB-induced factor homeobox 2 CLOCK clock homolog (mouse) BCL2L2 BCL2-like 2 RAB10 RAB10, member RAS oncogene family PRKAR2A protein kinase, cAMP-dependent, regulatory, type II, alpha PSKH1 protein kinase H1 CCND2 D2 RASGEF1B RasGEF domain family, member 1B USP14 specific peptidase 14 (tRNA-guanine transglycosylase) CDK5R1 cyclin-dependent kinase 5, regulatory subunit 1 (p35) CCNT2 cyclin T2 ACTR2 ARP2 actin-related protein 2 homolog (yeast) MTMR4 myotubularin related protein 4 SLC12A2 solute carrier family 12 (sodium/potassium/chloride transporters), member 2; Nkcc1; mBSC2 TLE4 transducin-like enhancer of split 4 (E(sp1) homolog, Drosophila) PAFAH1B1 platelet-activating factor acetylhydrolase, isoform Ib, alpha subunit 45kDa GORASP2 golgi reassembly stacking protein 2, 55kDa FBXO33 F-box protein 33 E2F7 E2F transcription factor 7 WEE1 WEE1 homolog (S. pombe) RYBP RING1 and YY1 binding protein LMAN2L , mannose-binding 2-like EZH1 enhancer of zeste homolog 1 (Drosophila) SPRY4 sprouty homolog 4 (Drosophila) SH3GL2 SH3-domain GRB2-like 2 BTG2 BTG family, member 2 CCNE1 cyclin E1 ITPR1 inositol 1,4,5-triphosphate receptor, type 1 CLDN12 claudin 12 VAMP1 vesicle-associated membrane protein 1 (synaptobrevin 1) PISD phosphatidylserine decarboxylase GHR growth hormone receptor YWHAQ tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide NAV1 neuron navigator 1 LRIG1 leucine-rich repeats and immunoglobulin-like domains 1 SOCS6 suppressor of cytokine signaling 6 PDCD4 programmed cell death 4 (neoplastic transformation inhibitor) SCOC short coiled-coil protein DMTF1 cyclin D binding myb-like transcription factor 1 MAPK3 mitogen-activated protein kinase 3 PRDM4 PR domain containing 4 SMYD5 SMYD family member 5 OGT O-linked N-acetylglucosamine (GlcNAc) (UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase) EIF2C4 eukaryotic translation initiation factor 2C, 4 RNF111 ring finger protein 111 BTRC beta-transducin repeat containing RAP2C RAP2C, member of RAS oncogene family LYPLA2 lysophospholipase II PEX5 peroxisomal biogenesis factor 5 GLUD1 glutamate dehydrogenase 1 HOXA3 homeobox A3 ZSWIM3 zinc finger, SWIM-type containing 3 PPP6C protein phosphatase 6, catalytic subunit ELL elongation factor RNA polymerase II HDGF hepatoma-derived growth factor (high-mobility group protein 1-like) TBPL1 TBP-like 1 CUL2 cullin 2 GGA3 golgi associated, gamma adaptin ear containing, ARF binding protein 3 RASL12 RAS-like, family 12 SMARCD2 SWI/SNF related, matrix associated, actin dependent regulator of , subfamily d, member 2 LRRN3 leucine rich repeat neuronal 3

Friedman et al. www.pnas.org/cgi/content/short/0812446106 24 of 28 Target gene Description

HIRA HIR histone cell cycle regulation defective homolog A (S. cerevisiae) CHORDC1 and histidine-rich domain (CHORD)-containing 1 RNF138 ring finger protein 138 RAD23B RAD23 homolog B (S. cerevisiae) SLC20A2 solute carrier family 20 (phosphate transporter), member 2 STX1A syntaxin 1A (brain) RECK reversion-inducing-cysteine-rich protein with kazal motifs EPHA7 EPH receptor A7 KPNA4 karyopherin alpha 4 ( alpha 3) RBM6 RNA binding motif protein 6 SGK serum/glucocorticoid regulated kinase PLEKHC1 pleckstrin domain containing, family C (with FERM domain) member 1 PCBP4 poly(rC) binding protein 4 PLXNA2 plexin A2 OMG oligodendrocyte myelin glycoprotein ENAH enabled homolog (Drosophila) UBE4B ubiquitination factor E4B (UFD2 homolog, yeast) CBX4 chromobox homolog 4 (Pc class homolog, Drosophila) RANBP3 RAN binding protein 3 VAT1 vesicle amine transport protein 1 homolog (T. californica) LRP6 low density lipoprotein receptor-related protein 6 PSME3 (prosome, macropain) activator subunit 3 (PA28 gamma; Ki) VAV2 vav 2 oncogene SUPT16H suppressor of Ty 16 homolog (S. cerevisiae) LATS2 LATS, large tumor suppressor, homolog 2 (Drosophila) PCDHA1 protocadherin alpha 1 MMD monocyte to macrophage differentiation-associated PPM1D protein phosphatase 1D magnesium-dependent, delta isoform CHEK1 CHK1 checkpoint homolog (S. pombe) POLR3F polymerase (RNA) III (DNA directed) polypeptide F, 39 kDa XAB1 XPA binding protein 1, GTPase DYRK1B dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1B TAF15 TAF15 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 68kDa PBX3 pre-B-cell leukemia homeobox 3 RARB retinoic acid receptor, beta AKT3 v-akt murine thymoma viral oncogene homolog 3 (protein kinase B, gamma) ARHGAP20 Rho GTPase activating protein 20 B4GALT1 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1 BDNF brain-derived neurotrophic factor CDC42EP2 CDC42 effector protein (Rho GTPase binding) 2 ACSL1 acyl-CoA synthetase long-chain family member 1 ACSL4 acyl-CoA synthetase long-chain family member 4 ARHGAP12 Rho GTPase activating protein 12 APP amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease) ALS2CR2 amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 2 CD164 CD164 molecule, sialomucin DLL4 delta-like 4 (Drosophila) ABCG4 ATP-binding cassette, sub-family G (WHITE), member 4 BCL2 B-cell CLL/lymphoma 2 DHDDS dehydrodolichyl diphosphate synthase ELMO2 engulfment and cell motility 2

Predicted miR-15a targets selected for further study labeled in bold. The predicted targets are sorted from high to low probabilities, according to PicTar scores.

Friedman et al. www.pnas.org/cgi/content/short/0812446106 25 of 28 Table S8. Predicted targets for miR-18a expressed in P2 mouse inner ear sensory epithelia Target gene Description

TRIM2 tripartite motif-containing 2 NEDD9 neural precursor cell expressed, developmentally down-regulated 9 CDC42 cell division cycle 42 (GTP binding protein, 25kDa) PTGFRN prostaglandin F2 receptor negative regulator KCNA1 potassium voltage-gated channel, shaker-related subfamily, member 1 (episodic ataxia with myokymia) SON SON DNA binding protein SIM2 single-minded homolog 2 (Drosophila) BTG3 BTG family, member 3 DPP10 dipeptidyl-peptidase 10 FBXO34 F-box protein 34 BHLHB5 basic helix-loop-helix domain containing, class B, 5 HSF2 heat shock transcription factor 2 HIF1A hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor) CTGF connective tissue growth factor IGF1 insulin-like growth factor 1 (somatomedin C) SOCS5 suppressor of cytokine signaling 5 AEBP2 AE binding protein 2 IRF2 interferon regulatory factor 2 RAB5C RAB5C, member RAS oncogene family

The predicted targets are sorted from high to low probabilities, according to PicTar scores.

Friedman et al. www.pnas.org/cgi/content/short/0812446106 26 of 28 Table S9. Predicted targets for miR-30b expressed in P2 mouse inner ear sensory epithelia Target gene Description

MKRN3 makorin, ring finger protein, 3 EED embryonic ectoderm development NEDD4 neural precursor cell expressed, developmentally down-regulated 4 PTGFRN prostaglandin F2 receptor negative regulator GALNT7 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7 (GalNAc-T7) GNAI2 guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 2 RHEBL1 Ras homolog enriched in brain like 1 PIK3R2 phosphoinositide-3-kinase, regulatory subunit 2 (p85 beta) SNAI1 snail homolog 1 (Drosophila) GPR124 G protein-coupled receptor 124 NEFL neurofilament, light polypeptide 68kDa GFPT2 -fructose-6-phosphate transaminase 2 SMARCD2 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 2 DDIT4 DNA-damage-inducible transcript 4 GLCCI1 glucocorticoid induced transcript 1 CADPS Ca2ϩ-dependent secretion activator FBXO34 F-box protein 34 PHTF2 putative homeodomain transcription factor 2 TIA1 TIA1 cytotoxic granule-associated RNA binding protein GALNT3 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3) SYNGR3 synaptogyrin 3 NDEL1 nudE nuclear distribution gene E homolog (A. nidulans)-like 1 GALNT2 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) NAV1 neuron navigator 1 ARHGEF6 Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6 EDNRA endothelin receptor type A HERC2 hect domain and RLD 2 GALNT1 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1) STAG2 stromal antigen 2 DPYSL2 dihydropyrimidinase-like 2 ACVR1 activin A receptor, type I TMEFF1 transmembrane protein with EGF-like and two follistatin-like domains 1 CPEB3 cytoplasmic polyadenylation element binding protein 3 MAT2A methionine adenosyltransferase II, alpha STC1 stanniocalcin 1 ELL elongation factor RNA polymerase II SSX2IP synovial sarcoma, X breakpoint 2 interacting protein KCTD5 potassium channel tetramerisation domain containing 5 MAML1 mastermind-like 1 (Drosophila) ELOVL5 ELOVL family member 5, elongation of long chain fatty acids (FEN1/Elo2, SUR4/Elo3-like, yeast) TIMP3 TIMP metallopeptidase inhibitor 3 (Sorsby fundus dystrophy, pseudoinflammatory) GJA1 gap junction protein, alpha 1, 43kDa MMD monocyte to macrophage differentiation-associated ARID4B AT rich interactive domain 4B (RBP1-like) EAF1 ELL associated factor 1 BCL2L11 BCL2-like 11 ( facilitator) PIGA phosphatidylinositol glycan anchor biosynthesis, class A (paroxysmal nocturnal hemoglobinuria) RARG†‡ retinoic acid receptor, gamma RARB†‡ retinoic acid receptor, beta ATP2B2† ATPase, Caϩϩ transporting, plasma membrane 2 PAX3†‡ paired box gene 3, Waardenburg syndrome 1 HOXA1† homeobox A1 EDNRB† endothelin receptor type B JAG2† jagged 2 DMD† dystrophin, muscular dystrophy, Duchenne and Becker types BDNF† brain-derived neurotrophic factor

The predicted targets are sorted from high to low probabilities, according to PicTar scores. †Predicted by only 2 target prediction algorithms, but also linked with hereditary hearing loss in mice. All other targets are predicted by 3 algorithms: TargetScan, miRanda and PicTar. ‡Two binding sites with high scores.

Friedman et al. www.pnas.org/cgi/content/short/0812446106 27 of 28 Table S10. Predicted targets for miR-99a expressed in P2 mouse inner ear sensory epithelia Target gene Description

ADCY1 adenylate cyclase 1 (brain) MBNL1 muscleblind-like (Drosophila) HS3ST3B1 heparan sulfate (glucosamine) 3-O-sulfotransferase 3B1 BAZ2A bromodomain adjacent to zinc finger domain, 2A HS3ST2 heparan sulfate (glucosamine) 3-O-sulfotransferase 2 CTDSPL CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase-like SMARCA5 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 5 FGFR3 fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism) ZZEF1 zinc finger, ZZ-type with EF-hand domain 1 EIF2C2 eukaryotic translation initiation factor 2C, 2 FZD8 frizzled homolog 8 (Drosophila) MTMR3 myotubularin related protein 3 ICMT isoprenylcysteine carboxyl methyltransferase

The predicted targets are sorted from high to low probabilities, according to PicTar scores.

Friedman et al. www.pnas.org/cgi/content/short/0812446106 28 of 28