Supporting Information Friedman et al. 10.1073/pnas.0812446106 SI Results and Discussion intronic miR genes in these protein-coding genes. Because in General Phenotype of Dicer-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-gene 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 unpublished work).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 humans 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 encode 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 human 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 zebrafish and mice feeding problems (2). (6, 7), is included in a human deafness-related locus, 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 (brain-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.