Malformed CNC in P0 <i>Dicer1^Egr2</i> mice.

<p><b><i>A,B,</i></b> Nissl stained sections of the cochlear nucleus complex of wild type (<b><i>A</i></b>) or <i>Dicer1^Egr2</i> mice aged P0 (<b><i>B</i></b>) at anterior (<b><i>A1,B1</i></b>), middle (<b><i>A2,B2</i></b>) or posterior (<b><i>A3,B3</i></b>) levels. All three subnuclei, i.e. the AVCN, PVCN, and DCN display a reduced size. AVCN, anteroventral cochlear nucleus; DCN, dorsal cochlear nucleus; PVCN, posterior ventral cochlear nucleus. Dorsal is up, lateral is to the right.</p

Severe disruption of the SOC in <i>Dicer1^Egr2</i> mice.

<p><b><i>A–F</i></b> Vglut1 immunoreactivity (<b><i>A,B</i></b>), Calb1 immunoreactivity (<b><i>C,D</i></b>), or Nissl stained sections (<b><i>E,F</i></b>) in the superior olivary complex of adult (P22– P30) wild type (<b><i>A,C,E</i></b>) or <i>Dicer1^Egr2</i> mice (<b><i>B,D,F</i></b>). Only weak Vglut1 staining is observed in the SOC of <i>Dicer1^Egr2</i> mice and principal nuclei such as the MNTB or the U-shaped LSO cell group are absent in these animals. Calb1 labeling is observed in somata of MNTB neurons and in the neuropil of the LSO, MSO, and SPN in wild type mice. No labeling is observed in <i>Dicer1^Egr2</i> mice. In Nissl stained sections, the cell groups of the MNTB and LSO are recognizable in control mice, but not in <i>Dicer1^Egr2</i> mice. The cell group at the ventral part in <i>Dicer1^Egr2</i> mice corresponds to the olivocochlear neurons. <b><i>G–J</i></b> Vglut1 immunoreactivity (<b><i>G,H</i></b>), or Nissl stained sections (<b><i>I,J</i></b>) in the superior olivary complex of P0 wild type (<b><i>G,I</i></b>) or <i>Dicer1^Egr2</i> mice (<b><i>H,J</i></b>). Vglut1 staining is restricted to the olivocochlear neurons in the SOC of <i>Dicer1^Egr2</i> mice, and principal nuclei such as the MNTB or the LSO cell group are absent in these animals. In Nissl stained sections, the cell groups of the MNTB and LSO are recognizable in wild type mice, but not in <i>Dicer1^Egr2</i> mice. The cell group at the ventral part in <i>Dicer1^Egr2</i> mice corresponds to the olivocochlear neurons. LSO, lateral superior olive; MNTB, medial nucleus of the trapezoid body; MSO, medial superior olive; OCN, olivocochlear neurons; SPN, superior paraolivary nucleus. Dorsal is up, lateral is to the right.</p

Presence of the olivocochlear neurons in <i>Dicer1^Egr2</i> mice.

<p><b><i>A,B</i></b> ChAT immunolabeling was detected throughout the LSO of wild type mice (<b><i>A</i></b>), whereas in <i>Dicer1^Egr2</i> mice, ChAT positive cells were restricted to a densely packed ventrolateral cell group (<b><i>B</i></b>). (<b><i>C–F</i></b>) The same SOC area was labeled by Vglut1, as revealed in the overlay (<b><i>E–F</i></b>). Two animals aged P15–P20 were analyzed per genotype. LSO, lateral superior olive; OCN, olivocochlear neurons. Dorsal is up, lateral is to the right.</p

Reduced size of the nuclei of the lateral lemniscus is attributable to secondary effects of <i>Dicer1</i> loss.

<p><b><i>A–D</i></b>, <i>Egr2::Cre</i> mice were crossed with <i>ROSA26R</i> mice, resulting in expression of β-galactosidase after Cre-mediated recombination. Only few X-gal positive cells were detected in the nuclei of the lateral lemniscus in the overview (<b><i>A,B</i></b>) or at high magnification (<b><i>C,D</i></b>). <b><i>E,F,</i></b> Vglut1 labeling of the nuclei of the lateral lemniscus in adult (P29, 2 animals per genotype) wild type (<b><i>E</i></b>) or <i>Dicer1^Egr2</i> mice (<b><i>F</i></b>) revealed reduced size of the nuclei in <i>Dicer1^Egr2</i> mice. <b><i>G,H</i></b>, Diminished size of the nuclei in <i>Dicer1^Egr2</i> mice (<b><i>H</i></b>) was confirmed by Nissl staining (P29, 2 animals per genotype) (<b><i>G</i></b>). NLL, nuclei of the lateral lemniscus. Dorsal is up, lateral is to the right.</p

Quantitative RT-PCR analysis of miR-96 in mouse brainstem.

<p>qRT-PCR analysis reveals that expression of miR-96 increases in the mouse brainstem with age, when comparing E18, P0 and P25. At least 3 biological repeats were done in triplicates (<i>t</i>-test,* P<0.05, ** P<0.01).</p

Smaller size of <i>Dicer1^Egr2</i> mice. Compared to wild type littermates, <i>Dicer1^Egr2</i> mice (black arrows) were smaller and the difference in size increased with age.

<p>P, postnatal day.</p

Normal CNC in adult <i>Dicer1^Atoh7</i> mice. <i>A,B,</i>

<p>Nissl stained CNC sections of adult (P42, 3 animals) wild type (<b><i>A</i></b>) or <i>Dicer1^Atoh7</i> mice (P42, 3 animals) (<b><i>B</i></b>) at anterior (<b><i>A1,B1</i></b>), middle (<b><i>A2,B2</i></b>) or posterior (<b><i>A3,B3</i></b>) levels. No difference in shape or size was observed between wild type and <i>Dicer1^Atoh7</i> mice. <b><i>C,D</i></b>, Nissl stained sections revealed normal formation of the SOC in <i>Dicer1^Atoh7</i> mice. <b><i>E,</i></b> Genotyping PCR of DNA extracted from the CNC of indicated mouse lines. In homozygous <i>Dicer1^Atoh7</i> mice, two alleles were present: 429 bp (recombined allele) and 767 bp (floxed allele), whereas in the CNC of wild type mice, a 560 bp fragment was amplified corresponding to the wild type allele. <b><i>F,</i></b> RT-PCR analysis of mRNA extracted from the CNC of a wild type or <i>Dicer1^Atoh7</i> mouse. In wild type, only the full length sequence (1,500 bp) was amplified, whereas in <i>Dicer1^Atoh7</i> mice, also the truncated sequence (360 bp) was amplified. Data are representative examples of 2 to 3 biologically independent experiments. These data confirm Cre-mediated recombination in the AVCN. fl, floxed allele; rec, allele after Cre-mediated recombination; wt, wild type allele.</p

Malformed CNC in adult <i>Dicer1^Egr2</i> mice.

<p><b><i>A,B,</i></b> Vglut1 immunoreactivity in the cochlear nucleus complex of adult wild type (P22– P30) (<b><i>A</i></b>) or <i>Dicer1^Egr2</i> mice (<b><i>B</i></b>) at anterior (<b><i>A1,B1</i></b>), middle (<b><i>A2,B2</i></b>) or posterior levels (<b><i>A3,B3</i></b>). <b><i>C,D</i></b><i>,</i> Nissl staining of CNC sections of wild type (<b><i>C</i></b>) or <i>Dicer1^Egr2</i> mice (<b><i>D</i></b>) at anterior (<b><i>C1,D1</i></b>), middle (<b><i>C2,D2</i></b>) or posterior levels (<b><i>C3,D3</i></b>) levels. All three subnuclei, i.e. the AVCN, PVCN, and DCN display a reduced size. <b><i>E–H</i></b> Nissl stained sections of the DCN (<b><i>E,F</i></b>) reveal absence of the microneuronal shell (black arrow), but presence of fusiform cells (black arrows) (<b><i>G,H</i></b>) in <i>Dicer1^Egr2</i> mice. <b><i>I,</i></b> The volume of the CNC was determined at P0 or adult stage from Nissl stained serial sections. A significant decrease in volume of the CNC was observed in <i>Dicer1^Egr2</i> mice at both ages (2 animals per genotype, aged P0 or P22 and P30) (Mann-Whitney U-test, *, P<0.05). <b><i>J,</i></b> The relative decrease in volume in <i>Dicer1^Egr2</i> mice was similar between P0 and adult mice. AVCN, anteroventral cochlear nucleus; DCN, dorsal cochlear nucleus; PVCN, posterior ventral cochlear nucleus. Dorsal is up, lateral is to the right.</p