Egr2::cre mediated conditional ablation of dicer disrupts histogenesis of mammalian central auditory nuclei.

Histogenesis of the auditory system requires extensive molecular orchestration. Recently, Dicer1, an essential gene for generation of microRNAs, and miR-96 were shown to be important for development of the peripheral auditory system. Here, we investigated their role for the formation of the auditory brainstem. Egr2::Cre-mediated early embryonic ablation of Dicer1 caused severe disruption of auditory brainstem structures. In adult animals, the volume of the cochlear nucleus complex (CNC) was reduced by 73.5%. This decrease is in part attributed to the lack of the microneuronal shell. In contrast, fusiform cells, which similar to the granular cells of the microneural shell are derived from Egr2 positive cells, were still present. The volume reduction of the CNC was already present at birth (67.2% decrease). The superior olivary complex was also drastically affected in these mice. Nissl staining as well as Vglut1 and Calbindin 1 immunolabeling revealed that principal SOC nuclei such as the medial nucleus of the trapezoid body and the lateral superior olive were absent. Only choline acetyltransferase positive neurons of the olivocochlear bundle were observed as a densely packed cell group in the ventrolateral area of the SOC. Mid-embryonic ablation of Dicer1 in the ventral cochlear nucleus by Atoh7::Cre-mediated recombination resulted in normal formation of the cochlear nucleus complex, indicating an early embryonic requirement of Dicer1. Quantitative RT-PCR analysis of miR-96 demonstrated low expression in the embryonic brainstem and up-regulation thereafter, suggesting that other microRNAs are required for proper histogenesis of the auditory brainstem. Together our data identify a critical role of Dicer activity during embryonic development of the auditory brainstem

Cryg-immunreactivity in the rat, mouse, and gerbil SOC at P4.

<p>All image series show Cryg-ir (α-Cryg) in the first, VGluT1-ir (α-VGluT1) in the second, and an overlay of both immunoreactivities in the third column. This order applies also to subsequent Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161140#pone.0161140.g003" target="_blank">3</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161140#pone.0161140.g005" target="_blank">5</a>. (<b>A)</b> Cryg-ir is clearly seen in the MNTB and in the ventral acoustic stria of rat. (<b>B)</b> The MSO and a subpopulation of the LSO show also prominent Cryg-ir. (<b>C,D)</b> The mouse displays a weaker labeling in the SOC. (<b>E-F)</b> MNTB, LSO and MSO of the gerbil show no Cryg-ir above background. MNTB, medial nucleus of the trapezoid body; MSO, medial superior olive; LSO; lateral superior olive. MNTB, medial nucleus of the trapezoid body, designated by a asterisk MSO, medial superior olive, designated by a star; () LSO; lateral superior olive, designated by a diamond. The abbreviations and symbols also apply to subsequent figures. Dorsal is up and medial to the left. n = 3, scale bar is 100 μm.</p

Altered auditory brainstem responses (ABR) in <i>Crygn</i>^<i>Egr2</i> mice.

<p>(<b>A</b>) Auditory brainstem response (ABR) thresholds for click, noise burst, and pure tone stimuli for control (white) and <i>Crygn</i>^<i>Egr2</i> mice (red) (n = 4 and 5 mice for controls and <i>Crygn</i>^<i>Egr2</i>, respectively). No difference was observed for ABR thresholds in response to click stimuli (left) or noise burst stimuli (middle panel, n.s., not significant, p > 0.05). Threshold for pure tone stimuli were slightly but significantly better in <i>Crygn</i>^<i>Egr2</i> mice (<i>p</i> = 0.0279, 2-way ANOVA comparing the genotype). (<b>B</b>) Outer hair cell function measured by distortion product otoacoustic emission (DPOAE) growth function (left), maximal signal strength (middle, max. amplitude) and threshold for the 2f1-f2 distortion product (right). <i>Crygn</i>^<i>Egr2</i> mice had slightly improved 2f1-f2 distortion products indicated by increased amplitudes at 45 dB SPL stimulation (left, <i>p</i> = 0.1048, 2-way ANOVA) and small though non-significant improvements of DPOAE thresholds (right, <i>p</i> = 0.1013, 2-way ANOVA). This indicated intact outer hair cells and cochlear amplification in both genotypes. Arrows in left panel illustrate how threshold and amplitude were determined from the individual growth functions. (<b>C</b>) Processing of fast temporal modulation was measured by auditory steady state responses (ASSR) to amplitude modulated stimuli of increasing modulation speed (left, modulation frequency), as function of the modulation index (middle, modulation depth in %,) and for increasing level of the carrier (right, -20 to 60 dB hearing level, HL). For both genotypes, responses dropped for stimulation speeds above 1,024 Hz and were lost for faster modulation (2,048 Hz). Detection thresholds for modulated stimuli were at ca. 3–4% modulation, and response strength increased with carrier level. There was a tendency for <i>Crygn</i>^<i>Egr2</i> signals to level off at lower signal strength than the control (right panel, 55–60 dB hearing level), though this was not statistically significant. Insets schematically illustrate the used stimuli. (<b>D</b>) Changes of average peak amplitude for ABR wave I to IV (defined as peak to peak amplitudes, illustrated in the inset showing an example of an ABR recording with marked negative (n) and positive (p) peaks of either wave. The growth function shows significantly increased ABR amplitudes at wave IV at higher stimulus levels (> 50 dB, <i>p</i> < 0.001, 2-way ANOVA). Growth functions for ABR wave I and II amplitudes also were significantly changed, corroborating the results from the slightly improved ABR thresholds and DPOAE functions in <i>Crygn</i>^<i>Egr2</i> mice. Data for controls are shown as open bars, symbols and black lines, data for <i>Crygn</i>^<i>Egr2</i> mice are shown in red. Data represent mean and standard deviation (A,B) or standard error of the mean (C,D). The number of measured ears is indicated in each panel. n.s., not significant, *: p < 0.05, ***: p < 0.001, ****: p < 0.0001 in Holm-Sidak's multiple comparisons test. (*) indicates statistical results from uncorrected single comparison with <i>p</i> < 0.05 in 2-sided t-test.</p

Cryg-ir in the mouse SOC at P25.

<p>All three antibodies against crystallins, i.e. Cryg (<b>A,B</b>), Crygd/e (<b>C,D</b>) or Crygn (<b>E,F</b>) gave no signals above background in the MNTB, the LSO and the MSO of mice aged P25. Dorsal is up and medial to the left. n = 3, scale bar is 100 μm.</p

Immunohistochemical and anatomical analysis of the SOC in <i>Crygn</i>^<i>Egr2</i> mice.

<p>(<b>A</b>) Normal gross anatomy of the SOC in <i>Crygn</i>^<i>Egr2</i> mice. GlyT2 and VGluT1 immunoreactivity in coronal brainstem sections of P25 <i>Crygn</i>^<i>Egr2</i> mice and control litter mates indicate normal gross morphology of SOC nuclei. (<b>B</b>) Structural alterations in nuclei of the SOC. In <i>Crygn</i>^<i>Egr2</i> mice, the volumes of the LSO and MNTB were significantly decreased. The MNTB of <i>Crygn</i>^<i>Egr2</i> mice displayed also lower cells number but normal cross sectional area of neurons. At P4, MNTB cell number was not affected by the genotype. Volume and cell number were analysed in Nissl-stained serial sections of the respective nucleus (6 nuclei from 3 animals/genotype). As a test for statistical significance, a two-tailed student’s <i>t</i>-test was used. Color coding (black: control control litter mice; red: <i>Crygn</i>^<i>Egr2</i> mice). Dorsal is up and medial to the left. * <i>p</i> ≤ 0.01; ***<i>p</i> ≤ 0.001.</p

Crygd/e-ir in the rat, mouse, and gerbil SOC at P4.

<p><b>(A)</b> Crygd/e labeling is observed in the rat MNTB and fibers of the acoustic stria. (<b>B)</b> LSO and MSO display moderate Cryge-ir. In the mouse, MNTB (<b>C</b>), LSO and MSO (<b>D</b>) are also labeled. (<b>E-F)</b> The gerbil MNTB, LSO and MSO show Crygd/e-ir similar to background. Dorsal is up and medial to the left. n = 3, scale bar is 100 μm.</p

Generation of a spatially restricted <i>Crygn</i> knockout mouse in the auditory hindbrain.

<p><b>(A)</b> Scheme of the knockout strategy, consisting of crossing a mouse line with a floxed allele of exon 2 of <i>Crygn</i> (<i>Crygn</i>:<i>tm1a</i> (EUCOMM)) and the <i>Egr2</i>::<i>Cre</i> driver line. Primers for probing recombination are depicted as black arrows. (<b>B)</b> Validation of the spatial ablation in the SOC. Left side: Genotyping of the floxed <i>Crygn</i> locus. In wt, a 524 bp long PCR product is amplified, whereas the mutant locus results in a 207 bp product. Right site: Confirmation of recombination in the SOC. Upon recombination, a 604 bp long product is amplified. The non-recombined locus is 3,411 bp in length and not amplified under the PCR conditions used. (<b>C)</b> RNA <i>in situ hybridization</i> analysis in the MNTB. An RNA probe complementary to exon 2 yields only signals in MNTB section of control mice whereas no signal is observed in <i>Crygn</i>^<i>Egr2</i> mice. In contrast, an RNA probe complementary to exons 2–4 still yields signals in the MNTB of <i>Crygn</i>^<i>Egr2</i> mice. This indicates transcription of the truncated <i>Crygn</i> gene. Scale bar is 200 μm.</p

Crygn-ir in the rat, mouse, and gerbil SOC at P4.

<p>Crygn clearly labels the MNTB (<b>A</b>), the LSO and the MSO (<b>B</b>) of rat. (<b>C)</b> The mouse MNTB shows a moderate labeling and the LSO and MSO a weak immunoreactivity (<b>D</b>). (<b>E)</b> Gerbils show a similar pattern of Crygn-ir as the mouse with the MNTB being strongest labeled. Dorsal is up and medial to the left. n = 3, scale bar is 100 μm.</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