Fig 1: Gpsm2 mutation leads to hypoplasia of the corpus callosum and affects growth cone outgrowth.(a) Hematoxylin staining of coronal sections from P6 Gpsm2Emx1 control (upper panel) and cKO brains (lower panel) at a caudal levels. Note the abscence of corpus callossum (CC) in the cKO mouse (inset). Scale bar, 1 mm, n=4 independent experiments. (b,c) In cultured hippocampal neurons at DIV2, N-cadherin-dependent outgrowth was reduced in Gpsm2 cKOs, but not in Gnai3 cKOs (b). The reduction in outgrowth was maintained on a laminin substrate in Gpsm2 cKOs (c). Data from three to six independent experiments are presented as whisker box plots (min/max) (n=number of growth cones). ***P<0.001 with unpaired Student’s t-test. (d) Images from three time points (0, 15 and 30 min) of a time-lapse movie from 2 DIV control and Gpsm2 cKO hippocampal neurons plated on N-cadherin-coated glass and showing the difference in distance covered (dotted lines and double-headed arrows). Scale bar=10 μm. (e) Quantification of the average number of pauses (±s.e.m.) during a 30 min period of growth cones from control (n=31 neurons) compared with Gpsm2 cKO (n=51 neurons), from four independent experiments. The Gpsm2 cKO growth cones pause more than controls. ***P<0.001, *P<0.05 with unpaired Student’s t-test. (f) Cumulated distance covered by control and Gpsm2 cKO growth cones over a 30 min period. Some pauses are indicated with blue arrows.
Fig 2: Gpsm2 and Gnai3 mutations inhibit stereocilia elongation.(a–c) SEM of cochlear inner hair cells (IHC) from controls (a), Gpsm2 (b) or Gnai3 cKOs (c) in P5 mouse. The kinocilium is indicated with yellow stars. In Gpsm2 (b) and Gnai3 (c) cKOs, the staircase pattern is almost absent with stereocilia of similar length and width. Lateral links between adjacent stereocilia are preserved (magenta arrows, insets). Scale bars, 1 μm. (d,e) Quantifications at P5 show reduced length of IHC tallest stereocilia and supernumerary stereocilia in both Gpsm2 and Gnai3 cKOs. Quantifications are presented as whisker box plots (min/max), *P<0.05; ***P<0.001 with one-way ANOVA (post hoc Bonferonni’s test). (f–h) SEM of basal cochlear IHC from controls (f), Gpsm2 (g) or Gnai3 cKOs (h) in P21 mouse. In Gpsm2 cKOs, stereocilia are short, typically with more than four rows and severely reduced staircase pattern. A similar, but weaker phenotype was observed in Gnai3 cKOs, with occasional longer stereocilia in a bundle with overall shorter stereocilia (h, magenta brackets). Scale bars, 1 μm. (i,j) Quantifications at P21 are consistent with the above illustrations with a severely reduced length of IHC stereocilia and supernumerary stereocilia in Gpsm2 cKOs, and a similar but milder phenotype in Gnai3 cKOs. Quantifications are presented as whisker box plots (min/max), *P<0.05; ***P<0.001 with one-way ANOVA (post hoc Bonferonni’s test). (k) 3D rendering of the surface of a control (top) and a Pou4f3-Gpsm2 mutant (bottom) at P8. Mutants exhibit supernumerary rows of abnormally short stereocilia, sometimes split or fragmented (yellow asterisks). Scale bars, 4 μm. (l) SEM of postnatal cochlear explants treated or not for 8 days (DIV8) with 100 ng ml−1 PTX. In the PTX-treated samples (bottom panels), the stereocilia are shorter than in controls (upper pannels), with similar widths. Scale bars, 1 μm. (m) Quantification of the length of the tallest stereocilia cultures treated with PTX (blue line=mean). n=34 (control) and 76 stereocilia (PTX). Cultures repeated three times. ***P<0.0001 with unpaired Student’s t-test.
Fig 3: Gpsm2 and Gαi3 depend upon myosin 15 to reach stereocilia tips but not for their targeting at HC apical membrane.(a,b) Immunocytochemistry for Gpsm2 (a) or Gαi3 (b) shows both protein localization at the tips of stereocilia (green asterisks), and at the apical membrane of HC as a crescent-shape (yellow dashed-line) from controls of shaker 2 (sh2/+). Homozygous mutation (sh2/sh2) leads to a loss of Gpsm2 and Gαi3 stereocilia tip staining, whereas the apical crescent is maintained. (c) Immunocytochemistry for Gpsm2 (green) shows protein localization at the tips of stereocilia in IHCs from controls of Gnai3, but absent from Gnai3 cKO. (d) Reciprocally, immunocytochemistry for Gαi3 (green) shows protein localization at the tips of stereocilia in IHCs from controls of Gpsm2, but absent from Gpsm2 cKO. Scale bars, 4 μm. The immunostainings were repeated four times.
Fig 4: Gpsm2 and Gnai3 mutations affect cochlear and vestibular function.(a,b) Hearing tests on 4-week-old mice reveal severe threshold increases in Gpsm2 cKO (a), compared with high frequency loss only in Gnai3 cKOs (b). Arrow in a indicates ABR thresholds exceeding the maximum testable intensity. Mean±s.d. click-evoked ABR (click-ABR) and tone-burst-evoked ABR (f-ABR). Mean threshold values (in dB SPL) of click-ABR of control mice are shown above corresponding bars. ***P<0.001 (Grey shaded area: P<0.05) by two-way ANOVA (post hoc Bonferonni's multi comparisons test). f-ABR: Control (Ctr) and cKO, n=8 ears from eight mice click-ABR: Control and cKOs: n=16 ears from eight mice. (c,d) Left panel: Gpsm2 cKOs (green traces) display increased circling activity in a representative open-field during the first 30 s and at the end of the track (10 min) compared with control littermates, whereas Gnai3 cKOs (magenta traces) are unaffected (right panel). (e,f) Gpsm2 cKOs mice cover more distance and rotate more than Gnai3 cKOs mice (each circle is an individual mouse). Open white circles are controls. (g) Top: heat map of force swim test occupancy for control and Gpsm2 cKOs. Bottom: during the 2 min test, Gpsm2 cKO mice showed less immobility and more body axis rotation compared with controls. (h) SEM of the surface view of the macula of the utricle of P11 mice in control (left) and Gpsm2 cKO (right). Stereocilia elongation in the mutant is dramatically reduced compared with control. Scale bars, 1 μm.
Fig 5: Gpsm2 and Gαi3 localization at stereocilia tips is dependent on myosin 15 and whirlin.(a–d) Immunocytochemistry for Gpsm2 (a,b) or Gαi3 (c,d) and staining with phalloidin (Ph, magenta) shows protein localization at the tips of stereocilia in IHCs from shaker 2 (sh2/+, a,c) and whirler (wi/+, b,d) control mice at P8. Homozygous mutations (sh2/sh2 and wi/wi) lead to a loss of Gpsm2 and Gαi3 stereocilia tip staining. The immunostainings were repeated four times. (e,f) Immunocytochemistry for myosin 15 (green) and staining with phalloidin (Ph, magenta) shows protein localization at tips of stereocilia in IHCs from controls of Gpsm2 (e) and Gnai3 (f) cKO P8 mice. Myosin 15 protein is still present at the tip of shortened stereocilia of both cKOs, though in reduced amounts. (g,h) Immunocytochemistry for whirlin (green) and staining with phalloidin (Ph, magenta) shows protein localization at tips of stereocilia in IHCs from controls of Gpsm2 (g) and Gnai3 (h) P8 mutant mice. Gpsm2 and Gnai3 mutation leads to a strong reduction of whirlin stereocilia staining. Scale bars, 4 μm. The immunostainings (e–h) were repeated six times and imaged blindly.
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