Transcription

1 DOI: /ncb2819 Gαi3 / Actin / Acetylated Tubulin Gαi3 / Actin / Acetylated Tubulin a a Gαi3 a Actin Gαi3 WT Gαi3 WT Gαi3 WT b b Gαi3 b Actin Gαi3 KO Gαi3 KO Gαi3 KO # # Figure S1 Loss of protein expression in Gα i3 knockout. Surface views of cochleae from Gnα i3 wild-type (Gα i3 WT) (a a ) and Gnα i3 knockout mice (Gα i3 KO) (b b ). The strong acetylated tubulin between IHCs and OHC1 rows (b) is due to labeling of the inner pillar cell heads. Labeling of (green) is absent from the cochlear epithelium of the knockouts. IHC (#) and OHC (,,). Scale bar: 10 mm (a-b). 1

2 Figure S2 Acetylated tubulin and pericentrin labeling correlates after G- protein signaling disruption. (a-b ) Representative surface views of HCs from mpins Ctrl (a) and mpins KO mice (b) labeled with pericentrin (green), acetylated tubulin (red) and phalloidin (blue), and the corresponding diagram on the right. Kinocilium and pericentrin coordinates were determined according to a grid applied to each HC as depicted on the HC schemes. The Y axis on the histograms correspond to the pericentrin position (x or y coordinates), the X axis correspond to the kinocilium position (x and y coordinates). Scale bar: 2 mm (a-b). (c,d ) Plots illustrating the correlation between the x and y coordinates from the base of the kinocilium and the pericentrin labeling in (c, c ) Gα i3 WT (n = 102 OHCs, n = 42 IHCs) and (d, d ) Gα i3 KO (n = 108 OHCs, n = 35 IHCs) mice. (e,f ) Plots illustrating the correlation between the x and y coordinates in (e, e ) mpins Ctrl (n=77 OHCs, n=34 IHCs) and (f, f ) mpins KO (n=77 OHCs, n=33 IHCs) mice. (g,h ) Plots illustrating the correlation between the x and y coordinates in (g,g ) PTX-untreated (n=101 OHCs, n=30 IHCs) and (h, h ) PTX-treated (n=110 OHCs, n=47 IHCs) cochlear explants. 2

3 Figure S3 Generation of mpins conditional mutant and localisation of apical asymmetrical complexes in the organ of Corti. (a) Schematic representation of the mouse mpins gene targeting strategy. Exons 5, 6, and 7 of the gene were excised following Cre and Flpe-mediated recombination. Regions of homologous recombination are indicated by light gray shading. AseI (A) and SacI (S) and the locations of the probes (5, 3 ) used for Southern blotting analysis are indicated. (b) Southern blot of recombinant ES cells showing homologous integration. (c) PCR genotyping to detect wild-type (170 bp) and targeted (250 bp) alleles. Cre-mediated excision was confirmed in cochlea by the presence of a 300 bp product. (d) Western blotting analysis of the mpins protein (75 kda) in control and mpins cko inner ear. In the cko, the protein levels are strongly reduced. (e-f ) Surface views of cochleae from mpins controls (Ctrl, e-e ) and mpins conditional knockouts (mpins cko, f-f ) from P0/P1 mice processed for immunocytochemistry. Labeling of mpins (green) is absent from the cochlear epithelium of the conditional knockouts.(g,g ) Surface view (g) and Z-stack (g ) confocal cross-section showing the apical localization of Par-6b (green) compared to β-catenin (red). (g ) Lateral view of (g) at the level indicated by the dashed line. (h, h ) Gα i3 (green) is located on the distal-abneural side of the HCs, opposite Vangl2 (red). (h ) High magnification of the inset in h. (i, i ) Gα o (green) labeling reveals weak cytosolic staining in HCs and in the non-sensory region, but no asymmetrical accumulation. Hair bundles and cell junctions were labeled with phalloidin (blue). (j) Representative immunoblot of brain (expressing Gα o ) and liver (not expressing Gα o ) membranes using Gα o antibody. Purified Gα o1 protein served as a positive control. IHC (#) and OHC (,,). Scale bar: 10 mm (e-i). 3

4 Figure S4 Gα i inhibition leads to loss-of-cilium-like morphological defects in the hair bundle. (a) In cultures treated with 5 pg/ml PTX for 48 hours (T3) the distributions of the orientations follow a gradient of severity from OHC1 to OHC3. HCs numbers, out of 2 cochleae per condition, are as follows: PTX T3 (n = 65,68,64 respectively for OHC1, OHC2, OHC3) (b, b ) Vangl2 asymmetry (green) is maintained in mpins cko. (c-d ) In cultures treated with 5 pg/ml PTX for 24 hours (T1), we observe hair bundles exhibiting round (c, c ) or flat shapes (d, d ). (e-f ) The apkc expression domain is more extended at the membrane of HCs in cultures treated with high doses (1 ng/ml) of PTX (f f ) compared to untreated (Unt) cultures (e e ). Note the long cilium in PTX-treated cultures. (g-i) High magnifications of a representative HC from (e) and (f). (h, j) Circular histograms of the distribution of apkc staining in Unt and PTX-treated condition. Each histogram represent data from 30 HCs. Statistical analysis via a two-tailed t test (P< ). Error bars show s.e.m. Scale bars: 10 µm (in b, e-f), 2 µm (in b -d, g -i ). 4

5 Figure S5 Gα i3 is recruited asymmetrically prior to kinocilium migration. (a c ) Luminal surface views of a cochlear epithelium at E15.5 at different locations along the duct. In the apical region of the sensory epithelium, only a few scattered HCs (future IHCs) have started to undergo differentiation, as indicated by MyoVI expression (a, arrowheads) and an apical actin ring (a, arrowheads). (b-b4) In a more basal region of the cochlea, and in the distal-abneural OHC region, Gα i3 starts to accumulate centrally in a few differentiating cells (b, white arrow). In the absence of asymmetrical Gα i3 localization, the kinocilium does not migrate in these cells (b, b1, b2, white arrowhead). By comparison, a line of differentiated IHCs is present, expressing Gα i3 asymmetrically and showing a distal kinocilium (b, b3-b4, yellow arrow and arrowhead). The white lines indicate the relative midline of every HC. (c c ) Luminal surface views of the basal region of cochlear epithelia at E15.5. Gα i3 is asymmetrically localized at the distal-abneural side of IHCs but absent from the prospective OHC region. Insets: higher magnification view of IHCs (yellow arrow) and future OHCs (white arrow) from (c) revealing that the kinocilium is always localized distally in IHCs (yellow arrowhead) but not in future OHCs (white arrowhead). (d-e ) Luminal surface views of the mid-basal to apical region of cochlear epithelia at E16.5 labeled with apkc (d-d ) and Par-6b (e,e ). The proteins are not asymmetrically distributed. (fg) HC differentiation is completed by P0, with every HC expressing MyoVI (f, green), and Gα i3 is asymmetrically localized to the distal-abneural side along with the kinocilium (g and g1-g4, green). Scale bars: 10 µm (in a-f). (h-i ) The asymmetric distribution of mpins (h-h, green) and Par-6B (i, i ) are inverted across the striola in the P0 utricular epithelium (dashed line). β2- spectrin immunostaining labels the cuticular plate (red). Arrows indicate HC polarity with respect to the lateral (Lat) or medial (Med) side of the utricle. (j) Scatter plots of the localization of the kinocilium relative to the center of the Gα i3 expression domain reveals a strong association between Gα i3 and the kinocilium position in wild type (n = 218 OHCs and 68 IHCs), Vangl2 Lp/ Lp (n = 237 OHCs and 73 IHCs) and PTK7 mutants (n = 124 OHCs and 33 IHCs). The dashed line indicates a perfect correlation (r = 1). (k, k ) Ectopic HCs induced by Atoh1-IRES-GFP expression (green) in the non-sensory epithelium display asymmetric cortical apkc (red) localization opposite to the kinocilium side. The dashed line indicates the boundary between the sensory and nonsensory epithelium. (l) Schematic representation of (k). 5

6 Figure S6 Full scans 6