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Supplementary material
Fig. S1. Misorientation and dysmorphology of the hair bundles and aberrant position of the
kinocilium are observed in the aberrantly attached Nectin-3–/– hair cells (HCs) at P5. (A and
B) Misorientation and dysmorphology of the hair bundles and abnormal positioning of the
kinocilium in the aberrantly attached HCs in the same and different rows. F-Actin and γ-tubulin
were doubly stained in the basal turn of the cochlea at P5. Scale bars, 5 µm.
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Fig. S2. Consecutive sectional transmission electron microscopic images of HCs in Nectin-3–
/–
mice at P1. (A) Lower magnification image of the image shown in Fig. 2C. (B) A consecutive
image 0.5 µm apart from A. Note that SC2 locates between HC2 and HC3. HC, hair cell; SC,
supporting cell. Arrows indicate the hair bundles on HCs. Scale bars, 10 µm.
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Fig. S3. Phenotypes of HCs do not apparently change in Nectin-3–/– saccules. (A) Double
staining of Nectin-1, -2 or -3 with F-actin in Nectin-3+/– saccules at P1. (B) Double staining of
Nectin-3 with F-actin in Nectin-3+/– and Nectin-3–/– saccules at P1. Arrows indicate deviation of
hair bundles, while the dotted lines indicate the striolar reversal zone. Scale bars, 5 µm (A) and
10 µm (B).
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Fig. S4. Summary of the position of the kinocilium in HCs at E16.5. (A) The position of the
kinocilium is shown in red. Data of Nectin-3+/– (n = 30, apical turn; n = 30, middle turn) and
Nectin-3–/– (n = 60, apical turn; n = 60, middle turn) are shown. (B) The results shown in (A)
were divided into three groups: two attached HCs in the same rows (upper); two attached HCs in
different rows (apicolateral and basomedial HCs) (middle); and two attached HCs in different
rows (basolateral and apicomedial HCs) (lower). Data are for n = 20 each.
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Fig. S5. Misorientation and dysmorphology of the hair bundles and aberrant position of the
kinocilium are observed in the aberrantly attached Nectin-1–/– HCs at P1. (A) Misorientation
and dysmorphology of the hair bundles and abnormal positioning of the kinocilium in the
aberrantly attached HCs. F-Actin and γ-tubulin were doubly stained in the basal turn of the
cochlea. (B) Lack of the concentration of the immunofluorescence signals for nectins at the
contact sites between the aberrantly attached HCs. F-Actin and either Nectin-2 or -3 were doubly
stained in the apical-middle turn of the cochlea. Note that at the contact sites between the
aberrantly attached HCs, the signal for Nectin-2 is not concentrated, while that for Nectin-3 is not
observed. Arrows indicate the contact sites between the aberrantly attached HCs. Scale bars, 10
µm.
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Fig. S6. Extension of the AJC and ABP components toward the basal side at the aberrantly
attached sites. The fluorescence intensity of the x-z sectional images of the Nectin-3–/– auditory
epithelial cells used in Fig. 7 was measured to show the localisation of the AJC (A) and ABP (B)
components at the boundary between the aberrantly attached HCs (brown dotted line) and at the
boundary between HCs and supporting cells (SCs) (green dotted line). The x-z sectional image
(left) and the quantification of the fluorescence intensity (right) are shown. The x axis of the
graph represents the distance from the apical to the basal side along the dotted line and the y axis
of the graph represents the fluorescence intensity. Scale bars, 5 µm.
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Fig. S7. Localisation of Pals1 and Par-3 changes in the aberrantly attached Nectin-3–/– HCs
at P1. Triple staining of Pals1 (A) and Par-3 (B) with F-actin and Nectin-1 in the apical-middle
turn of the cochlea. The dotted lines in the optical x–y sectional views in the upper rows
correspond to the sites of the x–z sections in the lower rows. Arrows indicate the position of the
concentration and extension of the signal for Nectin-1 at the boundary between the aberrantly
attached HCs. Arrowheads indicate the position of the boundary between HCs and SCs. Scale
bars, 10 µm.
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