Figure 1. Loss of synaptic ribbons and piccolino from the AZs of RIBEYE-deficient IHCs.

(A) Simplified schematic representation of an IHC with the afferent and efferent connectivities. (B) Maximal projection of confocal sections from organs of Corti immunolabeled for CtBP2 and RIBEYE, present in the nuclei and the ribbons, respectively. The RBE^WT/WT staining (top row) shows small puncta in the outer hair cell (OHC) and IHC rows representing the synaptic ribbons, which are completely absent in the RBE^KO/KO hair cells (bottom row). Scale bar = 50 µm. Zoom into the IHC row (right column), emphasizes the complete disappearance of CtBP2-labeling at the basolateral part of RBE^KO/KO IHCs. Scale bar = 10 µm. (C) Maximal projection of confocal sections from organs of Corti co-labeled for the presynaptic marker and anchor of the ribbon, bassoon (left column), and the postsynaptic marker, PSD-95 (middle column), in RBE^WT/WT and RBE^KO/KO IHCs. The merged picture (right column) shows the juxtaposition of bassoon (magenta) with PSD-95 (green), indicating its presence both at RBE^WT/WT and ribbonless RBE^KO/KO IHC synapses. Scale bar = 1 µm. (D) Maximal projection of confocal sections from organs of Corti co-labeled for the presynaptic marker RIM2 (left column) and the postsynaptic marker PSD-95 (middle column). The merged picture (right column) shows the co-localization of RIM2 (magenta) with PSD-95 (green) meaning its presence at the ribbonless IHC pre-synapses (scale bar = 5 µm). (E) Maximal projection of confocal sections from organs of Corti co-labeled for piccolino, a specific short splice variant of piccolo found at ribbons of RBE^WT/WT IHC synapses (left column), co-labeled with PSD-95 (middle column). The merged pictures (right column) show PSD-95 (green) immunofluorescence lacking juxtaposed piccolino signal (magenta) in RBE^KO/KO (bottom row), indicating absence of piccolino from afferent synapses of mutant IHCs. The punctate labeling for piccolo, away from PSD-95, most likely represents labeling of piccolo at conventional efferent synapses (schematically shown in Figure 1A). Scale bar = 5 µm. (F) Quantification of the area of PSD-95 immunofluorescent spots. The PSD-95 spots are siginificantly bigger in the RBE^KO/KO IHCs (p<0.0001, Mann-Whitney-Wilcoxon test, n = 178 spots, N = 3 for RBE^KO/KO and n = 163 spots, N = 3 for RBE^WT/WT). Box plots show 10, 25, 50, 75 and 90^th percentiles with individual data points overlaid; means are shown as crosses.

Figure 1—figure supplement 1. Gene-dosage dependent expression of the RIBEYE at IHC AZs.

(A) Maximal projection of organs of Corti immunolabeled for the C-terminal domain of both CtBP2 and RIBEYE, present in the nuclei and the ribbons. The number of ribbons does not differ between RBE^WT/KO (n = 50 IHCs, N = 4) and RBE^WT/WT IHCs (n = 39 IHCs, N = 3). Scale bar = 10 µm. (B) Box plot highlighting significantly reduced immunofluorescence intensities for the ribbons in the heterozygous condition (n = 600 spots for 40 RBE^WT/KO IHCs, N = 3 and n = 411 spots for 29 RBE^WT/WT IHCs, N = 3, p<0.0001, Mann-Whitney-Wilcoxon test). Box plots show 10, 25, 50, 75 and 90^th percentiles with individual immunofluorescent spots overlaid; means are shown as crosses.