Fig 1.
Inner ear transduction efficiency of AAV-PHP.eB and AAV-DJ administered at the neonatal stage.
(A) P8 cochlea injected at P3 with AAV-PHP.eB through the round window membrane. The image on the left shows F-actin (red) and transduced cells expressing GFP (green) in maximum projection in the same cochlea. Upper panels on the right show representative close-ups of the sensory epithelium at the cochlear base, mid, and apex; the graph below shows the corresponding longitudinal transduction profile (fraction of transduced sensory cells as a function of position) along the cochlear axis, the latter being oriented from base, x = 0, to apex, x ≃ 5700 μm). IHC: Inner hair cells; OHC1-3: Outer hair cells from row 1–3. (B) P8 cochlea injected at P3 with AAV-DJ through the round window membrane. The image on the left shows F-actin (red) and transduced cells expressing GFP (green), in maximum projection in the same cochlea that was cut into two pieces. Panels on the right show representative close-ups of the sensory epithelium (base, mid and apex); the corresponding longitudinal transduction profile (shown below) was obtained by combining the partial profiles obtained in the respective cochlear fragments. Note that AAV-DJ also transduces the inner sulcus and mesenchymal cells. IPhC: Phalangeal cells; DC: Deiter cells; PC: pillar cells; IBC: inner border cells. Scale bar 200 μm.
Fig 2.
Inner ear diffusion of FastGreen administered at embryonic stage E13.5.
(A) Photo of an E13.5 embryo inside the uterine horn. Notice the capillary pointing towards the external ear, located bellow the ‘V’-shaped veins corresponding to the transverse and sigmoid sinus veins. (B-C) Micrographs of E13 mouse embryos after the inner ears were injected with FastGreen. (B) The injected (left) ear. (C) The contralateral ear, which shows a higher concentration of dye. Scale bar 1 mm.
Fig 3.
Inner ear transduction efficiency of AAV-PHP.eB administered at embryonic stages E13.5 and E14.5.
(A-B) Whole-mount P0 cochleas injected with AAV-PHB.eB::GFP at E13.5 (A) and at E14.5 (B). Images on the left show stitched and projected views of the whole-mount cochleas imaged by mosaic confocal microscopy. Inset images below show the transduced sensory cells in more detail. Scale bars in overview images, 200 μm, in the insets, 25 μm. The graphs on the right show representative longitudinal transduction rate profiles measured in the right and left cochleas of 3 pups injected at E13.5 (A), and 3 pups injected at E14.5 (B). See also S3 Fig, which contains additional transduction rate profiles for 3 pups injected at E13.5 and 3 pups injected at E14.5. IHC, inner hair cells, OHC1, OHC2, and OHC3, first, second and third row of outer hair cells, respectively.
Table 1.
Total numbers of GFP-positive hair cells per individual: AAV-PHP.eB injected at E13.5.
Table 2.
Total numbers of GFP-positive hair cells per individual: AAV-PHP.eB injected at E14.5.
Fig 4.
Inner ear transduction efficiency of AAV-PHP.eB administered at embryonic stages E15.5 and E16.5.
(A) Confocal views (maximum intensity projections) of the cochlear epithelium of mice subjected to unilateral injection of AAV-PHB.eB::GFP at E13.5, and imaged at E15.5 (upper panels) or at E16.5 (lower panels). Panels from left to right show different cochlear locations from base to apex. Staining for F-actin (red) and GFP (green). Scale bars 25 μm (upper panels) and 50 μm (lower panels). (B) Longitudinal transduction rate profiles observed in cochleas dissected at either E15.5 (upper panels) or E16.5 (lower panels) after unilateral injection of AAV-PHP.eB at E13.5. IHC, inner hair cells, OHC1, OHC2, and OHC3, first, second and third row of outer hair cells, respectively.
Fig 5.
Inner ear transduction efficiency of AAV-DJ administered at embryonic stages.
(A) Whole-mount P0 cochleas injected with 1.2μl AAV-DJ at E13.5, E14.5, E15.5, E16.5. Scale bar 200 μm. (B) Close-ups of the mid-basal, mid, and apical regions of P0 cochleas injected either at E15.5 (upper panels), or at E16.5 (lower panels). Scale bar 25 μm. (C) Longitudinal transduction rate profiles measured in the right and left cochleas of three different pups injected at E15.5 (pup 1) or at E16.5 (pups 2 and 3). IphC, phalangeal cells, DC, Deiter cells, PC, pillar cells, IBC, inner border cells.