1. Foxo3 is expressed in the adult mouse cochlea.
Foxo3 is expressed in the adult mouse cochlea. A, Drawing of a cross-section of the adult organ of Corti. Type I spiral ganglion neurons resident in Rosenthal's canal (green) project to the mechanosensory single IHC (pink). Three OHCs (pink) are also seen. Supporting cells (gray) surround the hair cells. B, Surface view and cross-section of organ of Corti whole mount. Interphalangeal supporting cells (IP) are medial (left) to the IHCs. The apical tops of pillar cells form a line between inner and OHCs. Hensen supporting cells (Hen) are lateral (right) to the OHCs. C, Q-PCR for Foxo3 in cochlear extracts at different stages of postnatal development as indicated. Each time point is an average of 3–4 organ preparations, with error bars indicating SEM. D, Western blotting with an anti-Foxo3 antibody (first lane), compared with MW markers (second lane), with kDa values indicated (numbers), on a cochlear extract from 2-month-old mice (1/8 cochlea equivalents loaded). The Foxo3 antibody detected a band at 74 kD (arrow). Anti-β-act was used as a positive control (third lane) on a duplicate protein sample from the same gel (MW, fourth lane). E–G, Cryosections of 2-month-old spiral ganglion neurons stained with anti-acetylated tubulin (“TuJ1,” E, G, green) and anti-Foxo3 (F, G, red) antibodies. H, Whole-mount preparation of the 30 kHz region of a 2-month-old cochlea stained with anti-parvalbumin (white, IHCs), anti-oncomodulin (green, OHCs), and DAPI (blue, all nuclei). I, Same view as H stained with anti-Foxo3 antibody (red). Anti-Foxo3 labels hair cells and pillar cells. J, Colocalization of H with I; both hair cell markers are rendered in green and anti-Foxo3 staining is in red. DAPI is omitted in this view. K–P, Identical staining and preparation as in E–J using 4-month old cochlear tissue. Note how Foxo3 immunoreactivity in older spiral ganglion neurons (L, M) is excluded from the nucleus. Q–V, Identical staining and preparation as in K–P using cochlear tissue from a 4-month-old mouse exposed to 15 min of 75 dB filtered noise 5 h before being euthanized. R, S, Foxo3 immunoreactivity detected in the nucleus. Inset shows side views of a Foxo3+ neuron from confocal microscopy stacks. Scale bars: Q, 20 μm; T, 10 μm. W–BB, Enlarged view of hair cells from T–V, with hair cell markers (Pvalb, Ocm) rendered in green to better demonstrate anti-Foxo3 localization. DAPI is included in this view (blue).
Felicia Gilels et al. J. Neurosci. 2013;33:
©2013 by Society for Neuroscience