Fig 1.
Prickle1 is expressed in the spiral ganglion but not the organ of Corti by in situ hybridization during development.
(A-D) Prickle1 and Vangl2 mRNA expression was analyzed by whole mount in situ hybridization in wild-type cochleae. (A-B) An overview of the cochlea showing Prickle1 (A) and Vangl2 (B) expression at E15.5. (C-D) A higher magnification of the cochlea showing Prickle1 mRNA at E15.5 (C) and P0 (D). (E) β-gal staining was performed in Prickle1LacZ/+ cochlea to analyze Prickle1 expression at P30. Only the apex is shown. SG, spiral ganglion; SV, stria vascularis; OC, organ of Corti. Scale bar, A, B and E, 200 μm; C and D, 100 μm.
Fig 2.
Prickle1 mutation impairs type II fiber outgrowth.
(A-B”‘) The outgrowth of type II SGN afferents was analyzed by immunocytochemistry in Prickle1C251X mutants at P0. Cochleae were immuno-labeled with antibodies against β-tubulin. Overview of the Prickle1+/+ (A) and Prickle1C251X/C251X (B) cochleae, and the type II afferents from the corresponding basal (A’ and B’), middle (A” and B”), and apical (A”’ and B”’) turns are shown. (C-D”) The outgrowth of type II SGN afferents analyzed by immunocytochemistry in Prickle1f/f; Pax2-cre mice. Cochleae were immune-labeled with antibodies against Myo7a and β-tubulin, and the outgrowth of type II SGN afferents are shown in control mice (C-C”) and in Prickle1f/f; Pax2-cre mutants (D-D”) at P33. Arrow, type II afferents turning towards the apex; filled triangle, type II afferents still developing; empty triangle, fibers that grew past the outer hair cells. Scale bar, 100 μm.
Fig 3.
Loss of Prickle1 leads to aberrant afferent outgrowth in the apical cochlea.
A select population of type II fibers was labeled by dye tracing in Prickle1C251X/C251X mutants and their littermate controls at E18.5 and P0. (A) In the base, both control and Prickle1C251X/C251X mutants formed three rows of type II fibers growing towards the base. (B-F) In the apex of the Prickle1C251X/C251X mutant cochlea, outgrowth of some type II afferents was disrupted. (G and H’) Some afferents were not in the same focal plane as the radial fibers growing towards the hair cells (HCs). (H’) A higher magnification view of (H). Filled triangle, fibers that branched; arrow, fibers growing toward the apex; empty triangle, fibers that grew past HCs.
Fig 4.
Central projections from apical afferents are expanded in the cochlear nuclei in Prickle1C251X/C251X mutants.
Different colors of lipophilic dyes were applied to apex and the base of the cochlear (A, B), and their central projection were analyzed (A’, B’, C-E). (A and B) Overview of the cochlea showing the application of red dye to the base and green dye to the apex in control (A) and Prickle1C251X/C251X mutant (B). After 3 days of diffusion, there was partial overlap of the dye. (A’ and B’) Selective bundles of afferents and olivocochlear efferents (OCE) passed along the vestibular ganglion (VG). Only in Prickle1C251X/C251X mutant (B’), OCE separated into several bundles. In addition, several vestibular ganglion neurons (VG) were labeled. (C-E) Projections to the cochlear nucleus of the control (C) and the Prickle1C251X/C251X mutant (D, E). (C) In controls, afferent bundles from the apex and the base segregated and formed distinct fascicles. (D, E) In the mutant, although afferents from the base projected normally to the dorsal cochlear nucleus complex DCN, afferents from the apex formed collaterals that spread out throughout the DCN and the anterior-ventral cochlear nuclei (AVCN). (E) Higher magnification of the DCN of (D), showing details of apical afferents passing basal turn afferents to branch in the most dorsal aspect of the cochlear nucleus complex. Arrow, afferents innervating vestibular nuclei. Scale bars, 100 μm.
Fig 5.
Central projections from apical afferents are expanded in the cochlear nuclei in Prickle1C251X/C251X mutants.
Different colors of lipophilic dye were applied to the apex (green) and the base (red) of the cochlea. The brainstem was sectioned coronally. (A, C, E) In controls, the afferents from base and apex of the cochlea and the olivocochlear efferents (OCE) segregated into bundles and innervate distinct parts of the cochlear nuclei. (B, D, F) In Prickle1C251X/C251X mutants, the afferents from the apex expanded to the regions where normally basal afferents were. In addition, some apical afferents even projected to the vestibular ganglion (arrow). DCN, dorsal cochlear nuclei. Scale bar, 100 μm.
Fig 6.
Prickle1 mutation has no discernible effect on hair cell polarity.
(A-B) Hair cell polarity was analyzed by the hair cells with phalloidin, which did not stain the fonticulus (black dot within the hair cell surface). At P0, in Prickle1C251X/C251X mutant (B) compared with Prickle1+/+ (A), most hair cells had fonticulus organized at the lateral side (red arrows and circles). There were a few cells that are slightly mis-oriented in either Prickle1C251X/C251X or Prickle1+/+ OC (white arrows and white circles). (C-D) Hair cell polarity as analyzed by SEM Prickle1f/f; Pax2-cre mutant at P9 (C) or P33 (D). (E-F) The degree of fonticular rotation was quantified at various locations along the cochlea in both Prickle1+/+ (E) and Prickle1C251X/C251X (F) mice at P0, with positive degree meaning rotation towards the apex and negative meaning rotation towards the base. The frequency distribution of the individual row of hair cells was plotted against the degree of rotation. 5 degrees were binned into one bar. About 130 hair cells were analyzed for each row of hair cells. 3 Prickle1+/+ and 3 Prickle1C251X/C251X mice were analyzed. ANOVA analysis was performed. Scale bar, 10 μm.
Table 1.
Normalized gene expression in the cochlea from RNA-seq data in multiple studies.