Fig 2: DARC, atypical chemokine receptor, is expressed in cochlear hair cells, supporting cells, and spiral ganglion neurons. Cochlear whole mounts of control mice (A,B,Ca-d,i-l) and age-matched mice exposed to PTS-causing noise (103 dB SPL) (Ce–h,m–p) at 6 weeks of age and sacrificed 6 h later were stained with anti-DARC (magenta), anti-CXCL1 (green), and anti-Myo7A antibody (red). (A) DARC is brightly expressed in the outer pillar cells (OPCs), inner hair cells (IHCs) and spiral ganglion neurons (SGNs), and moderately expressed in outer hair cells (OHCs) and Deiters cells (DCs). Scale bar, 100 µm. (B) A high magnification view of the whole mount shown in A and a corresponding 3D reconstruction illustrate DARC expression in the columnar part of OPCs and DARC coexpression with CXCL1 in the basilar part of OPCs (bOPCs) (Ba–d,a'–d'). Three-dimensional reconstruction of CXCL1 expression confirms the finding illustrated in the Figure 5, highlighting the main localization of CXCL1 in the apical part of PCs (aPCs) followed by the basilar parts of inner PCs (bIPCs) and outer PCs (bOPCs) (Bc'). Scale bar, 25 µm. (C) PTS-causing acoustic trauma did not appreciably affect DARC positivity while causing reduction in CXCL1 positivity in the basal turn of PCs. Scale bar, 25 µm. The slides represent maximum projections of confocal z-stacks through whole mounts in the apical and basal region of the cochlea (~8 and 32 kHz region, respectively). Representative images are based on biological replicates in 4 control and 3 noise-exposed animals.

Fig 3: Within the crista ampullaris, CXCL1 localizes to supporting cells while DARC preferentially localizes to a subset of hair cells. Whole mounts of the crista ampullaris of control mice stained with anti-CXCL1 (green) (A), anti-DARC (magenta) (B), and anti-Myo7A (red) (C) antibodies. CXCL1 is expressed in the polygonally-shaped supporting cells of the crista ampullaris (A, D'- an empty white arrow). Myo7A-positive hair cells are separated by intervening CXCL1-positive supporting cells. DARC-positive cells are scattered within the crista's epithelium, with predominant localization in the central zone of each hemicrista (B), sometimes surrounded by CXCL1-positive supporting cells (a solid white arrow) (D'). Scale bars, 100 µm (A–D), 25 µm (D'). The pictures represent maximum projections of confocal z-stacks through whole mounts, based on biological replicates in 4 control cochleae.