Intermediate Cells of Dual Embryonic Origin Follow a Basal to Apical Gradient of Ingression Into the Lateral Wall of the Cochlea

Abstract

Intermediate cells of the stria vascularis are neural crest derived melanocytes. They are essential for the establishment of the endocochlear potential in the inner ear, which allows mechanosensory hair cells to transduce sound into nerve impulses. Despite their importance for normal hearing, how these cells develop and migrate to their position in the lateral wall of the cochlea has not been studied. We find that as early as E10.5 some Schwann cell precursors in the VIIIth ganglion begin to express melanocyte specific markers while neural crest derived melanoblasts migrate into the otic vesicle. Intermediate cells of both melanoblast and Schwann cell precursor origin ingress into the lateral wall of the cochlea starting at around E15.5 following a basal to apical gradient during embryonic development, and continue to proliferate postnatally.

Keywords: cochlea; inner ear; inner ear development; intermediate cells; melanoblast; neural crest; schwann cell precursor; stria vascularis.

FIGURE 1

Neural crest contributions to the inner ear during embryonic development from E12.5 to E17.5. Lineage tracing of neural crest cells using Wnt1-Cre2 mice crossed to an EYFP reporter line was visualized by staining with the anti-GFP antibody in green and DAPI for nucleus in blue. Arrowheads: intermediate cells of the stria vascularis; Arrows: melanoblast of the vestibule; CVN: Cochleo-vestibular neurons; SGN: Spiral ganglion neurons; S: saccule; U: utricle. Scale bars: 100 µm.

FIGURE 2

Intermediate cells in the developing stria vascularis follow a base-to-apical gradient of ingression and undergo morphological maturation at early postnatal days. (A) Immunostaining of E16.5 Wnt1cre-YFP mouse with white dashed square magnified in insets. Future intermediate cells migrate (arrows) then line up on top of the cochlear duct and ingress in the lateral wall of the cochlea following a basal to apical gradient. CD44 (in red) is a cell membrane marker of melanoblast that shows the protrusion in the marginal cells layer (Arrowheads). 1: apex, 2: mid apex, 3: mid base, 4: base. (B) Schematic representation of the base to apex semi-ingression process. Melanoblast are represented in dark pink at the surface of the lateral wall. (C). Immunostaining of P0, P6 and P32 Wnt1Cre-YFP mouse with white dashed square magnified in insets. Green cells in the stria vascularis are intermediate cells labeled by Wnt1-CRE2 crossed to a EYFP reporter. KCNQ1 and Connexin 26, markers of marginal and basal cells respectively, are both in purple to delimit the intermediate cell region in the stria vascularis.

FIGURE 3

Proliferation in the stria vascularis decreases 2 weeks after birth. (A) Schematic of the experimental using Edu injection at different time points during development. (B) Immunostaining of Wnt1Cre-YFP mice from P0 to P14 with EdU labeling (dark pink) and DAPI, a nuclear marker in blue. Some Edu positive marginal, intermediate and basal cells are shown by arrows, stars and arrowheads respectively. Anti-activated caspase 3 labeling (in turquoise) shows no significant cell death in the stria vascularis during development (right panel). (C) Graphic of the average proliferation in the three different layers of the inner ear from E16.5 to P14. For each stage n = 3.

FIGURE 4

Dual embryonic origin of intermediate cells. Immunostaining for melanoblast with MITF labelling (in red) and neural crest cells with P75 (in turquoise) shows at E11.5 the presence of melanoblast between the dermis and the otic vesicle and between the otic vesicle and the neural tube (Arrows). Lineage tracing of Schwann cell precursors using Plp1-YFP reporter line at E12.5 and P6. Immunostaining for melanoblast with the MITF antibody (red) and DAPI for nucleus (blue) show cells with both markers (arrowheads) or only melanoblast marker (arrows). Most intermediate cells express the glial cell marker PLP (green) and melanocyte markers DCT (in purple) at P6 except one cell that only express DCT (arrow). CVG: cochleaovestibular ganglion, NT: neural tube, OV: otic vesicle, V: vestibule.

FIGURE 5

Early migratory neural crest cells give rise to both glial cells and melanocytes cells in the inner ear. Inducible Wnt1-CreER shows no late-migrating neural crest contribution to the inner ear. (A) Tamoxifen was administered to pregnant females via oral gavage at the ages indicated in the experimental design and collected and analyzed at P0. (B) Immunostaining of Wnt1CreER-YFP mice following the experimental design at P0 with YFP (green) and DAPI for nuclear labelling. Arrows indicate the melanoblast of the vestibules, the arrowheads the intermediate cells of the stria vascularis and the stars the missing labelling in the melanoblasts and intermediate cells. (C) Graph shows the average number of labeled intermediate cells after administration of tamoxifen at each stage. n = 3. CVN: Cochleo-vestibular neurons; VN: vestibular neurons; SGN: Spiral ganglion neurons; S: saccule; U: utricle.