Transcriptomic analysis and ednrb expression in cochlear intermediate cells reveal developmental differences between inner ear and skin melanocytes

Abstract

In the inner ear, the neural crest gives rise to the glia of the VIII ganglion and two types of melanocytic cells: The pigmented cells of the vestibular system and intermediate cells of the stria vascularis. We analyzed the transcriptome of neonatal intermediate cells in an effort to better understand the development of the stria vascularis. We found that the expression of endothelin receptor B, which is essential for melanocyte development, persists in intermediate cells long after birth. In contrast, skin melanocytes rapidly downregulate the expression of EdnrB. Our findings suggest that endothelins might have co-opted new functions in the inner ear during evolution of the auditory organ.

Keywords: EdnrB; cochlea; development; intermediate cells; melanocytes; stria vascularis.

FIGURE 1

RNAseq of intermediate cells of the stria vascularis at P1. a. Experimental design. b. Section of a P1 cochlea from a Wnt1‐Cre x Ai3 reporter mouse, which was used in the experiment. c. Dissected lateral wall of the cochlea shown in b. d. FACS report showing the purity of the EYFP + cells. e, e′. bright field and fluorescence fields of the sorted intermediate cells

FIGURE 2

In situ validation of the RNAseq screen. The schematic shows a section through a P1 cochlea and the different layers of the stria vascularis are labeled. Expression of selected genes on cochlear section of cochlea at P1 by in situ hybridization. Inset shows negative control in situ hybridizations using sense probes. MC: marginal cells, IC: intermediate cells BC: basal cells

FIGURE 3

Expression of the endothelin family from E16.5 to P14 in the mouse cochlea. The gene expression of Et‐1, Et‐2, Et‐3, EdnrA, and EdnrB was analyzed by in situ hybridization. Inset shows a higher magnification of the selected area. At E16.5 and P0, detail of the cochlear turn and P6‐P14 detail of the stria vascularis. P0 sense column shows in situ hybridizations done with sense probes as negative controls. Square bracket: organ of corti, arrows: melanoblasts migrating in the stria vascularis, arrowheads: stria vascularis, stars: spiral ganglion. Scale bar: 200 µm, inset scale bar: 50 μm

FIGURE 4

Semi‐quantitative analysis of the endothelin receptor B expression shows a difference in EdnrB regulation between the melanocytes of the skin and the melanocytes of the ear at post‐natal stages. a: RNAscope of EdnrB (red) with immunolocalization of melanocytes using DCT (turquoise or purple) and neural crest cell derivatives using GFP expression in Wnt1Cre‐EYFP mice (green). Nucleus are labeled in blue by DAPI. Arrowheads mark the stria vascularis thickness, and arrows indicate the spiral ganglion containing glial cells originating from neural crest cells. Dashed lines mark the epidermis, dotted lines mark the hair follicle, and the star indicates the hair follicle in formation. NT: neural tube, OV: otic vesicle, SM: scala media. E11.5 panel, general scale bar: 200 µm, inset scale bar: 20 µm. All other panels, general scale bar: 20 µm, inset scale bar: 10 µm. Note: the transverse section in 3 months hair follicles is through the differentiated melanocytes only. b: Schematic representation of melanocytic cells development in the cochlea and skin. c: After the RNAscope assay was performed, the average mRNA expression was quantified by dividing the total spot count by the number of cells count per section. 3 animals were used for each stage (except E16.5 skin (2)). *=p < 0.05 based on the Kruskal–Wallis test