Figure 5. Subcellular localization of Yap in organotypic bubble cultures.

(A) In a schematic representation of a cross-section through an E17.5 Atoh-nGFP utricle, red lines represent optical sections through the apices of hair and supporting cells (Apex; shown in panel B) and through supporting cell nuclei (SCN; shown in panel C). Hair cells are marked in green. (B) In surface views of utricles maintained for 3 d as organotypic bubble cultures in collagen gels of Young's modulus 640 Pa (top panels) or 40 Pa (bottom panels), cytoplasmic Yap protein occurs in supporting cells but is nor observed in hair cells (green). The scale bar represents 25 μm. (C) In the same organotypic bubble cultures, Yap protein is excluded from the nuclei of supporting cells in a 640 Pa collagen gel (top panels). In contrast, Yap is translocated into the nuclei of supporting cells in a 40 Pa gel (bottom panels). The insets in the merge panels are magnified fourfold. The scale bar represents 25 μm. (D) Quantification of the density of Sox2-positive cells demonstrates a significant decrease in 40 Pa gels as compared to 640 Pa gels (means ± SEMs; p<0.001; N = 3 for each; Figure 5—source data 1). (E) The mean fluorescence intensity of antibody labeling for Yap is unchanged in utricles cultured for 3 d in 40 Pa gels as compared to 640 Pa gels (value range 0–85; means ± SEMs; p>0.05; N = 5 for 640 Pa and N = 6 for 40 Pa; Figure 5—source data 2). (F) The percentage of supporting cells showing Yap nuclear translocation is significantly higher in 40 Pa gels than in 640 Pa gels (means ± SEMs; p<0.0001; N = 4 for each; Figure 5—source data 3). (G) qPCR analysis of utricle bubble cultures maintained for 4 d reveals significant increase in the expression of the Yap target genes Ctgf, Cyr61, and Ankrd1 in 40 Pa gels as compared to 640 Pa gels (means ± SEMs; p<0.001 for each; N = 10 for each; Figure 5—source data 4).

DOI: http://dx.doi.org/10.7554/eLife.25681.027

Figure 5—figure supplement 1. Subcellular Yap localization in organotypic bubble cultures.

In utricles maintained for 4 d as organotypic bubble cultures in 640 Pa collagen gels (top panels), nuclear Yap labeling (green) occurs in Sox2-positive supporting cells (red) at the periphery of the macula. EdU incorporation (white) demonstrates cell proliferation. In similar preparations of the utricles maintained in 40 Pa gels (lower panels), nuclear Yap labeling and EdU incorporation occur in the Sox2-positive supporting cells throughout the sensory epithelium. The scale bar represents 25 μm.

DOI: http://dx.doi.org/10.7554/eLife.25681.032