Figure 2. Ex vivo utricular culture system.

(A) A schematic drawing portrays the sensory epithelia (green) of the six receptor organs of the murine inner ear. The red lines delineate the cuts introduced during the dissection of a utricular bubble. The mesenchymal stroma that normally surrounds the organ is shown in light blue; the chondrocytes that surround the inner ear are shown in dark blue. (B) In a diagram of a section through a utricular bubble, hair cells are shown in magenta, supporting cells in green, non-sensory cells in white, and mesenchyme in light blue. The bubble is embedded in a gel of collagen I (orange) containing chondrocytes (dark blue). (C) A sequence of surface views portrays the development of two utricular bubbles from E17.5 Atoh-nGFP mice over a period of 5 d. The sensory epithelia are marked in the first and final images by GFP expression (green; delineated by green dashed line). In a gel of 640 Pa stiffness (middle panels), the utricular sensory epithelium reaches the normal adult size (in vivo; upper panels) by 5 d, corresponding to developmental stage P2. In a collagen gel of 40 Pa stiffness (lower panels) the area of the sensory epithelium almost doubles by 5 d. The scale bar represents 50 μm. (D) Areal measurements, represented as means ± SEMs, demonstrate that the utricular sensory epithelium in a 640 Pa gel develops at a rate similar to that of the epithelium in vivo. By 5 d in culture the area of the sensory epithelium is similar to that of an adult utricle (p<0.05, N = 5 for both; Figure 2—source data 1). In a 40 Pa gel, the area of the sensory epithelium expands significantly faster and reaches 180% of the control value by 5 d (p<0.0001, N = 4 for 40 Pa; N = 5 for in vivo measurements; Figure 2—source data 1). (E) After 4 d in culture, quantification of EdU-positive cells in 0.1 mm² of the utricular sensory epithelium, represented as means ± SEMs, demonstrates a tenfold increase in the number of EdU-positive cells in a 40 Pa gel as compared with a 640 Pa gel. This difference is significant (p<0.001, N = 3 for both; Figure 2—source data 2) (F) A surface view of utricular bubbles after 4 d in culture demonstrates that the expansion of the macula in a 40 Pa gel stems from the re-entry of Sox2-positive supporting cells (green) into the mitotic cycle throughout the sensory epithelium, as demonstrated by the incorporation of EdU (white). In the identical preparation of the utricular bubble cultured in a 640 Pa gel, supporting-cell proliferation is limited to the organ’s periphery. The scale bar represents 50 μm.

DOI: http://dx.doi.org/10.7554/eLife.25681.012

Figure 2—figure supplement 1. Characterization of the ex vivo utricular culture system.

(A) The slopes of the stress-strain relations for individual gels, corresponding to the elastic moduli of the materials, rise as the number of chondrocytes increases from zero to 3·10⁶. (B) The stiffness of the collagen gel increases linearly with the number of chondrocytes. The relation is fit by the linear function ${\displaystyle E}$ = 206·${\displaystyle N_C}$ +15, in which ${\displaystyle E}$ is the elastic modulus and ${\displaystyle N_C}$ is the number of chondrocytes (R = 0.99, p<0.001).

DOI: http://dx.doi.org/10.7554/eLife.25681.015

Figure 2—figure supplement 2. Chondrocyte-secreted factors do not affect supporting-cell proliferation.

(A) In a brightfield image of a utricular bubble culture established at E17.5, the utricle and ampullae semicircular canals are labeled. A piece of cartilage was dissected from the same E17.5 inner ear and positioned near the edge of the utricle, indicated by the magenta line. (B) In a utricular bubble preparation after 24 hr in culture, immunolabeling for Sox2 (green) reveals the size and position of the sensory epithelia of the utricle; EdU (white) demonstrated the distribution of proliferating cells adjacent to (pink line) and distant from the cartilage. The scale bar represents 100 μm.

DOI: http://dx.doi.org/10.7554/eLife.25681.016