Reinforcement of cell junctions correlates with the absence of hair cell regeneration in mammals and its occurrence in birds

Abstract

Debilitating hearing and balance deficits often arise through damage to the inner ear's hair cells. For humans and other mammals, such deficits are permanent, but nonmammalian vertebrates can quickly recover hearing and balance through their innate capacity to regenerate hair cells. The biological basis for this difference has remained unknown, but recent investigations in wounded balance epithelia have shown that proliferation follows cellular spreading at sites of injury. As mammalian ears mature during the first weeks after birth, the capacity for spreading and proliferation declines sharply. In seeking the basis for those declines, we investigated the circumferential bands of F-actin that bracket the apical junctions between supporting cells in the gravity-sensitive utricle. We found that those bands grow much thicker as mice and humans mature postnatally, whereas their counterparts in chickens remain thin from hatching through adulthood. When we cultured utricular epithelia from chickens, we found that cellular spreading and proliferation both continued at high levels, even in the epithelia from adults. In contrast, the substantial reinforcement of the circumferential F-actin bands in mammals coincides with the steep declines in cell spreading and production established in earlier experiments. We propose that the presence of thin F-actin bands at the junctions between avian supporting cells may contribute to the lifelong persistence of their capacity for shape change, cell proliferation, and hair cell replacement and that the postnatal reinforcement of the F-actin bands in maturing humans and other mammals may have an important role in limiting hair cell regeneration.

Figure 1

The width of circumferential F-actin bands in utricular supporting cells of the sensory epithelium and cells of the nonsensory epithelium increases in mice as they develop and mature. A-C) The images are high resolution (63x 1.4NA objective, 0.3 μm thick confocal sections) 2-D z-projections of a stack of images starting at least 5 μm above and ending at least 5 μm below where the bands were detectable from utricles of mice ages embryonic day 18 (E18), postnatal day 8 (P8), and P83. The transition from sensory epithelium (S) to nonsensory epithelium (NS) shows the difference in width of F-actin bands between these two regions as mice age. Circumferential F-actin bands extend further from the cell junctions towards the center of the cell in the S than in the NS as mice develop and mature. Scale bar for A-C in C, 30 μm. D-F) A high magnification image of the S in the frames in A shows that the width of F-actin bands (green arrows) in supporting cells of the S increases almost to the extent of covering the entire apical surface area of the supporting cells by P83. Scale bar for D-F in F, 10 μm. G-I) An overlay of the image in B shows the circumferential F-actin bands of supporting cells (blue) in relation to the actin-rich cuticular plates of hair cells (tan). A gap between the F-actin bands of adjacent supporting cells becomes resolvable as mice age, and as a result, the outlines of individual supporting cells are seen in the P8 and P83 mouse frames. Scale bar for G-I in I, 10 μm.

Figure 2

A space between the circumferential F-actin bands of adjacent supporting cells which becomes resolvable as mice age is at the level of the adherens junctions. A-C) Images of N-cadherin immunohistochemistry (red), phalloidin staining (green), and their overlaid image show there is a visible gap between the F-actin bands at the intercellular adherens junction joining supporting cells in a postnatal day 8 mouse utricle. These frames are a single 0.3 μm thick confocal slice at the z-depth where the fluorescence intensity of N-cadherin immunohistochemistry was at its highest. A large accumulation of F-actin was visible at this depth. Scale bar for A-C in C, 5 μm. D-F) High magnification images of cells from the nonsensory epithelium from Fig. 1A show the width of circumferential F-actin bands increases in cells of the nonsensory epithelium as mice age, but not to the extent that it does in supporting cells of the sensory epithelium. Scale bar for D-F in F, 10 μm. (A magenta-green version of this figure is provided in the supplementary material)

Figure 3

The width of apical junction regions (AJRs) can be measured by plotting intensity profiles for lines drawn perpendicular to the junctions and analyzing them with a 1-D edge detector. A-C) An AJR was defined as the area containing the intercellular junction (J) of adjoined supporting cells and the circumferential F-actin (CA) extending from both sides of the junction into the intracellular domain of each cell. Circumferential F-actin is shown in green, and N-cadherin immunohistochemistry shows the junction in red. Scale bar in A, 5 μm; scale bar in B and C, 0.4 μm. D) Stacks of confocal images were collected of whole mount utricles stained with phalloidin. Scale bar, 150 μm. E) The stacks were converted to 2-D projections, and lines (red) were drawn perpendicular to the AJRs (designated by numbers and white bars). Scale bar, 10 μm. F) The 12-bit intensity values for each pixel in the lines were saved and plotted versus distance in Matlab R2007a (blue plot). The distance between the edges of local peaks on the intensity profile corresponded to the width of individual AJRs (numbers and black lines). The forward difference of the intensity profile, an approximation of the derivative, was calculated and plotted over the intensity profile (red plot). The intensity peak widths were then measured by calculating the distance between the local maxima and minima on the forward difference plot, which corresponded to the edges of each AJR. (A magenta-green version of this figure is provided in the supplementary material)

Figure 4

Quantification of apical junction region (AJR) width shows there is exponential growth of AJRs in supporting cells of the sensory epithelium and cells of the nonsensory epithelium as mice develop and mature from embryonic day 18 (E18) to postnatal day 83 (P83). Measurements of the width of AJRs from ≥ 400 junctions in fifteen different age groups of mice for the sensory epithelium and six different age groups for the nonsensory epithelium were taken. AJR width increases 400% in the sensory epithelium (black squares) and 178% in the nonsensory epithelium (open squares). Von Bertalanffy growth curves (red lines) fit the data with high r-squared values, and the data points fall within the 95% confidence intervals (C.I.; blue lines). The parameters for the curve fits all had squared error values less than 0.2. W: AJR width; W_∞: AJR width at infinity; C: scaling factor to prevent initial width from equaling zero; k: curvature parameter; t: time in days.

Figure 5

Ultrastructural analysis of longitudinal and transverse sections of utricles from mice ranging in age from embryonic day 18 (E18) to postnatal day 52 (P52) demonstrate circumferential F-actin bands increase in width, not intercellular junctions; and there is a coinciding increase in the height of F-actin bands. A-B) Increases in the vertical dimension of circumferential F-actin bands parallel to the lateral membrane are seen in longitudinal sections from an E18 and P52 mouse. Scale bar for A and B in B, 10 μm. C-D) High magnification images of the boxes outlined with black dashes in A and B show the intercellular junction (arrow heads) does not change in width while the F-actin bands extend further from the junction as mice age. Scale bar for C and D in D, 2 μm. E-F) Transverse sections from an E18 mouse (E) and a P52 mouse (F) provide a view identical to that seen in confocal images. Circumferential F-actin bands (width shown by black bars) almost cover the entire apical planar area of the supporting cells by P52. The size of the intercellular junctions, however, remains the same at both ages. Vesicles (arrow heads) are visible in the F-actin bands in the transverse section from the P52 mouse. Scale bar for E and F in F, 2 μm.

Figure 6

The width of circumferential F-actin bands in utricular supporting cells of the sensory epithelium and cells of the nonsensory epithelium remains narrow throughout life in chickens. A-C) 2-D projections were taken of stacks of high resolution confocal images (63x 1.4 NA objective) from phalloidin-stained utricles of chickens at postnatal day 0 (P0), P13, and P365. The transition from sensory epithelium (S) to nonsensory epithelium (NS) shows that the width of F-actin bands between these two regions apparently remains narrow as chickens age. Scale bar for A-C in C, 30 μm. D-I) Higher magnification images of the S and NS from the frames in A-C show that the bands (white arrows) remain within close spatial proximity to the junctions in both regions. Scale bar for D-F in F, 5 μm. Scale bar for G-I in I, 5 μm.

Figure 7

Quantification of apical junction region (AJR) width shows there is apparently no growth of AJRs in supporting cells of the sensory epithelium and cells of the nonsensory epithelium as chickens develop and mature from postnatal day 0 (P0) to P365. The width of AJRs from ≥ 400 junctions from chickens from five different age groups was quantified. AJR width in supporting cells of the sensory epithelium (black squares; solid line is the average of all the age groups) and cells of the nonsensory epithelium (open squares; dotted line is the average of all the age groups) remains narrow throughout life (x-axis is on a logarithmic scale).

Figure 8

Ultrastructural analysis of longitudinal and transverse sections of utricles from chickens ranging in age from postnatal day 0 (P0) to P365 demonstrates that circumferential F-actin bands remain narrow in the vertical dimension in addition to width. A-D) The vertical dimensions of F-actin bands (arrows in C and D) at the adherens junctions (arrowheads in C and D) in TEM images of longitudinal sections from a P0 and P365 chicken are thin and comparable. Scale bar for A and B in B, 2 μm. Scale bar for C and D in D, 0.5 μm. E-F) Transverse sections provide a view identical to that seen in confocal images, and the width (black bars) of the F-actin bands in P0 (E) and P365 (F) chickens is similar. Scale bar for E and F in F, 2 μm.

Figure 9

The spreading of mammalian utricular epithelia gradually decreases with the age of the animal from which the tissue was harvested throughout the culture period, while the spreading of avian utricular epithelia occurs extensively and independently of the animal's age. A) Outlines of representative images of mammalian utricular sensory epithelia derived from embryonic day 18, postnatal day 1 (P1) and P15 mice at 1, 24, 48 and 72 hr in culture illustrate the gradual reduction in tissue spreading with age (Davies et. al, 2007). B) Outlines of representative images of avian utricular sensory epithelia derived from P0, P60 and P180 chickens at 1, 24, 48 and 72 hr in culture illustrate the age-independent extensive tissue spreading. Scale bar, 1 mm.

Figure 10

The rate of spreading and proliferation of avian utricular epithelia on fibronectin (FN) is maintained at similar levels throughout life. A) Rates of spreading shown as fold increase in area throughout the culture period were similar in neonates (postnatal day 0 (P0), n=41 sheets) and adult chickens (P60, n= 45 and P180, n= 44). B) Inhibition of cell division with the DNA polymerase inhibitor aphidicolin (25 μM) yielded a similar degree of tissue spreading in utricular epithelial sheets from P0 (n=9 sheets) and P180 (n=5 sheets) chickens at 24, 48 and 72 hr in culture. C) S-phase entry after 72 h in culture shown as the percentage of BrDU positive cells was maintained at similar levels in chickens ranging in age from P0 to P420 (number of epithelia sheets analyzed; P0, n=27; P6, n=27; P12, n=26; P24, n=23; P60, n= 24; P180, n=27; P420, n= 11). CBT and SPAFAS are the names of the two different suppliers of chickens utilized in this experiment.

Figure 11

The width of circumferential F-actin bands increases in the utricular supporting cells of developing humans and other murine balance organs. A) A high resolution confocal image of the sensory epithelium of a utricle stained with phalloidin from a 60 year-old human cadaver shows the extent to which the F-actin bands cover supporting cell apical areas. Scale bar, 15 μm. B and C) A zoomed in region of the sensory epithelium (box outlined with red dashes in A) shows the location of the bands in supporting cells (blue) in relation to the cuticular plates of hair cells (tan). Very little area remains free of F-actin in the supporting cell apical areas. Scale bar in B and C, 5 μm. D) A high resolution confocal image of the sensory epithelium in the cristae ampullaris from a postnatal day 120 mouse shows that the circumferential F-actin bands of supporting cells have almost an identical morphology to that seen in the utricles of adult mice. Scale bar, 5 μm.

Figure 12

A schematic of transverse and longitudinal sections through the utricular sensory epithelium compares the thickness of circumferential F-actin bands in chickens of all ages, immature mammals, and adult mammals. F-actin is colored green, supporting cells are colored gray, and hair cells are colored light gray. The red dashed lines on the transverse sections correspond to the location of the longitudinal sections.