Figure 1.
Illustrations of a whole cochlea and cochlear sections (A and B). B is an illustration of a section through dotted line in A. The cochlea exhibits spiral structure with several turns (A). The maturation of its sensory epithelia occurs in the basal to apical direction (A). The cochlea includes three spaces within its duct; scala tympani (ST), scala media (SM), and scala vestibuli (SV) (B). Hematoxylin and eosin stained section of an E18.5 cochlear basal turn (C) and an illustration (D) are presented. These are enlarged images of the orange dotted box in panel B. On the SM side of basilar membrane (BM in D) there are sensory epithelia composed of inner hair cells (IHC in C and D) and outer hair cells (OHC in C and D). The organ of Corti includes one row of IHCs and three rows of OHCs (red cells ind D). Supporting cells (SC) including Claudius and Hensen's cells exist lateral to (yellow cells in D) or under the hair cells (pale orange cells in D). Tympanic border cells (green cells in B) cover the ST side of the basilar membrane. The lateral and medial regions of the organ of Corti are referred to as lesser epithelial ridge (LER) (yellow cells) and greater epithelial ridge (GER) (orange cells), respectively. Below the organ of Corti, there is a spiral modiolar artery (C and D) surrounded by the tympanic border cells. Tympanic lip is the medial border of BM. The scale bar in C indicates 50 µm.
Table 1.
Cellular types and patterns of proliferation markers expression.
Figure 2.
Section through an E13.5 murine cochlear duct one hour after BrdU injection.
All panels are from one section. Double immunostaining was performed for BrdU (A, C, D, and F) and Ki-67 (B, C, E, and F) simultaneously, and nuclei were stained using DAPI (D, E, and F). C is a merged image of A and B. F is a merged image of D and E. We observed many BrdU and Ki-67-positive cells in the cochlear ducts (A and B) and almost all the BrdU-positive cells were Ki-67-positive (C). Both staining patterns clearly indicate a zone of non-proliferating cells (F) representing future sensory epithelial cells. The scale bar indicates 50 µm.
Figure 3.
Section through an E18.5 cochlea five days after BrdU injection.
All panels are from one section. This section is representative of the ten cochleae studied. Triple immunostaining was performed for BrdU (A, C, D, and F), Ki-67(B, C, E, and F), and Myosin 7a (Myo7a, hair cell marker) (F). Nuclei were stained using DAPI (D, E, and F). We observed BrdU-positive cells mainly lateral and medial to organs of Corti (A, C, D, and F) in the lesser epithelial ridge (LER) and greater epithelial ridge (GER) areas, respectively. In contrast, Ki-67-positive cells were found among the tympanic border cells lining the scala tympani (ST) side of basilar membrane, and among cochlear nerve fibers (B, C, E, and F). GER : greater epithelial ridge, LER : lesser epithelial ridge, SM : scala media, ST : scala tympani. The scale bar indicates 50 µm.
Figure 4.
Slow-cycling tympanic border and GER cells (E18.5).
All panels are from one section. This section is representative of the ten cochleae studied. Triple immunostaining was performed for BrdU (A, B, E, F, and H), Ki-67(C, D, E, F, and H) and Myosin7a (Myo7a, hair cell marker) (F and H). Nuclei were stained using DAPI (B, D, F, and H). The difference interference contrast image (G) and fluorescent signal detection image were taken at the same time and merged (H). We detected double-positive cells in the tympanic border cell populations (arrows) and the GER (arrowheads) in the E18.5 (BrdU administered at E13.5). GER: greater epithelial ridge, IHC: inner hair cell, OHC: outer hair cells, DIC: difference interference contrast image. The broken line in F indicates the basilar membrane. The scale bar indicates 50 µm.
Figure 5.
Postnatal slow-cycling cells of the tympanic border zone.
Panels A-H (P4) are from one secion. Panels I and J (P9) are from a second section. These sections are representative of the cochleae studied. Triple (A-H) or double (I and J) immunostaining was performed for BrdU (A, B, E, F, and H), Ki-67(C, D, E, F, H, I, and J) and Myosin7a (Myo7a, hair cell marker) (F, H and J). Nuclei were stained using DAPI (B, D, F, H, and J). The difference interference contrast image (G) and fluorescent signal detection image were taken at the same time and merged (H). We observed double-positive cells within the tympanic border zone (arrows) in the P4 mouse cochlea (BrdU administered at E18.5) (A-H). We did not detect double-positive cells in the P9 mouse cochlea although there were few Ki-67-positive cells in the tympanic border zone (I and J). IHC: inner hair cell, OHC: outer hair cells DIC: difference interference contrast image. The broken lines in F and J indicate the basilar membrane. The scale bar indicate 50 µm.
Figure 6.
Localization of slow-cycling cells within tympanic border cell region.
We counted the numbers of double-positive cells within three areas of the basilar membrane (A): from the region under the tympanic lip to under the medial side of the inner hair cells (medial region); from the region under the inner hair cells to the area under the outer hair cells (region below the HCs); and the region under the Hensen's and Claudius cells (lateral region) (B). Statistical analyses were performed using one-way analysis of variance (ANOVA) and multiple comparisons (Tukey-Kramer method). Roughly two-thirds of the observed slow-cycling cells (41/63) were located in the region below the hair cells where a cochlear spiral modiolar artery exists. The number of slow-cycling cells in the area below the HCs was significantly greater than was detected in other regions (A). * : p<0.01. Bars indicate the standard error of the mean. IHC: inner hair cell, OHC: outer hair cells.
Figure 7.
Slow-cycling cells in the supporting cell layer of the vestibular organ.
All panels are from one section. Triple immunostaining was performed for BrdU (A, B, F), Ki-67 (C, D, F), and Myosin7a (Myo7a, hair cell marker) (F), and nuclei were stained using DAPI (B, D, E, F). We detected double-positive cells (arrows) in the supporting cell layer (below Myosin7a-positive hair cells) in the E18.5 mouse vestibular organ (BrdU administered at E13.5). The scale bar indicates 50 µm.
Figure 8.
Nestin-positive tympanic border cells (E18.5).
Panels A and B are from the same section. Panels C-H are from a second section. Double immunostaining (A and B) for Nestin and Myosin7a (Myo7a, hair cell marker), or triple immunostaining (C-H) for BrdU (C, D, and H), Ki-67 (E, F, and H), and Nestin (G and H) was performed. Nuclei were stained using DAPI (B, D, and F). Tympanic border cells positive for Nestin (arrows in A and B). Most of the BrdU-Ki-67-double positive tympanic border cells are also positive for Nestin (arrows in C-H). The white arrowheads indicate a cochlear spiral modiolar artery. Asterisks in A and B indicate Nestin-positive cells in GER and LER. The scale bar indicates 50 µm.
Figure 9.
Nestin-positive tympanic border cells (P4).
All panels are from one section. This section is a representative result of the ten cochleae studied. Triple immunostaining was performed for BrdU (A, B, F, and H), Ki-67(C, D, F, and H) and Nestin (E, F and H). Nuclei were stained using DAPI (B, D, F, and H). The difference interference contrast image (G) and fluorescent signal detection image were taken at the same time and merged (H). We observed triple-positive cells in the tympanic border zone (arrows) in the P4 mouse cochlea (BrdU administered at E18.5). IHC: inner hair cell, OHC: outer hair cells, DIC: difference interference contrast image. The white arrowheads indicate a cochlear spiral modiolar artery. The scale bar indicates 50 µm.
Figure 10.
Proliferation status of tympanic border cells in the mature cochleae.
Triple immunostaining was performed on the whole mount cochlea of four-week-old mice for Myosin7a (Myo7a, hair cell marker) (A, C, D and E), BrdU (B-D, G and H), and Ki-67(B, C, E, J, and K). Nuclei were stained using DAPI (B-E, G, H, I, and K). Stacked images of the whole mount cochlea (A-C) showed that many BrdU-positive cells (asterisks in B and C, BrdU administered from P3 to P5) and one Ki-67-positive cell (white boxed cell in B) were located around hair cells. Re-sliced images of A-C (D and E, re-sliced at yellow and white dotted lines in A, respectively) showed that these BrdU-positive cell (yellow arrow in D) and Ki-67-positive cell (white arrow in E) were located at the level of tympanic border cells. Optical sections of the whole mount cochlea showed that BrdU- and Ki67- signals were those from nuclei (F-K). F-H and I-K were optical sections at the regions of yellow and white box in B, respectively. IHC: inner hair cells, OHC: outer hair cells. The scale bar in A indicates 30 µm. The other scale bars in D, F, and I indicate 5 µm.