Pattern formation in the basilar papilla: evidence for cell rearrangement

Abstract

The avian basilar papilla is composed of hair and supporting cells arranged in a regular pattern in which the hair cells are surrounded and isolated from each other by supporting cell processes. This arrangement of cells, in which the apical borders of hair cells do not contact one another, may be generated by contact-mediated lateral inhibition. Little is known, however, about the way in which hair and supporting cells are organized during development. Whole mounts double-labeled with antibodies to the 275 kDa hair-cell antigen and the tight junction protein cingulin were therefore used to examine the development of cell patterns in the basilar papilla. Hair cells that contact each other at their apical borders are seen during early development, especially on embryonic days (E) 8 and 9, but are no longer observed after E12. Hair and supporting cell patterns were analyzed in three different areas of the papilla at E9 and E12. In two of these regions between E9 and E12, the ratio of supporting cells to hair cells does not change significantly, whereas there is an increase in both the number of supporting cells around each hair cell and the number of hair cells that each supporting cell contacts. In the third region examined, there is a dramatic rise in the number of supporting cells around each hair cell, which although accompanied by a small, significant increase in the ratio of supporting cells to hair cells cannot be accounted for by an increase in supporting cell numbers. These data show that a rearrangement of hair and supporting cells with respect to one another may be a fundamental process underlying the development of a regular pattern in the basilar papilla.

Fig. 1.

Schematic drawings of E9 and E12 chick basilar papillae. Figure shows the three regions within which micrographs were taken for the analysis of contacts between hair and supporting cells.IP, Inferior-proximal; SP, superior-proximal; CD, central-distal.

Fig. 2.

Basilar papillar whole mounts stained with antibodies to the HCA. At E6 (a), hair cells are seen in a small patch at the distal end of the papilla. By E7 (b), the patch has enlarged and a few proximally located hair cells (arrows) can also be observed. Hair cells are seen along the entire length of the papilla at E8 (c), although those seen at the proximal end (arrows) are stained more brightly and appear distinct from the remainder.D, Distal; P, proximal; I, inferior; S, superior. Scale bar, 100 μm (applies to all micrographs).

Fig. 3.

Anti-HCA and cingulin staining in the early embryonic basilar papilla. Micrographs are from distal (a–c) and proximal (a′–c′) regions of E6 (a, a′), E7 (b, b′), and E8 (c, c′) papillae that have been stained with antibodies to both the HCA and cingulin. At E6, many distal cells stain only very weakly for the HCA (arrows in a). At E7 an increasing number of hair cells are seen in the distal region (b) and the first proximally located hair cells are seen at this stage (b′). At E8, in distal regions, the density of hair cells is very high, and the epithelium has a compact, crowded appearance (c). In the proximal end at this stage (c′), many hair cells are only just starting to differentiate (small arrows), but a small number appear to be much more mature (large arrow). Scale bar (located in c′): 10 μm (applies to all micrographs).

Fig. 4.

Graph of changes in number of HCA-positive cells observed in photomontages of the basilar papilla with developmental age. The number of HCA-positive hair cells increases most rapidly between E7.5 and E9.5. The means ± SE for each stage are as follows: E6, 94 ± 7.3 (n = 7); E7, 627 ± 50.1 (n = 7); E8, 2453 ± 253.4 (n = 7); E9, 9200 ± 140.0 (n = 2); E 9.5, 11,129 ± 181.4 (n = 3); and E12, 12,192 ± 165.0 (n = 3).

Fig. 5.

Whole mounts from the central-distal papilla at E9 and E12. Preparations have been double-labeled to reveal HCA and cingulin through one channel (a, b) and cingulin alone through the other channel (a′, b′). Note the presence of hair cell–hair cell contacts (arrows) at E9 (a′, a′). These are extremely infrequent by E12 (b′, b′). The overall appearance of the papilla does not change overtly between E9 and E12 in this region. Scale bar (shown inb′): 10 μm (applies to all micrographs).

Fig. 6.

Whole mounts from the superior-proximal papilla at E9 and E12. Preparations have been double-labeled to reveal HCA and cingulin through one channel (a, b) and cingulin alone through the other channel (a′, b′). Small arrows in a and a′ point to the same hair cell. By E12 each hair cell is surrounded by a number of roughly triangle-shaped supporting cells in this region. Big arrows in b and b′ point to the same hair cell. Scale bar (shown in b′): 10 μm (applies to all micrographs).

Fig. 7.

Whole mounts from the inferior-proximal papilla at E9 and E12. Preparations have been double-labeled to reveal HCA and cingulin through one channel (a, b) and cingulin alone through the other channel (a′, b′). At E9 (a), some supporting cells do not contact any hair cells (arrowheads); at E12 (b), virtually all supporting cells contact two or three hair cells. Note the large number of supporting cells around each hair cell at E12 compared with those at E9. Scale bar (shown in b′): 10 μm (applies to all micrographs).

Fig. 8.

Electron micrographs of E10 papillae that have been colloidal-gold labeled for the HCA. a, Two hair cells can be seen contacting at their apical border (arrow). b, c, Adjacent serial sections that show two gold-labeled hair cells separated by a supporting cell process (arrowhead in b), which come into contact at their apical ends (arrow inc). h, Hair cell; s, supporting cell. Scale bars: 500 nm (bar in c also applies to b).

Fig. 9.

Graph showing how hair cell–hair cell contacts change during papillar development. Values for the proximal, medial, and distal regions at each stage are indicated. Values above bars are the number of areas examined for each region. Counts were not made from the medial and proximal regions of E15 papillae because of the high degree of compaction of the supporting cell apical surfaces in these areas. Error bars represent SDM.

Fig. 10.

Double-labelling of cingulin and centrosome-associated material in papillar whole mounts.a, Photomontage of a medial region of the papilla at E12, spanning the entire width from the superior edge (left-hand side) to the inferior edge (right-hand side). Scale bar, 10 μm. b, b′, A medial region of an E10 papilla that has been double-labeled with antibodies to the centrosome-associated material and cingulin (b) and cingulin alone (b′). A single, discrete blob of staining can be seen within each cell (arrows, arrowheads) with the anti-centrosome-associated material antibody (b). Scale bar, 10 μm. c, Anti-centrosome-associated material labeling in the proximal region of a posthatch papilla. The antibody does not recognize centrosome-associated material of mature hair cells, and each brightly stained blob represents a single supporting cell. Arrows point to the unlabeled hair cells between each ring of supporting cell centrosomes. Note the greater number of centrosomes in the rings at the inferior edge of the papilla. Scale bar, 20 μm. S, Superior edge;I, inferior edge; arrows, hair cells;arrowheads, supporting cells.

Fig. 11.

Graphs of changes in contacts between hair and supporting cells from E9 and E12. a, c, e show changes in the number of supporting cells around each hair cell; b, d, f show changes in the number of hair cells that each supporting cell contacts. Graphs are for the central-distal (CD) region (a, b), superior-proximal (SP) region (c, d), and inferior-proximal (IP) region (e, f). Error bars represent SEM.