Planar and vertical signals control cellular differentiation and patterning in the mammalian cochlea

Abstract

The sensory epithelium of the mammalian cochlea is composed of a regular mosaic of sensory hair cells and nonsensory supporting cells. During development, differentiation occurs in a gradient that progresses along the axis of the cochlea from base to apex. To begin to identify some of the factors that regulate this developmental process, the potential roles of planar and vertical signals were examined during early stages of cochlear development. We demonstrate roles for both underlying mesenchymal cells and adjacent epithelial cells in the differentiation and patterning of the sensory epithelium, and in particular in the development of mechanosensory hair cells. As development proceeds, the requirements for both planar and vertical signals decrease, and development of the sensory epithelium becomes essentially independent from these cues. Finally, we demonstrate that the temporal gradient of cellular differentiation is not dependent on planar signals within the developing sensory epithelium.

Figure 1.

Differential interference contrast image of an intact E13.5 cochlear epithelium after removal of the underlying mesenchymal cells by treatment with thermolysin. Apical (Apex) and basal (Base) ends of the cochlear duct are indicated. The solid line represents the location for separation of the single apical piece. Dashed lines represent subsequent subdivisions to create basal pieces that are of approximately equivalent sizes to the single apical piece.

Figure 2.

Mesenchymal cells are not required for development of hair cells or cellular patterning. A, Low-magnification image of an intact cochlear epithelium isolated on E13.5 and maintained in the absence of mesenchymal cells (-mes) for 5 DIV (E13.5 + 5 DIV). A band of myosin VI-positive hair cells (red) extends along the entire length of the explant. The inner hair cells are separated from the outer hair cells by a line of pillar cell heads, labeled with the actin marker phalloidin (green). Inset, High-magnification image of the basal region of a living E13.5 + 7 DIV/-mes intact epithelium. Hair cells have been labeled by rapid uptake of FM1-43, a dye that is thought to enter hair cells through active transduction channels (see Results for details). Note that the hair cells are arranged into a characteristic pattern of a single row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs). The row of pillar cell heads (PCs) located between the row of inner hair cells and first row of outer hair cells is devoid of staining. B, Low-magnification view of an intact E13.5 + 5 DIV/-mes cochlear epithelium. A characteristic pattern of inner and outer hair cells is present. Hair cells and pillar cells are labeled as in A. C, Low-magnification image of an intact E12.5 + 6 DIV/-mes cochlear epithelium. Hair cells and pillar cells are labeled as in A. A characteristic cellular pattern is present in the basal region of the explant (Base), but supernumerary outer hair cells are present in the apex. Inset, High-magnification view of the boxed region illustrating the cellular pattern of the organ of Corti, including one row of IHCs separated from the three or more rows of OHCs by the PCs. D, Low-magnification view of an intact E12.5 + 6 DIV cochlear duct maintained with mesenchymal cells (+ mes). Hair cells and pillar cells are labeled as in A. Note the similar development of supernumerary outer hair cells in the apical region of the explant. Scale bars: A-D, 100 μm; A, C, insets, 50 μm.

Figure 3.

Hair cell differentiation and alignment in basal and apical pieces of cochlear epithelium isolated from mesenchyme and other regions of the cochlea on E13.5 or E12.5. A, Basal cochlear epithelial piece from an E13.5 + 5 DIV/-mes explant. Hair cells (labeled with anti-myosin VI in red) are arranged in a single row of inner hair cells (arrows) and three rows of outer hair cells (brackets) separated by a single row of pillar heads (labeled with phalloidin in green). B, Basal epithelial piece from an E12.5 + 6 DIV/-mes explant. Hair cells (labeled with anti-myosin VI in red) are arranged in a line, but specific rows of inner and outer hair cells have not developed. C, Cross section of the sensory epithelium in a basal E13.5 + 3 DIV/-mes explant. A single inner hair cell (IHC) and two outer hair cells (OHCs) are labeled with anti-myosin VI. A third outer hair cell (star) is also present but is stained less intensively. D, Basal epithelial piece from an E13.5 + 5 DIV/-mes explant. The pillar cell marker p75^ntr (green) is expressed in the region between the inner (arrows) and outer hair cells (stained in red with anti-myosin VIIa). E, Basal epithelial piece from an E13.5 + 5 DIV/-mes explant. Prestin, a specific marker for outer hair cells, is expressed in outer hair cells (bracket) but not in inner hair cells (arrowheads). F, Apical epithelial piece from an E13.5 + 5 DIV/-mes explant labeled with phalloidin. Stereociliary bundles (arrows) are intensely labeled with phalloidin (green) and have a “(” morphology that is characteristic of cochlear hair cells. In addition, the presence of stereociliary bundles indicates that the hair cells have become polarized along the luminal-basal axis. G, Basal epithelial piece from E13.5 + 5 DIV/-mes explant. An arc of hair cells (labeled with anti-myosin VI) extends along the entire length of the piece, and a disrupted but recognizable cellular pattern is present. The apparent increase in the total number of hair cells by comparison with basal pieces maintained in medium with serum is probably caused by a lack of spreading of the epithelial piece. Scale bars: A, B, 50 μm; C-F, 25 μm; G, 100 μm.

Figure 4.

Associated mesenchymal cells are instructive for hair cell differentiation and patterning. A, Apical epithelial piece from an E13.5 + 5 DIV/-mes. An arc of hair cells (labeled with anti-myosin VI in all panels) extends along the length of the explant. B, Apical epithelial piece from an E12.5 + 6 DIV/-mes explant. The number of hair cells is significantly reduced by comparison with an apical piece from E13.5 + 5 DIV/-mes (Table 1), and no cellular pattern is evident. C, Apical piece from an E12.5 + 6 DIV/+mes explant. By comparison with apical pieces cultured without mesenchyme (B), there is a significant increase in the number of hair cells (Table 1) and a marked improvement in the level of cellular patterning (rows), including the formation of specific rows of hair cells and a row of pillar heads. D, Apical epithelial piece from an E12.5 + 6 DIV/-mes explant that was maintained on top of a monolayer of mesenchymal cells (visualized by labeling with phalloidin in green) that were isolated from an E13.5 cochlea and maintained for 7 DIV before introduction of the apical epithelial piece. The number and pattern of hair cells (red) appear similar to apical epithelial pieces maintained without mesenchymal cells. E, Apical epithelial piece from an E12.5 + 6 DIV explant that was reassociated with its endogenous underlying mesenchyme (see Results for details). There is a significant increase in the number of hair cells, and a characteristic pattern of hair cells and supporting cells is present. Scale bars, 100 μm.

Figure 5.

Expression of Math1 in E12.5 + 6 DIV epithelia. A, Math1-positive cells (red) are present in an arc that extends along the length of the explant in an intact E12.5 + 6 DIV/+mes. To aid in visualization of the sensory epithelium, filamentous actin has been labeled in green. Orientation of the explant is as indicated. B, C, Isolated E12.5 + 6 DIV/-mes apices labeled and oriented as in A. The region of high cell density in each explant corresponds with the area that would normally develop as the sensory epithelium. Only a few Math1-positive cells were detected in either sample (C, arrows). D, High-magnification image of the sensory epithelium from an intact E12.5 + 6 DIV explant with mesencyme labeled as in A. Note the single row of Math1-positive inner hair cells and the three rows of Math1-positive outer hair cells. E, High-magnification image of the high cell density region from an isolated E12.5 + 6 DIV apex. Few Math1-positive cells are present (E, arrow). Scale bar: (in A) A-C), 100 μm; (in E) D, E, 50 μm.

Figure 6.

Differentiating hair cells are present in the cochlear epithelium at E13.5 in vivo. A, Whole mount of the entire cochlear duct at E13.5 in vivo. A single line of myosin VI-positive cells extends along the basal 75% of the cochlea. B, High-magnification image of the boxed region in A, illustrating the single line of myosin VI-positive presumptive inner hair cells. Scale bars: A, 100 μm; B, 25 μm.

Figure 7.

The basal-to-apical gradient of cellular differentiation is not dependent on planar cues. E12.5 cochleas were separated into an apical (25% of total length) and basal (75% of total length) piece and maintained in vitro for the indicated time periods. A, Basal and apical pieces after 1 DIV. For the basal piece, the extreme base of the cochlea (star) and the separation site (dashed line) are indicated. For the apical piece, the separation site (dashed line) is indicated. No hair cells are present in either piece. B, Apical and basal pieces after 2 DIV. Both pieces are oriented as in A. In the basal piece, a band of myosin VI-positive hair cells (arrows) extends from the base of the cochlea toward the separation site. No myosin VI-positive cells are present in the apical piece. C, Apical and basal pieces after 3 DIV. Both pieces are oriented as in A. The band of myosin VI-positive cells in the basal piece now extends along the length of the basal piece. Myosin VI-positive cells are also present in the apical piece at this time point. Note that in the apical piece expression of myosin VI is most intense in the region closest to the separation site (arrowhead). Inset, High-magnification image of the band of myosin VI-positive cells in the apical piece after 3 DIV. D, Apical and basal pieces after 4 DIV. Myosin VI-positive hair cells now extend along the full length of both the apical and basal pieces. Scale bar: (in D) A-D, 100 μm; inset, 50 μm.