THE JOURNAL OF COMPARATIVE NEUROLOGY 3143614-625 (1991)

Hair Cell Differentiation in the Developing Chick Cochlea and in Embryonic Cochlear Organ Culture JENNIFER S. STONE AND DOUGLAS A. COTANCHE Department of Anatomy and Neurobiology, Boston University School of Medicine, Boston, Massachusetts 02118

ABSTRACT We have defined a method for growing chick embryonic cochleae in organ culture that preserves many aspects of hair cell differentiation. Cochlear ducts were isolated from embryonic day 8 chicks, placed in organ culture, and incubated for 48 hours (to a point equivalent to embryonic day 10). The cultured ducts were then fixed and processed for scanning electron microscopy. As controls, cochlear ducts at embryonic days 8 and 10 were dissected and immediately fixed and processed for scanning electron microscopy. We chose this period to culture cochleae because at the corresponding time in vivo hair cells undergo a dynamic phase of differentation. During this time, the number of stereocilia in the stereociliary bundle increases, and two to three rows of stereocilia nearest the kinocilium elongate, initiating the staircase pattern of the bundle. Also, the orientation of many hair cells shifts from nonpolarized at embryonic day 8 to polarized toward the inferior edge of the basilar papilla at embryonic day 10. Many of these aspects of hair cell differentiation proceed normally in organ culture. The appropriate distal-to-proximal gradients of hair cell density, apical surface area, and stereociliary number are preserved. Elongation of the 1-2 stereociliary rows next to the kinocilium continues, and more stereociliary bundles are oriented toward the inferior edge in cultured cochleae than in embryonic day 8 chicks. It appears that cochlear organ culture can serve as an effective method with which to study how hair cell differentation is regulated. Key words: stereocilia,hasilar papilla, scanning electron microscopy, hearing, tectorial membrane

The differentiation of chick cochlear hair cells is a superb example of the acquisition of form. Embryonic hair cells build an array of long actin-filled stereocilia on their apical surfaces (Flock and Cheung, '77; Tilney et al., '80),which they later use to transduce mechanical disturbances in the fluid of the scala media into neural signals (Lowenstein and Wersall, '59). In the adult, these stereocilia are arranged in rows in a staircase pattern, with the longest row oriented toward the kinocilium and embedded in the overlying tectorial membrane. From one end of the epithelium to the other, there exists a gradient in hair cell surface area and density and in the number and length of stereocilia (Tilney and Saunders, '83).Hair cells of the proximal basilar papilla have larger surfaces, are distributed more diffusely, and have more stereocilia than distal cells. Mammalian hair cells also display a proximal to distal gradient in stereociliary number and length (Lim, 'SO), but they have similar surface areas in all regions (Pujol, '91). The chick cochlear duct contains the auditory sensory epithelium known as the basilar papilla, which is thought to be equivalent to the mammalian organ of Corti. The precursor of the mature basilar papilla consists of a pseudo-

o 1991 WILEY-LISS. INC.

stratified layer of epithelial cells that are morphologically homogeneous and possess numerous microvilli and one kinocilium. By embryonic day 9 (E91, bundles of stereocilia have formed in most regions of the epithelium (Cotanche and Sulik, '84). These stereocilia are the same length as microvilli on adjacent supporting cells, but they are thicker in diameter. After E9, the stereocilia elongate and increase in number according to their position along the length of the basilar papilla (Tilney et al., '86, '88). The mechanisms by which hair cells become distinguishable from supporting cells and attain their morphological gradients along the basilar papilla are poorly understood. However, these structural modifications must involve the regulated synthesis of actin and its assembly into filaments (Tilney and DeRosier, '86; Tihey et al., '88).One of our interests is the identification of location-specific influences that direct stereociliary differentiation. Accepted September 16, 1991 Preliminary accounts of this study were presented at the Northeast Regional Developmental Biology Conference, April 1990 and at the American Society Cell Biology Meeting, December 1990.

ORGAN CULTURE OF EMBRYONIC COCHLEAR HAIR CELLS Results from previous experiments suggest that the chick inner ear is capable of establishing many of its morphological properties when removed from extrinsic inputs. The initial studies that demonstrated the capacity of the chick otocyst to differentiate into components of the inner ear in culture were performed by Fell ('28) and Friedman ('56). In 1989, Corwin and Cotanche showed that chick hair cells can develop position-specific properties such as stereociliary number and length when otocysts were grown on the chorioallantoic membrane, in the absence of continuous neural inputs. Swanson et al. ('90) found that when they denuded chick otocysts of mesenchyme and implanted them in limb bud mesenchyme, the normal epithelial derivatives differentiated but the global pattern of inner ear epithelia was disrupted. Despite these advances, the autonomy of chick hair cell differentiation in culture has not been thoroughly demonstrated. A culture system in which chick cochlear hair cells develop normal gradients and which also permits experimental manipulation would assist investigators who wish to examine the factors that guide hair cell differentiation. In order to determine whether hair cell differentiation can continue normally in isolated basilar papillae, we cultured cochlear ducts for 2 days beginning at E8. During this period in vivo many notable changes in hair cell morphology occur. The number of stereocilia increases, and the stepwise elongation of stereocilia is initiated (Cotanche and Sulik, '84; Tilney et al., '88). Also, hair cells adjust the orientation of their apical bundles (Cotanche and Corwin, '91). After examining the basilar papillae of E8 and E l 0 controls, we found that both hair cell density and apical surface area increase during this period. We compared the spatial and temporal progress of hair cell differentiation in cultured cochleae to controls. Our results show that chick hair cells retain their position-specific features and proceed with several steps of differentiation while in organ culture.

MATERIALS AND METHODS Dissection of the cochlear ducts White leghorn chick embryos at E8 (stage 34, Hamburger and Hamilton, '51) were removed from the egg and decapitated, and the temporal bones were isolated in sterile Hanks' buffered saline solution at pH 7.4 and 4°C. Cochlear ducts were removed from the cartilaginous precursor of the temporal bone, and the connective tissue surrounding the organ was teased away by using microforceps. This dissection usually necessitated opening the scala media in the proximal and distal ends of the cochlea. Large portions of the vestibulocochlear nerve often remained attached to the basilar papilla. Two cultured cochleae were embedded in plastic, sectioned for light microscopy, and stained with toluidine blue. Examination of these sections showed that there were very few, if any, ganglion cells left in the cultures. However, we can not rule out the possibility that ganglion cells were present.

Cochlear organ culture The culture apparatus that permitted sustenance and growth of the cochlear ducts for 48 hours combined materials used by Van De Water et al. ('731, Orr ('81), and Cotanche (unpublished data). Each duct was laid on Millipore filter paper soaked in Matrigel (Collaborative Research Inc), an artificial extracellular matrix. The filter paper was placed on a metal grid suspended in culture solution in an

615

organ culture Petri dish. The cochlear duct is shaped like a pea pod; the tegmentum vasculosum comprises one side and the basilar papilla another. Thus, it can rest on the filter paper in two positions, with either the tegmentum vasculosum or the basilar papilla contacting the Matrigel. Neither position seemed to affect growth of the cochlear duct adversely. The culture solution consisted of 10% fetal calf serum in L-glutamine-supplemented serumless medium, pH 7.0 (Gibco)and 1%antibiotic-antimycotic (Gibco). The culture apparatus was placed in a humid incubator with 5% CO,/95% air at 38°C for 48 hours.

Scanning electron microscopy Twelve of the fifteen cochleae we placed in culture remained uncontaminated and showed signs of significant growth. These cochleae were fixed in 2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4, 4°C) for 24 hours and postfixed with 1% osmium tetroxide in 0.1 M sodium phosphate buffer (pH 7.4,4"C) for 1 hour. The tegmentum vasculosum and tectorial membrane were removed from each cochlea, thus leaving the basilar papilla with its sensory epithelium exposed. We left the tectorial membrane intact in two cochleae for analysis. The basilar papillae were critical-point-dried (Samdri 780A, Tousimis), mounted on adhesive stubs, sputtercoated with goldipalladium (SEM Coating Unit, Polaron), and examined in either an IS1 60 or an Amray lOOOB scanning electron microscope. Additional cochlear ducts (10 at E8 and 10 at EIO) were dissected and fixed immediately after dissection to serve as controls. These tissues were processed for analysis as above. In four cases, the control (E8) and cultured cochleae were from the same animal (i.e., one cochlear duct was fixed immediately and the other was placed in organ culture). E l 0 controls are considered to be age-matched, or isochrorzzc, to cultured cochleae. We will refer to the control cochleae as E8 and E l 0 samples and the cultured cochleae as ElOx samples.

Morphological analysis We examined scanning electron micrographs of all control and cultured samples (a total of 32 cochleae) in a general qualitative manner. Three cochleae from each group (i.e., 3 E8's, 3 ElO's, and 3 ElOx's) were selected for analysis because they retained sufficient structural integrity throughout the dissections, thus enabling us to analyze hair cell morphologies in all three regions of the same ear. In some ears, there was structural damage due to tissue processing, and in others, portions of the sensory epithelium remained covered by tectorial membrane. Therefore, these ears were not used for quantitative analysis. One control (E8) and one cultured cochleae were from the same animal. Quantitative data were collected from scanning electron micrographs of proximal, middle, and distal regions of these cochleae. We considered the proximal region to be 5-lo%, the middle region to be 50%, and the distal region to be 90-95% of the total length from the proximal tip of the basilar papilla. We examined the following properties of these cochleae: hair cell apical surface area, stereociliary number, hair cell density, stereociliary elongation, and orientation of the stereociliary bundles. Techniques for each analysis will be covered individually. Apical surface area. Apical profiles of hair cells were traced on acetate sheets from scanning electron micrographs ( 3 , 0 0 0 ~of ) three regions of the basilar papillae for each experimental group. The regions we examined were midway along the width of the basilar papilla at proximal,

616 middle, and distal locales along the length. Six cells were measured from each micrograph. Cell tracings were digitized with the aid of a Hamamatsu C2400 video camera and a ComputerEyes video digitizer (Digital Vision) interfaced to an Apple Macintosh IICX. The apical surface areas of the cells were computed by using Image 1.21 (NIH Research Services branch), a digital image processing and analysis program. Stereociliary number. To determine the average number of stereocilia for hair cells, two scanning electron micrographs (7,000X ) were taken of hair cells in proximal, middle, and distal regions of control and cultured cochleae, mid-way between the inferior and superior edges of the epithelium. The photos were taken parallel to the apical surfaces of the hair cells so that tips of the stereociliary bundles were visible. With this arrangement, it was possible to count the individual stereocilia in each bundle. Stereocilia on six cells from each region were counted, and stereociliary numbers from these cells were averaged to obtain mean regional values for each cochlea. Hair cell density. Scanning electron micrographs ( 3 , 0 0 0 ~were ) taken from control and cultured cochleae at proximal, middle, and distal regions along the length of the basilar papilla. The average number of hair cells for a given area (100 bm2) in each region was calculated in the following manner. The photos were taken at three midwidth loci in each region (i.e., roughly halfway between the superior and inferior edges). A 6 x 6 cm square (comprising a 400 bm2area) was drawn on acetate and placed over each micrograph, and the number of hair cells within the square was determined. These numbers were then divided by 4 to give the number of hair cells in a 100 pm2 area, or hair cell densities. Next, hair cell densities for the three mid-width loci were averaged to obtain proximal, middle, and distal regional values. Stereociliary bundle orientation. Stereociliary bundle orientations were determined by the system described by Cotanche and Corwin ('91). Scanning electron micrographs ( 3 , 0 0 0 ~were ) taken of the inferior third of the sensory epithelium in proximal, middle, and distal regions of control and cultured cochleae. A small circle with 360" marks was drawn on acetate and placed over micrographs of hair cells so that the loo", 180", 260", and 360" marks pointed toward the proximal end, the inferior edge, the distal end, and the superior edge of the basilar papilla, respectively. Next, the kinocilium for the bundle was located and its orientation in degrees was determined. The degree value for each cell was then plotted on a graph to create a histogram for each region of the sample. On the X axis, 180" represented an orientation toward the inferior edge of the cochlea and 270" represented an orientation toward the distal end. The number of hair cells with each orientation was plotted on the Y axis. Stereociliary elongation. In order to determine whether elongation of stereocilia in rows near the kinocilium proceeds normally during the culture period, we examined hair ) cells from scanning electron micrographs ( 1 0 , 0 0 0 ~from proximal, middle, and distal regions of control and cultured epithelia. Tilney et al. ('88) have shown that until around E l 0 all of the immature stereocilia within each bundle are the same height. They observed that one to two rows of stereocilia nearest the kinocilium begin to elongate around E10. This elongation proceeds sequentially in adjacent rows until hatching, at which point stereociliary bundles from all regions of the basilar papilla have attained a full staircase

J.S. STONE AND D.A. COTANCHE configuration. We chose not to measure stereociliary heights because at this stage of development the stereocilia are only slightly higher t,han the microvilli of supporting cells, and it is difficult to visualize their entire length. Instead, we qualitatively determined the extent of stereociliary elongation in cultured and control cochleae by surveying hair cells along the entire sensory epithelium with the samples oriented at an angle so that the length of the stereocilia could be assessed.

Statistical analysis The data we obtained from our measurements of the three hair cell properties (apical surface area, stereociliary number, and cell density) were subjected to age versus position (2 age groups x 3 regions) multivariate analyses of variance (MANOVAs) using SYSTAT version 5.1. The MANOVAs compared the two age groups, the three regions, and the degree of interaction between groups and positions. The interaction value indicates whether a significant difference between groups is uniform across the three regions. Thus, a significant interaction value indicates that one of the three regions changes with a greater proportion than the other two regions. For the MANOVAs, differences between groups were considered significant if confidence levels exceeded 95% ( P < ,051. Post-hoc t-tests with Bonferoni adjustments were performed to determine differences between individual regions or groups. For these tests, differences between regions were considered significant if confidence levels exceeded 99.5% ( P < .005).

RESULTS Development of the cochlear duct in organ culture During normal development between E8 and E10, the chick basilar papilla elongated and changed its shape from a squat crescent to an oblong crescent (Cotanche and Sulik, '84; Tihey et al., '86). The maximum width of the basilar papilla (ie. the distance from the superior edge to the inferior edge) did not appear to change between E8 and E l 0 in middle and distal regions. However, the proximal end of the epithelium became more tapered as the basilar papillae grew in length. As a result, portions of the proximal end were narrower at E l 0 than they were at E8. These aspects are apparent in low power scanning electron micrographs of E8 and E l 0 controls (Fig. la,b). Fifteen cochlear ducts were dissected from chick embryos at E8 and placed in organ culture for 48 hours. Of these fifteen ducts, only three became contaminated or necrotic. The remaining 12 cochlear ducts grew significantly while in culture and displayed many normal morphological features. In most cultured cochleae, the width of the sensory epithelium appeared narrower in the proximal end and wider in the distal end, and the interposed region was tapered (Fig. lc). This gradient is typical of control cochleae at E10. However, cultured basilar papillae were usually wider than controls in all regions, and in many cases they displayed unusual shapes, often shorter or more curved than controls. In one cultured cochlea the epithelium appeared rippled, causing some hair cells to lie in troughs. Cochleae cultured for 2 days had tectorial membranes with characteristics of E l 0 controls (Fig. 2a,c). The tectorial membrane has two components which are formed by different populations of cells within the basilar papilla. The supporting cells begin to secrete the deeper, columnar

ORGAN CULTURE OF EMBRYONIC COCHLEAR HAIR CELLS

Fig. 1. Growth of the cochlea in culture. Scanning electron micrographs of cochlear ducts from controls at E8 (a)and at E 10 (b)and from 48 hour cultures ( c ) show the luminal surface of the basilar papilla (BP). All cochleae are oriented with their proximal (P) ends on the left and their distal (D) ends to the right; their superior edges are toward the top of the page. In viva, cochlear ducts grew in length and changed

617

shape between E8 and E10. Cochlear ducts that were grown in culture appear also to have elongated, although we have not taken formal measurements. Here, the cultured cochlear duct is supported by a piece of filter paper (arrowheads) that is concave as a result of dehydration. Thus, the curvature of the BP makes it appear much shorter than E8 and E l 0 control cochleae. Bar = 100 km.

618

J.S. STONE AND D.A. COTANCHE

Fig. 2. Development of the tectorial membrane and the tegmentum vasculosum in culture. a: Chick basilar papillae appear to have formed normal tectorial membranes while in culture. This micrograph shows a cultured cochlear duct with its tectorial membrane left intact. Bar = 100 bm. b: This tegmentum vasculosum was removed from a cultured cochlear duct. Its lateral folds are typical of an E l 0 tegmentum. Bar =

100 Fm. c: The amorphous and filamentous components of the tectorial membrane and the line that delimits the two components (arrowheads) are evident in this high power micrograph. Bar = 10 bm. d: The normal surface structure is apparent in this cultured tegmentum vasculosum. Large oval light cells are the predominant cell type at E10, the equivalent point during normal development. Bar = 10 pm.

component of the tectorial membrane at E7 (Cohen and Fermin, '85; Ganeshina, '85). The amorphous component, formed by the homogene cells, covers the columnar component at E9. The border of the two components is marked by a line that is initially inferior and slowly advances toward the superior basilar papilla as the tectorial membrane matures (Sheil and Cotanche, '90). Both the columnar and the amorphous components were apparent in the two cultured cochlear ducts from which we did not remove their tectorial membranes. The tegmentum vasculosum also appears to have developed normally while in culture. Between E8 and E l 0 in vivo, the tegmentum vasculosum evolves from a simple flat sheet of cells to a complex folded sheet of cells (Cotanche and Sulik, '82; Cotanche et al., ' 8 7 ) . Dark cells become interspersed between the originally uniform population of light cells. In culture, the tegmentum vasculosum became folded and displayed light cells, typical of an E l 0 cochlea (Fig. 2b,d).

also structurally intact. Hair cells and supporting cells were arranged in normal configurations; microvilli of supporting cells surrounded the stereociliary bundles of hair cells. The positional gradients in the appearance of hair cells and their distribution were evident in scanning electron micrographs (Fig. 3). To ensure that hair cells in fact developed properly while in culture for the 48 hour period, we examined five specific hair cell properties: hair cell apical surface area, stereociliary number, hair cell density, stereociliary elongation, and orientation of the stereociliary bundles. We

Development of the sensory epithelium in culture In cultured cochleae that appeared healthy at the macroscopic level, the organization of the sensory epithelium was

Fig. 3. Hair cells within control and cultured sensory epithelia. These scanning electron micrographs were taken from embryonic and cultured basilar papillae. In this series of micrographs, the inferior edges of all basilar papillae are toward the bottom of the page. At E8 (a,d,g),hair cells in the proximal (P),middle (MI, and distal (D) regions had relatively few stereocilia that were uniform in length. In E l 0 (b,e,h)and ElOx (c,f,i)cochleae, the number of stereocilia per hair cell had increased in all regions, and a few rows of stereocilia closest to the kinocilium had grown so that they were longer than the rest (arrowheads point to kinocilia). In both developmental stages and in cultures, these gradients in apical surface area, stereociliary number, and hair cell density are evident in this series of micrographs. Bar = 1 bm.

J.S. STONE AND D.A. COTANCHE a. Apical Surface Area

TABLE 1. MultivariateAnalysis o f Variance"

Developmental group

E8

El0

a. Apical surface area Pairing E8 vs. E l 0 E8 vs. ElOx E l 0 vs. ElOx b. Stereociliary number Pairing E8vs. E l 0 E 8 v s ElOx E l 0 vs. ElOx c. Hair cell density Pairing E8 vs. E l 0 E8 vs. ElOx E l 0 vs. ElOx

ElOx

Sample

b. S t e r e o c i l i a r y Number

t

f

P

1.48 41.45 10.37

2.88

84.94 117.92 2.78

,001 ,000

9.88 20.90 .812

Region f

Interaction P

f

P

21 27 5306 18.60

,001 ,000 ,001

.552 .192

596 799 829

171

102.44 29 78 50.24

.OOO ,000 .OOO

9.96 2.88 .148

007 115 865

,035 ,010 ,418

91.53 45.05 90.69

,000 ,000 ,000

.003 032

-230

17.46 3.53 3.9R

001 080 063

*Significant (P < .05) 200

L Y

2

Proximal 150

0

Middle

(5.407 pm2at proximal, 4.660 pm' at middle, and 3.690 pm' distal). 50 With respect to developmental changes, apical surface e areas increased slightly between E8 and E l 0 in each of the $ 0 E8 El0 ElOx three regions of the basilar papilla (Fig. 4a). Cultured hair cells also had greater apical surface areas than E8 samples Sample in all regions of the basilar papilla; in fact, their apical surface areas exceeded the values of E l 0 controls that were measured in corresponding areas. n The graphs of the surface area data in Figure 4a suggest E c. H a i r Cell Density a that a proximal-to-distal gradient in surface area exists in all three developmental groups we studied. MANOVA comparisons indicate that statistically significant effects of 3 15 I position are in fact present at each stage of development and in culture (Table 1).Post-hoc t-tests which compare c 10 v1 regional surface areas for all three developmental groups show that the positional difference that exists between the 2 5 r three regions is present mainly between the middle and Y u n distal regions (Table 2). When surface areas are statistically e E8 El0 E l Ox compare2 between different developmental groups, there [s i Sample no significant difference between apical surface areas at E8 and E l 0 (Table 1).In contrast, apical surface areas differ Fig. 4. Developmental differences in hair cell morphologies during significantly between E8 and ElOx samples and between development and in culture. These graphs illustrate the changes in (a) E l 0 and ElOx samples. When interaction between age and apical surface area, (b)stereociliary number, and fc) hair cell density that occurred in cochlear epithelia between E8 and E l 0 and while in position is examined, no significant interactions exist in any of the three pairings (E8 vs. E10, E8 vs. ElOx, and E l 0 vs. culture. Morphological data for each of the three regions o f the basilar ElOx). This suggests that hair cell surface areas changed in papilla (proximal, middle, and distal) are provided. a: For each bar, n = 18 cells (6 cells/regionianimal). b: For each bar, n = 18 cells (6 culture so that the proportionate regional gradients present cellsiregionianimal). c: For each bar, n = 9 separate 100 km2areas ( 3 in E8 and E l 0 cochleae were retained. areasiregionianimal). Error bars represent standard errors of the Stereociliary number. Region-specific gradients in stemean. reociliary number were apparent in E8, E10, and ElOx basilar papillae (Fig. 4b). At E8, proximal, middle, and distal hair cells had an average of 90,57, and 38 stereocilia, compared these data to results obtained from similar respectively. In contrast, at E10, proximal, middle, and distal hair cells displayed an average of 180, 97, and 81 examinations of E8 and E l 0 control cochleae. Apical surface area. The apical surface areas of hair stereocilia, respectively. Cultured basilar papillae also demcells appeared to vary systematically along the length of the onstrated a proximal-to-distal gradient in stereociliary numbasilar papilla in both E8 and E l 0 cochleae (Fig. 4a). At ber; proximal hair cells averaged 196 stereocilia, middle each developmental stage, the mean apical surface area in cells averaged 125 stereocilia, and distal cells averaged 100 the proximal region (3.615 pm2at E8 and 4.263 pm2at E10) stereocilia. The number of stereocilia per hair cell showed an agewas greater than the mean surface area in the middle (3.007 pm2 at E8 and 3.211 pm2 at E10) and the distal related increase in all three regions when E8 cochleae were (1.809 pm2 at E8 and 2.542 pm2 at E10) regions. Apical compared to E l 0 cochleae (Fig. 4b). Hair cells grown in surface areas of hair cells that were grown in culture culture had more stereocilia than hair cells from correspondmaintained region-specific values within the basilar papilla ing regions of E8 control cochleae. Moreover, cultured hair t

I L L

. Y

100

Distal

621

ORGAN CULTURE OF EMBRYONIC COCHLEAR HAIR CELLS TABLE 2. Post-hoc t-Tests' a. Apical surface area (developmental groups combined)

t

X

P

4.428 1 j 3 . 0 9

M

3 625

D (df = 161

2 677

>

3.65*

b. Stereociliary number

E8

El0 t

X

-~

P M

90.23 ~.. /=4.18* 57.16 \_ 2.41 38.0 ~

D = 101 (df c. Hair cell density

M

X

t

17" '> 96.7 --- ....

9.60' /Z=-

D (df = 101

t

X

P

Ig5>

M

124 lo()

D

3.55'

11 20 _---

(df = 10)

D

M t

~~

1.87

80.5

P X

E8

P

ElOx

X

t

X

t

~~

El0

El0

ElOx (df = 7)

ElOx (df = 7)

'P = proximal; M = middle; D *Significant (P < ,0051.

=

E8

E8

3.30

117 -0.37

6.78

El0 ElOx (df = 7)

10.75

distal

cells from proximal, middle, and distal regions had greater numbers of stereocilia than isochronic, E l 0 controls. When the data for stereociliary number are subjected to MANOVAs, a strongly significant effect of position on the number of stereocilia per cell for each of the developmental group pairings is demonstrated (Table 1).Post-hoc t-tests for the positional effect of stereociliary number in each of the three developmental groups show that, at E8, middle hair cells have significantly different numbers of stereocilia than proximal cells but not distal cells (Table 2). In cultured and E l 0 control epithelia, middle hair cells have significantly fewer numbers of stereocilia than those in the proximal region, but are not significantly different from hair cells in the distal region. When stereociliary numbers amongst the developmental groups are compared, there is a significant difference between E8 and E l 0 groups and between the E8 and ElOx groups. However, no significant difference between E l 0 and ElOx groups can be demonstrated. An interaction between group and position is present only in the comparison of E8 and E l 0 stereociliary numbers. Hair cell density. The density of hair cells, defined as the number of hair cells per 100 km2, increased progressively from the proximal to the distal end of the basilar papilla in E8, E10, and ElOx cochleae (Fig. 4c). At E8, the mean cell density was 3.34 cells per 100 kmZ,5.94 cells per 100 pm2,and 7.33 cells per 100 km2in the proximal, middle, and distal regions, respectively. This gradient was also evident in E l 0 samples where there were roughly 5.42 cells per 100 pm2 in the proximal end, 7.03 cells per 100 km2 in the middle region, and 13.39 cells per 100 km2 in the distal end. In culture, hair cells maintained the location-specific cell density pattern typical of isochronic controls with 5.42 cells per 100 km2,6.78 cells per km', and 10.75 cells per 100 km2 in the proximal, middle, and distal regions, respectively.

With respect to developmental changes, hair cell density increased between E8 and E l 0 in all regions of the basilar papilla. The most prominent changes occurred in the proximal and distal regions, while the number of hair cells per 100 p,mZ increased only slightly in the middle region (Fig. 4c). Cultured basilar papillae exhibited greater hair cell densities than E8 controls primarily in the proximal and distal regions. The density values for cultures were similar to E l 0 controls in the proximal and middle regions, but the distal region did not attain the densities present in the distal region of E l 0 controls. Strong positional gradients are present at all three developmental stages following MANOVAs (Table 1).There are significant age-related differences between E8 and E 10 and between E8 and ElOx, but not between the E l 0 and ElOx. A significant interaction between the group and position values exists only between E8 and EIO, and from the graph, it is apparent that this is caused by the large increase in density in the distal region at E10. This is reinforced by post-hoc t-tests which compare developmental differences in cell density in the three regions (Table 2). The only significant differences are seen in the distal region, where E8 densities are significantly smaller than both E l 0 and ElOx densities, but E l 0 values are not significantly different from ElOx values. Orientation of stereociliary bundles. Hair cells from all regions of E8 epithelia had haphazardly oriented stereociliary bundles, whereas E l 0 epithelia displayed more uniformly aligned hair cells. Specific changes in hair cell orientation can be seen in the histograms (Fig. 5 ) . At E8, hair cells in all regions had orientations that were widely splayed on the X axis of each histogram. Many hair cells in the middle and distal regions pointed toward 270", the distal end. In contrast, the distributions of E l 0 hair cells in all regions were narrower, and more hair cells pointed away from 270" and toward 180", the inferior edge. Thus, hair

622

20[P,.,* ;!Lj-;;[,T-sh J.S. STONE AND D.A. COTANCHE

E10

EIOX

P

=,,,

10

20

100

180

260

360

M

20 10 [, 20

100

180

260

360

20

100

180

260

360

M

, - , 100

,

260

'I,, - , 100

::b,, ,A,,

-11

180

360

D

20

20

180

260

,

20

180

260

360

20

100

180

260

, 360

100

180

260

360

20

D

D

: 360

100

100

180

260

360

20

Fig. 5. Quantification of hair cell reorientation in control and cultured basilar papillae. Hair cell orientations in proximal (PI, middle iM), and distal iD) regions of three samples at each age iE8, E10, and ElOx) were determined, and histograms were created (see Materials and Methods). On the X axis of each histogram, loo", N O " , 260", and

360" represent hair cell orientations toward the proximal end, the inferior edge, the distal end, and the superior edge, respectively. The number of hair cells analyzed is represented on the Y axis of each histogram. The histograms illustrate data collected from one sample at each age.

cells at E l 0 were less haphazardly oriented than those at E8, and tended to point toward the inferior edge of the basilar papilla as opposed to its distal end. Cultured hair cells had narrower distributions of orientation in middle and proximal regions, demonstrating that reorientation took place in cochlear cultures (Fig. 5). However, in the distal ends of cultured basilar papillae, reorientation did not appear to occur. Here, hair cells displayed a wider range of orientations than isochronic controls and tended to point toward the distal end of the basilar papilla. Elongation of stereocilia. Hair cells from E8 epithelia had bundles with stereocilia that were uniform in length in all regions of the sensory epithelium (Fig. Sa,d,g). At this developmental stage, hair cell orientation was indicated only by kinociliary location. At E10, however, at least 1-2 rows of stereocilia nearest the kinocilium had elongated on many hair cells in all regions of the epithelium (Fig. Sb,e,h). The degree of stereociliary elongation appeared equal for most hair cells in each region, although regional differences were evident. For example, distal hair cells displayed more elongated rows of stereocilia than middle and proximal hair cells. These results support data from previous studies which demonstrated that hair cells initiate formation of the staircase configuration of their stereociliary bundles between E8 and E l 0 (Tilney et al., '88). In cultured basilar papillae, we observed a similar pattern of stereociliary growth (Fig. 3c,f,i). Hair cells in proximal, middle, and distal regions had 1-2 rows of elongated stereocilia situated close to their kinocilia. As in E l 0 controls, the development of stereociliary elongation was synchronous throughout the

majority of the cultured hair cells within each region of the cultured epithelia, and differences among the regions existed. In fact, hair cells at E l 0 and those that have grown for 2 days in culture are difficult to distinguish morphologically.

DISCUSSION We chose to grow cochlear ducts in culture from E8 to E l 0 because these 48 hours constitute a dynamic developmental period during which the sensory epithelium expands and hair cells attain several distinct and positionspecificmorphological features. We examined the acquisition of these features in chick cochleae that were grown in organ culture in order to determine whether they form in the absence of continuous extrinsic inputs, such as neural or humoral growth factors. Previous experiments utilized cell culture and explantation to demonstrate that the embryonic chick cochlea is capable of developing gross characteristics of the mature sensory epithelium while relying primarily on intrinsic guidance (Fell, '28; Friedmann, '56; Orr, '81; Corwin and Cotanche, '89; Swanson et al., '90). As of yet, it has not been demonstrated that chick hair cells grown in culture are capable of developing region-specific features in the absence of extrinsic inputs. During the period between E8 and E10, the basilar papilla increases in length but maintains the maximal width attained by E8 (Cotanche and Sulik, '85).In addition, the basilar papilla changes its shape from crescentic to spatula-like. The basilar papilla continues to grow in organ

ORGAN CULTURE OF EMBRYONIC COCHLEAR HAIR CELLS culture, although it attains shapes and dimensions that differ from those formed in vivo. These phenomena suggest that the morphogenesis of the chick cochlea is influenced by the growth of the temporal bone. By isolating the cochlear duct in organ culture, this influence is removed and the epithelium may be free to attain new forms. Our findings agree with Swanson et al. (’go), who showed that transplanted cochlea continue to grow despite their displacement and with Corwin and Cotanche (’891, who found that transplanted cochleae exhibited variable, especially shorter, basilar papillae. It is interesting to note, however, that the relative position-dependent changes in the width of the basilar papilla occur in cochlear organ culture since distal regions are easily distinguished from the narrower proximal regions. In our cochlear organ culture apparatus, hair cells continue to differentiate the following morphologic features in a manner that parallels normal development: apical surface area, stereociliary number, hair cell density, reorientation of the stereociliary bundle, and elongation of stereocilia. Hair cells appear to increase their apical surface areas slightly between E8 and E10, although this increase is not statistically significant. This may be attributable to the small number of cochleae we sampled at each age, the small number of hair cells we examined in each region, or a high variability in surface area in all regions of the basilar papilla at E10. This increased variability is probably due to additional differentiation of hair cells in each region between E8 and E10. Position-specific gradients in hair cell surface area exist in basilar papillae at both developmental stages. In culture, hair cells maintain a positional gradient in surface area, yet their surface areas are larger than isochronic controls in all regions. This finding is not surprising since cultured basilar papillae are usually wider than controls throughout their length. It is possible that hair cells expand in culture because there is less spatial restraint. It is also conceivable that, since hair cell surfaces are still expanding at E l 0 (Tilney et al., ’861, the increased surface areas seen in E lox cultures represent a more advanced developmental state. At the point we introduce cochlear ducts into organ culture (E8), differentiation of stereociliary bundles is underway. Previous studies have shown that hair cells are first detectable in the distal region at E6, in the middle region by E7.5, and in the proximal region by E9 (Cotanche and Sulik, ’84; Tilney et al., ’86). At these early stages of hair cell differentiation, a region-specific gradient in the stereociliary number is already evident. Yet, the number of stereocilia per cell is not final, for it continues to increase in all regions of the epithelium as development progresses until the mature numbers in the gradient are achieved. The stereociliary numbers we observe at E8 and E l 0 concur with previous developmental studies on stereociliary differentiation. In cochlear organ culture, hair cells increase the number of their stereocilia as they grow in a positionspecific manner. This suggests that the factors that regulate the region-specific budding of stereocilia are intrinsic to the cochlear duct, or that they have had their inductive effect on the cochlear epithelium prior to E8. Although the sensory epithelium of the cochlea grows in length between E8 and E10, our data indicate that hair cell density increases significantly during this period in the proximal and distal ends. This is not surprising since many new hair cells differentiate within these regions between E8

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and E10. But it is not clear why hair cell density remains constant in the middle region. Perhaps hair cell differentiation is completed between E7.5 and E8 in the middle region, and the hair cell density remains unchanged there as a result. In this case, new hair cells would only emerge in areas that flank the middle region after E8. This developmental trend is reminiscent of mammals, in which hair cell differentiation is completed in the mid-basal region first and is followed by progression of maturation toward either end (Bredberg, ‘68). Another explanation for the static nature of hair cell density in the middle region is that the middle portion of the basilar papilla, being intermediate to the proximal and distal ends, has a continuum of hair cell densities along its length. Hence, to sample the same “middle” site in different individuals is a difficult task to accomplish with precision. In culture, hair cell density changes as it does in vivo; it increases in the proximal and distal ends and remains the same in the interposed region. However, in the distal end of cultured epithelia, the number of hair cells per 100 pm2tends to be somewhat less than in the corresponding region of E l 0 epithelia. This is not surprising since basilar papillae grown in culture are often wider in the distal region than isochronic controls, and one would assume that if the same number of hair cells were to become distributed in a wider epithelium, their density would be diminished. Despite the temporal difference in their emergences in vivo, stereociliary bundles in all regions of the cochlea begin to elongate at the same t i m r a r o u n d E l 0 (Tilney et al., ’88). Elongation starts in the one to two rows of stereocilia nearest the kinocilium. This process involves addition of actin monomers to the plus end of the existing actin filaments in a spatially and temporally controlled manner. Factors that regulate actin polymerization in the basilar papilla have not been established. Tilney and collaborators (’86,’88) have suggested that actin capping proteins, which bind the plus end of the filament and prevent monomer addition, may be sequentially released from the filament at different sites within the stereociliary bundle. As a result of this regulated uncoupling, actin polymerization (and hence, stereociliary elongation) would occur in the characteristic stepwise manner. These influences appear to be effective in culture since stereociliary elongation occurs normally and parallels in vivo development. Between E9 and P3, hair cells in all regions of the basilar papilla become aligned toward its inferior edge (Cotanche and Corwin, ’91). They are oriented in many different directions at E8, the point at which we put cochlear ducts in culture. Hair cells that are grown in culture for two days starting at E8 begin to reorient their stereociliary bundles in proximal and middle regions. However, there is no evidence of reorientation in the distal ends of cultured basilar papillae. It is possible that the curvatures of the cultured basilar papillae may have affected our analysis of hair cell orientations in the distal region. For example, many cultured cochleae have unusual contours of the inferior edge at the distal ends. It is possible that our estimation of the inferior edge’s direction was imprecise; this would have shifted the histograms in one direction. The mechanism which guides reorientation has not been established. Hair cells may remodel their apical membranes so that the stereociliary bundle’s direction is adjusted or the entire cell may rotate to face the inferior edge. Corwin and Cotanche (’86) have postulated that the tectorial mem-

J.S. STONE AND D.A. COTANCHE

624 brane, which is attached to the tallest row of stereocilia (Engstrom and Engstrom, '781, may guide reorientation as it moves across the basilar papilla. Since the tectorial membrane appears to have formed normally in cultured cochleae, it is plausible that this mechanism may also direct reorientation in culture. Our results support previous findings that the cochlear duct need not be an intact structure to differentiate appropriately. Russell and Richardson ('87)demonstrated that if cochleae from postnatal mice were bisected, isolating the distal end from the proximal end and exposing the sensory epithelium to solutions with a different ionic composition than endolymph, hair cells still developed most of their normal characteristics. Swanson et al. ('90) discovered when they transplanted only the ventromedial half of the chick otocyst in limb bud mesenchyme, sensory epithelial structures still formed. In our cultures, position-specific hair cell differentiation proceeded in a normal fashion although the dissection of the cochlear duct necessitated opening the scala media and bathing the basilar papilla with culture medium. Many investigators have demonstrated the importance of mesenchyme in directing normal development of the sensory epithelium of the inner ear (Orr, '76; Van De Water et al., '80; Anniko, '85; Swanson et al., '90). Our study did not address this influence since we paid little attention to the frequency with which mesenchyme remained attached to the cochlear ducts. It is interesting to note, however, that the basilar papilla appeared to develop normally no matter whether the tegmentum vasculosum or the basilar papilla contacted the Matrigel, a substance with much the same composition of extracellular matrix. Our results confirm the notion that the cochlear duct contains the appropriate information and/or has already received the necessary external stimulus at E8 to autonomously direct differentiation of hair cell stereociliary bundles. This complex process necessitates precise temporal and spatial regulation by factors which have not been pinpointed. Cochlear organ culture is an excellent system with which to examine these developmental influences since it renders the cochlear duct accessible to chemical and mechanical manipulation. With these tools, one can explore molecular mechanisms that influence the differentiation of hair cells. Of considerable interest is the identification of location-specific influences that direct the gradients in stereociliary differentiation. There are many examples of molecules that behave as morphogens toward cells within their proximity such as cell adhesion molecules in the cochlea (Richardson et al., '87; Raphael et al., '88) and retinoic acid in the limb bud mesenchyme (Tickle et al., '82; Summerbell, '83).These molecules must establish a specific pattern or gradient within a tissue to be effective in regulating a specific cell or group of cells. It would be of great value to demonstrate the presence of a similar gradient in the basilar papilla that correlates with the position-specific expression of hair cell morphologies.

ACKNOWLEDGMENTS We would like to thank Dan Picard for his excellent technical assistance, Wende Reenstra for her guidance in tissue culture techniques, Dr. Linda Wright for her assistance with the image analysis system, and Drs. Linda Wright, Lynne Werner, and Edwin Rube1 for their assistance in setting up and interpreting the statistical analysis of our results.

This work was supported by NIDCD grant DC00412 (D.A.C.)and NINDS training grant T32NS07152 (J.S.S.).

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