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SEM analysis of the developing tectorial membrane in the chick cochlea
Hearing Research, Elsevier
HEARES
47 (1990) 147-158
147
01397
SEM analysis of the developing tectorial membrane in the chick cochlea Monica Jean Shiel and Douglas A. Cotanche Department
ofAnatomy, Boston University School of Medicine Boston, Massachusetts, (Received
16 August
1989; accepted
10 March
U.S.A.
1990)
The development of the tectorial membrane in the embryonic chick cochlea was studied using scanning electron microscopy. Chick embryos ranged in age from embryonic day 7 (E7) to post-hatching day 15. Our studies revealed that a fine filamentous matrix arose on the apical surface of the basilar papilla at approximately E7. This matrix was secreted by the supporting cells which encircled the hair cells. By E9, the early matrix had increased in vohrme but remained filamentous in structure, except at the inferior edge of the basilar papilla where it was condensed into a layer of laterally-oriented columns. At E9 the TM exhibited an additional layer of matrix, called the amorphous component. It appeared to originate from the homogene cell population, and attached to the early columar matrix at the inferior edge of the basilar papilla. The two components of the TM were separated by a longitudinal ridge, called the ‘track’, which marked the inferior edge of the amorphous component. As the cochlea developed, the basilar papilla increased in width, the columnar component elongated and the track appeared to recede. These morphological findings point to separate developmental origins for the two components of the tectorial membrane. Tectorial
Membrane,
Development,
Cochlea,
Basilar
Papilla,
Chick
The tectorial membrane (TM) is a hydrated extracellular matrix located over the lumenal surface of the basilar papilla in the avian cochlea. It is currently thought to function as a point of insertion for the tallest stereocilia of the hair cells, and thus provide the necessary shearing forces needed to open ion channels in the hair cell stereocilia. Opening these channels initiates signal transduction within the hair cell. The TM is situated with its superior edge anchored to the homogene cells, which are located in the superior wall of the cochlear duct. The inferior edge of the TM is anchored to the supporting cells along the inferior edge of the basilar papilla (BP). In cross section, it is seen to have a wedged shape, with its thicker edge in contact with the homogene cells, and its tapered edge in
Correspondence to: Douglas A. Cotanche, Department of Anatomy, Boston University School of Medicine, 80 East Concord Street, Boston, MA. 02118, U.S.A.
0378-5955/90/$03.50
0 1990 Elsevier Science Publishers
contact with the inferior side of the BP (Jahnke et al., 1969; Hoshino, 1973; Tanaka and Smith, 1975). The lumenal surface of the TM appears smooth along its superior border, while the remainder of its surface is segregated into laterally oriented colu~s. The underside of the TM exhibits a honeycombed pattern, with each cylinder in the honeycomb encircling a single hair cell. The undersurface of the TM is attached to the longest row of stereocilia on each hair cell and anchored to the supporting cells surrounding the hair cells by a fine fibrillar matrix of extracellular material (Jahnke et al., 1969; Dohlman, 1971; Rosenhall, 1971). The biochemical composition of both the mammalian and avian TM is mainly glycosaminoglycans and glycoproteins (Richardson et al., 1987; Khalkhali-Ellis et al., 1987; Thalmann et al., 1987). However, the details of the polypeptide composition of the avian TM remain obscure. One dimensional gel electrophoresis followed by band digestion with bacterial collagenase showed that Type II collagen, a glycoprotein, is found in abundance in the mammalian TM, but does not appear
B.V. (Biomedical
Division)
148
to be present in avian TM (Thalmann et al., 1987). It has not been documented as to whether types of collagen other than Type II exist in the avian TM. Development of the chick TM was initially studied using light and transmission electron microscopy (Cohen and Fermin, 1985; Ganeshina, 1985). It was reported in these studies that TM secretion began on E7 and the majority of the TM appeared to be secreted from the homogene cell population, with the supporting cells in the BP responsible for the secretion of a lesser or “subtectorial’ TM. Observations of the events involved in TM regeneration following noise damage led us to further investigate the embryonic development of the TM. When the chick cochlea was exposed to noise damage, the TM was destroyed over the region of hair cell damage, but after a period of recovery, it was regenerated. New TM was secreted exclusively from the supporting cell population to form a honeycomb surrounding the newly regenerated cells, with no cont~bution from the homogene cells (Cotanche, 1987b). In order to examine the relationship between regeneration in the TM and its original production in the embryo, we have examined the normal development of the TM using scanning electron microscopy. By using gentle fixation procedures, as well as viewing living, unfixed tissue, we were able to observe the developing TM in a state comparable to its in vivo state. Methods Fertilized white leghorn chicken eggs were obtained from SPAFAS Hatcheries Inc. (Norwich, CT). The eggs were incubated at 37.5OC and 40% hu~dity in a laboratory incubator. Embryonic chicks were dissected out of the egg and staged according to the specifications of Hamburger and Hamilton (1951). Embryos used in this study ranged from stage 32 (embryonic day 7, E7) to stage 46 (E21, hatching). Hatchlings were obtained from SPAFAS on post-hatching day 1 (Dl ) and allowed to mature in a heated brooder until post-hatting day 15 (D15). At least ten embyos and hatchlings were examined per stage. For fixation of the cochleas the temporal bones were isolated and placed in HEPES-buffered Hanks’ balanced salt solution (HHBSS). The
buffer was oxygenated and maintained at pH 7.4 at 4°C. The initial steps in the dissection isolated the cochlea from the surrounding tissue of the cranial cavity. The superior portion of the labyrinth was dissected to expose the Scala media to the fixative. The tympanic membrane was then removed, the columella (stapes) extracted, and the round window was punctured to allow the fixative to permeate the Scala vestibuli, Scala tympani and Scala media, thus accessing the tectorial membrane. For each chick embryo both ears were dissected in under ten minutes, and immediately immersed in fixative. Primary fixation was achieved in 2% glutaraldehyde in 0.1 M sodium phosphate buffer at pH 7.4 and 4°C for 24 h. The tissues were then postfixed in 1% Osmium tetroxide in 0.1 M sodium phosphate buffer at pH 7.4 for 45 minutes at 4°C. In preparation for SEM analysis, the tissues were dehydrated up to 70% &OH, and the apical surface of the tectorial membrane was fully exposed by dissection of the overlying tegmentum vasculosum. After the final dissections, the tissues were dehydrated to 100% EtOH, and the samples were critical point dried with CO, as a transition fluid in a Tousimis Samdri Model 780A. Tissues were sputtercoated with gold/palladium in a Polaron E5000 sputtercoater and then examined with an ISI 60 scanning electron microscope at 30 kV. In order to ensure that our SEM observations were representative of structures present in the living, unfixed cochlea. the TM was isolated from unfixed, living cochleae at E13. The cochleae were rapidly removed from the temporal bones by a technique described in a previous publication (Tilney et al., 1989) and placed in oxygenated HHBSS. The tegmentum vasculosum was removed from each ear and the BP was immersed in Protease Type XXVII (Sigma, No. P4789) at a concentration of 0.05 mg/ml for 2 min and then placed back in protease-free HHBSS. The tectorial membrane was dissected off the BP and placed on a clean glass slide. The structure of the unfixed embryonic TM was examined on a Zeiss Axioskop equipped with differential-interference-contrast (DIC) optics and viewed with video-enhanced contrast through a Hammamatsu C2400 video camera.
149
Results
The mature TM (IX5) covers the lumenal surface of the BP (Fig. la) and a higher magnification view of the TM demonstrates the fine structure of its lumenal surface (Fig. lb). A thin line is
present at the superior edge of the TM and is located lon~tudinally from the proximal to distal ends of the BP. This line serves to divide the TM into two morphologically distinct components. We have named the two components according to their textures observed using the SEM. The smooth, ‘amo~hous’ matrix exists superior to the line or ‘ track’, and is anchored to homogene cells. The striated, or Lcolumnar’ matrix is inferior to the track, and is divided into discrete columns of matrix which elongate as the embryo matures. A second dimension of the TM exists, since the columnar matrix also lies below, or deep to, the amorphous matrix. Initially, the columnar matrix is fibrillar, and it alone covers the surface of the BP. At a later developmental stage, the amo~hous layer of matrix is secreted over the developing columnar layer. In Fig. lc, at E18, the amorphous layer has been dissected away to reveal the underlying columnar matrix. The columnar component of the TM is first seen at E7 and appears as clumps of fibrillar, extracellular matrix-like material on the lumenal surface of the BP (Fig. 2a). Prior to this developmental stage, no matrix was discernable in the SEM preparations. This early secretion of the matrix is localized over the supporting cell microvillae. By E8 (Fig. 2b), a larger volume of matrix is secreted and it covers both the hair and supporting cell surfaces in a thick, matted layer. The amorphous component of the TM appears at E9 as a second layer which covers the initial columnar matrix, and it exhibits a longitudinal
Fig. 1. The structure of the avian tectorial membrane. (a) For orientation purposes, the mature tectorial membrane (TM) is shown covering the BP in the cochlea of a D15 chick. The wider, distal BP is to the left. The proximal end is located at the right in this SEM photograph. Superior (Sup) and inferior (Inf) are designated. Homogene cells (Ho), are located uniformly along the superior border of the BP. At this magnification the track is faintly distinguishable (arrows). Bar = 500 pm. (b) At a higher magnification of the mid-apical surface of the BP, the TM exhibits a track (arrows), which separates the inferior columnar TM from the superior amorphous TM. Bar =lOO am. (c). The underlying nature of the matrix which makes up the second dimension of TM structure is easily seen in this SEM photograph from an El8 cochlea. The track remains evident as well (arrows). Bar =lO am.
Fig. 2. Early development of the avian tectorial membrane. (a) The BP is shown here at E7 with the supporting cell population secreting the initial matrix-like material. This matrix later forms the columnar portion of the tectorial membrane. Bar = 10 pm. (b) By E8, a larger volume of matrix has been secreted, and the hair cells are now covered by a thick mat of TM, which has been dissected away in order to visualize the underlying BP. An underlying hair cell (HC) is identified by its stereociliary bundle. Bar =lO pm. Both SEM photographs were taken from the midpoint of the developing BP.
line, the track, at its most inferior edge. The track extends longitudinally from the proximal to distal end of the BP, and continues to do so from E9 through D15. Since it defines the inferior border of the amorphous component, it also acts to divide the amorphous and columnar components. At E9 (Fig. 3a), the amorphous matrix covers the entire surface of the BP and the original fibrillar matrix, which eventually becomes columnar, is sand-
wiched between the hair cells and the amorphous matrix. Initially, the columnar matrix secreted by the supporting cells shows only a slight indication of the columns which later arise from it. As development progresses, the inferior edge of the matrix forms thick, laterally oriented columns, which elongate with age while the track appears to recede (Fig. 3b). By El1 (Fig. 3c), the columns are longer and thinner, with the matrix above the track remaining amorphous, and having a more longitudinally oriented direction, as if tensile forces were acting to stretch it from proximal and distal directions. By El2 (Fig. 3d), the track extends down the center of the TM, and each component occupies half of the entire TM width. By El3 and El4 (Fig. 3e-f), the track is located in the upper third of the TM, and by hatching (E21), it exists at the upper border of the superior edge of the BP. At D15, (see Fig. l), the track is located at the most superior edge of the TM, with the amorphous component of the TM being very slender, and the columnar component making up the majority of TM surface area. Since the supporting cells appeared responsible for the secretion of the columnar component of the TM beginning at E7, the cells which secrete the amorphous component were sought. Previous reports had stated that the majority of the TM was secreted by the homogene cells (Cohen and Fermin, 1985; Ganeshina, 1985). In our study, it appears that the homogene cells are responsible for secreting only the amorphous component. The amorphous component is first observed at E9, and is securely attached to the homogene cell population which borders the BP at its superior edge. It is bordered at its inferior edge by the track (Fig. 4). This figure also clearly shows the amorphous component overlying the columnar component below it. In our SEM studies, the direction of the forces being applied to the TM appears to differ for each component (Fig. 5). The amorphous component is a continous, amorphous matrix, which is oriented longitudinally, its surface texture appearing different from the columnar TM. The columnar fibers appear to be stretched taut by tensile forces acting perpendicular to those imposed on the amorphous component. Deep to the surface of the amorphous layer, the TM forms a honeycombed matrix encir-
Fig. 3. Developmental series of the developing tectorial membrane from E9 to E14. This series of SEM micrographs is of embryonic days E9(a), ElO(b), Eli(c), ElZ(d), E13(e), and E14(f), in which we have documented the existence, and initial appearance of a raised line of extracellular matrix which is the inferior edge of the superior, amorphous TM component. We have named this edge the ‘track’. At E9 (a), the track is located at the inferior edge of the BP, and is fused with the underlying early columnar matrix. Through E9 to E14, the track appears to recede, and in later stages resides at the most superior edge of the TM. At El2 (d), the track separates the TM into approximately equal halves with amorphous TM overlying the tall hair cells, and columnar TM overlying the short hair cells. El2 is characterized by the onset of auditory function. Although it appears that the track exists at the upper edge of the TM, in actuality it resides at the middle of the TM due to the fact that all photographs were taken at the uniform magnification. Bar = 50 ~.rrnfor a-f.
amorphous layer on the upper surface of the TM which extends from the track up to the superior edge of the TM. When the plane of section of the light microscope is focused on the bottom surface of the TM, the honeycomb organization of the TM can be seen as it surrounds each hair cell. These observations of the unfixed, unstained TM indicate that our SEM observations represent real structures present in the living cochlea. Discussion
Fig. 4. The amorphous component of the TM is suspended from the homogene cell population at E12, located at the neural edge of the BP. This supports the idea that the amorphous component is secreted from the homogene cells. The track is designated by arrows. Bar = 50 pm.
cling each hair cell. Hollow vertical cylinders of matrix arise from the initial honeycomb. The top of this growing cylinder elongates in a lateral direction during development, as if it is being pulled toward the superior edge of the BP. Inferior to the track, and visible at the apical surface of the BP, the honeycombs are continuous with the laterally-oriented fibers of the columnar component. Condensation of each TM component increases with advancing development. With progressive condensation, the apical surface of the hair cells can no longer be seen, and the TM appears increasingly dense. At its surface, the TM appears to be more homogenous, yet it does not possess a fully solid surface. Small pores are present in a random pattern throughout the amorphous component, while the pores of the columnar component flank the laterally oriented columns in a uniform manner (Fig. 5). The TM underlying the amorphous component can be exposed by dissection, and a porous, honeycombed matrix, which gives rise to laterally directed columns is still evident (Fig 6). Video-enhanced DIC examination of the unfixed TM at El3 indicates the presence of laterally-oriented columns extending from the inferior edge of the BP up to the track (Fig. 7). There is an
These results show that the tectorial membrane develops as two temporally, and structurally distinct components, the columnar and amorphous layers. The two components appear to be secreted by two separate cell populations, and are oriented in two different directions. At early developmental stages the amorphous layer lies over the columnar component and extends from the superior to the inferior borders of the BP. As the cochlea grows in width, the columnar component elongates between the track and the inferior edge of the BP, so that it eventually comprises the majority of the surface of the TM. The track which marks the inferior border of the amorphous component appears to be receding toward the superior edge of the BP as the columnar component grows. These results indicate that there are at least two cell populations which are individually responsible for secreting each component of the TM. The supporting cells apparently secrete the columnar component beginning at E7, while the homogene cells presumably secrete the amorphous component starting at E9. It is possible that since the TM is secreted from separate cell types that each component is unique in chemical composition and/or function. This difference in chemical composition could cause the TM secreted by supporting cells to become columnar, while the amorphous remains less rigidly structured. component Another option is that the columnar component contains other types of collagen, exclusive of Type II collagen, which would be more conducive to the formation of a rigid, less amorphous structure than would a non-collagenous matrix (Thalmann et al., 1987). Further biochemical analysis should clarify any difference in chemical composition between the TM produced by each cell type.
Fig. 5. Orientation of the two components of tectorial membrane are shown at ElO, El1 and E16. The presence of amorphous (AC) and columnar components (CC) of the TM are seen in samples at developmental ages ElO(A), Eli(B), and E16(C). The two components are oriented perpendicular to each other when viewed with SEM. Specifically, the amorphous component is oriented longitudinally, parallel to the length of the BP, while the columnar component runs laterally across the width of the BP. Figure 5D shows a closeup of the columns being secreted from the supporting cells which surround the hair cells (HC). Bars in a, b, c = 10 pm. Bar in d = 5 pm.
These SEM results correlate well with data which describe the secretion of the TM from supporting cells using transmission electron microscopy (Cotanche and Sulik, 1984; Cohen and Fermin, 1985; Ganeshina, 1985). The evidence that the homogene cells appear responsible for partial secretion of the TM also correlates with the TEM data of Cohen and Fermin (1985). However, the results reported here are in contradiction with those of Cohen and Fermin, where it was observed that homogene cells initiate TM secretion at E12E14. In the study reported here, the superior amorphous component is well established by El0 (Fig. 4) indicating that homogene cells secrete this
layer earlier than previously thought, or that cells other than homogene cells are contributing to the amorphous layer of the TM early in development. Other possible contributors might include the cells of the tegmentum vasculosum, which lie directly above the amorphous component during early development. From the data reported here, we have postulated the model shown in Fig. 8, with a comparison to that posed by Cohen and Fermin. Cohen and Fermin postulated that the homogene cells are the major secretors of the TM, with the supporting cells only secreting the minimal ‘subtectorial’ portion of the TM. Data presented in this paper indicate that initially, it is the support-
Fig. 6. The tectorial membrane does not display a homogenous apical surface. Here, at E17, it can be noted that the developing TM does not display a homogenous, solid surface. Instead, the amorphous component contains pores which appear random in their placement, while the columnar component displays pores which initially flank the columns of matrix, giving rise later to defined spaces between each individual column. Arrows identify the track in this micrograph. Bar = 50 pm.
ing cells which are responsible for the secretion of the TM, while the homogene cells later secrete a second layer over the first. Differences in the morphology of the two components may be related to the function of the avian TM. One indication that they may function differently is the direction of their orientation. The amorphous layer is oriented in a longitudinal direction, paralleling the length of the BP. The columnar layer is oriented in a lateral direction, parallel to the width of the BP, thus perpendicular to the amorphous component. By E12, the TM is separated into two equal parts by the longitudinally-extending track. This separation of the TM overlies the division of the BP into two populations of hair cells; tall and short. Tall hair cells are located below the area covered by the amorphous TM, while short hair
Fig. 7. Video-DIC image of the unfixed TM at E13. These three photomicrographs were taken from a video tape of a preparation of an unfixed TM at E13. The three photomicrographs were taken at three levels through the TM: the top (A), the middle (B) and the bottom (C) of the TM. On the top layer the columnar (C) and amorphous (A) components can be seen separated by the track (arrowheads). Bar = 100 pm.
155
cells are below the area covered by the columnar TM. Tall hair cells are predominantly afferently innervated (Takasaka and Smith, 19711, and are thought to be similar to the inner hair cells found organ of Corti. Short hair cells in the mammalian and are contacted by smaller afferent endings,
a
b
d
e
endings thought to be efferent in nature (Whitehead and Mores& 1985). It may be that each TM component is responsible for exerting a different tensile force on the underlying hair cell populations. The initiation of hearing begins with the chick BP functioning as early as Ell-El2 (Saunders et al., 1973; Jackson and Rubel, 1978). Future studies in which the tensile forces exerted by the TM upon developing hair cells can be measured may help to elucidate the role of the TM in the onset of hearing function. Our initial analysis of the events in TM development was that the amorphous component appeared to pull the underlying columnar TM into thin columns as the TM developed. It seemed to us that the amorphous component was receding in a superior direction because the track which marks its inferior border grew further away from the inferior edge of the BP with age. An alternative interpretation is that the growing width of the BP could be pulling the columns away from their attachment to the overlying amorphous TM. Preliminary measurements of the TM at six developmental stages ranging from E9 to El4 show that while both the columnar component of the TM and the TM as a whole are increasing in width, the amorphous component remains uniform in its width (Shiel and Cotanche, unpublished results). These results support the idea that the track is not actually receding during development, rather, it remains stationary and marks the early fusion point of the amorphous component of TM with the columnar component. Thus, the increasing
Fig. 8. Schematic model of the development of the two components of the tectorial membrane. A schematic model of the amorphous and columnar components in the developing tectorial membrane at four stages during development representing E9 (a); El1 (b); El3 (c) and E21 (d), as seen in a cross-section through the BP. The dark layer represents the amorphous component with its connection to the homogene cells (Ho). The cross-hatched area represents the columnar component arising from the supporting cells in the BP. As the TM and the BP grow in width, the columnar component is stretched into laterally-oriented columns extending between the inferior border of the amorphous layer and the inferior edge of the BP. In (d) the TM connecting to the tall hair cells is indicated by the dark hatching, (e) A previous interpretation of TM development in which most of the substance of the TM is produced by the homogene cells (adapted from Cohen and Fermin, 1985).
156
length of the columns may be correlated with the increase in width of the entire BP. The width of the BP remains stationary through E9 but doubles between E9 and E15, the most significant increase occurring between El1 and El5 (Cotanche and Sulik, 1985; Tilney et a., 1986). From these preliminary results, we speculate that the elongation of the columnar TM is brought about by an increase in the width of the entire BP. However, this hypothesis requires further experimentation. One way to test this assumption will be to disrupt the secretion of one, or both TM components, and determine what consequences this has on the development of the entire structure. Events which may be influenced by the developing TM also include reorientation of the hair cell stereocilia, and stereociliary growth. Both events are necessary for proper hair cell function. The stereociliary bundles are attached to the mature TM via the tallest stereocilia (Engstrom and Engstrom, 1980), but undergo important changes during development. By E9, stereocilia are found uniformly on hair cells across the BP (Cotanche and Sulik, 1984), having differentiated in a distal to proximal manner. In the embryonic cochlea. the stereociliary bundles are initially non-uniformly oriented. They then undergo a shift in orientation such that the tallest row of stereocilia faces away from the superior edge of the BP in the adult cochlea (Cotanche, 1987a; Tilney et al., 1987). By Eli, all stereociliary bundles in the proximal l/3 and the inferior edge of the distal 2/3 of the BP are oriented as seen in the adult cochlea. It is also during this time that the columnar component of the TM is undergoing a significant increase in growth. It may be that the TM is exerting the necessary pulling force on the hair cell stereocilia required for reorientation (Corwin and Cotanche, 1986), especially as the initial reorientation is localized to the inferior edge of the TM, which is also where the columnar TM is hypothetically exerting its action early in development. Stereocilia initiate elongation soon after reorientation. and continue lengthening until E12, when they halt all growth in length, possibly due to the existence of a ‘capping factor’ (Tilney et al., 1986, 1988). At El2 to E16, the stereocilia again resume their growth, this time in width. and fi-
nally, they resume their growth in length from El6 to E21. A further speculation is that during this same developmental stage, the TM acts as an accessory to release the ‘capping factor’ (Tilney et al., 1988), by exerting a tensile force which opens channels in the stereocilia and allows ions to flow into hair cells and release the capping factor. One consideration of this SEM study was that perhaps our observations were artifactual due to the fixation procedures necessary for SEM analysis of the TM. The results of Runhaar (19X9), suggest that the chick TM is a highly homogenous structure when viewed with video enhanced differential interference contrast microscopy. Runhaar also challenges the efficacy of the SEM as a tool for providing an accurate representation of the TM in the chick. There are three reasons why we feel our results accurately represent the developing TM. First, twelve stages of development were studied. and at least ten animals were examined at each stage. Structural patterns were consistent in all of these samples. Second, two separate fixation protocols were used. Initially, the fixation procedure included osnlication only. We found that this was not ideal for embryonic tissues. Consequently, the protocol outlined in the methods section was adopted. In either case, the general structures were maintained. Third, in order to ensure that structures were not products of fixation alone, we used video-enhanced DIC microscopy to examine unfixed living preparations. Again, we were able to identify the amorphous and columnar components, as waI1 as the track which separates them. These measures strongly suggest that the results presented here are not due to fixation artifact, but rather, a real representation of the developing structure of the TM in the chick cochIea. Acknowledgements We would like to thank Wende Reenstra, Ken Rhodes, Daniel Picard, and Dr. Kathy Svoboda for their help in preparing this manuscript. This study was part of a Masters Thesis submitted to Boston University in partial fulfillment of the requirement for a Master of Arts degree. This work was supported by NIDCD Grant No. DC0041 2.
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References Cohen, G.M. and Fermin, C.D. (1985) Development of the embryonic chick’s tectorial membrane. Hear. Res. 18, 2939. Corwin, J.T. and Cotanche, D.A. (1986) Tectorial filaments in living cochleae: a possible traction mechanism for the embryonic reorientation of differentiating hair cell cilia bundles. Assoc. Res. Otolaryngol. Abstr. 9, 32. Cotanche, D.A. and Sulik, K.K. (1984) The development of stereociliary bundles in the cochlear duct of chick embryos. Dev. Brain Res. 16, 181-193. Cotanche, D.A. and Sulik, K.K. (1985) Parameters of growth in the embryonic and neonatal chick basilar papilla. Scanning Electron Microscopy/l985/1, 407-419. Cotanche, D.A. (1987a) Development of hair cell stereocilia in the avian cochlea. Hear. Res. 28, 35-44. Cotanche, D.A. (1987b) Regeneration of the tectorial membrane in the chick cochlea following severe acoustic trauma. Hear. Res. 30, 197-206. Dohlman, G.F. (1971) The attachment of the cupulae, otolith and tectorial membranes to the sensory cell areas. Acta Otolaryngol. 71, 89-105. Engstrom, H. and Engstrom, B. (1978) Structure of hairs on cochlear sensory cells. Hear. Res. 1, 49-66. Ganeshina, O.T. (1985) Formation of the tectorial membrane of the cochlear canal in the avian embryogenesis. Arkiv. Anatomii, Gistologii i Embriologii 12, 11-20. Hamburger, V. and Hamilton, H.L. (1951) A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. Hoshino, T. (1973) Tectorial membrane and otolithic membrane of pigeon inner ear: an SEM study. Arch. Histol. Jpn 35, 141-152. Jackson, H. and Rubel, E.W. (1978) Ontogeny of behavioral responsiveness to sound in the chick embryo as indicated by electrical recordings of motility. J. Comp. Physiol. Psychol. 92, 682-696. Jahnke, V., Lundquist, P.G. and Werslll (1969) Some morphological aspects of sound perception in birds. Acta Otolaryngol. 67, 583-601. Khalkhai-Ellis, Z., Hamming, F.W. and Steel, K.P. (1987) Glycoconjugates of the tectorial membranes. Hear. Res. 25, 185-191.
Richardson, G., Russel, I.J., Duance, V.C. and Bailey, A.J. (1987) Polypeptide composition of the mammalian tectorial membrane. Hear. Res. 25, 45-50. Rosenhall, U. (1971) Morphological patterns of the organ of Corti in birds. Arch. Klin. Exp. Ohr.-. Nas.- Kehlk. Heilk. 200, 42-63. Runhaar, G. (1989) The surface morphology of the avian tectorial membrane. Hear. Res. 37, 179-188. Saunders, J.C., Coles. R.B. and Gates, G.R. (1973) The development of auditory evoked responses in the cochlear nuclei of the chick. Brain Res. 63, 59-74. Takasaka, T. and Smith, C.A. (1971) The structure and innervation of the pigeon’s basilar papilla. J. Ultrastructural Res. 35, 20-65. Tanaka, K. and Smith, C.A. (1975) Structure of the avian tectorial membrane. Ann. Oto-Rhino-Laryngol. 84, 287296. Thalmann, I., Thallinger, G., Crouch, E.C., Comegys. T.H., Barrett, N. and Thalmann, R. (1987) Composition and supramolecular organization of the tectorial membrane. Laryngoscope 97. 357-367. Tilney, L.G., Tilney, M.S., Saunders. J.C. and DeRosier. D.J. (1986) Actin filaments, stereocilia, and hair cells of the bird cochlea. III. The development and differentiation of hair cells and stereocilia in embryos. Dev. Biol. 116. 100-118. Tilney. MS., Tilney, L.G. and DeRosier, D.J. (1987) The distribution of hair cell bundle lengths and orientations suggests an unexpected pattern of hair cell stimulation in the chick cochlea. Hear. Res. 25, 141-151. Tilney, L.G., Tilney, M.S. and Cotanche, D.A. (1988) Actin filaments, stereocilia, and hair cells of the bird cochlea. V. How the staricase pattern of stereociliary length is generated. J. Cell Biol. 106, 355-365. Tilney. MS., Tilney, L.G., Stephens, R.E., Merte. C., Drenckhahn, D., Cotanche, D.A. and Bretscher, A. (1989) Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea. J. Cell Biol. 109, 1711-1723. Whitehead M.C. and Morest, D.K. (1985) The development of innervation patterns in the avian cochlea. Neuroscience 14, 255-276.
HEARES
47 (1990) 147-158
147
01397
SEM analysis of the developing tectorial membrane in the chick cochlea Monica Jean Shiel and Douglas A. Cotanche Department
ofAnatomy, Boston University School of Medicine Boston, Massachusetts, (Received
16 August
1989; accepted
10 March
U.S.A.
1990)
The development of the tectorial membrane in the embryonic chick cochlea was studied using scanning electron microscopy. Chick embryos ranged in age from embryonic day 7 (E7) to post-hatching day 15. Our studies revealed that a fine filamentous matrix arose on the apical surface of the basilar papilla at approximately E7. This matrix was secreted by the supporting cells which encircled the hair cells. By E9, the early matrix had increased in vohrme but remained filamentous in structure, except at the inferior edge of the basilar papilla where it was condensed into a layer of laterally-oriented columns. At E9 the TM exhibited an additional layer of matrix, called the amorphous component. It appeared to originate from the homogene cell population, and attached to the early columar matrix at the inferior edge of the basilar papilla. The two components of the TM were separated by a longitudinal ridge, called the ‘track’, which marked the inferior edge of the amorphous component. As the cochlea developed, the basilar papilla increased in width, the columnar component elongated and the track appeared to recede. These morphological findings point to separate developmental origins for the two components of the tectorial membrane. Tectorial
Membrane,
Development,
Cochlea,
Basilar
Papilla,
Chick
The tectorial membrane (TM) is a hydrated extracellular matrix located over the lumenal surface of the basilar papilla in the avian cochlea. It is currently thought to function as a point of insertion for the tallest stereocilia of the hair cells, and thus provide the necessary shearing forces needed to open ion channels in the hair cell stereocilia. Opening these channels initiates signal transduction within the hair cell. The TM is situated with its superior edge anchored to the homogene cells, which are located in the superior wall of the cochlear duct. The inferior edge of the TM is anchored to the supporting cells along the inferior edge of the basilar papilla (BP). In cross section, it is seen to have a wedged shape, with its thicker edge in contact with the homogene cells, and its tapered edge in
Correspondence to: Douglas A. Cotanche, Department of Anatomy, Boston University School of Medicine, 80 East Concord Street, Boston, MA. 02118, U.S.A.
0378-5955/90/$03.50
0 1990 Elsevier Science Publishers
contact with the inferior side of the BP (Jahnke et al., 1969; Hoshino, 1973; Tanaka and Smith, 1975). The lumenal surface of the TM appears smooth along its superior border, while the remainder of its surface is segregated into laterally oriented colu~s. The underside of the TM exhibits a honeycombed pattern, with each cylinder in the honeycomb encircling a single hair cell. The undersurface of the TM is attached to the longest row of stereocilia on each hair cell and anchored to the supporting cells surrounding the hair cells by a fine fibrillar matrix of extracellular material (Jahnke et al., 1969; Dohlman, 1971; Rosenhall, 1971). The biochemical composition of both the mammalian and avian TM is mainly glycosaminoglycans and glycoproteins (Richardson et al., 1987; Khalkhali-Ellis et al., 1987; Thalmann et al., 1987). However, the details of the polypeptide composition of the avian TM remain obscure. One dimensional gel electrophoresis followed by band digestion with bacterial collagenase showed that Type II collagen, a glycoprotein, is found in abundance in the mammalian TM, but does not appear
B.V. (Biomedical
Division)
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to be present in avian TM (Thalmann et al., 1987). It has not been documented as to whether types of collagen other than Type II exist in the avian TM. Development of the chick TM was initially studied using light and transmission electron microscopy (Cohen and Fermin, 1985; Ganeshina, 1985). It was reported in these studies that TM secretion began on E7 and the majority of the TM appeared to be secreted from the homogene cell population, with the supporting cells in the BP responsible for the secretion of a lesser or “subtectorial’ TM. Observations of the events involved in TM regeneration following noise damage led us to further investigate the embryonic development of the TM. When the chick cochlea was exposed to noise damage, the TM was destroyed over the region of hair cell damage, but after a period of recovery, it was regenerated. New TM was secreted exclusively from the supporting cell population to form a honeycomb surrounding the newly regenerated cells, with no cont~bution from the homogene cells (Cotanche, 1987b). In order to examine the relationship between regeneration in the TM and its original production in the embryo, we have examined the normal development of the TM using scanning electron microscopy. By using gentle fixation procedures, as well as viewing living, unfixed tissue, we were able to observe the developing TM in a state comparable to its in vivo state. Methods Fertilized white leghorn chicken eggs were obtained from SPAFAS Hatcheries Inc. (Norwich, CT). The eggs were incubated at 37.5OC and 40% hu~dity in a laboratory incubator. Embryonic chicks were dissected out of the egg and staged according to the specifications of Hamburger and Hamilton (1951). Embryos used in this study ranged from stage 32 (embryonic day 7, E7) to stage 46 (E21, hatching). Hatchlings were obtained from SPAFAS on post-hatching day 1 (Dl ) and allowed to mature in a heated brooder until post-hatting day 15 (D15). At least ten embyos and hatchlings were examined per stage. For fixation of the cochleas the temporal bones were isolated and placed in HEPES-buffered Hanks’ balanced salt solution (HHBSS). The
buffer was oxygenated and maintained at pH 7.4 at 4°C. The initial steps in the dissection isolated the cochlea from the surrounding tissue of the cranial cavity. The superior portion of the labyrinth was dissected to expose the Scala media to the fixative. The tympanic membrane was then removed, the columella (stapes) extracted, and the round window was punctured to allow the fixative to permeate the Scala vestibuli, Scala tympani and Scala media, thus accessing the tectorial membrane. For each chick embryo both ears were dissected in under ten minutes, and immediately immersed in fixative. Primary fixation was achieved in 2% glutaraldehyde in 0.1 M sodium phosphate buffer at pH 7.4 and 4°C for 24 h. The tissues were then postfixed in 1% Osmium tetroxide in 0.1 M sodium phosphate buffer at pH 7.4 for 45 minutes at 4°C. In preparation for SEM analysis, the tissues were dehydrated up to 70% &OH, and the apical surface of the tectorial membrane was fully exposed by dissection of the overlying tegmentum vasculosum. After the final dissections, the tissues were dehydrated to 100% EtOH, and the samples were critical point dried with CO, as a transition fluid in a Tousimis Samdri Model 780A. Tissues were sputtercoated with gold/palladium in a Polaron E5000 sputtercoater and then examined with an ISI 60 scanning electron microscope at 30 kV. In order to ensure that our SEM observations were representative of structures present in the living, unfixed cochlea. the TM was isolated from unfixed, living cochleae at E13. The cochleae were rapidly removed from the temporal bones by a technique described in a previous publication (Tilney et al., 1989) and placed in oxygenated HHBSS. The tegmentum vasculosum was removed from each ear and the BP was immersed in Protease Type XXVII (Sigma, No. P4789) at a concentration of 0.05 mg/ml for 2 min and then placed back in protease-free HHBSS. The tectorial membrane was dissected off the BP and placed on a clean glass slide. The structure of the unfixed embryonic TM was examined on a Zeiss Axioskop equipped with differential-interference-contrast (DIC) optics and viewed with video-enhanced contrast through a Hammamatsu C2400 video camera.
149
Results
The mature TM (IX5) covers the lumenal surface of the BP (Fig. la) and a higher magnification view of the TM demonstrates the fine structure of its lumenal surface (Fig. lb). A thin line is
present at the superior edge of the TM and is located lon~tudinally from the proximal to distal ends of the BP. This line serves to divide the TM into two morphologically distinct components. We have named the two components according to their textures observed using the SEM. The smooth, ‘amo~hous’ matrix exists superior to the line or ‘ track’, and is anchored to homogene cells. The striated, or Lcolumnar’ matrix is inferior to the track, and is divided into discrete columns of matrix which elongate as the embryo matures. A second dimension of the TM exists, since the columnar matrix also lies below, or deep to, the amorphous matrix. Initially, the columnar matrix is fibrillar, and it alone covers the surface of the BP. At a later developmental stage, the amo~hous layer of matrix is secreted over the developing columnar layer. In Fig. lc, at E18, the amorphous layer has been dissected away to reveal the underlying columnar matrix. The columnar component of the TM is first seen at E7 and appears as clumps of fibrillar, extracellular matrix-like material on the lumenal surface of the BP (Fig. 2a). Prior to this developmental stage, no matrix was discernable in the SEM preparations. This early secretion of the matrix is localized over the supporting cell microvillae. By E8 (Fig. 2b), a larger volume of matrix is secreted and it covers both the hair and supporting cell surfaces in a thick, matted layer. The amorphous component of the TM appears at E9 as a second layer which covers the initial columnar matrix, and it exhibits a longitudinal
Fig. 1. The structure of the avian tectorial membrane. (a) For orientation purposes, the mature tectorial membrane (TM) is shown covering the BP in the cochlea of a D15 chick. The wider, distal BP is to the left. The proximal end is located at the right in this SEM photograph. Superior (Sup) and inferior (Inf) are designated. Homogene cells (Ho), are located uniformly along the superior border of the BP. At this magnification the track is faintly distinguishable (arrows). Bar = 500 pm. (b) At a higher magnification of the mid-apical surface of the BP, the TM exhibits a track (arrows), which separates the inferior columnar TM from the superior amorphous TM. Bar =lOO am. (c). The underlying nature of the matrix which makes up the second dimension of TM structure is easily seen in this SEM photograph from an El8 cochlea. The track remains evident as well (arrows). Bar =lO am.
Fig. 2. Early development of the avian tectorial membrane. (a) The BP is shown here at E7 with the supporting cell population secreting the initial matrix-like material. This matrix later forms the columnar portion of the tectorial membrane. Bar = 10 pm. (b) By E8, a larger volume of matrix has been secreted, and the hair cells are now covered by a thick mat of TM, which has been dissected away in order to visualize the underlying BP. An underlying hair cell (HC) is identified by its stereociliary bundle. Bar =lO pm. Both SEM photographs were taken from the midpoint of the developing BP.
line, the track, at its most inferior edge. The track extends longitudinally from the proximal to distal end of the BP, and continues to do so from E9 through D15. Since it defines the inferior border of the amorphous component, it also acts to divide the amorphous and columnar components. At E9 (Fig. 3a), the amorphous matrix covers the entire surface of the BP and the original fibrillar matrix, which eventually becomes columnar, is sand-
wiched between the hair cells and the amorphous matrix. Initially, the columnar matrix secreted by the supporting cells shows only a slight indication of the columns which later arise from it. As development progresses, the inferior edge of the matrix forms thick, laterally oriented columns, which elongate with age while the track appears to recede (Fig. 3b). By El1 (Fig. 3c), the columns are longer and thinner, with the matrix above the track remaining amorphous, and having a more longitudinally oriented direction, as if tensile forces were acting to stretch it from proximal and distal directions. By El2 (Fig. 3d), the track extends down the center of the TM, and each component occupies half of the entire TM width. By El3 and El4 (Fig. 3e-f), the track is located in the upper third of the TM, and by hatching (E21), it exists at the upper border of the superior edge of the BP. At D15, (see Fig. l), the track is located at the most superior edge of the TM, with the amorphous component of the TM being very slender, and the columnar component making up the majority of TM surface area. Since the supporting cells appeared responsible for the secretion of the columnar component of the TM beginning at E7, the cells which secrete the amorphous component were sought. Previous reports had stated that the majority of the TM was secreted by the homogene cells (Cohen and Fermin, 1985; Ganeshina, 1985). In our study, it appears that the homogene cells are responsible for secreting only the amorphous component. The amorphous component is first observed at E9, and is securely attached to the homogene cell population which borders the BP at its superior edge. It is bordered at its inferior edge by the track (Fig. 4). This figure also clearly shows the amorphous component overlying the columnar component below it. In our SEM studies, the direction of the forces being applied to the TM appears to differ for each component (Fig. 5). The amorphous component is a continous, amorphous matrix, which is oriented longitudinally, its surface texture appearing different from the columnar TM. The columnar fibers appear to be stretched taut by tensile forces acting perpendicular to those imposed on the amorphous component. Deep to the surface of the amorphous layer, the TM forms a honeycombed matrix encir-
Fig. 3. Developmental series of the developing tectorial membrane from E9 to E14. This series of SEM micrographs is of embryonic days E9(a), ElO(b), Eli(c), ElZ(d), E13(e), and E14(f), in which we have documented the existence, and initial appearance of a raised line of extracellular matrix which is the inferior edge of the superior, amorphous TM component. We have named this edge the ‘track’. At E9 (a), the track is located at the inferior edge of the BP, and is fused with the underlying early columnar matrix. Through E9 to E14, the track appears to recede, and in later stages resides at the most superior edge of the TM. At El2 (d), the track separates the TM into approximately equal halves with amorphous TM overlying the tall hair cells, and columnar TM overlying the short hair cells. El2 is characterized by the onset of auditory function. Although it appears that the track exists at the upper edge of the TM, in actuality it resides at the middle of the TM due to the fact that all photographs were taken at the uniform magnification. Bar = 50 ~.rrnfor a-f.
amorphous layer on the upper surface of the TM which extends from the track up to the superior edge of the TM. When the plane of section of the light microscope is focused on the bottom surface of the TM, the honeycomb organization of the TM can be seen as it surrounds each hair cell. These observations of the unfixed, unstained TM indicate that our SEM observations represent real structures present in the living cochlea. Discussion
Fig. 4. The amorphous component of the TM is suspended from the homogene cell population at E12, located at the neural edge of the BP. This supports the idea that the amorphous component is secreted from the homogene cells. The track is designated by arrows. Bar = 50 pm.
cling each hair cell. Hollow vertical cylinders of matrix arise from the initial honeycomb. The top of this growing cylinder elongates in a lateral direction during development, as if it is being pulled toward the superior edge of the BP. Inferior to the track, and visible at the apical surface of the BP, the honeycombs are continuous with the laterally-oriented fibers of the columnar component. Condensation of each TM component increases with advancing development. With progressive condensation, the apical surface of the hair cells can no longer be seen, and the TM appears increasingly dense. At its surface, the TM appears to be more homogenous, yet it does not possess a fully solid surface. Small pores are present in a random pattern throughout the amorphous component, while the pores of the columnar component flank the laterally oriented columns in a uniform manner (Fig. 5). The TM underlying the amorphous component can be exposed by dissection, and a porous, honeycombed matrix, which gives rise to laterally directed columns is still evident (Fig 6). Video-enhanced DIC examination of the unfixed TM at El3 indicates the presence of laterally-oriented columns extending from the inferior edge of the BP up to the track (Fig. 7). There is an
These results show that the tectorial membrane develops as two temporally, and structurally distinct components, the columnar and amorphous layers. The two components appear to be secreted by two separate cell populations, and are oriented in two different directions. At early developmental stages the amorphous layer lies over the columnar component and extends from the superior to the inferior borders of the BP. As the cochlea grows in width, the columnar component elongates between the track and the inferior edge of the BP, so that it eventually comprises the majority of the surface of the TM. The track which marks the inferior border of the amorphous component appears to be receding toward the superior edge of the BP as the columnar component grows. These results indicate that there are at least two cell populations which are individually responsible for secreting each component of the TM. The supporting cells apparently secrete the columnar component beginning at E7, while the homogene cells presumably secrete the amorphous component starting at E9. It is possible that since the TM is secreted from separate cell types that each component is unique in chemical composition and/or function. This difference in chemical composition could cause the TM secreted by supporting cells to become columnar, while the amorphous remains less rigidly structured. component Another option is that the columnar component contains other types of collagen, exclusive of Type II collagen, which would be more conducive to the formation of a rigid, less amorphous structure than would a non-collagenous matrix (Thalmann et al., 1987). Further biochemical analysis should clarify any difference in chemical composition between the TM produced by each cell type.
Fig. 5. Orientation of the two components of tectorial membrane are shown at ElO, El1 and E16. The presence of amorphous (AC) and columnar components (CC) of the TM are seen in samples at developmental ages ElO(A), Eli(B), and E16(C). The two components are oriented perpendicular to each other when viewed with SEM. Specifically, the amorphous component is oriented longitudinally, parallel to the length of the BP, while the columnar component runs laterally across the width of the BP. Figure 5D shows a closeup of the columns being secreted from the supporting cells which surround the hair cells (HC). Bars in a, b, c = 10 pm. Bar in d = 5 pm.
These SEM results correlate well with data which describe the secretion of the TM from supporting cells using transmission electron microscopy (Cotanche and Sulik, 1984; Cohen and Fermin, 1985; Ganeshina, 1985). The evidence that the homogene cells appear responsible for partial secretion of the TM also correlates with the TEM data of Cohen and Fermin (1985). However, the results reported here are in contradiction with those of Cohen and Fermin, where it was observed that homogene cells initiate TM secretion at E12E14. In the study reported here, the superior amorphous component is well established by El0 (Fig. 4) indicating that homogene cells secrete this
layer earlier than previously thought, or that cells other than homogene cells are contributing to the amorphous layer of the TM early in development. Other possible contributors might include the cells of the tegmentum vasculosum, which lie directly above the amorphous component during early development. From the data reported here, we have postulated the model shown in Fig. 8, with a comparison to that posed by Cohen and Fermin. Cohen and Fermin postulated that the homogene cells are the major secretors of the TM, with the supporting cells only secreting the minimal ‘subtectorial’ portion of the TM. Data presented in this paper indicate that initially, it is the support-
Fig. 6. The tectorial membrane does not display a homogenous apical surface. Here, at E17, it can be noted that the developing TM does not display a homogenous, solid surface. Instead, the amorphous component contains pores which appear random in their placement, while the columnar component displays pores which initially flank the columns of matrix, giving rise later to defined spaces between each individual column. Arrows identify the track in this micrograph. Bar = 50 pm.
ing cells which are responsible for the secretion of the TM, while the homogene cells later secrete a second layer over the first. Differences in the morphology of the two components may be related to the function of the avian TM. One indication that they may function differently is the direction of their orientation. The amorphous layer is oriented in a longitudinal direction, paralleling the length of the BP. The columnar layer is oriented in a lateral direction, parallel to the width of the BP, thus perpendicular to the amorphous component. By E12, the TM is separated into two equal parts by the longitudinally-extending track. This separation of the TM overlies the division of the BP into two populations of hair cells; tall and short. Tall hair cells are located below the area covered by the amorphous TM, while short hair
Fig. 7. Video-DIC image of the unfixed TM at E13. These three photomicrographs were taken from a video tape of a preparation of an unfixed TM at E13. The three photomicrographs were taken at three levels through the TM: the top (A), the middle (B) and the bottom (C) of the TM. On the top layer the columnar (C) and amorphous (A) components can be seen separated by the track (arrowheads). Bar = 100 pm.
155
cells are below the area covered by the columnar TM. Tall hair cells are predominantly afferently innervated (Takasaka and Smith, 19711, and are thought to be similar to the inner hair cells found organ of Corti. Short hair cells in the mammalian and are contacted by smaller afferent endings,
a
b
d
e
endings thought to be efferent in nature (Whitehead and Mores& 1985). It may be that each TM component is responsible for exerting a different tensile force on the underlying hair cell populations. The initiation of hearing begins with the chick BP functioning as early as Ell-El2 (Saunders et al., 1973; Jackson and Rubel, 1978). Future studies in which the tensile forces exerted by the TM upon developing hair cells can be measured may help to elucidate the role of the TM in the onset of hearing function. Our initial analysis of the events in TM development was that the amorphous component appeared to pull the underlying columnar TM into thin columns as the TM developed. It seemed to us that the amorphous component was receding in a superior direction because the track which marks its inferior border grew further away from the inferior edge of the BP with age. An alternative interpretation is that the growing width of the BP could be pulling the columns away from their attachment to the overlying amorphous TM. Preliminary measurements of the TM at six developmental stages ranging from E9 to El4 show that while both the columnar component of the TM and the TM as a whole are increasing in width, the amorphous component remains uniform in its width (Shiel and Cotanche, unpublished results). These results support the idea that the track is not actually receding during development, rather, it remains stationary and marks the early fusion point of the amorphous component of TM with the columnar component. Thus, the increasing
Fig. 8. Schematic model of the development of the two components of the tectorial membrane. A schematic model of the amorphous and columnar components in the developing tectorial membrane at four stages during development representing E9 (a); El1 (b); El3 (c) and E21 (d), as seen in a cross-section through the BP. The dark layer represents the amorphous component with its connection to the homogene cells (Ho). The cross-hatched area represents the columnar component arising from the supporting cells in the BP. As the TM and the BP grow in width, the columnar component is stretched into laterally-oriented columns extending between the inferior border of the amorphous layer and the inferior edge of the BP. In (d) the TM connecting to the tall hair cells is indicated by the dark hatching, (e) A previous interpretation of TM development in which most of the substance of the TM is produced by the homogene cells (adapted from Cohen and Fermin, 1985).
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length of the columns may be correlated with the increase in width of the entire BP. The width of the BP remains stationary through E9 but doubles between E9 and E15, the most significant increase occurring between El1 and El5 (Cotanche and Sulik, 1985; Tilney et a., 1986). From these preliminary results, we speculate that the elongation of the columnar TM is brought about by an increase in the width of the entire BP. However, this hypothesis requires further experimentation. One way to test this assumption will be to disrupt the secretion of one, or both TM components, and determine what consequences this has on the development of the entire structure. Events which may be influenced by the developing TM also include reorientation of the hair cell stereocilia, and stereociliary growth. Both events are necessary for proper hair cell function. The stereociliary bundles are attached to the mature TM via the tallest stereocilia (Engstrom and Engstrom, 1980), but undergo important changes during development. By E9, stereocilia are found uniformly on hair cells across the BP (Cotanche and Sulik, 1984), having differentiated in a distal to proximal manner. In the embryonic cochlea. the stereociliary bundles are initially non-uniformly oriented. They then undergo a shift in orientation such that the tallest row of stereocilia faces away from the superior edge of the BP in the adult cochlea (Cotanche, 1987a; Tilney et al., 1987). By Eli, all stereociliary bundles in the proximal l/3 and the inferior edge of the distal 2/3 of the BP are oriented as seen in the adult cochlea. It is also during this time that the columnar component of the TM is undergoing a significant increase in growth. It may be that the TM is exerting the necessary pulling force on the hair cell stereocilia required for reorientation (Corwin and Cotanche, 1986), especially as the initial reorientation is localized to the inferior edge of the TM, which is also where the columnar TM is hypothetically exerting its action early in development. Stereocilia initiate elongation soon after reorientation. and continue lengthening until E12, when they halt all growth in length, possibly due to the existence of a ‘capping factor’ (Tilney et al., 1986, 1988). At El2 to E16, the stereocilia again resume their growth, this time in width. and fi-
nally, they resume their growth in length from El6 to E21. A further speculation is that during this same developmental stage, the TM acts as an accessory to release the ‘capping factor’ (Tilney et al., 1988), by exerting a tensile force which opens channels in the stereocilia and allows ions to flow into hair cells and release the capping factor. One consideration of this SEM study was that perhaps our observations were artifactual due to the fixation procedures necessary for SEM analysis of the TM. The results of Runhaar (19X9), suggest that the chick TM is a highly homogenous structure when viewed with video enhanced differential interference contrast microscopy. Runhaar also challenges the efficacy of the SEM as a tool for providing an accurate representation of the TM in the chick. There are three reasons why we feel our results accurately represent the developing TM. First, twelve stages of development were studied. and at least ten animals were examined at each stage. Structural patterns were consistent in all of these samples. Second, two separate fixation protocols were used. Initially, the fixation procedure included osnlication only. We found that this was not ideal for embryonic tissues. Consequently, the protocol outlined in the methods section was adopted. In either case, the general structures were maintained. Third, in order to ensure that structures were not products of fixation alone, we used video-enhanced DIC microscopy to examine unfixed living preparations. Again, we were able to identify the amorphous and columnar components, as waI1 as the track which separates them. These measures strongly suggest that the results presented here are not due to fixation artifact, but rather, a real representation of the developing structure of the TM in the chick cochIea. Acknowledgements We would like to thank Wende Reenstra, Ken Rhodes, Daniel Picard, and Dr. Kathy Svoboda for their help in preparing this manuscript. This study was part of a Masters Thesis submitted to Boston University in partial fulfillment of the requirement for a Master of Arts degree. This work was supported by NIDCD Grant No. DC0041 2.
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