Hearrng Research, 18 (1985) 29-39

Elsevier

29

HRR 00593

Development of the embryonic chick’s tectorial membrane

Glenn M. Cohen and Cksar D. Fermin * Lkyxutmm~ of Bmlogical Sciences. Florrdu Inst~/u~e of Technolog~~, Melhournc~. FL .l~YOI. L;.S.A

(Received 4 September 1984; accepted 27 March 198s)

The nascent tectorial membrane (TM) is identifiable as early as stage 33 (7th day) as thin, wispy material. By stage 37 (I 1 th day),

the dense mesh of the immature TM and fibrous webs (subtectorial threads) that attach the TM to the basilar papilla are distinct hut

scanty. The TM condenses slightly in its upper face. The growth of the columnar cells and basilar papilla during the following days

pulls the TM, lifting it upward, and resembling the cables on a suspension bridge in cross-section. As a result, a large hollow wedge

forma. During stages 40 -44 (14thL18th days). the columnar cells secrete large amounts of fibrous material. which fills the hollow

wedge and condenses into the dense meshes. The honeycombed patterns appear at this time. The supporting cells secrete the fihrous

webs. Their secretory activity closely corresponds to that of the columnar cells. The secretory material from both cell types remains

attached to the apical ends of their respective cells after secretory activity ends. By hatching (stage 46 - 21 days), the columnar cells

have filled with fibrous material and their cytoplasmic organelles are restricted to the apices, The cytoplasm of supporting cells is

relatively clear, with few cytoplasmic remnants of their Intense secretory activity earlier.

tectorial membrane. inner ear development, chick inner ear, columnar cell. supporting cell

Introduction

The mammalian tectorial membrane has a fusi- form shape. It is composed of two protofibrillar types [3] that form a continuous (non-cavitated) fibrous mesh [13]. The stereocilia of outer hair

cells touch the tectorial membrane, as do those of inner hair cells of some mammalian species [18].

By comparison, the avian tectorial membrane dif- fers in size, structure and attachments from its

mammalian counterpart. It is wedge-shaped and composed of dense meshes. The latter are cavi-

tated, giving the avian tectorial membrane its dis- tinctive honeycombed appearance. The honey- combs are positioned above the hair cells, so that the tallest stereociliary tips embed into the inner

margins of their hexagonal walls [27]. Fibrous webs (subtectorial threads) securely hold the tec-

torial membrane in place. The webs fasten the tectorium to the basilar papilla by attaching to

* Presrnr address: Department of Otorhinolaryngology and

Communicative Sciences, Baylor College of Medicine, Hous-

ton, TX 77030. U.S.A.

both the walls of the honeycombs above and the microvilli of the supporting cells below [4,10].

The objectives of the present study were first to describe the development of the chick’s tectorial

membrane and its constituent parts. the dense meshes, honeycombs, and fibrous webs and sec- ond to identify the secretory cells and their contri-

butions to the nascent tectorial membrane. Por- tions of this study have been presented in abbrevi- ated form [7,24].

Materials and Methods

The white leghorn eggs used for this study were obtained from Mussel-White and Cal-Maine hatcheries located in Maitland and Apopka, Florida, respectively. The eggs were incubated at 36%37.5”C and staged according to Hamburger

and Hamilton’s scheme. We followed the same dissection, fixation, electron microscopic proce- dures described previously [2,5.6].

The embryos were decapitated, fixed in 3.5% glutaraldehyde that was buffered in 0.1 M cacodylate, pH 7.2-7.4, at 4°C. Embryos from 5.5

037%5955/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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days of incubation (stage 29) to hatching (21 days _ stage 46) were used. Lagenae were embedded in Araldite 502, and sectioned alternately at 2 and 5 pm, starting at the proximal end and progressing through the distal (apical) end. Every 100 pm, thin sections were obtained for transmission electron microscopy (Zeiss 9S-2). Light and electron micro- graphs were used for diagrammatic reconstruc- tions of developmental events.

We estimated the secretory contributions of the supporting and columnar cells by their cytological appearances, their spatial relation to the develop- ing tectorial membrane, and mature associations with these structures, i.e., direct connections of their secretions with various portions of the tec- torial membrane.

For staining mucopolysaccharides, specimens were first embedded in methacrylate (JB-4) and then sectioned between 5 and 10 pm. Sections were annealed to microscope slides by gentle heat- ing. Then Spicer’s PAS-Alcian blue procedures were followed [14]. Slides were divided into two groups and stained at either pH 1 or 2.5 to reveal different chemical groups.

Results

We used the anatomical terminology of Dohl- man [4] and Smith et al. [26,27] throughout. For descriptive purposes, they distinguished between the tectorial membrane (TM) proper (dense mesh) and fibrous webs (subtectorial threads).

The TM was sensitive to fixation, even when other cells were well preserved. Shrinkage dis- torted the TM and tempered some conclusions

about spatial relations with hair cells, the basilar papilla and lagenar duct. Moreover, the TM was also sensitive to mechanical manipulation during dissection of the lagena, sometimes pulling loose from the basilar papilla and/or columnar cells. We described cytoplasmic and nuclear changes of columnar and supporting cells to relate cytological appearances to secretory activities.

7th-8th days (stages 33-34) The nascent tectorial membrane is composed of

a single layer of fine fibrillar material. It overlies the microvilli of the supporting cell (Fig. l), which may have secreted some of it. Their k&cilia and microvilli are attached to this fine matrix, but those of hair cells are not. In both hair and sup- porting cells, the kinociliary basal bodies are en- circled by conspicuous satellite bodies [2]. Their nuclei have migrated subcentrally en route to their final basal positions. Rough endoplasmic reticula (rER) are more numerous and prominent in sup- porting than in hair cells. The apical ends of supporting and hair cells are coupled by tight junctions that are fortified by cytoplasmic anchors.

Columnar cells are pseudostratified and their cytoplasm loses electron density. Their nuclei are close to the basement membrane. They lack true ciliary structures, though a few cytoplasmic processes extend from their apical surfaces.

9th day (stage 35) The rER begin to fill with protein as they form

extensive tubular networks throughout the sup- porting cells. Columnar cells are no longer pseudo- stratified.

Fig. 1. Nascent tectorial membrane. The fibrils attach to the supporting cells (S) and arch over the hair cells (H). The amorphous

fibrils fan out. 7 days (stage 33). X 12000.

Fig. 2. Early tectorial membrane. The future dense mesh begins to collect over stereocilia, forming a canopy, but has not yet

condensed. The fibrous webs (subtectorial threads) have almost the same density as the dense mesh at this early stage. 11 days (stage

37). x24000.

Fig. 3. Fibrils of tectorial membrane attaching to kinocilium. Tips of kino- and stereocilia embed into the developing tectorial

membrane. 11 days (stage 37). X 37000.

Fig. 4. Cross-section of early tectorial membrane. Once the tectorial membrane lifts up, it leaves a large, wedgeshaped hollow. Columnar cells (C) secrete the dense mesh (D) in massive quantities for the next several days and fill the hollow wedge. 12 days (stage

38). x200.

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llth-12th duys (stages 37-38)

The TM matrix consists almost entirely of un-

condensed fibrillar material (Fig. 2). Its fibrils

remain attached to the kino- and stereocilia (Fig.

3). The fibrous webs, whit :h arch over the h air cells, connect the microvilli of supporting cells to the nascent TM. Because the : TM is relatively Iii mP at this stage, it conforms to the broad contours Of

Fig. 5. Supporting cells during secretory phase. The copious rough endoplasmic reticulum, dilated with

will form fibrous webs, reflects the intense secretory activity. 14 days (stage 40). X 24000.

Fig. 6. Early honeycombed tectorial membrane. The dense mesh (D) has condensed. Columnar cells (C) matrix as secretory activity subsides. Because of shrinkage from fixation, the tectorial membrane has torr

and has lifted up (t). 16 days (stage 42). X 200.

proteinaceous material that

/ begin to fill with a fibt ‘OILS

L free from the taB hair ( :ells

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Fig. 7. Columnar ceils secreting and remodeling. The columnar cells secrete the dense mesh of the tectorial membrane (TM).

Numerous particles (b) are embedded within the TM. but they are rarely attached to the inner surfaces of the honeycombs. 16 days

(stage 42). X 3250.

the underlying basilar papilla and columnar cells. However, their growth during the following days pulls the TM, lessening its slack and lifting it

upward (see Figs. 1, 2 and 4). In the process, a hollow wedge forms below the TM, which will become filled with dense material from the col-

umnar cells. The supporting cells transform from their col-

umnar shape to the characteristic flask-shape, with their long necks extending between the hair cells. The nuclei have descended basally into the ex- panded bases. The rER are now coiled and mainly

positioned at the basal end, and those positioned apically are dilated. Mitochondria are scarce and smaller than those in adjacent hair cells, but are similarly shaped.

Columnar cells are distinguishable from the ad- joining epithelium by their elongated shape and density. Although the organelles are still dispersed throughout the cytoplasm, some rER and mitochondria have migrated to the apical ends and have begun to collect there. Nuclei continue mov-

ing toward the center of the cells. Up to this time, columnar cells have contributed little to the TM: a minimal secretion of dense (upper surface) material. The latter is attached to their luminal

processes. By the 12th day, the upper face of the TM is

well defined. The TM consists of a continuous sheet that covers the basilar papilla, but in cross- section resembles a cable on a suspension bridge

(Fig. 4).

13th-14th duys (stages 39-40) As the TM condenses slightly in its upper layer.

the honeycombs begin to form. The TM seems to attach to the fibrous webs above the short and intermediate hair cells first. Multivesicular bodies appear in the supranuclear region of supporting cells.

By the 14th day, the supporting cells’ cisternae distend and their population peaks, but shdrtly thereafter they fragment and relocate (Fig. 5).

Columnar cells secrete the bulk of the TM, as

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represented by its dense, fibrous substance (Figs. 4 and 6). Columnar cells begin their massive secre- tions within a day after the TM has lifted. The secretions are deposited inside the hollow wedge rather than externally upon the original upper face. The condensed secretions are first identifia-

ble attached to the columnar cells and to short and intermediate hair cells where they rapidly fill the narrow space below the TM’s upper face. The larger area above the tall hair cells requires several days to fill (Fig. 4).

Fig 8. Honeycombed tectorial membrane. The stereocilia embed into the undersurface of the dense mesh (D). Fibrous webs (F) anchor the tectorial membrane in place. 3 days after hatching. x 5700. Inset, tectorial membrane before hatching has developed the

honeycombs and other mature features. 18 days (stage 44). x 500.

Fig. 9. Columnar cells with apical organelles. As the columnar cells end their secretory activity, the cytoplasm collects in the apical

(luminal) ends; fibrous proteins that appear ultrastructurally similar to the tectorial membrane fill the cells. The tectorial membrane

has pulled away and is not visible. 3 days after hatching. X 2400.

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16th day (stage 42) The TM contains both loose and condensed

fibrillar material (Fig. 6). The honeycombed pat- tern of the TM is now obvious. Small, dense

particles 500 nm in diameter are scattered within

and on the walls of the honeycombs. There are few intercellular spaces except at their apical regions

because the supporting cells are joined to each other by long, tight junctions. The necks of sup- porting cells have narrowed. At their bases, rER

have fragmented. Large numbers of Golgi com-

plexes appear. Intracellular spaces are now more

common. The cytoplasmic electron density is

steadily declining. In columnar cells, organelles have begun to collect in their apices and the cytoplasm has become fibrous (Fig. 7).

19th day (stage 45) In supporting cells, cisternae have disappeared

and abundant Golgi complexes have replaced them. The Golgi complexes align themselves along the vertical axis in the apical region, where they

Fig. 10. Schematic diagram of developing supporting cells. As they differentiate, supporting cells change in size, shape and density, Supporting cells secrete the proteins that comprise the fibrous webs. F, fibrous webs; D, dense mesh; H, hair cells.

Fig. 11. Schematic diagram of differentiating columnar cells. Their cytoplasmic organization and appearance change as the columnar

cells secrete the dense mesh of the tectorial membrane. The secretory material (SM) binds tightly to the apices ( --, ). Organelles

migrate to the apices (4) during differentiation and secretion. 30-33 (5.5-7 days); 37-39 (11-13 days): 40-46 (14-21) days).

Fig. 12. Schematic summary. Columnar cells (C) secrete most of the dense mesh of the tectorial membrane (TM). Supporting cells

(SC) secrete the fibrous webs. The process is progressive and is largely completed before hatching. A, 8 days; B, 12 days; C, 16 days; D, 21 days (hatching).

remain thereafter. In columnar cells, rootlets ap- proximately 100-200 nm in diameter and with microtubules approximately 20 nm in diameter extend from the apical cytoplasm into the fibrillar material which forms the dense portion of the TM. Smooth ER cisterns occasionally extended into these apical processes.

20th-21st days (stages 45-46 - hatching) andpost- hatched

The honeycombs are well defined. The TM now resembles its adult form, although maturation is not completed until after hatching. The TM is composed predominantly of thick, dense meshes.

The honeycombs above the short hair cells have changed from an off-centered tilt to a sharply angled, oblique tilt, while those above the tall hair cells remain vertical.

In the fibrous webs, individual fibrils extend almost vertically from the microvilli of the sup- porting cells to attach to the underside of the TM.

Because supporting cells encircle the hair cells, the fibrous webs form canopies around the apical ends of individual hair cells. As a result, each hair cell is encased in a chamber that opens upward into the honeycombs of the TM (Figs. 8,10,12).

The fibrous webs and dense meshes distinctly differ in their light and electron microscopic ap- pearances. When stained with PAS-Alcian blue at pH 2.5, the fibrous webs stained blue and the dense meshes maroon, indicating that they contain different mucosubstances.

Supporting cells contain many microtubules that extend from their bases to apices. Their basal rER are now completely fragmented and Golgi com- plexes are found only at their necks. The nuclei are euchromatic nuclei instead of heterochromatic. The apical centrioles may represent remnants of basal bodies and kinocilia that are usually shed a few days earlier.

In columnar cells, organelles are packed at the apex, with a few scattered peripherally in the

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cytoplasm (Fig. 9). The columnar cells are filled with a fibrous matrix that reinforces them for

bearing the forces of anchoring the TM. The ma- trix resembles the texture of the TM ultrastructur-

ally. Summaries of the developmental events are

shown in Figs. 10-12.

Discussion

General configuration

The avian tectorial membrane (TM) resembles

the catenary curve of a suspension bridge in

cross-section (Figs. 4 and 6). Its geometry suggests that the TM bears a slight lateral tension across its

upper face. Because of its massive size, the TM gives the impression of imposing a load upon the basilar papilla ([27], p. 287). However, its thickest wedge is firmly anchored onto the columnar cells, thereby reducing the potential load on the basilar

papilla (Figs. 6-8, 11, 12). Moreover, the avian TM, along with its mammalian counterpart, is highly hydrated [14,25], giving it a buoyant density

similar to the endolymph, i.e., neutral. Thus, the TM, even at its thickest wedge, may float above rather than rest directly upon the basilar papilla.

Onset of auditory function

Structural and functional events in the auditory

periphery are closely linked and temporally coor- dinated. They collectively contribute to the in- creased sensitivities and broadened bandwidths [21,22]. For example, cochlear (lagenar) micro-

phonics are first recordable the 13th day [30] and are quickly followed by eighth nerve action poten-

tials the 16th embryonic day [22]. During this interval, the TM transforms from a thin sheet to a honeycombed wedge, the latter resembling its adult form. Secretory activities of columnar and sup- porting cells coincide with the formation of the

TM (Figs. 10-12). Concurrently, stereociliary tips are embedding into the growing TM, dendrites and perikarya are myelinating [6], afferents and efferents are establishing their mature synaptic loci on hair cells [2,5,6,20], the tympanic and vestibular scalae are opening and enlarging [5], the middle ear is clearing [22], and the columella is ossifying

[3,1 II.

Honeycombs Columnar cells secrete most of the fibrils that

form the TM. Their development and secretory activity are closely linked to the maturation of

other cell types of the lagenar duct and basilar

papilla [2,5,6,20]. Columnar cells undergo a series of cytological changes before, during and after secretory activity. As secretory activity subsides,

the cytoplasm collects in narrow bands in the apical (luminal) ends and the columnar cells fill

with a dense, fibrous matrix that is ultrastructur- ally similar to the TM [27]. This material

strengthens the columnar cells for their post-secre- tory function of anchoring the TM (Fig. 11). The

avian TM, along with its mammalian counterpart

]9,13,251, is proteinaceous and also contains

mucosubstances. The fibrils of the dense meshes differ in density and staining affinities from those of the fibrous webs.

The avian TM is cavitated and has a honey-

combed pattern [27]. The latter appears as soon as the fibrils begin to condense into the dense mesh,

shortly after the columnar cells secrete them (Fig. 10). The honeycombs conform to the surface

geometry and topography of the hair cells that they encircle, i.e., honeycombs above short hair cells are larger than those above the tall [27].

Dohlman [14] suggested that the honeycombs

form as a result of enzymatic digestion: lysosomal enzymes dissolved the mesh to form the cavities.

Although small, dense granules appeared as the dense mesh formed and some clung to the inner

surfaces of the honeycomb walls (Figs. 7 and 8), a lytic function seems unlikely, for granules are also embedded within the walls themselves and are also present in the mammalian TM [28,29], which is

not cavitated. Instead, the honeycombs may self- assemble from subunits that fit together in specific combination, as occurs with microtubules, cell membranes, and contractile filaments of muscle, etc. [15].

Honeycombs continuously change their tilt across the width of the basilar papilla. For exam- ple, they are positioned almost vertically above the tall hair cells, but tilt obliquely and show the sharpest tilts above the short hair cells (Fig. 6). The graded tilts, if a structural feature of the TM, could serve an auditory function by stimulating stereocilia of short and tall hair cells by different

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shearing angles. However, tilts may be artifactual and occur during fixation as the shrinking TM pulls the thin, free segment above the short hair cells toward the much thicker and firmly anchored wedge above the tall.

The tallest stereociliary tips of all the chick’s hair cells are embedded into the inner margins of the honeycombs; the shorter stereocilia, though not in contact, might have been embedded but pulled free during fixation. The tips are the densest parts of the stereocilia (Fig. 9) and probably repre- sent both attachment sites and reinforcements for stronger linkages to the TM. The stereociliary tips of outer hair cells in the human, rhesus monkey, squirrel monkey, cat, guinea pig, bat and mouse show similar densities [12]. Moreover, intestinal microvilli, close structural counterparts to stereo- cilia and also filled with actin fib&s [S], have dense tips and are embedded in a mucous layer. Their dense tips contain a-actinin [23], which is involved in initiating actin polymerization and at- tachment.

Fibrous webs

The fibrous webs tether the TM to the basilar papilla (Fig. 9) and apparently maintain the pre- cise register of the honeycombs to the stereociliary tips [4,27]. The fibrous webs form strong, perma- nent attachments between the supporting cells and TM, which begin during development and remain through adulthood [2,4,7,10,27]. They traverse the entire length and width of the TM and encircle every hair cell. Fibrous webs are composed of thin, stable fibrils. We detected no fibrillar degenera- tion, loss, or ‘shedding’, as Dohlman [4] suggested.

The cytoplasm of supporting cells changes from a dense granular texture during the secretory phase to a permanently clear cytoplasm as secretory ac- tivity subsides shortly before hatching (Figs. 10-12). They undergo a narrowing of their necks, which accentuates their flask-shape [2,5].

In the mammal, the attachments of the TM to the spiral limbus are strong and permanent [l], but the insertion of stereocilia of outer hair cells into Hardesty’s membrane, and the linkage of trabecu- lae to Corti’s organ at both Hensen’s stripe and at the marginal band [16] are weaker, less numerous and less widely distributed than the bird’s These attachments, which may not exist in some animals

[18], usually tear as the TM shrinks during fixation and/or from exposure to an unsuitable ionic environment [14]. Thus, many questions will re- main about precise spatial relationships within the inner ear until the TM and adjacent cellular struc- tures can be preserved without shrinkage or tear- ing.

Acknowledgements

This research was supported by NIH Grant RR09032-01, and department funding to G.M.C. and time for manuscript revision to C.D.F. by NINCDS Grant NS-10940.

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