Anat Embryol (1993) 187:425432

Anatomyand

Embryology

9 Springer~Verlag 1993

Development of the tectal cells in the mouse cochlea Joaquin Rueda, Jorge J. Prieto, Maria E. Rubio, Angel Guti6rrez, Jaime A. Merchfin Departamento de Histologia, Instituto de Neurociencias, Universidad de Alicante, Apdo. Correos 374, E-03080 Alicante, Spain Accepted: 2 February 1993

Abstract. Tectal cells appear at birth in the outer part of the developing organ of Corti. At first they are attached to the basilar membrane, but later they ascend through the auditory epithelium. During the 1st postnatal week (coinciding with the development of the minor tectorial membrane), the newly formed tectal cells show several cytological characteristics suggesting increased metabolic and secretory activities, which include: (1) a large Golgi complex, (2) abundant amorphous material inside the cisterns of rough endoplasmic reticulum, and (3) dense granules inside the mitochondrial matrix. All these features gradually disappear, and by the 14th postnatal day the tectal cells show a dark cytoplasm and few and short microvilli. In addition, tectal cells were stained selectively by some lectins. These findings suggest that tectal cells may participate in the secretion of some components of the minor tectorial membrane, different from those produced by Deiters' cells, Hensen's cells and pillar cells. Key words: Inner ear - Tectorial membrane - Organ of Corti - Glycoconjugates

Introduction The mammalian auditory receptor, or organ of Corti (OC) is a neuroepithelium formed by supporting cells and two types of sensory cells: inner hair cells (IHC) and outer hair cells (OHC). The tectorial membrane (TM) is an extracellular gel-like matrix composed of proteins and glycoconjugates. It is closely associated with the auditory epithelium, contacting the stereocilia of the hair cells (Lira 1986). The inner sulcus is a space delimited between the TM and the top of the neuroepithelium, and is occupied by endolymph. In the organ of Corti there are several intraepithelial spaces, named tunnel of Corti (lined by the pillar cells), Nuel's spaces (between Deiters' cells) and outer tunnel (formed by the outermost Deiters' cell and the innermost Hensen's cell), all of which contain a perilymphatic-type fluid. Correspondence to: J. Rueda

A different type of supporting cell, named tectal cell, has been described recently. These cells form the lateral wall of the outer tunnel of the cochlea in the moustached bat (Henson and Henson 1979; Henson etal. 1983). They were initially described by several authors as similar to Hensen's cells or Deiters' cells (Retzius 1884; Bredberg et al. 1972; Hunter-Duvar 1978). The current point of view, however, is that they constitute a separate population with a number of morphological features different from those found in Hensen's or Deiters' cells, including (1) lack of contact with the basilar membrane, (2) a sparse population of short microvilli on their endolymphatic surface, and (3) a cytoplasm that is more electrondense than that of the adjacent Hensen's cells, and which contains comparatively more organelles (Henson et al. 1983). Nothing is known about the structural development of the tectal cells, but their location in the OC, next to other cell types which participate in crucial developmental processes, such as the secretion of the external portion of the TM, suggest that they may also be involved in some of these processes. The cochlea develops from the otic placode, which is a thickening of the ectoderm on both sides of the rhombencephalon in the early embryo. Later, the ectoderm interacts with the mesoderm and rhombencephalon to form the otocyst (Van de Water and Ruben 1976). The otocyst is a spherical sac of undifferentiated cells that forms the membranous labyrinth through a number of complex organogenetic and histogenetic changes (Whitehead 1986). The cochlear duct develops from the ventral portion of the otocyst. The epithelium of the cochlear duct in newborn mice and rats consists of tall columnar ceils, which form the so Called greater and lesser epithelial ridges (GER and LER, respectively) (Held 1926). During the first 2 weeks of life of the animal, the maturation of the cells forming the GER and LER leads to the opening of several fluid-filled intercellular spaces (inner sulcus, tunnel of Corti, Nuel's spaces and outer tunnel) (Hinojosa 1977; Lim and Anniko 1985). In addition, both GER and LER cells have been ira-

426 plicated in the formation of the T M (Lira 1972). The T M develops in stages and is formed by two different parts: the major and minor T M (Lira 1972). The major T M is first seen covering the tall columnar epithelial cells of the spiral limbus (Lim and Anniko 1985), extending rapidly over the G E R . The L E R is initially devoid of TM, but is gradually covered by the advancing major T M and the newly formed minor TM. Thus, during early stages of development (E16-PN7), the major T M covers only the G E R and IHCs, while the minor T M covers the developing OHCs. It is generally accepted that the two parts of the T M (major and minor) are secreted by the cells of the G E R and LER, respectively, and contain large amounts of glycoconjugates (Lira 1972, 1977; Lim and Anniko 1985; Gil-Loyzaga et al. 1985; Lim and Rueda 1990a, 1992; Prieto et al. 1990a; Rueda and Lim 1993). However, it is not known what role the tectal cells play in the secretory processes of the components of the T M during development. The aim of the present work is to study the histogenesis of these cells so as to acquire a better understanding of their role in the secretion of the TM.

Materials and methods Forty CBA mice, ranging from the 18th gestational day to the 21st postnatal day were used in this study. Gestational age was determined by the vaginal plug technique, considering as day 1 the first day that the vaginal plug was observed. The animals were given an overdose of chloral hydrate (0.3 mg/kg of body weight), then they were decapitated and their cochleas rapidly removed. A hole was made in the apex and the round window membrane was removed. Through this opening the cochleas were perfused with an ice-cold solution containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3. The specimens were kept for 2 h in the fixative and, after a brief wash in buffer, they were postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer, for 2 h, at 4~ C. For transmission electron microscopy (TEM), the cochleas were dehydrated in graded ethanols and embedded in Epon. Thin sections were made in a Reichert Ultracut ultramicrotome and examined in a Zeiss EM-10C electron microscope. For scanning electron microscopy (SEM), the cochleas were dehydrated, critical point dried in CO2, coated with gold, and examined in a JEOL JSM-840 scanning electron microscope. For the lectin study, 1-pro-thick sections were cut from the Epon-embedded specimens. The lectin-binding protocol used was that of Hsu and Raine (1982). Briefly, tissue was exposed by removing the epoxy resin by immersing the sections in a saturated solution of sodium hydroxide in ethanol for 10 rain. After blocking the endogenous peroxidase with hydrogen peroxide, the sections were incubated for 24 h at 4~ C with biotinylated lectins (Vector Labs., Burlingame, Calif.), diluted in 0.05 M TRIS-buffered saline (TBS). The lectins used, as well as their sugar specificity and optimal concentration, are shown in Table 1. After a wash in 0.05 M TBS, the sections were incubated in avidin-biotin peroxidase complex (Vector Labs., Burlingame, Calif.) and lectin binding was revealed by incubating the slides in 3-amino-9-ethylcarbazole (AEC) in 0.05 M TBS containing 0.015% hydrogen peroxide. After washing, the sections were counterstained with Harris' haematoxylin, rinsed again, and mounted in glycerin jelly. Controls were made by omitting the lectin conjugate in the corresponding step. Because of the delay in maturation (1-2 days) of the cochlear apex with respect to the base, we chose the medium coil of the cochlea in all the specimens in order to obtain consistent results when comparing data from different developmental stages.

Table 1. Lectins used, sugar specificity for each lectin and their optimal concentration Lectins

Specificity

Con A PHA-E

c~-D-mannose Galactose N-aceyl-glucosamine N-acetyl-galactosamine Mannose ~-D-galactose N-acetyl-galactosamine N-acetyl-glucosamine N-acetyl-neuraminic acid N-acetyl-glucosamine

RCA II SBA WGA succ-WGA

Optimal concentration 7.5 pg/ml 5 gg/ml

7.5 pg/ml 10 gg/ml 10 gg/ml 10 gg/ml

Results At the 18th gestational day, the auditory primordium appeared as a compact epithelium formed by tall columnar cells attached to the basilar membrane. A noticeable exception to this pattern appeared in the two or three cells immediately external to the outermost Deiters' cell (Fig. 1); the cytoplasm of these cells had a watery aspect, with large empty vacuoles. The nucleus was pale and had coarse chromatin clumps. Very often, these cells had completely degenerated, and the morphological signs of cell death included the appearance of fragments of nuclei and cytoplasmic organelles located over the apical surface of the epithelium. At birth, these degenerating cells had been replaced by others whose cytoplasm showed numerous cisterns of rough endoplasmic reticulum, with the cisternal lumen almost filled by an a m o r p h o u s flocculent material (Fig. 2). The apical m e m b r a n e of these new cells, which were often swollen, was already covered by the minor TM, that was anchored to the adjacent Hensen's cells. The basal surface of these cells rested on the basilar membrane, although this contact seemed to be transitory, as we failed to find any conclusive EM image o f these cells attached to the basilar m e m b r a n e in sections from the 5th-6th postnatal day. Beside the detachment from the basilar membrane, other structural changes took place in these cells during the 1st postnatal week, such as the development of a prominent Golgi complex, which could be arranged in several dictyosomes (Fig. 3). The cisterns of the rough endoplasmic reticulum became larger and more abundant, and the material inside them closely resembled, ultrastructurally, the extracellular material lining the apical surface of the inner hair cell and pillar cells at the free end of the major T M (Figs. 3, 4). Moreover, this material is also similar, although less compact, to that observed in the endoplasmic reticulum of the third row of Deiters' cell, which began to secret the transitory marginal pillar of the T M at the 5th-7th postnatal day (Fig. 4). Another important cytoplasmic feature was the existence of small, granular electrondense inclusions inside the mitochondrial matrix (Fig. 3), from the 3rd to the 9th postnatal day. At the 10th postnatal day the organ of Corti showed an almost adult aspect, although the Nuel's spaces were

427

Fig. 1A-C. Developing organ of Corti at the 18th gestational day. A SEM photograph showing the apical surface of the medial coil of the cochlea. The tectorial membrane does not cover the OHC (stars) and it is anchored at the head of the pillar cell (arrow). x 1100. B TEM picture showing a degenerated nucleus over the

K611iker's organ, close to the developing major TM (MTM). x 2500. C TEM picture of the outer zone of the organ of Corti. External to the third row of Deiters cells (DC), two cells are expelled out to the endolymphatic space (arrows). OHC, outer hair cells, x 2500

n o t yet fully developed. The cells located external to the third r o w o f Deiters' cells did n o t show any swelling o f the apical m e m b r a n e , and they h a d a d a r k c y t o p l a s m containing just a few profiles o f cisterns o f endoplasmic reticulum (Fig. 5). The dense granules present in the mit o c h o n d r i a l matrix h a d already disappeared.

By the 14th postnatal day these cells could be readily distinguished f r o m the n e i g h b o u r i n g Hensen's cells, as they showed few and short microvilli and a darker cytoplasm (Fig. 6). Their m o r p h o l o g i c a l features therefore resemble those described by H e n s o n et al. (1983) for the tectal cells.

428 in the pillar cells), but in the adult animal only the tectal cells appeared labelled, while the Hensen's or the Claudius' cells were not (Fig. 7). Discussion

Fig. 2. TEM photograph of the developing tectal cells at the day of birth. The cells contain abundant rough endoplasmic reticulum (asterisks) with the cisternal lumen filled with an amorphous material. The apical membrane is swollen over the cell (arrows). x 6800

Lectin labelling The lectins used in this experiment stained distinctly the tectal cells both during development and in the adult animal. Three of the lectins (RCA, succ-WGA and SBA) stained only the apical surface of the tectal cells, but did not produce intracellular labelling. The staining with SBA and succ-WGA was only noticeable during the period in which the minor TM was attached to the LER cells. On the other hand, RCA labelling of the endolymphatic surface of the tectal cells remained faint until the adult stage (Fig. 7). The other three lectins (WGA, PHA-E and Con A) showed distinct patterns of intracellular labelling. The WGA stained some scarce cytoplasmic granules, both at early stages and in adult animals; the reaction was similar in all the supporting cells. The labelling with PHA-E was very strong on the apical and basolateral membranes of the supporting cells. The cytoplasmic labelling was diffuse in all the supporting cells, and there were no differences between them in early stages, but in adult animals tectal cells appeared slightly more stained than Hensen's and Claudius cells. Con A lectin showed the most distinct labelling between the tectal and neighbouring cells: at the 14th postnatal day the cytoplasmic reaction was strong in the region of the tectal cells, as well as in other neuroepithelial cells (e.g.

Two different processes occur in the LER during cochlear development, involving the cells placed external to the third row of Deiters' cells. These processes take place in the perinatal period, when the organ of Corti undergoes major developmental transformations (Lim and Anniko 1985). The first process is the sloughing off of the primordial epithelial cells occupying the tectal cell area; this may represent a morphogenetic degeneration. The inner sulcus is opened by the regression of the epithelial cells of the GER, which show many cytoplasmic vacuoles, considered to be autophagic in nature (Hinojosa 1977). On the other hand, the opening of the tunnel of Corti and the Nuel's spaces, which requires the transformation of the developing pillar and Deiters' cells, is not consistent with the existence of evident cytoplasmic vacuoles in these cells (Lim and Anniko 1985). Our results suggest that the cell death in the tectal cell area is a degeneration process different from those described in the forementioned areas. Many different mechanisms of cell death have been described in various systems (Oppenheim 1991). However, all of them fall into one of two categories (Kerr et al. 1987): one is necrosis, which appears in many pathological processes, and the other is called apoptosis, which is most characteristic of embryonic cell death and normal tissue turnover. Apoptosis involves a loss of cell volume with an initial preservation of cytoplasmic organelles and a later fragmentation of the cells, which are phagocytosed by adjacent cells (Oppenheim 1991). All the morphogenetic changes described in the development of the auditory receptor, including those affecting the tectal cell area, probably fall in the category of apoptosis. The second process that LER cells undergo during development is related to the genesis of the cells which replace the degenerated ones. The new cells appear at birth, and are attached to the basal membrane; in the following days, they ascend through the auditory epithelium, losing their contact with the basal membrane by the 5th or 6th postnatal day. These new cells have several cytological characteristics which suggest high metabolic and secretory activities, such as a large Golgi complex, abundant amorphous material inside the cisterns of rough endoplasmic reticulum, and dense granules inside the mitochondrial matrix. The transitory presence o f dense granules in the mitochondrial matrix has been related to certain physiological and pathological changes in cell metabolism (Smith and Ord 1983). Moreover, in other organs the small granular inclusions in the mitochondrial matrix contain calcium (Normann and Hall 1978), a finding which indicates a mechanism of cation sequestration by the mitochondria. It is known that the activation of different cell receptors may produce a transient increase in free cytoplasmic Ca 2 § which, among other things is able to

429

Fig. 3A-C. The auditory receptor at the 5th postnatal day. A SEM photograph of the medial coil of a cochlea in which the TM has been removed. The arrows point to the tectal cell area. x2000. B T E M picture of the cytoplasm of the developing tectal cells, showing small granular electron-dense inclusions within the mitochondrial matrix, x 8000. C T E M picture of the tectal cell. The

apical membrane ~s swollen, protruamg into me enaolympnauc space (arrows). A b u n d a n t cisterns of the Golgi apparatus can be observed (arrowheads). The dilated cisterns of endoplasmic reticulum appear filled with an amorphous material (asterisks), similar to that observed at the free end of the newly formed M T M in Fig. 4. x 2500

430

Fig. 4. TEM photograph of the organ of Corti at the 7th postnatal day. The major TM (MTM) covers the inner hair cells (IHC) while the minor TM (rnTM) is anchored to the Hensen's cells (arrow), external to the tectal cells (TC). Note that the newly formed mar-

ginal pillar (arrowheads)is located over the third row of Deiters' cell. Asterisks indicate the stereocilia of the outer hair cells. PC, pillar cells, x 1200

trigger some secretory processes. The presence of dense granules in the mitochondrial matrix of tectal cells might be related to high levels of free Ca 2 + in the cytoplasm and, in fact, mitochondrial granules are present when the tectal cells show morphological evidence of secretory activity but, after the 9th postnatal day, once the morphology of the cells has changed, the mitochondrial granules are no longer seen. It is well known that the major T M is secreted by the cells of K611iker's organ (Lim 1977, 1987; Hinojosa 1977; Gil-Loyzaga et al. 1985; Lim and Anniko 1985; Lira and Rueda 1990 a; Prieto et al. 1990 a; Rueda and Lim 1993), while the minor T M is produced by the L E R cells (Lira 1977; Lim and Anniko 1985; Rueda and Lim 1988; Lim and Rueda 1992). Therefore, it seems reason-

able to relate the secretory activity of the immature tectal cells, which are part of the LER, to the synthesis of components of the minor TM. The adult T M is composed of two types of fibrils: type A and type B (Kronester-Frei 1978). Type A fibrils are packed in bundles throughout the main body and the basal layer of the TM. Type B fibrils are heavily concentrated in peripheral structures known as the marginal band, cover net and Hensen's stripe (Steel 1986). By means of various techniques involving histochemical and immunocytochemical methods, it has been shown that different parts of the T M differ in composition. Type II collagen has been suggested as a component of type A fibrils (Lim 1987; Richardson etal. 1987), while glycoconjugates have been identified both in type

431

Fig. 5. TEM photograph of the outer zone of the organ of Corti at the 10th postnatal day. The organ of Corti is like that of the adult, although the Nuel's spaces are not open yet. The marginal pillar (asterisks) contributes to anchor the TM (TM). The tectal cell (TC) has dark cytoplasm with a few cisterns of endoplasmic reticulum. Note that no swelling is present at the apical surface of the cell. The mitochondrial matrix does not contain granules. DC, Deiters' cells. OHC, outer hair cells. • 3300

Fig. 7A-D. Photomicrographs of 1-gin-thick sections of the auditory receptor showing lectin labelling at the 4th postnatal day (A, B) and in the adult stage (C, D). RCA (A, C) strongly stains the tectorial membrane (TM), as well as the endolymphatic surface of the cells (arrows). Con A shows intracytoplasmic reaction at the 4th postnatal day (B) in the tectal cell area (7), the pillar

Fig. 6. TEM photograph of a tectal cell (TC) at the 16th postnatal day. The cell shows few and short microvilli and dark cytoplasm, different from that of the neighbouring Hensen's cells (HC). x 7000

cells (P) and the K611iker's organ (KO). In adult (D), besides the reaction of the tectorial membrane (TM), intracellular staining is evident in tectal cells (arrowhead) but not in neighbouring cells. IS, inner sulcus; T, tunnel of Corti; /, inner hair cell; O, outer hair cell. A x250; B a n d D x320; C x512

432 A fibrils, by means o f cationic dyes (Hasko and Richardson 1988; Prieto et al. 1990b), and in type B fibrils by means o f lectin labelling at the ultrastructural level (Lim and R u e d a 1990 b). The nature o f the material secreted by the developing tectal cells is currently u n k n o w n , as they react differently using different histochemical techniques. The alcian blue/PAS staining m a r k s the pillars and Dciters and Hensen's cells, but n o t the tectal cells (Lim and R u e d a 1990a, Figs. 3, 4). O n the other hand, results o f lectin staining suggest that Hensen's cells are responsible for the p r o d u c t i o n o f the cover net and marginal band, while Deiters' cells and pillar cells are involved in the p r o d u c tion o f the m i n o r T M (Rueda and Lira 1993). In our present experiments, tectal cells were labelled with some lectins different f r o m those staining Hensen's cells. Moreover, when the development o f the L E R is experimentally impaired (as in the congenitally h y p o t h y r o i d cochlea), the m i n o r T M is n o t f o r m e d (Prieto et al. 1990a). This lends s u p p o r t to the idea that L E R cells have a role in the p r o d u c t i o n o f the m i n o r TM. The different reaction o f the tectal cells to b o t h alcian blue/ P A S and lectin staining suggests that tectal cells participate in the secretion o f some c o m p o n e n t s o f the m i n o r T M which are different f r o m those p r o d u c e d by Deiters' cells, Hensen's cells and pillar cells. This idea is also supported by the presence inside the cisterns o f the endoplasmic reticulum o f a material that shows ultrastructural similarities with that f o u n d in the T M . Further studies involving lectin labelling at ultrastructural level, as well as a u t o r a d i o g r a p h i c experiments using 3H-labelled carbohydrates are needed for a better u n d e r s t a n d i n g o f the role o f the tectal cells during the development o f the auditory receptor.

Acknowledgements. This study was supported in part by the Spanish Government (DGICYT PB89-0485 and PB91-0752). The authors wish to thank Dr. Jos6 M. Juiz for helpful comments, Maria Dolores Segura for technical assistance, Margarita Castro for typing and Emilio Guti6rrez and Mercedes Garcla Encinas for illustrations.

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