maturation of junctional complexes during embryonic and early postnatal development of inner ear...

Am J Otola~'ngol 3:242- 253, 1982

Maturation Complexes and Early Postnatal Development of Inner Secretory Epithelia

of Junctional during Embryonic

Ear

MATTI ANNIKO, M.D., PH.D., AND DAN BAGGER-SJ6BKCK, M.D., PH.D.

The development and maturation of tight junctions in secretory epithelia of the inner ear, i.e., the stria vascularis in the cochlea and the dark cell epithelium around the vestibular organs, have been analyzed with the freeze-fracture technique. Inner ears were followed from the otocyst stage to the mature stage (mouse; gestational age 20 to 21 days). Before epithelial cells differentiate into specific tissues, tight junctions have a loose network of sealing elements with zero to four sealing strands. During and after cytodifferentiation, the numbers of strands increase both in the marginal cells of the stria vascularis and in the dark cells around the vestibular organs. Large intra- and intercellular variations in the numbers of sealing strands of the tight junction occur prior to birth in both types of secretory epithelia. In the dark cells the mature structure of the tight junction with five to eight sealing elements is reached at birth. Maturation of marginal cell junctions occurs during the first few days postnatally. Thus, the tight junctions are morphologically mature before the development of the high potassium concentration in the endolymph. (Key words: Secretory epithelia; Inner ear; Freeze-fracturing; Tight junction; Endolymph maturation.)

Different types of epithelia vary with regard to transepithelial permeability. Claude and Good- enough j correlated these functional properties with junctional morphology. "Leaky" junctions, such as those found in the proximal convoluted tubule of the mammalian kidney, are composed of on ly one or two strands, while the phys- iologically " t igh t" tight junctions, e.g., those that seal the amphibian urinary bladder, have four to 11 strands. Tight junctions vary from tissue to tissue in the numbers of sealing strands and in the geometric organizations of networks that are formed by the cross-linking of individual strands. The extent to which the component sealing strands are interconnected and the direc- tion in which the resultant linkage groups are oriented relative to the cell surface appear to

From the Department of Otolaryngology, Karolinska Hos- pital and King Gustaf V Research Institute, Karolinska Insti- tute, S-104 01 Stockholm, Sweden. Received January 12, 1982. Accepted for publication February 23, 1982. Supported by grants from the Swedish Medical Research Council proj- ect no, 12X-720, and the Foundation Tysta Skolan.

Address correspondence and reprint requests to Dr. Anniko.

be characteristic of tight junctions in a given tissue, 'z-4 Even within a given tissue, the pack- ing of the individual strands and the general orientation of the network can vary w i th the physiologic state of the tissue. 5

The ionic c o m p o s i t i o n of e n d o l y m p h is unique for an extra-cellular fluid with its very high potass ium concentra t ion and very low sodium concentration. ~ This composition of endolymph has been considered essential for the initiation of hearing. 7 Recent studies show that an endolymphatic hydrops can occur in both the cochlear and vestibular parts of the inner ear after experimental blocking of the communication between the endolymphatic duct and the cochlea, indicating that there are probably two independent systems for the production of inner ear fluids in the cochlear and vestibular parts of the labyrinth, s In the mouse, the maturat ion of endolymph occurs during the first week postnatally :~ in parallel with the morphologic maturation of the stria vascularis ~~ but after the vestibular dark cells have reached maturity. '~ The electrophysiologic maturation of the cochlea is

0196-0709182/070010242 $02.40 ~) W. B. S a u n d e r s Co,

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ANNIKO AND BAGGER-SJOB,~CK

Figure 1. A (above), electron micrograph (EM) showlng a columnar cell from the twelfth gestational day otocyst epithelium. The cell surface is covered with several microvilli (MV). The tight junction complex (shown in higher magnification in B), below, is composed of irregular strands with zero to four sealing elements. The lack of sealing strands is indicated with arrows. The tight junction is im- mature, with a shal low network of strands that are not always cont inuous . The membrane contains a large number of particles. Gap junctions are not apparent, but one is sus- pected (double mTOW). A x2,800; B, x6,100,

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JUNCTIONAL COMPLEX MATURATION

Figure 2. Electron micrograph, fourteenth gestational day inner ear, The anatomic location is the region close to or in the secretory ceils. The cells are covered with a thick layer of microvilli (MV). The endolymphatic space (ES) contains aggregates similar to cell arganelles (asterisks}. The tight junction (arrow) is composed of two to three sealing elements loosely connected. •

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reached during the second to third weeks postnatally. 12,L3

The present study was undertaken to analyze the epithelial junctions (tight junctions and gap junctions) in the secretory epithelia (stria vascularis in the cochlea and dark cells around the vestibular organs) of the developing inner ear, from the otocyst stage until the time of the mature composition of endolymph, which in the mouse occurs postnatally. 9

MATERIAL AND METHODS

CBA/CBA mice were used as the experimental animals. The gestational age was determined with the vaginal plug technique by considering day I the day when a mucoid plug was observed. Embryonic inner ears were taken for fixation on gestational days 12, 14, 15, 16, 19 and 21 (day of birth), and two, four, six, and eight days after birth. Specimens were fixed in 3 per cent glutaraldehyde in 0.133 M sodium phosphate buffer. After being rinsed in buffer, the specimens were transferred to a buffered solution of 30 per cent glycerol for two hours. The specimens were thereafter microdissected: prenatal inner ears were divided into the vestibular and cochlear parts of the labyrinth, except for the twelfth gestational day inner ear anlage, which

is cystic and was left intact. Concerning the postnatal inner ears, the vestibular organs with surrounding membranous structures and the stria vascularis (mainly from the basal coil) were dissected free from surrounding structures. The total material consisted of 108 specimens.

The specimens were mounted on gold speci- men holders and fractured in a Balzers 360 M freeze-fracturing unit. The resultant replicas were transferred to copper mesh grids and ex- amined in a Philips 400 electron microscope. In addition, the junctions in the developing stria vascularis were analyzed in thin, eponembed- ded sections of the one- to four- day postnatal inner ear (36 specimens). This period had, according to the freeze-fractured specimens, the most active stages in tight junctional maturation. These additional inner ears were fixed in 3 per cent glutaraldehyde in 0.133 M sodium phosphate buffer. After being rinsed in buffer, the specimens were postfixed in 2 per cent osmic tetroxide, dehydrated in increasing concentra- tions of alcohol, and embedded in epon mixture.

RESULTS

The twelfth gestational day inner ear anlage is composed of an otocyst with a columnar, in part pseudostratified, epithelium lining the otocyst

ANNIKO AND BAGGER-SJOBKCK

Figure 3. Elect ron micrograph, f i f teenth ges- rational day, Cell at the anatomic location of the stria vascularis. The surface is covered with microvilli of different shapes: long and slender with a club-shaped top (unfilled arrow) or of an inverted pear shape (asterisk). Pos- sibly the latter s tructure represents secretion of or- ganic mater ial from the cell into the endalymphat- ic space, The tight junc- t ion comprises approximately five parallel strands that are incomplete and loosely connected, x4,600,

Figure 4, Elect ron micrograph, the same conditions as in Figure 3. This t i g h t j unc t i on at f i rs t glance appears extensive for this embryonic stage. However, the strands are i n c o m p l e t e and not always connected with each other. These characteris- tics classify the junction as "very leaky" according to Claude and Goodenough. 1 Notice that the strands are not confined to the apical part of the cell but also go fu r t he r down the cel l (arrows). A gap junction is close to a s t rand in the t i g h t j unc t i on c o m p l e x (unfilled arrow), x 11,000.

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Figure 5. Electron micrograph, apical part of a marginal cell (MC) on the sixteenth gestational day. Most marginal cells have this type of tight junction with zero to three sealing elements. The area with- out strands is indicated by an arrow, x13,600.

Figure 6. Elect ron micrograph, nineteenth ges- rat ional day. The t ight junctions of the marginal cells have three or more seal ing e lements . The network of strands is ex- tending down on the cell surface {arraws). x8,100.

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Figure 7. Elect ron micrograph, day of birth (twenty-first gestational day). The marginal cell has five to seven parallel s t rands of seal ing elements compr i s ing the tight ]unction. Two large gap junc t ions (arrows) occur be low the t ight junction. This maturity of tight junctions occurs frequently also on tile nineteenth ges ta l iona l day. x8,000.

ANNIKO AND BAGCER-SJOBA.CK

Figure 8 (left). Electron micrograph, four days after birth (DAB). The tight junction has about the same morphologic charac- teristics as in the eight-DAB marginal cell and as described in the literature for mature marginal ceils from other species. Compared with embryonic tight junctions, the network of strands has a greater depth but not a larger number of parallel strands, x 8,800. Figure 9 (right). Electron micrograph, four days after birth. The tight junction of this marginal cell is extremely "tight" according to the classification of Claude and Goodenough.1 Approximately ten strands occur, The network of sealing elements is of a considerable depth. Tight junctions with this extension are also observed in elder animals, x18,400.

lumen. There is no spec ia l iza t ion of cells except that cells reaching the surface are covered with m i c r o v i l l i . D u r i n g s u b s e q u e n t days , m o r - phogenes i s occurs and is largely ended on the s ix teenth gesta t ional day, w h e n the inner ear has r eached m o r p h a l o g i c matur i ty on gross exami- na t ion . C y t o d i f f e r e n t i a t i o n of cells into spe- c ia l ized epi thel ia can be ident i f ied as early as the four teen th ges ta t ional day, ma in ly in the early fo rmat ion of hair cells, cupula , and otoconia, j'~ Cells at the ana tomic site for the stria vascularis are ident i f ied on the fif teenth to sixteenth gesta- t ional days. is The locat ion for the dark cell epi- t he l ium can be ident i f ied one to two days ear- lier. H

The freeze-fracture part of the present s tudy w a s f o c u s e d on the j u n c t i o n s of t hese two ep i the l i a after the i r ana tomic local izat ion be-

came apparent. Prior to these stages references are made to support ing cells in general.

Freeze-fractured Specimens

TWELFTH GESTATIONAL DAY. The co lumnar cells were covered wi th microvil l i . The t ight junctions were composed of one to three sealing elements, which were often rather fragmented and sha l l ow . The re was no r egu l a r n e t w o r k of strands. At several sites sealing strands were lacking [Fig, 1). A small number of gap junctions could be seen. The particle density of the cell membrane was high.

FOURTEENTH GESTATIONAL DAY. A t t h i s stage of embryonic deve lopment the gross morphologic features of the cristae ampullares had started to form. Epithelial cells were covered with a th ick


247


Figure I0 Cleft}. Electron micrograph, fifteenth gestational day. This future dark cell has an irregular network of sealing elements with a range from two to eight strands within the same junction. The strands comprise a loosely connected network. The cell membrane has a large number of small particles. • Figure 11 (right). Electron micrograph, day of birth. This dark cell has a mature structure of the tight junction with regard to the number of strands (five to eight) and the depth of the network. The cell membrane has fewer particles than during embryonic development, Compare with Figure 10. )<8,600.

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layer of microvilli. It was still difficult to distin- guish the epithelial layer of the crista itself from cells developing into the secretory epithelium surrounding the vestibular organs (Fig. 2). The general features of the tight junctions were approximately the same as those observed on the twelfth gestational day except for an increase to one to four sealing elements.

Epithelial cells in the cochlea (non-hair cells) had a structure of t ight junctions similar to that described for the vestibular epithelium.

Stria Vascularis

FIFTEENTH GESTATIONAL DAY. Cells at the lateral wa l l o f the scala med ia were covered w i t h numerous microvilli. The tight junctions were composed of two to five strands but had a very i r regu la r ne twork and were no t con t i nuous (Figs. 3 and 4). Large gap junctions occurred, sometimes in close connect ion wi th the tight junct ional complex. The cell membrane contained a large number of particles.

SIXTEENTH TO TWENTY-FIRST GESTATIONAL

DAYS. This period was character ized by reg- ularization of the network of sealing strands, which became cont inuous and often parallel. The number of strands increased from about three on the sixteenth gestational day to five to six on the nineteenth to twenty-first gestational days. Large gap junctions connected adjacent cells (Figs. 5, 6, and 7). The cell membranes contained large numbers of particles. Although the general trend was to gain morphologic ma- turi ty of t ight junctions sealing the marginal cells towards the endolymphat ic space, intra- and intercellular variations in the numbers of sealing strands occurred during the embryonic stage. This became however, less apparent with increasing age. From the n ine teenth gestational day on, strands occurred directed toward the basal part of the cell (Fig. 6).

Two TO EIGHT DAYS AFTER BIRTH (DAB). The number of strands did not increase postnatally as compared with previous developmental stages. The network of sealing elements, however, increased in depth (Fig. 8). Mature condi t ions

ANNiKO AND BAGGER-SJOBJ~CK

Figure 12, Electron micrograph, eight days after birth. The dark cell has a mature configuration of the tight junction. How- ever, gap junctions are few. The area with cyto- plasmic projections from the dark cell is indicated by an asterisk. •

were present in the 4-day-old stria vascularis (basal coil). Occasionally, an extensive network with eight to ten sealing strands was found (Fig. 9).

Dark Cell Epithelium

The developmental sequence for the maturation of tight junctions was the same as that described for the marginal cells of the stria vascularis. At the fifteenth gestational day, the tight junctions were more mature than those in the stria vascularis. However , great variation occurred not only between adjacent cells but also within the same tight junction (Fig. ]0). The

numbers of sealing strands ranged from two to nine at the same cell m e m b r a n e . While the downward extension of the network of strands in the stria vascularis occurred mainly postnatally, this extension was found on the future dark cells prior to birth.

Mature conditions with five to eight sealing elements were present in most cell at birth, including the general organization of the tight junction (Fig. 11). As in the marginal cells of the stria vascularis, a small variat ion in the numbers of sealing strands occurred between ad}a- cent cells. Mature dark cel[s had fewer gap junctions than did marginal cells of the stria vascularis (Fig. 12).


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Figure 13 (above, left). Electron micrograph, stria vascularis of the basal coil of a 1-day-old mouse. The marginal cells (MC) are closely apposed in their apical parts (arrows), indicating a tight junction. The intercellular space below the tight junction contains some granular material. At two regions (unfilled arrows) the two MCs are more closely together, indicating the formation of cell junctions. In the adult stria vascularis desmosames are frequently found at these locations, x21,000. Figure 14 (above, right}. Electron micragraph, stria vascularis of the basal coil of a 1-day-old mouse. The marginal cells (MC) become closely opposed at various levels from the endolymphatic surface (arrows). This micragraph shows the basal portion of MGs, as is indicated by the location of the cell nuclei. At this level of the MCs these appositions can possibly indicate the formation of gap junctions (unfilled arrow), x 14,100. Figure I5 (below, left). Electron mtcrograph, stria vascularis of the basal coil of a 3-day-old mouse, The cell membranes of two marginal cells (MC) are close together at a slightly greater distance than at previous stages of postnatal maturation. Compare with Figure 13. The tight junction (arrows) is morphologically mature in thin sections. Below the tight junction other junctional complexes are forming (unfilled arrows). The maturation of these junctions is similar to that found at the 1-day-old stage. Compare with Figure 13. The MCs still show morphologic signs of high metabolic activity: distended rough endoplasmic reticulum (asterisk) and clusters of ribosomes (polysomes). • 19,900. Figure 16 (below, right). Electron micrograph, stria vascularis of the basal coil of a 4-day-old mouse. Below the tight junction (arrows) the marginal cells (MC) are considerably closer together than at previous postnatal stages, However, the intercellular distances vary greatly, and the formation of cellular digitations has started (unfilled arrows). The MCs still show signs of high metabolic activity: distended rough endoplasm[c reticulum, polysomes and microcytotic vesicles at the cell surface, x19,000.

ANNIKO AND BAGGER-SJOB~,CK

Figure 17 (above, left). Electron micrograph, the same conditions as in Figure 16. In this region of the stria vascularis the cell membranes of the two marginal ceils (MC) follow in parallel for a long distance below the t ight junctiarL (arrows). Compare with Figures 13, 15 and 16. This intercellular area has a mature configuration. Compare wi th Figure 20. x 15,100.

Figure 18 (above, right). Electron micrograph, stria vascularis of the basal coil of a 3-day-old mouse. The intercellular distances between adjacent marginal cells (MC) vary greatly, but a large number of junctional complexes are unde r development (arrows). At the level of the cell nucleus shown here, gap junctions are frequently found in the adult stria vascularis, x 15,000.

Figure 19 (below, left). Electron mierograph, stria vascularis of the basal coil of a 4-day-old mouse. Cellular digitations from marginal cells (MC) are under development, as are junctional complexes (filled arrows). The arrows indicate what is possibly the lower end of the apically located tight junction, x 19,800. Figure 20 (below, right). Electron micrograph, stria vascularis of the basal coil of a 2-month-old mouse. The cell junctions are morphologically mature. The t ight junction is indicated by arrows. Several desmosomes are marked with unfi l led arrows. The marginal ceils (MC) have their cell membranes in parallel below the desmosomes, x16,800.


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Ultrathin Sections of the Postnatal Stria Vascularis

The junctional maturation of marginal cells is illustrated in a series of transmission electron micrographs (Figs. 13-20).

The tight junction was short at birth but rapidly increased in length during the following two to four days. Similar findings were observed in freeze-fractured specimens, where the depth of the sealing increased. Thin sections showed that adjacent marginal ceils became attached to each other below the tight junction at different levels. These areas were recognized in the mature stage as zonulae adherens (desmosomes). Freeze-fractured specimens showed gap junctions close to the tight junctions.

DISCUSSION

The sequence of the maturation of tight junctions of the inner ear secretory epithelia is in agreement with the morphologic maturation of the two cell types, i.e., the vestibular structures become mature before the cochlear structures. The dark ceils and the marginal cells have similar compositions of mature tight junctions with five to eight sealing strands. These findings concerning the marginal cells of the mouse are in agreement with corresponding studies of the mature stria vascularis of the guinea pig ("intermediate to tight" type, i.e., four to seven sealing strands). 16'1~ However, occasionally we also found junctions of the very tight type, with ten or more sealing strands.

In comparison, in the cochlea of the chinchilla the apical band of tight junctions between supporting cells and between supporting and hair cells consists of four to 18 cIosely spaced, parallel tight junctions that seldom branch. TM Con- cerning the guinea pig, Jahnke 19 showed that the zonulae occludentes of the cochlear and vestibular sensory epithelia consist of strands of the "very tight" type. The junctional complexes likewise create a barrier against flow of ions and maeromolecules.'."~

During embryonic development there is a rapid proliferation of cells, and cell-to-cell communication is of great importance. 2~,''~ Revel et al. 24 showed that in the early chick embryo large gap junctions frequently occur during early stages of embryonic development, whereas the formation of tight junctions is a sign of later maturation and organization. These general principles are also valid, according to our observations for the secretory epithelia of the mare-

malian inner ear. Although dark cells and marginal ceils are present at their anatomic locations early during embryonic development, the maturation of the tight junctions takes approximately a week, and occurs in parallel with the general morphologic development of the two cell types. '~ The tight junctions are mature before the development of the specific elemental composition of endolymph." Gap junctions frequently occurred between marginal ceils but were fewer between dark cells. In general, gap junctions are found where one can demonstrate physiologic coupling.

Different epithelia vary with respect to transepithelial permeability. Because of the specific composition of mature endolymph, the scala media must be sealed from its environment in order to maintain its integrity. In general, the inner ear epithelia have an "intermediate-to- tight" type of tight junction, which thus requires an active process to preserve the ionic composition of endolymph. Such a function in die cochlea is considered to occur in the stria vascularis. The marginal and dark cells showed deep branching of the sealing strands. According to Pricam et at., 2'~ it is not always the number of sealing strands but sometimes the degree of branching and the depth of a given tight junction that can be the most important features to corre- late with epithelial permeability, at least when using extraeellular tracers. In the endolymphatic sac of the guinea pig considerable differences in the numbers of strands occur between the proximal, intermediate, and distal portions. 's Differences in the ionic compositions of endolymph have been found between cochlear and vestibular endolymphs as well as between endolymph in the endolymphatic sac and other endolymphs. 27 It seems probable that the number of sealing strands and the depth of the tight junction contribute to the permeability of the tissue.

References

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2. Staehalin LA- Further observations on the fine structure of freeze-claaved tight junctions, J Call Sci 13:763- 786, 1973

3. Staehalin LA: Structura and fuactian of intercellular junctions. Int Rev Cytol 39:191-283, 1974

4. Hull BE, Staehelin LA: Functional significance of the variations in the geometrical organization of tight junction networks. J Cell Biol 68:888-704, 1976

5. Pitelka DR, Hamamoto ST, Duafala JG, at ah Cell contacts in the mouse mammary gland. J Cell Biol 56:797-818, 1973

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6. Smith CA, Lowry OH, Wu ML: The electrolytes of the labyrinthine fluids. Laryngoscope 64:141-153, 1954

7. Basher SK, Warren RL: A study of the electrochemistry and osmotic relationships of the cochlear fluids in the neonatal rat at the time of the development of tha en- docochlear potential. J Physiol (Lend) 212:739-761, 1971

8. Kimura RS, Schuknecht I-IF, eta CY, et ah Obliteration of the ductus reunions. Acta Otolaryngol (Stoekh) 89:295-309, 1980

9. Anniko M, WrCblewski R: Elemental composition of the developing inner ear. Ann Otol Rhinol Laryngol 9Q:25-32, 1981

10. Anniko M, Nordemar H: Embryogenesis of the inner ear. IV. Postnatal maturation of the secretory epithelia of the inner ear in correlation with the elemental composition in the endolymphatic space. Arch Oto-Rhino- Laryngol 229:281-288, 1980

11. Anniko M: Embryologic development in viva and in vitro of the dark cell region of the mammalian crista ampullaris. Acta Otolaryngol (Stockh) 90:106-114, 1980

12. Mikaelian D, Ruben RJ: Development of hearing in the normal CBA-J mouse. Correlation of physiological observations wi th behavioral responses and with cochlear anatomy. Acta OtolaryngaI (Stockh) 59:451-461, 1965

13. Alford BR, Ruben RJ: Physiological, behavioral and an- atomical correlates of the development of hearing in mouse, Ann Otol Rhinol Laryngol 72'.237-247, 1963

14, Annlko M: Embryonic development of vestibular sense organs and their innervation, in: Remand R ted,): De- velopment of Auditory and Vestibular Systems. New York, Academic Press, 1982, in press

15. Sher AE; The embryonic and postnatal development of

the inner ear of the mouse. Acta Otolaryngal (Stockh) suppl 285, 1-77, 1971

16. Reale E, Luciana L, Franke K, eta]: Intercellular junctions in the vascular stria and spiral ligament. ] U1- trastruct Res 53:284-297, 1975

17. Jahnke K: Die Feinstruktur gefriarge~tzter Zellmembran- Haftstellen der Stria vascularis. Anat Embryo] 147: 189-201, 1975

18. Gulley RL, Reese TS: Intercellular junctions in the re- ticular lamina of the organ of Corti. J Neurocytol 5:479-507, 1976

19. Jahnke K: The fine structure of freeze-fractured intercellular junctions in tke guinea pig inner ear. Acta Otolaryngol (Stockh) suppl 336:1-40, 1975

20. Farquahar MG, Palade GE: Junctional complexes in various epithelia. ] Cell Biol 17:375-412, 1963

21. Frbmter E, Diamond 1: Route of passive ion permeation in epithelia. Nature (New Biol) 235:9-13, 1972

22. Bennett MVL: Function of electrotonic junctions in embryonic and adult tissues. Fed Proc 32:65-75, 1973

23. Lowenstein WR: Membrane junctions in growth and differentiation. Fed Proa 32:60-64, 1973

24. Revel JP, Yip P, Chang LL: Cell junctions in the early chick embryo--a freeze etch study, Devel Biol 35:302-317, 1973

25. Pricam C, Humbert F, Perrelet A, et ah A freeze-etch study of the tight junctions of the rat kidney tubules. Lab Invest 30:286-291, lg74

26. Bagger-Sj/sb~ick D, Lundquist P-G, Rask-Andersen H: In- tercellular junctions in the epithelium of the endolymphatic sac, in:Vosteen K-H, Schuknacht H, Pfalz CR st al (eds.): M~ni~re's Disease. Berlin, Georg Thieme, 1981, pp 127-140

27, Miayamoto H, Morgenstern C: Potassium level in endolymphatic sac of guinea pig in vivo. Arch Oto- Rhino-Laryngol 222:77-78, 1979


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