The Early Development of the Otic Vesicle in Staged Human Embryos

J. Embryol. exp. Morph., Vol. 11, Part 4, pp. 741-755, December 1963 Printed in Great Britain

by RONAN O'RAHILLY1 From the Department of Anatomy, St. Louis University School of Medicine, and the Department of Embryology, Carnegie Institution of Washington

WITH TWO PLATES

INTRODUCTION EVER since Huschke first recorded, in 1831, that the membranous labyrinth develops from the surface ectoderm (Streeter, 1918), a number of studies of otic development have been made (as reviewed by Altmann, 1950). The introduction during the present century, however, of embryonic staging in all vertebrate classes, and its use especially during the past two decades, have enabled precise correlations of morphogenesis and histogenesis with external embryonic appearances to be made. In the opinion of the present writer, the term 'stage' should not be utilized unless a staging system has been employed, and particularly not where mere time or measurements have been used. Expressions such as 'the 39-day stage' or 'the 18-mm. stage' are thus to be deprecated. In the present study of the otic vesicle, particular attention has been directed to the basement membranes, which have been almost totally ignored in previous accounts both of the developing, and indeed even of the adult, labyrinth.

MATERIAL The present account is based on an examination of early, sectioned embryos in the collection of the Department of Embryology, Carnegie Institution of Washington. The planes of section and the stains varied; sixteen of the specimens were stained by Mallory's 'azan' procedure. The thirty-six embryos, which extend from stage 9 (3 post-ovulatory weeks) to stage 18 (6 post-ovulatory weeks), are listed in Table 1. The material is classified in stages according to the 'developmental horizons' devised by Streeter. The ages of the embryos can be estimated from their stages, according to the data provided by Olivier & Pineau (1962), as summarized in Table 2. 1 Author's address: Department of Anatomy, Saint Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis 4, Missouri, U.S.A. 742 RONAN O'RAHILLY

TABLE 1 Human embryos utilized in the present study of the otic vesicle

Stage No. Somites Stage No. Mm. 9 1878 2 13 836 40 10 3709 4 7889 4-2 391 8 9297 4-5 1201 8 8581 4-8 5074 10 8372 5-6 3710 11 14 8308 5-85 3707 12 8999 60 9870 12 6503 6-3 11 7611 16 8552 6-5 470 17 8314 80 5072 17 15 8997 90 8116 17 16 8773 130 12 8943 22 17 8998 110 8505b 23 8789 11 -7 9154 24 18 8355 150 6097 25 9247 150 7852 25 8942 25 5923 28 7724 29

TABLE 2 Approximate length and probable embryonic age of staged human embryos Post-ovulatory Stage Somites Mm. days 9 1-3 20 10 4-12 22 11 13-20 24 12 21-29 26 13 30- 4-6 28 14 5-7 32 15 7-9 33 16 7-11 37 17 11-14 41 18 12-17 44

OBSERVATIONS Stage 9 (1-3 somites) (Plate 1, fig. A) The otic region can first be distinguished in the embryo (No. 1878) of two somites as a thickening on the lateral aspect of the neural fold (Text-fig. 1). It is lined by a basement membrane that is continuous with that of the rhombencephalic neural groove. THE OTIC VESICLE IN HUMAN EMBRYOS 743 Comment In an embryo of two or 'possibly three' somites, Wilson (1914) recorded 'a diffuse and rather extensive thickening of the head ectoderm', which 'may possibly foreshadow the appearance of the 'auditory areas'. In the two- somite embryo (No. 1878) referred to above, Ingalls (1920) described a moderate thickening 'about opposite the middle of the rhombencephalon'. He identified it as the 'otic plate' and remarked that its basal surface 'is cleaner and sharper than in the adjacent body-wall and hence more like the neural folds'. The otic

TEXT-FIG. 1. Human embryo (No. 1878) of two somites. The left-hand drawing shows the dorsal aspect of the cephalic neural folds; the otic region is shown as a black area on each side. The middle drawing represents the left lateral aspect; the left otic region is indicated in black. The right-hand diagram shows, after median section, the right half of the embryo seen from the medial side. The location of the underlying otic zone is marked by a boundary line on the medial aspect of the right neural fold. zone apparently involves more ectoderm than is eventually incorporated into the otic vesicle. Groth (1939), from his study of the rabbit, preferred to speak of a lateral ridge, or Seitenwulst, from a part {Labyrinthzone) of which the otic plate (Labyrinthplatte) develops.

Stage 10 (4-12 somites) (Plate 1, fig. B) The nuclei of the otic plate occupy mostly a basal position, leaving a superficial cytoplasmic zone termed the marginal velum. The velum appears to be covered by a terminal bar net (as is the lens at a later stage) and a brush border. Mitotic figures, in this and subsequent stages, are found in the superficial 744 RONAN O'RAHILLY (later central) portion of the plate (as is also the case in the wall of the neural groove and tube). The first indication of invagination of the otic plate is observed at ten somites. Neural crest cells, recognizable in this region first at four somites, delaminate from the wall of the neural groove and proceed laterally and ventrally. They are presumed to be the facial, or the so-called acousticofacial, crest. Comment At four somites (No. 3709), Bartelmez (1922) recognized the otic plate and, medial to it, an enlargement in the medial wall of the rhombencephalic neural fold. This swelling was identified as the acousticofaciai division of the neural crest, or the acousticofacial ganglion, which 'arises near, but not exactly at the dorsal edge of the open neural fold' and not 'from cells intermediate between neural and somatic ectoderm'. At eight somites (No. 1201), the otic plate was distinguished by a clear, peripheral zone, the marginal velum. As a result of 'the lateral migration of the ganglionic Cells en masse' from the neural fold, an 'otic sulcus' (Bartelmez, 1922) was observed in the ventricular surface of the neural fold. At about nine somites ('Du Ga' embryo), Evans (according to Bartelmez, 1922) noted that the otic plate displayed a peripheral brush border. Moreover, the ganglionic anlage was found to be delaminating from the neural fold. Baxter & Boyd (1938) described an embryo of ten somites in which the 'acousticofacial crest' was continuous across the median plane, an appearance attributed to precocious closure of the neural tube. At twelve somites (No. 8970), invagination of the otic plate was just commenc- ing. The nuclei of the pseudostratified epithelium were basal in position, and

PLATE 1 The numeral at the lower right-hand corner of each photograph indicates the embryonic stage. FIG. A. Stage 9. Embryo No. 1878. Section 12-5-7. x270. The otic region constitutes the lateral (right-hand) aspect of the rhombencephalic neural fold. The basement membrane is visible. FIG. B. Stage 10. Alum cochineal. Embryo No. 5074. Section 2-1-2. x270. The nuclei of the otic plate can be seen to occupy mostly a basal position, leaving a marginal velum superficially. FIG. C. Stage 11. Embryo No. 7611. Haematoxylin and eosin. Section 3-19. x270. The otic pit has formed, and appears to be lined by a terminal bar net and a brush border. FIG. D. Stage 12. Embryo No. 9154. Iron haematoxylin and phloxine. Section 2-2-10. x 270. The otic vesicle is forming and its cavity communicates with the surface by a narrowing pore. In the lower half of the photograph, the wall of the vesicle appears to be contri- buting to the vestibulocochlear crest. FIG. E. Stage 13. Embryo No. 836. 'Azan.' Section 2-2-3. x200. A projection of the surface ectoderm indicates the site of the connecting stalk which became largely obliterated on closure of the otic pit from the surface. FIG. F. Stage 13. Embryo No. 9297. 'Azan.' Section 3-2-3. x 270. A basement-membrane-covered projection of the wall of the otic vesicle constitutes the remains of the connecting stalk. /. Embryol. exp. Morph. Vol. 11, Part 4

RONAN O'RAHILLY (Facing page 744) THE OTIC VESICLE IN HUMAN EMBRYOS 745 the marginal velum was observed. The mitotic figures were found near the free surface of the plate, that is, adjacent to the brush border. Bartelmez (1922) detected 'a well-developed internal reticular apparatus' in the form of 'a series of clear spaces in the cytoplasm'. Bartelmez & Evans (1926) considered that 'there is some evidence of migration from the otic disc', and it is even possible that the ' seeming spread of the crest cells is really a union of the crest with elements previously proliferated from the ectoderm', including the otic disc. In another twelve-somite embryo (No. 3707) also, an indication of initial invagination of the otic plate was noted. It is now realized that the nuclei of columnar epithelia in vertebrate embryos move temporarily to the free surface before a division. This intermitotic, nuclear migration, which occurs in the neural tube (Sauer & Chittenden, 1959) and its derivatives, in the otic plate and vesicle (Sauer, 1936), and also in the lens plate and vesicle, accounts for the appearance of mitotic figures in a juxta- luminal position. The so-called acousticofacial crest, or the caudate Kopfganglienleiste of Veit, appears to be still the object of considerable controversy. In mammals, some workers, e.g. Adelmann (1925), in the rat, have maintained that the vestibulocochlear ganglion 'is a derivative of the common acoustico-facial ganglionic mass, owing its origin entirely to neural-crest proliferation'. Bartelmez & Evans (1926), in the human, believed that the acousticofacial crest 'is concerned with the development of the geniculate ganghon and probably the acoustic ganglia'. On the other hand, according to a number of more recent authors, e.g. Van Campenhout (1935), in the pig, and Groth (1939), in the rabbit, the acousticofacial crest is purely facial, and the vestibulocochlear ganglion is derived from the otic vesicle. Similarly, Theiler (1949), in the human, found that, 'only with the constriction of the otic vesicle from the surface epithelium, do cells bud out from its basal circumference and unite with the foregoing ganglion'. Stage 11 (13-20 somites) (Plate 1, fig. C) The otic pit forms in the otic plate during this period, in a manner reminiscent of the formation of the lens pit three stages later (Text-fig. 2). Comment The progressive invagination of the otic plate has been well illustrated by Streeter (1942, his Fig. 8, xi). Between the middle of stage 10 and the middle of stage 11, Bartelmez & Evans (1926) found that 'the otic disc comes to lie progressively farther caudally' in relation to the neural tube. According to the same authors, the characteristic position of the otic disc dorsal to the second pharyngeal cleft is attained by sixteen (actually seventeen) somites. Also during this stage, it is maintained that additions from the surface ectoderm ('hyoid epibranchial placode') to the facial crest may possibly be made. It was pointed out by Sharp (1885) that 'invagination seems to be the most simple, as well as one of the commonest, methods by which organs are formed 746 RONAN O'RAHILLY in the animal series. . .. The formation of the eye (both lens and optic vesicle), ear, and nose, form no exception to this rule.' Moreover, the lens has even been considered 'in its own right a sensory structure', i.e., that 'the lens is an independent organ derived from an anterior placode of the epibranchial series' (Duke-Elder, 1958). A certain histogenetic similarity between the developing otic and lens vesicles is referred to several times in the present paper (Text-fig. 2). From another point of view, namely, that of its initial origin, a comparison may be made between (1) the developing otic plate, and (2) the medullary plate, together with its derivative, the retinal plate. Thus, the specialized appearance of the otic ectoderm has prompted some authors, e.g. Streeter (1942), to the view that it is to be regarded 'as a detached island of neural ectoderm, related

(a) (b)

Otic vesicle Lens vesicle Stage 13

11 «

15 10

TEXT-FIG. 2. A comparison between (a) the formation of the otic plate (stage 10), pit (stages 11 and 12), and vesicle (stage 13), and (b) the formation of the lens plate (stage 13), pit (stage 14), and vesicle (stage 15). Note the behaviour of the basement membranes.

to but not identical with the brain-wall ectoderm'. Furthermore, Groth (1939) maintained that the otic plate, the retinal plate, and the olfactory plate all arise from 'similar parts of the brain anlage', namely, the lateral ridge, or Seiten- wulst, at the border of the medullary plate. According to his scheme (his Fig. 28), an initially similar origin is envisioned, after which both the otic and the olfactory epithelium remain outside the developing neural tube, whereas the early retinal epithelium (i.e., prior to its invagination) becomes incorporated in the wall of the neural tube.

Stage 12 (21-29 somites) (Plate 1, fig. D) The otic vesicle is forming and its cavity communicates with the surface by a narrowing pore. In one specimen (No. 7724), the pore appears to have closed THE OTIC VESICLE IN HUMAN EMBRYOS 747 already and the vesicle is connected with the surface ectoderm by a solid cord of cells. Contributions from the ventral wall of the otic pit to the vestibulocochlear crest occur (e.g. in No. 9154 and No. 8944) but appear to be less conspicuous than the corresponding crest migrations from the optic primordium to the sheath of the optic vesicle (as described by Bartelmez & Blount, 1954). Comment It has been noted that the otic pit deepens and expands, and that its opening on the surface becomes progressively narrower.

Stage 13 (30 or more somites; approximately 4-6 mm) (Plate 1, figs. E, F, and Plate 2, figs. G, H) The otic pit becomes closed from the surface to form the otic vesicle, or otocyst, the dorsomedial portion of which can be distinguished as the endolymphatic appendage. In closing from the surface, the otic vesicle, like the lens, becomes surrounded by its basement membrane (Text-fig. 2). The remains of the connecting stalk may be seen in some specimens as a projection (covered by basement membrane) on the wall of the otic vesicle (e.g., in No. 9297, Plate 1, fig. F) and/or on the surface ectoderm (e.g. in No. 836, Plate 1, fig. E). In one specimen (No. 7889, Plate 2, fig. G), in which a continuous stalk is present, the continuity between the basement membrane of the surface ectoderm and that of the otic vesicle can be traced clearly along the stalk. As soon as the otic vesicle has separated from the adjacent surface ectoderm, however, the former is completely covered by, and the latter is completely lined by, a basement membrane. A few cells may be found in the cavity of the otic vesicle, but the nuclei and cell remnants characteristic of the cavity of the developing lens vesicle do not appear to have a counterpart in the ear. Over an appreciable area, the basement membrane of the otic vesicle and that of the rhombencephalon are seen to be in contact (Plate 2, fig. H), an arrangement reminiscent of that seen later (at stage 15) between the lens vesicle and the inverted lamina of the optic cup. An occasional indication of cellular migration from the wall of the otic vesicle may still be found (e.g. in No. 8372). The formation of the otic vesicle is summarized in Text-fig. 3. Comment The temporary stalk and its remnants were illustrated by several authors (e.g. Fineman, 1915; Perovic & Aust, 1915), and a detailed investigation of this feature (based partly on the Carnegie collection) was published by Anson & Black (1934), who did not, however, refer to the basement membrane. Accord- ing to Bast, Anson & Gardner (1947), the nodule-like remnant of the point of closure of the otic pit comes to be ' situated on the lateral aspect of the endolymphatic appendage at the junction of its distal and middle thirds'. Streeter (1945) found that a capillary network was laid down around the 748 RONAN O'RAHILLY vesicle (Plate 2, fig. H), particularly ventrally and laterally, where, in the more advanced embryos, the mesoderm was beginning to become condensed as the otic capsule.

Postovulatory days TEXT-FIG. 3. The formation of the otic vesicle in the human embryo. The ordinate represents embryonic length in mm., the abscissa shows age in postovulatory days, and the black rectangles indicate embryonic stages.

PLATE 2 The numeral at the lower right-hand corner of each photograph indicates the embryonic stage. FIG. G. Stage 13. Embryo No. 7889. Haematoxylin and eosin. Section 6-4-1. x200. The connecting stalk, which is still present in this embryo, shows the continuity of the basement membrane of the surface ectoderm and that of the otic vesicle. FIG. H. Stage 13. Embryo No. 7889. Haematoxylin and eosin. Section 6-3-12. x200. The basement membrane of the otic vesicle (left) and that of the rhombencephalon (right) are seen to be in contact over an appreciable area (above). FIG. I. Stage 17. Embryo No. 8789. 'Azan.' Section 9-2-3. x270. The thickened area of epithelium from which the wall of a semicircular duct will be formed. FIG. J. Stage 18. Embryo No. 9247. 'Azan.' Section 41-1-1. x 50. Low-power view of the membranous labyrinth, to show one of the projecting hollow shelves (right) which gives rise to a semicircular duct. FIG. K. Stage 18. Embryo No. 9247. 'Azan.' Section 41-2-2. x270. The epithelial layers adjacent to a developing semicircular duct (fig. L) fuse, lose their basement membrane, and sacrifice their identity, so that a portion of the otic cavity (within the curve of the semicircular duct) becomes obliterated. FIG. L. Stage 18. Embryo No. 9247. 'Azan.' Section 41-1-1. x270. Higher-power view of the developing semicircular duct shown in fig. J. /. Embryol. cxp. Morph. Vol. 11, Part 4

RONAN O'RAHILLY THE OTIC VESICLE IN HUMAN EMBRYOS 749 The basement membranes of both the otic vesicle and the lens vesicle have been shown to be PAS-positive in the chick embryo (Meyer & O'Rahilly, 1961;' O'Rahilly & Meyer, 1960).

Stage 14 {approximately 5-7 mm) As usual, the mitotic figures are placed centrally, that is, near the cavity of the vesicle. Remnants of the connecting stalk may still be seen in some specimens. The endolymphatic appendage is set off from the future utricular portion of the vesicle by a developing fold. Comment Streeter (1945) pointed out that the endolymphatic appendage is becoming tapered, and that models reveal that the ventral portion of the vesicle is becoming elongated to form the cochlear duct.

Stage 15 {approximately 7-9 mm.) The utriculo-endolymphatic fold becomes more pronounced. Ventro- medially, the tip of the vesicle represents the cochlear duct. The thickness of the wall of the vesicle varies from one portion to another; the wall of the endolymphatic appendage is becoming thinner. Comment It has been noted that the endolymphatic appendage is distinctly demarcated from the remainder (utriculosaccular portion) of the otic vesicle.

Stage 16 {approximately 7-11 mm.) Thickenings in the wall of the main, or vestibular, portion of the vesicle presage the appearance of the semicircular ducts.

Stage 17 {approximately 11-14 mm) (Plate 2, fig. I) Portions of the wall of the vestibular part of the. otic vesicle become thinner and approximated as a prelude to the cellular disintegration that takes place during the formation of the semicircular ducts at the next stage.

Comment Bast & Anson (1949) found that 'the endolymphatic appendage in the embryo of 12 mm. is divided into the proximally placed ductus and the distal expansion or saccus'. Stage 18 {approximately 12-17 mm) During the formation of each of the semicircular ducts from thickened areas of epithelium, the adjacent portions of the epithelium become thinned, the cells lose their individuality, and their basement membrane disappears, so that 48 750 RONAN O'RAHILLY the tissue merges with the underlying mesoderm (Plate 2, fig. K). Obliteration of the corresponding portions of the cavity of the otic vesicle ensues, so that the hollow ledges are converted into semicircular tubes, and so that continuity of the remaining epithelium and also of the remaining basement membrane is immediately restored. The wall of the semicircular duct opposite the site of sealing is thickened considerably (Plate 2, fig. L).

Comment During this stage, from one to three semicircular ducts are formed. The order of formation is anterior, posterior and lateral (Bast, Anson & Gardner, 1947). The mode of development was aptly described by Rathke in 1839 as follows (Hasse, 1873a): 'Each membranous semicircular duct arises when the membranous vestibule at one place makes a fold convex outwards, and then both leaves of the fold grow together at its base, and finally its substance is re- absorbed in a manner such that the newly arising duct is, as it were, .split off from the vestibule.' Hasse (1873a), in his discussion of the turtle, described a white Striation (Streiferi)> formed by columnar epithelium, along the concave side of the semicircular ducts of various vertebrates. From its appearance in the adult, he referred to this as a raphe, and it is now sometimes called the raphe of Hasse (mistakenly mentioned as being 'on the outer side' by Kolmer, 1927). Hasse believed that the raphe represents the site of closure of the developing duct, as described by Rathke. Kolmer, however, had some reservations in that he found that the raphe was not very clear in his embryonic material, and that its characteristic epithelium first appeared in later life (im spdteren Alter). The curious histogenesis of the semicircular ducts was studied further by Streeter (1906), who found that, in one embryo (No. 175) of stage 18, 'absorp- tion of the epithelium was going on before the ... walls of the vesicle had actually come together. So it is possible that during this process the vesicle cavity is in some cases left temporarily in open communication with the spaces of the adjacent mesoderm.' One is perhaps reminded here of the pre-emphy- sematous histological arrangement believed by some workers to play a role in the formation of alveoli in the lung. By about this time, growth changes in the utricular and saccular portions of the vesicle result in 'first, a rotation so that the endolymphatic (otic) duct and sac come to lie in a medial position . .. and, second, a shifting of the long axis of the otic vesicle from a vertical to a more and more horizontal position. Thus the semicircular ducts gradually come to lie laterally and the cochlea medially' (Bast & Anson, 1949). The L-shaped cochlear duct develops its characteristic spiral during the next five stages (Streeter, 1951). The details of the membranous labyrinth, with particular emphasis on the saccule, from approximately stage 19 until the end of the first trimester of intra-uterine life, have been described in some detail THE OTIC VESICLE IN HUMAN EMBRYOS 751 (Brunner, 1934). Furthermore, an account of the fetal development of the entire labyrinth has been provided in an important monograph (Bast & Anson, 1949).

DISCUSSION Particular attention to the behaviour of the basement membranes during the formation of the otic vesicle reveals (1) that, as in the case of the lens, although two stages earlier, a part of the basement membrane of the surface ectoderm becomes invaginated to form the basement membrane surrounding the otic vesicle; and (2) that, during the subsequent formation of each of the semicircular ducts, portions of the basement membrane of the otic vesicle disintegrate, thereby allowing the development of a hollow loop of membranous labyrinth to take place. After each of the above occurrences, (1) and (2), immediate restoration of the continuity of the basement membrane is accom- plished. The disintegration of portions of basement membrane during development, referred to under item (2) above, may be regarded as an instance of the general plasticity of the subepithelial supporting tissue, both basement membrane and lamina propria. Even in maturity, fluctuations in the thickness, the visibility, and the staining reactions of a basement membrane under varied conditions may be encountered. Thus, in the striated tubules of the submandibular gland of the guinea-pig, it was found that atropine caused an increase in the PAS reactivity of the basement membrane, whereas pilocarpine had the opposite effect (Miiller, 1954). In the case of ovariectomized mice, oestrogens resulted in a thinning and a heightened aniline blue intensity of the basement membrane of the vaginal epithelium (Bartoszeqicz & Dux, 1961). In the case of the embryonic eye (human), it has been shown (O'Rahilly, 1962) that the basement membrane of (1) the optic vesicle, and (2) the lens plate, persist into adulthood as, at least portions of, respectively (1) the internal limiting membrane of the retina and the basal lamina of the uvea, and (2) the lens capsule. The question arises, therefore, whether, in the case of the ear, the basement membrane of the otic vesicle also remains throughout life, in this instance as a membrane surrounding the various components of the membranous labyrinth. That a basement membrane is found in certain portions of the membranous labyrinth of the adult seems to be definite. Thus, Wislocki & Ladman (1955) observed that the tectorial membrane (mouse and human) stained intensely with the periodic acid-Schiff reaction, as did also 'a basement membrane upon which the epithelium of the maculae and cristae rests'. In addition, the basilar membrane (bat; guinea-pig) was found to be positive, as was also the basement membrane between the two cellular layers of the vestibular membrane (Plotz & Perlman, 1955; Mangabeira-Albernaz, 1961). In the semicircular ducts, Hasse (18736) figured a basement membrane (crocodile), and Sieber & Schmidt 752 RONAN O'RAHILLY (1962) detected (in the human) 'a narrow argyrophil membrane of the type of a basal membrane'; it was, however, 'visible not regularly or only here and there'. Even if we avoid a discussion of the relationship of basement membranes to ' submicroscopic adepithelial membranes' (Salpeter & Singer, 1960), it is of interest to note that 'basement membranes' have also been described with the aid of the electron microscope, e.g. in the utricle (Smith, 1956) and spiral prominence (Smith, 1957) of the guinea-pig, and in the cultivated spiral organ of the fowl (Friedmann, 1962). The basilar membrane (in the rat), which is frequently regarded as'a particularly thick, differentiated basement membrane', is actually separated from the epithelium of the spiral organ by a membrane that is 200A in thinness (Iurato, 1962). It can be concluded, therefore, that, although the existence of a continuous and intact basement membrane throughout the various ramifications of the adult membranous labyrinth appears to be still an open question, at least certain areas do possess a basement membrane, which may reasonably be presumed to be derived from that found surrounding the otic vesicle in the embryo.

SUMMARY 1. The early development of the otic vesicle has been investigated in thirty- six human embryos from stage 9 to stage 18 (3 to 6 post-ovulatory weeks). 2. The otic zone was first distinguished at stage 9 and invagination had com- menced in the more advanced specimens of stage 10. The otic pit deepened progressively during stage 11, and certain resemblances to the developing lens were noted. It is believed that contributions from the wall of the otic pit to the vestibulocochlear crest occurred, e.g. at stage 12, but they were not as conspicuous as the corresponding crest migrations from the optic primordium to the sheath of the optic vesicle. The otic pit became closed from the surface at stage 13 to form the otic vesicle, the dorsomedial portion of which became recognizable as the endolymphatic appendage. 3. As in the case of the lens, a part of the basement membrane of the surface ectoderm became invaginated (stages 11 to 13) to form the basement membrane surrounding the otic vesicle. 4. Although their precursors were detectable earlier, the cochlear and the semicircular ducts were clearly denned by the end of stage 18. During the formation of the semicircular ducts, portions of the epithelium lost their identity and their basement membrane disappeared, so that the epithelium merged with the underlying mesoderm, and obliteration of portions of the cavity of the otic vesicle ensued. 5. It has been established previously that the various basement membranes associated with the embryonic eye persist into adulthood. Moreover, it is known that at least certain portions of the adult membranous labyrinth possess THE OTIC VESICLE IN HUMAN EMBRYOS 753 a basement membrane, derived presumably from that surrounding the otic vesicle in the embryo. The possible existence of a continuous and intact basement membrane throughout the adult membranous labyrinth, however, seems to require further investigation. RESUME Les premiers stades du developpement de la vesicule auditive chez Vembryon humain 1. On a etudie le developpement precoce de la vesicule otique chez 36 embryons humains des stades 9 a 18 (3 a 6 semaines apres l'ovulation). 2. La zone otique a ete distingue en premier lieu au stade 9, et son invagination etait commencee chez les specimens plus avances du stade 10. La depres- sion otique s'approfondit progressivement pendant le stade 11, et on a note certaines ressemblances avec le cristallin en cours de developpement. On pense que surviennent des apports de la paroi de la cupule otique a la crete vestibulo- cochleaire, par exemple au stade 12, mais ils ne sont pas aussi evidents que les migrations correspondantes de la crete a partir de l'ebauche optique vers la gaine de la vesicule optique. La cupule otique s'est fermee superficiellement au stade 13 pour former la vesicule otique, dont la partie dorso-mediane se trouve reconnaissable en tant qu'appendice endolymphatique. 3. Comme dans le cas du cristallin, une partie de la membrane basale de l'ectoderme superficiel s'est invaginee (stades 11 a 13) pour former la membrane basale entourant la vesicule otique. 4. Bien que leurs precurseurs aient ete decelables plus tot, les canaux cochleaire et semi-circulaires etaient nettement definis a la fin du stade 18. Pendant la formation des conduits semi-circulaires, des portions d'epithelium ont perdu leur identite et leur membrane basale a disparu, de sorte que l'epithelium s'est fusionne avec le mesoderme sous-jacent, et que s'est ensuivie Pobliteration de parties de la cavite de la vesicule otique. 5. II a ete etabli anterieurement que les diverses membranes basales associees a l'oeil embryonnaire persistent chez l'adulte. De plus, on sait que certaines parties au moins du labyrinthe membraneux adulte possedent une membrane basale, derivee probablement de celle qui entoure la vesicule otique del'embryon. Cependant, il semble necessaire d'examiner ulterieurement la possibility de l'existence d'une membrane basale continue et intacte d'un bout a l'autre du labyrinthe membraneux de l'adulte.

ACKNOWLEDGEMENTS The assistance of a Special Research Fellowship (BT-744) from the United States Public Health Service is gratefully acknowledged. The writer is indebted to Dr James D. Ebert, Director of the Department of Embryology, Carnegie Institution of Washington, for the use of departmental facilities, and to Dr Mary E. Rawles for her interest. The photomicro- graphy is the work of Mr Richard D. Grill. 754 RONAN O'RAHILLY

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(Manuscript received 20th June 1963)