Development of the otolith organs and semicircular canals in the Japanese red-bellied newt, Cynops pyrrhogaster

HBIRIrlG RES[alKH

ELSEVIER

Hearing Research 84 (1995) 41-51

Development of the otolith organs and semicircular canals in the Japanese red-bellied newt, Cynopspyrrhogaster Michael L. Wiederhold

a,b, *

Masamichi Yamashita c, Kristin A. Larsen a, Jeffrey S. Batten Hajime Koike d, Makoto Asashima d

a,b,

Department of Otolaryngology-Head and Neck Surgery, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7777, USA b Audie L. Murphy Memorial Veterans Hospital, San Antonio, TX 78284-7777, USA c Institute of Space and Astronautical Science, Sagamihara, Japan d Tokyo University, Tokyo, Japan Received 23 April 1994; revised 19 November 1994; accepted 20 December 1994

Abstract

The sequence in which the otoliths and semicircular canals and their associated sensory epithelia appear and develop in the newt are described. Three-dimensional reconstruction of serial sections through the otic vesicle of newt embryos from stages 31 through 58 demonstrate the first appearance, relative position and growth of the otoliths. A single otolith is first seen in stage 33 embryos (approximately 9 days old); this splits into separate utricular and saccular otoliths at stage 40 (13 days). The lateral semicircular canal is the first to appear, at stage 41 (14 days). The anterior and posterior canals appear approximately one week later and the vestibular apparatus is essentially fully formed at stage 58 (approximately 5 weeks). The data reported here will serve as ground-based controls for fertilized newt eggs flown on the International Microgravity Laboratory-2 Space Shuttle flight, to investigate the influence of microgravity on the development of the gravity-sensing organs.

Keywords: Otoconia; Otolith; Utricle; Saccule; Semicircular canals; Development

I. Introduction Amphibians are favorable species in which to study development, since their vestibular systems are similar to those of mammals but they develop much more rapidly. The Japanese newt, Cynops pyrrhogaster, offers the additional advantage that adult females hibernate after mating in the fall, keeping sperm and eggs separate through the winter. Specimens collected during hibernation can be stored under refrigeration. When these are warmed and injected with hormone, they shed their eggs, activate the sperm and produce fertilized eggs within a few days of injection. Thus, embryos of any desired developmental stage can be readily obtained.

* Corresponding author. Tel: (210) 567-5655; Fax: (210) 567-3617. 0378-5955/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 3 7 8 - 5 9 5 5 ( 9 5 ) 0 0 0 1 2 - 7

As in all vertebrate animals, the newt gravity sensors include a utricle and a saccule. In the adult newt, the otoconia in the utricle have the familiar triplanar facets at each end, characteristic of mammalian otoconia. These have been shown in other amphibians to be composed of CaCO 3 in the calcite crystal form (Marmo et al., 1983a). The otoconia in the saccule are either of a prismatic or fusiform form. In frogs, these otoconia are composed of CaCO 3 in the aragonite form (Marmo et al., 1983a, Marmo et al., 1983b). Pote and Ross (1991) refer to the fusiform, or football shaped otoconia as 'pinacoid'. We demonstrate here that the newt has otoconia similar to those of other amphibians. Adult lizards also contain calcitic otoconia in the utricle and aragonitic otoconia in the saccule (Marmo et. al., 1981). Amphibians, fish and birds also have a third otolith organ, the lagena. This organ appears later than the utricle and saccule in the newt (Koike et al., 1995).

42

M.L. Wiederhold et al. / Hearing Research 84 (1995) 41-51

A

pc

Fig. 1. Schematic drawing of an amphibian vestibular system. AC: anterior semicircular canal; PC: posterior canal; LC: lateral canal; CC: common crus of anterior and posterior canals; MU: macula of utricle; MS: macula of saccule; ML: macula of lagena; ED: endolymphatic duct; ES: endolymphatic sac. Modified from M a r m o et al., 1983.

Fig. 1 is a schematic drawing of an amphibian vestibular system, showing the relative positions of the anterior, posterior and lateral semicircular canals and the three otolith organs. The maculae of the utricle, saccule and lagena are shown. Also shown are the endolymphatic duct and sac which connect the inner ear with the surface of the brain and spinal cord in amphibians (Paterson, 1949). As noted often in the comparative literature, the non-auditory portions of the amphibian vestibule are similar to those in other vertebrates, including mammals. We report here the form and early development of the otic vesicle, the otoliths and the semicircular canals. T h e d e v e l o p m e n t and growth of the sensory m a c u l a e / c r i s t a e in the otolith organs and semicircular canals are also quantified and described.

2. M e t h o d s

Adult female newts, weighing approximately 6 grams, judged to be one to two years old, were collected between October and April from rice paddies in Japan and stored at 4° C. In the laboratory, they were warmed

to 25°C one day before hormone injection. One hundred international units (I.U.) of human chorionic gonadotropin ( H C G ) was injected subcutaneously twice, separated by 2 days. Alternatively, a single injection of 200 I.U. H C G also produced viable eggs. Fertilized eggs were laid over several days, beginning 3 days after the first injection. Individual females produced from zero to over 100 eggs, averaging approximately 15 viable eggs per newt. Developmental stages were determined by observation through a dissecting microscope and comparison with the sequence and stage descriptions reported by O k a d a and Ichikawa (1947) and Okada (1989). Embryos were fixed by immersion in mixed aldehydes (0.5% paraformaldehyde, 1.0% glutaraldehyde) with 0.1M cacodylate buffer for 18 hrs. Specimens were dehydrated with increasing concentrations of ethanol (35% to 100%, 15 min. each), propylene oxide (15 min.) and infiltrated with Medcast plastic. After overnight curing at 60 ° C, sections were cut at 5 /xm thickness and stained with 1% toluidine blue and 1% sodium borate. Mounted and cover-slipped sections were observed with Nomarski optics. Serial sections were traced with a 'camera lucida' attachment and reconstructed graphically, using image analysis software (PC3D, Jandel Scientific Corporation) run on a DOS system. To measure and display the otoliths, the area in each section containing otoconia was outlined. Volume was then computed by multiplying the area on each section by the 5 /xm section thickness and summing the volumes for each section which contained otoconia. The area of sensory structures was computed by tracing a line along the portion of the lumen containing hair cells in the section with the greatest extent of macula, multiplying this length by the anterior-posterior extent of the macula or crista and dividing by 4~-. This calculation assumes that the profile of each macula or crista is an ellipse. This procedure produced areas similar to those obtained by measuring the length of macula in each section and avoided the uncertainty in determining macular extent in sections near the edge of the macula. For scanning electron microscopy, the utricle and saccule were exposed in freshly sacrificed adult preparations. The saccule was removed and opened over a specimen stub so that the otoconia flowed onto the stub. Utricular otoconia were removed with forceps and similarly placed. Both were rinsed briefly in distilled water, which was blotted away from the edge of the mass of otoconia within 30 seconds. Dried specimens were coated with 60:40 gold - palladium and examined in a J E O L 35M SEM. The care and use of the animals reported in this study were approved by the University of Texas Health Science Center as San Antonio's Institutional Animal Care and Use Committee.

43

M.L. Wiederhold et al./ Hearing Research 84 (1995) 41-51

50

3. Results

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In adult newts, the utricular macula is covered by a tightly packed cluster of large otoconia. Fig. 2a is a scanning electron micrograph of three utricular otoconia removed from an adult. Each otoconium has a barrel shape and three planar surfaces which usually meet at a point at the end of each stone. This shape is similar to that seen in all mammalian otoconia. In adult newts, the utricular otoconia range from 20 to 50 /.Lm long and 12 to 20 # m wide. In contrast to the utricle, the saccule contains primarily prismatic otoconia with obliquely sheared ends (Fig. 2b), and a smaller number of football-shaped, or 'fusiform' otoconia (Fig. 2c). The prismatic otoconia range from 1.5 to 25 p,m long and 0.5 to 6 / ~ m wide. More detailed morphometric data on otoconial size and shape at different stages are given in the accompanying paper (Steyger et al., 1995.) The embryonic development of C y n o p s from fertilization to hatching is divided into 42 stages (Okada and Ichikawa, 1947). The precursor of the inner ear, the otic vesicle, can first be seen as a small spherical structure in stage 25 embryos, approximately 5 days after the eggs are laid. At stage 26, the otic vesicle is approximately 240 ~ m in diameter with an 80 /zm lumen. Okada's studies, performed at 18°C, indicate that stage 26 occurs at 6 days and stage 32 occurs 10 days after the eggs are laid (Okada, 1989). However, our studies, performed at 25 °C, find that all stages develop significantly earlier. As shown in Fig. 3, stage 26 occurs between day 5 and 6 and stage 32 at day 8. Drawings of the larvae at several stages are included in Fig. 8, below. From stage 26 to stage 32, the otic vesicle is spherical with a relatively thick wall. Serial sections through

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the entire otic vesicle have been obtained from embryos at stages 31 to 58. Fig. 4 illustrates representative sections through the otic vesicle at stages 33 to 57. At stage 32 (not illustrated), the otic vesicle is an empty sphere. There is no obvious specialization of the ceils constituting the wall of the vesicle. At stage 33, a small number of otoconia are seen (Fig. 4a). Even a single otoconium is easy to identify; their birefringence causes them to appear bright and multicolored in the polarized light used in Nomarski optics. No specialization of the cells of the vesicle wall is apparent at this stage either. The number of otoco-

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Fig. 2. Scanning electron micrographs of adult newt otoconia, a. Three large barrel-shaped otoconia from the utricle, b. Prismatic otoconia from the saccule, c. Two large fusiform otoconia from the saccule. Scale bar = 10/xm for each.

I

M.L. Wiederhold et aL / Hearing Research 84 (1995) 41-51

nia and volume of the otolith increases from this stage onward throughout the stages of development we have studied. At stage 42 (Fig. 4c), a columnar epithelium has developed below the lumenal surface of the developing macula. H a i r cells can first be distinguished at approximately stage 43, indicating the early development of a sensory macula, although a saccule and utricle cannot be distinguished at this stage. The maximum diameter of the hair ceils and the spacing between cells is 10 to 12 p~m. As will be demonstrated below, the utricular and saccular maculae have separated at this stage. Ciliary bundles on the hair cells are first seen at approximately stage 48. Hair cells and their ciliary bundles are present in the stage 50 and 57 photomicrographs in Figs. 4e and 4f. At stages 33 and 34, the single macula and otolith lie in a nearly horizontal position. During subsequent development, the saccular otolith and underlying macula move to a more medial position, with the surface of the macula becoming more nearly vertical. The angle of the macula with respect to horizontal was determined in photomicrographs of sections through the single otolith or, from stages 40 on, the saccule. In each case, the angle was determined at the midpoint of the anterior-posterior extent of the saccule or the single otolith. As illustrated in Fig. 5, there is a progression, if irregular, from an orientation with the medial aspect of the macula raised 10 ° from horizontal at stage 33, to one with the medial aspect raised approximately 70 ° at stages 57 and 58. The inset in Fig. 5 illustrates how the angle was determined at stage 52. The data were fit, using a least-squares procedure, with a second-order polynomial (Fig. 5). The regression coefficient of the fit is 0.800, indicating that this relationship accounts for 64% of the variability of data points. (The large angle at stage 42 is due to the greater amount of curvature in the macula in the section at this stage (see Fig. 4c). This caused uncertainty in where to draw the tangent line, which may in turn be due to uncertainty in which section to chose as the midpoint when the single otolith is separating into saccular and utricular portions.) The lateral semicircular canal is the first to appear. Fig. 6 illustrates sections through the developing canals, taken at the center of each crista or its precursor, at stages 42, 48 and 57. Fig. 7 shows higher-magnification views of the developing cristae from the same sections shown in Fig. 6. At stage 42, a well-defined crista of the lateral canal is evident adjacent to the utricular

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Developmental Stage Fig. 5. Plot of angle of inclination of the saccule with respect to horizontal. Inset illustrates, with a schematic drawing of a stage 52 section, similar to those in Fig. 4, how the angle was determined. For stages 33 to 39, when a macula is not easily identified, the angle was de t e rmi ne d from the tangent to the luminal face of the otic vesicle at the closest approach of the otolith.

macula. The precursor of the anterior canal crista at stage 42 appears as a slight dilation on the lateral aspect of the otic vesicle (Figs. 6b and 7b). Not even a precursor of the posterior canal crista is seen at stage 42. Figs. 6c and 7c show the portion of the otic vesicle where the posterior crista will appear, and the only structure resembling a sensory epithelium is the saccular macula (indicated by S). Only the lateral canal forms a complete torus by stage 42. At stage 48, anterior and posterior canals have formed, but no differentiation of hair cells can be seen in their cristae. At stage 57, a clearly defined crista is seen in the posterior canal (Fig. 6i). Although not very clear in the example shown, the organization of a crista is also seen in the anterior canal at stage 57 (Fig. 7h). Three-dimensional reconstructions of serial sections through the otic vesicle at various stages are shown in Fig. 8. These demonstrate how the otoliths develop, as well as how the semicircular canals first emerge from the otic vesicle. At stage 31, no otolith is seen; the stage-33 otolith shown is only 25 ~ m (5 sections) in anterior-posterior extent. At stage 42 (approximately five days after stage 33), separate utricular and saccular otoliths can be seen and they are much larger than the combined otolith at stage 33. Also, a small but

Fig. 4. Photomicrographs of transverse sections through the otic structures of newt larvae at stages 33 to 57. All sections shown are at the midpoint of the anterior - posterior extent of the otic vesicle with a single otolith (stages 33 and 39, a and b) or of the saccule (stages 42 - 57, c f). Otoconia are seen in the lumen in each section. Hair bundles are visible on the hair cells at stages 50 (e) and 57 (f) and are indicated by arrowheads. Scale bar = 100 /~m.

46

M.L. Wiederhold et al. / Hearing Research 84 (1995) 41-51

c o m p l e t e lateral semicircular canal, with a crista approximately 55 /xm long, can he seen at stage 42. No a n t e r i o r or posterior canals n o r cristae can be seen at this stage. A t stage 44 (not shown) a small a n t e r i o r crista, a p p r o x i m a t e l y 15 /xm long, can be seen but n e i t h e r the a n t e r i o r n o r posterior canal are present. T h e posterior a n d a n t e r i o r canal cristae can first be d i s t i n g u i s h e d at a b o u t stage 50. By stage 58, a complete adult-like v e s t i b u l a r system is present. W e l l - d e v e l o p e d otoliths a n d sensory m a c u l a e are p r e s e n t for the utricle, saccule a n d lagena. All t h r e e semicircular canals, with w e l l - d e v e l o p e d cristae are also seen in the r e c o n s t r u c t i o n at stage 58. T h e e n d o l y m p h a t i c sac a n d duct are seen on the d o r s o - m e d i a l aspect of the saccule at stage 58. As a p r e c u r s o r of the sac, a small p r o t u b e r a n c e of the otic vesicle is seen at this position as early as stage 31. A

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s e p a r a t e space f o r m i n g the sac is first seen at stage 41, while a true e n d o l y m p h a t i c duct, c o n n e c t i n g the sac to the saccule, a p p e a r s b e t w e e n stages 48 a n d 53. T h e duct has grown significantly by stage 58. T h e v o l u m e of the otic vesicle capsule a n d l u m e n a n d of the otolith(s), for stages 31 - 58 are p l o t t e d in Fig. 9. Note that over these stages, the otic vesicle v o l u m e increases by approximately a factor of 160 w h e r e a s the otolith first a p p e a r s at stage 33 and increases n e a r l y 400-fold by stage 58. F r o m stage 35 (5 days before hatching) t h r o u g h at least stage 58, b o t h the v o l u m e of the e n t i r e vestibular system a n d that of the c o m b i n e d otoliths c o n t i n u e to grow logarithmically with d e v e l o p m e n t a l stage. A single otolith is seen from stages 33 to 37, which separates into the utricular a n d saccular otoliths b e t w e e n stages 39 a n d 41. W e have also used a non-invasive X-ray microfocus

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Fig. 6. Photomicrographs of 5 ~m transverse sections through the otic vesicle, illustrating the cristae of the semicircular canals for larvae at stages 42 (a -c), 48 (d - f) and 57 (g - i). Each section illustrated is through the midpoint of the anterior - posterior extent of the crista for the lateral (a, d, g), anterior (b, e, h) and posterior canals (c, f, i). All sections are oriented with dorsal upward and lateral to the left. a. shows both lateral canal crista and the utricular macula (at ventral aspect of otic vesicle). Panel b shows precursor of anterior canal crista and a portion of the utricular macula, at the ventral aspect. Panel c shows only the saccular macula (S). In d, the lateral crista (L), utricular (U) and saccular (S) maculae are seen. Note sensory epithelium at all stages in the lateral canal crista (a, d, g); anterior and posterior canal cristae contain clear sensory epithelium only at stage 57 (h and i). Scale bar in i = 500 ~m applies to all panels.

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system to measure otolith size in live developing newt larvae, which is reported separately (Koike et al., 1995). In Fig. 10, the otolith volumes from Fig. 9 are replotted, along with the area of each otolith seen in dorsoventral view radiographs at comparable stages. Several points in this plot should be noted. The utricular and saccular otoliths a p p e a r separate at stage 37 in the radiographs, whereas they cannot be distinguished until stages 41 or 42 in the reconstructions. Both the saccular and utricular otoliths grow steadily from stages 42 to 52 in the reconstructions, but the saccular otolith area in the dorso-ventral radiographs grows very little between stages 45 and 52. In lateral radiographs, the saccular otolith is seen to grow more rapidly at these stages. In an attempt to determine the stages at which the various sensory structures in the newt vestibular system might become functional, we have calculated the area of the sensory maculae for the otolith organs and of the cristae of the three semicircular canals. These data are plotted in Fig. 11. At stage 33, only the precursor of a single macula is seen; it is not clear if this can be

identified as a precursor of either the utricle or saccule. As indicated in Fig. 11, separate utricular and saccular maculae can be clearly identified from stage 41 on. The lateral semicircular canal is the first to appear, growing from the lateral aspect of the utricle at stage 42 (Figs. 6a and 7a). The area of the lateral canal crista grows very rapidly between stages 42 and 44 and thereafter grows at a rate similar to that for all of the other sensory structures. As the anterior canal grows from the anterior-lateral aspect of the utricle, the precursor of its crista appears before that of the posterior canal (Figs. 6b, c and 7b, c), but an identified sensory epithelium develops somewhat earlier in the posterior canal, which develops from the posteriorlateral aspect of the saccule (Fig. 11).

4. D I S C U S S I O N Earlier

descriptions of the development of C. showing histological sections through the brain of embryos up to stage 42 do not illustrate or

pyrrhogaster

M.L. Wiederhold et al./ Hearing Research 84 (1995) 41-51

48

even mention the otoliths (e.g., O k a d a and lchikawa, 1947). The same is true for Xenopus lael,is (Paterson, 1949). We have found that it is necessary to rapidly fix, dehydrate and embed specimens to maintain the otoconia. Specimens stored in buffer for one week were found to be lacking otoconia. Acid fixatives, such as Bouin's, rapidly dissolve the otoconia. Restricting each alcohol and buffer step to 15 min., appears to optimize preservation of the otoconia. Thus, in earlier reports, otoconia may have been dissolved during preparation. The fact that the otoliths appear at the same stage in both sectioned material and in the radiographs of live specimens (Fig. 10) indicates that, with the techniques used here, the otoconia are retained during histological preparation. The ability to distinguish the saccular and utricular otoliths at earlier stages in the radiographs is likely due to the 5 / z m section thickness; if there is less than a 10 p~m space between the two otoliths, there

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could be no section without otoconia and if the space is less than 5 ~m, all sections through the otoliths will contain some otoconia. In dorsal-ventral radiographs, the two otoliths can be distinguished at stage 36 (Koike et al., 1995) and in SEM of the in situ otolith, a continuous otolith with distinct dilations of the anterior utricle and posterior saccule are clearly seen at stage 41 with separate otoliths usually seen by stage 45 (Steyger et al., 1995). It may be that the thin 'isthmus' connecting the utricular and saccular otoliths before stage 41 are not sufficiently radio-opaque to be detectable in the radiographs between stages 36 and 41. In some SEM's of in situ preparations at stage 45, the isthmus can still be seen (Steyger et al., 1995). Thus, there is considerable variation between techniques, and between specimens of the same stage, in the point at which the two otoliths are seen as separate. The fact that the saccular and utricular otoliths contain similar

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Fig. 8. Three dimensional reconstruction of serial sections through the developing otic vesicle of newt embryos at stages 31 - 58. Note separate 100/xm calibration bar for each stage. All stages are shown at the same orientation, indicated in the center of the figure. Drawings of larvae at each stage are from Okada, 1989. Abbreviations: C: capsule; L: lumen; O: otolith; UO: utricular otolith; MU: macula utriculi; SO: saccular (and lagenar, for stage 58) otolith; MS: macula sacculi; AC: anterior semicircular canal; ACC: anterior canal crista; LC: lateral semicircular canal; LCC: lateral canal crista; PC: posterior semicircular canal; PCC: posterior canal crista; D: dorsal; V: ventral; M: medial; L: lateral; A: anterior; P: posterior.

49

M.L. Wiederhold et al. / Hearing Research 84 (1995) 41-51 Growth 108

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Fig. 9. Plot of otic vesicle and otolith volume from stages 31 to 58. Otic vesicles from both sides were measured for specimens at each stage. The capsule volume (open circles) includes all volume within the outer wall of the otic vesicle, while lumen volume (filled circles) is that inside the luminal wall of the otic vesicle. Combined otolith (open triangle) is the volume of the single otolith seen before stage 40. Volumes of utricular otolith (filled triangles) and saccular otolith (filled squares) are plotted separately for stages 44 - 58.

o t o c o n i a a n d a p p e a r c o n n e c t e d over several stages suggests that the two otoliths arise from the same source.

I n o t h e r a m p h i b i a n species, as well as reptiles, utricular o t o c o n i a have b e e n shown to consist of calcite (as are all o t o c o n i a in m a m m a l i a n a n d avian species) w h e r e a s the saccular o t o c o n i a are m a d e of aragonite, as are all t h r e e otoliths in most a q u a t i c species G r o w t h of O t o l i t h s 10 5

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and eristae

of the three semicircular canals as a function of developmental stage. Maculae were defined as areas in which hair cells could be identified and distinguished from supporting cells. Area was computed as ~r/4 (anterior - posterior length) x (medial - lateral width). Utricular macula: filled triangle; Saccular macula: filled square; Lateral canal crista: open circle; Posterior canal crista: open square; Anterior canal crista: open triangle.

(Carlstrom, 1963, Shichiri, 1986, G a u l d i e , 1993, M a r m o et al., 1992). T h e t h r e e shapes of o t o c o n i a described here are similar to those described in frogs ( M a r m o , 1983b, Pote a n d Ross, 1991), a l t h o u g h the fusiform o t o c o n i a a p p e a r m o r e p r e v a l e n t in frogs t h a n in the newt. F o u r i e r t r a n s f o r m i n f r a r e d spectroscopy of adult saccular o t o c o n i a indicates that they are at least 99% a r a g o n i t e ( W i e d e r h o l d et al., 1994, W i e d e r h o l d et al., 1995). X-ray diffraction confirms this a n d that o t o c o n i a in the a d u l t newt utricle are calcite. In larvae prior to stage 52, both u t r i c u l a r a n d saccular o t o c o n i a are calcite. T h e e n d o l y m p h a t i c sac a n d duct in adult newts are also filled with m a n y small stones identical in form to the prismatic o t o c o n i a in the saccule (Steyger et al., 1995). T h e otic vesicle first a p p e a r s in 6-day old embryos. T h e single otolith a p p e a r s at stage 33 a n d grows rapidly t h r o u g h stage 39. T h e data shown in Fig. 3 indicate that this period c o r r e s p o n d s to 8 to 12 days after eggs are laid. F r o m stages 39 t h r o u g h 58, both the otic vesicle l u m e n a n d the otolith c o n t i n u e to grow logarithmical, with a p p r o x i m a t e l y the same e x p o n e n t . In the toadfish, Sokolowski (1986) a n d Sokolowski a n d P o p p e r (1987) d e m o n s t r a t e that the p r i m o r d i a of the u t r i c u l a r a n d saccular otoliths a p p e a r at the same time a n d s e p a r a t e d from o n e a n o t h e r . Fish otoliths are a r a g o n i t e a n d form a solid c o n c r e t i o n , r a t h e r t h a n the loose collection of o t o c o n i a f o u n d in a m p h i b i a n s a n d terrestrial animals. F r o m Figs. 6, 7 a n d 11, it is a p p a r e n t that the otolith organs develop well before the semicircular canals. T h e

50

M.L. Wiederhold et al./ Hearing Research 84 (1995) 41-51

lateral semicircular canal is the first to develop, at stage 42. The rapid growth (in the logarithmic plot) of the lateral canal crista between stages 42 and 44 suggests the initial growth from a small precursor to an early functional state. Hair cells in the larval maculae are approximately 10 /xm apart (see Fig. 4). Assuming hexagonal packing, each hair cell then occupies approximately 87/xm 2 of the sensory epithelium. Thus, at stage 42, the lateral canal macula contains only about 23 hair cells, while at stage 44 it contains approximately 57 hair cells. Although we are aware of no data indicating how many hair cells a macula must contain to become functional, if this change in growth rate and a population of about 57 hair cells does indicate achievement of such function, the posterior canal would be expected to become functional at stage 52 and the anterior canal at stage 53. The growth of the lateral canal crista beyond stage 44 is logarithmic, with an exponent (slope in Fig. 11) similar to that observed for the otic vesicle and otolith volumes and the areas of the otolith organ maculae beyond their initial rapidgrowth phase (i.e., after stage 42.) In a previous study (Wiederhold et al., 1992b), we observed differences in swimming behavior of newt larvae during hypo-gravity on parabolic flights. Larvae at stages 42 to 44 made only brief attempts at swimming at the onset of hypo-g in light. In the dark, they made no significant swimming attempts during hypo-g. In contrast, stage 54 - 57 larvae exhibited vigorous, if intermittent, swimming throughout a 20-sec period of hypo-g, both in light and in the dark. At stage 42 - 44, the larvae moved minimally and then only if they had visual input, which was interpreted to give them spatial information lacking from the otoliths in near microgravity. Later-stage larvae, with the anterior and posterior semicircular canals to sense pitch and roll (Figs, 6, 7 and 11) and the saccular macula situated to encode vertical displacement (Fig. 5), the animals are much more active in hypo-g. These results can be interpreted to indicate that the larvae will explore the novel environment of hypo-g only if they have adequate sensory inputs to the central nervous system to compute their current position. This interpretation is consistent with the assumption that cristae become functional when they contain approximately 50 to 60 hair cells. The order of development of the semicircular canals in the newt begins first with the lateral canal, followed by the posterior and then the anterior canals. This is the reverse of the order reported in fetal humans (Anson, 1973). In man, all three semicircular canals are developed by 7 1 / 2 weeks of gestation and are clearly functional at the time of birth, when rotational movement in any plane can be experienced. In the newt, larvae usually hatch from the egg near stage 42. In the first few days after hatching, the larvae remain sedentary at the bottom of a container and only make

swimming movements in the horizontal plane. They use their balancers, anterior ventral projections from the side of the head, to maintain a level position on the bottom of the container, by resting with the distal tip of both balancers just touching the bottom. Thus, the combination of the balancers, two horizontally oriented otolith organs and only the lateral semicircular canal all appear suited to sense movement within the horizontal plane between stages 42 and 50. The appearance of 3-dimensional swimming after the mid-50 stages correlates with the acquisition of the receptors to sense these movements. The rate of otolith growth, assessed by the X-ray system (Koike et al., 1995) appears quantitatively different from that determined by reconstruction of serial sections (Fig. 10) at stages 42 to 55. As indicated in Figs. 5, the saccule moves from a horizontal to a nearly vertical orientation over these stages, which would reduce its projection in a dorso-ventral radiograph. However, if the saccular area is measured in a lateral radiograph, the rate of growth over these stages is much greater (Koike et al., 1995). Even though the area of the otolith image in radiographs is being compared to the volume computed from a reconstruction, the growth of the otoliths assessed by these two methods is remarkably similar at the stages when the otoliths are close to horizontal, where area seen in a dorsoventral radiograph would be proportional to otolith volume. Previous reports are contradictory regarding potential effects of gravity on otolith development. Howland and Ballarino (1981) raised chick embryos on a centrifuge at 2 g from stage E7 and found that the utricular otolith was heavier in control animals. In a repeat study (Ballarino and Howland, 1984), there was no difference in otolith weight between control and 2 g animals. However, the otolith has already begun to mineralize by stage E7 in chicks. Lim et al., (1974) found no difference in the rat saccule in animals reared on a centrifuge. Despite earlier reports that the saccule and utricle developed 'normally' in Xenopus embryos reared in space, Lychakov and Lavrova (1985) reported that the utricular otolith in the space-reared animals was 30% larger than in control animals, whereas the size of the saccular otolith was not different from control. This result indicates that the weight, rather than just mass, of the utricular otolith might be controlled during development. Similarly, Pedrozo and Wiederhold (1994) report that the statolith in Aplysia californica is smaller in embryos reared at 2 g. Effects of rearing in microgravity on otolith growth and development of their associated sensory structures are being evaluated in newt larvae flown on the International Microgravity Laboratory - 2 Space Shuttle flight in 1994. The eggs were 4 to 6 days old at the launch of the 15-day mission, so they were in microgravity before

M.L. Wiederhold et al. / Hearing Research 84 (1995) 41-51

otolith mineralization began and continued in microgravity through stage 45 - 50, covering the most rapid growth period of the vestibular system. The data presented here will serve as normal, ground-based control studies for comparison with space-reared specimens.

Acknowledgements We would like to thank Dr. Peter Steyger for his help with photography and in the preparation of several figures and Dr. Francesco Marmo for his original drawing used in Fig. 1. Supported by NASA, V A Medical Research Funds and the Japanese Ministry of Education.

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