Evidence Concerning the Morphogenesis of Saccular Receptors in the Bullfrog (Rana catesbeiana)

E. R. LEWIS AND c. w. LI Department of Electrical Engineering and Computer Sciences and the Electronics Research Laboratory, University of California, Berkeley, California 94720

ABSTRACT A dichotomy of hair-cell types has been found in the bullfrog sacculus, and considerable evidence supports the view that one type (“periph- eral”) is transformed during macular growth to the other type (“central”). Between the periphery and the center of the macula, one finds a gradation of form from “peripheral” to “central” type. Occasionally in adults and more often in stage-26 tadpoles one finds the presumably younger peripheral type of hair cell occurring well beyond the limits of the macula proper. The appar- ent morphogenic sequence for saccular hair cells is (1) development of a kinocillum on an endolymphatic epithelial cell, (2) gradual transformation of microvilli into stereocilia, (3) growth of the stereocilia and development of kinociliary bulb, (4) achievement of final size and form.

The scanning electron microscope has in scanning micrographs by Hillman and been employed quite successfully in stud- Lewis (‘71). ies of the surfaces of ciliated cells in gen- eral (Barber and Boyde, ’68) and of audi- MATERIALS AND METHODS tory and vestibular receptors in particular (Bredberg, Lindeman, Ades, West and Eng- Twenty adult bullfrogs (Rana catesbei- strom, ’70; Hillman and Lewis, ’71; Lim ana) and ten stage-26 tadpoles (Gosner ’60) and Lane, ’69; Marovitz, Arenberg and were used in this study. The animals were Thalman, ’70). In a recently published killed by decapitation and then pithed to scanning microscopy paper, Lewis and prevent movement during dissection and Nemanic (‘72) reported two distinct types the early stages of fixation. The otic cap- of hair cells in the saccular macula of the sules were exposed from the roof of the mudpuppy (Necturus maculosus). One mouth and perfused with osmium tetroxide type, which occurs at the periphery of the solution in the manner employed by Hill- macula, is very similar to hair cells of the man (‘69). Proper osmolarities for the os- mudpuppy utricular macula. The other mium solutions were determined by pre- type, occurring throughout the center of liminary experiments. The effects of the macula, is quite distinct. Following hyperosmotic and hyposmotic solutions on that study, we found a similar dichotomy microvilli and cilia were quite marked, among hair cells of the bullfrog saccular and the optimal osmolarities (measured on macula. One of us (C.W. Li) undertook a a Fiske Osmometer) were bracketed very scanning microscopy examination of the easily. As others have found (Lombard development of the saccular macula dur- ’70), the optimal osmolarity for tadpoles ing metamorphosis, and soon found evi- was approximately one half that for adult dence that the peripheral hair cells were frogs. involved in macular growth. Continuing After fixation, the otolith and gelati- this study, we have expanded the evidence. nous membrane were removed mechanical- The work was greatly facilitated by an ad- ly. Only the very tips of the kinocilia are ditional distinction, not found in the mud- attached directly to the gelatinous mem- puppy, between the peripheral and central types. The central type of hair cell exhib- 1 Research sponsored by the Joint Services Electronics Program, Contract F44620-71-C-0087 and the United ited the very obvious kinociliary bulb first States Public Health Service, NationalInstituteofHealth, reported by Hillman (‘69) and later shown grant GM-17523.02.

J. MORPH.,139: 351-362. 351 352 E. R. LEWIS AND C. W. LI

Fig. 1 Endolymphatic surface of the saccular macula of an adult bullfrog. The gelatinous membrane was removed mechanically, but a few otoconia can be seen scattered over the sur- face. The hair cells, whose stereocilia are apparent as small white tufts, are very regularly spaced over the entire macula. See figure 2 for the position of the line across which hair-cell orientation reverses. X 260. brane, and the stereocilia of the receptors microvilli when the membrane is removed. as well as the microvilli of supporting cells Following osmium fixation on the other apparently are connected to the membrane hand, all of the connections apparently by very thin strands (Hillman, '69). Alde- are rather weak. During subsequent re- hyde fixation appears to strengthen these moval of the gelatinous membrane, the connections of the gelatinous membrane, strands break leaving the microvilli and leading to disarray or tearing of cilia and stereocilia behind; and the connections be- MORPHOGENESIS OF SACCULAR RECEPTORS 353 scanning electron microscope (Cambridge Instrument Stereoscan 11). Quantitative measurements were made on calibrated micrographs of essentially flat, horizontal surfaces. Means, root-mean- A&P VL A square deviations from those means, and ranges of luminal surface areas of hair cells and supporting cells were computed from measurements on all such cells with- in randomly selected areas (approximately 1000 ~2)on each of three adult maculae and four tadpole maculae. The mean areas of nonmacular endolymphatic epi- thelial cells were estimated from counts of all such cells in randomly selected areas (approximately 1000 pz) of the endolym- phatic surfaces from three tadpole sacculi and four adult sacculi. Microvillar densi- ties were computed from counts over ran- domly selected areas (Approximately 4 p2) from six or more preparations for each cell type. Sample sizes (n) are given with each computed value in the results.

RESULTS Fig. 2 The orientation of the macula with re- spect to the saccular branch of the VIIIth nerve Gross geometry of the muculu (A) and the shapes of some saccular maculae from bullfrog stage 26 tadpoles (B,C, and D) and from In both the adult bullfrog and the stage- adult bullfrogs (E,F, and G). In diagram A, DM, 26 bullfrog tadpole, the saccular macula P, VL and A indicate dorsomedial, posterior, ven- is located on the endolymphatic surface trolateral and anterior respectively. In each of the of the medio-ventral wall of the sacculus, other diagrams, the line of hair-cell reversal is shown as a dashed line and the numbers around and is markedly tilted from the vertical so the perimeters of E,F, and G indicate the numbers that its ventral extremity is lateral and its of rows of hair cells clearly identifiable either as dorsal extremity is medial. The saccular the peripheral type or as transitional between pe- branch of the VIIIth nerve approaches the ripheral and central types. macula from above and slightly to the rear. The macula itself is kidney shaped, with tween the kinocilium and the membrane the concave portion of its perimeter facing yields, leaving the kinocilium behind. The the direction from which the nerve ap- gelatinous membrane has ports over the proaches. The boundaries of the macula receptors (Hillman and Lewis, '71) and we are quite well defined not only by the pres- have viewed the kinocilia and arrays of ence of ciliated receptors within the mac- stereocilia through these ports with the ula but also by the transition from the gelatinous membrane in place over the squamous epithelium of the surrounding macula. In addition, Hillman ('69) has stud- endolympatic surface to the elevated, col- ied transmission micrographs of the mac- umnar epithelium of the macula (fig. 1). ula with the membrane in place. The The epithelium of the macuIa not only ap- geometrical arrangements of microvilli, pears to be elevated, but the luminal sur- stereocilia and kinocilia observed with the face areas of the macular supporting cells membrane in place are not noticeably dif- are consistently smaller than the luminal ferent from those we observed after remov- surface areas of the nonmacular epithelial ing it. cells. We observed areas of 7 +- 2 p* (4 - The tissue was dried using the critical 15, n = 100) and 14 t 4 pz (6 - 23, point method with freon (Cohen, Marlow n = 100) for macular supporting cells of and Garner, '68) and coated with a thin the tadpole and adult respectively. In both layer of evaporated gold for viewing in the adult and tadpole, we observed mean areas 3 54 E. R. LEWIS AND C. W. LI MORPHOGENESIS OF SACCULAR RECEPTORS 355

Fig. 3 Hair cells at the periphery of the saccular macula of an adult bullfrog, showing the gradation in size and shape from peripheral type (lower right) to central type (upper left). X 3800. Fig. 4 Cilia-like projections from two adjacent endolymphatic epithelial cells just outside the present perimeter of an adult macula. X 16,000 (line = 1 pm). Figs. 5-9 Gradation of size and shape of peripheral hair cells of an adult macula. x 16,000 (lines = 1 Fm). 356 E. R. LEWIS AND C. W. LI of approximately 30 pz for nonmacular per p2 (over 7 sample areas of 4 p2, cho- epithelial cells [29, n = 71 for the tad- sen at random from 6 sacculi); whereas on pole; 31, n = 125 for the adult]. the supporting cells of the macula we ob- The density of hair cells is quite con- served a microvillar density of 35 f 5 per stant over the macular surface (fig. 1); p2 (over 10 sample areas of 4 p2, chosen so that no striola (line of higher hair-cell at random from 8 maculae). Thus in the density) is apparent from casual SEM ob- adult bullfrog, a further distinction ex- servation of the entire macula (see Linde- ists between macular supporting cells and man, '69). As they are in other vertebrates, the epithelial cells surrounding the mac- the hair cells of the bullfrog are polarized. ula. This distinction is sharpened even The stereocilia are graded in length and further by remnants of the fine strands arranged much like a stand of organ pipes, reported by Hillman, which occur only on with succeeding rows being occupied by the macular supporting cells (fig. 3). progressively longer stereocilia. The kinocil- In the stage-26 tadpole, remnants of ium occurs directly behind the row of long- the fine strands occur on the supporting est stereocilia. Thus each hair cell can be cells of the macula; but the observed mean assigned an orientation. The orientation microvilli density of the supporting cells of hair cells within the macula of the bull- (24 * 1 per pz over 6 sample areas from frog sacculus is almost identical to that in 6 maculae) was essentially indistinguish- the mudpuppy sacculus, whose macula also able from the mean microvilli density on is kidney-shaped (Lewis and Nemanic, the surrounding squamous epithelium (23

'72). As in the mudpuppy, the line across _+ 2 per p2 over 6 sample areas from 6 sac- which hair-cell orientation reverses runs culi). Even without this added distinction, more-or-less parallel to the concave and the boundaries of the tadpole macula were convex edges of the macula and approxi- quite sharp (fig. 13). mately halfway between them (fig. 2). The microvilli are arrayed in a remark- The mudpuppy differs from the bullfrog, ably orderly fashion (fig. 6), with very dis- however, in that the entire macula is ro- tinct rows. Occasionally, at the periphery tated slightly counterclockwise relative to of the macula, the microvilli are interrupt- those shown in figure 2; and the concave ed by large processes, possibly emerging edge faces a slightly more posteriad direc- cilia (fig. 4). tion, corresponding to a slightly more On the surfaces of the hair cells in the posterior approach of the saccular branch center of the macula, microvilli form a of the VIIIth nerve. semi-circle around the shortest stereocilia, Microvilli just as they do in mudpuppy sacculus (Lewis and Nemanic, '72). On the hair In the 4 to 5 pm space between the sup- cells at the perimeter of the macula, the porting cells of the macula and the gelati- microvilli appear to merge with the stereo- nous membrane, Hillman ('69) has found cilia, and it often is impossible in the scan- very thin electron-dense strands, which ning electron micrographs to tell one from emanate from the supporting-cell micro- the other (figs. 5, 6, 7). villi and which apparently condense to Two types of saccular hair cell form the gelatinous membrane. This sug- gests that the supporting-cell microvilli The hair cells throughout the broad might be involved in secretion of the gela- central region of the macula have been tinous membrane. In the mudpuppy, Lewis Fig. 10 Hair cells intermediate between pe- and Nemanic ('72) found that the micro- ripheral and central, showing graded size of kino- villi of the supporting cells were markedly ciliary bulb. X 9000 (line = 1 pm). more dense than those of the surrounding Fig. 11 An anomolous intermediate hair cell, with its kinocilium emerging at the side of the squamous endolymphatic epithelium. In stereociliary tuft rather than behind it. Similar the adult bullfrog, a similar but not nearly anomolies have been reported by Flock ('64). so marked difference exists; but in the X 16,000(line = 1 pm). stage-26 bullfrog tadpole, the difference Fig. 12 A small area in the center of a saccu- lar macula which was fixed in slightly hyperos- is almost negligible. On the adult bullfrog motic osmium tetroxide. The supporting cells are squamous endolymphatic epithelium, we well delineated by terminal bars, making the cell observed a microvillar density of 26 f 2 distribution quite easy to observe. X 3500. MORPHOGENESIS OF SACCULAR RECEPTORS 357 358 E. R. LEWIS AND C. W. LI described very well by Hillman (‘69) and their mean luminal surface areas was ob- have been shown in scanning electron served. The hair-cell area in the center of micrographs by Hillman and Lewis (‘71). the adult macula was 24 * 7 p2 (6-43, These hair cells are unusual in that the n = 100); while the corresponding values kinocilium, which is approximately equal for the tadpole were 23 f 11 p2 (2-43, in length to the longest stereocilia, termi- n = 100). However, a marked difference nates in a large bulb. We found hair cells was seen in the macular supporting cells, of this type not only in adult bullfrogs, which in the adult had a luminal surface but also in the stage-26 tadpoles. area of 14 * 4 pz (6-23, n = 100) and The second type of hair cell, previously in the tadpole an area of 7 * 2 p2 (4 - 15, unreported for the frog but found by n = 100). Consistent with these differ- Lewis and Nemanic (‘71) in mudpuppy, ences in supporting-cell and hair-cell sizes, occurs around the entire perimeter of the the density of hair cells on the surface of frog and tadpole maculae. The most pe- the tadpole saccular macula was 0.023 * ripheral of these hair cells have very short 0.001 per p2 (over 5 sample areas of 4,000 stereocilia and a long kinocilium with no pL2chosen at random from 5 maculae); bulb. As one moves from the extreme pe- while the corresponding value in the adult riphery toward the center of the macula, was 0.015 k 0.0015 per p2 (over 11 sam- he observes a gradual change from this ple areas of 15,000 p2 chosen at random peripheral hair cell type to the central from 7 maculae). This represented an ob- type reported by Hillman (fig. 3). Figures served hair-cell density decrease of approx- 4-11 show this succession in detail. The imately one-third in going from tadpole to width of the peripheral band over which adult. In spite of this decrease in density, this gradation occurs is not constant, the observed total number of saccular- but is consistently greater on the concave macula hair cells increased markedly from side (nerve side) of the macula than it is tadpole to adult. A 5 gm tadpole, for exam- on the convex side (fig. 2). One also finds ple, had approximately 850 hair cells; hair cells of the peripheral type distrib- while a 312 gm adult had approximately uted randomly but very sparsely through- 2500. Thus the increased macular area out the entire central macula. of the adult more than compensated the Beyond the edges of the macula proper decreased hair-cell density; and macular in adult frogs one often finds single out- growth consisted, at least in part, of cell lying hair cells (of the peripheral type), proliferation. with ten or more squamous epithelial cells between them and their nearest neigh- DISCUSSION bors at the edge of the macula. In the macula proper, hair cells are separated by Macular growth and the formation only one or two supporting cells (fig. 12), of new hair cells and these have distinctly smaller luminal surfaces than the squamous epithelial Taken as they stand, our results imply cells and generally exhibit remnants of the that as the area of the saccular macula fine strands reported by Hillman (see un- increases several fold during the growth der microvilli). In stage-26 tadpoles, one and maturation of the bullfrog from stage- finds even more outlying peripheral hair 26 tadpoles to large adults, the number of cells, and one finds them at even greater hair cells increases, but apparently not in distances from the macula proper (figs. direct proportion to macular size. Thus 13-15). In fact, we have found them at dis- Fig. 13 The saccular macula and surrounding tances greater than 100 pm from the edge endolymphatic epithelium from a stage-26 tadpole. of a 170 pm macula. Most of these out- Note the relatively large luminal surface areas of the epithelial cells surrounding the macula and lying hair cells occur on the concave side the dichotomy of peripheral and central hair-cell (i.e., the side toward the saccular branch types. X 450. of the VIIIth nerve). Fig. 14 Higher magnification of an area near Qualitatively, the hair cells of the tad- the edge of a tadpole macula, showing two out- lying receptors (indicated by arrows). x 1080. pole saccular macula were essentially Fig. 15 Close-up view of one of the two out- identical to those of the adult saccular lying receptors of figure 14. X 10,800 (line = macula; and no significant difference in 1 pm). MORPHOGENESIS OF SACCULAR RECEPTORS 359 360 E. R. LEWIS AND C. W. LI the hair-cell density decreases slightly, eral type to central type suggests transfor- corresponding to an increase of the mean mation rather than replacement. luminal surface area of supporting cells. This in turn suggests that the gradation It is reasonable to suspect, however, that of hair-cell types shown in figures 3 to 11 apparent differences in cell densities were might represent a temporal sequence dis- artifactual results of differences in the played in spatial coordinates. Newly formed tissues as reflected in the requirement for saccular hair cells may be of the periph- lower osmolarities in the fixative for the eral type, with a kinocilium as Iong as tadpole. Thus it is conceivable that the 5 pm or more and stereocilia less than 2 tadpole macula underwent more shrinkage pm long (fig. 9). The stereocilia then than the adult macula. This possibility is could grow both in length and diameter accentuated by the fact that Proebsting (figs. 5-11); and as the longest stereocilia (‘24) reported that macular growth in lar- approach the length of the kinocilium, the vae Triturus was due primarily to increase kinociliary bulb could begin to form (figs. in cell number and that cell size remained 8-11), becoming fully formed as the long- essentially constant. On the other hand, est stereocilia reach the tip of the kino- the fact that both the nonmacular epithe- cilium. Thus we might conclude that hair lial cells and the hair cells in our prep- cells of the peripheral type are in fact im- arations had the same mean luminal sur- mature, whiIe those of the central type are face area in both tadpoles and adults tends mature. Our observation of a very few to indicate that differential shrinkage may scattered peripheral and intermediate type not have occurred in our preparation and hair cells in the interior of the macula that the observed hair-cell density de- would indicate that some hair-cell prolif- creases from tadpole to adult may not have eration and macular growth also occurs been artifactual. there. On the other hand, these may be Two conclusions are much more defi- peripheral cells whose transformation nite. First, the distribution of hair-cell somehow was arrested as the perimeter types does not change with growth. From move past them, or they may be replace- the smallest to the largest maculae ob- ments for hair cells somehow damaged or served in this study, hair cells of the pe- destroyed in the central macula. ripheral type always occupied the entire Furthermore, if this hypothesis is cor- perimeter and surrounded a large area oc- rect, then the occurence of widely scat- cupied by the hair cells of the central type tered peripheral hair cells beyond the with their kinociliary bulbs. Second, dur- concave side of the tadpole macula proper ing macular growth many new hair cells and the occurrence of the widest band of are formed. peripheral and intermediate hair-cell types The presence of outlying hair cells in on the concave side of the macula proper adults and their presence in even greater in both tadpoles and adults would imply numbers and at even greater distances that macular expansion is directed pri- from the macula proper in tadpoles, indi- marily toward the saccular branch of the cates that hair cells apparently can form VIIIth nerve. among squamous endolymphatic epithe- Assuming that the peripheral hair cells lium cells well beyond the macula proper. were in fact newly formed, we searched This implies a tendency toward outward the perimeter of the macula for even ear- expansion of the macula by addition of lier signs of hair cell development. Among hair cells at its perimeter. the epithelial cells close to the edge of the If in fact the macula does grow in this adult macula, but not elsewhere on the manner, then the peripheral type of hair endolymphatic epithelium, we found many cell either must be replaced by or be trans- otherwise normal appearing epithelial cells formed to the central type as the macular with what appeared to be single cilia perimeter moves on. Otherwise the width whose diameters were essentially identical of the area occupied by peripheral types to the kinocilium diameter of the hair cells would continually increase; whereas it (fig. 4). It is possible that these cells are actually remains fairly constant at one undergoing differentiation to receptor to six receptors wide. The consistent cen- cells. Furthermore, in the smallest clearly tripetal gradient of hair cells from periph- identifiable receptors, the stereocilia have MORPHOGENESIS OF SACCULAR RECEPTORS 361 diameters essentially the same as the physiological studies as well as additional neighboring microvilli. It seems possible, microscopy. therefore, that microvilli in the differen- tiating epithelial cell become modified to LITERATURE CITED form stereocilia. Barber, V. C., and A. Boyde 1968 Scanning Geisler, van Bergeijk and Frishkopf (‘64) electron microscopic studies of cilia. Z. Zell- forsch., 84: 269-284. reported that “hair cells gradually fade Bredberg, G., H. H. Lindeman, H. W. Ades, R. out into undifferentiated cells” near the West and H. Engstrom 1970 Scanning elec- lateral edge of the bullfrog amphibian tron microscopy of the organ of corti. Science, papilla; while along the medial edge the 170: 861-863. Cohen, A. L., D. P. Marlow and G. E. Garner 1968 macula ends abruptly. They suggested that A rapid critical point method using fluorocar- the lateral edge is a zone of growth of the bons (“freons”) as intermediate and transitional papillar macula. Interestingly, the papillar fluids. J. Microscopie, 7: 331-342. branch of the VIIIth nerve approaches the Flock, A. 1964 Structure of the macula utriculi with special reference to the directional inter- macula from the lateral side. Further- play of sensory responses as revealed by mor- more, in preliminary scanning electron phological polarization. J. Cell Biology, 22: microscope studies of the amphibian pa- 413-431. pilla, both in tadpoles and in adult frogs, Geisler, C. D., W. A. van Bergeijk and L. S. Frish- kopf 1964 The Inner Ear of the Bullfrog. J. we found hair cells with long kinocilia and Morph., 114: 43-58. short stereocilia along the entire lateral Gosner, K. L. 1960 A simplified table for staging edge. As we progressed in a medial direc- anuran embryos and larvae with notes on iden- tion, we found the same succession we had tification. Herpetologica, 16: 182-190. Hillman, D. E. 1969 New ultrastructural findings observed at the periphery of the saccular regarding a vestibular ciliary apparatus and its macula. The stereocilia became longer; possible significance. Brain Research, 13: 407- bulbs began to appear at the tips of the 412. kinocilia; and we finally came to a broad Hillman, D. E., and E. R. Lewis 1971 Morpho- logical basis for a mechanical linkage in otolithic area of hair cells whose surface appearance receptor transduction in the frog. Science, 174: was essentially identical to that of the 416-419, central hair cell of the saccular macula. Lewis, E. R., and P. Nemanic 1972 Scanning This type of hair cell occurred right up to electron microscope observations of saccular ultrastructure in the mudpuppy (Necturus mac- the medial edge of the papillar macula. ulosus), Z. Zellforsch, 123: 441-457. No gradient of hair-cell type occurred on Lim, D. J., and W. C. Lane 1969 Cochlear sen- the medial edge. Thus our observations sory epithelium, a scanning electron microscop- and conclusions concerning the morpho- ic observation. Ann. Otol., 79: 1-5. Lindeman, H. H. 1969 Studies on the morphol- genesis of saccular hair cells are consistent ogy of the sensory regions of the vestibular ap- with the observations and conclusions of paratus. Springer-Verlag, Berlin. Geisler et al. concerning the hair cells of Lombard, R. E. 1970 A comparative morpho- the amphibian papilla. logical analysis of the inner ear of salamanders. Regardless of whether or not the pe- Doct. Diss., Univ. of Chicago. Marovitz, W. F., .I, K. Arenberg and R. Thalman ripheral type of hair cell in the saccular 1970 Evaluation of preparation technique for macula is an immature version of the cen- the scanning electron microscope. The Laryngo- tral type, one is left with the question of scope, 80: 1680-1700. Proebsting, G. 1924 Zellenzahl and Zellgrosse whether or not there is a functional dif- in Labyrinthorgan der Tritonen, nebst anderen ference between the two types. The an- damit zusammenhangenden Fragen. 2001. Jahr., swer to this question will require electro- Abt. Zool. Physiol. Tiere, 41 : 425-488.