Hearing Research, 45 (1990) 33-40 Elsevier
HEARES
33
01337
Growth of a fish ear II. Locations of newly proliferated sensory hair cells in the saccular epithelium of Astronotus ocellatus Arthur N. Popper 1 and Becky Hoxter 2 ’ Department of Zoology, University of Maryland College Park, Maryland, U S.A. and 2 Department of Anatomy and Cell Biology, Georgetown University School of Medicine, Washington, District of Columbia, U.S.A. (Received
2 September
1989; accepted
25 October
1989)
Tritiated thymidine was used to investigate the sites of postembryonic hair cell addition in adult fish species known to add large numbers of hair cells for several years after hatching. Several types of proliferated sensory hair cells, were found throughout the saccular epithelium. with the majority of the the edges of the epithelium. There was no evidence for an ‘annular’ addition of sensory hair cells in Fish; Saccule;
Hair Cell; Proliferation;
Growth
Introduction The normal occurrence of postembryonic proliferation of inner ear sensory hair cells has been demonstrated in elasmobranchs (Corwin, 1981; 1983; 1985b), bony fishes (Platt, 1977; Popper and Hoxter, 1984), amphibians (Corwin, 1985a) and in all avian otic endorgans other than the basilar papilla (Jorgensen and Mathiesen, 1988). Postembryonic proliferation has also been demonstrated in the bird basilar papilla in response to hair cell loss as a result of trauma (Corwin and Cotanche, 1988; Ryals and Rubel, 1988). Embryonically, hair cell addition occurs along the edges of the growing epithelium (Ruben, 1967; Lewis and Li, 1973; Li and Lewis, 1974; Li, 1978; Sans and Chat, 1982; Lim and Arm&o, 1985; Katayama and Corwin, 1989). However, proliferation appears to occur throughout the sensory region of the crista ampullaris and possibly other endorgans during later stages of development in the mouse (Lim and Anniko, 1985). Embryonic
Correspondence to: Dr. Arthur N. Popper, Department of Zoology, University of Maryland, College Park, MD 20742, U.S.A. 0378-5955/90/$03.50
AsfronotuF ocellatuF (oscar), a labeled cells, including newly cells some distance away from Astronotus.
0 1990 Elsevier Science Publishers
hair cell addition is also likely to occur throughout the developing saccule in the toadfish, Opsanus tau (Sokolowski and Popper, 1988). Postembryonic studies have demonstrated that hair cell addition occurs primarily at the edges (margins) of the sensory epithelium in elasmobranchs (Corwin, 1981, 1983, 1985b) and amphibians (Corwin, 1985a), although in amphibians, a small proportion of added hair cells are found within the epithelium. The vast majority of newly proliferated hair cells occur away from the edges of the sensory epithelia in birds (Jorgensen and Mathiesen, 1988). Post-trauma hair cell replacement in birds occurs at the site of damage (Corwin and Cotanche, 1988; Ryals and Rubel, 1988). We have demonstrated that hair cell proliferation is extensive in a cichlid fish, Astronotus ocellatus (the oscar). An estimate indicates that Astronotus adds on the average of 150 cells per day over one or two years after hatching (Popper and Hoxter, 1984). However, our earlier study did not demonstrate whether the newly proliferated cells occurred only at epithelial margins, or whether they occurred throughout the epithelium. The current investigation was directed at determining the locations of newly proliferated cells. Preliminary
B.V. (Biomedical
Division)
34
results were reported Hoxter, 1985).
in abstract
form (Popper
and
Materials and Methods Tritiated thymidine labeling was used to locate post-emb~o~cally proliferated sensory hair cells. Six Asrrunotus were used in the study (Table I). Five animals were from 4.5 to 5.7 cm in standard length (4.0 to 8.4 g) while one animal was 7.9 cm and 23.8 g, The animals were injected with 1 pCi/gm body weight of methyL3H thy~~ne one or five times on a once-daily basis several hours after the lights came on in the aquarium room. Animals were allowed to survive for one to 21 days (Table I) and then sacrificed under deep anesthetic (ethyl-aminobenzoate, Sigma). Following anes~eti~tion, the crania were quickly opened, the brains aspirated away, and the cranial cavity flooded with 2% glutaraldehyde in phosphate buffer. The heads were left in fixative for 24 to 48 hours. The saccular epithelium was then dissected out, post-fixed in 1% osmium tetroxide in phosphate buffer, dehydrated to propylene oxide, embedded in araldite 502, serially sectioned at 1 to 2 pm thickness and placed on gelatin-cl-nom alum coated slides. The slides were dipped in NTB-3 emulsion and stored at 4’C for three to five weeks. After developing the slides in Kodak D19, the tissue was stained in toluidine blue. Results Newly proliferated cells were identified presence of labeled thymidine over their
TABLE
by the nuclei.
Several types of labeled cells were found in each animal. Of the labeled cells, 23% to 47% were clearly sensory hair cells (Table I; Fig. 1). The hair cells were usually located a substantial distance from the edges of the saccular epithelium (Fig. IA). Sites of new sensory hair cells were deter~ned by mapping their locations from serial sections of the epithelia. Data for each fish, giving the number of labeled hair cells at 10% intervals from dorsal to ventral edges of the epithelium, are presented in Table 2. The Table also shows the total number of labeled hair cells for each 10% interval summarized for all animals, as well as the percent of total hair cells at each interval. The percent data are represented graphically in Fig. 2. The figure demonstrates that most of the labeled hair cells were located away from the dors~-ventral margins of the epithelium. To demonstrate the positions of newly proliferated sensory hair cells on the saccular epithelium, the positions of all sensory cells of one fish (No. 40) are plotted in Fig. 3A. (The figure has been normaiized to be the same dorsal-ventral size along its entire length.) The figure demonstrates a relatively even distribution of labeled hair cells across the total length and width of the saccular epithelium. There is no indication that cells were located particularly close to the epithelial margins (e.g., right along the X- and Y-axes) or there was any one focus for labeled cells. Similar data were obtained for each of the other animals used in the study. Each saccular epithelium also had many labeled cells that were not hair cells (Table 2b; Figs. lA, lB, and 1E). Some of these cells had characteris-
I
ANIMALS
USED IN LABELING
Fish Number
Number
37 40 44 46 48 50
1 5 5 5 5 2
Inject
STUDY
AND THE NUMBER
OF LABELED
CELLS
FROM
EACH
SPECIMEN
Number of days post injection to sacrifie
Total Number labeled cells
Number labeled hair cells
Labeled hair ceils as % of total labeled cells
14 1 1 14 21 1
145 232 416 632 643 80
68 73 84 169 181 18
47% 31% 20% 27% 28% 23%
Fig. 1. A. epithelium epithelium (n) labeled
Cross-section of the saccular epithelium from (the edges of the hair cell region are indicated (between arrowheads). B. Higher power of the non-hair cells. C. and D. Other labeled sensory cells located in regions
fish No. 46 showing all the cells from dorsal to ventral margins of the by arrows). Several labeled cells are located towards the center of the labeled cells in the region between arrowheads in 1A. (h) labeled hair cell, hair cells from the same fish. E. Labeled non-hair cells. F. Several labeled just outside of the sensory epithelium.
36
Distribution of Labeled Hair Cells Along Dorsal-Ventral Axis of Epithelium
n c
14%
_
tl
0%“; -10
0
m
Hair cells
I
I
I
,
I
10
20
30
40
50
DORSAL-VENTRAL
60
POSITION
m
70 OF
HAIR
80
Non-halr
cells
90
110 120
100
CELLS
Fig. 2. Graph showing per cent of each labeled cell type (hair cell, non-hair cell) at 10% intervals along the dorsal-ventral saccular epithelium for all animals (Table 2). Cells located at - 10 and + 110 represent the complete pool of extramacular in Astronotus.
tics of support cells (Fig. lB), while others were neither support nor hair cells. The non-sensory cells were found throughout the epithelium and 10.8% were outside of the sensory region (Table 2b, Figs. lF, 2, 3B). Discussion Our data demonstrate that newly proliferated sensory hair cells in the saccular epithelium of Astronotz~ are found throughout the saccular epithelium (Fig. 2, 3A). Moreover, labeled nonsensory cells, some of which are likely to be potential precursors to sensory hair cells, are also located throughout the epithelium (Figs. 1, 2, 3B). There is also a relatively small population of extramacular cells that were labeled (Fig. 1F) It is not clear whether these cells are potential sensory hair cells or other types of cells. There are several possible hypotheses regarding site(s) for initial proliferation of new sensory hair cells. One hypothesis is that cells proliferate around
axis of the cells found
the edges of the epithelium and then migrate into the epithelium. Alternatively, cells proliferate throughout the epithelium and there is little or no migration. A third possible hypothesis is that both mechanisms are operative. If all (or the greater proportion) of the cell proliferation occurred at the edges of the epithelium we would have expected to see some evidence of migration of cells into the epithelium since mature but newly proliferated hair cells are found throughout the epithelium. Such evidence might include one or more peaks of labeled cells at, or close to, the edges of the epithelium in animals sacrificed within 24 hours after their last injection (animals 40, 44 and 45, Table 1). No such evidence was encountered. Instead, the distribution of hair cells and precursor cells tends to be lower at the epithelial margins (Fig. 2), with a relatively even distribution through the rest of the epithelium. We suggest that the data support the hypothesis that cells arise throughout the epithelium dur-
37 TABLE
II
POSITIONS OF LABELED CELLS FOR EACH LABELED CELLS THAT WERE NOT SENSORY
EXPERIMENTAL HAIR CELLS
ANIMAL.
A. LABELED
SENSORY
HAIR
CELLS.
B.
A. Labeled sensory hair cells Position
0 10 20 30 40 50 60 70 80 90 100
Total
Number 31
40
44
46
0 3 13 9 8 6 4 6 9 5 4
0 5 8 10 6 13 5 10 7 6 3
0 11 4 12 4 8 10 4 12 13 5
0 18 20 18 10 15 20 16 17 17 10
B. Labeled non-sensory cells. Cells located margins of the epithelium. Position
-10 0 10 20 30 40 50 60 70 80 90 100 110
48
% Total
Variance
50 0 1 3 6 2 1 1 0 3 0 1
0 9 13 21 23 24 27 23 22 11 6
at the - 10 and the + 110 position
are extraepithelial
0
0.0 8.0% 10.4% 13.0% 9.1% 11.5% 11.5% 10.1% 12.0% 8.9% 5.0%
0 47 61 76 53 67 67 59 70 52 29
cells located
5.1 5.9 5.2 6.8 7.3 9.3 1.7 6.3 5.6 2.8 anywhere
outside
Number
Labeled
31
40
44
46
48
50
Total
% Tot
Variance
10 0 16 21 13 18 20 7 14 17 9 10 0
22 2 14 17 27 24 45 23 25 27 14 13 0
35 2 36 50 47 45 52 55 40 31 33 21 28
57 4 69 12 75 45 49 68 64 67 48 56 45
50 1 64 62 79 71 105 19 51 59 44 28 11
1 0 5 7 6 14 10 8 9 8 9 2 0
175 9 204 229 247 217 281 240 203 209 157 130 84
7.3% 0.4% 8.6% 9.6% 10.4% 9.1% 11.8% 10.1% 8.5% 8.8% 6.6% 5.5% 3.5%
20.2 1.4 24.8 24.4 28.4 19.7 30.3 28.7 19.7 21.3 16.2 17.4 17.1
of the
Cells
In each case, cells were pooled in increments of 10% of the distance from the dorsal margin of the saccular epithelium. Data for individual animals shows the total number of cells at each increment. The ‘Total’ column gives the total number of cells at a particular position for all the animals. The ‘% Total’ column shows the per cent of all the labeled cells that are at a particular position.
ing postembryonic hair cell proliferation. In addition to our data showing broad distribution of labeled hair cells and other cells on the epithelium even after only 24 hours following a single injection of thymidine, other studies in our laboratory support the hypothesis. Presson et al. (1988), sacrificed Astronotus 15 min after thymidine injection and found labeled cells, including cells that are likely to be precursors to sensory hair cells, throughout the epithelium. Presson et al. also found labeled hair cells throughout the epithelium
within 24 h of a single thymidine injection. It is unlikely that cells could have migrated through the epithelium from the edges in 24 h had they been produced at the periphery. Less direct evidence supporting this hypothesis are data from growth studies in Astronotus (Popper and Hoxter, 1984). These studies showed that the density of sensory hair cells does not change appreciably even as the saccular epithelium grows from 5000 to over 150,000 hair cells. If cell addition was annular, as suggested in the first hy-
38
Distribution
of Labeled
100
8 ; E
* ***
*
So
a*
Hair
*
*
x
**
x
* *
* Y
;
*
***
.Y
*
f
*
x
*
* *
*
w”
**
40
Cells
x Y
60
c H
*
*
D
Ceils - Fish #40
2O f
a
0 0
60
100
160
RDSTRAL-CAUDAL
i
140
;
120
i
200 POSITION
260
Non-Hair
300
Cells
n
0
-20
c
::
I
q
-40 0
60
160 100 ROSTPAL-CAUDAL
200 POSITION
260
300
Fig. 3. Distribution on the saccular epithelium of all labeled cells for fish X40. Each mark shows the rostral-caudal (X-axis) and dorsal-ventral (Y-axis) position of an individual cell. The dorsal-ventral axis of the epithelium (Y-axis) has been normalized to 100% while the X-axis is represented as section number from the rostra1 end of the epithelium. a. Labeled sensory hair cells b. Labeled non-hair cells.
pothesis, we would have expected to find a large number of cells labeled in the annular position and few, if none, labeled in the center of the epithelium. Marginal vs. distributed addition of hair cells Investigations of postembryonic sensory hair cell proliferation in other vertebrates gives a variable picture of where such cells are added. Some of the differences may result from using different species and inner ear endorgans in various studies. The only anamniotic data are for the macula neglecta of an elasmobranch (Corwin, 1983) and the saccule of the amphibian Bufo marinus (Corwin, 1985a). In both species, some hair cell addition occurs in the central portion of the epi-
thelium, although Corwin demonstrates that the predominant cell addition is at the margins. In the only demonstration of hair cell addition in normal amniotes, Jorgensen and Mathiesen (1988) demonstrated that the adult budgerigar (Mefopsittacus undulatus) adds hair cells in all otic endorgans other than the basilar papilla. Moreover, 80% of all labeled cells in this avian species (125 of 133 sensory hair cells) were located central to the edge of the epithelia. Our data parallel those for addition of rods in the eyes of the goldfish (Raymond, 1985). The teleost eye, as ear, continues to add sensory cells throughout life, and rod addition is found most extensive centrally, although some addition occurs peripherally as well (Johns, 1982; Kock, 1982; Stell and Kock, 1984). Significance of cell addition It is not yet clear why fishes add large numbers of sensory hair cells, although it has been suggested that hair cell addition may be required for the teleost ear to continue to do the computations for signal analysis as the animal grows and the relative position between the ear and the sound detector, the swimbladder, changes (Popper et al. 1988; Rogers et al, 1988). Though one might argue for maintenance of hair cell addition at the epithelial margin in postembryonic animals, random addition of hair cells may be more useful in animals that add extensive numbers of hair cells postembryonically. If cells were added marginally, as in embryonic growth, there would either be significant differences in patterns of innervation of hair cells in different epithelial regions or there would have to be a constant breaking and making of hair cell innervations by innervating neurons as hair cells are added as has been reported in the macula neglecta of the ray Raja ocellata (Corwin, 1985b). By adding hair cells throughout the epithelium, as in Astronotus, all of the innervating neurons would potentially be able to increase the number of hair cells they innervate, thus keeping some relative allometry of input to neurons. A pattern similar to that proposed here has been reported for innervation of newly differentiated rod photoreceptors in goldfish (Carassius auratus) (Johns and Easter, 1977; Johns, 1982; Kock and Stell, 1985).
39
Although it is not yet clear there is any functional topographic pattern in the fish saccule (e.g., for frequency, intensity or temporal parameters of the signal), marginal addition of sensory hair cells to the ear would potentially result in constant changes in the function of different ear regions as the ear grows. For example, were the margin responsive to low frequencies and the center of the epithelium to higher frequencies, the frequency response characteristics of cell regions would change as the margin was extended in a system that added cells only marginally. In contrast, by having cell addition throughout the epithelium, the potential roles of ear regions would remain stable. While this may not be a problem in an amphibian where hair cell addition is limited to a few cells per day, the problem is of a greater magnitude in Astronotus where hair cells in a single saccular macula may increase from 5000 cells to more than 150,000 cells in a year or two. Acknowledgements
We are grateful to Dr. William M. Saidel and Dr. Joelle Presson for reading and commenting upon earlier versions of the MS The work reported here was supported by NIH grants DC00140 and NS-21646. References Corwin, J.T. (1981) Postembryonic production and aging of inner ear hair cells in sharks. J. Comp. Neurol. 201,541-553. Corwin, J.T. (1983) Postembryonic growth of the macula neglecta auditory detector in the ray, Ruja clauata: Continual increases in hair cell number, neural convergence, and physiological sensitivity. J. Comp. Neurol. 218, 345-356. Corwin, J.T. (1985a) Perpetual production of hair cells and maturational changes in hair cell ultrastructure accompany postembryonic growth in an amphibian ear. Proc. Natl. Acad. Sci. 82, 3911-3915. Cot-win, J.T. (1985b) Auditory neurons expand their terminal arbors throughout life and orient toward the site of postembryonic hair cell production in the macula neglecta in elasmobranchs. J. Comp. Neurol. 239, 445-452. Corwin, J.T. and Cotanche, D.A. (1988) Regeneration of sensory hair cells after acoustic trauma. Science 240, 1772-1774. Johns, P.R. (1982) Formation of photoreceptors in larval and adult goldfish. J. Neurosci. 2, 178-198.
Johns, P.R. and Easter, Jr., S.S. (1977) Growth of the adult goldfish eye. II. Increase in retinal cell number. J. Comp. Neurol. 176, 331-342. Jorgensen, J.M. and Math&en, C. (1988) The avian inner ear: Continuous production of hair cells in vestibular sensory organs, but not in the auditory papilla. Natu~iss~schaften 75, 319-320. Katayama, A. and Corwin, J.T. (1989) Cell production in the chicken cochlea. J. Comp. Neurol. 281, 129-135. Kock, J.-H. (1982) Neuronal addition and retinal expansion during growth of the crucian carp eye. J. Comp. Neurol. 209, 264-2’74. Kock, J.-H. and Stell, WK. (1985) Formation of new rod photoreceptor synapses onto differentiated bipolar cells in goldfish retina. Anat. Rec. 211, 69-74. Lewis, E.R. and Li, C.W. (1973) Evidence concerning the morphogenesis of saccular receptors in the bullfrog (Rana c~te~~eiana~. J. Morph. 139, 351-362. Li, C.W. and Lewis, E.R. (1974) Mo~hogenesis of auditory receptor epithelia in the bullfrog. In: 0. Johari and I. Corvin (Eds.) SEM/1974 Chicago, IITRI, pp. 792-798. Li, C.W. (1978) Hair cell development in the inner ear. In: Scanning Electron Microscopy, Vol. II, SEM, Inc., O’Hare, IL, pp. 967-974. Lim, D.J. and Anniko, M. (1985) Developmental morphology of the mouse inner ear: A scanning electron microscopic observation. Acta Oto-Laryngol. suppl. 442, l-69. Platt, C. (1977) Hair cell distribution and orientation in goldfish otolith organs. J. Comp. Neurol. 172, 283-297. Popper, A.N., Hoxter, B. (1984) Growth of a fish ear: I. ~antitative analysis of sensory hair cell and ganglion cell proliferation. Hearing Res. 15, 133-142. Popper, A.N. and Hoxter, B. (1985) Post-embryonic hair cell production in the saccule of a teleost fish. Sot. Neurosci. Abst. 11, 451. Popper, A.N., Rogers, P.H., Saidel, W.M. and Cox, M. (1988) The role of the fish ear in sound processing. In: J. Atema, R.R. Fay, A.N. Popper, W.N. Tavolga (Eds.), Sensory Biology of Aquatic Animals, Springer-Verlag, New York, pp. 687-710. Presson, J. C., Popper, A.N. and Hoxter, B. (1988) Proliferating neuroepithehal cells in the saccule of an adult fish: source of new hair cells. Abst. Assn. Res. Otolaryngol., 11, 155-156. Rogers, P.H., Popper, A.N., Cox, M, and Saidel, W.M. (1988) Processing of acoustic signals in the auditory system of bony fish. J. Acoust. Sot. Amer., 83, 338-349. Raymond, P.A. (1985) The unique origin of rod photoreceptors in the teleost retina. Trends Neurosci. 8, 12-17. Ruben, R.J. (1967) Development of the inner ear of the mouse: a radioautographic study of terminal rnitoses. Acta OtoLaryngol. Suppl. 220, l-44. Ryals, B.M. and Rubel, E.W. (1988) Hair cell regeneration after acoustic trauma in adult Coturnix quail, Science 240, 1774-1776. Sans, A. and Chat, M. (1982) Analysis of temporal and spatial patterns of rat vestibular hair cell differentiation by triti-
40
ated thymidine radioautography. J. Comp. Neurol. 206, l-8. Sokolowski, B. and Popper, A.N. (1988) The gross and ultrastructural development of the saccule of the toadfish, Opsanw tau. J. Morphology 194, 323-348.
Stell, W.K. and Kock, J.-H. (1984) Structure, development and visual acuity in the goldfish retina. In: S.R. Hilfer and J.B. Sheffield, (Eds.), Molecular and Cellular Basis of Visual Activity, Springer-Verlag, New York, pp. 79-106.
HEARES
33
01337
Growth of a fish ear II. Locations of newly proliferated sensory hair cells in the saccular epithelium of Astronotus ocellatus Arthur N. Popper 1 and Becky Hoxter 2 ’ Department of Zoology, University of Maryland College Park, Maryland, U S.A. and 2 Department of Anatomy and Cell Biology, Georgetown University School of Medicine, Washington, District of Columbia, U.S.A. (Received
2 September
1989; accepted
25 October
1989)
Tritiated thymidine was used to investigate the sites of postembryonic hair cell addition in adult fish species known to add large numbers of hair cells for several years after hatching. Several types of proliferated sensory hair cells, were found throughout the saccular epithelium. with the majority of the the edges of the epithelium. There was no evidence for an ‘annular’ addition of sensory hair cells in Fish; Saccule;
Hair Cell; Proliferation;
Growth
Introduction The normal occurrence of postembryonic proliferation of inner ear sensory hair cells has been demonstrated in elasmobranchs (Corwin, 1981; 1983; 1985b), bony fishes (Platt, 1977; Popper and Hoxter, 1984), amphibians (Corwin, 1985a) and in all avian otic endorgans other than the basilar papilla (Jorgensen and Mathiesen, 1988). Postembryonic proliferation has also been demonstrated in the bird basilar papilla in response to hair cell loss as a result of trauma (Corwin and Cotanche, 1988; Ryals and Rubel, 1988). Embryonically, hair cell addition occurs along the edges of the growing epithelium (Ruben, 1967; Lewis and Li, 1973; Li and Lewis, 1974; Li, 1978; Sans and Chat, 1982; Lim and Arm&o, 1985; Katayama and Corwin, 1989). However, proliferation appears to occur throughout the sensory region of the crista ampullaris and possibly other endorgans during later stages of development in the mouse (Lim and Anniko, 1985). Embryonic
Correspondence to: Dr. Arthur N. Popper, Department of Zoology, University of Maryland, College Park, MD 20742, U.S.A. 0378-5955/90/$03.50
AsfronotuF ocellatuF (oscar), a labeled cells, including newly cells some distance away from Astronotus.
0 1990 Elsevier Science Publishers
hair cell addition is also likely to occur throughout the developing saccule in the toadfish, Opsanus tau (Sokolowski and Popper, 1988). Postembryonic studies have demonstrated that hair cell addition occurs primarily at the edges (margins) of the sensory epithelium in elasmobranchs (Corwin, 1981, 1983, 1985b) and amphibians (Corwin, 1985a), although in amphibians, a small proportion of added hair cells are found within the epithelium. The vast majority of newly proliferated hair cells occur away from the edges of the sensory epithelia in birds (Jorgensen and Mathiesen, 1988). Post-trauma hair cell replacement in birds occurs at the site of damage (Corwin and Cotanche, 1988; Ryals and Rubel, 1988). We have demonstrated that hair cell proliferation is extensive in a cichlid fish, Astronotus ocellatus (the oscar). An estimate indicates that Astronotus adds on the average of 150 cells per day over one or two years after hatching (Popper and Hoxter, 1984). However, our earlier study did not demonstrate whether the newly proliferated cells occurred only at epithelial margins, or whether they occurred throughout the epithelium. The current investigation was directed at determining the locations of newly proliferated cells. Preliminary
B.V. (Biomedical
Division)
34
results were reported Hoxter, 1985).
in abstract
form (Popper
and
Materials and Methods Tritiated thymidine labeling was used to locate post-emb~o~cally proliferated sensory hair cells. Six Asrrunotus were used in the study (Table I). Five animals were from 4.5 to 5.7 cm in standard length (4.0 to 8.4 g) while one animal was 7.9 cm and 23.8 g, The animals were injected with 1 pCi/gm body weight of methyL3H thy~~ne one or five times on a once-daily basis several hours after the lights came on in the aquarium room. Animals were allowed to survive for one to 21 days (Table I) and then sacrificed under deep anesthetic (ethyl-aminobenzoate, Sigma). Following anes~eti~tion, the crania were quickly opened, the brains aspirated away, and the cranial cavity flooded with 2% glutaraldehyde in phosphate buffer. The heads were left in fixative for 24 to 48 hours. The saccular epithelium was then dissected out, post-fixed in 1% osmium tetroxide in phosphate buffer, dehydrated to propylene oxide, embedded in araldite 502, serially sectioned at 1 to 2 pm thickness and placed on gelatin-cl-nom alum coated slides. The slides were dipped in NTB-3 emulsion and stored at 4’C for three to five weeks. After developing the slides in Kodak D19, the tissue was stained in toluidine blue. Results Newly proliferated cells were identified presence of labeled thymidine over their
TABLE
by the nuclei.
Several types of labeled cells were found in each animal. Of the labeled cells, 23% to 47% were clearly sensory hair cells (Table I; Fig. 1). The hair cells were usually located a substantial distance from the edges of the saccular epithelium (Fig. IA). Sites of new sensory hair cells were deter~ned by mapping their locations from serial sections of the epithelia. Data for each fish, giving the number of labeled hair cells at 10% intervals from dorsal to ventral edges of the epithelium, are presented in Table 2. The Table also shows the total number of labeled hair cells for each 10% interval summarized for all animals, as well as the percent of total hair cells at each interval. The percent data are represented graphically in Fig. 2. The figure demonstrates that most of the labeled hair cells were located away from the dors~-ventral margins of the epithelium. To demonstrate the positions of newly proliferated sensory hair cells on the saccular epithelium, the positions of all sensory cells of one fish (No. 40) are plotted in Fig. 3A. (The figure has been normaiized to be the same dorsal-ventral size along its entire length.) The figure demonstrates a relatively even distribution of labeled hair cells across the total length and width of the saccular epithelium. There is no indication that cells were located particularly close to the epithelial margins (e.g., right along the X- and Y-axes) or there was any one focus for labeled cells. Similar data were obtained for each of the other animals used in the study. Each saccular epithelium also had many labeled cells that were not hair cells (Table 2b; Figs. lA, lB, and 1E). Some of these cells had characteris-
I
ANIMALS
USED IN LABELING
Fish Number
Number
37 40 44 46 48 50
1 5 5 5 5 2
Inject
STUDY
AND THE NUMBER
OF LABELED
CELLS
FROM
EACH
SPECIMEN
Number of days post injection to sacrifie
Total Number labeled cells
Number labeled hair cells
Labeled hair ceils as % of total labeled cells
14 1 1 14 21 1
145 232 416 632 643 80
68 73 84 169 181 18
47% 31% 20% 27% 28% 23%
Fig. 1. A. epithelium epithelium (n) labeled
Cross-section of the saccular epithelium from (the edges of the hair cell region are indicated (between arrowheads). B. Higher power of the non-hair cells. C. and D. Other labeled sensory cells located in regions
fish No. 46 showing all the cells from dorsal to ventral margins of the by arrows). Several labeled cells are located towards the center of the labeled cells in the region between arrowheads in 1A. (h) labeled hair cell, hair cells from the same fish. E. Labeled non-hair cells. F. Several labeled just outside of the sensory epithelium.
36
Distribution of Labeled Hair Cells Along Dorsal-Ventral Axis of Epithelium
n c
14%
_
tl
0%“; -10
0
m
Hair cells
I
I
I
,
I
10
20
30
40
50
DORSAL-VENTRAL
60
POSITION
m
70 OF
HAIR
80
Non-halr
cells
90
110 120
100
CELLS
Fig. 2. Graph showing per cent of each labeled cell type (hair cell, non-hair cell) at 10% intervals along the dorsal-ventral saccular epithelium for all animals (Table 2). Cells located at - 10 and + 110 represent the complete pool of extramacular in Astronotus.
tics of support cells (Fig. lB), while others were neither support nor hair cells. The non-sensory cells were found throughout the epithelium and 10.8% were outside of the sensory region (Table 2b, Figs. lF, 2, 3B). Discussion Our data demonstrate that newly proliferated sensory hair cells in the saccular epithelium of Astronotz~ are found throughout the saccular epithelium (Fig. 2, 3A). Moreover, labeled nonsensory cells, some of which are likely to be potential precursors to sensory hair cells, are also located throughout the epithelium (Figs. 1, 2, 3B). There is also a relatively small population of extramacular cells that were labeled (Fig. 1F) It is not clear whether these cells are potential sensory hair cells or other types of cells. There are several possible hypotheses regarding site(s) for initial proliferation of new sensory hair cells. One hypothesis is that cells proliferate around
axis of the cells found
the edges of the epithelium and then migrate into the epithelium. Alternatively, cells proliferate throughout the epithelium and there is little or no migration. A third possible hypothesis is that both mechanisms are operative. If all (or the greater proportion) of the cell proliferation occurred at the edges of the epithelium we would have expected to see some evidence of migration of cells into the epithelium since mature but newly proliferated hair cells are found throughout the epithelium. Such evidence might include one or more peaks of labeled cells at, or close to, the edges of the epithelium in animals sacrificed within 24 hours after their last injection (animals 40, 44 and 45, Table 1). No such evidence was encountered. Instead, the distribution of hair cells and precursor cells tends to be lower at the epithelial margins (Fig. 2), with a relatively even distribution through the rest of the epithelium. We suggest that the data support the hypothesis that cells arise throughout the epithelium dur-
37 TABLE
II
POSITIONS OF LABELED CELLS FOR EACH LABELED CELLS THAT WERE NOT SENSORY
EXPERIMENTAL HAIR CELLS
ANIMAL.
A. LABELED
SENSORY
HAIR
CELLS.
B.
A. Labeled sensory hair cells Position
0 10 20 30 40 50 60 70 80 90 100
Total
Number 31
40
44
46
0 3 13 9 8 6 4 6 9 5 4
0 5 8 10 6 13 5 10 7 6 3
0 11 4 12 4 8 10 4 12 13 5
0 18 20 18 10 15 20 16 17 17 10
B. Labeled non-sensory cells. Cells located margins of the epithelium. Position
-10 0 10 20 30 40 50 60 70 80 90 100 110
48
% Total
Variance
50 0 1 3 6 2 1 1 0 3 0 1
0 9 13 21 23 24 27 23 22 11 6
at the - 10 and the + 110 position
are extraepithelial
0
0.0 8.0% 10.4% 13.0% 9.1% 11.5% 11.5% 10.1% 12.0% 8.9% 5.0%
0 47 61 76 53 67 67 59 70 52 29
cells located
5.1 5.9 5.2 6.8 7.3 9.3 1.7 6.3 5.6 2.8 anywhere
outside
Number
Labeled
31
40
44
46
48
50
Total
% Tot
Variance
10 0 16 21 13 18 20 7 14 17 9 10 0
22 2 14 17 27 24 45 23 25 27 14 13 0
35 2 36 50 47 45 52 55 40 31 33 21 28
57 4 69 12 75 45 49 68 64 67 48 56 45
50 1 64 62 79 71 105 19 51 59 44 28 11
1 0 5 7 6 14 10 8 9 8 9 2 0
175 9 204 229 247 217 281 240 203 209 157 130 84
7.3% 0.4% 8.6% 9.6% 10.4% 9.1% 11.8% 10.1% 8.5% 8.8% 6.6% 5.5% 3.5%
20.2 1.4 24.8 24.4 28.4 19.7 30.3 28.7 19.7 21.3 16.2 17.4 17.1
of the
Cells
In each case, cells were pooled in increments of 10% of the distance from the dorsal margin of the saccular epithelium. Data for individual animals shows the total number of cells at each increment. The ‘Total’ column gives the total number of cells at a particular position for all the animals. The ‘% Total’ column shows the per cent of all the labeled cells that are at a particular position.
ing postembryonic hair cell proliferation. In addition to our data showing broad distribution of labeled hair cells and other cells on the epithelium even after only 24 hours following a single injection of thymidine, other studies in our laboratory support the hypothesis. Presson et al. (1988), sacrificed Astronotus 15 min after thymidine injection and found labeled cells, including cells that are likely to be precursors to sensory hair cells, throughout the epithelium. Presson et al. also found labeled hair cells throughout the epithelium
within 24 h of a single thymidine injection. It is unlikely that cells could have migrated through the epithelium from the edges in 24 h had they been produced at the periphery. Less direct evidence supporting this hypothesis are data from growth studies in Astronotus (Popper and Hoxter, 1984). These studies showed that the density of sensory hair cells does not change appreciably even as the saccular epithelium grows from 5000 to over 150,000 hair cells. If cell addition was annular, as suggested in the first hy-
38
Distribution
of Labeled
100
8 ; E
* ***
*
So
a*
Hair
*
*
x
**
x
* *
* Y
;
*
***
.Y
*
f
*
x
*
* *
*
w”
**
40
Cells
x Y
60
c H
*
*
D
Ceils - Fish #40
2O f
a
0 0
60
100
160
RDSTRAL-CAUDAL
i
140
;
120
i
200 POSITION
260
Non-Hair
300
Cells
n
0
-20
c
::
I
q
-40 0
60
160 100 ROSTPAL-CAUDAL
200 POSITION
260
300
Fig. 3. Distribution on the saccular epithelium of all labeled cells for fish X40. Each mark shows the rostral-caudal (X-axis) and dorsal-ventral (Y-axis) position of an individual cell. The dorsal-ventral axis of the epithelium (Y-axis) has been normalized to 100% while the X-axis is represented as section number from the rostra1 end of the epithelium. a. Labeled sensory hair cells b. Labeled non-hair cells.
pothesis, we would have expected to find a large number of cells labeled in the annular position and few, if none, labeled in the center of the epithelium. Marginal vs. distributed addition of hair cells Investigations of postembryonic sensory hair cell proliferation in other vertebrates gives a variable picture of where such cells are added. Some of the differences may result from using different species and inner ear endorgans in various studies. The only anamniotic data are for the macula neglecta of an elasmobranch (Corwin, 1983) and the saccule of the amphibian Bufo marinus (Corwin, 1985a). In both species, some hair cell addition occurs in the central portion of the epi-
thelium, although Corwin demonstrates that the predominant cell addition is at the margins. In the only demonstration of hair cell addition in normal amniotes, Jorgensen and Mathiesen (1988) demonstrated that the adult budgerigar (Mefopsittacus undulatus) adds hair cells in all otic endorgans other than the basilar papilla. Moreover, 80% of all labeled cells in this avian species (125 of 133 sensory hair cells) were located central to the edge of the epithelia. Our data parallel those for addition of rods in the eyes of the goldfish (Raymond, 1985). The teleost eye, as ear, continues to add sensory cells throughout life, and rod addition is found most extensive centrally, although some addition occurs peripherally as well (Johns, 1982; Kock, 1982; Stell and Kock, 1984). Significance of cell addition It is not yet clear why fishes add large numbers of sensory hair cells, although it has been suggested that hair cell addition may be required for the teleost ear to continue to do the computations for signal analysis as the animal grows and the relative position between the ear and the sound detector, the swimbladder, changes (Popper et al. 1988; Rogers et al, 1988). Though one might argue for maintenance of hair cell addition at the epithelial margin in postembryonic animals, random addition of hair cells may be more useful in animals that add extensive numbers of hair cells postembryonically. If cells were added marginally, as in embryonic growth, there would either be significant differences in patterns of innervation of hair cells in different epithelial regions or there would have to be a constant breaking and making of hair cell innervations by innervating neurons as hair cells are added as has been reported in the macula neglecta of the ray Raja ocellata (Corwin, 1985b). By adding hair cells throughout the epithelium, as in Astronotus, all of the innervating neurons would potentially be able to increase the number of hair cells they innervate, thus keeping some relative allometry of input to neurons. A pattern similar to that proposed here has been reported for innervation of newly differentiated rod photoreceptors in goldfish (Carassius auratus) (Johns and Easter, 1977; Johns, 1982; Kock and Stell, 1985).
39
Although it is not yet clear there is any functional topographic pattern in the fish saccule (e.g., for frequency, intensity or temporal parameters of the signal), marginal addition of sensory hair cells to the ear would potentially result in constant changes in the function of different ear regions as the ear grows. For example, were the margin responsive to low frequencies and the center of the epithelium to higher frequencies, the frequency response characteristics of cell regions would change as the margin was extended in a system that added cells only marginally. In contrast, by having cell addition throughout the epithelium, the potential roles of ear regions would remain stable. While this may not be a problem in an amphibian where hair cell addition is limited to a few cells per day, the problem is of a greater magnitude in Astronotus where hair cells in a single saccular macula may increase from 5000 cells to more than 150,000 cells in a year or two. Acknowledgements
We are grateful to Dr. William M. Saidel and Dr. Joelle Presson for reading and commenting upon earlier versions of the MS The work reported here was supported by NIH grants DC00140 and NS-21646. References Corwin, J.T. (1981) Postembryonic production and aging of inner ear hair cells in sharks. J. Comp. Neurol. 201,541-553. Corwin, J.T. (1983) Postembryonic growth of the macula neglecta auditory detector in the ray, Ruja clauata: Continual increases in hair cell number, neural convergence, and physiological sensitivity. J. Comp. Neurol. 218, 345-356. Corwin, J.T. (1985a) Perpetual production of hair cells and maturational changes in hair cell ultrastructure accompany postembryonic growth in an amphibian ear. Proc. Natl. Acad. Sci. 82, 3911-3915. Cot-win, J.T. (1985b) Auditory neurons expand their terminal arbors throughout life and orient toward the site of postembryonic hair cell production in the macula neglecta in elasmobranchs. J. Comp. Neurol. 239, 445-452. Corwin, J.T. and Cotanche, D.A. (1988) Regeneration of sensory hair cells after acoustic trauma. Science 240, 1772-1774. Johns, P.R. (1982) Formation of photoreceptors in larval and adult goldfish. J. Neurosci. 2, 178-198.
Johns, P.R. and Easter, Jr., S.S. (1977) Growth of the adult goldfish eye. II. Increase in retinal cell number. J. Comp. Neurol. 176, 331-342. Jorgensen, J.M. and Math&en, C. (1988) The avian inner ear: Continuous production of hair cells in vestibular sensory organs, but not in the auditory papilla. Natu~iss~schaften 75, 319-320. Katayama, A. and Corwin, J.T. (1989) Cell production in the chicken cochlea. J. Comp. Neurol. 281, 129-135. Kock, J.-H. (1982) Neuronal addition and retinal expansion during growth of the crucian carp eye. J. Comp. Neurol. 209, 264-2’74. Kock, J.-H. and Stell, WK. (1985) Formation of new rod photoreceptor synapses onto differentiated bipolar cells in goldfish retina. Anat. Rec. 211, 69-74. Lewis, E.R. and Li, C.W. (1973) Evidence concerning the morphogenesis of saccular receptors in the bullfrog (Rana c~te~~eiana~. J. Morph. 139, 351-362. Li, C.W. and Lewis, E.R. (1974) Mo~hogenesis of auditory receptor epithelia in the bullfrog. In: 0. Johari and I. Corvin (Eds.) SEM/1974 Chicago, IITRI, pp. 792-798. Li, C.W. (1978) Hair cell development in the inner ear. In: Scanning Electron Microscopy, Vol. II, SEM, Inc., O’Hare, IL, pp. 967-974. Lim, D.J. and Anniko, M. (1985) Developmental morphology of the mouse inner ear: A scanning electron microscopic observation. Acta Oto-Laryngol. suppl. 442, l-69. Platt, C. (1977) Hair cell distribution and orientation in goldfish otolith organs. J. Comp. Neurol. 172, 283-297. Popper, A.N., Hoxter, B. (1984) Growth of a fish ear: I. ~antitative analysis of sensory hair cell and ganglion cell proliferation. Hearing Res. 15, 133-142. Popper, A.N. and Hoxter, B. (1985) Post-embryonic hair cell production in the saccule of a teleost fish. Sot. Neurosci. Abst. 11, 451. Popper, A.N., Rogers, P.H., Saidel, W.M. and Cox, M. (1988) The role of the fish ear in sound processing. In: J. Atema, R.R. Fay, A.N. Popper, W.N. Tavolga (Eds.), Sensory Biology of Aquatic Animals, Springer-Verlag, New York, pp. 687-710. Presson, J. C., Popper, A.N. and Hoxter, B. (1988) Proliferating neuroepithehal cells in the saccule of an adult fish: source of new hair cells. Abst. Assn. Res. Otolaryngol., 11, 155-156. Rogers, P.H., Popper, A.N., Cox, M, and Saidel, W.M. (1988) Processing of acoustic signals in the auditory system of bony fish. J. Acoust. Sot. Amer., 83, 338-349. Raymond, P.A. (1985) The unique origin of rod photoreceptors in the teleost retina. Trends Neurosci. 8, 12-17. Ruben, R.J. (1967) Development of the inner ear of the mouse: a radioautographic study of terminal rnitoses. Acta OtoLaryngol. Suppl. 220, l-44. Ryals, B.M. and Rubel, E.W. (1988) Hair cell regeneration after acoustic trauma in adult Coturnix quail, Science 240, 1774-1776. Sans, A. and Chat, M. (1982) Analysis of temporal and spatial patterns of rat vestibular hair cell differentiation by triti-
40
ated thymidine radioautography. J. Comp. Neurol. 206, l-8. Sokolowski, B. and Popper, A.N. (1988) The gross and ultrastructural development of the saccule of the toadfish, Opsanw tau. J. Morphology 194, 323-348.
Stell, W.K. and Kock, J.-H. (1984) Structure, development and visual acuity in the goldfish retina. In: S.R. Hilfer and J.B. Sheffield, (Eds.), Molecular and Cellular Basis of Visual Activity, Springer-Verlag, New York, pp. 79-106.
