J. Embryo!. exp. Morph. Vol. 17, 3, pp. 533-41, June 1967
533
With 2 plates
Printed in Great Britain
The neural crest and the acoustic ganglion
By M. S. DEOL 1
M.R.C. Experimental Genetics Research Unit,
Department of Animal Genetics, University College London
INTRODUCTION
Although the origin of the acoustic ganglion has been the subject of numerous
studies there is no unanimity of opinion about it. Most of the earlier investigators (Bartelmez, 1922; Adelman, 1925), using mammalian embryos, believed
that it arose from the neural crest, but the experiments of Campenhout (1935)
and Yntema (1937) on amphibian embryos led them to the view that it was largely,
if not wholly, of placodal origin. This view was supported by Halley (1955), who
worked on the cat, and later by Batten (1958), who worked on the sheep. In
fact Batten stated categorically that the otic placode was the sole source of the
acoustic ganglion. It was thought that an entirely new approach to the problem,
namely the use of mutant genes, might help to resolve the difference of opinion.
The most suitable mutant for the present purpose seemed to be piebald-lethal
(symbol s1; Lane, 1966). This locus is known to be concerned with the development and differentiation of the neural crest. Mayer (1965) cultivated neural crest
and skin from various regions of piebald (s/s) and normal embryos in different
combinations in the coelom of the chick embryo, and came to the conclusion
that the s gene caused spotting primarily through its effect on the neural crest.
Beilschowsky & Schofleld's (1962) discovery of megacolon, associated with a
marked reduction of the myenteric ganglion, in a considerable proportion of
piebald mice indicated that the gene affected the neural crest before its differentiation into melanoblasts and ganglionic primordia. The reason for using the s1
allele in the present study, instead of the s with which these investigators worked,
was that large parts of the coat of s/s mice, particularly in the head region, are
normally pigmented, whereas s1^1 mice are almost entirely white, suggesting that
this allele affects the whole of the neural crest. This view was strengthened by
Lane's (1966) discovery that megacolon and reduction of the myenteric ganglion
occurred in all s1^1 animals. The argument behind this study was that if
the neural crest contributed to the formation of the acoustic ganglion then this
ganglion in s^js1 mice might be expected to be abnormal in some way. As the
abnormalities of the ganglion cells themselves are generally not easy to see by
1
Author's address: Department of Animal Genetics, University College London, Gower
Street, London, W.C.I, U.K.
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M. S. DEOL
means of the standard histological techniques, it was decided to examine in great
detail the neural epithelium and the closely related supporting structures in the
inner ear. The neural epithelium is dependent on the ganglion during its formation, and owing to its extreme complexity is ideally suited for bringing to light
any deficiencies of the ganglion cells. Abnormalities of the neural epithelium
were indeed found in all s1^1 animals examined, their description and possible
significance being the subject of this paper.
MATERIAL AND METHODS
This study is based on the sections of the inner ear of eighteen s1^1 and six
normal mice. The normal animals came from the same litters as some of the
s1^1 ones, and since they conformed to the text-book picture of the normal mouse
with regard to the inner ear, this number was regarded as sufficient. The s1^1
mice ranged in age from 14 days, when the fine differentiation of the inner ear is
just complete, to 36 days, which is nearly the maximum age these animals usually
reach. Their coat was either entirely unpigmented or a small patch of coloured
hair occurred on the head, at the top or to one side. They were roughly tested
for their hearing ability before being fixed. Some of them appeared to be deaf,
but the majority clearly responded by means of the pinna reflex. In view of the
impending histological examination of the organ of Corti elaborate hearing
tests were not considered necessary.
The mice were fixed in Witmaack's fluid by perfusion. The head was removed,
decalcified in 1 % nitric acid, neutralized in 5 % sodium sulphate, and washed
overnight in running water. The ears, along with the adjoining parts of the brain,
were then cut off with a razor blade. They were embedded first in celloidin and
then in paraffin. Serial sections were usually cut at 10 /ti in a plane parallel to the
modiolus in the cochlea (in two cases the plane was transverse). The sections
were stained with Ehrlich's haematoxylin and orange G containing a trace of
erythrosin.
OBSERVATIONS
The inner ear was found to be normal on both sides in all six control animals
with regard to gross morphogenesis as well as fine differentiation. The organ of
Corti had the text-book appearance, with the nuclei of the outer hair cells
arranged in a row at a higher level and those of Deiter's cells at a lower one
PLATE 1
Figs. A-C: Transverse sections of the left cochlear duct (scala media) of a 16-day-old normal
mouse (A) and two piebald-lethal mice of the same age (B,C), all from the same litter. CT,
Corti's tunnel; DC, Deiter's cells; OHC, outer hair cells; RM Reissner's membrane; SG,
spiral ganglion; SV, stria vascularis; TM, tectorial membrane, x 120.
Figs. D-F. High magnification views of the outer hair cells and Deiter's cells shown in figs. A
(D), B (E) and C (F). x 606.
/. Embryo/, exp. Morph., Vol. 17, Part 3
M. S. DEOL
PLATE 1
facing p. 534
/. Embryol. exp. Morph., Vol. 17, Part 3
M. S. DEOL
PLATE 2
Neural crest and acoustic ganglion
x
535
x
(Plate 1,figs.A, D). In all eighteen s js animals, on the other hand, the inner ear
was found to be abnormal on both sides. Its gross morphogenesis appeared to
have taken place along normal lines, but the fine structure was severely affected.
This was true in particular of the neural epithelium in certain regions. The range
and severity of the abnormalities varied a great deal from animal to animal, and
even the two sides of the same animal were frequently dissimilar. The one part
of the ear that was invariably and unmistakably affected was the cochlea, and
within the cochlea the structures most severely affected were the organ of Corti
and the stria vascularis. In some cases these organs were affected in their entirety,
in others they appeared to be normal in part, but in no case were they normal
along their whole length.
In six sxjsx animals all structures contained in the cochlear duct (scala media)
were abnormal, either on one side only or both (Plate 1,fig.B). A very large number
of the hair cells were missing, and the remainder were nearly always abnormal,
mostly reduced in size, so that their nuclei lay close to the top of the organ of
Corti (Plate 1, fig. E). Deiter's cells were present but abnormally formed, with
their nuclei at different levels. The tunnel of Corti was missing in large parts of
the cochlea, particularly near the apex. The cells lining the external and internal
sulci were loosely spread out, sometimes forming hollow masses. The arrangement of the cells of Hensen, Claudius and Boettcher was seriously disturbed.
The tectorial membrane was much thicker than normal, and usually attached to
Reissner's membrane along most of its dorsal aspect. It was thus kept well
separated from the hair cells. Reissner's membrane was sometimes in contact
with the organ of Corti, and in rare instances even with the stria vascularis at its
basal border, near the external spiral sulcus. The stria vascularis was very
poorly organized, and reduced in width and thickness. The distal end of
Reissner's membrane was frequently found to have differentiated into the stria
vascularis, appearing as if the apical border of the stria were bent along the
membrane (Plate 1,figs.B, C). All these structures were affected in their entirety,
there being no normal region.
In the other twelve sxjsx animals the cochlear abnormalities were largely confined to the hair cells, Deiter's cells and the stria vascularis (Plate 1,figs.C, F). In
general the abnormalities were of the same kind as in the first group, but they were
not uniform. Normal and abnormal hair cells and Deiter's cells alternated in
patches of various sizes without any clear pattern in the same ear. The stria
vascularis was much more constantly affected. In fact only two or three
PLATE 2
Figs. G-J: Stria vascularis from the left ear of a 31-day-old normal mouse (G), from the left
ear of its piebald-lethal littermate (H), from another part of the same ear (I) and from the
right ear of the same mouse (J). x 255.
Figs. K-M. Right saccular maculae of a 21-day-old normal mouse (K) and two piebaldlethal mice of the same age (L,M), all from the same litter. NE, Neural epithelium; O,
otoliths. x306.
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M. S. DEOL
1 1
small areas were observed in all s ^ animals where the appearance of the stria
could be described as normal. The usual abnormality of the stria was thinness
(Plate 2, figs. G, H), sometimes so extreme that it was barely existent (Plate 2,
fig. J), but occasionally it was much thicker than normal (Plate 2, fig. 1). The
transition from very thin to very thick areas was usually quite abrupt. In rare
instances cellular degeneration was also observed in the stria vascularis
(Plate 2, fig. I).
The macula of the saccule was also affected in all s1^1 mice. In two or three
animals the abnormalities were only slight, but in others they ranged from
moderate to severe. The normal arrangement of the neural epithelium, with
the somewhat columnar hair cells tightly packed together and the hairs topped
by a fairly uniform layer of otoliths (Plate 2, fig. K), was lacking. The hair cells
were far fewer and frequently rounded, and the thickness of the epithelial layer
was considerably reduced, and at times even irregular (Plate 2, figs. L, M).
The hair cells often lacked hairs. The otolith granules were either greatly reduced
(Plate 2, fig. L) or extraordinarily abundant (Plate 2, fig. M). They seldom
formed a layer of uniform thickness, sometimes occurring in large masses. The
free wall of the saccule was frequently in contact with the otoliths, and cellular
connexions between the wall and the neural epithelium, running through the
otolith layer, were not uncommon. In places the otolith layer was seen to be
hanging well above the hair cells, instead of resting on them. This particular
type of saccular abnormality occurred in all ears in which the entire contents
of the cochlear duct were abnormal, as illustrated in Plate 1, fig. B, and in
two others in which the cochlear abnormalities were of the type shown in
Plate 1, fig. C.
The macula of the utricle appeared to be normal in the majority of ears, and
only slightly abnormal in the rest. Minor abnormalities in the organization of
the neural epithelium and the otolith layer were observed, but as they also
occasionally occur in normal mice it is not possible to say what importance, if
any, should be attached to them. No clear differences were seen between the
cristae of normal and s1^1 mice. The same applies to the ganglion cells.
CONCLUSIONS AND DISCUSSION
The foregoing observations leave little doubt that the piebald-lethal (s1) gene
causes abnormalities in the inner ear. The asymmetrical nature of many of these
abnormalities is an indication that they are probably rather remote effects of the
gene, but however variable they may be the neural epithelium is always involved.
It is not unreasonable to assume that the abnormalities of the neural epithelium
are reflexions of deficiencies in the acoustic ganglion. In view of the demonstrable
influence of the piebald locus on the neural crest before its differentiation into
melanoblasts and ganglionic primordia (see Introduction) and the principle of
the unity of gene action, the most plausible explanation of the abnormalities
Neural crest and acoustic ganglion
537
observed in the inner ear of piebald-lethal mice is that the neural crest contributes to the formation of the acoustic ganglion. This is not to say that the whole
of this ganglion is derived from the neural crest. Indeed, it is virtually certain
from the work of Campenhout (1935), Yntema (1937), Halley (1955), Batten
(1958) and others that a substantial part of it originates in the otic placode. The
question is whether the placodal and neural crest derivatives are mixed in the
innervation of each sensory organ or whether they innervate different sensory
organs. The abnormalities observed appeared to be confined to the cochlea and
the saccule, the involvement of the utricle being uncertain. This would seem to
suggest that ganglion cells innervating these structures are largely if not wholly
of neural crest origin. It is hoped that further work along similar lines will
help in the elucidation of this point.
Piebald-lethal is not the only mutant in which defects of pigmentation are
associated with abnormalities of the inner ear. In his study of the mutant
surdescens (su), Kocher (1960) reported the occurrence of two types of inner ear
abnormalities in the inbred strain C57BL and the stock carrying the white
(Miwh) gene. These, designated as type 1 and type 2 abnormalities, were believed
to be caused by the same su gene. As the type 2 abnormality was found only in
the white stock, it occurred to the present author that it might be confined to
animals carrying the Miwh gene. A reinvestigation was undertaken, and it was
found that the type 2 abnormality was indeed an attribute of the Miwhj + genotype; all eighteen animals showing this abnormality were carrying the white
gene. This finding acquires significance when it is remembered that white is a
major spotting gene, and the type 2 abnormality bears a close resemblance to
the effects of the piebald-lethal gene as shown in Plate 1, figs. B and C, and
Plate 2, fig. M.
Another mutant in which both the inner ear and pigmentation are affected is
pallid (Lyon, 1951, 1953). The otoliths in the saccular and utricular maculae in
pallid mice are frequently reduced or absent, either unilaterally or bilaterally. As
the otoliths are believed to be secreted by the macula itself (Lyon, 1955; Deol &
Lane, 1966), this suggests some deficiency of the neural epithelium, which in
turn may implicate the ganglion cells innervating the saccule and the utricle. It
is significant that in piebald-lethal animals also the otoliths were frequently
affected, unilaterally as well as bilaterally, although the result was not always
their reduction or absence. If this view of the effects of the pallid gene is correct,
the correlation between the absence of the otoliths and the tilted position of the
head (Lyon, 1951) takes on a somewhat different complexion. It is possible that
the head is tilted to one side not because the otoliths are asymmetrically absent,
but because the maculae, and possibly also the ganglia, are asymmetrically
abnormal. This interpretation is more compatible with Lyon's (1951) observation that pallid mice with asymmetrical otoliths hold the affected side higher,
whereas in animals with experimentally eliminated labyrinth it is the normal side
that is held higher. If in both cases it was just a question of presence or absence of
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M. S. DEOL
something the tilt would be expected to occur in the same direction. But if in the
pallid animal there is an abnormality of the neural epithelium, and possibly also
of the ganglion, which constitutes a change in function, then the tilt may well
occur in a direction different from that resulting from a unilaterally eliminated
labyrinth.
Another animal in which an inherited defect of pigmentation is associated with
abnormalities of the inner ear is the cat. Bosher & Hallpike (1965) have reviewed
the literature on the subject and given an account of the abnormalities. They
occur only in the cochlea and the saccule, and in many ways are similar to those
observed in s1^1 (and also Miwh/+) mice. The main difference is that whereas
the inner ear was affected in all s1^1 mice examined, it was apparently normal in
about 20 % of white cats. This difference becomes less significant when it is remembered that in several s1^1 animals normal and affected areas occured side
by side. It seems therefore that in the cat also the neural crest plays a part in the
formation of the acoustic ganglion. The occurrence of deafness in piebald or
white bull terriers and fox terriers (Wright, 1918) suggests that the same may be
true of the dog.
An essential difference between albinism and white spotting is of some interest here. Albino mice have melanocytes which cannot form pigment on account
of a chemical block, whereas white spots are believed to be devoid of any kind of
melanocyte (Billingham & Silvers, 1960). Since the coat of the s1^1 mouse in
this respect is just one large white spot, it would seem that the s1 gene has
resulted in the elimination of all melanocytes. However, the ganglion cells,
although deficient in some ways, are present in fairly normal numbers, at least
in the spiral ganglion region. This apparent discrepancy may simply mean that
the gene affects the two derivatives of the neural crest in different ways, or it may
mean that placodal and neural crest cells are mixed throughout the acoustic
ganglion, and what we see are cells of placodal origin. There is another possibility which should also be taken into consideration. The concept of the white
spot as an area devoid of melanocytes is based on the fact that no cells resembling
the clear melanocytes of albino animals are found in it. But if the shape of the
melanocyte were to be radically affected at the same time as its pigment-forming
properties the result may be the same, for the identification of the clear melanocyte is largely dependent on its characteristic shape. It is possible that something
like this happens in the piebald-lethal mouse, and the melanocytes are in fact
present in the coat, but so altered as to be unidentifiable.
Besides the neural epithelium the only other structure in the inner ear that was
always affected in the piebald-lethal mouse was the stria vascularis. This indicates that the same factors govern the development of both of them, a conclusion
reached in an earlier study (Deol, 1966) on the basis of completely different
considerations. The fact that whereas the macula of the saccule was fairly
uniformly affected the hair cells in the organ of Corti could be normal in one part
of the cochlea and abnormal in another suggests that the developmental
Neural crest and acoustic ganglion
539
relationship between the ganglion cells and neural epithelium is different in the
saccule and the organ of Corti.
This study has [some bearing on the manner of gene action in the circling
mutants of the mouse (Deol, 1966). The abnormalities of the inner ear in many
piebald-lethal animals are comparable in their extent and severity to those seen
in some of the circling mutants, but they do not cause circling behaviour. This
supports an earlier conclusion (Deol, 1966) that the cause of the circling behaviour does not lie in the inner ear. The presence of long stretches of histologically and functionally normal hair cells in Corti's organ when the whole of
the stria vascularis is clearly abnormal indicates that in the circling mutants the
abnormalities of the hair cells cannot be consequent on those of the stria
vascularis.
SUMMARY
1. There is no agreement about the origin of the acoustic ganglion. Some
investigators maintain that it originates in the neural crest, and others that it
arises from the otic placode. It was thought that mutant genes might help in
resolving the doubt.
2. The gene piebald-lethal (s1) affecting pigmentation in the mouse was
selected for this purpose. There was strong evidence that the piebald locus
influenced the development of the neural crest before its differentiation into
melanoblasts and ganglionic primordia.
3. Histological examination revealed striking abnormalities in the inner ear of
all piebald-lethal mice. The neural epithelium was invariably affected. In view
of the known action of the piebald locus on the neural crest and the intimate
developmental relationship between the acoustic ganglion and the neural epithelium, the most plausible explanation of these abnormalities is that the neural
crest contributes to the formation of the acoustic ganglion in the mouse.
4. This conclusion is supported by studies on two other mutants (white and
pallid) in which defects of pigmentation and the inner ear occur together. The
presence of similar abnormalities, either observed or deduced from deafness, in
the inner ear of white cats and piebald dogs suggests that the acoustic ganglion
in these animals has a similar origin.
RESUME
La crete neurale et le ganglion acoustique
1. L'origine des ganglions acoustiques est controversee. Certains chercheurs
soutiennent qu'ils proviennent des cretes neurales, d'autres qu'ils derivent de la
placode otique. On pense que l'etude de certains mutants peut aider a resoudre
cette alternative.
2. Le gene 'piebald-lethal' (s1) affectant la pigmentation de la souris est
choisi dans ce but. II y a de bons arguments pour penser que le locus 'piebald'
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M. S. DEOL
influence le developpement des cretes neurales avant leur differentiation en
melanoblastes et en primordia ganglionnaires.
3. L'examen histologique revele des malformations importantes de l'oreille
interne de toutes les souris 'piebald-lethal'. L'epithelium neural est toujours
affecte. Connaissant Faction du locus 'piebald' sur les cretes neurales et les
relations etroites entre les developpements des ganglions acoustiques et de
l'epithelium neural, l'explication la plus plausible de ces malformations est que
les cretes neurales contribuent a la formation de ganglion acoustique chez
la souris.
4. Cette conclusion est confirmee par des etudes sur deux autres mutants
('white' et 'pallid') dans lesquels des alterations de la pigmentation et de l'oreille
interne apparaissent conjointement. La presence de malformations analogues,
soit observees, soit deduites de la surdite, dans l'oreille interne de chats ' white'
et de chiens 'piebald', suggere que le ganglion acoustique, chez ces animaux, a
aussi pour origine les cretes neurales.
The author is greatly indebted to Mrs Priscilla W. Lane of The Jackson Laboratory, Bar
Harbor, Maine, U.S.A., for supplying the animals used in this study.
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{Manuscript received 1 February 1967)