/. Embryol. exp. Morph. Vol. 71, pp. 97-108, 1982
97
Printed in Great Britain © Company of Biologists Limited 1982
Visuotectal projections following
temporary transplantation of embryonic eyes
to the body in Xenopus laevis
ByN. S. MUNRO1 AND L. D. BEAZLEY1
From the Neurobiology Laboratory, Department of Psychology,
The University of Western Australia
SUMMARY
Eye anlagen of Xenopus laevis at stages 22-26 were transplanted, 180° rotated, into the
flank of similarly staged embryos and 24-36 h later were placed into the orbits of final hosts
at stages 32-36. After metamorphosis visuotectal projections were mapped electrophysiologically. In one series, eyes were normally oriented in the final host and visuotectal projections were found to be normal. A second series, in which eyes were placed in a rotated
orientation in the final host, had developed rotated projections. The results suggest that at
stage 22 the eye already contained positional information in terms of the organization of
central connections which was not overridden by cues from surrounding tissue on the flank
or in the final host orbit. Earlier experiments by Hunt and Jacobson (1972), which indicated
that body tissues impose positional information on the developing eye between stages 29 and
31, are discussed in relation to the present study.
INTRODUCTION
In the visual system, retinal ganglion cells make specific and ordered connections within the visual centres. Sperry (1951) introduced the now well known
hypothesis of neuronal specificity to explain how such precision of neural
circuitry could develop. It was suggested that ganglion cells become cytochemically specified early in development, send out axons into the brain, and
choose partner tectal cells of matching label. By analogy with ear and limb
development (Harrison, 1921), Sperry suggested that the axial determination of
the eye might be a two-stage phenomenon, occurring first in the anteroposterior
and then the dorsoventral axes.
The results of embryonic eye rotations (Jacobson, 1968) suggested that from
stage 28-31 (Nieuwkoop & Faber, 1956) periocular tissue imposed positional
information irreversibly on the eye, such that the retinal axes were aligned
with the major axes of the body. This was thought to take place first in the
anteroposterior and later in the dorsoventral axes. Later, the results of temporary
1
Authors' address: Neurobiology Laboratory, Department of Psychology, University of
Western Australia, W.A. 6009, Australia.
98
N. S. MUNRO AND L. D. BEAZLEY
Specified
at outset
Specified
on body
Normal
Rotated
Normal
Normal
Normal
Rotated
Specified
in final host
transplantation of the developing eye to the flank (Hunt & Jacobson, 1972)
were taken as evidence that polarizing information is also present on the side of
the body.
Recently, however, the studies of Jacobson and Hunt have been brought into
question by experiments in which the embryonic eyes were rotated in the orbit
(Sharma & Hollyfield, 1974, 1980; Gaze, Feldman, Cooke & Chung, 1979).
The resulting visuotectal projections indicated that from the earliest stages of
eye development the eye carried positional information which could not be
overridden by extraocular influences. The conflicting results of these recent
experiments with those of Jacobson and Hunt and their colleagues have led us
to perform an eye-to-body transplant experiment. In Xenopus laevis embryos,
eyes were transplanted, 180° rotated, on to the flanks of intermediate host
embryos and then retransplanted 24-36 h later into the orbits of final hosts.
In series 1, the second eye manipulation was to derotate the eye such that it was
placed in a normal orientation in the head, while in series 2 the eye was returned
from the flank to the head with no or only partial derotation, thus maintaining a
rotation relative to the position of the eye within the original donor. As indicated
in Figure 1, a rotated map in series 1 would indicate that information had
become stamped on the eye during its sojourn on the flank. Alternatively, a
normal map would suggest that the eye was either programmed at the time of its
original transfer to the flank or had become programmed only after it was
transplanted into the final host orbit. Series 2 would distinguish between these
possibilities since a rotated map could only be explained by the eye being
programmed at the outset. Since Gaze et al. (1979) have suggested that the use
of low ionic strength operating medium by Jacobson and co-workers may
partially explain the discrepant results, we have performed some embryonic
operations in a solution of low ionic strength. The resulting visuotectal projections were assessed electrophysiologically and the anatomy of the visual
pathways examined histologically. These results have been presented elsewhere
in abstract form (Munro & Beazley, 1981).
Temporary transplantation of embryonic eyes
99
Fig. 2. A TS head of stage-22 Xenopus embryo. The optic vesicles are evaginating
from the neural tube. The approximate level of surgical section to remove the eye
anlage is indicated by the broken line. Haematoxylin and eosin. B Side view of
stage-24 Xenopus tadpole onto which an eye enlage (<-) had been grafted at stage 22.
C TS flank of a stage-35/36 tadpole showing an eye which had been transplanted
at stage 22. Eye appears normal, with lens and with retina beginning to separate into
nuclear and plexiform layers; pigment epithelium surrounds the eye which is
embedded in ectoderm. Haematoxylin and eosin. D Side view stage-35/36 tadpole with an 180° rotated eye which was transplanted to the body at stage 22.
The transplanted eye has a dorsally situated choroid fissure and pigmentation is
progressing from the pole now ventrally placed; these features are reversed in
comparison with the unoperated eye. Scale bars represent 0-1 mm in Fig. 2 A,
005 mm in Fig. 2C and 0-5 mm in Figs 2B and D.
METHODS
Operations. Xenopus laevis embryos were obtained by induced ovulation
with gonadotrophin.
Embryos at stages 22-26 (Nieuwkoop & Faber, 1956) were removed from
their gel coats using fine forceps, and washed several times in 50% Holtfreter's
solution. They were then transferred to an operating dish and accommodated
in shallow depressions in the plasticine base. Either 100% or 10% Holtfreter's
solution containing 1:5000 MS222 (Sandoz) was used for the operating medium.
Various operating media have been used by different research groups; we chose
100% Holtfreter's as a standard solution since it has been considered by tradi-
100
N. S. MUNRO AND L. D. BEAZLEY
NORMAL XENOPUS
S
LEFT TECTUM
ROSTRAL
LATERAL I 10 9
Fig. 3. Projection of the right visual field to the left optic tectum in a normal
Xenopus mapped with the right eye centred. For each tectal position the corresponding field position is indicated by the appropriate number on the perimetric
chart representation of the visual field. N = nasal; T = temporal; S = superior;
I = inferior. Same conventions apply to Figs. 4 and 5.
tional embryologists to promote healing and was in line with studies of Sharma
& Hollyfield (1974, 1980). The eye with its overlying ectoderm was excised with
electrolytically sharpened tungsten needles and fine forceps. Fig. 2 A shows the
developing eye anlage at stage 22 and indicates the tissue included in the
transplant. In an attempt to include all elements of the eye the cuts were made
approximately at the point of junction of the eye anlage and neural tube
(Fig. 2 A). It seems likely therefore that we transplanted cells of the optic stalk
(Holt, 1980). However, some cells with eye-forming potential must have
remained since in about one third of donor embryos examined 18-20 days later,
a small but complete eye was seen in the site from which the eye primordium
had been removed.
Immediately after removal, the eye was grafted (Fig. 2B), rotated 180°, on
the ipsilateral flank of a similarly staged host embryo, from which a small flap
of ectoderm had been removed. At least thirty minutes were allowed for healing,
with a very light glass bridge being used to hold the eye gently in place. The
embryos were transferred to 50 % Holtfreter's solution for a further 30 minutes
and then into spring water. Animals were re-anaesthetized 24-36 h later at
stages 32-36. The transplanted eye was cut out, any adhering tissue carefully
removed, and finally transplanted into the ipsilateral socket of a similarly staged
embryo from which the eye had been removed. The operating solutions in all
cases corresponded to that used in the initial operation. Fig. 2D shows an
animal just prior to re-implantation of the eye into the orbit. Some of the
embryos were fixed at this point in Zenker's solution, wax embedded, serially
sectioned (10 /im), and stained with haematoxylin and eosin. All the eyes
developed normally (Fig. 2C and D). Pigmentation of the epithelial layer
develops progressively in normal animals from the dorsal part of the eye to the
Temporary transplantation of embryonic eyes
101
Table 1. Summary of results in animals with organized visuotectal projections
Rotation of eye
Animal
Series 1
Series 2
Control
XEB11
XEB14
XEB15
XEB17
XEB18
XEB29
XEB16
XEB1
XEB3
XEB23
XEB24
XEB13
XEB20
Stage eye On flank of In orbit
transplanted intermediate of final
to flank
host
host
24
24
24
24
24
25
26
22/23
24
24
25
26
24
180°
180°
180°
180°
180°
180°
180°
180°
180°
180°
180°
180°
0°
0°
0°
0°
0°
0°
0°
0°
135°
180°
320°
180°
240°
0°
projection to Site of entry
contralateral of optic nerve
tectum
to brain
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Rotated
Rotated
Rotated
Rotated
Rotated
Normal
135°
180°
320°
180°
240°
Chiasma*
Chiasmaf
Chiasmaj
—
ChiasmaJ
Chiasma*
Chiasmaf
—
Abnormalf
ChiasmaJ
Chiasmaj
Chiasma*
ChiasmaJ
* Pathway demonstrated by HRP.
f Pathway demonstrated by solochrome cyanin.
% Pathway demonstrated by dissection only.
— Not investigated.
In all animals embryonic operating medium was 100% Holtfreter's solution, except
XEB16 and XEB29 in which 10% Holtfreter's solution was used. All rotations refer to a
clockwise direction.
ventrally placed choroid fissure. In eyes rotated 180° on the flank, the wave of
pigmentation proceeded from ventral to the developing choroid fissure, now
dorsally placed with reference to the body axes.
Animals to be reared to maturity were kept at 22 ± 2 °C on a 12 h light/dark
cycle and fed daily with a mixture of spinach and soya bean powders. Following
metamorphosis animals were given chopped liver twice weekly. .
Electrophysiological mapping. Within three months of metamorphosis
animals were set up for electrophysiological mapping using standard techniques
(Beazley, 1975). To summarize the mapping procedure, frogs were anaesthetized
with ether and immobilized with subcutaneous injections of 0-0025 mg alloferin.
Skin was reflected and a portion of skull and outer meninges were removed to
expose the tecta. The animal was then positioned at the centre of an Aimark
visual perimeter with the optic axis of the operated eye coincident with the
fixation point. Electrical activity on the tectal surface was recorded with a
tungsten electrode (tip diameter 1-3 /im) in response to small black discs moved
slowly in the visual field. Action potentials were amplified and displayed on an
oscilloscope and relayed over a loudspeaker. Recordings were made serially
from tectal loci on a 200 fim grid and the projection of the operated eye to its
102
N. S. MUNRO AND L. D. BEAZLEY
XEB 16
(10% HOLTFRETERS)
RIGHT TECTUM
ROSTRAL
Fig. 4. XEB16, in which the left eye anlage was transplanted 180° rotated to the
body at stage 26 and placed with 0° rotation in the orbit of the final host at stage
35/36. Embryonic operating medium was 10% Holtfreter's solution. The map is
essentially normal with a duplication of responses to tectal position 6.
XEB I
LEFT TECTUM
ROSTRAL
LATERAL I
6
?IGHT VISUAL FIELD^
r
Fig. 5. XEB1, in which right eye anlage was transplanted 180° rotated to the body
at stage 22/23 and replaced 135° clockwise rotated in orbit of the final host at
stage 35/36. Embryonic operating medium was 100% Holtfreter's solution. The
map is 135° clockwise rotated.
contralateral tectum was mapped. Care was taken to ensure the unoperated eye
was covered throughout. After mapping, the skull was replaced and the skin
held in position with isobutyl cyanoacrylate in those animals to be used for
axonal transport studies.
Histology. (1) Horseradish peroxidase (HRP). Either before recovery from
mapping or 24 h later under MS222 anaesthesia the experimental eye was
enucleated and a 25 % solution of HRP in phosphate buffer (0-067 M, pH 7-4)
applied to the cut end of the optic nerve within the orbit. After 48-96 h brains
were removed and processed histochemically for HRP localization using the
Temporary transplantation of embryonic eyes
103
Fig. 6. A-C TS through brain of XEB11, in which the right eye was transplanted
180° rotated to the body at stage 24 and placed in the orbit of the final host with 0°
rotation at stage 35/36. The visuotectal projection was normal. Optic pathways of the
experimental eye are demonstrated by HRP transport. Stain: Cresyl violet. A At the
level of the anterior diencephalon, fibres ascend the lateral margin of the optic
tract bilaterally and terminate in the neuropil of Bellonci, contralaterally (<-) and
ipsilaterally (—). B Immediately caudal to the optic chiasma: Fibres ascend the
contralateral optic tract en route to the tectum. Neuropils of Bellonci are labelled
as in Fig. 6 A. C Lateral edge of left tectum and tegmentum showing termination
zones in the contralateral tectum (—) and contralateral basal optic nucleus (<-).
Scale bars represent 0-25 mm in Fig. 6 A, B; 01 mm in C.
para-phenylenediamine/pyrocatechol method (Hanker, Yates, Metz & Rustioni,
1977). They were then wax embedded, serially sectioned (10/*m) and lightly
stained with cresyl violet.
(2) In other animals, heads were fixed in buffered formalin, wax embedded,
serially sectioned (10 /on) and stained with cresyl violet and solochrome cyanin.
RESULTS
Electrophysiology. Ten normal animals were mapped and a representative
projection from the right eye (centred) to the left tectum is shown in Figure 3.
The projection is essentially uniform in that equal steps of electrode position in
either the rostrocaudal or mediolateral axes result in approximately equal shifts
in the location of the maximal visual response. Nasal, temporal, superior, and
inferior fields project to rostral, caudal, medial, and lateral areas respectively.
Visuotopically ordered projections were found in 13 experimental animals
(summarized in Table 1). Seven of these animals were in series 1, having an eye
that had been rotated 180° while on the body but derotated and placed in a
normal orientation in the head. The resultant maps were normal in every case,
including two animals operated in 10% Holtfreter solution (Fig. 4). In five
104
N. S. MUNRO AND L. D. BEAZLEY
animals, constituting series 2, the eye had been placed 180° rotated on the body
and at various orientations (135°, 180°, 240°, and 320°) relative to normal in the
final host orbit. The orientation of the map always corresponded to the final
orientation of the eye within the head (Fig. 5). The remaining animal which had
developed a retinotopic map, Xenopus XEB20, served as a control, the eye being
placed unrotated on the body and in the orbit. A normal map was recorded.
In a further four experimental animals maps were disorganized, consistent
with extensive retinal ganglion cell death (Beazley, 1981), and in eight others no
projection was recordable.
Anatomy. In animals which had developed retinotopically ordered projections, eyes appeared normal macroscopically and microscopically. Although
optic nerves on the experimental side often took tortuous routes through the
extraocular muscles, in every case examined except XEB3, they entered the
brain via the chiasma. Animals with transported HRP showed that optic nerve
fibres then took a normal route (Steedman, Stirling & Gaze, 1979) to reach the
contralateral tectum and basal optic nucleus, and the neuropil of Bellonci
bilaterally (Fig. 6 A, BandC).
DISCUSSION
In this experiment we have temporarily transplanted Xenopus eye anlage
(stages 22-26), 180° rotated, to the flank of similarly staged intermediate hosts.
24-36 h later at stages 32-36, the transplanted eyes were placed in final host
orbits. This was done either after a further 180° rotation to return the eye to the
same rotation it had occupied in the original donor (series 1) or with no derotation (or only partial derotation) thus maintaining the eye in a rotated position
relative to its original position (series 2). The development of retinal specificity
in terms of the connections subsequently formed in the brain has been assessed
by electrophysiological mapping of visuotectal projections. Twelve experimental
animals which had developed retinotopically organized projections (seven in
series 1, five in series 2) are directly relevant to the question of the development
of retinal specification and subsequent discussions are restricted to them. All in
series 1 gave normal maps, a finding compatible with the eye already containing
positional information at the time of its initial transfer to the body and remaining
uninfluenced by its sojourn there. The result is not compatible with the eye being
programmed on the flank since, had this happened, the map would have been
rotated (see Fig. 1). However, the finding of a normal map leaves open the
possibility that the eye might have been reprogrammed while in the final host
orbit. This can be ruled out however by series 2 in which all animals had developed rotated maps, a result explicable by the eye being programmed before
initial surgery. Had the eye been programmed either on the flank or in the final
host normal maps would have formed. These two experimental series, taken
together, therefore suggest that at the time of initial transfer (stages 22-26) the
Temporary transplantation of embryonic eyes
105
eye already contained positional information Which could not be overridden by
body cues.
This conclusion is in accord with the results of eye rotation experiments, but
without temporary displacement of the eye, in various amphibia including the
Pipid Xenopus laevis (Gaze et al. 1979•; Sharma & Hollyfield, 1980), the Ranid,
Ranapipiens (Sharma & Hollyfield, 1974) and the Leptodactylid, Limnodynastes
dorsalis (Humphrey, Dunlop & Beazley, 1980). All these studies were consistent
with the developmental programme of the eye being intrinsically programmed
and not subject to repolarization by surrounding periocular tissue.
Such conclusions are however in sharp contrast to earlier studies of Hunt and
Jacobson (reviewed by Jacobson, 1978) using experimental paradigms such as
eye rotation (Jacobson, 1968) and temporary transplantation of the eye to the
flank (Hunt & Jacobson, 1972). They suggested that cellular specificities remain
labile in the eye until stage 28 in Xenopus and then became irrevocably stamped
on the eye between stages 29-31 by surrounding tissue, these cues being available
not only in periocular tissue but also on the flank. Gaze et al. (1979) have
suggested that use of operating media of low ionic strength by the Hunt and
Jacobson group was at least a partial explanation of the discrepant eye rotation
results. Evidence from grafts between albino and pigmented animals was
presented which clearly showed that such media could lead to death of tissue in
the operated eye and regrowth in part or whole from unoperated underlying
tissue which would necessarily be unrotated. As these authors point out this
suggestion cannot account for all the eye-to-body transplant results of Hunt and
Jacobson since there is no underlying tissue with eye-forming potential on the
flank. The evidence for specification of the eye on the flank relates to two
separate eye-to-body transplant experiments. One, described in review format
by Jacobson (1978, p. 385), involved temporary transplantation of eyes at stage
28 to the ipsilateral flank of similarly staged embryos with 180° rotation and
reimplantation later into final host orbits at stages 31/32 with a further 180°
rotation. The projections were reported to be 180° rotated, suggesting that the
eye had become specified on the flank. The experiment is essentially similar to
series 1 of the present study yet we consistently recorded normal maps. However,
there would appear to be a discrepancy between the review (Jacobson, 1978,
p. 385) and the original experiment to which it relates (Hunt & Jacobson, 1972).
The full paper did not include the experimental series shown as III in the review.
Rather, the eye rotation performed in 1972 involved long-term transplants of
eyes to the body at stages 31/32 (not 28) since the aim was to investigate the
retention of specificity by cells deprived of tectal tissue. The transplants were
thus performed too late to be relevant to the question of the acquisition of specificity. In the absence of a full description of the eye-to-body rotation experiment
shown in the review, the evidence concerning specificity of eyes on the body
therefore rests with the other experimental paradigm, that of contralateral eye
transposition.
106
N. S. MUNRO AND L. D. BEAZLEY
In such an experiment (Hunt & Jacobson, 1972), at stage 28 an eye was placed
on the opposite side of the body of a similarly staged intermediate host, thus
necessarily reversing the nasotemporal axis of the eye relative to body axes,
and later at stages 32-34 the eye was placed in the orbit of the final host on the
same side it had occupied in the intermediate host. Had the eye remained
unaffected by body cues while on the flank the resultant map would have been
nasotemporally inverted. In the three animals successfully recorded by Hunt
and Jacobson, normal maps had formed, suggesting that the nasotemporal axis
of the eye had become respecified on the body of the intermediate host. Another
explanation of this result, however, is that tissue of the underlying optic stalk in
the final host contributed cells of normal specificity to the eye and responses
were mapped only from that region. To exclude this possibility a parallel
experiment, not performed by Hunt and Jacobson, is required in which after
transfer of the eye to the contralateral flank of an intermediate host, the eye is
later placed in a final host orbit on the same side as that from which the eye
originated. A map reversed in the nasotemporal axis would provide positive
evidence that the eye had acquired its specificities while on the flank. However, a
series of experiments (Feldman, Gaze & Keating, personal communication)
involving this and other contralateral eye transposition experiments supported
the concept that the eye was not specified while on the flank but was irrevocably
programmed at the time of initial transfer. Nevertheless, there may be other
explanations for the apparently discordant results between the present experiment and those of Hunt & Jacobson (1972). One could be differences in the
extent of healing between tissue of the transplanted eye and its surrounding
flank. Another is the length of optic stalk attached to the eye when transplanted.
However, the uniformity of our results despite the unavoidable variation between
surgery on individual animals, the use of animals at several developmental
stages (22-26) and the choice of two operating media at widely different ionic
strengths might argue against these possibilities.
It is interesting that one experimental series by Hunt & Jacobson (1973) does
give results compatible with this study. They found that when the eye was
placed onto the ventral midline it was not reprogrammed. They considered that
at this site axial cues would not have influenced the eye. However, we interpret
the results differently. Although the body cues along the dorsoventral axis would
be complex at this site, the anteroposterior cues would operate normally, thus,
using their argument, we would have expected a map rotated in the anteroposterior axis.
Our present results also suggest that morphological features such as the
position of the choroid fissure and the pattern of eye pigmentation are specified
in the early eye vesicle and are not influenced by temporary transposition in a
rotated position to the body. Indeed, these features seemed inseparably linked
to the development of retinal specificity in terms of the central organization of
connections. Recent eye rotation experiments (Beach & Jacobson, 1979) also
Temporary transplantation of embryonic eyes
107
support the concept that programming of choroid fissure development is
related to events within the eye itself and is not influenced by surrounding tissue.
Other studies involving embryonic, eye rotation have reported the formation of
double choroid fissures in some animals (Sharma & Hollyfield, 1980) and the
development of 'geological faulting', a misalignment of retinal layers (Gaze
et al. 1979). The contribution of underlying tissue to eyes rotated in the orbit
has been convincingly demonstrated to be the explanation for the development
of such abnormalities (Gaze et al. 1979).
We also found that, with one exception, the optic nerves of experimental eyes
entered the brain along the normal route (optic chiasma and optic tract),
whereas Gaze et al. (1979) reported that optic nerves often entered the brain at
abnormal sites after eye rotation at stages 21/22-30. Our relatively late transfer
of the eye (stages 32-36) to replace an existing eye, which would have already
sent out axons (Grant & Rubin, 1980) and thereby may have provided a guide
for fibres, possibly explains the conservative nature of the optic nerve trajectories.
In summary, the present experiment suggests that at early stages of eye
development (stages 22-26) the retina contains information in terms of the
specific neural connections to be made in the brain. This information was not
overridden by cues from surrounding tissue while the eye was temporarily
transplanted to the body, as has been suggested by an earlier study of Hunt &
Jacobson (1972). Rather the results are in accord with more recent eye rotation
experiments (Sharma & Hollyfield, 1974, 1980) and Gaze and his coworkers
(Gaze et al. 1979) that the eye is irrevocably specified from very early in its
development.
L. D. Beazley is a Senior Research Fellow, National Health and Medical Research Council,
Australia. The research was supported by an NH & MRC Australia Grant No. 77/2087,
and the Muscular Dystrophy Research Association of Western Australia. We are grateful
to Drs A. H. Lamb and S. A. Dunlop for helpful comments on the manuscript. We thank
Mr Peter Meyer and Mr Owen Walker for skilled laboratory assistance, and Mr Herb
Jurkiewicz for invaluable help with the photography.
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{Received 13 November 1981, revised 16 March 1982)