/ . Embryol. exp. Morph. Vol. 31, 1, pp. 123-137, 1974
Printed in Great Britain
123
The retinotectal projection from a double-ventral
compound eye in Xenopus laevis
By K. STRAZNICKY, 1 R. M. GAZE 2 AND M. J. KEATING 2
From the National Institute for Medical Research, London, and the
Department of Physiology, University of Edinburgh
SUMMARY
The retinotectal projection was mapped in 22 post-metamorphic Xenopus in which the
eye under investigation had been made double-ventral by operation at stage 32. The contralateral retinotectal projection from a double-ventral eye is neither normal nor does it show
the type of abnormality predicted from previous work on double-nasal and double-temporal
eyes. In the case of double-ventral eyes, the nasal part of the field projection tended to be
reduplicated about the horizontal midline and those field positions corresponding to lateromedial rows of electrode positions on the tectum ran ventrodorsally in the field. As the
electrode rows on the tectum progressed more caudally, so the corresponding rows of
stimulus positions in the field tended to curl in a temporal direction. These observations
have been interpreted as indicating that the nasotemporal and dorsoventral polarities of the
eye are not irreversibly determined at stage 32 and that the mechanisms generating the
nasotemporal and dorsoventral axes of the eye may interact with each other.
INTRODUCTION
In a normal Xenopus there is a well-ordered fibre projection from the retinal
ganglion cells to the contralateral optic tectum. The ordering of synaptic
connexions in this projection is believed to reflect processes of axial polarization
of the retina (Sperry, 1943; Stone, 1944; Szekely, 1954, Jacobson, 1968) and
what has been called the 'specification' of retinal ganglion cells. Some insights
into these processes may be gained by observing the effects of surgical interference with the developing eye.
In Xenopus with one 'compound eye' (Gaze, Jacobson & Szekely, 1963;
1965; Gaze, Keating, Szekely & Beazley, 1970) made up of two nasal (NN)
or two temporal (TT) half-retinae, the projection pattern differs from that seen
in the normal animal. The projection from each half of the 'compound retina'
spreads out evenly to cover the entire dorsal surface of the tectum, instead of
being restricted to the rostral (TT eye) or caudal (NN eye) half of the tectum,
as might be expected from the normal pattern. Although it is important to
1
Present address: Department of Anatomy, School of Medicine, The University of Zambia,
P.O. Box RW110, Lusaka, Zambia.
2
Authors' address: National Institute for Medical Research, London NW7 1AA, U.K.
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K. STRAZNICKY, R. M. GAZE AND M.J.KEATING
account for these findings an unequivocal interpretation has not yet been possible
(Straznicky, Gaze & Keating, 1971).
It has been inferred from earlier experiments that the nasotemporal and
dorsoventral retinal axes become polarized at separate times during development (Szekely, 1954; Jacobson, 1968). This led us to hope that some light might
be shed on the problem of the specification of retinal ganglion cells by investigating the projections formed by other varieties of compound eye. Accordingly
we constructed compound eyes made up from two ventral halves or two dorsal
halves in Xenopus embryos. The present paper reports on the nature of the
retinotectal projection from these eyes. An abstract of some of this work has
been published elsewhere (Gaze, Keating & Straznicky, 1971) and a short
discussion of parts of it was included in another work (Gaze, 1970).
METHODS
The animals used were Xenopus laevis, bred in the laboratory and staged
according to the normal tables of Nieuwkoop & Faber (1956). After operation
the animals were reared at 20 °C and were fed, while tadpoles, on strained
Heinz baby soup (beef and liver) and, after metamorphosis, on chopped heart
or liver.
The operations to produce double-ventral (VV) eyes were performed in
Holtfreter's solution with minimal addition of MS222 (Tricaine methanesulphonate, Sandoz) to prevent movement of the animal. Two embryos at
stage 32 were placed head-to-head in an operating dish. With tungsten needles
the dorsal half of one eye aniage was excised and discarded and was replaced
by the ventral half of the eye aniage of opposite laterality taken from the other
animal. In this way a VV eye was produced in which both the normal and the
transplanted ventral half-retina had normal nasotemporal polarity. In a
comparable fashion double-dorsal (DD) eyes were produced.
Operated animals were raised through metamorphosis and, when they had
reached a body length of 3-5 cm, were used for electrophysiological analysis
of the retinotectal projection. The techniques used for this procedure have
been described in previous papers (Gaze et al. 1963, 1970). At the end of the
recording experiment the head of the animal was fixed in Susa fixative and
15/im sections, stained by Holmes' silver method, were prepared for histological analysis.
RESULTS
Both DD eyes and VV eyes developed in a fashion that appeared, by external
criteria, normal. In all DD eyes, however, the optic nerve failed to form; a
failure that may be attributed to the absence of a ventral fissure in these eyes.
DD eyes therefore did not form a retinotectal projection.
Histological examination of VV eyes shortly after operation showed that
Retinotectal projection in Xenopus
3B
Fig. 1. Double optic nerve leaving the back of the retina in a Xenopus larva which
had been given a double ventral eye as described in the methods. Bar, 100/tm.
Fig. 2. Eye showing two ' ventral' fissures. This eye gave the map illustrated in Fig. 7.
Fig. 3 (A) Normal optic nerve head, adult Xenopus. Bar, 100/tm. (B) Residual
reduplication of the optic nerve head shown in an animal with a compound double
ventral eye. Bar, 100 /^m.
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K. STRAZNICKY, R. M. GAZE AND M.J.KEATING
19
IS
17
16
15
14
13
Fig. 4. Normal contralateral retinotectal projection in adult Xenopus, right visual
field to left tectum. The other diagram represents the tectum seen from above. The
midline is to the left, rostral (R) in front and caudal (C) behind. The numbers on the
diagram represent electrode positions. The lower diagram is a chart of the right
visualfieldextending from the centre of thefieldout to 100°. N, Nasal; T, temporal;
S, superior; I, inferior. The numbers on thefieldchart indicate the optimal response
positions for the corresponding electrode positions on the tectum. Figs. 5-9 use the
same conventions.
Retinotectal projection in Xenopus
127
Fig. 5. Projection from the right eye (double ventral) to the left tectum
in Xenopus VV 16.
there was, in some cases, a double optic nerve leading from the eye (Fig. 1),
the two parts of which united to form one nerve shortly after leaving the eye.
At the time of recording W eyes commonly (but not always) had two ' ventral'
fissures (Fig. 2). A second 'ventral' fissure in the dorsal half of the eye was not
seen, however, in all the animals which showed a reduplicated projection of the
nature described below. Again, at the time of recording, VV eyes often showed
residual histological evidence of the earlier operation, in the form of a double
optic nerve head (Fig. 3).
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K. STRAZNICKY, R. M. GAZE AND M.J.KEATING
31
30
29
28 27
26
25
24
23
22 2
20
19
IS
17
16 15
14
13
12
II
10
9
R
Fig. 6. Projection through the right eye (double ventral) to the left tectum
in Xenopus VV 18.
Electrophysiological mapping of the visual projection was restricted to the
exposed dorsal surface of the optic tectum. In normal animals this part of the
tectum receives input from the inferior two-thirds of the retina and thus receives
the projection of the superior two-thirds of the visual field (Fig. 4). The fibres
from the most dorsal or superior retinal regions (most ventral or inferior field)
project round the lateral edge of the tectum in a position that is not readily
accessible for electrode placing.
The projection from the W eye to its contralateral (left) optic tectum was
mapped in 22 animals. In 18 of these there was clear electrophysiological
Retinotectalprojection
30
23
16
29
22
15
2S
21
14
27
20
13
129
in Xenopus
26 25
19 18
12 11 10
Fig. 7. Projection through the right eye (double ventral) to the left tectum
in Xenopus VV 12.
evidence that the transplanted ventral retina (or retina occupying this site, dorsal
in these animals) had formed connexions with the dorsal surface of the tectum.
In the remaining four animals the projection from the VV eye only arose from
the superior visual field, i.e. from the original ventral retina.
In the first group (18 animals) the pattern obtained is illustrated in Fig. 5,
with minor modifications of this pattern being seen in some animals of the
group. With the exception of its most nasal part the entire visual field projects
to the dorsal surface of the tectum. In a normal Xenopus (Fig. 4) a lateromedial
row of tectal recording positions gives a row of corresponding field positions
that runs ventrodorsally; and the projection from a double-nasal or double9
EMB 31
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K. STRAZNICKY, R. M. GAZE AND M.J.KEATING
- N
Fig. 8. Projection through the right eye (double ventral) to the left tectum
in Xenopus VV 10.
temporal eye (Gaze et al. 1963, 1965) shows reduplication of these rows of field
positions, the tectal projection from the two halves of the retina being mirrorsymmetrical about the vertical midline. Thus for a VV eye it could have been
expected that for each lateromedial row of tectal positions there would be
two rows of field positions, running ventrodorsally in the superior field and
dorsoventrally in the inferior field, the two rows being mirror images of each
other about the horizontal meridian. Fig. 5 shows that this relatively simple
state of affairs does not obtain in the actual projection from a VV eye.
Retinotectal projection in Xenopus
c
S
131
20
Fig. 9. Projection through the right eye (double ventral) to the left tectum
in Xenopus VV 8.
In general the nasal portion of the field projection follows prediction but
the orientation of the rows of positions in the temporal field progressively
departs from the dorsoventral, so that the field rows projecting to the most
caudal tectum run in the nasotemporal direction rather than dorsoventral ly.
In addition the field projection to caudal tectal areas does not show reduplication of field positions to one tectal site. The absence of field reduplication to
the caudal one-third of the tectum is seen more clearly in Fig. 7. The sequential
arrangement of the field rows in the temporal field is such as to indicate that
9-2
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K. STRAZNICKY, R. M. GAZE AND M.J.KEATING
the original ventral retina (dorsal field) is 'predominating' over the transplanted
ventral retina. In no case did we observe the opposite effect.
Of the 18 animals in which the transplanted half-retina (or retina occupying
this dorsal position) was shown to project to the tectum, 12 gave a projection
pattern closely approximating to that in Fig. 5. The other six animals showed
a generally similar pattern with some modifications. Thus four animals did
show reduplication of field positions in the projection to more caudal tectum
(Fig. 6). Even in Fig. 6 it may be seen that the most caudal tectal row does not
show field reduplication but this is probably not significant because the expected double field positions would be so close as to be inseparable. The
remaining two animals showed an additional variation from the pattern of
Fig. 5 in that the ' cartwheeling' of field row orientation was seen not only in
the temporal field but also in the nasal field (Fig. 7). There was, however, no
sign in the nasal field of the dominance of one or other half-retina.
The second group (four animals) comprised those experiments in which the
field projection arose from only the dorsal field (original ventral retina). The
transplanted half-retina in the dorsal position did not appear to connect with
the contralateral optic tectum. Two of these four animals showed a pattern
equivalent to the projection from the dorsal field in Fig. 5. Thus in the nasal
field the rows run ventrodorsally but in the temporal field the rows curl round
to run nasotemporally (Fig. 8). The other two animals of this group showed
'cartwheeling' in both nasal and temporal fields (Fig. 9).
DISCUSSION
The response of the retinotectal system to the surgical creation of relative
size-disparities between the retina and the tectum can tell us something of the
mechanisms responsible for the ordering of neuronal connexions in this system.
Recently, therefore, there has been a number of studies using this experimental
design. Each study has produced results which are fairly consistent but the
conclusions emerging from the various studies have not been uniform. Some
experiments indicate a 'plasticity 'in the tectal site at which retinal axons
will terminate following tectal or retinal lesions, while other experiments yield
results showing a form of fixed 'place-specificity' in that retinal neurones connect at the same tectal loci as they would have done if the size disparity had not
been created. Even worse, there is no obvious reason for the partitioning of the
experimental situation into those yielding one type of result or the other. Thus
'fixed place specificities' has been the conclusion of some studies involving
retinal ablations in adult goldfish (Attardi & Sperry, 1963) and embryonic chick
(DeLong & Coulombre, 1965,1967) and after tectal lesions in post-metamorphic
Xenopus (Straznicky, 1973). The spreading, compression or translation of connexions has been described after the construction of compound double-nasal
or double-temporal eyes in Xenopus (Gaze et al. 1963, 1965), after tectal
Retinotectai projection in Xenopus
Fig. 10 (A). The retinal (not field) projection to the tectum in normal Xenopus
showing the disposition of the horizontal meridian. (B) The retinal projection to
the tectum that would be expected from a double ventral eye on the assumption that
the normal retinotopic organization was preserved, and no spreading occurred.
That part of the tectum lateral to the projection of the horizontal meridian should
give no responses as indicated by the cross-hatched area. (C) The retinal projection to the tectum as recorded in animals with a double ventral eye. The tectum
gives responses right out to the lateral edge and the projection of the horizontal
meridian is displaced laterally.
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K. STRAZNICKY, R. M. GAZE AND M.J.KEATING
lesions in adult goldfish (Gaze & Sharma, 1970; Yoon, 1971-1972; Sharma,
1972 a, b) and following retinal lesions in goldfish (Horder, 1971; Yoon, 1973).
In normal Xenopus the dorsal surface of the tectum receives input from ventral
retina (dorsal field) and a considerable part of the dorsal retina (ventral field).
The tectal projection of the horizontal meridian of the field therefore runs
rostrocaudally across the tectal surface, some distance (several hundred microns)
from the lateral edge (Fig. 10 A). If the construction of a VV eye results in a
compound eye possessing neuronal specificities found normally only in ventral
retina and if the projection from such a VV eye preserves the topography of the
normal projection, then those tectal positions lateral to the normal projection of
the horizontal meridian should give no response (Fig. 10B). In fact the projections from a VV eye in all cases extends right up to the most lateral part of
the tectum, with an accompanying displacement laterally of the projection of
the horizontal meridian (Fig. IOC). It seems, therefore, that the half-retinae
deriving from both the original and transplanted ventral retinal primordia (or
retinal tissue occupying the latter dorsal position, whatever its origin) have
spread their connexions across a greater extent of the tectum than would
normally be innervated by ventral retina. We do not know the nature of the
field projection to the most infero-lateral aspects of the tectum because this
part of the brain is inaccessible to the electrode under our conditions of experiment. We can say, however, that the 'spreading' of the projection in the case of
a VV eye is non-linear in that there is a much expanded representation of the
central field compared with the peripheral field (Figs. 7-9). Since there are no
obvious consistent gaps in the visual field projection to the tectum in these
experimental animals we would guess that, if there is indeed a retinal input to
the inaccessible lateral tectal areas, it would also arise from the central retina,
which in VV eyes appropriates an increased tectal area.
In NN and TT eyes the spreading of the projection from each half-retina was
distributed evenly along the rostrocaudal tectal axis (Gaze et ah 1963, 1965).
We have discussed this phenomenon in detail in a previous paper (Straznicky
et al. 1971) in which we considered three possible mechanisms underlying the
spreading. We argued that the spreading was more likely to be due to a plasticity
in fibre connexions than to some form of regulation of the embryonic retina
whereby each half-retina somehow reconstitutes the range of neuronal specificity
found in a normal eye. We concluded, therefore, that each half-retinal anlage
generated, during growth subsequent to the operation, retinal ganglion cells
with neuronal specificities appropriate only to that half-retina. Surgical manipulation of retinal fragments at stage 32 did not appear, in the cases of NN and
TT eyes, to alter radically the specificities of the fragments eventually produced.
The 'cartwheeling' effect in the rows of temporal (and sometimes nasal) field
positions in W projections was a completely unexpected finding in the present
experiments and it must cast some doubt on the validity of our earlier conclusions. This cartwheeling effect is sufficiently different from the expected pattern
Retinotectalprojection
in Xenopus
135
to warrant a critical examination of the hypotheses on which our predictions
were based. The pattern we expected to find showed vertically running rows
of field positions across the visual field, arranged in a mirror-reduplication
about the horizontal meridian. This prediction was based on the following
three assumptions: first, that the polarization of the retina is about two orthogonal axes, nasotemporal and dorsoventral; secondly, that the mechanisms
responsible for this axial polarization operate independently about the two
axes; and thirdly, that the surgical operation on the developing retina, since it
takes place after the establishment of retinal polarity, does not alter the polarity
of the fragments of the reconstituted eye.
Goodwin (1971) has carried out a theoretical analysis of some of the results
reported in this paper, in fact concentrating on the type of pattern seen in Fig. 5.
He concluded that the pattern was explicable if one postulated that the two
retinal axes were not truly orthogonal, because of an interaction between the
axis-generating mechanisms. This conclusion is plausible; it does not, however,
account for the apparent predominance of the original ventral retina over the
transplanted ventral (now dorsal) retina described in the results. Goodwin's
predictions display mirror symmetry about the horizontal meridian and this
symmetry was not usually observed in the projection from the temporal field
to the caudal tectum. It is interesting to note that the pattern predicted by
Goodwin for the projection from a VV eye, on the basis of two non-interacting
orthogonal gradients, showed the 'cartwheeling' phenomenon in both nasal
and temporal field rows. This pattern was found in four animals (Figs. 7-9).
The reasons for this variation in results is not apparent to us.
Goodwin thus questioned two of the three assumptions described above, the
orthogonality of the axes and the independence of the mechanisms generating
the axes. If we now question the third assumption, we can say from the present
results and from the observations of Hunt & Jacobson (1973) that this assumption is certainly invalid, at least under the circumstances of these experiments.
The abnormal orientation of the field rows from the temporal field of VV eyes
indicates that the polarity of nasal retina in these eyes is different from that of
the nasal retina which would have developed from the two ventral fragments if
they had been left in situ. This alteration of the polarity of transplanted fragments in compound eyes may perhaps be shown even more dramatically in the
experiments of Hunt & Jacobson (1973). If we assume, in these compound
eye experiments, that the 'transplanted' retina did indeed derive from the
transplanted retinal primordium rather than by regeneration from the original
half-retina (such enantiomorphic twinning, as described by Harrison (1921) in
limb-bud development, has been seen in surgically produced half-eyes under
comparable circumstances - Feldman & Gaze, unpublished), then the polarity of
the transplanted fragment can undergo complete reversal of both nasotemporal
and dorsoventral axes.
Thus surgical interference with the developing retina may lead to alterations
136
K. STRAZNICKY, R. M. GAZE AND M. J. KEATING
in the polarity of the retina deriving from the reconstituted fragments. This
means, unfortunately, that experiments involving the construction of compound
eyes cannot at the moment tell us much of the rules governing the formation of
retinotectal connexions. The rationale of such experiments has involved the
assumption that the operation did not interfere with the range of neuronal
specificities produced by the reconstituted fragments. We now find that not
only this assumption is unjustified, but further, we cannot even assume that the
polarity of such subsequently generated neuronal specificity will be unaltered.
If one accepts that selective neuronal connexions reflect the acquisition, during
development, of neuronal specificities and that these are imposed upon neuronal
populations by polarized fields (Sperry, 1943,1944.1945), then the value of compound eyes will lie in what they can tell us of this process of polarization. When
this latter process is more completely understood then the retinotectal projections formed by compound eyes may tell us something more definitive about
the rules by which polarized neuronal arrays interconnect. Results already
obtained indicate that the polarity of subdivisions of the retina is not irreversibly
determined at stage 32. They may also indicate that the axial polarization which
in the intact eye appeared to be laid down orthogonally and independently
(Szekely, 1954; Jacobson, 1968) are in fact neither exactly orthogonal nor
independent.
The work described in this paper was carried out in the Department of Physiology,
University of Edinburgh, during the tenure by K. Straznicky of a Wellcome Research
Fellowship.
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