/. Embryol. exp. Morph. Vol. 53, pp. 39-66, 1979
Printed in Great Britain © Company of Biologists Limited 1979
39
The orientation of the visuotectal map in Xenopus:
developmental aspects
By R. M. GAZE, 1 JOAN D. FELDMAN, 2 J. COOKE 1
AND S-H. CHUNG 1
This paper is dedicated to the memory of Mai tin Pristige
SUMMARY
Rotations and translocations of the eye anlage were performed in Xenopus embryos of
stages ranging from 21/22 to 30. Some of the operations involved grafting wild-type eye
anlagen into albino host orbits. Operations were performed under a variety of operating
media and conditions. In later larval life, or after metamorphosis, the visuotectal maps
from the operated eyes were recorded electrophysiologically. Results fell into two classes.
In the majority, the orientation of the visuotopic map corresponded to the orientation of
the eye at the time of recording. In the minority the visuotopic maps were 'compound',
consisting of two parts each with its own independent orientation. The organization of the
compound maps was such that one component was oriented in correspondence with the
orientation of the eye, while the other component was normally oriented. Histological
analysis and observations on genetically marked grafts indicated that the component parts
of the compound eye were of dual cellular origin. The component giving the rotated (or
translocated) map belonged to the originally operated eye tissue; whereas the component
giving the normally oriented map was derived from newly grown eye tissue coming from
the optic stalk. In no case was a normally oriented map obtained from a rotated or translocated eye. The results are discussed in relation to mechanisms proposed to account for
the determination of map-related retinal specificity.
INTRODUCTION
In amphibians, visually guided behaviour, such as the ability to locate and
catch small prey-objects in the visual field, is dependent on the existence of an
orderly map of the retina, formed by terminals of the optic nerve fibres, on
the surface of the optic tectum. The visual world is mapped optically onto the
retina and this map is then transferred, via the optic nerve fibres, to the surface
of the main visual centre of the brain, the optic tectum. In normal animals the
fibre projection from retina to tectum is so arranged that temporal retina sends
fibres to rostral tectum, nasal retina to caudal tectum, dorsal retina to lateral
tectum and ventral retina to medial tectum. Thus, because of the optical
inversion of the image in the eye, nasal visual field maps rostrally on the
1
Authors' address: National Institute for Medical Research, The Ridgeway, Mill Hill,
London NW7 1AA, England.
2
Author's address: Department of Zoology, University College, London, Gower Street,
London, WC1E 6BT, England.
40
R. M. GAZE AND OTHERS
c
c
Fig. 1. The orientation of the visuotopic map on the tectum in a normal Xenopus
(left) and in an animal with a 180° rotated eye (right). The bottom diagrams
represent the left visual field and the crossed arrows indicate the naso-temporal
and dorso-ventral meridians. The middle diagrams represent the left retina and
show how the field meridians are optically reversed. Notice that in the rotated retina
(right diagram) the visualfieldmeridians are now oriented differently in comparison
with the normal retina (left diagram). The top diagrams represent the right optic
tectum and show (left diagram) the orientation of the normal visuotectal map and
(right diagram) the rotated orientation of the map after eye rotation.
tectum, temporal field caudally, dorsal field maps medially and ventral field
maps laterally (Fig. 1).
In this paper we are concerned with the orientation of the visuotopic map
on the tectum (and thus, of the retino-tectal fibre projection) and when this
becomes determined during development. If an eye is rotated by 180° about
the (future) optic axis in a Xenopus embryo of stage 32 (Nieuwkoop & Faber,
1956), before any nerve connexions have formed between the developing eye
and brain, the visuotopic map that develops later is comparably rotated
(Fig. 1). Thus the mapping orientation between retina and tectum is already
determined by stage 32. It has been reported (Jacobson, 1967, 1968) that the
eye anlagen in Xenopus embryos are undetermined, with respect to the later
orientation of the visuotopic map, up to stage 28; that the anlage is polarized
along the naso-temporal (or rostro-caudal) axis by stage 30; and that the eye
is completely determined in both naso-temporal and dorso-ventral axes by
Visuotectal map orientation in Xenopus
41
stage 32. Later work involving explantation of the embryonic eye into a neutral
culture environment (Hunt & Jacobson, 1973) suggested that, while the stages
at which determination occurred were as mentioned above, the eye anlage
already possessed a presumptive axial orientation by stage 22.
The developing eye in Xenopus is a disc- or cup-shaped organ and possesses
various asymmetrically situated anatomical markers, such as the ventral
choroid fissure, the shape of the iris and a graded distribution of pigmentation.
The pattern-values of the cells which generate these anatomical markers are
already determined in very early eye anlagen since, after early eye rotation,
these anatomical markers develop in rotated position. Anatomically re-oriented
structures of this sort are nevertheless reported to correlate with a normal
visuotectal map following eye rotations prior to stage 28 (Jacobson & Hunt,
1973). Thus a temporal dissociation was postulated between anatomical determination and determination of map-related cellular specificities, since the
latter appeared to remain undetermined until stage 28.
The present experiments grew out of attempts, started several years ago
(Feldman & Gaze, unpublished), to confirm the undetermined state of the
stage-28 eye in Xenopus. In these previous experiments we had been unable
to obtain any evidence that stages 28 and 29 were significant in relation to
the determination of map-related cellular specificities; and we were thus left
with an unsatisfactory conflict of results.
In the present work eye operations (mainly rotations) have been performed
at various stages from as early as stage 21/22; and in several cases genetic
tissue markers were used. These experiments lead us to conclude that the eye
anlage in Xenopus is determined, in relation to the later orientation of the
retinotectal map, from at least as early as stage 21/22 and that repolarization
of the anlage does not occur. Moreover our results suggest a reasonable interpretation of why it was formerly thought that the eye was undetermined until
stage 28. And finally, we discuss what we consider to be the significance, for
developmental neurobiology, of these results. Results of eye rudiment rotations,
essentially in accord with the present ones, have been reported by Sharma &
Hollyfield (1978) in another anuran species.
METHODS
Embryonic operations
Xenopus embryos were produced in the laboratory by induced ovulation.
Larvae were reared in 10% Steam's solution (Rugh, 1962) in large plastic
aquaria, at a temperature of approximately 20 °C and with a normal light/
dark cycle. Tadpoles were fed with strained Heinz Beef and Liver baby soup
and after metamorphosis animals were fed on Tubifex worms and, later, minced
beef heart. Animals were used for electrophysiological recording of the visuotectal projection from the operated eye either during larval life or after
metamorphosis.
42
R. M. GAZE AND OTHERS
Fig. 2. Transverse sections showing the eye anlage in a stage-24 Xenopus embryo
(A), a stage-26 embryo (B) and a stage-28 embryo (C). The heavy black line in the
top photograph outlines the tissue included in the operation. In each photograph
ventral is to the left and lateral is upwards. Embryos were embedded in Araldite,
cut at 2/tm and sections were stained with toluidine blue. Calibration for all
photographs is 100 [im (bottom photograph).
Visuotectal map orientation in Xenopus
43
Operations, performed between stages 21 and 30, involved moving the
excised eye anlage, including a small patch of overlying ectoderm with a
developing lens precursor and a very few head mesenchyme or neural crest
cells. These operations included rotation of the eye through 180° in situ (to
invert both naso-temporal and dorso-ventral axes), translocation of the eye
from right to left orbit (to invert only the naso-temporal axis) and translocation
of the eye from right to left orbit with 180° rotation (to invert only the dorsoventral axis). Figure 2 shows the eye anlagen in embryos of stages 24, 26 and
28 and indicates the cellular elements included in the operation. An attempt
was made to include all the cells of the anlage, back to the isthmus of its
junction with the brain; but some cells with the competence to form eye must
have sometimes been left in situ, since in 40% (6/15) of control operations
where excised neural tissue was not replaced, a minute but complete eye
(less than 1/3 normal diameter) was present after a week.
Operations were performed with sharpened tungsten needles and a mounted
hair loop, on embryos lying in shallow depressions in a wax bed. Variations
in a few cases involved leaving the overlying epidermis unrotated, and in some
other cases, omitting the very light glass bridge which normally holds the
eye gently in place for some 10 min after the operation.
In one group of animals a genetic cell marker was used. In these cases, the
operations involved grafting rotated wild-type eye anlagen into the prepared
orbits of albino hosts. Both donor and host were at stage 25. The albino
stock was made available by courtesy of Dr J. B. Gurdon.
Animals operated in early 20s stages were externally indistinguishable from
normally developing embryos (except for eye position and, in the case of
albino hosts, a small group of wild-type epidermal cells) by 2 h after operation.
They were then transferred to 1/10 strength saline until the onset of feeding at
5 days. Depending on the stage of operation, development from operation to
stage 28 occupied up to 15 h. Two days after operation larvae were screened
for gross maldevelopment of the eye or for de-rotation and some 10 % were
then discarded.
Operating solutions
In order to ensure that the results obtained did not depend critically on the
composition of the operating media, operations were performed, in various
cases, in the following solutions (see Rugh, 1962):
1. 66 % Niu-Twitty solution, pH 7-6.
2. 50 % Niu-Twitty solution but with calcium and magnesium reduced to
25 % strength, pH 7-4.
3.* Saline equivalent to 20 % Steinberg's solution plus 5 % Holtfreter's
solution, pH 7-4.
* We are indebted to Dr R. K. Hunt for suggesting this solution to us.
44
R. M. GAZE AND OTHERS
Electrophysiological recording of the visuotopic projections
The visuotopic maps were recorded from operated animals at various stages
of development from early stage 50s to shortly after metamorphosis.
Animals were anaesthetized by immersion in MS 222 (Tricaine methane
sulphonate, Sandoz), 1:3000 in Niu-Twitty solution. The skin and membranous
skull and meninges were removed with a tungsten needle and watch-maker's
forceps. The tecta were then photographed with a Polaroid camera at a magnification of 50 x, except in the case of albino tadpoles where the lack of
pigment made photography useless.
The animal was set up on a Plasticine plinth at the centre of a Perspex sphere
filled with oxygenated Niu-Twitty solution containing MS 222 at a concentration
of 1:15000. A hole in the top of the sphere permitted introduction of the
Niu-Twitty solution and the recording and indifferent electrode.
The sphere containing the animal was set up at the centre of an Aimark
projection perimeter with the eye under investigation directed towards the
fixation point of the perimeter arc. The eye was centred by visual estimation
and the optic nerve of the normal eye was then cut.
Recording positions on the tectum were chosen under direct vision through
a stereo microscope, in relation to positions previously marked on the tectal
photograph; or, in the case of albino animals, in relation to an outline drawing
of the tectum.
Recording electrodes were glass micro-pipettes filled with a Wood's metal
and indium mixture tipped with platinum. Tip diameter was 10-15 /«n and
impedance did not exceed 100 kQ. at 1000 Hz. Responses (mainly multi-unit)
were amplified via an FET pre-amplifier of band width 100-10000 Hz, displayed on an oscilloscope and monitored over a loudspeaker.
The microelectrode was placed serially on different positions on the tectum
by one experimenter and for each tectal position the corresponding field
position(s) was determined by a second experimenter who was ignorant of
the tectal position chosen. Responses were evoked by movement of a black
disc, subtending 10°, against an illuminated grey background. Mapping was
performed in muted room lighting. In most cases, at the end of mapping, the
position and orientation of the eye were recorded photographically.
Histology
Most animals were finally fixed in Heidenhain's Susa fixative and serial
transverse paraffin sections, cut at 15/*m, were stained by Holmes' silver
method. Embryos of various stages were fixed in half-strength Karnovsky at
pH 7-4, embedded in Araldite and cut transversely at 2 /*m. Sections were
stained with toluidine blue. In several experiments the operated eyes were
reconstituted from histological sections, with the aid of a computer.
Visuotectal map orientation in Xenopus
45
Table 1. Distribution of results according to stage at operation
Rotated
Compound
21-23
24-26
12
6
6
11
27-28
30
Fig. 3. Visuotectal map through left (operated) eye recorded just after metamorphosis. The eye had been in 180° rotated position from operation at stage 23
until the stage 40s, and had later de-rotated. Inset diagram shows the orientation
of the eye and choroid fissure at the time of recording. Even though the eye was
rotated up to the 40s stages, this map is normal and its orientation accords with
that of the fissure. In this and all other maps shown, the top diagram represents
the dorsal surface of the right tectum with the small arrow pointing rostrally
along the mid-line and the bottom diagram represents the left visual field. Numbers
on the tectal diagram indicate positions of the recording electrode and corresponding
numbers on the visual field diagram represent optimal stimulus positions. The
animal's eye is to be considered to be on the far side of the chart, looking out at
the observer through the centre of the chart. The field chart extends for 100°
outwards from the centre point. N, S, T, I: nasal, superior, temporal and inferior
field; n, d, t, v: nasal, dorsal, temporal and ventral retina. In this map the large
arrow on the visualfieldchart indicates the ordering offieldpositions corresponding
to rostro-caudal rows (large arrow) on the tectum.
4
EMB
53
46
R. M. GAZE AND OTHERS
Fig. 4
Fig. 5
Fig. 4. Visuotectal map from an animal in which the left eye was rotated at stage 23
and the map was recorded at stage 57. Both eye and map are rotated 180°. In this
map the large arrow on the visual field chart indicates the order of positions corresponding to rostro-caudal rows (large arrow) on the tectum.
Fig. 5. Visuotectal map from an animal in which the left eye was rotated at stage 23
and the map was recorded at stage 58. Both eye and map are rotated 180°. In this
map the large arrow on the visual field chart indicates the order of positions
corresponding to rostro-caudal rows (large arrow) on the tectum.
RESULTS
Electrophysiological recording was carried out on 77 operated animals, of
which 61 (79 %) had formed retino-tectal connexions. Maps were uninterpretable
in 22 animals, either because connectivity was poor and responses were hard
to obtain, showing no apparent relationship of tectal and field positions, or
because there were too few points for analysis. These cases will not be considered
further.
Interpretable maps were obtained in 39 animals (64 % of those connected)
and it is with these that the rest of this paper is concerned.
47
Visuotectal map orientation in Xenopus
11 12 1 3 1 4 15 16
5
6 7 8 9
Fig. 6
10
Fig. 7
Fig. 6. Visuotectal map from an animal in which the left eye was rotated at stage
27/28 and the map was recorded after metamorphosis. In this map the large
arrow on the visual field chart indicates the order of positions corresponding to
latero-medial rows (large arrow) on the tectum. Both eye and map are rotated by
180°.
Fig. 7. Visuotectal map from an animal in which a right eye had been translocated
into the left orbit, with 180° rotation, at stage 25. The map was recorded at stage 57.
The large arrow on the visual field chart indicates order corresponding to rostrocaudal rows (large arrow) on the tectum. The eye is inverted dorso-ventrally. The
orientation of the map corresponds to that of the eye, being inverted dorsoventrally and normal naso-temporally.
In all 39 successful cases the results of recording the visuo-tectal projections
fell into one of two categories. These are (1) rotated maps, where the orientation
of the map reflects accurately the altered orientation of the eye, and (2)
'compound' maps, where the map is made up of two partial maps with differing
orientation. In no case did we find a normally oriented map following eye
rotation or translocation. The distribution of the two categories of result,
according to stage of operation, is shown in Table 1. We found no evidence
4-2
48
R. M. GAZE AND OTHERS
that any of the variables surrounding the operation, described in the Methods
section, affected the pattern of results or led to results other than those to be
described.
Rotated maps
The larger class of visuotectal maps obtained (22/39) showed an orientation
completely in accord with the altered anatomical orientation of the eye in the
head at the time of recording, when this latter orientation was deduced from
the nature of the original operation (rotation, or translocation with or without
rotation) together with the position of the choroid fissure at the time of
recording. Figures 3-7 show five such maps and the eyes that produced them.
A variable amount of de-rotation occurred between the initial operation
and the time of mapping; as a result of screening after operation it was known
that the de-rotation had occurred well after the putative stages (29-31) of
irreversible axial determination. Figure 3, shown for comparative purposes, is
completely equivalent to the normal map from a left eye but came in fact
from an eye which had been 180° rotated until the late 40s stages and which
had subsequently de-rotated to the normal position.
Agreement between orientation of the map and that of the anatomical
markers on the eye was always within some 20°, which is the limit of resolution
of the mapping technique. In other words, both the anatomical asymmetries
and the map orientations accorded with the orientation of the eye after operation.
Histological examination of such eyes showed in most cases normal retinal
structure apart from reduplication or other abnormality of the optic nerve
head in six cases, the presence of a cellular inclusion in the inner plexiform
layer in one case, the presence of an accessory retinal structure in the form of
a rosette near the optic nerve head in two cases and localized retinal deformation
FIGURE 8
Retinal abnormalities accompanying simple, rotated maps.
(A) Reduplicated optic nerve head from an eye rotated at stage 30. The map, made
at stage 59, was 180° rotated, corresponding to the rotation of the eye. Arrows
indicate the two optic nerve heads. Calibration, 100/tm.
(B) Cellular inclusion in the inner plexiform layer of a retina from an eye which
had been rotated at stage 23 and mapped just after metamorphosis. The eye
eventually de-rotated to normal position (after stage 40) and gave a normal map
(Fig. 3). Calibration, 50 /on.
(C) Large rosette structure (arrow) behind retina and close to optic nerve head
in an animal where the eye had been rotated at stage 22. The map, made at stage
65, was 180° rotated, as was the eye. Calibration as in A.
(D) Small rosette structure (arrow) at exit of optic nerve in an animal where the
eye had been rotated at stage 27/28. The map, made after metamorphosis, was
180° rotated, as was the eye. Calibration as in A.
Visuotectal map orientation in Xenopus
49
50
R. M. GAZE AND OTHERS
17 18
19 20
12 13 14
15 16
N-
Fig. 9
Fig. 10
Fig. 9. Compound visuotectal map from an animal in which a left eye had been
rotated at stage 23 and the map recorded shortly after metamorphosis. The map
comprises two partial maps, overlapping each other in the part of the field indicated
by the solid triangle. The partial map from the lower part of thefield(with numbered
positions joined by continuous lines) is produced by the rotated part of the eye.
The partial map from upper field (with numbered positions joined by dashed lines)
has an almost normal orientation and comes from newly grown eye tissue, as
described in the text. The two partial maps have thus opposite senses, as indicated
by the large arrows in the visual field and on the tectum. The retina of this eye
showed obvious signs of its dual origin (Fig. 12 A). Despite the compound nature
of the map and of the retina, the eye shows only a single, rotated, fissure.
Fig. 10. Compound visuotectal map from an animal in which the left eye had been
rotated prior to stage 26 and the map was recorded at stage 56. The eye shows two
fissures. The dorsalfissureis the original, rotated one while the ventral fissure represents new eye tissue. The naso-dorsal temporo-ventral axis formed by these two
fissures suggests that the eye underwent some de-rotation after the newfissurehad
been formed. The map is composed of two partial maps, the orientation of which
accurately reflects the orientation of the two parts of the eye, as indicated by
the fissures. The orientation of the maps is indicated by the large arrows in the
visual field and on the tectum.
Visuotectal map orinetation in Xenopus
51
in two cases (Fig. 8). Despite the general normality of the retina the subsequent
fibre pathway between eye and tectum was often bizarre.
'Compound' maps
The second, smaller, class of visuotectal maps was of a type best described
as 'compound'. In this category, which comprised 17 out of 39 maps, the
visual field (and hence the retina) is divided into two territories, each territory
forming its own map across part or all of the tectum, but with its own independent orientation. Electrode positions, over part or all of the tectum, therefore record visual activity evoked by stimulation of two separate parts of the
retina. Figures 9-11 show three such maps and the eyes that produced them.
It is characteristic of these compound projections that one of the component
maps is organized as from normally oriented retina whereas the other map is
re-oriented as would be expected from the nature of the original operation.
There is also a tendency for whichever component of the projection comes
from the smaller portion of the retina to show abnormal 'sliding' or 'curving'
of the rows of visual field positions represented in successive straight rows of
tectal positions, and to cover the tectum less completely than the larger map.
The compound type of result for a 180° rotation is shown in Figs. 9 and 10
and that for a right/left translocation in Fig. 11.
All except two of the eyes giving these compound maps showed one or
more of three types of evidence that they are of anatomically compound origin.
The layered structure of the retina may exhibit 'geological faulting' (Fig. 12 A)
and signs of abuttment of two cell populations; there are frequently two optic
nerve heads despite the single, normal, optical system (Fig. 12B, C); and in
cases where the eye was transplanted between genotypes, genetic chimaerism
across a continuous boundary was seen in the pigment epithelium (Fig. 13F).
To external inspection, there is often one fissure in or close to normal orientation
and another more nearly corresponding to that expected from the nature of
the operation (Fig. 10), while the pigmentation on the eye may also show
a mirror image pattern that accords with the positions of the two fissures.
The map shown in Fig. 9 offers particularly striking evidence that what
appears as a single eye may be composed of two territories with quite independent sets of positional cues. Figure 9 shows that a particular region of the
retina (ie. visual field positions) must send axons to two quite separate parts
of the tectum in connexion with separate maps; the solid triangle shows a
position in the visual field which was represented at two separate tectal sites.
In this animal part of the retina was doubled, at the region where the two
parts of the compound retina meet (Fig. 12 A).
It was the histological evidence suggesting incipient physical reduplication
of the retina (reduplicated optic nerve head, 'geological faulting') observed
early in the present series of experiments, that prompted us to employ genetically
marked grafts of rotated eyes. The histological observations, together with the
52
R. M. GAZE AND OTHERS
Fig. 11A and B. For legend see opposite.
Visuotectal map orientation in Xenopus
53
orientation of the two components of the compound map, which implied
that one component came from rotated (or translocated) tissue and the other
from normally oriented tissue, suggested to us that these eyes were anatomically
compound. They appeared to comprise two sets of tissue elements, one deriving
from the operated eye itself and the other from the 'host'. It thus became
necessary to demonstrate, unequivocally, the anatomically compound nature
of the eye.
Eyes produced by grafts of ostensibly complete wild-type optic vesicles into
the empty orbits of albino hosts confirm, in a striking manner, that the mature
eyes can be of compound origin after such operations. Such eyes are shown
in Fig. 13A-D, and the existence of an extensive albino (host) component is
evident. Maps from genetically chimaeric eyes (Fig. 14) were themselves compound, with their components behaving autonomously according to presumptive
orientation (host, original; donor, re-oriented).
An interesting observation is that several animals which in later tadpole
life showed this extensive host-derived component, came from a batch of
animals where no eye showed more than a very restricted ventro-nasal host
albino segment for about 48 h after operation. There thus appears to be a
possibility of disproportionate growth by the unrotated tissue, leading to a
partial takeover of the eye. This preliminary evidence for selective growth by
unrotated cell groups, subsequent to their initial recruitment into the eye,
would suggest that the proportion of the visual world that is seen through
unrotated retina in the operated eye may increase as development proceeds.
This point is taken up in the Discussion.
The evaluation of the results of eye operations of the sort described in this
paper depends heavily on detailed histological analysis. Thus, while most
compound maps are associated with the presence of obvious secondary choroid
fissures one of the compound maps in the present series came from an eye which
showed, to external inspection, only one fissure, in rotated position (Fig. 9).
Fig. 11. (A) Compound visuotectal map from an animal in which a right eye had
been translocated (without rotation) to the left orbit at stage 24. The map was
recorded at stage 57. As would be expected from the nature of the operation the
compound nature of the map manifests itself in the naso-temporal axis but not
dorso-ventrally. The nasal half of the map is reversed naso-temporally and comes
from the originally transplanted eye. The temporal half of the map is normally
oriented and represents new eye tissue. Map orientations are indicated by large
arrows in the visual field and on the tectum. (B) A stereo pair showing a computeraided reconstruction of the eye, based on serial sections cut transversely to the
animal's main axis. The retina has two separate optic nerves. The reconstruction
has been arbitrarily subdivided such that half the retina (continuous lines) feeds
one optic nerve and the other half (dashed lines) feeds the other optic nerve. In the
plane of the sections, dorsal is to the left and ventral to the right. The nasal pole
of the eye is closer to the observer, when the figure is viewed stereoscopically. The
line forming a right angle close to the eye was for reference purposes during the
reconstruction. The two optic nerves from the eye are shown in Fig. 12.
54
R. M. GAZE AND OTHERS
Fig. 12. (A) Retinal reduplication in the eye which produced the compound map
shown in Fig. 9. The arrow indicates a region of 'geological faulting' where the
retinal layers are out of register. Calibration 100 fim. The two lower photographs
(B, C) show the two separate optic nerves leaving the eye which produced the map
shown in Fig. 11. Calibration 100 /«n for both.
Visuotectal map orientation in Xenopus
55
Accessory fissure-like structures in rotated eyes may not extend however to
the retinal margin (Goldberg, 1976a). In this animal the anatomically compound nature of the retina was obvious histologically (Fig. 12A). This experiment thus provides evidence of a partial normally oriented map coming from
an eye which was, to external inspection, apparently simply a rotated eye. The
possible significance of this will be taken up in the Discussion.
The converse situation also occurs; whilst most maps classed as rotated
came from eyes with a single, correspondingly inverted fissure, in two cases
the eyes showed secondary fissures. In one animal the map came from the
greater part of the field (Fig. 15) and the eye showed a fissure ventrally and
one in a rotated position corresponding to the map (Fig. 13C). No retinal
histology was available for this animal. In another animal the eye was rotated
by about 150° anti-clockwise, as was the map (Fig. 16). The eye showed two
blood vessels ventrally, straddling the normal position of the fissure. Some
points towards the centre of the field were reduplicated and histology showed
a reduplicated nerve head and 'geological faulting' centrally in the fundus,
suggesting that an abortive attempt at the formation of a compound retina
had been made.
Even when the map is rotated according to the position of the (single,
rotated) fissure, histological analysis can yield useful information. The map
shown in Fig. 17 appears simple. It is rotated in correspondence with the
position of the fissure, but the map is confined to the nasal half of the field.
In this animal the retina had two optic nerves, a thin one leaving the eye in
the central fundus and a thick one exiting much further temporally. It is
thus possible that this map is half of what should have been a compound
map; the other half (temporal field) is missing since nasal retina did not connect with the tectum. In another animal the map came from nasal field and
was rotated 170° clockwise (Fig. 18). The choroid fissure on the other hand
was rotated some 30° clockwise and may represent new eye tissue corresponding
to the position of the missing half-map. The dorsal surface of the eye showed
'ventral' silver pigmentation and carried a small blood vessel in the position
where the rotated fissure should have been. Two points in the temporal field
were reduplicated. Histological analysis showed retinal abnormality, with reduplication, at the optic nerve head. It is likely, therefore, that this result also
represents half of a compound map, again with nasal retina (temporal field)
mainly not connected.
DISCUSSION
We shall discuss the presented results from three points of view. First, we
derive from our data what seems to us to be a coherent interpretation of the
genesis, in the precursor tissues of the Xenopus eye, of specificity for the later
formation of tectal connexions. Next, we discuss some possible causes for the
disparity in the apparent behaviour of eye-cup specificities, in our own and
56
R. M. GAZE AND OTHERS
Visuotectal map orientation in Xenopus
57
in other hands, following ostensibly similar surgical experiments; and we
suggest a possible resolution of these differences. Finally we draw attention
to some major consequences of our own interpretation, if it should be upheld,
for theories of the genesis of selective nerve connexions.
The results of the present experiments are best accounted for by supposing
that there is no communication of information to the optic vesicle from surrounding tissue, and little subsequent intercellular interaction with respect to
polarity within eye tissue itself. We suggest that all aspects of eye patterning,
both those relating to anatomical asymmetries and those relating to orientation
of the map, are determined from very early vesicle stages or before. Our
experiments indicate that, over a wide range of operating conditions, the
developing eye does not become 'repolarized' after rotation or translocation
before stage 28. The majority class of our results shows that, from as early as
stage 21/22, eye rotation gives later a corresponding map rotation. In no
case was a normal map observed after eye rotation before stage 28.
The minority class of our results showed compound maps. The structural
observations on the eyes giving these compound visuotectal maps, both in the
main series and in the genetic chimaeras, make us confident that they are
always of dual cellular origin. The configurations of pigmented tissue in the
chimaeric eyes, and the positions of histological retinal faulting and accessory
choroid fissures in relation to the boundaries between autonomously mapping
domains in the visual field, reveal an early autonomy of positional properties
in areas of the developing eye anlage, both for anatomical marker development
and for orientation of the retino-topic neuronal projection to the tectum.
We thus believe that our compound maps come from eyes where the cell
layer that controls these aspects of pattern (whether this is actually neural
FIGURE
13
(A, B, C) Chimaeric eyes from animals in which a wild-type eye anlage was grafted,
in rotated orientation, into an albino host orbit at stage 25. Photographs were
made at stage 56 (A), stage 55 (B) and stage 54 (C). In each photograph dorsal is
upwards and nasal is to the left. Each eye shows a well-demarcated host (albino)
segment.
(D) Chimaeric eye from an animal in which a wild-type eye anlage had been transplanted, in rotated orientation, into an albino host orbit at stage 24/25. The
photograph, made at the end of mapping at stage 55, shows the eye to have a
fissure dorsally (upwards in photograph) and a restricted host (albino) segment
ventrally. The map from this animal is shown in Fig. 14.
(E) Eye which had been rotated at stage 23. The animal was mapped at stage 59.
The eye has two fissures, one ventral (downwards in photograph) and the other
naso-dorsal. The map in this case (Fig. 15) was simple and rotated by about
130° clockwise, thus corresponding in orientation to the more dorsal fissure.
(F) Section through retina of a chimaeric eye, showing the characteristic abrupt
transition (arrow) from normal wild-type pigment epithelium to albino pigment
epithelium. Calibration, 100/tm.
58
R. M. GAZE AND OTHERS
Fig. 14A and B. For legend see opposite.
Visuotectal map orientation in Xenopus
59
retina or the pigmented epithelium adjacent to it) consists of a tract of cells
descended from the originally re-oriented eye anlage and another tract descended
from those cells having remained behind in the forebrain margin at operation
and therefore in their original configuration. Whether or not the later emergence
and participation of these cells is a special regulatory phenomenon provoked
by the healing together of positionally disparate cells (French, Bryant &
Bryant, 1976), can only be answered by future grafting operations between
genotypes.
We would emphasize that in our view the two series, of rotated and compound maps, manifest a single tendency, which is for the operated eye to be
replaced, to a variable extent depending on surgical trauma, by new eyeforming cells from the optic stalk. Thus some of our autonomously behaving
rotated maps came from eyes which showed signs of incipient or abortive
reduplication; while a few of our compound maps showed little overt sign of
retinal abnormality. The simplest way of accounting for these relationships is
to assume that there is a continuous spectrum of retinal abnormality, from
minimal signs such as cellular inclusions in the inner plexiform layer to frank
'geological faulting' of the retina, and that only the more marked abnormalities
are picked up electrophysiologically.
What we never observe is a disjunction between the orientation of the
anatomical pattern in a particular part of the eye and the orientation of the
tectal map of the visual world as seen through that part of the eye. Our assumption
that all aspects of eye patterning are determined from very early vesicle stages
leaves open the possibility that no specificity lies within neural retina itself
at any stage. It could be that specificity within the pigment epithelial tissue
coming to underlie any region of the neural retina is the sole determinant of
both the connectivity properties and the anatomy of that eye region, since
this is the correlation we observe. The actual primary location of specificity
information in the developing eye is another question and awaits future
analysis.
We now draw attention to various features of experimental method which
have (in the past) been held to influence the possibility of respecification of
Fig. 14. (A) Compound visuotectal map from an animal with a chimaeric eye
(see Fig. 13D). At stage 24/25 a wild-type eye anlage was transplanted in rotated
orientation into an albino host orbit. The animal was mapped at stage 55. Dorsal
field positions 4, 5, 6 and 7 are approximately correct and would excite the narrow
strip of host (albino) retina, see Fig. 13 D and the retinal reconstructions here.
Other field positions are misplaced, with positions 1-3 and 5-9 tending to be
rotated according to the position of the choroid fissure. (B) Stereo pair showing a
computer-aided reconstruction of the eye. The segment of albino tissue is shown
by a dashed line that extends from the optic nerve head out to the ventral margin
of the eye. The lines and symbols outside the eye were for reference purposes
during the reconstruction.
60
R. M. GAZE AND OTHERS
Fig. 15
Fig. 16
Fig. 15. Visuotectal map from an animal in which the left eye had been rotated
180° at stage 23. The map was made at stage 59, by which time the eye had partly
de-rotated and showed a secondary fissure ventrally (Fig. 13E). Despite the
external appearance of a secondary fissure, the map in this case is simple and
rotated to an extent corresponding with the orientation of the rotated (nasodorsal) fissure. Orientation of the map is shown by the large arrows in the visual
field and on the tectum.
Fig. 16. Visuotectal map from an animal in which the left eye had been rotated
180° at stage 23. The map was made at stage 58. The choroid fissure points dorsotemporally and there is evidence of another fissure ventro-nasally. The map however is simple, apart from a couple of reduplicated field positions, and is rotated
in correspondence with the orientation of the rotated fissure. Orientation of the
map is indicated by the large arrows in the visual field and on the tectum.
the rotated eye by the surrounding tissues, and thus to provide possible
explanations for our failure to observe such respecification at any stage.
In a previous series of early eye rotation experiments (Feldman & Gaze,
unpublished), referred to in the Introduction, rotations were mainly performed
at stages 28-31. In these experiments no evidence was found to indicate that
stage 28 was in any way significant for the orientation of the map later recorded.
Visuotectal map orientation in Xenopus
Fig. 17
61
Fig. 18
Fig. 17. Visuotectal map from an animal in which the left eye had been rotated
170° at stage 22. The map was made at stage 55. Essentially, the map is rotated in
accord with the orientation of the fissure and is largely confined to the nasal half
of the field. Field position 4a was obtained with electrode tip deeper than at
position 4. In a normal animal, 4a would be ventral to 4 in the field, since deeper
penetration of the electrode at the lateral edge of the dorsal tectum allows the tip
to record from 'hidden' tectum which received input from ventral field.
Fig. 18. Visuotectal map from an animal in which the left eye anlage had been
rotated before stage 25. The map was made 6 months after metamorphosis. The
map is essentially rotated 170° clockwise and comes from the nasal and ventral
fields. The map thus comes from temporal and dorsal retina. The only sign of the
originally ventral nature of this part of the eye is dorsal silver pigmentation and
a small blood vessel at the dorsal pole. The fissure just nasal to the ventral pole
of the eye presumably indicates new eye tissue which in this case is largely unconnected with the tectum. Orientation of the map is indicated by large arrows
in the visual field and on the tectum.
EM IB 53
62
R. M. GAZE AND OTHERS
Since the interval between stages 28 and 31 in Xenopus is only some 5 h, it
could be argued that imprecise staging of the embryos might have been the
cause of our failure to demonstrate the previously reported eye repolarization.
It was for this reason, and to give rotated eyes plenty of time to heal into
the orbit before stage 28, that the present series of operations was extended
backwards to stage 21/22. In view of the present results, we can say that
imprecise staging, or failure of the operated eye to heal within the time required,
are not the reasons for our inability to obtain retinal respecification.
A further possible reason for the different results of eye rotations in our
own hands and in those of others, lies in the nature of the operating medium.
We have in the past, in general, used standard embryological operating solutions
such as full-strength Holtfreter or Niu-Twitty. Hunt and Jacobson, on the
other hand, have used solutions (R. K. Hunt, personal communication) of
much lower ionic strength. The earlier operations of the present series were
performed under ionic strengths of the medium equivalent only to some 50 %
Niu-Twitty with low calcium and magnesium (equivalent to 25 % Niu-Twitty
solution), while the wild-type/albino transplants were peformed using a solution
with an ionic strength equivalent to less than 25 % Niu-Twitty (see Methods).
All the present operations were transferred to TV strength saline within 2 h,
whereupon a variable period of up to 15 h ensued before the reported stages
of irreversible eye polarization.
In view of the small diffusion distances involved, the rapid healing of the
epidermis with its presumed regulatory properties, and the fact that none of
our variations in the operating solution influenced the results we describe, we
are confident that such variables are irrelevant to any cell interactions after
operation, within the limits of cell viability. We have observed, however, that
the use of low ionic strength medium, which is not one that would be employed
at operation by traditional embryologists wishing to promote graft cell survival,
causes more collapse, distortion and incipient cell death in eyecups during the
operative period than does the solution we have tended mostly to use, of the
order of half strength Niu-Twitty.
The following comments on the anatomy of the developing eyecup in Xenopus
may offer the beginning of a resolution of the apparently conflicting results
obtained in the present experiments and in previous work. The uniform
correlation we have observed is between eyecup anatomy and the orientation
of the map on the tectum. The former feature is undoubtedly determined
among vesicle cells at very early 20s stages. The only way that structures
characteristic of the ventral region of the normal eye can develop in their
normal orientation, after a rotation operation which includes the proximal zone
of the vesicle, is by de-rotation (a common feature in embryological work), or
by outgrowth of a ventral, originally oriented component. Our grafts between
wild-type and albino animals demonstrate this latter phenomenon.
From stage 28 on, the proximal epithelial wall of the eye becomes spotted
Visuotectal map orientation in Xenopus
63
with differentiating melanophores from the neural crest and is easy to see in
the microscope and to include entirely in the operation. In the early stage 20s,
when the entire vesicle is relatively thick-walled, the proximal epithelial wall
is also easy to visualize (Fig. 2); but then uncertainty as to the precise boundary
of the eye-component region within the brain-base may lead to difficulties,
Around stages 26 and 27 the inner, presumptive pigmented moiety of the eye
vesicle becomes thin and papery whereas the thick, pad-like presumptive retina,
etc., form a body resembling the entire eye vesicle of a few hours previously.
It is then easy to perform a rotation or transfer which leaves much presumptive
pigment epithelium in situ, with its capacity to direct anatomical patterns, to
give rise to new neural retinal tissue (Stone, 1949) and perhaps even to regenerate
an entire eye structure. We suggest that the map from a rotated eye is either
itself rotated or else comes from newly grown unrotated tissue. We have shown
that the external appearance of an eye may be misleading. The compound
nature of eyes is not always reflected in the possession of two fissures, and
close histological examination can reveal a dual anatomical origin for the
retina even when only one retinal component appears to connect with the
tectum (see pages, 53 & 55).
Finally, as mentioned previously, we have observed that the use of low
ionic strength operating medium tends to lead to less viable tissues than does
the use of normal media. Cell deaths at this time might be expected to lead
to the regenerative cell replacement behaviour that presumably underlies the
formation of compound eyes of the sort described here. The relative overgrowth of the originally oriented, host-derived, component of these eyes could
then easily be misinterpreted, on mapping, as being due to repolarization.
In relation to what we have just argued, we must now comment on some
earlier work (Szekely, 1954; Stone, 1960) where behavioural studies have led
to claims for a repolarization of the urodele eye. Detailed reading allows the
criticism that the series of eye rotation stages, the results of which were investigated by Stone (1960), was not extensive enough for the distinction which he
drew between a stage marking the end of anatomical regulative ability and
one marking the later end of functional repolarizability. No clear instances of
an eye with simply rotated anatomy, but mediating normally guided behaviour,
are reported. In his study on the time of determination of the position of the
choroid fissure, Stone (1966) described one animal in which eye rotation
resulted in the appearance of two fissures (see also Sato, 1933). In view of our
own experience of extremely early anatomical specification in amphibians, and
the confused behaviour that might be expected from a cryptically compound
eye, we feel that Stone's results are compatible with his having observed the
decrease, with age at operation, of the probability of such phenomena as
de-rotation in initially rotated eye anlagen, or the formation of compound eyes.
The behavioural criteria whereby Szekely (1954) was led to postulate behavioural
adaptation in the dorso-ventral axis and not in the naso-temporal axis are
not entirely satisfactory.
5-2
64
R. M. GAZE AND OTHERS
The only case, to our knowledge, where a neural precursor has been shown
to undergo genuine repolarization (not by de-rotation) of a developing structural
pattern, is that of small distal eye vesicle (thus, neural retinal) grafts in chicks
(Goldberg, 19766). This author claimed a developmental time distinction
between repolarizability in two 'Cartesian' axes, with respect to ganglion cell
axonal patterns in the plane of the retinal surface. On close consideration of
this carefully reported work, where transfers of retina were carried out just
at the time of its normal invagination onto the prospective pigment epithelium,
we feel that it is consistent with retinal fibre orientation, in its naso-temporal
and dorso-ventral aspects, being passively responsive to guidance factors in
the newly contacting prospective pigment epithelium.
We must conclude by saying that our experiments give us no reason to
suppose that specification of the eye for map formation involves polarization
of the anlage across two Cartesian axes (naso-temporal and dorso-ventral),
either separately or simultaneously. Indeed, while our observations have
nothing specific to say on the matter, they allow us to start considering the
idea that eye specification may be organized, from the onset of development,
according to polar rather than Cartesian co-ordinates (see McDonald, 1975).
Such an idea would fit naturally with our understanding of the mode of growth
of the eye from the group of precursor cells in which determination of orientation
properties initially occurs. The retina in Xenopus grows by radial accretion of
cells, to all cell layers, at the ciliary margin (Straznicky & Gaze, 1971). Thus
cells close to the optic nerve head at the back of the eye are the 'oldest' cells
in the retina, whereas cells at the ciliary margin of the retina are the 'youngest'
cells, at any stage of development.
Thus although we remain ignorant of cellular mechanism, the relative
positions of origin of axons within the eye could naturally be coded by a
circumferential component, determined by clonal derivation from a particular
precursor cell position in the initial array, together with a radial component
equivalent to physiological time or to radial position within a cell lineage.
Local averaging interactions among an expanding population of cells thus
derived could ensure overall coherence of a set of specificities in construction
of the retinotopic projection, as well as the asymmetric aspects of the eye's
anatomy.
We suggest that both our classes of result, that is rotated maps and compound
maps, point in the same direction, which is towards very early determination
of orientation properties in the eye anlage followed by a preservation of these
early properties according to cell lineage relationships thereafter. The distorted
arrangement of map positions, often seen within the less extensive of the two
components of a compound map (Fig. 9), are indeed what might be expected
on the hypothesis of extreme conservation of relative mapping values according
to clonal history on the part of small groups of cells. The geometrical
distortion of normal clone shapes and/or the alteration of clonal histories by
Visuotectal map orientation in Xenopus
65
intercalatory growth stimulation, caused perhaps by the abnormal joining of
positionally disparate cells after the operation, might lead to just such distorted
projections on a rather extreme cell autonomy hypothesis. Such a mode of
co-ordinated growth and pattern control, with intercalatory growth as the
main expression of cellular interactions after surgical rearrangement of tissue,
is being recognized as characteristic of a number of developing systems whose
modes of growth have features in common with that of the eye. French et ah
(1976), in reviewing these systems, point out the evidence that pattern in
second order morphogenetic fields (i.e. those occurring later in development
than that determining the organism's body pattern as a whole) is specified in
ways closely related to the way the eye here appears to be specified. Examples
are vertebrate limbs and insect imaginal discs; and the way in which these
systems resemble the eye is that true cellular growth, as opposed to embryonic
cleavage, is occurring coincidentally with the laying down of pattern values
in the anlagen.
If it is accepted that eye specification for map formation is determined early,
as we suggest, there is no longer a need to postulate separate mechanisms of
cellular response controlling, on the one hand, cell fates as precursors of
particular anatomical structures, and on the other hand the propensities of
their descendants for the later formation of topic nerve projections. We are
left with no evidence that these two aspects of cellular participation in pattern,
for that best-studied neural anlage, the eye, are determined by separate developmental events. It is important to stress that, since the determinative events
occur while the eye domain is a group of, at the most, a very few hundred
cells in the forming neural plate or tube, these events do not directly establish
the differentiated pattern and connectivity properties among the definitive eye
cells. Rather, they fix the overall orientation of specificities for construction
of a future eye among a group of precursor cells.
The experiments described in this paper seem to be able to account, in a
coherent and parsimonious fashion, for the previously reported (Jacobson,
1967, 1968) apparent lack of polarization in the eye before stage 28. We suggest
that the eye, which in our experiments shows autonomy of specification for
map orientation from as early as stage 21/22, may appear to be undetermined
before stage 28 if, in an eye rotation, only part of the anlage is rotated; or if
the treatment of the tissue is such as to encourage cell death and thus cell
replacement from the optic stalk.
These results call into question the interpretation of two other phenomena
reported previously. These are the two-stage polarization of the eye (Jacobson,
1967, 1968) and the repolarization of the eye by information from the flank
(Hunt & Jacobson, 1972; Hunt & Platt, 1978). Further experiments along
these lines are in progress.
We would like to thank Mrs June Colville for her excellent histological assistance.
66
R. M. GAZE AND OTHERS
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