J. Embryol. exp. Morph. Vol. 58, pp. 79-91, 1980
Printed in Great Britain © Company of Biologists Limited 1980
79
The retinotectal projections from surgically
rounded-up half-eyes in Xenopus
By C. STRAZNICKY, 1 R. M. GAZE 2
AND M. J. KEATING 2
From the National Institute for Medical Research, Mill Hill, London
SUMMARY
In Xenopus embryos of stage 32 half of one eye anlage was removed and the remaining
half was surgically rounded-up. The visuotectal projections through such half-eyes, recorded
after metamorphosis, showed in most cases a deformation of the map which corresponded
to the deformation imposed on the half-eye at operation. Some eyes gave normal maps and
some showed mirror-reduplication.
INTRODUCTION
In two recent papers (Straznicky & Gaze, 1980; Gaze & Straznicky, 1980)
we have shown that when various types of 'compound eyes' are formed
surgically in Xenopus embryos, the retinal fragments that form the compound
eye project to the tectum with the orientations that they would have shown,
had they been normally positioned as parts of normal eyes. The partial visuotopic map from each fragment is thus oriented, in relation to a normal map, as
are the retinal fragments in relation to a normal eye. Thus the developmental
programme among cells in the developing eye, that relates to the orientation
of the retinotectal map, is stable after these types of operations.
If the nasal or temporal half of an eye in a Xenopus embryo of stage 32
(Nieuwkoop & Faber, 1956) is removed, the residual half-eye eventually
produces, in most cases, what appears to be a full-size normal eye with a
normal retinotectal projection (Berman & Hunt, 1975; Feldman & Gaze, 1975).
Although this has not been investigated in detail, it seems likely that the
restitution of a normal eye from a half-eye, with the eventual development of
a normal retinotectal map, is related to extra mitosis following the operation,
probably occurring at the unapposed cut edge (Horder & Spitzer, 1973),
whereby the missing positional values of the half-eye are replaced.
We have noticed an interesting relationship between growth and pattern
formation in half-eyes. If the nasal, temporal or dorsal half of an eye is removed
1
Author's address: School of Medicine, Flinders University of South Australia, Bedford
Park, South Australia 5042.
2
Authors' address: National Institute for Medical Research, The Ridgeway, Mill Hill,
London NW7 1AA, U.K.
80
C. STRAZNICKY, R. M. GAZE AND M. J. KEATING
Fig. 1. Schematic diagrams showing the nature of the operations performed. Half
of the right-eye anlage (interrupted outline) was removed and the remaining half
(shaded) was rounded-up. Top, i TR; middle, \ NR; bottom, £ VR.
in a Xenopus embryo of stage 32, and the residual half-eye is then surgically
rounded-up, by partially freeing it from the surrounding mesenchyme and
folding the fragment so that the cut edges are apposed, the half-eye heals up
to form an eye which is at all stages smaller than normal and which may show
distortion of the retinotectal map which reflects the distortion imposed on the
developing eye at operation.
We describe in this paper the nature of these distortions and discuss the
results in the light of current ideas on the question of stability versus modifiability of the programme for retinal development. Some of the results presented
come from the original experiments performed in 1969 while others are recent.
The findings have been consistent throughout the entire series of experiments.
METHODS
Xenopus embryos of stage 32 were operated in full-strength Niu-Twitty
solution containing MS 222 (tricaine methane sulphonate, Sandoz) at a
concentration of 1:5000.
The right eye of an embryo was exposed and the nasal, temporal or dorsal
half of the eye anlage, together with the lens, was freed by use of glass needles
and then removed by suction with a Spemann pipette. The residual half-eye
anlage was partially separated from the surrounding mesenchymal tissue and
the edges of the eye rudiment were surgically apposed. After the rounding-up
of the eye the line of fusion was directed rostrally in temporal halves, caudally
Retinotectal projections from Xenopus rounded-up half-eyes
Table 1
Eye
$TR
&VR
Result
No.
Map oriented according to eye deformation
NN reduplication
Approximately normal
Unclassifiable
Map oriented according to eye deformation
TT reduplication (+residual distortion of
part-map)
Approximately normal
Unclassifiable
Map oriented according to eye deformation
Approximately normal
5
4
1
1
11
4
4
1
11
2
Fig. 2. Predicted visuotectal projection from rounded-up half-eyes, based on the
assumption that (1) the half-eye retained its original developmental programme
relating to map orientation, and (2) the projection from the half-eye was spread
across the whole tectum.
81
82
C. S T R A Z N I C K Y , R. M. GAZE AND M. J. K E A T I N G
Fig. 3. 'Normal' visuotectal projection. This map was obtained from a £ TR eye.
For this and all succeeding maps the conventions are as follows.
Top: dorsal surface of left tectum showing rows of electrode positions. The black
arrow to the left of the diagram points rostrally along the midline. The large open
arrow on the tectum is included to give a sense of the orientation of the projection.
Bottom: perimeter chart of the right visual field. The animal is to be thought of as
being behind the chart, looking out through the centre of the chart towards the
observer. The chart covers 100° from centre to periphery. N, nasal; D, dorsal; T,
temporal; V, ventral.
Rows of response positions are shown, corresponding to the rows of tectal
positions. The large open arrow, relating to the similar arrow on the tectum, is to
give a sense of the orientation of the projection.
In other figures of i NR and £ TR eyes, an inset diagram shows the orientation
of the eye fissure, when this had been noted at the time of recording.
in nasal halves and dorsally in ventral halves (Fig. 1). The approximated edges
of the eye were held together for about 30 minutes by using splinters of coverslip
glass as weights, after which the cut edges remained fused permanently. Animals
were then transferred to fresh Niu-T witty solution and 24 h after the operation
each of the operated eyes was checked under a stereo microscope to determine
the success of the operation. Animals which revealed a 10° or wider gap between
Retinoteclal projections from Xenopus rounded-up half-eyes 83
(b)
(a)
T-
Fig. 4. (a, b). Visuotectal maps from rounded-up nasal half-eyes. The characteristic
distortion of the projection, described in the text, is seen.
the approximated edges of the operated eye were discarded. The remaining
animals were reared to metamorphosis and beyond, and visuotectal projections
from the rounded-up half-eyes were mapped electrophysiologically. The
technique of mapping and the histology used have been described in a previous
paper (Straznicky & Gaze, 1980).
RESULTS
Visuotectal maps through the operated eye to the contralateral tectum were
recorded from 44 animals (Table 1). In 11 of these animals the operated eye
was a nasal half (-J- NR), in 20 a temporal half-eye (£ TR) and in 13 a ventral
half-eye ( | VR). The largest class of results from each type of operation was
that in which the orientation of the visuotectal map paralleled the deformation
imposed on the eye at operation. Figure 2 shows the nature of the map to be
expected from rounded-up half-eyes, on the basis that the programme relating
84
C. S T R A Z N I C K Y , R. M. GAZE AND M. J. KEATING
(b)
(a)
-N
T-
Fig. 5. (a, b) Reduplicated projections from £ NR eyes. In (a) the orientation of the
eye fissure was not commented on at the time of recording; the map closely resembles
that from an NN compound eye. In (6) the eye fissure was rotated 90° clockwise,
as is the reduplicated map.
to the orientation of the projection is stable and that the projection spreads to
fill the available tectal space, as it does with compound eyes.
A smaller class of results from \ NR and \ TR experiments was one where
the maps showed complete or partial mirror-reduplication, resembling that
seen in maps from compound eyes. A few results, from each type of operation,
gave normal maps. In all cases the projection extended across the whole of the
mappable surface of the tectum.
Normal maps, even from normal eyes, differ from one another, and the
decision as to what can be called a normal map is subjective. Figure 3 illustrates
what we have called a 'normal map', in this case coming from a rounded-up
temporal half-eye. Essentially, in a normal map, latero-medial rows of tectal
recording positions give corresponding ventro-dorsal rows of stimulus positions
in the visual field. To lessen the selective and subjective elements inherent in
-N
Retinotectal projections from Xenopus rounded-up half-eyes 85
the classification of the experimental results, we have chosen to illustrate the
findings with several examples from each class of map so that the reader can
decide whether or not the descriptions are valid.
Rounded-up nasal half-eyes
Five out of 11 maps were oriented according to the nature of the operation
(Fig. 4). Rows of stimulus positions in the temporal field (corresponding to the
least affected part of the retina) are normally oriented. In the nasal field,
corresponding to the most distorted part of the retina, the rows of stimulus
positions curl towards the nasal pole of the field. These eyes were smaller than
the normal eyes.
In four animals the operated eye gave a mirror-reduplicated projection
resembling that from a surgically formed compound double-nasal eye (Fig. 5).
In one of these the dorso-ventral alignment of the projection was normal and
in the other the map was rotated 90° clockwise, in accord with the position of
the fissure, which pointed caudally.
One animal gave an approximately normal visuotectal map; and the map
from the remaining animal was unclassifiable, in that the tectum was covered
with a chaotic projection from a small region of central visual field.
Rounded-up temporal half-eyes
Eleven out of 20 maps were oriented according to the nature of the operation
(Fig. 6). Rows of stimulus positions in the nasal field, corresponding to the
least affected part of the retina, are normally oriented. In the temporal field,
corresponding to the most distorted part of the retina, the rows of stimulus
positions curl towards the temporal pole of the field. The eyes were small.
Four animals gave maps showing mirror-reduplication resembling that seen
with surgically produced double-temporal compound eyes (Fig. 7). In both the
cases shown the reduplication is seen in dorsal field, while temporoventral
field (naso-dorsal retina) is not reduplicated but shows instead the distortion
typical of this operation.
Four maps were classified as approximately normal and one showed a
chaotic projection from a small region of central field to the whole tectum.
Rounded-up ventral half-eyes
Eleven out of 13 eyes gave maps which were oriented according to the nature
of the operation (Fig. 8). These maps show 'barrelling' of the visual field
contour lines, from dorsal to ventral, in both nasal and temporal parts of the
visual field. The field projection also tends to extend much further ventrally
than it does in a normal animal. The operated eyes were smaller than normal
and all showed gold pigmentation dorsally with a normally positioned ventral
fissure. Two animals gave an approximately normal projection.
T-
-N
T-
(c)
V
V
V
Fig. 6. (a, 6, c) Visuotectal maps from rounded-up temporal half-eyes, (a) and (b) show the characteristic distortion described in the text.
(c) shows a similar distortion and, in addition, points 1 and 2 are reduplicated.
N
(b)
-N
z
o
H
w
>
w
N
o
v*
O
N
c/3
ON
00
Retinotectal projections from Xenopus rounded-up half-eyes
(a)
(b)
24
-N
87
T-
Fig. 7. (a, b) Reduplicated projections from £ TR eyes. In both maps the reduplication is partial and confined to the temporo-dorsal sector of the field.
DISCUSSION
Previous observations on compound eyes have shown that the retinal
fragments retain their original programming for (1) generating within the eye
the particular cell characteristics responsible for the orientation of the visuotectal map (Straznicky & Gaze, 1980; Gaze & Straznicky, 1980); (2) carrying
out the characteristically timed pattern of retinal histogenesis (Straznicky &
Tay, 1977); and (3) forming selectively nerve fibre tracts which are positioned
appropriately according to the nature of the retinal fragment (Straznicky, Gaze
& Horder, 1979). Further evidence for the existence of stable programming in
retinal fragments has come from study of the regeneration of optic nerve fibres
from compound eyes, where it has been shown that, when such fibres regenerate
to the ipsilateral tectum, they initially innervate only part of it; and the part
innervated corresponds to the nature of the compound eye (Gaze & Straznicky
1979).
-N
C. STRAZNICKY, R. M. GAZE AND M. J. KEATING
(a)
-N
T-
Fig. 8. (a, b, c and d) Visuotectal projections from rounded-up ventral half-eyes,
showing the characteristic distortions described in the text. Field positions labelled
a in (c) and (d) come from deeper at the correspondingly numbered tectal positions.
If we assume that the rounded-up half-eyes in the present experiments also
maintain intact their developmental programmes for map orientation, an
assumption which implies that retinal cell properties relating to map orientation
are, in this situation, related to cell lineage, we would expect the maps to
reflect the distortions imposed on the retina at operation (Fig. 2) and the
results presented show that this is so. Since each operated eye will initially
contain only half the positional values of a complete eye, we might expect the
initial tectal projection to be restricted to the corresponding half of the tectum,
as occurred in the study on regeneration quoted above (Gaze & Straznicky,
1979). We know, however, that each (similar) part of a conventional compound
eye will eventually spread across the whole of the contralateral tectum during
development (Straznicky, Gaze & Keating, 1971), and the present results
indicate that such a spreading also occurs with the projections from rounded-up
half-eyes.
Retinotectal projections from Xenopus rounded-up half-eyes 89
30
(c)
(.d)
-N
Fig. 8 (c and d). For legend see opposite.
The nature of the operation suggests that the distortions found in the
visuotopic projections from rounded-up half-eyes should be symmetrical about
the vertical meridian for ventral half-eyes and symmetrical about the horizontal
meridian for nasal and temporal half-eyes (Fig. 2). This prediction is fulfilled
for ventral half-eyes but not for the other varieties, where the distortion seen
is confined mainly to the dorsal part of thefield.This is perhaps because more
ventral parts of the field would project too far laterally on the tectum to be
recorded. It is also possible that the injury to the region of the ventral fissure,
which must occur in operations for nasal or temporal half-eyes but which does
not occur with ventral half-eyes, may disturb the retinotopic arrangement of
fibres as they form the optic nerve, and this may be reflected in the nature of
the map.
Four out of 11 \ NR eyes, and 4 out of 20 \ TR eyes, gave maps showing
mirror-reduplication resembling that seen in maps from conventional NN and
TT compound eyes respectively. The occasional recurrence of mirror-redupli-
90
C. STRAZNICKY, R. M. GAZE AND M. J. KEATING
cation in unrounded-up half-eyes has been described previously (Gaze, 1970;
Berman & Hunt, 1975; Feldman & Gaze, 1975); and in the previous paper of
this series (Gaze & Straznicky, 1980) we have shown that mirror-reduplication
may be induced when compound eyes are formed from retinal fragments that
would not be expected to give this type of pattern, provided that the operations
are performed in a medium of low ionic strength. We have suggested, therefore,
that mirror-reduplication should be regarded as a form of tissue response to
injury, particularly when the injury is aggravated by the use of operating
solutions that damage the cells and slow down healing.
Perhaps the most interesting aspect of the reduplications seen in the present
work is that several of them are partial. Figure la and b illustrate maps from
\ TR eyes; in each case the main projection is reduplicated in TT style and the
small region of ventro-temporal field shows an additional, non-reduplicated,
partial projection which has the orientation characteristic of maps from a nonreduplicated \ TR eye. Figure 6 c shows a projection from a \ TR eye in which
two points are reduplicated. This combination of reduplicated projection with
a distorted, partial, non-reduplicated projection, was not seen in the maps from
\ NR eyes. Moreover, no map from a \ VR eye showed reduplication. The
cellular events underlying reduplication of this sort are unknown, and the
phenomenon will repay extensive study.
Surgically rounded-up half-eyes are smaller than half-eyes that have not
been rounded-up in this way. The difference in size persists into adult life. We
have previously shown that the Xenopus eye grows by mitosis at the ciliary
margin (Straznicky & Gaze, 1971). It is probable that when a half-eye is left
unrounded-up, a new complete ciliary margin is rapidly formed by mitosis at
the cut edge of the residual half-eye. Furthermore, regeneration of a new halfeye from tissue in the optic stalk is a likely possibility (Gaze, Feldman, Cooke
& Chung, 1979) and in this case the eye eventually formed might be expected to
be normal in all respects.
It seems that the process of surgically rounding-up the half-eye, whereby the
cut ciliary margins are apposed, prevents in some way the re-formation of the
missing ciliary margin; and the eye so treated is thereafter stunted in its growth,
never regains its full cell complement, and remains a half-eye in terms of its
cellular positional values. Further evidence for this last point comes from the
work of Steedman, who has shown, by the method of cobalt impregnation,
that the optic pathway from a rounded-up ventral half-eye forms only a medial
tract; the lateral tract, which normally carries retinal fibres of dorsal origin, is
missing (Steedman, in preparation).
Further work is needed to reveal the nature of the cellular mechanisms
responsible for the different behaviour of half-eyes that are rounded-up and
those that are not. The present experiments, however, provide further evidence
for the stability of the retinal developmental programme in various abnormal
situations.
Retinotectal projections from Xenopus rounded-up half-eyes 91
These experiments were partly performed during the tenure by C.S. of a Wellcome
Research Fellowship.
REFERENCES
BERMAN, N.
& HUNT, R. K. (1975). Visual projections to the optic tecta in Xenopus after
partial extirpation of the embryonic eye. /. comp. Neurol. 162, 23-42.
FELDMAN, J. D. & GAZE, R. M. (1975). The development of half-eyes in Xenopus tadpoles.
/. comp. Neurol. 162, 13-22.
GAZE, R. M. (1970). The Formation of Nerve Connections. London: Academic Press.
GAZE, R. M., FELDMAN, J. D., COOKE, J. & CHUNG, S-H. (1979). The orientation of the
visuotectal map in Xenopus: developmental aspects. /. Embryol. exp. Morph. 53, 39-66.
GAZE, R. M. & STRAZNICKY, C. (1979). Selective regeneration of optic fibres from a compound eye to the ipsilateral tectum in Xenopus. J. Physiol., Lond. 293, 58 P.
GAZE, R. M. & STRAZNICKY, C. (1980). Stable programming for map orientation in disarranged embryonic eyes in Xenopus. J. Embryol. exp. Morph. 55, 143-165.
HORDER, T. J. & SPITZER, J. L. (1973). Absence of cell mobility across the retina in Xenopus
laevis embryos. / . Physiol., Lond. 233, 33-34P.
NIEUWKOOP, P. D. & FABER, J. (1956). Normal Table o/Xenopus la.evis(Daudiri). Amsterdam:
North-Holland.
STRAZNICKY, K. & GAZE, R. M. (1971). The growth of the retina in Xenopus laevis: an
autoradiographic study. J. Embryol. exp. Morph. 26, 67-79.
STRAZNICKY, C. & GAZE, R. M. (1980). Stable programming for map orientation in fused
eye fragments in Xenopus. J. Embryol. exp. Morph. 55, 123-142
STRAZNICKY, C , GAZE, R. M. & HORDER, T. J. (1979). Selection of appropriate medial
branch of the optic tract by fibres of ventral retinal origin during development and in
regeneration: an autoradiographic study in Xenopus. J. Embryol. exp. Morph. 50, 253-267.
STRAZNICKY, C , GAZE, R. M. & KEATING, M. J. (1971). The retinotectal projections after
uncrossing the opta chiasma in Xenopus with one compound eye. /. Embryol. exp.
Morph., 26, 523-542.
STRAZNICKY, C. & TAY, D. (1977). Retinal growth in normal, double dorsal and double
ventral eyes in Xenopus. J. Embryol. exp. Morph. 40, 175-186.
(Received 16 January 1980, revised 15 February 1980)
EMB 58