/ . Embryol. exp. Morph. Vol. 68, pp. 9-21, 1982
Printed in Great Britain © Company of Biologists Limited 1982
The innervation of a virgin tectum by a doubletemporal or a double-nasal eye in Xenopus
By C. STRAZNICKY1 AND R. M. GAZE*
From the National Institute for Medical Research,
Mill Hill, London
SUMMARY
In Xenopus embryos at stage 32/33 one eye anlage was removed and the other mfcde
into a compound double-nasal or double-temporal eye. At metamorphosis the optic nerve
was cut and the compound eye was permitted to regenerate fibres both to its own contralateral tectum and to the ipsilateral 'virgin' tectum. One month later the projection from
the compound eye to the virgin tectum was assessed autoradiographically by use of tritiated
proline. Projections from double-temporal eyes were found to be restricted to rostrolat^ral
tectum, whereas projections from double-nasal eyes covered the entire tectal surface. It was
concluded that the results did not suggest that positional markers existed on the tectum
before the arrival of the optic fibres.
INTRODUCTION
In a recent series of papers (Straznicky & Gaze, 1980; Gaze & Straznicjcy,
1980 a, b; Straznicky, Gaze & Keating, 1981) we have examined the development
and regeneration of retinotectal fibre projections from surgically constructed
'compound eyes' in Xenopus. The evidence presented shows that each half
of such a reconstructed eye retains some attributes, or markers, whjch
identify it as a half-eye. When a compound double-nasal .(NN), doubletemporal (TT) or double-ventral (VV) eye is caused, after metamorphosis, to
innervate its ipsilateral tectum along with the projection from the normal eye,
the compound eye projection restricts itself to the appropriate part of the
tectum. NN fibres innervate caudomedial tectum, TT fibres innervate rostrolateral tectum and VV fibres innervate medial tectum. This shows that the
retinal fibres are recognizing something which constrains them to terminate
in particular regions of the tectum. These 'markers' on the tectum could be
produced and carried by the tectal cells themselves, as supposed by Sperry
(reviewed 1951, 1963, 1965), or could be related to the fibre projection, either
1
Author's address: Centre for Neuroscience, School of Medicine, Flinders University of
South Australia, Bedford Park, South Australia 5042.
2
Author's address: National Institute for Medical Research, The Ridgeway, Mill Hill,
London NW7 1AA, England.
10
C. STRAZNICKY AND R. M. GAZE
as the fibres themselves or as a trace left on the tectum by the fibres (Schmidt,
1978).
If the projection from the normal eye is removed before the fibres from the
compound eye reach this tectum, the projection from the compound eye is
no longer restricted but spreads at once to cover the entire tectum (Straznicky
& Tay, 1981). This suggests that the fibres from the compound eye are recognizing the fibres from the normal eye, rather than the tectal cells themselves,
or that any markers deposited by the nerve fibres are very short lived. Since
an optic fibre projection can mark the tectum in a way that is recognizable to
another incoming optic fibre projection, the question arises whether the original
establishment of the retinotectal projection, in early development, requires the
existence of tectal markers. If so, tectal markers must exist on the tectum
before the arrival of the optic fibres, as suggested by Sperry. If not, then
some mechanism other than target affinity must be responsible for the first
establishment of the projection.
In this paper we describe another approach to the question of whether or
not tectal positional markers exist before the first arrival of optic fibres. We
have arranged for one tectum to remain without optic innervation ('virgin')
during development, by removal of the contralateral eye in embryonic life,
before the fibres grow to the brain. Then, after metamorphosis, we caused the
fibres from the other eye (which was compound, TT or NN) to innervate the
virgin tectum, and we studied the resulting projections by means of autoradiography with tritiated proline. The consistent result was that TT projections
to the virgin tectum were restricted to rostrolateral tectum but NN projections
covered the entire tectum. A brief account of this work has appeared elsewhere
(Gaze & Straznicky, 1980c).
METHODS
Xenopus laevis were obtained from laboratory breeding pairs.
Surgery
Under MS 222 anaesthesia (tricaine methane sulphonate, Sandoz 1:3000),
the right and left eyes were operated on at stages 32-33 (Nieuwkoop & Faber,
1967). The nasal half of the right eye anlage of the host embryo was removed
and replaced by a temporal half of the left eye of a donor embryo to form a
double-temporal (TT) eye. Similarly in other embryos double-nasal (NN) eyes
were formed. One to two hours after the first surgery the left eye of the operated
embryos was removed. The TT and NN eye animals were kept separately
under standard laboratory conditions and reared past metamorphosis.
One week after metamorphosis the remaining right optic nerve was exposed
through the pharynx, under MS 222 anaesthesia (1:1000), and cut close to
the optic chiasma to facilitate bilateral tectal regeneration. Tn a few animals
with right compound eye the optic nerve was not cut, and these animals served
11
Innervation of virgin tectum
-N
Fig. 1. The visuotectal projection from a control NN eye. The upper diagram
represents the dorsal surface of the left tectum with the large open arrow pointing
rostrally along the midline. Numbers and filled circles represent rows of tect^l
recording positions.
The lower diagram represents the visual field of the right, NN, eye. The eye js
to be considered as being on the far side of the chart, looking out at the observer
through its centre. The chart covers 100° outwards from the centre. Numbers and
filled circles indicate rows of optimal stimulus positions corresponding to the
tectal recording position. N, S, T, I: nasal, superior, temporal, inferior.
Central regions of field project rostrally on the tectum, and the nasal and
temporal extremities of the field project caudally. The autoradiographic reconstruction of this projection is shown in Fig. 3e. Most-rostral tectum was n0t
investigated at recording.
as controls. Four to six weeks after the optic nerve section, or metamorphosis
in the case of control animals, the resultant optic fibre projections from the
right compound eyes were assessed autoradiographically and, in some cases,
electrophysiologically.
;
12
C. S T R A Z N I C K Y AND R. M. GAZE
T-
Fig. 2. The visuotectal projection from a control TT eye. The conventions are
as in Fig. 1, with the addition that open circles on the tectum represent positions
from which no response was obtained.
In this case the nasal and temporal extremities of the visual field project rostrally
on the tectum, and the vertical midline of the field projects caudally. The autoradiographic reconstruction of this projection is shown in Fig. 3(c). Caudal and
medial tectum was not investigated at recording.
Histology
Twenty-four hours before sacrifice 10/*Ci [3H]proline (3H-P; 21 Ci/mmol
Amersham) in 0-25 /i\ solution was injected into the vitreous. The head of the
animal was fixed in Bouin's solution, the dissected brain was embedded in
paraffin and serially sectioned at 10 /tm. The sections were mounted on slides
and coated with Ilford K2 nuclear emulsion, exposed at 4 °C for 14 days,
developed in Kodak Dektol and counterstained with Harris's haematoxylin.
Innervation of virgin tectum
RTTL
13
RNNL
Fig. 3. The reconstructed autoradiographic distributions of the projections from
the compound eyes in control animals.
For each example serial transverse sections through the tecta were processed
for autoradiography. The outline of every fifth section was drawn with the aid of
a camera lucida and the mediolateral extent of the tectum and of the labelled
region, as estimated by visual inspection, was measured with a map measurer. The
resulting measurements were marked on graph paper and the overall outlines
joined up. Thus each diagram represents a flattened view of the tecta seen from
dorsally. The mediolateral extent of each diagram is a measured dimension (bar
= 1 mm) while the rostrocaudal dimension is arbitrary.
In each case the right eye was compound and the left eye had been removed
in embryonic life, (a-d) represent TT projections while (e) and (/) represent NN
projections. In each diagram the open arrowhead is situated rostrally in the midline. The tectum on the right in each diagram is contralateral to the eye and the
tectum on the left is ipsilateral.
In (a), (c) and (e) no label is shown ipsilaterally. In (b), (d) and (/) a small
amount of atypically distributed label is shown rostrally and medially in the
ipsilateral tectum. The amount of this label is greatly over-emphasized by the
all-or-none nature of the line diagram. Projection c came from the animal which
also provided the map shown in Fig. 2, and projection f came from the animal
giving the map shown in Fig. 1.
The extent of the tectum and the tectal projection/s from the right eye were
measured on camera-lucida drawings.
Electrophysiology
In a few animals (control group) the visuotectal projection from the right
operated eye was mapped in order to check the success of the embryonic eye
14
C. STRAZNICKY AND R. M. GAZE
Innervation of virgin tectum
; 15
operation. Visuotectal recordings were carried out according to standard
procedures (Straznicky & Gaze, 1980; Gaze & Straznicky, 1980a, b).
RESULTS
VT controls
The left (normal) eye was removed at stage 32/33, and the right eye was
made NN in two animals and TT in four animals.
One week after metamorphosis, and twenty-four hours before the anitnals
were killed, the compound eye was labelled with 3 H-P. One animal with an
NN eye and two with TT eyes were mapped electrophysiologically. Each animal
recorded showed the reduplication of the visuotopic map characteristic of the
nature of the compound eye. The NN eye projected to the tectum so that the
vertical midline of the field was represented on rostral tectum, while nasal and
temporal field extremities were represented caudally (Fig. 1). The maps from
the TT eyes showed that the nasal and temporal extremities of the field projected
to rostral tectum while the vertical midline of the field projected cau<ially
(Fig. 2).
The distributions of the projections from the control compound eye$, as
revealed by autoradiography, are summarized in Fig. 3. It may be seen that
the two NN projections gave complete coverage of the tectum contralateral
to the compound eye (Fig. 3e,f) and that the tecta ipsilateral to the compound
eye are empty of label except for a small amount of faint and atypical labelling
rostromedially in Fig. 3/. Three of the four TT projections (Fig. 3 a, b, d) $how
a deficit in the projection to the caudomedial part of the contralateral tectum,
as has been noted previously for TT eyes (Straznicky et al. 1981). One animal
(Fig. 3 c) gave a complete coverage of the contralateral tectum. The tecta
ipsilateral to the compound eye were devoid of label in two cases (Fig. 3d and
c) and show only a minimal amount of atypical labelling rostrally in Fig. 3 b
and d. In Fig. 3 b the fibre projection is unusual in that the optic nerve fibres
Fig. 4. Bright-field and dark-field photographs of transverse sections from control
brains, showing the relative intensity of the contralateral and the aberrant ipsilateral
label.
,
(a, b) NN projection. The reconstruction of this projection is shown in Fig. 3(/)
and Fig. 1 shows the map obtained, (a) is a low-power view of the mid-tect&l
region. The contralateral projection is heavily labelled but ipsilateral label is ndt
visible in this bright-field micrograph. A dark-field micrograph of the same
section (b) shows a small amount of label ipsilaterally in the region of the medi$l
optic tract (arrow). Bar for (a) and (b) = 500 /*m.
(c, d) TT projection. The reconstruction of this projection is shown in Fig. 3(d).
Again, the bright-field micrograph shows heavy labelling rostrally in the contralateral tectum but not in the ipsilateral tectum. A dark-field micrograph of the
same section shows some ipsilateral label (arrow). Bar for c and d = 500 /«n. ,
16
C. STRAZNICKY AND R. M. GAZE
RNN L
Fig. 5. Autoradiographic reconstruction of the projections from compound eyes
to both tecta, after 28 days regeneration.
(a-e) TT projections; (f-j) NN projections. Bar = 1 mm. For each diagram
the contralateral tectum is on the right, the ipsilateral tectum is on the left and
the open arrowhead is situated rostrally on the midline.
TT projections (a-e) may be seen to have a greater deficit caudomedially on the
ipsilateral (virgin) side than contralaterally, whereas NN projections (/-;) in
most cases cover the entire ipsilateral tectum.
reach contralateral tectum over two quite separate pathways - one via the
chiasma and the other as a separate fibre bundle entering the rostral tectum
directly from the cranial cavity.
The nature of the small amount of ipsilateral labelling in Fig. 3b, d a n d / i s
not known; its relative intensity is shown, for two cases, in Fig. 4. Apart from
the aberrant label the ipsilateral tectum appears to be virgin in each case.
Compound-eye projections to the virgin tectum
The projections from the TT eye to both tecta 28 days after optic nerve
section are shown, for all five animals investigated, in Fig. 5 a-e. On both
contralateral and ipsilateral (virgin) sides the tectal coverage is incomplete
Innervation of virgin tectum
17
RNNL
Fig. 6. Autoradiographic reconstructions of the projections from compound eye$
to both tecta after 42 days regeneration. The picture is essentially similar to that
shown in Fig. 5 for 28 days, (a-d) TT projections, (e-g) NN projections. Conventions as in Fig. 5.
caudomedially, and this deficit is more marked on the virgin tectum. The
projections from NN eye to both tecta, 28 days after optic nerve section, are
shown, for all five animals investigated, in Fig. 5f-j. With one exception
(Fig. 5g) the tectal coverage is virtually complete on both contralateral, and
ipsilateral (virgin) sides. And with the same exception the label is more dense
rostrally on the virgin tectum in all cases.
The projections from TT and NN eyes after 42 days of regeneration are
shown in Fig. 6. There is no obvious consistent difference between these
projections and those at 28 days regeneration shown in Fig. 5.
!
DISCUSSION
In the previous paper of this series we described the development of the
retinotectal projection from TT, NN and VV eyes (Straznicky et al. 1981).
It was found that TT projections initially restricted themselves to rostrolateral
tectum and spread slowly across the tectum, so that tectal coverage was nearly
complete shortly after metamorphosis. In a comparable fashion, VV projections
were initially restricted to medial tectum and took time to spread acro$s the
18
C. STRAZNICKY AND R. M. GAZE
tectum laterally. NN projections, on the other hand, covered most of the
tectum from the start, although these projections were most dense caudally.
In normal Xenopus the retina grows by the addition of rings of cells at the
ciliary margin (Straznicky & Gaze, 1971) whereas the tectum grows from
rostrolateral (oldest tissue) to caudomedial (youngest tissue; Straznicky &
Gaze, 1972). The retina sends a fibre projection to the tectum from around
stage 45, when both retina and tectum are small in comparison with the
adult; and shortly thereafter the retinotectal map may be shown electrophysiologically to have the normal orientation and order (Gaze, Keating & Chung,
1974). From this time retina, tectum and the retinotectal projection all increase
in extent while the ordering of the retinotectal map is maintained (Gaze,
Keating, Ostberg & Chung, \919b). These findings have led to the proposal
that the retinotectal connexions shift progressively during normal development
(Gaze et al 1919b).
The topological problem of fitting the retinal projection on to the tectum,
when both retina and tectum are growing differently, is made yet more difficult
in the case of compound eyes. These have been shown to grow like normal
eyes by cellular addition at the ciliary margin (Feldman & Gaze, 1972); and
the pattern of the projection, particularly for TT eyes, is such that the shift
of connexions that occurs during growth is much greater than for normal eyes
(Straznicky et al. 1981). Thus for normal eyes, and even more for compound
eyes, the observed modes of growth of the projections do not readily fit the
idea that localized tectal positional markers exist prior to the arrival of the
optic nerve fibres. Not, that is, if the tectal markers are stable and the differential
affinities between the various retinal markers and the various tectal markers
are also stable.
The present paper describes a different approach to the question of whether
or not tectal positional markers exist before the arrival of optic fibres. The
virgin tectum is one that has had its normal source of optic fibre input removed
in embryonic life, before the optic fibres have grown from the eye to the
tectum. We have shown that the virgin tecta in these experiments are indeed
virgin in that they possess virtually no autoradiographically demonstrable
projection from the remaining (compound) eye after metamorphosis. It is
conceivable that there was a transient projection to these tecta in early life
but this is rendered unlikely by the observations (Steedman, Stirling & Gaze,
unpublished; Fawcett, Hirst & Gaze, unpublished) that cobalt or HRP impregnation of fibres from a residual eye shows the ipsilateral tectum to be
uninnervated in tadpole life. These virgin tecta, therefore, grow up with no
experience of optic fibres, certainly over the greater part of the tectal surface.
It is of course obvious that such tecta have extensive connexions with other
parts of the nervous system, including, probably, visually related regions such
as the nucleus isthmi, some of which send terminals to those tectal layers
normally occupied by optic fibres. Even so it remains true that the tecta are
Innervation of virgin tectum
19
devoid of optic fibres and we can then ask how the system behaves when optic
fibres are first supplied to the tectum after metamorphosis.
When a compound eye is caused to innervate both tecta after metamorphosis,
we find that the TT projections are restricted to rostrolateral tectum to a
greater extent on the virgin side than is the case on the contralateral side. NN
projections to the virgin tecta, on the other hand, show complete tectal coverage
and, in four out of five cases, the autoradiographic density of the projection
was greater rostrally than caudally.
The projections from TT eye to virgin tecta are thus compatible with the
idea that tectal positional markers already exist on the tectum when the fibres
arrive from the compound eye. The projections from NN eyes to virgin tecta,
however, do not support this idea. Fibres from NN eyes, which under other
circumstances have been shown to restrict themselves to caudomedial tectum
(Gaze & Straznicky, 19806), covered the entire surface of the virgin tectum
within 28 days of nerve section. There was no rostral deficit such as might
have been expected if the nasal fibres restricted themselves initially to caudal
tectum.
In a previous paper (Straznicky et al. 1981) we have argued that the
arrangement of fibres within the optic tract could play a major role in the
establishment of the normal retinotectal projection and might also account for
the observed differences in the development of projections from TT, NN and
W eyes. Any mapping mechanism must provide for the proper internal
ordering of the map, its proper extension across the tectum and its proper
orientation. All these characteristics of the normal map could be accounted
for if the development mechanism used in the establishment of the map
involved selective recognition between individually labelled retinal and tectal
units, as previously proposed by Sperry (see 1951). The evidence for positional
labelling of retinal fibres is strong (Gaze, Feldman, Cooke & Chung, 1979#;
Straznicky et al. 1981) but the evidence for the existence of tectal labels ab
initio is weak, as we show here and in the previous paper of this series
(Straznicky et al 1981).
Since we are trying to identify an alternative mapping mechanism, other
means for establishing the three prime requisites of a normal map must be
found. The internal ordering of the map could probably result from interactions between labelled retinal fibres leading to the preservation of retinal
neighbourhood characteristics in the tectal distribution of the optic fibre
endings. Evidence for some kind of fibre/fibre interactions is strong although
the mechanism is obscure. What controls the overall extent of the retinal
fibre projection on the tectum is unknown, but a generalized recognition of
tectum (and other optic termination areas elsewhere in the brain) is a strong
likelihood. Given the chance, retinal fibres will terminate in the tectum rather
than in adjacent foreign tissue.
The provision of a correct orientation for the map, in the absence of a
20
C. STRAZNICKY AND R. M. GAZE
target-affinity mechanism, requires a form of control distinct from that ordering
the map internally. Orientation could be provided by a weak system of polarity
markers across the tectum, recognizable to incoming optic fibres (Straznicky,
1978; Willshaw & von der Malsberg, 1979). This could represent the tectal
part of a general system of rostrocaudal and mediolateral axial cues across
the whole brain (Sharma, 1981) or a polarization particular to the tectum,
perhaps induced early in embryogenesis by the influence of the diencephalic
anlage (Scalia & Fite, 1974; Chung & Cooke, 1978). On the other hand,
orientation could be provided by the optic fibres being led on to the tectum
from the correct parts of the tectal margin (Straznicky et al. 1981).
In this context we may mention that fibres from different parts of the retina
appear to behave differently in the optic tract. Ventral retinal fibres select the
medial branch of the optic tract during development, and this positional
requirement is absolute rather than relative (Straznicky, Gaze & Horder, 1979;
Steedman, 1981). Temporal retinal fibres normally approach the rostral tectal
margin in a coherent bundle, and again they seem constrained to do this in
an absolute fashion; nasal retinal fibres, on the other hand, are widespread
in the optic tract as they approach the tectum (Steedman, 1981).
Since the results of the present experiments do not consistently support the
idea that the optic fibre projections to a virgin tectum are established under
the influence of pre-existing tectal positional markers, it will be helpful to
know how the fibres innervating a virgin tectum are arranged in the tract.
Experiments to determine this are in progress.
We thank Mrs June Colville for expert histological assistance.
REFERENCES
S.-H. & COOKE, J. (1978). Observations on the formation of the brain and of nerve
connections following embryonic manipulation of the amphibian neural tube. Proc. R.
Soc. Lond. B 201, 335-373.
FELDMAN, JOAN D. & GAZE, R. M. (1972). The growth of the retina in Xenopus laevis: an
autoradiographic study. II. Retinal growths in compound eyes. /. Embryol. exp. Morph.
27, 381-387.
GAZE, R. M., FELDMAN, JOAN D., COOKE, J. & CHUNG, S.-H. (1979O). The orientation of
the visuotectal map in Xenopus: developmental aspects. J. Embryol. exp. Morph. 53,
39-66.
GAZE, R. M., KEATING, M. J. & CHUNG, S.-H. (1974). The evolution of the retinotectal
map during development in Xenopus. Proc. R. Soc. Lond. B 185, 301-330.
GAZE, R. M., KEATING, M. J., OSTBERG, A. & CHUNG, S.-H. (19796). The relationship
between retinal and tectal growth in larval Xenopus: implications for the development
of the retinotectal projection. /. Embryol. exp. Morph. 53, 103-143.
GAZE, R. M. & STRAZNICKY, C. (1980a). Stable programming for map orientation in disarranged embryonic eyes in Xenopus. J. Embryol. exp. Morph. 55, 143-165.
GAZE, R. M. & STRAZNICKY, C. (19806). Regenerating optic nerve fibres from a compound
eye to both tecta in Xenopus: evidence relating to the state of specification of the eye and
the tectum. J. Embryol. exp. Morph. 60, 125-140.
GAZE, R. M. & STRAZNICKY, C. (1980C). The innervation of a virgin tectum by a compound
eye in Xenopus. J. Physiol. Lond. 301, 20P.
CHUNG,
Innervation of virgin tectum
21
P. D. & FABER, J. (1967). Normal Table of Xenopus laevis (Daudin). Amsterdam:
North-Holland.
SCALIA, F. & FITE, K. (1974). A retinotopic analysis of the central connections of the Optic
nerve in the frog. /. comp. Neurol. 158, 455-477.
SCHMIDT, J. T. (1978). Retinal fibers alter tectal positional markers during the expansion
of the half retinal projection in goldfish. / . comp. Neurol. 177, 279-299.
SHARMA, S. C. (1981). Retinal projection in a non-visual area after bilateral tectal ablation
in goldfish. Nature, 291, 66-67.
SPERRY, R. W. (1951). Mechanisms of neural maturation. In Handbook of Experimental
Psychology (ed. S. S. Stevens), pp. 236-280. New York: Wiley.
SPERRY, R. W. (1963). Chemoaffinity in the orderly growth of nerve fiber patterns and
connections. Proc. natn. Acad. Sci., U.S.A. 50, 703-710.
SPERRY, R. W. (1965). Embryogenesis of behavioural nerve nets. In Organogenesis (ed.
R. L. De Haan & H. Ursprung). New York: Holt, Rinehart & Winston.
STEEDMAN, J. G. (1981). Pattern formation in the visual pathways of Xenopus laevis. Ph.D.
thesis, Faculty of Science, University of London.
STRAZNICKY, K. (1978). The acquisition of tectal positional specification in Xenopus. Nfuroscience Letts. 9, 177-184.
STRAZNICKY, K. & GAZE, R. M. (1971). The growth of the retina in Xenopus laevis: an
autoradiographic study. /. Embryol. exp. Morph. 26, 67-79.
STRAZNICKY, K. & GAZE, R. M. (1972). The development of the tectum in Xenopus laevis:
an autoradiographic study. /. Embryol. exp. Morph. 28, 87-115.
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, 253267.
STRAZNICKY, C , GAZE, R. M. & KEATING, M. J. (1981). The development of the retinotectal projections from compound eyes in Xenopus. J. Embryol. exp. Morph. 62, 13—35.
STRAZNICKY, C. & TAY, D. (1981). Spreading of neuroretinal projections in the ipsilateral
tectum following unilateral enucleation: a study of optic nerve regeneration in Xenopus
with one compound eye. / . Embryol. exp. Morph. 61, 259-276.
WILLSHAW, D. J. & VON DER MALSBERG, C. (1979). A marker induction mechanism for the
establishment of ordered neural mappings: its application to the retinotectal problem.
Phil. Trans. R. Soc. Lond. B 285, 203-243.
NIEUWKOOP,
(Received 28 September 1981, revised 2 December 1981)