/ . Embryol. exp. Morph. Vol. 27, 2, pp. 381-387, 1972
Printed in Great Britain
381
The growth of the retina in Xenopus laevis,
an autoradiographic study
II. Retinal growth in compound eyes
By JOAN D. FELDMAN 1 AND R. M. GAZE 1
From the National Institute for Medical Research, London
SUMMARY
The retina of Xenopus laevis has previously been shown, using autoradiographic methods,
to develop in the normal animal by the annular addition of cells at the ciliary margin.
The development of the retina in animals with surgically produced 'compound eyes' was
subsequently studied. In these animals the eye cup was split along the dorsoventral axis and
the resulting half-eyes were recombined so as to form animals with a double-nasal eye.
The retina in experimental animals was found to develop as in the normal animal. No
labelling of cells with radioactive thymidine was seen along the cut edge of each half-eye;
thus in terms of cell division each half of the compound eye remains a half.
INTRODUCTION
We are concerned with the mode of growth of the retina and with the mode of
development of the connexion pattern between retinal ganglion cells and the
tectum in Xenopus. In a normal Xenopus the connexions that form between
ganglion cells in the retina and the tectum lead to the existence, in the adult, of
the well-organized retinotectal projection which has previously been described
(Gaze, Jacobson & Szekely, 1963; Gaze, Keating, Szekely & Beazley, 1970).
During normal development in Xenopus the eye connects with the contralateral tectum in such a way that, in the adult, most-nasal retina sends fibres to
most-caudal tectum while most-temporal retina connects with most-rostral
tectum; central retina projects to central tectum. In normal animals the retina
increases in size throughout larval development by the addition of cells at the
ciliary margin (Straznicky & Gaze, 1971). This process continues until after
metamorphosis. Thus the retinal cells that project, in the adult, to the rostral
and caudal poles of the tectum are among the youngest cells in the retina, having
differentiated late in development; whereas the retinal cells which project, in the
adult, to the central regions of the tectum are among the oldest cells in the retina,
having differentiated early in development.
In Xenopus embryos at developmental stage 32 (Nieuwkoop & Faber, 1967)
1
Authors' address: Division of Developmental Biology, National Institute for Medical
Research, Mill Hill, London NW7 1AA, U.K.
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J. D. FELDMAN AND R. M. GAZE
it is possible to make 'compound eyes' by, for instance, removing the temporal
half of the eye cup and replacing it, in dorso-dorsal orientation, with a nasal
half-eye taken from the opposite side of another embryo at the same stage of
development (Gaze, Jacobson and Szekely, 1963, 1965; Gaze, 1970) so that a
double-nasal (NN) compound eye results. Other varieties of compound eye can
be constructed in a comparable fashion.
In Xenopus with such surgically produced NN or TT\ (double-temporal)
compound eyes, the projection from the compound eye to its contralateral
tectum in the adult animal is abnormal (Gaze, Jacobson & Szekely, 1963,1965).
If the eye is NN, then both nasal and temporal extremities of the retina project
to the caudal pole of the tectum, while cells along the vertical midline of the
retina project to the rostral tectal pole. These midline retinal cells in an NN eye
thus connect to that part of the tectum which, in a normal animal, receives the
projection from the most temporal retinal cells.
The midline retinal cells in an NN eye thus behave, in relation to the connexions
they form, as do cells from the temporal pole of a normal eye. A comparable
statement can be made about the entire ganglion-cell population of each half
of the compound retina, in that its tectal projection resembles that from a whole
normal eye in terms of tectal extent and the ordering of the connexion pattern
(Gaze, Jacobson & Szekely, 1963, 1965).
Two alternative mechanisms have been put forward to account for the
connexion pattern formed by compound eyes (Straznicky, Gaze & Keating,
1971). In one, each half-retina comprising the compound eye is deemed to
undergo pattern regulation in that the cells at the midline take on the ' specificity
characteristics' of the cells at the missing pole of the eye; in the other view,
regulation of this sort is held not to occur and the connexion pattern actually
formed is determined by a matching of the polarity and the extent of the available
retina to the available tectum.
The normal developing Xenopus retina is surrounded by a ring of precursor
cells at the ciliary margin which mitose during development and thus add to the
extent of the neural retina (Straznicky & Gaze, 1971). Since each half of the
compound eye projects to the tectum as if it were a whole normal eye, and in
view of the possibility that each half of a compound eye is a regulated system in
terms of cellular positional information, we thought that it would be worthwhile
to find out whether each half of the compound eye also resembles a normal eye
in being surrounded by a ring of retinal precursor cells. Cells from the temporal
pole of the normal eye take part in the growth of the eye whereas midline cells do
not. We have therefore investigated the mitotic history of cells at the cut edge of
the compound NN eye, using tritiated thymidine as a marker.
Fig. 1. Autoradiograph of the eyes of a Xenopus larva. [3H]thymidine was given at stage 47 and the tissues were prepared for autoradiography 24 hours later.
(A) Normal eye; the eye is unlabelled except for the ciliary margins, which are shown in higher magnification in B and C.
(D) Compound (NN) eye; the eye is unlabelled except for the ciliary margins, which are shown in higher magnification in E and F. The
darkly staining cells in the fundal part of the retina are degenerate cells; they are not labelled.
In each eye the plane of section passes through the optic nerve head. The bar in each photograph represents 50 /*m.
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J. D. FELDMAN AND R. M. GAZE
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Retinal growth in compound eyes 0/Xenopus
385
MATERIALS AND METHODS
The methods employed have been described in earlier papers (Gaze, Jacobson
& Szekely, 1963; Straznicky & Gaze, 1971).
In brief, double-nasal compound eyes were made by removing the temporal
hah0 of an eye in a stage 32 (Nieuwkoop & Faber, 1967) Xenopus laevis embryo
and replacing it with a nasal half-eye from the opposite side of another stage 32
embryo. At stage 45 or 47, 5 /tCi of tritiated thymidine (specific activity 20 Ci/
mmole) were injected into the ventral surface of the larvae in the gut region, and
8 animals were killed 24 h after injection. Tissues were fixed in Carnoy's fixative
and serial sections were cut at 4 fim. Deparaffinized sections were then coated
with Ilford Nuclear Research Emulsion G5 and were exposed for 3-9 weeks
before being developed. Sections were then stained with haematoxylin and eosin.
RESULTS
The retinae of the normal eye (Fig. 1 A, B, C) were similar to those reported by
Straznicky & Gaze (1971). In summary, at this stage there are about 40 ganglion
cells across the retinal equator (as seen in transverse sections) and these are all
unlabelled, since they had undergone their final DNA synthesis before administration of the thymidine. Labelled cells are to be found at the ciliary margins,
where active incorporation of thymidine was going on at the time the label was
given.
The findings in the retinae of compound eyes were essentially similar to those
Fig. 2. Schematic representation of the growth of the normal eye and the compound
(NN) eye in Xenopus.
(A) Growth of a normal eye. On the left is shown the small eye of a stage 32 larva.
Although such an eye would have several ganglion cells along the naso-temporal
axis of the retina, in this figure the eye is divided into only two parts, temporal and
nasal, and these are labelled Tl and N l . Growth of the eye involves adding on
successive rings of new cells at the retinal margin, as shown in the diagrams on the
right. The highest numbers indicate the most-nasal or most-temporal cells.
(B) Growth of a compound NN eye in which regulation is assumed not to occur.
Each half-eye is considered to consist of nasal (initially N l , Nl) cells only. The
addition of new cells occurs in rings as in the normal eye. The eye ends up with all its
ganglion cells 'nasal' and none 'temporal'.
(C) Growth of a compound NN eye in which regulation is assumed to occur, followed
by mitosis along the cut edge of each half-eye. The addition of new cells follows, for
each half-eye, the pattern shown for a normal eye (A). In this case two apposed
'normal' eyes with opposite nasotemporal polarity would result.
(D) Growth of a compound NN eye in which regulation is assumed to occur and
there is no mitosis at the line of junction between the two half-eyes. In this case, since
the cells established originally as N1, T1 in each half-eye remain present throughout
the further growth of the eye, a continuous manifestation of retinal regulation will be
needed to provide the pattern shown for the adult. The 'name' of each ganglion cell
must thus change each time a new ring of cells is added at the margin.
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J. D. FELDMAN AND R. M. GAZE
in the normal eyes. Some disorganization of structure was apparent within the
retina, but labelling of cells remained confined to the periphery of the retina and
no labelled cells were seen at the position of the cut edges of the two half-eyes
(Fig. 1 D , E , F ) .
DISCUSSION
The results of the present experiments show that the cells at the midline of an
NN compound eye, in contradistinction to the cells at the temporal pole of a
normal eye, do not take part in the cell division leading to the growth of the eye.
Each half of a compound eye is thus not equivalent to a whole eye in terms of its
mitotic pattern.
This experimental finding adds support to our previous arguments (Straznicky,
Gaze & Keating, 1971) that retinal pat-tern regulation is unlikely as an explanation for the connexion pattern found in compound eyes. Retinal pattern regulation is a hypothesis which has been invoked to account for the production of a
full scale of' specificity' values along half the normal extent of the appropriate
retinal axis (compare Fig. 2C and D with Fig. 2 A and B). In the case of NN
eyes, retinal pattern regulation would result in two complete scales of such values,
one covering the half-axis of each half-eye.
In this case, the absence of mitosis around the cut edge of each half-eye makes
it necessary to postulate not only an initial retinal regulation, but also a
continual manifestation of retinal regulation throughout the period of growth
of the eye; that is, until after metamorphosis. In Fig. 2C initial regulation of the
compound eye, followed by the addition of new rings of cells as in two normal
juxtaposed eyes, has been shown diagrammatically. This can be contrasted with
the picture of continuous regulation which must be hypothesized if the idea of
retinal pattern regulation is to be maintained in the light of the experimental
finding that mitosis only occurs at the free edge of the compound eye. This
process demands a continuous change of 'specificity label' of all retinal cells
each time that a new ring of cells is added to the retinal margin. We have therefore concluded that it seems more likely that retinal pattern regulation does not
occur and that retinotectal connexions are formed by some mechanism which
takes into account the polarity and extent of available retina (in the present
circumstances, a half-retina) and tectum.
REFERENCES
GAZE, R. M. (1970). The Formation of Nerve Connections. London: Academic Press.
GAZE, R. M., JACOBSON, M. & SZEKELY, G. (1963). The retinotectal projection in Xenopus
with compound eyes. /. Physiol., Lond. 165, 484-499.
GAZE, R. M., JACOBSON, M. & SZEKELY, G. (1965). On the formation of connexions by
compound eyes in Xenopus. J. Physiol. (Lond.) 176, 409-417.
GAZE, R. M., KEATING, M. J., SZEKELY, G. & BEAZLEY, LYNDA. (1970). Binocular interaction
in the formation of specific intertectal neuronal connections. Proc. R. Soc. B 175, 107147.
Retinal growth in compound eyes of Xenopus
387
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NIEUWKOOP,
(Manuscript received 14 July 1971)