/. Embryol. exp. Morph. Vol. 26, 1, pp. 67-79, 1971
Printed in Great Britain
57
The growth of the retina in Xenopus laevis:
an autoradiographic study
By K. STRAZNICKY 1 and R. M. GAZE 2
From the Neurobiological Research Unit, Department of Physiology, University
of Edinburgh and the Department of Anatomy, University of Pecs, Hungary
SUMMARY
The growth of the retina has been studied in Xenopus by use of autoradiography with
tritiated thymidine. At the time when retinal polarization first occurs (around stage 30) there
are only some 20 ganglion cells across the retinal equator and the rest of the retina develops
later, by annular addition of cells at the ciliary margin. This process continues beyond metamorphosis.
INTRODUCTION
The regeneration of retinotectal connections has been extensively studied in
amphibians and it is well established that a fibre projection of approximately
normal retinotopic arrangement may be re-established across the tectum when
the optic nerve fibres have been cut and allowed to regenerate. The most widely
accepted hypothesis which has been proposed to account for these findings is
the hypothesis of neuronal specificity (Sperry, 1943, 1944, 1945, 1951, 1965).
According to this hypothesis the retinal ganglion cells each acquire a specific
cytochemical individuality during neurogenesis, the tectal neurons also become
cytochemically specified in a comparable and matching fashion, and the
eventual outgrowth of axons from the developing retina allows similarly specified
retinal and tectal neurons to link synaptically.
Such a hypothesis obviously tends to focus our interest on the events taking
place in retina and tectum during the critical stages of embryonic and larval
life. We need to know more about the mode of development or retina, tectum
and the optic nerve joining them. The nature of the retinotopic fibre projection
in the adult amphibian is well known from electrophysiological analysis (Gaze,
1958; Gaze & Jacobson, 1963; Gaze, Jacobson & Szekely, 1963; Cronly-Dillon,
1968). But whereas in the adult animal we are dealing with a fibre projection that
is, if not static in terms of its connectivity pattern, at least quiescent, this is by
no means the case during larval development. During larval life the eye and the
tectum both grow in size and acquire more cells. And it seems likely that a study
1
Author's address: Department of Anatomy, University Medical School, Szigeti ut 30,
Pecs, Hungary.
2
Author's address: National Institute for Medical Research, Mill Hill, London, N.W. 7,
U.K.
5-2
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K. STRAZNICKY AND R. M. GAZE
Retinal growth in Xenopus
69
of the mode of this growth and the temporally ordered sequence of extra connexions that must form between the expanding retina and the expanding tectum,
could help us to understand the mechanisms that exist to control the development of the ordered connexions found in the adult. As a first step in this direction this paper examines the mode of growth of the retina, studied by means of
autoradiography with tritiated thymidine (3H-T).
METHODS
Eggs were produced by appropriate treatment of Xenopus with chorionic
gonadotrophin and the resulting embryos were reared in the laboratory, in some
cases until after metamorphosis. Single injections of 3H-T were made into the
belly region of young larvae and intraperitoneally in older larvae and juveniles.
Larvae were staged according to the normal tables of Nieuwkoop & Faber
(1956). At stage 30 or younger, 1 /tCi was injected per animal; at stages 33 and
35, 2/tCi; at stages 40 and 45, 5/tCi; at stages 53, 58, 61 and after metamorphosis, 10 /tCi. The 3H-T had a specific activity of 5 Ci/m mole. Injections into
older larvae and juveniles were made using an ordinary 1 ml tuberculin syringe
with a 25 G hypodermic needle, while injections into young larvae were made
using a glass micropipette with a plunger driven by a micrometer screw. Animals
were injected at stages 30, 33, 35, 40, 45, 53, 58, 61, 66 (metamorphosis) and 3
weeks after metamorphosis (juvenile). In the Results section we consider
animals injected at stages 30, 35, 45, 58 and 3 weeks after metamorphosis, as
these were representative of the complete series. Several animals were injected
at each stage and these were then killed at selected intervals after injection. The
stages at which animals were killed for autoradiography corresponded to those
given above. In some animals cumulative labelling with two to three injections
between stages 27 and 29 was performed. The animals were reared at approximately 20° C.
Tissues were fixed in Carnoy's fixative for 3-24 h, then rapidly processed,
cleared in CHC13, embedded in paraffin wax, cut at 3-6 /im and mounted on
slides. Deparaffinized sections were coated with Ilford Nuclear Research
Emulsion G5 and were exposed at 5°C for 3-9 weeks before being developed.
The sections were then stained with cresyl fast violet.
In all the illustrations the bar represents 100/*m
Fig. 1. Eye from an animal labelled at stage 30 and killed 2 h later.
Fig. 2. Eye from an animal labelled at stage 30 and killed at stage 35.
Fig. 3. Fundus of the eye from an animal labelled at stage 30 and killed at stage 66
(metamorphosis). The arrows indicate the regions of labelled cells adjacent to the
optic nerve head (ON). The rest of the retina was unlabelled.
Fig. 4. Optic nerve head (ON) from an animal labelled at stage 30 and killed
3 months after metamorphosis. Arrows point to groups of labelled cells. The rest
of the retina was unlabelled.
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K. STRAZNICKY AND R. M. GAZE
N „.
Retinal growth in Xenopus
71
RESULTS
The mode of growth of the eye has been studied mainly from sections cut
vertically so as to include the ciliary margins of the retina above and below the
lens and at the same time to include the maximal diameter of the eye posteriorly.
In such sections the number of ganglion cells can be counted across the equator
of the eye, from the ciliary margin on one side to that on the other.
Label given at stage 30
At stage 30 the eye has only some 20 ganglion cells across the equator. If the
animal is given 3H-T at stage 30 and killed for autoradiography 2 h later the
eye is found to be extensively labelled (Fig. 1). If an animal is labelled at stage
30 and killed at stage 35, autoradiography reveals massive labelling near the
ciliary margins, the relative distribution of the label indicating that the cells
farthest away from the margin have undergone fewer divisions than those at the
margin (Fig. 2). Most of the ganglion cells to be seen across the equator are
unlabelled and thus were probably formed prior to the administration of the
3
H-T. Animals labelled at stage 30 and autoradiographed at or 3 months after
metamorphosis show that the retinal label is then confined to a small region
around the exit of the optic nerve at the back of the eye (Figs. 3, 4). Thus the
entire extent of the retina of the stage 30 eye eventually comes to comprise only
this small disk of retina around the optic nerve head in the postmetamorphic
animal, and all the rest of the juvenile retina has developed later than stage 30.
There are some 300-400 ganglion cells across the equator of the retina in a
6-month juvenile.
Label given at stage 35
If an animal is labelled at stage 35 (when there are 20-30 ganglion cells across
the equator) and killed 2 h later, autoradiography shows heavy labelling at the
ciliary margins while the central 20 or so ganglion cells in the section are unlabelled (Fig. 5). By stage 45 the continuing cell division at the ciliary margin
has diluted the label somewhat in this region and the mass of labelled cells
appears farther towards the fundus (Fig. 6). In the animal illustrated there were
some 40 ganglion cells across the retinal equator, of which the central 20 were
mostly unlabelled and had thus been formed before administration of the label.
The labelled edges of the retina had been formed between stages 35 and 45.
The retina grows very rapidly at this period in the animal's development and
Fig. 5. Eye from an animal labelled at stage 35 and killed 2 h later.
Fig. 6. Eye from an animal labelled at stage 35 and killed at stage 45.
Fig. 7. Eye from an animal labelled at stage 35 and killed at stage 48.
Fig. 8. Optic nerve head (ON) from an animal labelled at stage 35 and killed three
months after metamorphosis. Arrows indicate groups of labelled cells. The rest
of the retina was unlabelled.
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K. STRAZNICKY AND R. M. GAZE
Retinal growth in Xenopus
73
Fig. 7 shows the distribution of retinal label at stage 48. Here there are some
66 ganglion cells across the retinal equator. The central 20 cells are mostly
unlabelled, having undergone their final DNA synthesis before administration
of the thymidine at stage 35. The two fringes of labelled ganglion cells now
appear to be displaced considerably towards the fundus, due to the extensive
mitosis which has occurred at the ciliary margin and has added on new, unlabelled (and dilutely labelled) cells at the edge of the retina. By 3 months after
metamorphosis the retina is completely free of label except for a small group of
cells gathered round the optic nerve head (Figs. 8, 9).
Label given at stage 45
When H-T is given at stage 45 and the animal is killed 2 h later, the distribution of retinal label is as shown in Fig. 10. Essentially the labelled cells are
confined to the ciliary margin, where mitosis and DNA synthesis are occurring.
Occasionally labelled cells can be found in the fundus of the retina; these may
represent glial elements developing later than neural cells (Jacobson, 19686).
At this stage there are some 40 ganglion cells across the retinal equator and they
are all unlabelled. By stage 61 extensive further growth of the retina gives a
result such as that shown in Figs. 11, 12 and 13. The two pools of labelled cells
indicate what part of the retina was actively proliferating when the label was
given at stage 45; the central, unlabelled, part of the retina (37 cells across) had
been formed prior to administration of the label and the unlabelled edges,
comprising the greater part of the retina, have developed since the thymidine
was given. In Fig. 11 the dorsal unlabelled edge of the retina contains 80
ganglion cells while the ventral unlabelled edge contains 65. In this section there
were approximately 200 ganglion cells across the whole equator of the retina.
3
Label given at stage 58
Growth of the eye in Xenopus continues up to and after metamorphosis,
although the rate of addition of new retinal cells slows down in the later phases
of growth. Fig. 14 is taken from an animal to which 3H-T had been given at
stage 58, shortly before metamorphic climax, and which had been killed 24 h
later. The retinal margin is seen to be actively proliferating. If the animal has
been labelled at stage 58 and allowed to survive until 3 months after metamorphosis, the retinal label is then found to occupy a position some short way in
Fig. 9. Section through fundus, close to optic nerve head, from a different animal,
labelled at stage 35 and killed 3 months after metamorphosis. Arrows indicate
labelled regions of retina.
Fig. .10. Eye from an animal labelled at stage 45 and killed 2 h later. Arrows indicate
groups of labelled cells at the ciliary margins of the retina.
Fig. 11. Eye from an animal labelled at stage 45 and killed at stage 61. Arrows
indicate bands of labelled cells in the fundus.
Fig. 12. Higher magnification of the fundus shown in Fig. II.
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K. STRAZNICKY AND R. M. GAZE
Retinal growth in Xenopus
75
from the ciliary margin (Fig. 15), thus indicating again that further proliferation
has occurred at the edge of the retina. Fig. 15 also illustrates a common finding,
which is that the labelled cells in the bipolar and receptor layers extend farther
towards the fundus than do the labelled cells in the ganglion cell layer.
Fig. 17. Enlargement of box A from Fig .16. Arrows indicate the site of labelled cells
in the ganglion cell layer and in the bipolar cell layer.
Fig. 18. Enlargement of box B from Fig 16. Arrow shows the site of labelled cells.
Fig. 13. Fundus and optic nerve head (ON) from an animal labelled at stage 45 and
killed at stage 6.1. Arrows indicate narrow bands of labelled cells, one on each side of
the optic disk.
Fig. 14. Ciliary margin of the retina from an animal labelled at stage 58 and killed
24 h later. A group of labelled cells is shown at the ciliary margin. The rest of the
retina was unlabelled.
Fig. .15. Segment of retina from an animal labelled at stage 58 and killed 3 months
after metamorphosis. The part of retina shown is some distance in from the ciliary
margin, which is beyond the top of the photomicrograph. The arrows indicate the
positions of the labelled cells farthest from the ciliary margin in each of the three
layers of the retina.
Fig. 16. Eye from an animal labelled at stage 58 and killed 3 months after metamorphosis. The labelled regions of retina are indicated by boxes A and B and these
regions are shown, at higher magnification, in Figs. 17 and 18.
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K. STRAZNICKY AND R. M. GAZE
Throughout its development the eye grows in a fashion which is asymmetrical
about the optic nerve head. This is well shown in Figs. 7, 10 and 11 and is
further illustrated in Fig. 16. In this case the label was given at stage 58 and the
animal killed 3 months after metamorphosis. The labelled regions on each side
of the retina are shown in Figs. 17 and 18. On the dorsal limb of the retina
(Fig. 17) the labelled cells are some way from the ciliary margin, while on the
ventral limb (Fig. 18) they are much closer to the edge of the retina. More cells
have thus been added to the dorsal edge than to the ventral edge of the retina.
It is perhaps worth mentioning that cells labelled at any particular time form
more or less distinct groups in the retina (Figs. 3, 12 and 15). This may indicate
that cells which had undergone their final mitosis at a given time kept their
relative positions and did not move farther away from each other or spread over
a large sector of the retina.
M
S58
23-36 W
Fig. 19. Synoptic diagram indicating the temporal mode of growth of the retina in
Xenopus. The stage of the retina and the size of the subdivisions are only approximately correct. S, stage of development (Nieuwkoop & Faber, 1956); M, metamorphosis; M + 3, 3 months after metamorphosis; D, days; W, weeks.
Further observations
Cumulative labelling performed between stages 27 and 29 resulted in those
cells developing before stage 30 being massively labelled. Autoradiography of
such animals at various stages of larval life suggested to us that the numbers of
labelled cells did not decrease significantly over a period of up to 12 weeks. This
persistence of labelling was also found consistently in the other groups of animals
(Figs. 2-3, 7-9) and indicates that no considerable proportion of these cells later
degenerate and disappear.
Retinal growth in Xenopus
77
The growth of Xenopus eye continues after metamorphosis for an undetermined period. Animals given 3H-T 3 weeks after metamorphosis (at stage 66 + )
and killed 48 h later, show labelled cells present at the ciliary margin of the retina.
COMMENT
The Xenopus sensory retina grows throughout larval and into postmetamorphic life by the addition of cells to all three layers at the ciliary margin. The
development of the retina is shown diagrammatically in Fig. 19, which represents
a vertical section through the eye, lens and optic nerve.
It can be seen that the ontogenetically oldest cells are grouped around the
exit of the optic nerve. This group of cells is followed by progressively younger
cells as we go towards the retinal margin. These results are in accord with previous autoradiographic studies on developing amphibian (Jacobson, 1968 b),
chick (Fujita & Horii, 1963), and mouse (Sidman, 1961) eye, which showed that
mitosis first ceases in the central zone of the retina. The present results show that
after stage 35 in Xenopus, newly formed retinal cells originate entirely from the
retinal margin. The situation thus resembles that found in the regenerating adult
newt retina (Gaze & Watson, 1968) and in the developing retina of Rana
(Hollyfield, 1968). This latter author has shown labelled cells moving from the
retinal margin into the inner nuclear towards the fundus of the eye. Apart from
the sort of situation shown in Figs. 15 and 17, we have found no evidence of
intraretinal cell movement of Xenopus.
Glticksmann described cell degeneration in the early stages of the developing
frog eye (Gliicksmann, 1965) and assumed that new cells would form to replace
the degenerated ones. Our observations do not support the idea of any considerable degeneration of differentiated cells in the retina of Xenopus; if this were to
happen, noticeable numbers of cells labelled at an early larval stage should have
disappeared by the time of or after metamorphosis. Indeed, it would be surprising if any significant amount of late cell degeneration, or of intraretinal cell
movement were to occur, since this would upset the topological relationship
between retina and tectum.
From the present analysis of retinal growth two problems become obvious.
The first has to do with the' specification' of retinal ganglion cells and the second
with the way in which the developing retina connects with the developing brain.
During development the eye connects with the brain in an orderly fashion. In
Xenopus, this orderly fibre projection is such that the nasal part of the retina
connects with the caudal part of the optic tectum; temporal retina connects with
rostral tectum; inferior retina connects with dorsal tectum and superior retina
connects round the lateral edge of the tectum. This orderly projection may be
restored when an adult optic nerve is cut and allowed to regenerate and there is
much evidence (reviewed in Gaze, 1970) to suggest that each ganglion cell
'knows' where it is in the retina and 'knows' when its axon has got to the right
78
K. STRAZNICKY AND R. M. GAZE
place on the tectum. The system behaves, in other words, as though the ganglion cells of the retina are specified in a positional sense. Furthermore, this
positional labelling of the retinal ganglion cells occurs at a known stage of
development. Jacobson (1968 a) has shown that the eye becomes polarized across
the nasotemporal axis at stage 30 and across the dorsoventral axis a few hours
later. If an eye from a stage 28 embryo is rotated, the animal later shows
normal vision. If the eye is rotated in a stage 32 embryo, the animal later shows
visual behaviour that is upside-down and back-to-front. Thus at about stage 30
polarization of the eye takes place and thereafter the ganglion cells will normally
only connect with their appropriate regions of tectum; i.e. they behave as if they
have been specified.
The initial formation of ganglion cells in Xenopus, which occurs at about stage
30, was studied autoradiographically by Jacobson (19686), who showed that the
ganglion cell precursors underwent their final DNA synthesis shortly before the
retina became polarized across the nasotemporal axis. However, at stage 30 only
a minute part of the cells that will make up the adult retina have been formed
(Fig. 19). Jacobson (19686) claimed that cumulative labelling (of Xenopus eye)
started at stage 30 and continued over several days gave no labelling of ganglion
cells; and furthermore, he stated that by stage 35 DNA synthesis had ceased in
all the cells which later differentiated into receptor cells. Both these statements
would appear to be incorrect in view of the results described in the present
paper. Simple consideration of the differences between the size of the eye at
stage 35 and in the adult must indicate that all layers of the stage 35 retina
receive many new cells to permit adult proportions to be achieved and this
addition of cells is illustrated in Figs. 15, 17 and 18. Jacobson (19686) held
that, at stage 29, DNA replication ceases in all the neuroblasts which later
differentiate into ganglion cells, with the exception of a small percentage at the
periphery of the retina. This ' small percentage' is what we are concerned with in
the present paper; and it gives rise to almost the whole of the adult retina, which
develops after retinal polarization has occurred. And since the rest of the eye
develops with a polarity consistent with the initially polarized part (even if
rotated), it would seem that the 'specification' of the later developing ganglion
cells is transmitted to the new cells somehow as they appear. The details of this
process of specification are completely unknown but may be related to the
differing life histories of the two daughter cells that result from the division of a
cell at the retinal margin. Thus, the autoradiographic evidence suggests that,
of two such daughter cells, the one nearer the fundus heads in the direction of
differentiation, while the cell nearer the margin of the retina will divide further,
and so on.
The second problem to be raised by this study is that of how the developing
retina connects with the brain. The adult projection from retina to tectum has
already been mentioned; the edges of the retina project in order round the edges
of the tectum and the centre of the retina projects (approximately) to the centre
Retinal growth in Xenopus
79
of the dorsal surface of the tectum. But when optic nerve fibres first reach the
tectum (shortly after stage 35), only the central regions of the retina have yet
been formed. All the rest of the retina is added later, at the edges. Thus, if the
initially innervated piece of tectum is the place to which the central retinal
fibres project later in the animal's life, then the tectum should also grow at the
edges, to accommodate, in a proper order, the newly arriving fibres from the
retinal margins. The mode of growth of the tectum in relationship to the development of the retina is presently being investigated.
We would like to thank Miss E. M. Forrest for her expert histological assistance. K.
Straznicky was a Wellcome Research Fellow for J969.
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