/. Embryol. exp. Morph. Vol. 28, l,pp. 87-115, 1972
Printed in Great Britain
87
The development of the tectum in Xenopus
laevis: an autoradiographic study
By K. STRAZN1CKY 1 AND R. M. GAZE 2
From the National Institute for Medical Research,
Mill Hill, London
SUMMARY
The development of the optic tectum in Xenopus laevis has been studied by the use of
autoradiography with tritiated thymidine. The first part of the adult tectum to form is the
rostroventral pole; cells in this position undergo their final DNA synthesis between stages
35 and 45 or shortly thereafter. Next, the cells comprising the ventrolateral border of the
tectum form. These cells undergo their final DNA synthesis at or shortly after stage 45.
Finally the cells comprising the dorsal surface of the adult tectum form, mainly between
stages 50-55. This part of the tectum originates from the serial addition of strips of cells
medially, which displace the pre-existing tissue laterally and rostrally. The formation of the
tectum is virtually complete by stage 58.
The tectum in Xenopus thus forms in topographical order from rostroventral to caudomedial. The distribution of labelled cells, several stages after the time of injection of isotope,
indicates that, at any one time, a segment of tectum is forming which runs normal to the
tectal surface and includes all layers from the ventricular layer out to the surface. In Xenopus,
therefore, the times of origin of tectal cells appear to be related not to cell type or tectal layer
but to the topographical position of the cells across the surface of the tectum.
INTRODUCTION
The factors controlling the establishment of orderly neuronal connexions
between the eye and the brain have been investigated extensively by study of
the regeneration of the retinotectal projection in adult amphibians and fishes
(for references, see Gaze, 1970). Comparatively little work has so far been done
on the original formation of these connexions during neurogenesis: and the
reasons for this neglect are partly practical and partly historical. It has been
easier to investigate the regeneration of fibres in the adult visual system than to
use embryonic or larval material. A main aim of studies on optic nerve regeneration, however, has been to reveal mechanisms that may be concerned in the
original establishment of connexions in part of the nervous system during
development: and one of the foremost hopes (more or less explicit) of the
investigators working in this field has been that information obtained from the
1
Author's address: Anatomy Department, University of Zambia, Box 2379, Lusaka,
Zambia.
2
Author's address: The National Institute for Medical Research, Mill Hill, London,
NW7 1AA, U.K.
K. STRAZNICKY AND R. M. GAZE
S
C
R
Fig. 1. Diagram to show the suggested mode of retinotectal connexion, on the
assumption that the tectum, like the retina, grows in rings. Retina is on the left;
tectum is on the right. Stages of development shown range from 1, the first appearance of a retinotectal. connexion, to 4, the adult projection. N, S, T, I: nasal,
superior, temporal, inferior; C, R: caudal, rostral. The arrows represent the
retinotectal connexions.
study of nerve regeneration in adult animals may be immediately relevant to the
investigation of how the nerve connexions form in the first place.
Development and regeneration are different things however: and it remains
no more than a reasonable guess that similar mechanisms may be at work in
the two situations. Therefore it is a good idea to study the development of nerve
interconnexion by the most direct means possible: and the present paper is one
of a series in which we attempt to do this.
Development of the tectum in Xenopus
89
In adult Xenopus (as in other vertebrates) there is an orderly projection of
retinal fibres to the optic tectum. This projection is such that the nasal extremity
of the visual field (temporal extremity of the retina) projects to rostral tectum;
temporal field projects to caudal tectum: superior field to medial tectum and
inferior field projects round the lateral edge of the tectum. The centre of the
field (and thus the centre of the retina) projects more-or-less to the centre of the
tectal surface.
We have recently shown (Straznicky & Gaze, 1971) that the retina in Xenopus
grows by the addition of rings of cells at the ciliary margin: and moreover this
process continues until after metamorphosis. Thus the mode of growth of the
retina is concentric; and the oldest ganglion cells in the retina are those which,
in the adult, are gathered round the optic nerve head. We also know (Gaze &
Peters, 1961) that visuomotor responses may be elicited in Xenopus at stage 49
(Nieuwkoop & Faber, 1956) of larval life; so some form of ordered connexion
must exist between eye and brain at this stage. But the retina at stage 49 is
small and the greater part of the adult retina develops later than stage 49. Since
the retina grows by the addition of concentric rings of cells and since, in the adult,
concentric rings of retina project to concentric rings of tectum (centred on the
tectal mid-point), it becomes of considerable interest to find out how the tectum
grows.
The simplest assumption would perhaps be that the tectum should also grow
in rings (Fig. 1) such that the original piece of retina to connect with the
tectum would do so with that piece of tectum which, in the adult, would be
central tectum. Newly developing rings of retina could then project, in order,
to newly developing rings of tectum and in this way the orderly nature of the
projection could be maintained from the start.
In the present paper we describe a study of the development of the optic
tectum in Xenopus, using autoradiography with tritiated thymidine. The results
indicate that the tectum differs from the retina in that it does not grow in rings.
The implications of this are considered in the Discussion.
METHODS
Larvae of Xenopus laevis of various stages were each given a single injection,
into the belly, of methyl-tritiated thymidine (3H-TdR), specific activity 20 Ci/mM.
The youngest animals each received 1 /tCi, older tadpoles 5 /*Ci, the oldest
larvae 10/*Ci and metamorphosed forms 15/tCi. In a few cases the larvae
received each two injections between the stages 27-29. A complete list of all the
stages analysed in the present experiments is given in Table 1.
Larvae were kept at approximately 20 °C, were fed with filtered Heinz baby
soup and were staged according to the Normal Table of Nieuwkoop & Faber
(1956). After administration of the 3H-TdR, larvae were killed for immediate
autoradiography within 2 h (stages 35 and 45) or within 24 h or 36 h for later
90
K. STRAZNICKY AND R. M. GAZE
Table 1. The distribution of experimental animals in the series, with stage of
labelling (L), stage at which the animals were killed (K), and number of animals
in each class
WPM: weeks post metamorphosis; MPM: months post metamorphosis.
L
K
No.
27-29
35
40
45
60
2 WPM
1
1
1
1
2
30
35
2
30
45
45
48
52
55
61
66
3 MPM
No.
2
2
1
1
4
2
2
2
40
3
45
58
3
1
61
1
66
1
33
33
37
45
2
1
2
35
35
3
40
45
48
40
K
L
51
51
66
2
2
52
60
9 MPM
1
3
3 MPM
6 MPM
1
1
54
55
55
3
4
7
2
58
58
3 MPM
2
1
64
1
62
62
2
40
45
61
2
3
1
3 WPM
7 WPM
2
1 MPM
1 MPM
1
stages. Otherwise the animals were kept until they had reached a predetermined
stage before being killed.
Up until stage 48 whole embryos were fixed in Carnoy's fixative. From stage 50
upwards the heads of the embryos and juveniles were fixed in Susa for 3-24 h
according to the size of the specimen. Specimens from stage 58 were decalcified
or the brain of juvenile animals was removed from the skull. Tissues were
rapidly processed and embedded in paraffin wax. Serial sections were cut at
5-10 /tm in the sagittal, horizontal or coronal plane. Deparaffinized sections
were coated with Ilford G5 emulsion and were exposed at 4 °C for 4-12 weeks.
Autoradiographs were then processed in Kodak D76 developer and afterstained
with cresyl fast violet or with hematoxyline-eosin, and mounted in synthetic
resin.
The distribution maps of labelled tectum (Figs. 11, 13, 19, 21, 25) were prepared in the following manner. The outlines of every fifth section were drawn
Development of the tec turn in Xenopus
91
1
t:::
o
)
Rostral
Fig. 2. (a) Expanded diagram showing three transverse sections through the tectum,
rostral, mid-caudal and caudal; to indicate the position and extent of labelling
following administration of thymidine in the early stage 50's and autoradiography
several stages later. Big dots represent heavy labelling, small dots light labelling.
(b) Transverse section through a labelled tectum to show the various distances that
were measured to permit the construction of the maps shown in Figs. 11, 13, 19,
21 and 25.
by the use of a camera lucida and on each outline the position of the labelled
'wedge' of tectal tissue was marked. Fig. 2a shows a diagrammatic representation of three such outlines from a series made in this way. Next, the various
distances shown in Fig. 2b were measured (in mm) from the outline drawings.
These distances were then transferred to mm graph paper. A line representing
the tectal midline was drawn and the measurements for each section placed the
measured number of mm to right or to left of the midline. Each section was
positioned 5 mm from the preceding one. Thus the measurements of the final
diagram are arbitrary and the magnification is not the same in both major axes.
The overall results are 'tectum-shaped' however and serve to show the distribution of label adequately.
RESULTS
In Xenopus the first retinal ganglion cells to cease DNA synthesis do so at
stages 28-29 (Jacobson, 1968). By stage 35 optic nerve fibres can be seen at
the region of the chiasma. The first histological evidence of the development of
a tectal structure in the midbrain may be seen between stages 40 and 45, when
cells may be seen apparently migrating out from the central cell mass to meet
the arriving optic nerve fibres (Fig. 3 a, b). In Xenopus, the development and
maturation of the layered tectal structure is dependent on the arrival of optic
nerve fibres. If one eye is removed by stage 30, before it has sent axons to the
brain, then the corresponding (contralateral) tectum does not develop properly
(Fig. 4): the superficial or opticus layer remains thin and the tectum in later
life appears smaller than normal.
92
K. STRAZNICKY AND R. M. GAZE
3a
Development of the tectum in Xenopus
93
Administration of3H-TdR at stage 35
Administration of 3H-TdR at stage 35, followed by autoradiographic preparation of the tissues within 2 h, shows that at this time most cells in the region
that will form the tectum are incorporating the label (Fig. 5). Grains are
sparsely distributed over the 'tectal' roof and are present in greater quantities
at the ventrolateral margin of the 'tectal' region. We may follow the eventual
distribution of these labelled cells, or of their immediate progeny, by labelling
at stage 35 and autoradiographing at various intervals thereafter. Thus by
stage 45 the rostral pole of the tectum is marked by the appearance of cells in
the white matter (largely optic fibres) which have presumably migrated out from
the central cell mass. If label is given at stage 35, these rostral tectal cells are
labelled at stage 45 (Fig. 6a, b). The cells of the central cell mass in the tectal
region are also labelled, and with a distribution that shows the most heavily
labelled cells along the ventrolateral margin of the tectum and the less heavily
labelled cells over the tectal roof. This distribution pattern can only be seen
where the general level of heaviness of labelling is light: in animals where the
level of labelling is heavy, most cells in the tectal central cell mass appear
labelled, including the ventricular layer.
At stage 48 labelled cells are to be found at the rostral pole of the tectum, in
the white matter. Labelled cells also exist in the central grey matter and again
these show a graded distribution - most heavy labelling being ventrolateral,
with the degree of labelling tailing off dorsally and caudally (Fig. la, b).
Fig. 3. (a) Transverse section through rostral 'tectum' in a stage-41 Xenopus.
Many of the fibres comprising the white matter are optic afferents. A few cells can
be seen separating from the central cell mass as the fibres pass into the dorsal
mesencephalon. 15 /tm section, Holmes's silver method. The bar represents 50 /tm.
(b) Transverse section through the rostral tectum in a stage-46 Xenopus. The earliest
formation of tectal layering may be seen. Holmes's silver method. Bar represents
50 /tm.
Fig. 4. Transverse section through both tecta to show the differences that result
from early enucleation of one eye. The eye contralateral to the tectum on the right
of the photograph was enucleated at stage 29/30, before any neuronal connexion had
formed with the brain. The animal was then killed for histological examination at
stage 49/50. The superficial (opticus) layer of the normally innervated tectum (left
in the photograph) is of normal thickness whereas the superficial layer of the
deprived tectum is thin. Holmes's silver method. Bar represents 100 /im.
Fig. 5. Autoradiograph of transverse section of 'tectum' from a tadpole injected
with 3H-TdR at stage 35 and killed 2 h later. The midline is to the left of the
photograph. There is extensive labelling in the ventrolateral 'tectum' and scattered
grains over the cells of the 'tectal' roof. There is no proper tectal structure at this
early stage. Bar represents 50 /tm.
Fig. 6. (a, b) Transverse sections through the dorsolateral part of the rostral pole of
the tectum in two tadpoles, each injected at stage 35 and killed at stage 45. In (a) the
surface of the tectum is to the left of the photograph; in (b) it is to the right. Labelled
cells are seen in white matter and in grey matter. Bar represents 50 /*m.
94
K. STRAZNICKY AND R. M. GAZE
Development of the tectum in Xenopus
95
By stage 64, just before the end of metamorphosis, label given at stage 35 is
still to be found at the rostroventral pole of the tectum (Fig. 8 a, b). No other
cells in the tectum are labelled. Thus cells taking up the label at, or shortly after,
stage 35 are found later at the rostroventral pole of the tectum and they do not
further migrate.
Administration of3H-TdR at stage 45
If 3H-TdR is given at stage 45 and the animal is killed 2 h later, labelled cells
are found throughout the ventricular lining and among cells adjacent to the
ventricle across the entire tectum. Fig. 9a shows the rostral tectal pole in such
an animal. The cells in the tectal white matter are unlabelled and labelled cells
are confined to the ventricular region. Further caudal in the same preparation
the optic ventricle can be seen and this also shows label in the ventricular and
adjacent cells (Fig. 9b).
By stage 48 the label which was administered at stage 45 is to be found
rostrally in the tectum (Fig. 10 a, b). Transverse sections show that cells in both
grey and white matter are labelled at the rostral pole at this stage (Fig. 10c) and
that as we go caudally the localized region of heavy labelling moves away from
the midline, out laterally (Fig. 10d, e). In each case the heavily labelled region
comprises a wedge of tectum from the innermost grey region to the outermost
white region: and in each case there is evident a graded distribution of label
such that heaviest label is most lateral, with lighter label towards the midline.
The distribution of tectal label, in dorsal view, is shown diagrammatically in
Fig. 11.
At stage 52 a comparable distribution of labelled cells is seen, with the
rostral pole of the tectum labelled (Fig. 12a) and the 'full-thickness' wedge of
Fig. 7. (a) Parasagittal section through rostral pole of tectum of a tadpole injected
at stage 35 and killed at stage 48. Dorsal is upwards and rostral is to the left. Labelled
cells may be seen in white and grey matter near the rostral pole of the ventricle. Bar
represents 100/tm. (b) Transverse section through rostral pole of tectum of a tadpole injected at stage 35 and killed at stage 48. Dorsal is upwards and the midline
is to the right. Heavy labelling is seen in white and grey matter, ventrally in tectum.
A graded diminution in the heaviness of labelling is seen as we go dorsally from
the ventral wedge of heavily labelled tissue. Bar represents 100 [im.
Fig. 8. (a) Parasagittal section through the optic tectum in a tadpole injected at
stage 35 and killed at stage 64. Dorsal is upwards and rostral is towards the left.
The only labelled cells to be seen in the tectum are at the rostroventral pole, in the
region shown by the inset. This region is shown at higher magnification in 8 (/>). Bar
represents 200 /tm. (b) High-power view of labelled cells from region shown in the
inset of Fig. 8a. Bar represents 50/*m.
Fig. 9. (a) Transverse section through rostral tectum in a tadpole injected at stage 45
and killed two hours later. Only the ventricular layer and some adjacent cells are
labelled. Bar represents 50 jim. (b) The same animal, further caudal. The optic ventricle is shown and is surrounded by labelled cells. Bar represents 50 fim. In both
photographs dorsal is upwards and medial is to the right.
96
K. STRAZNICKY AND R. M. GAZE
10b
Development of the tectum in Xenopus
97
Rostral
Fig. 11. Distribution map of labelled tectal tissue in a tadpole injected at stage 45
and killed at stage 48. The diagram, prepared as described in the section of
'Methods', shows the tecta in dorsal view. The heavy black line represents the
distribution of labelled cells as determined from measurements made on serial
transverse sections (see Fig. 2 a, b).
labelled tectum becomes more lateral as we go caudally in the tectum (Fig. \2b,
c). At stage 52 the tectal distribution of labelled cells is shown diagrammatically
in dorsal view in Fig. 13 which shows that the caudal half of the tectum is completely unlabelled.
At stage 55 and later stages, label given at stage 45 is found at the rostral pole
of the tectum, ventrally. This is shown for stage 55 in Fig. 14 a and b; for stage 61
in Fig. 15 a and b; for stage 66 in Fig. 16 a and b; and for the 3-month postmetamorphic juvenile in Fig. 17. In each of these cases the labelled cells are
situated at the rostroventral pole of the tectum. Dorsal tectum is unlabelled and
there is a gradient of distribution of label such that the most heavily labelled
cells are rostral and the degree of labelling tails off as we go more caudally
round the ventrolateral border of the tectum (Fig. 16). Thus we can say that
cells incorporating label at, or shortly after, stage 45 come eventually to occupy
the rostroventral pole of the tectum and to form part of its ventrolateral
margin.
Fig. 10. Tectal autoradiographs of animals labelled at stage 45 and killed at stage 48.
(a) Parasagittal section through tectum; dorsal is upwards and rostral towards the
left. The only labelled cells to be seen are enclosed in the inset. This region, at the
rostral pole of the tectum, is shown at higher magnification in (b). Bar represents
100 /tm. (b) High-power view of the inset region in (a). Labelled cells are to be seen
in white and grey matter. Bar represents 50/tm. (c, d, e) Transverse sections
through the tectum of a different animal. Dorsal is upwards and medial towards
the left, c, Most rostral; d, further caudal; e, more caudal still. The labelled region
lies more ventrolateral the more caudal the section. Bar represents 100 /tm.
Fig. 12. (for Fig. 12 b, c, see p. 98). Transverse sections through the tectum of a
tadpole injected at stage 45 and killed at stage 52. In all photographs dorsal is
upwards and medial is towards the left, (a) Rostral pole. Labelled cells in both
white and grey matter. Bar represents 50 /tm. (b) Further caudal. The band of
labelled cells is more ventrolateral than in (a). Bar represents 50/tm. (c) Most caudal.
The labelled tissue is more ventrolateral than in (b). Bar represents 50 /tm.
EMB
28
98
K. STRAZNICKY AND R. M. GAZE
12c
15a
99
Development of the tectum in Xenopus
Rostral
Fig. 13. Distribution map of labelled tectal tissue in a tadpole injected at stage 45 and
killed at stage 52. The conventions for this and the following maps are the same as
for Fig. 11. The dotted outline represents the ventricle.
Administration ofzH-TdR at stage 51
If 3H-TdR is injected at stage 51 and the animal is killed 36 h later the labelled
cells are found only in the ventricular and adjacent cells of the tectum (Fig. 18 ad). No other cells in the tectum are labelled at this stage.
If such an animal is kept until stage 66 (end of metamorphosis) before being
autoradiographed, we may find the eventual distribution of the cells which were
labelled at, or shortly after, stage 51. It may be seen that the rostral pole of the
tectum is unlabelled and that there is a more-or-less linear distribution of
labelled cells, running from near the midline rostrally to far lateral caudally
(Fig. 19<7, b). The labelled region comprises a wedge of cells extending from the
ventricular surface to the outer surface of the tectum. Transverse sections
show the labelled region to be well-localized across the tectal surface and there
is a graded distribution of label such that most heavily labelled cells are lateral
and less heavily labelled cells are closer to the midline (Fig. 20).
It is thus possible to say, from the position and graded distribution of the
labelled cells, that the rostrolateral tectum was formed before the label became
available at stage 51; the labelled cells were forming at about that time; and the
caudomedial tectum was formed after stage 51.
For description of Fig. \2b, c see p. 97.
Fig. 14. (a) Horizontal section showing distribution of labelled cells in an animal
injected at stage 45 and killed at stage 55. Rostral is towards the top of the photograph and medial is to the left. The section is ventral in the tectum. The labelled
cells are enclosed in an inset and this region is shown at higher magnification in
(b). The heavy black mark in the tectum just caudal to the inset is an artifact. Bar
represents 100 /am. (b) Higher magnification of the labelled region of rostroventral
tectum shown in (a). Bar represents 50 /tm.
Fig. 15. (a) Horizontal section showing the distribution of labelled cells in an
animal injected at stage 45 and killed at stage 61. Rostral is towards the top of the
photograph and medial is towards the right. The labelled cells are at the rostral
pole of this ventral section and are enclosed in an inset which is shown at higher
magnification in (b). Bar represents 100/*m. (b) Higher magnification of the region
of inset in (a) (rotated 90° clockwise). Bar represents 50 /tm.
7-2
100
K. STRAZNICKY AND R. M. GAZE
Development of the tectum in Xenopus
Rostral
101
Rostral
Fig. 19. (a, b) Distribution maps of labelled tectal tissue from two animals injected at
stage 51 and killed at stage 66 (metamorphosis).
Administration of*H-TdR at stage 52
A distribution comparable to that described for stage 51 may be seen if the label
is given at stage 52 and the animal is kept alive until stage 60. In this case the
distribution (in dorsal view) of the full-thickness wedge of labelled tectum is
shown in Fig. 21 and the nature of the labelled wedge of tissue, as well as the
graded distribution of label in it, is shown in Fig. 22a-c. If an animal is
labelled at stage 52 and kept alive until 9 months after metamorphosis the
terminal distribution is similar to the previous case in that the group of labelled
cells is still to be found in the dorsal tectum some variable way out from the
midline (Fig. 23a, b).
Whereas the 'full-thickness' wedge of labelled tectum indicates clearly that
Fig. 16. (a) Horizontal section showing distribution of labelled cells in an animal
injected at stage 45 and killed at stage 66 (metamorphosis). Rostral is towards the
top of the photograph and medial is towards the right. The most heavily labelled
cells are at the rostral pole. The region enclosed in the inset is shown at higher
magnification in (b). Bar represents 100 /tm. (b) Higher magnification of inset in (a).
Bar represents 100/tm.
Fig. 17. Horizontal section showing distribution of labelled cells in an animal
injected at stage 45 and killed 3 months after metamorphosis. Rostral is towards
the right of the photograph and the lateral surface is uppermost. The labelled
cells are at the rostral pole of the tectum. Bar represents 100 /tm.
Fig. 18. The distribution of labelled cells in the tectum of an animal injected at
stage 51 and killed 36 h later, (a) Transverse section through rostral tectum. The
labelled cells are in and adjacent to the ventricular layer. The inset region is shown
at higher magnification in Fig. 18 b. Bar represents 100 /tm. (b) Higher magnification
of the inset region in (a). Bar represents 50 /tm. (c) Further caudal in the same preparation. The inset region is shown at higher magnification in (d). Bar represents
100/tm. (d, p. 102) Higher magnification of inset region in Fig. 18c. Bar represents
50/tm.
102
K. STRAZNICKY AND R. M. GAZE
Development of the tectum in Xenopus
103
Rostral
Fig. 21. Distribution map of labelled tectal tissue from an animal injected at
stage 52 and killed at stage 60.
the tectum comprising the labelled area is forming at one time, without any
apparent difference between the various tectal layers, yet a significant difference
can be found between those animals labelled at stage 52 and those labelled at
stage 51. In the former, in addition to the serial addition of new tectum caudomedial to the labelled wedge, there occur frequent labelled cells distributed
fairly widely in the superficial opticus layer lateral to the edge of the main
labelled region. These sporadic labelled cells may be lightly labelled, as if they
had undergone several label-diluting divisions. The distribution of labelled
cells to lateral and to medial of the lateral border of the main labelled wedge is
such that lateral to it, few cells are labelled, while medial to it, virtually all
cells are labelled; the edge of the region is thus very obvious to the eye
(Fig. 24a-d).
Administration ofzH-TdR at stage 54
Animals labelled at stage 54 and killed at 3 months after metamorphosis
show a wedge of labelled cells, of full tectal thickness, running from near the
midline rostrally to somewhat lateral, caudally (Fig. 25). As with animals
For description of Fig. \%d see p. 101.
Fig. 20. Transverse section showing part of the lateral edge of the optic tectum
from an animal injected at stage 51 and killed at stage 66. Dorsal is upwards and
lateral is to the left. A wedge of labelled cells extends from the ependyma to the
outer tectal surface. Bar represents 50 /tm.
Fig. 22. Transverse section showing labelled tectum from an animal injected at
stage 52 and killed at stage 60. (a) Low-power view. There is a 'full-thickness wedge'
of labelled tissue enclosed in the inset. The most heavily labelled cells are laterally
placed in this wedge and the heaviness of labelling decreases towards the tectal
midline. Bar represents 100/*m. (b) Higher power view of the inset region in (a).
Lateral to the wedge of heavily labelled cells, most cells are unlabelled; medial to
the wedge, virtually all cells are labelled and the labelling decreases in a graded
fashion medially. Bar represents 50 /tm. (c) High-power view of the medial part of
the tectum on the other side in the same preparation. The distribution of label is
similar to that of (6) but the direction of the gradient is now reversed. Bar represents
50 /tm.
104
K. STRAZNICKY AND R. M. GAZE
22 c
For description of Fig. 22c see p. 103.
Fig. 23. (a) Transverse section through the tectum in an animal injected at stage 52
and killed nine months after metamorphosis. Dorsal is upwards and lateral is to the
right. The only labelled cells to be found are enclosed in the inset which is shown at
higher magnification in (b). Bar represents 100 /tm. (b) Higher magnification of the
inset region in (a). Bar represents 50 /on.
Development of the tectwn in Xenopus
105
labelled at stage 52, those labelled at stage 54 show many sporadically distributed labelled cells in the outer tectal layer, lateral to the main wedge of
labelled tissue (Fig. 26).
Administration ofzH-TdR at stage 55
Animals labelled at stage 55 and killed 36 h later show labelled cells confined
to the ventricular and adjacent region of the optic tectum, most prominent
caudally (Fig. 27).
Administration of3H-Td.R at stage 58
Animals labelled at stage 58 and killed 24 h later show tectal labelling which
is sparse and confined to the ventricular layer and adjacent cells (Fig. 28). As
late as 3 months after metamorphosis, label given at stage 58 is still confined to
cells in the ependyma of the tectum, except for a small number of cells near the
caudal midline, which show label from ependyma out to superficial white
matter (Fig. 29).
Administration of^H-TdR one month after metamorphosis
Label injected at this time results in the appearance, when autoradiography
is initiated within 24 h of the injection, of a very few scattered labelled cells
in the tectal ependyma.
DISCUSSION
The observations described in the present paper are concerned with the
anatomical position taken up by cells undergoing their final DNA synthesis
at various stages of development. Factors relevant to the interpretation of
3
H-TdR autoradiography, especially with reference to the determination of cell
birthdays, have been discussed by Sidman (1970), LaVail & Cowan (1971) and
Fujita (1964) and some of these factors may usefully be considered here, before
we attempt to assess the meaning of the observations on tectal growth in
Xenopus.
The pulse-labelling technique, as used in this investigation, is a useful way of
determining cell birthdays. We assume that those cells synthesizing DNA at the
time the label is made available to them will incorporate it. Since, at any given
time of administration of the label, some of these cells will be undergoing their
final DNA synthesis and others will be destined to go through the cell cycle
once or more times before their final DNA synthesis, we would expect to find in
the adult, as a result of the administration of a pulse of 3H-TdR at some stage
of development, a distribution of cells showing variously heavy, moderate and
light labelling.
In the present experiments we find just such a distribution of heavily labelled,
moderately labelled and lightly labelled cells (Figs. 7, 10, 16, 22, 24) and we
are entitled to assume that, in most cases, relative density of labelling reflects
106
K. STRAZNICKY AND R. M. GAZE
Development of the tectum in Xenopus
107
Rostral
Fig. 25. Distribution map of labelled tectal tissue from an animal injected at stage 54
and killed three months after metamorphosis.
relative time of birth of the cells involved. This assumption is supported by
the following factors:
(1) Injections have been made on a comprehensive series of stages, ranging
from before the first appearance of the tectum until adult life. Autoradiographic
analysis has also been performed, in many cases, at a series of developmental
stages after the time of injection.
(2) This study accounts for the existence of all cell types in the tectum. Indeed,
the work shows clearly that in Xenopus the time-order of cell birthdays is
mainly related not to cell types but to cell position - that is, to the surface topography of the tectum.
(3) Most important of all, the evidence is internally consistent. There is
a coherent, changing relationship between heavily and lightly labelled cells as
we compare the whole range of results from animals labelled at various stages
and those auto radiographed at various times after administration of the label.
Fig. 24. (a) Transverse section through mid-caudal tectum in an animal injected at
stage 52 and killed at stage 60. There is a 'full-thickness wedge' of labelled tectum
enclosed in the two left-hand insets. The insets are shown at higher magnification
in (b, c and d). Bar represents 100 /*m. (b) Higher magnification of the left-hand
top inset in (a). This is the superficial region of tectum within the 'wedge'. Virtually
all cells are labelled. Bar represents 50 fim. (c) Higher magnification of the righthand top inset in (a). This is superficial tectum lateral to the main labelled area.
Sporadic labelled cells are seen. Bar represents 50 /tm. (d) Higher magnification
of the lower inset in (a), showing the sharp edge of the labelled region and the
graded diminution of label towards the midline. Bar represents 50/*m.
108
K. S T R A Z N I C K Y AND R. M. GAZE
Fig. 26. Transverse section through caudal tectum in an animal injected at stage 54
and killed three months after metamorphosis. Dorsal is upwards and lateral is to
the left. Bar represents 100/^m.
Fig. 27. Horizontal section through caudal tectum in an animal injected at stage 55
and killed 36 h later. Caudal is towards the top of the photograph. The labelled
cells are in and adjacent to the ventricular layer. Bar represents 100 [im.
Development
of the tectum in Xenopus
109
The time-resolution that may be achieved by pulse-labelling depends on
the width of the pulse: that is, on the time after injection for which the thymidine
is available for uptake by the cells. In mammals, this is variously reported as
being less than 4 h (Cronkite, Bond, Fliedner & Rubini, 1959; Messier &
Leblond, 1960). There is some uncertainty in the case of cold-blooded vertebrates. Hay & Fishman (1961) give a figure of 3 h for newts, following intraperitoneal (IP) injection; and this agrees with the results of Yamada & Roesel
(1968). On the other hand, Grillo, Urso & O'Brien (1965) concluded that, in
the newt, labelling may persist for more than a day and that the interval is dosedependent. Moreover, the work of O'Steen & Walker (1961) suggests that after
IP administration in the newt, 3H-TdR may remain available for 5 days. This
matter is obviously not settled; it would seem reasonable that the availability
time may vary with dosage and also with site of administration. In the present
experiments there is considerable doubt about both these factors, in that the
dosages mentioned in the section on methods are the intended doses and
represent a maximum. The actual dose in any animal would be some lower
(possibly considerably lower) figure, since noticeable quantities of the injection
could often be seen to leak out of the animal immediately after administration.
And whereas the site of the injection was meant to be 'intraperitoneal', the
precise location of the tip of the needle was not known in any case, nor was the
nature of the tissue actually receiving the injection. Our preliminary investigations on the time of availability of 3H-TdR in Xenopus larvae indicate that
incorporation of 3H-TdR into the acid-insoluble pool and its availability in the
acid-soluble pool may both continue for 48 h or so (P. Unrau & R. M. Gaze,
unpublished). Since this is the case we will not expect to be able to show much
in the way of time-resolution by this method, when considering young
larvae, because at the beginning of development the animals pass through
several stages per day. Luckily, as they grow the tadpoles slow down the rate
at which they change stages. Thus beyond stages 45-47 there is a period of
several days per stage and this permits an adequate time-resolution of events.
Fortunate also is the fact that, whereas in chick and mouse the development
of the tectum is complete within a matter of days, in Xenopus it takes weeks
or months. This again allows a degree of time-resolution in Xenopus which
would be difficult to achieve in the other two species.
The spatial resolution of the pulse-labelling method, i.e. the number of
labelled cells across the region showing a gradient distribution of grains, will
depend on various factors: (a) the number of further mitoses due to occur in
cells labelled at the time of administration; (b) the pulse-width; (c) the amount
of label injected, or rather the amount available to the cells; (d) the exposure
time of the autoradiographs. Thus in the present experiments a (relatively)
large dose together with a long exposure time will be expected to give a wide
distribution of labelled cells; whereas a small dose together with a short exposure time will be expected to give a very localized distribution of labelled cells.
110
28
K. STRAZNICKY AND R. M. GAZE
Development of the tectum in Xenopus
111
The present experiments give examples of labelling ranging from very localized
to widely distributed; all, however, agreeing with the topographical distribution
of label described in this paper. All the varieties of distribution seen in these
autoradiographs seem thus to be explicable in terms of the various factors
mentioned above.
At all stages studied the cells incorporating label within hours of the administration were in or close to the ventricular layer of the tectum. These cells and/or
their immediate progeny then migrate to take up the positions described in this
paper.
The first part of the tectum to form is the rostroventral pole. 3H-TdR administered at stage 35 or stage 45 becomes located, by stage 48, at this part of
the tectum, where it is found in cells of all tectal layers. These labelled cells
thereafter are found in the same position throughout tadpole life and after
metamorphosis. Thus once they have migrated to their positions in the tectal
roof they do not further migrate. And if label is administered at any of the
sampled times after stage 45, the rostral pole of the tectum does not become
labelled. Thus the cells forming the rostral pole undergo their final DNA
synthesis some time between stages 35 and 45 or shortly thereafter and no
further significant numbers of cells are added to the rostroventral pole after
this.
The next part of the tectum to form is the ventrolateral border. This is shown
by the graded distribution of label found at various stages after administration
of 3H-TdR at stage 45. The fact that, following a single pulse of 3H-TdR at
stage 45, graded labelling can be seen from the rostroventral pole right round
the ventrolateral margin to the caudal half of the tectum (Fig. 16) suggests that
all these labelled cells must have originated within a fairly short period, either
measured temporally or according to stage. Thus the cells of the ventrolateral
border originate at, or shortly after, stage 45; and 3H-TdR administered at any
of the sampled times after stage 45 does not result in this ventrolateral border
of the tectum becoming labelled. So no further significant numbers of cells are
added to this region much later than stage 45.
At around stage 50 and afterwards the cellular components of the dorsal
surface of the adult tectum are forming. The distribution maps (Figs. 19, 21, 25)
show that this happens in the form of a linear wave or wedge of newly formed
tectum, comprising the entire tectal thickness and running from near the midline rostrally to far lateral towards the caudal end of the tectum. The grainFig. 28. Parasagittal section through tectum of an animal injected at stage 58 and
killed 24 h later. Dorsal is upwards and caudal is to the left. Labelled cells are
confined to the ependyma and paraependymal region. Bar represents 100/im.
Fig. 29. Parasagittal section near the midline of the tectum, from an animal injected
at stage 58 and killed three months after metamorphosis. Some labelled cells are
found from the ependyma to the outer edge of the tectum (top of photograph).
Bar represents 50 /tm.
112
K. S T R A Z N I C K Y AND R. M. GAZE
Rostral
Fig. 30. Diagram to show the mode of growth of the tectum in Xenopus. The tectal
outline is seen from above. The heavy lines represent the distribution-contours of
the wedges of labelled tectum resulting from administration of 3H-TdR at the
stages indicated by the numbers. Each distribution-contour thus represents the wedge
of tectum that was forming at the time the label was administered. For each contour
we can say that the whole of the tectum rostrolateral to it was formed before the
time of administration of the label, while that part of the tectum caudomedial to the
line was formed after the time of labelling.
density distribution within the wedge of labelled tissue (Figs. 22, 24) is such as
to indicate that all that part of the tectum rostrolateral to the wedge was
formed before the time of administration of the label, whereas all that part of the
tectum caudomedial to the wedge was formed after it. Thus the final part of the
tectum to form is the caudomedial part of the dorsal roof, and this is virtually
complete by stage 58: The overall pattern of tectal development indicated by
these experiments can thus be summarized in the form of a composite diagram
as in Fig. 30. As may be seen, our results indicate that the formation of the
tectum takes place mainly between stages 45 and 55.
We can say, therefore, that the tectum in Xenopus forms in topographical
order from rostroventral to caudomedial by the serial addition of strips of cells
medially, which displace the pre-existing tissue laterally and rostrally, and
not according to a time-pattern wherein the various layers form separately,
each layer more-or-less complete, over the entire extent of the tectum, as has
been reported for the chick (LaVail & Cowan, 1971). In Xenopus our results
show that at any one time a segment of tectum is forming which runs normal to
the tectal surface and includes all layers from the ventricular layer to the
surface. Furthermore, since we do not find labelled cells elsewhere than in the
ventricular layer and adjacent region within a few hours of administration of
the label, it seems likely that thymidine incorporation and cell division take
place, at all stages, mainly or entirely in the innermost part of the tectum, with
later migration of the labelled cells outwards. This would agree with the
Development of the tectum in Xenopus
113
observation of Kollros (1953) that, in Rana, most of the tectal mitoses are confined to the ventricular layer except in the early stages of development.
The fact that, in Xenopus, at any one time a segment of tectum is forming
which extends from the ventricular surface out to the external surface, immediately brings to mind the columnar anatomico-physiological organization of
the mammalian cerebral cortex. The superficial part of the amphibian tectum
is also organized, physiologically, according to a columnar scheme; beneath
any one surface position on the tectum the various classes of retinal afferents,
all coming from the same region of the retina, end at different depths (Maturana,
Lettvin, McCulloch & Pitts 1960). The present results indicate that the cells
comprising such a tectal column all form at about the same time, possibly from
a common parent cell.
The gradient distribution of labelling that is found several stages after
administration of a pulse of 3H-TdR in the early stage 50's, would be compatible
with the idea that cells may mitose at all levels of the tectal segment, with localization of each mitosis after the first to the medial side of the 'wedge' accounting
for the direction of the tectal gradient. This interpretation is contradicted, however, by the observation that, for several hours after administration, the label
is restricted to the deepest layers of the tectum.
Thus it seems more likely that the formation of a segment of dorsal tectum
is achieved by the passage of a wave of mitosis through the ventricular layer
in the directon rostrolateral to caudomedial. Possibly each mitosing ventricular
cell forms not only the next adjacent ventricular cell towards the medial side,
but also forms a series of cells which then distribute themselves vertically
through this part of the tectum. It is clear that, during the later stages of tectal
development, the initial incorporation of 3H-TdR takes place in and close to
the ventricular layer; it is also clear that the later distribution of labelled cells
forms a full-thickness wedge or slice of tectal tissue. What is not at all clear is
how the initial ependymal distribution becomes converted into the later wedgedistribution.
An intriguing feature of tectal development in Xenopus is the appearance of
sporadic labelled cells in the superficial layer of the tectum lateral to the main
wedge of forming tissue, following administration of the label at stage 52 or
stage 54 in this series. The distribution of grain densities in these sporadically
labelled cells did not appear to fit any obvious pattern and their site of origin is
unknown. Conceivably they may have mltostd in situ; or perhaps they originated
in the ependymal region. It was frequently noticed that a labelled cell was
present in the ventricular layer opposite such a sporadic superficial cell.
The results of the present experiments indicate clearly that the tectum in
Xenopus does not grow in rings; it grows from front to back and from lateral to
medial. These conclusions are compatible with the observation of Kollros (1953)
that, at all larval stages of development, mitoses were more frequent in the
caudal half of the tectum in Rana.
8
E M B 28
114
K. STRAZNICKY AND R. M. GAZE
The mode of growth of the Xenopus tectum is thus very different from that of
the Xenopus retina, which grows by the serial addition of concentric rings of
cells at the ciliary margin (Straznicky & Gaze, 1971). As can be seen from
Fig. 30, central and caudal tectum, to which central and nasal retina project
in the adult, have not yet developed at stages 51-52. Yet central retina exists
at these stages; and it comprises then the same cells that constitute central
retina in the adult animal. These differing modes of growth of retina and tectum
thus pose an interesting problem in neurological topology: how can a sheet of
cells that grows in rings like the retina connect in a continuously expanding
fashion (for retina, tectum and their interconnexion all grow together) with
a sheet of cells that grows differently, like the tectum, and still give rise to the
ordered projection found in the adult? The autoradiographic evidence presented
in this paper, considered in conjunction with the previously published evidence
on the growth of the retina, requires us to say that //the initial retinotectal projection that forms during larval life is ordered in the adult sense, then during
growth of the retina and tectum we must have a continually shifting population
of retintectal connexions. Electrophysiological mapping of the developing
retinotectal projection in Xenopus tadpoles of various stages lends support to
this idea (Gaze, Chung & Keating, 1972).
REFERENCES
E. P., BOND, V. P., FLIEDNER, J. M. & RUBINI, J. R. (1959). The use of tritiated
thymidine in the study of DNA synthesis and cell turnover in haemopoetic tissues. Lab.
Invest. 8, 263-275.
FUJITA, S. (1964). Analysis of neuron differentiation in the central nervous system by tritiated
thymidine autoradiography. /. comp. Neurol. 122, 311-327.
GAZE, R. M. (1970). The Formation of Nerve Connections. London: Academic Press.
GAZE, R. M., CHUNG, S-H. & KEATING, M. J. (1972). The development of the retinotectal
projection in Xenopus. Nature, Lond. 236, 133-135.
GAZE, R. M. & PETERS, A. (1961). The development, structure and composition of the
optic nerve of Xenopus laevis (Daudin). Q. Jl exp. Physiol. 46, 299-309.
GRILLO, R. G., URSO, P. & O'BRIEN, D. M. (1965). The effect of dose on the incorporation
of 3H-thymidine in the nuclei of the liver capsule cells in the newt Triturus viridescens.
Expl Cell Res. 37, 683-685.
HAY, E. D. & FISCHMAN, D. A. (1961). Origin of the blastema in regenerating limbs of the
newt Triturus viridescens. Devi Biol. 3, 26-59.
JACOBSON, M. (1968). Cessation of DNA synthesis in retinal ganglion cells correlated with
the time of specification of their central connections. Devi Biol. 17, 219-232.
KOLLROS, J. J. (1953). The development of the optic lobes in the frog. (1) The effects of
unilateral enucleation in embryonic stages. /. exp. Zool. 123, 153-187.
LAVAIL, J. H. & COWAN, W. M. (1971). The development of the chick optic tectum. II.
Autoradiographic studies. Brain Res. 28, 421-441.
MATURANA, H. R., LETTVIN, J. Y., MCCULLOCH, W. S. & PITTS, W. H. (1960). Anatomy
and physiology of vision in the frog (Rdna pipiens). J. gen. Physiol. 43, Suppl. 129-175.
MESSIER, B. & LEBLOND, C. P. (1960). Cell proliferation and migration as revealed by
radioautography after injection of thymidine-H3 into male rats and mice. Am. J. Anat.
106, 247-285.
NIEUWKOOP, P. D. & FABER, J. (1956). Normal Table of Xenopus laevis {Daudin). Amsterdam : North Holland Publishing Co Ltd.
CRONKITE,
Development of the tectum in Xenopus
115
W. K. & WALKER, B. E. (1961). Radioautographic studies of regeneration in the
common newt. Anat. Rec. 139, 547-555.
SIDMAN, R. L. (1970). Autoradiographic methods and principles for study of the nervous
system with thymidine-H3. In Contemporary Research Methods in Neuroanatomy (ed.
W. J. H. Nouta & S. O. E. Ebbeson), pp. 252-274. Berlin: Springer-Verlag.
STRAZNICKY, K. & GAZE, R. M. (1971). The growth of the retina in Xenopus laevis: an
autoradiographic study. /. Embryol. exp. Morph. 26, 67-79.
YAMADA, T. & ROESEL, M. E. (1968). Labelling of lens regenerate cells grafted into the
newt optic chamber. A study of the availability time of tritiated thymidine. Expl Cell
Res. 50, 649-652.
O'STEEN,
{Manuscript received 21 February 1972)
8-2