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.

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 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.

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.

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.

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.

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.

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 μCi, older tadpoles 5 μCi, the oldest larvae 10μCi and metamorphosed forms 15μCi. 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.

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

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
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

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 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 μm in the sagittal, horizontal or coronal plane. Deparaffinized sections were coated with Ilford G 5 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 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.

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.

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.

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. 3a, 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.

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 μm section, Holmes’s silver method. The bar represents 50 μm. (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 represents50 µm

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 μm section, Holmes’s silver method. The bar represents 50 μm. (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 represents50 µm

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 µm.

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 µm.

Administration of 3H-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. 6 a, 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.

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 µm.

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 µm.

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 µm

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 µm

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. 7a, b).

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 100μm. (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 µm

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 100μm. (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 µm

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.

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 (b). Bar represents 200 μm. (b) High-power view of labelled cells from region shown in the inset of Fig. 8a. Bar represents 50µm

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 (b). Bar represents 200 μm. (b) High-power view of labelled cells from region shown in the inset of Fig. 8a. Bar represents 50µm

Administration of 3H-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).

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 µm. (b) The same animal, further caudal. The optic ventricle is shown and is surrounded by labelled cells. Bar represents 50 µm. In both photographs dorsal is upwards and medial is to the right.

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 µm. (b) The same animal, further caudal. The optic ventricle is shown and is surrounded by labelled cells. Bar represents 50 µm. In both photographs dorsal is upwards and medial is to the right.

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.

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 µm. (b) High-power view of the inset region in (a). Labelled cells are to be seen in white and grey matter. Bar represents 50µm. (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 µm.

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 µm. (b) Further caudal. The band of labelled cells is more ventrolateral than in (a). Bar represents 50µm. (c) Most caudal. The labelled tissue is more ventrolateral than in (b). Bar represents 50µm.

(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 µm. (b) Higher magnification of the labelled region of rostroventral tectum shown in (a). Bar represents 50µm.

(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 100µm. (b) Higher magnification of the region of inset in (a) (rotated 90° clockwise). Bar represents 50 µm.

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 µm. (b) High-power view of the inset region in (a). Labelled cells are to be seen in white and grey matter. Bar represents 50µm. (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 µm.

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 µm. (b) Further caudal. The band of labelled cells is more ventrolateral than in (a). Bar represents 50µm. (c) Most caudal. The labelled tissue is more ventrolateral than in (b). Bar represents 50µm.

(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 µm. (b) Higher magnification of the labelled region of rostroventral tectum shown in (a). Bar represents 50µm.

(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 100µm. (b) Higher magnification of the region of inset in (a) (rotated 90° clockwise). Bar represents 50 µm.

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).

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).

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.

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.

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 labelled tectum becomes more lateral as we go caudally in the tectum (Fig. 12 b, 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. 15a and b; for stage 66 in Fig. 16a and b; and for the 3-month post-metamorphic 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. 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 100µm. (b) Higher magnification of inset in (a). Bar represents 100µm.

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 100µm

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 µm. (b) Higher magnification of the inset region in (a). Bar represents 50 µm. (c) Further caudal in the same preparation. The inset region is shown at higher magnification in (d). Bar represents 100µm. (d, p. 102) Higher magnification of inset region in Fig. 18c. Bar represents 50 µm

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 μm. (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 μm. (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 μm.

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 μm.

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 100µm. (b) Higher magnification of inset in (a). Bar represents 100µm.

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 100µm

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 µm. (b) Higher magnification of the inset region in (a). Bar represents 50 µm. (c) Further caudal in the same preparation. The inset region is shown at higher magnification in (d). Bar represents 100µm. (d, p. 102) Higher magnification of inset region in Fig. 18c. Bar represents 50 µm

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 μm. (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 μm. (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 μm.

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 μm.

Administration of 3H-TdK 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 a – d). 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. 19a, 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).

Fig. 19.

(a, b) Distribution maps of labelled tectal tissue from two animals injected at stage 51 and killed at stage 66 (metamorphosis).

Fig. 19.

(a, b) Distribution maps of labelled tectal tissue from two animals injected at stage 51 and killed at stage 66 (metamorphosis).

Fig. 21.

Distribution map of labelled tectal tissue from an animal injected at stage 52 and killed at stage 60.

Fig. 21.

Distribution map of labelled tectal tissue from an animal injected at stage 52 and killed at stage 60.

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.

Administration of 3H-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).

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 μm. (b) Higher magnification of the inset region in (a). Bar represents 50 μm.

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 μm. (b) Higher magnification of the inset region in (a). Bar represents 50 μm.

Whereas the ‘full-thickness’ wedge of labelled tectum indicates clearly that 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. 24 a—d).

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 (6, c and d). Bar represents 100μm. (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 μm. (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 μm. (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 μm.

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 (6, c and d). Bar represents 100μm. (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 μm. (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 μm. (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 μm.

Fig. 25.

Distribution map of labelled tectal tissue from an animal injected at stage 54 and killed three months after metamorphosis.

Fig. 25.

Distribution map of labelled tectal tissue from an animal injected at stage 54 and killed three months after metamorphosis.

Administration of 3H-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 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).

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 μm.

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 μm.

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 μm.

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 μm.

Administration of 3H-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 of 3H-TdR 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).

Fig. 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 μm.

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 μm.

Fig. 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 μm.

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 μm.

Administration of 3H-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.

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 3H-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 investigation002C 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 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 autoradiographed at various times after administration of the label.

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.

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 grain-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.

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.

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.

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 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 wedge-distribution.

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 mitosed 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.

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 1 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 if 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).

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