the development of the diencephalon in xenopus

Anat Embryol (1982) 163:371-388 Anatomy and Embryology �9 Springer-Verlag 1982

The Development of the Diencephalon in Xenopus An Autoradiographic Study

David Tay and Charles Straznicky

Centre for Neuroscience and Department of Human Morphology, School of Medicine, The Flinders University of South Australia, Bedford Park, S.A. 5042, Australia

Summary. The development of the diencephalon and the time of origin of neurons of thalamic nuclei were determined in Xenopus with 3H-thymidine autoradiography. Isotope was administered into embryos, tadpoles and metamorphic animals and sacrificed after survival time between 24 hours and 5 months. The position and the number of heavily labeled cells, corresponding to terminal mitoses at the time of isotope injection were established on transverse and sagittal sections of the frog brain. Neurons in the diencephalon were distributed in a spatiotemporal manner such that cells generated earliest were located in the caudo-ventro-lateral portion of the diencephalon followed in a rostro-dorso-medial sequence by cells formed at later stages. The waves of cell generation resulted in three apparent developmental gradients in the caudo-rostral, latero-medial and in the ventro-dorsal directions in the diencephalon. Consequently neurons generated latest were found in the rostro-dorso-medial portion of the diencephalon. The overall rostro- dorso-medial diencephalic growth and the spatiotemporal generation of its neurons are the reverse of the tectal growth and cell generation reported in Xenopus which occurs in a rostrolateral to caudomedial direction.

The findings of the present observations appear to indicate that the mirror-image reversal of the retinotectal and retinodiencephalic projections along the temporo-nasal retinal axis is the consequence of the divergent growth of the diencephalon and the tectum from the common embryonic di-mesencephalic junction. These observations furthermore suggest that the orientation of the retinal maps is ensured by the differential maturity gradients in the tecturn and diencephalon, respectively, presumably expressed in molecular terms.

Key words: Autoradiography - Development - Diencephalon - Xenopus

Introduction

The retina projects to the optic tectum and the dieneephalic visual centres in an orderly manner in frogs. The retinotectal and retinodieneephalic maps

Offprint request and correspondence to: Dr. D. Tay

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372 D. Tay and C. Straznicky

have the following characteristics: i) they are ordered such that the neighbour- hood relationships of the retinal ganglion cells are preserved in the tectal and diencephalic distribution of their optic fibre terminals, ii) they cover the whole extent of the tectum and the visual neuropil areas of the diencephalon in adult normal animals and iii)they are oriented in a particular way such that the temporonasal retinal axis projects to the rostrocaudal tectal axis and to the caudorostral axis of the diencephalic neuropil. In contrast, the dorsoventral retinal axis is aligned with the lateromedial tectal and diencephalic axes. (Gaze 1958; Lflzflr 1971; Scalia and Fite 1974; Levine 1980). The retinotopic maps are, therefore, ordered in two dimensions in each of the visual centres. However, the orientation of the retinotectal and retinodiencephalic projections are mirror- image reversal in relation to the temporonasal retinal axis (Scalia and Fite 1974).

The results of previous studies in the retina and in the optic tectum of Xenopus, using 3H-thymidine have contributed to the understanding of the way in which the orderly retinotectal connections are formed. The retina grows by accretion of cells at the ciliary margin throughout the whole life of the animal (Straznicky and Gaze 1971). Retinal growth thus occurs concentrically, such that the oldest cells occupy the retinal centre followed by younger and younger rings of cells towards the retinal periphery. The rectum, on the other hand, is formed by the serial addition of strips of neurons along its caudomedial margin resulting in a rostroventral to caudomedial linear growth (Straznicky and Gaze 1972).

It is thus apparent that the retinal and tectal growth patterns are incongruent. These results suggest that the retinal and tectal spatial relations should undergo continuous readjustment during development in order to maintain the topo- graphical and ordered retinoteetal projections. It has, indeed, been demonstrated by electrophysiological and morphological tests that the retinal projection shifts caudomedially on the developing tectum (Gaze et al. 1974, 1979; Scott and Lflrflr 1976). In the early stages, the retinal projection is restricted to the rostrolateral part of the growing tectum and as development proceeds it spreads caudomedially. Optic fibres from the newly generated peripheral temporal part of the retina terminate in the rostral part of the rectum displacing the pre-existing optic fibres further caudomedially. Peripheral nasal optic fibres, on the other hand, terminate in the caudal part of the growing tectum. At any one time during larval life, therefore, the orderliness of the retinotectal map is maintained, whilst the projection is shifted caudomedially in the growing rectum.

Although there are substantial contralateral and ipsilateral retinodiencephalic projections in frogs, relatively little information is available on how these projections develop during larval life. In Rana pipiens (Currie and Cowan 1975) and in Xenopus laevis (Khalil and Sz6kely 1976) the contralateral retinodiencephalic projections are present from early larval stages in contrast to the delayed formation of ipsilateral retinodiencephalic projections (Currie and Cowan 1974).

The evolution of the retinodiencephalic projections in Xenopus, like that of the retinotectal projections, can be studied morphologically by two different approaches; i) by establishing the growth pattern, the time of origin of neurons in the diencephalon and ii) by studying the formation of the retinodiencephalic projections with autoradiographic tracing techniques. This paper deals with

Development of Diencephalon in Xenopus 373

t he r e su l t s o f t he f i r s t a p p r o a c h , n a m e l y w i t h t he e s t a b l i s h m e n t o f t h e b i r t h d a t e s

o f d i e n c e p h a l i c n e u r o n s a n d t he d e s c r i p t i o n o f t he d i e n c e p h a l i c g r o w t h u s i n g

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M a t e r i a l s a n d M e t h o d s

Xenopus laevis, tadpoles at various stages of development and young post-metamorphic animals were used in this study. Single injections of 3H-thymidine (3H-T) [Specific activity: 23 Ci/mmole, Amersham] were administered into the yolk sacs in embryos and intraperitoneally in older tadpoles and metamorphic toadlets. Animals were staged according to the normal tables of Nieuwkoop and Faber (1956). Several animals were injected at each stage and they were sacrificed after various post-injection survival times (Fig. 1). Animals were kept separately according to the stage at which

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Fig. 1. The different stages at which 3H-thymidine was injected and the time of sacrifice are indicated. M and 3M denote metamorphosis and three months after metamorphosis, respectively. Numbers in brackets give the age of the tadpoles in days after fertilisation. The crosses ( x ) indicate the time of sacrifice of the animals. Thickness of the sections is also given in gm


the isotope was administered and reared to metamorphosis and beyond under standard breeding conditions.

After sacrifice, the head of the tadpole up to stage 55 or the dissected brain of older animal was fixed in Bouins' solution for 24 h, dehydrated and embedded in paraffin. The brains were cut serially (thickness in Fig. 1), in the transverse or longitudinal plane. The closely spaced serial sections were mounted on acid cleaned gelatinized slides and processed for autoradiography. Depar- affinized sections were coated with ILFORD K2 Nuclear Emulsion and exposed at 5 ~ C in light-tight boxes for two weeks. Autoradiographic slides were developed in K O D A K D19 and counterstained with Harris's haematoxylin.

The position and number of the heavily labeled neurons on the autoradiograms were established on animals following isotope administration at various stages of development. Lightly or very sparsely labeled neurons, whose label was diluted by subsequent division/s were not included in the quantitative assessment. For easy comparison, the diencephalon was subdivided along its antero- posterior axis into six portions containing distinctive cytological features.

Abbreviations Used in the Text:

A D L Anterior dorsolateral thalamus O L N Olfactory nerve A E Anterior entopeduncular nucleus ON Optic nerve A S Aqueduct of Sylvius PC Posterocentral thalamus A V L Anterior ventrolateral thalamus PE Posterior entopeduncnlar nucleus CE Cerebellum PL Posterolateral thalamus DH Dorsal habenular nucleus P M Posteromedial thalamus DI Diencephalon PO Preoptic nucleus DL Dorsolateral thalamus POR Preoptic recess D M Dorsomedial thalamus P T Pretectal nucleus FB Forebrain RO Rotundus nucleus HA Habenular nucleus SC Spinal chord H R Hypothalamic recess S M Sulcus medius H Y Hypothalamus SU Suprachiasmatic nucleus IP Interpeduncular nucleus T Tectum MO Medulla oblongata VII Ventral habenular nucleus M S Medial septal nucleus VL Ventrolateral thalamus OC Optic chiasma Z L Zona limitans OL Olfactory lobe I I I Third ventricle

Results

1. The Anatomy of the Diencephalon

A short account on the normal anatomy of the adult Xenopus diencephalon is given here in order to serve as reference to the autoradiographic findings. With some minor modifications, the nomenclature used in the present description is based on the report of Frontera (1952) for the anuran diencephalon and Knapp et al. (1965) on the optic tract in Rana pipiens.

The diencephalon can conveniently be divided into anterior, middle and posterior regions each of them containing discernible rostral and caudal portions (Fig. 2A). In yaung frogs (3 months after metamorphosis, 2-3 cm body length), the whole diencephalon extends for about 660 gm beginning from the union of the third ventricle (III) with the preoptic recess (POR) (Fig. 2B) to the posterior thalamus indicated by the first appearance of the stratified tectal gray (T) (Fig. 2G). The anterior portion of the diencephalon includes the optic

Development of Diencephalon in Xenopus

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Fig. 2. Coronal sections of the diencephalon to demonstrate the positions and relationships of various thalamic nuclei. Figure A is a sketch of the dorsal view of the brain on which the sections (B-G) are taken. Sections on subsequent figures (4 to 9) are also taken at the same level of the brain. Bar represents 500 gm


chiasma (OC) (Fig. 2C), it extends for about 170 gm and ends at the most rostral pole of the hypothalamic gray (HY). The middle portion of the diencephalon encompasses those sections showing the outpocketing of the third ventricle (III) into the hypothalamic recess (HR) (Figs. 2D and 2E) and ends when they appear to be detached from each other (Fig. 2F). The sector from this point to the appearance of the rostral tectal pole (T) (Fig. 2G) is designated as the posterior diencephalon.

The sulcus medius (SM) and the zona limitans (ZL), separate the dorsal (thalamus) and the ventral (hypothalamus) parts of the diencephalon though this border is ill defined in the anterior third (Figs. 2B and 2C). Further subdivi- sion of the Xenopus diencephalon into different thalamic nuclei (Fig. 2) shows a similarity in the overall arrangement of the thalamus among the anurans (Frontera 1952). Owing to the smaller size of the Xenopus brain, these thalamic nuclei are, therefore, correspondingly smaller in extent than those of the Ranid frogs. The hypothalamus and the hypothalamic recess run caudally as far as the tegmental part of the middle third of the mesencephalon.

The thalamic neurons, receiving direct retinal inputs (Fite et al. 1977; Levine 1980), are located in the dorsolateral nucleus (DL), rotundus nucleus (RO), posterocentral (PC), posterolateral (PL) and in the ventrolateral nuclei (VL) of the middle and posterior regions of the thalamus. Neurons of the PC and VL as well as the pretectal nucleus (PT), assumed to project to the tectum (Wilczynski and Northcutt 1977) occupy the caudalmost part of the diencephalon (Figs. 2F and 2G).

2. The Early Development of the Diencephalon

In this section the early development (stages 20 50) of the diencephalon is described with special reference to the time and place of origin of its precursor neurons. Isotope administered to embryos soon after the closure of the neural tube (between stages 20-25), and sacrificed at stage 50 when the general appearance of the adult brain has been formed, resulted in heavy labeling of neurons in the border of the diencephalon and mesencephalon. Cells generated and labeled at stage 25 were located in the rostroventral part of the growing midbrain and in the caudoventral part of the diencephalon, indicating that these neurons had similar birthdates and a common origin (Fig. 3A). Only very few labeled cells were found in the hypothalamus and in the suprachiasmatic areas. Except for the habenular nuclei (HA), the more dorsal and anterior parts of the diencephalon remained unlabeled indicating that these cells were generated after stage 25. Following isotope administration between stages 30-40 labeled cells were located at a more rostral and dorsal position in the diencephalon and were more dispersed than those generated earlier (Fig. 3 B). Injections of 3H-T between stages 4047 resulted in labeling of neurons further rostral and rostrodorsal in the diencephalon. Labeled cells in the rectum were similarly displaced in a caudodorsal direction. Isotope administration at stage 50 with short survival time (5 h) gave very heavy labeling in the neurogenic zone of the diencephalon (Fig. 3C). It is interesting to note that only sporadic labeling was found in HA owing to their precocious development (Fig. 5).


Fig. 3A-D. Parasagittal sections of the brain showing the distribution of the 3H-thymidine labeled cells in the growing diencephalon and tectum. Isotope injection at stage 25 (A) and at stage 40 (B) and sacrificed at stage 50. Injection at stage 50 and sacrificed five hours later (C). Note in (C) that the labeled cells are located exclusively in the ependymal layer. Composite diagram (D) shows the growth pattern of the diencephalon between stages 25 and 50. Hatched area denotes cells generated at stage 25, dotted area between stages 25 and 40 and shaded area between 40 and 50. Arrow in (A) points at the di-anesencephalic junction (DMJ). TV=tectal ventricle. Bar represents 100 ~tm

The observations on the early labeling pattern indicate a sequential cell proliferation starting f rom the di-mesencephalic junct ion as early as f rom stage 20. The subsequent mitoses of the neurogenic cells resulted in a rost rodorsal growth of the diencephalon and a caudomedial growth of the mesencephalon. The time of origin o f neurons and their spatial a r rangement in the diencephalic- mesencephalic regions are indicated in Fig. 3D. The composi te diagram was drawn f rom serial sagittal sections o f stage 50 brains with stages 20-50 isotope administrations. It can be seen that the first g roup of stem cells giving rise to both the diencephalon and the mesencephalon derived f rom the di-mesencephalic junct ion of the developing brain.

3. The Growth of the Diencephalon

In the previous section it was shown that the progenitor cells o f the diencephalon originated f rom the embryonic di-mesencephalic junction. In order to determine the growth of the diencephalon and the birthdates o f its neurons th roughout larval life and after metamorphosis , isotope was injected f rom stages 20 to metamorphosis . The posit ion and distribution o f labeled cells were subsequently established in animals sacrificed at or after metamorphosis . It was generally difficult to resolve the precise regional differences in the distribution of labeled cells in the brains generated only a few developmental stages apart. As indicated


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Fig. 4A-F. Transverse sections of the diencephalon of an animal with stage 20 isotope injection and with metamorphic sacrifice. The left hand side of each picture shows the exact section from which the sketch on the right hand side was drawn. The location of the labeled cells on the autoradiogram was represented by crosses on the stetch to mark their corresponding positions. Note that crosses do not represent individual labeled cells. Sections (A F) were taken from the corresponding levels as shown in Fig. 2 (B G). Bar represents 500 gm

in Fig. 1, there were many over lapping stages at which the injections were carried out, therefore, only selected cases typifying the overall growth trends are to be considered.

4. Isotope Administration at Stages 2 0 ~ 5

Labeled cells were localized a long the lateral side of the rostral d iencephalon, in the lateral marg in of AVL, RO, SU and VL nuclei of the tha lamus (Figs. 4 A - 4C). Fur ther caudally, in the middle diencephalon, the ventrolateral par t of the RO and the D L nuclei of the tha lamus were heavily labeled (Fig. 4C).

Development of Diencephalon in J(enopus 379

Fig. 5. The position and number of labeled cells in the diencephalon after stage 25 injection and metamorphic sacrifice. The crosses in the transverse sections of the ~. diencephalon show the distribution of the labeled cells. The lower part ,~ of the figure shows a histogram

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Still further posteriorly, the entire lateral flank of DL and VL contained heavily labeled cells. These labeled cells formed an oblique band extending in the dorsolateral direction along the boundary of the thalamic gray and the white matter (Fig. 4D). Another group of labeled cells appeared on the dorsolateral part of the hypothalamus and this too formed a band extending almost perpendicular- ly to the other group of cells from the thalamus (Figs. 4D and 4E). Further caudal in the brain, labeled cells were distributed at the anterior pole of the tectum as well as in the ventrolateral half of PC (Fig. 4F).

Generally, many more cells were labeled in the posterior than in the anterior part of the diencephalon. In order to quantify the regional differences in the generation of diencephalic neurons, labeled cells were counted along the rostrocaudal axis of the brain following stages 25-30 injections. On each transverse section the number of heavily labeled cells, representing cells in terminal mitosis at the time of isoptope administration, was established and these numbers were correlated (Fig. 5). It can be seen that the peak of the labeled cell number occurred in the caudal third of the diencephalon from whence the number decreased anteriorly. All the labeled ceils were confined to the ventrolateral margin of the thalamic and to the dorsolateral margin of the hypothalamic gray. The only exception was the habenular complex (HA) in the rostrodorsal part of the diencephalon where labeled cells were located mostly in the DH nucleus (Figs. 4B, 4C and 5).

5. Isotope Administration at Stages 30-40

Most of the labeled cells were located in the lateral boundary of the thalamic gray slightly medially from cells formed before the isotope injection. Few labeled cells were also found more dorsally in the lateral portion of ADL, DM, DL nuclei of the thalamus (Fig. 6). The rest of the dorsal thalamus remained unlabeled but scattered labeling was still noticed in HA. Several labeled cells were present at the most dorsal part of the anterior hypothalamic nucleus (HY).


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The results of the counts of the labeled cells indicated that the peak of the mitotic activity shifted from the caudal to the middle third of the diencephalon (Fig. 6).

6. Isotope Administration at Stages 42 50

At or after metamorphosis, in this group of animals labeled cells occupied the ventromedial strip of the thalamus. Cells formed at these stages were located more medially than those generated between stages 20-40, though they were still some considerable distance away from the ependymal layer of the diencephalon. The central portions of PO, AVL, ADL, VL and DL contained labeled cells with a noticeable higher occurrence in the posterior part of the middle diencephalon. In contrast to earlier stages, PC contained less labeled cells which were dispersed in a more medial and dorsal location. The number of labeled ceils in the hypothalamus, especially in its posterior part, increased compared to earlier stages. Apart from the habenular nucleus, the rostrodorsal part of the diencephalon contained only few labeled cells.


Following isotope administration at stages 52-55 and sacrifice at or after metamorphosis, labeled cells were dispersed in a dorsomedial position along the axis of the diencephalon. In contrast to earlier labeling pattern, labeled cells were scattered over a much wider mediolateral range. This trend was apparent in the VL, PT and DM thalamic nuclei where the neurons were arranged in layers mediolaterally (Fig. 7). Relatively few labeled cells were found in the medial and central core of PC. Occasional labeling was visible in the VH nucleus. Cell counts along the rostrocaudal axis of the diencephalon showed that the highest number of labeled cells were distributed in the anterior third of the thalamus (Fig. 7). Thus, in comparison with earlier stages the site of the highest proliferative activity of the diencephalon shifted more rostrally in the midlarval stages.


Fig. 7. The position and number of labeled cells in the diencephalon after stage 52 injection and metamorphic sacrifice

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8. Isotope Administration at Stage 58

Stage 58 corresponds to the onset on metamorphic climax (Nieuwkoop and Faber 1956). The structural development of the visual centres is almost complete, however, sustained proliferative activity is still maintained in certain areas of the germinal epithelium (Staznicky and Gaze 1972; Tay and Straznicky 1980). As regards to the diencephalon, most of the labeled cells were located in the rostrodorsal part of the thalamus and in the PO. No labeled cells were found in the posterior and ventral parts of the thalamus. The cell production had


also slowed down in HY revealed by the smaller number of labeled cells than previously found (Fig. 8). Labeled cells retained their dorsomedial position three months after metamorphosis as indicated by the presence of heavy labeling of ADL, DM and the medial portion of DL thalamic nuclei. Similar to stages 52-55 isotope administration, labeled cells along the ventricular recess were widely dispersed. The number of labeled cells along the rostrocaudal axis of the diencephalon substantially increased in the rostral portion of the thalamus (Fig. 8).

Since the neurons generated at stage 58 were dispersed over a large part of the medio-lateral extent of the diencephalic gray, the place of generation and the probable active migration of such neurons appeared useful to be determined. Following spot labeling at stage 58 the germinal layer of the rostrodorsal thalamus, POR and the anterior HY and to a much lesser extent, the posterior and ventral thalamus contained labeled neurons. No extraependymal labeling of neurons were found at 6-12 h post-injection survival. The first cells that migrated from the ependymal out to the subependymal layer were seen 24 h after injection (Fig. 9A). Subsequently longer survival for 2, 5 and 10 days after injection resulted in lateralward migration of cells further away from the neurogenic zone (Figs. 9B and C) well into the central core of DM and PM thalamic nuclei (Fig. 9 D).


At the height of the metamorphic climax (stage 62), the proliferative activity of the germinal layer slowed down considerably, though cells were still being added to the diencephalon at its rostrodorsal margin adjacent to the HA area. Sparse labeling also occurred in the HYR. By the end of metamorphosis (stage 66), the mitotic activity of the germinal layer virtually ceased. Occasional labeled cells, assumed to be glial elements, were found along the vascular bed of the diencephalon and in the capillary endothelium.

The summary of results obtained on the spatiotemporal origin of diencephalic neurons is given in a series of composite diagrams (Fig. 10). From early developmental stages onwards the diencephalon clearly showed a caudorostral gradient of neuron generation with a resultant caudorostral spread of highest regional mitotic activity from embryonic stages to metamorphosis. Another time-position gradient was established dorsoventrally. Neurons generated earliest occupied a ventral position in the diencephalon followed dorsally by neurons produced at later stages. Owing to the outwards expansion of the gray, initially by lateral displacement of cells by younger elements, a lateromedial gradient of neuron generation was also observed. However, from midlarval stages many of the newly generated cells migrated outwards to more lateral layers of the diencephalic nuclei causing a substantial overlap in the generation time of cells destined for each set of layers (Figs. 9A-D). The approximate time for the onset and the cessation of neuron generation in major diencephalic nuclei is given in Table 1. These data show that thalamic nuclei situated caudoventrally are generated earlier then those occupying a more rostrodorsal position. The DH and VH nuclei in the epithalamic region are exception due to the very early onset of neuron generation especially in DH nucleus.


Fig. 9A-D. The distribution of heavily labeled neurons after various post-injection survival time following isotope administration into stage 58 tadpoles. All sections are from the middle of the diencephalon. (A) Twenty-four hours survival, labeled cells are in the ependymal and subependymal layers. The migration of labeled cells can be seen in (B) through (D) representing animals sacrificed after 2, 5 and 10 days survival times, respectively. Bar in (D) represents 250 gm applies to all four photographs


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Fig. 10. Composite diagrams showing the age distribution of 3H-thymidine labeled neurons in the diencephalon. Vertical lines in inset A show the planes of sectioning, r, denotes rostral; c, caudal; d, dorsal; v, ventral; and l, lateral. Boxes with distinctive markers represent cells generated at particular stages of development. Bar represent 500 gm

Table 1. The birthdates of neurons of the major diencephalic nuclei in Xenopus shown in developmental stages

Diencephalic nucleus Stages at which cells are labeled

Entopeduncular nucleus 32M5 Dorsolateral thalamic nucleus 32 55 Dorsomedial thalamic nucleus 50-62 Habenular nucleus 25-40 Hypothalamic nucleus 25-50 Interpeduncular nucleus 40 45 Posterocentral thalamic nucleus 32 45 Posterolateral thalamic nucleus 32-40 Posteromedial thalamic nucleus 35-42 Preoptic nucleus 25-58 Rotundus nucleus 30-45 Suprachiasmatic nucleus 32-45 Ventrolateral thalamic nucleus 30-45

Discuss ion

The results of the present experiments concern the spat io temporal generat ion of diencephalic neurons in Xenopus established with the use of 3H-T autoradiography. After injection, 3H- thymidine is available for uptake for about six hours by cells in the postmitot ic phase of D N A synthesis (Dziadek and Dixon 1977). Because of the long proliferative activity of the Xenopus bra in (from early embryogenesis to the beg inn ing of the metamorphosis ) the vast major i ty of cells, labeled at one par t icular stage, undergo several subsequent mitoses result-


ing in the dilution or the complete disappearance of isotope activity following long post-injection survival. In contrast, cells labeled during their terminal mitosis retain the whole amount of isotope activity causing the heavy silver grain deposition over the nuclei of such cells in the autoradiograms. The presence of heavy nuclear label thus corresponds to cells which were undergoing terminal mitosis (birthdates of cells) at the time of isotope administration.

Tritiated thymidine autoradiographic studies in Xenopus have shown a radial or circumferential generation of neurons in the retina from the center towards the periphery (Jacobson 1968, 1976; Straznicky and Gaze 1971; Straznicky and Tay 1977) and a caudomedial linear generation of neurons in the tectum (Straznicky and Gaze 1972). A similar sequential generation of neurons have been reported in the mesencephalic trigeminal neuron pool (Lewis and Straznicky 1979) and in the isthmic nucleus (Tay and Straznicky 1980). Our present results on the growth of the diencephalon show three gradients of which the caudorostral sequence of cell generation is the most apparent. Indeed the caudoventro- lateral portion of the diencephalon is formed first followed by cells generated later and consequently, located in a more rostral position. The heavy labeling in the caudal diencephalon and in the rostral tectum after stages 20-30 injections support the view that the early embryonic precursor cells, giving rise to these areas, originated from the common di-mesencephalic junction of the brain vesi- cles (Picouet 1975). The di-mesencephalic precursor cells subsequently initiate a rostro-medio-dorsal growth of the diencephalon and a caudo-medio-dorsal growth of the tectum (Straznicky and Gaze 1972). However, the direction of the diencephalic and tectal growth can be reversed when the di-mesencephalic region is transposed to the caudal part of the presumptive midbrain plate during early embryogenesis (Chung and Cooke 1978).

During mid-larval stages (stages 50-55) the overall growth of the tadpole speeds up. The eye doubles its size with the corresponding substantial increase of ganglion cells (Jacobson 1976, Straznicky and Tay 1977) and of optic fibres (Wilson 1971). The rapid growth of the tadpole at these stages coincides with an increased proliferative activity of the tectum (Straznicky and Gaze 1972) and with an apparent upsurge of diencephalic cell production seen in the present study.

The neurons of the habenular nuclei are generated from stage 20 onwards, earlier than the adjacent rostrodorsomedial part of the thalamus. The precocious development of the habenular nuclei partly obscures the caudorostral and ventrodorsal gradients of cell generation applicable to the rest of the diencephalon. The rapid expansion of the wall of the diencephalon is assured by two mechanisms; i)neurons generated sequentially in the germinal layer are displaced further and further laterally creating a gradient in the time of origin of cells in an outside-in direction and ii) an active migration of cells from the germinal layer which takes place around midlarval stages. The outward migration of cells in the dorsal thalamus is compatible with the well known inside-out sequence of neuron origin in the mammalian cortex (Berry and Rogers 1965) and with the growth pattern of the tectum (Straznicky and Gaze 1972). In the diencephalon, however, only some of the cells migrate further out, spreading over a wide area in the diencephalic gray. Since we did not attempt to identify the type of neurons reaching their target by active migration, the significance


of the regrouping of the thalamic neurons through postmitotic migration could not be elucidated.

It is worth noting that the spatiotemporal generation of diencephalic neurons in J(enopus has many common features with the development of the mammalian diencephalon. Angevine (1970) has described three gradients; caudorostral, ventrodorsal and lateromedial, in relation to the time of origin in the mouse dorsal thalamus. However, neither the secondary outward migration of neurons in the thalamus, nor the precocious development of the epithalamic region were noticed. Since the generation time of diencephalic neurons in the mouse is five days as compared to 6 weeks proliferative activity of the Xenopus diencephalon, such subtle regional differences in the birthdates of neurons and transitory cell migrations could only be resolved in the latter. Although the sequential generation of cells, especially along the caudorostral and ventrodorsal directions, are more marked in Xenopus than in the mouse, the common developmental patterning of the diencephalon in both animals indicates similar histogenetic trends across different classes of vertebrates.

The way that diencephalic neurons are generated has apparent consequences on the orientation of the retinodiencephalic map. The retina in frogs projects in a retinotopic fashion to the contralateral tectum, to the contralateral and the ipsilateral thalamic regions (corpus geniculatum thalamicum, nucleus of Bellonci), to the pretectal region and to the basal optic nucleus of the tegmentum (Scalia et al. 1968; L~zfir and Sz6kely 1969; L~z~r 1971; Scalia and Fite 1974). Optic fibres of the temporal hemiretina are represented on the rostral and optic fibres of the nasal hemiretina on the caudal part of the tectum. In each diencephalic region the temporal hemiretina projects posteriorly (caudally) and the nasal and ventral hemiretinae anteriorly (rostrally). The temporonasal axis of the retinodiencephalic maps, therefore, represent mirror-image reversal of the temporonasal axis of the retinotectal map.

Golgi studies on the developing tectum in Xenopus have shown that the caudomedial tectal growth is accompanied by a similar cell differentiation gradient (LS, z~r 1973). During development the less differentiated neurons are al- ways located in the caudomedial portion of the tectum. Although no such studies on the diencephalon have yet been carried out, it is reasonable to suppose that the gradient of cell differentiation, in line with the overall growth, is in the caudorostral direction. It is also probable that the rostrocaudal tectal and caudorostral diencephalic maturation gradients have similar time course. In view of this, we suggest the following possible mechanisms to account for the mirror-image reversal of the retinotectal and retinodiencephalic maps.

Direct experimental evidence shows that temporal fibres innervate the rostral and nasal fibres innervate the caudal portion of the developing tectum (Straz- nicky et al. 1981). However, it is not known what cellular or tissue properties of the tectum ensures preferential fibre termination. It could be that temporal retinal fibres prefer the more matured parts of the visual centres, hence, they terminate in the rostral part of the developing tectum and in the caudal parts of the developing diencephalic neuropil. Nasal retinal fibres, on the other hand, terminate in less matured areas in the caudal tectum and in the rostral parts of the diencephalic visual neuropil. We propose that the maturation gradients,


expressed presumably in molecular terms, representing a graded difference from rostral to caudal in the tectum and from caudal to rostral in the diencephalon serve as orientational cues for the ingrowing optic fibres. Although the exact nature of preferential fibre-substrate interactions is obscured at present, the end result of the interactions is well known. Both in normal and in experimental conditions the temporo-nasal retinal axis invariably projects to the rostrocaudal tectal and to the caudorostral diencephalic axes (Scalia and Fite 1974; Tay and Straznicky 1978; Straznicky 1981).

In conclusion, following a single injection of 3H-T at various gtage s of development, heavily labeled cells in the diencephalon of postmetamotph'f~~afii - mals were regionally distributed. The stages at which the isotope was administered are considered to be the birthdates of these particular neurons. With regard to the generation of diencephalic neurons three gradients ; caudorostral, lateromedial and ventrodorsal, were established. The sequential generation of neurons results in a rostro-dorso-medial overall growth of the diencephalon. We suggest that the mirror image reversal of the topographic retinodiencephalic projection with respect to the retinotectal projection may be due to the differential growth and maturation of the diencephalon and rectum from the common embryonic di-mesencephalic junction.

Acknowledgement. Authors gratefully acknowledge Mrs. Teresa Clark's skilled assistance with the autoradiography. D.T. is a Sir Colin and Lady Mackenzie Fellow.

References

Angevine JB Jr (1970) Time of neuron origin in the diencephaIon of the mouse. An autoradiographic study. J Comp Neurol 139:129 188

Berry M, Rogers AW (1965) The migration of neuroblasts in the developing cerebral cortex. J Anat 99:691 709

Chung SH, Cooke J (1978) Observations on the formation of the brain and of nerve connections following embryonic manipulation of the amphibian neural tube. Proc R Soc London Ser B 201:335 373

Currie J, Cowan WM (1974) Evidence'for late development of the uncrossed retinothalamic projections in the frog: Rana pipiens. Brain Res 71:133-139

Currie J, Cowan WM (1975) The development of the retinotectal projection in Rana pipiens. Dev Biol 46:103 119

Dziadek M, Dixon KE (1977) An autoradiographic analysis of nucleic acid synthesis in the presumptive primordial germ cells in Xenopus laevis. J Embryol Exp Morphol 37:13-31

Fite KV, Carey RG, Vicardo D (1977) Visual neurons in frog anterior thalamus. Brain Res 127:283 290

Frontera JG (1952) A study of the anuran diencephalon. J Comp Neurol 96:1 69 Gaze RM (1958) The representation of the retina on the optic lobe of the frog. Q J Exp Physiol

Cogn Med Sci 43:209-214 Gaze RM, Keating M J, Chung SH (1974) The evolution of the retinotectal map during development

in Xenopus. Proc R Soc London Ser B 185:30i 330 Gaze RM, Keating MJ, Ostberg A, Chung SH (1979) The relationship between retinal and tectal

growth in larval Xenopus: implications for the development of the retino-tectal projection. J Embryol Exp Morphol 53 : 103 143

Jacobson M (1968) Development of neuronal specificity in retinal ganglion cells of Xenopus. Dev Biol 17:20~218

Jacobson M (1976) Histogenesis of retina in the clawed frog with implication for the pattern of devleopment of retinotectal connections. Brain Res 103 : 541-545


Khalil SH, Sz~kely G (1976) The development of the ipsilateral retinotectal projections in Xenopus toad. Acta Biol Acad Sci Hungary 27:253-260

Knapp H, Scalia F, Riss W (I965) The optic tracts of Rana pipiens. Acta Neuroi Scand 41 : 325-355 Lfizfi.r G (1971) The projection of the retinal quadrants on the optic centers in the frog. A terminal

degeneration study. Acta Morphol Acad Sci Hungary 19:325-334 Lfiz~tr G (1973) The development of the optic tectum in Xenopus laevis: a Golgi study. J Anat

116:347 355 Lfizfir G, Sz~kely G (1969) Distribution of optic terminals in the different optic centres of the

frog. Brain Res 16:1 14 Levine RL (1980) An autoradiographic study of the retinal projection in Xenopus laevis with

comparisons to Rana. J Comp Neurol 189:1-29 Lewis S, Straznicky C (1979) The time of origin of mesencephalic trigerminal neurons in Xenopus.

J Comp Neurol 183:633-645 Nieuwkoop PD, Faber J (1956) Normal table of Xenopus laevis (Daudin). North Holland Publ.

Co., Amsterdam Picouet MJ (1975) Le developpement du thalamus des Anoures au cours de la metamorphose.

J Embryol Exp Morphol 33:313-333 Scalia F, Fite K (1974) A retinotopic analysis of the central connections of the optic nerve in

the frog. J Comp Neurol 158:455-478 Scalia F, Knapp H, Halpern M, Riss W (1968) New observations on the retinal projection in

the frog. Brain Behav Evol 1:324-353 Scott TM, Lfi.zS.r G (1976) An investigation into the hypothesis of shifting neuronal relationship

during development. J Anat 12t :485M-96 Straznicky C (1981) Mapping projections from double nasal and double temporal compound eyes

to dually innervated tectum in Xenopus. Dev Brain Res 1:139-152 Straznicky K, Gaze RM (1971) The growth of the retina in Xenopus laevis: An autoradiographic

study. J Embryol Exp Morphol 26:67 79 Straznicky K, Gaze RM (I 972) The development of the tectum in Xenopus laevis: An autoradiograph-

ic study. J Embryol Exp Morphol 28 : 87 115 Straznicky K, Tay D (1977) Retinal growth in double dorsal and double ventral eyes in Xenopus.

J Embryol Exp Morphol 40:175-185 Straznicky C, Gaze RM, Keating MJ (1981) The development of the retinotectal projections from

compound eyes in Xenopus. J Embryol Exp Morphol 62:13-35 Tay D, Straznicky K (1978) Retinodiencephalic projections from compound eyes in Xenopus.

Neurosci Lett 10 : 29-34 Tay D, Straznicky C (1980) The development of the nucleus isthmi in Xenopus: An autoradiographic

study. Neurosci Lett 16:313-318 Wilczynski W, Northcutt RG (1977) Afferents to the optic tectum of the leopard frog: An HRP

study. J Comp Neurol 173:219-230 Wilson M (1971) Optic nerve fibre counts and retinal ganglion cell counts during development

of Xenopus laevis (Daudin). Q J Exp Physiol 55:83-91

Accepted September 5, 1981

the development of the diencephalon in xenopus

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