Cell Tissue Res (1993) 272:273-287 Cell&Tissue

Research �9 Springer-Verlag 1993

Patterns of cellular proliferation and migration in the developing tectum mesencephali of the frog Rana temporaria and the salamander Pleurodeles wMtl Andrea Schmidt, Gerhard Roth

Institut fiir Hirnforschung, Universit/it Bremen-FB 2, Postfach 330440, W-2800 Bremen 33, Germany

Received: 9 July 1992 / Accepted: 27 October 1992

Abstract. The development of the tectum mesencephali was studied in the frog Rana ternporaria and the sala- mander Pleurodeles waltl by means of nuclear staining and by labeling of cells with bromodeoxyuridine (BrdU). The general spatial and temporal pattern of cell prolifer- ation and cell migration is the same in both species, despite drastic differences in overall tectal morphology. However, the salamander species differs from the frog species by (1)a generally lower cell proliferation rate, (2) a reduction in the activity of the lateral proliferation zone, and (3) a reduction in the formation of superficial cellular layers. Because point (3) affects processes that occur late in ontogeny, our experiments provide evidence that the simple morphology of the tectum of Pleurodeles waltl, compared with the multilayered tectum of Rana, is a consequence of a paedomorphic alteration of the ancestral developmental pattern of the amphibian tec- tum.

Key words: Cell migration Cell proliferation - Paedo- morphosis - Bromodeoxyuridine - Tectum mesencephali - Pleurodeles waltl (Urodela) - Rana temporaria (Anura)

Introduction

It is generally accepted in comparative neurobiology that the morphological complexity (e.g., the number of differ- ent morphological types of neurons, the number of mor- phologically distinct nuclei, the degree of lamination, the formation of columns, etc.) of the vertebrate brain has increased during evolution and that such an increase in complexity is paralleled by an increase in capacity of information processing and control of behavior (Bul- lock 1984). Therefore, brains showing a simple overall morphology are believed to represent a phylogenetically primitive (plesiomorphic) state.

Correspondence to .' A. Schmidt

This assumption is contradicted by the fact that groups and even classes of vertebrates are characterized by the phenomenon of secondary simplification of the entire brain or parts of the brain. This is true for the entire brain of lepidosirenid dipnoans (African and American lungfishes, Northcutt 1987) and for all extant amphibians (Lissamphibia), viz., frogs (Anura), sala- manders (Caudata), and caecilians (Gymnophiona) (Roth et al. 1992).

Among amphibians, frogs show the most complex brain morphology. The brain exhibits a number of mor- phologically distinct nuclei that are often found in a migrated position within the white matter. The tectum mesencephali of frogs is multiply laminated (see Fig. 1 b). It consists of nine alternating cellular and fiber layers (Potter 1969), and a substantial number of migrated cells (up to 30% of the total cell number) is found in the superficial part of the tectum (Sz6kely and L~zfir 1976; personal observations). Its overall organization resem- bles the tectum of most other groups of vertebrates, but it is less laminated and shows a smaller number of differ- ent morphological types of tectal cells than is found in teleost fishes, reptiles and birds [4-6 in amphibians com- pared with 14 in teleosts, Sz6kely and Litzfir (1976), Van- egas et al. (1984)].

The tectum mesencephali of salamanders (see Fig. 1 a) and caecilians has a much simpler morphology and con- sists essentially of a periventricular cellular layer and a superficial fiber layer. Few, if any, migrated cells are found in the superficial fiber layer (Roth et al. 1990; Schmidt and Wake 1991). In the rest of the brain, most nuclei are morphologically indistinct and are not found in a migrated position (Naujoks-Manteuffel and Man- teuffel 1988).

It has long been thought that the Lissamphibia are secondarily simplified vertebrates (e.g., Romer 1970). This simplification is usually seen in the context of pae- domorphosis (Wake 1966; Northcutt 1987; Roth et al. 1992). Here, paedomorphosis is defined as the retention of embryonic or larval traits (such as brain morphology) of ancestors in the adult stage of an descendant species.

(3

d

274

Fig. 1. Morphological differentiation of an urodele (a) and an anur- an (b) tectum. Transverse sections. Whereas the anuran tectum reveals a multilayered morphology (1-9), the urodele tectum is bilayered. Numbers within the salamander tectum indicate subdivi- sions made on the basis of Golgi- and HRP-studies (Roth 1987). A reversed sequence of numbers was used in a and b in order

to emphasize that the different layers in the urodele and anuran tectum are not strictly homologous. Schematic drawings of an uro- dele tectum (e) and an anuran tectum (d) demonstrating subdivi- sions of different tectal zones, m Medial zone; i intermediate zone; l lateral zone. Bar: 100 gm

The basis for paedomorphosis is a general slow-down of development including the disappearance of certain developmental stages, mainly those appearing late in on- togeny (Roth et al. 1992). In the case of tectal morpholo- gy, this implies that amphibians in general, and salaman- ders and caecilians in particular, have originated from ancestors that possessed a more highly laminated tectum and a larger number of tectal cell types. It also implies that the simple tectal morphology of salamanders corre- sponds with a situation present in embryonic or larval stages of their hypothetical ancestors.

Whereas this does not necessarily mean that the tec- turn of adult salamanders corresponds with the tectum of a frog larva, because salamanders did not originate from extant frogs, it is more reasonable to assume that the condition found in frogs is closer to the ancestral condition than that found in salamanders and caecilians. Therefore, we expect that the spatial pattern of cell pro- liferation, the constitution of cellular layers and the for- mation of the different morphological types of tectal neurons should exhibit the same general pattern in frogs and salamanders, but that salamanders should show signs of retardation with respect to development.

In order to test this hypothesis, detailed knowledge of the development of the tectum in frogs and salaman-

ders, particularly the pattern of cell proliferation and cell migration, is necessary. A number of studies exists on the development of the tectum of frogs, but their results are contradictory both with respect to the time course of cell proliferation and the constitution of tectal layers. Whereas Straznicky and Gaze (1972) have postu- lated a simultaneous constitution of the cellular layers, Dann and Beazley (1988) and Constantine-Paton (1988) describe a time-dependent constitution of the layers. Kollros (1988) postulates that an interpositioning of "' newborn cells" into previously formed tectal layers oc- curs during development. No studies on the development of the tectum exist in salamanders.

The main goal of our study was to reconstruct the temporal and spatial pattern of cell proliferation and cell migration during the development of the tectum in the frog Rana temporaria (Ranidae) and the salamander Pleurodeles waltl (Salamandridae) using the bromodeo- xyuridine (BrdU) method. These two amphibian species where chosen because they show a typical "amphib ian" developmental strategy that includes an aquatic larval stage, as opposed to the large groups of directly develop- ing frogs (e.g., the extremely speciose genus Eleuthero- dactylus) and salamanders (e.g., the plethodontid tribes Plethodontini and Bolitoglossini). This reconstruction is

275

the basis for testing our hypothesis that the differences in tectal morphology between frogs and salamanders are attributable to secondary simplification of the latter in the context of paedomorphosis. Because paedomorpho- sis is a phenomenon rather than a mechanism, the possi- ble developmental mechanisms underlying secondary simplification of the brain of salamanders, should be investigated.

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

Animals

Fifty-four larvae of Pleurodeles waltI (Salamandridae, Caudata) and 42 tadpoles of Rana temporaria (Ranidae, Anura) were used. These animals were reared in our laboratory at room temperature in plastic containers. There was an artificial cycle of 12 h light/12 h dark. Tadpoles of R. temporaria were fed on dried stinging nettles. Pleurodeles larvae were fed on brine shrimp and beef heart.

Nuclear staining

Two to 3 animals/stage were fixed at different ontogenetic stages (R. temporeria 21, 24, 29, 31, 37, 41, 43; P. waltl 34, 37, 38, 44, 48, 52, 53, 55a, 55c) in Bouin's solution modified after Dubosq (Romeis 1968). Whole animals (early larval stages) or brains (later stages) were embedded in Histosec and cut serially in transverse sections at 10 gm. The sections of R. temporaria were stained ac- cording to the Klfiver-Barrera method (Romeis 1968), sections of P. waltl where stained with nuclear red (Romeis 1968). In sections stained with these methods, regions of notable cell proliferation can be distinguished from those with no or low proliferation by dark staining and the appearance of elongated cells.

T a b l e 1. Time of labeling of tectal cells with BrdU in Rana tempor- aria and Pleurodeles waltl and time of sacrifice. Numbers in brackets number of animals investigated

Species Time of Time of sacrifice labeling

Rana temporaria stage 31 - stage 31 (7) - stage 32 (4) - stage 37 (3) - metamorphosis (3)

stage 41 - stage 41 (3) - 2 weeks after

metamorphosis (2)

Pleurodeles waltl stage 37 - stage 48 (3) - 5 months after

metamorphosis (3) stage 38 - stage 41 (3)

- stage 44 (3) stage 44 - stage 47 (3)

- 5 months after metamorphosis (2)

stage 52 - 5 months after metamorphosis (2)

stage 55a - 5 months after metamorphosis (3)

stage 55c - 5 months after metamorphosis (2)

Bromodeoxyuridine (BrdU) labeling

The tadpoles were incubated at different ontogenetic stages for 16 h in a 10 mM solution of 5-bromo-2'-deoxyuridine (BrdU, Sig- ma, Deisenhofen, Germany) in tap water. Studies on the optimal time of incubation revealed that there are differences between R. temporaria and P. waltl. Whereas a minimal time of t h is required in order to achieve labeling in R. temporaria, the minimal time for P. waltl is about 6 h. The first labeling was performed at stages in which the caudal tectum still is within an early developmental stage while cell migration already occurs in the rostral tectum. The latest labeling was performed shortly before metamorphosis. In P. waltl, cells were labeled at early stages 37, 38, in midlarvai stage 44, and at later stages 52, 55a, and 55b (staging according to Gallien and Durocher 1957). In R. temporaria, cells were labeled at stages 31, and 41 (staging according to Gosner 1960). After incubation, animals were rinsed several times in order to wash out any BrdU that was not incorporated. The tadpoles were al- lowed to develop in small plastic containers at room temperature. They were sacrificed at different developmental stages (Table 1). In order to guarantee sufficient time for cell migration animals of all groups were allowed to survive until metamorphosis. A study on the time sequence of cellular migration was done in animals in which cells were labeled at early and midlarval ontogenetic stages. (P. waltl: 37, 38, 44; R. temporaria: 31). In these animals migrating cells were localized not only after metamorphosis but also at earlier stages (Table 1). BrdU-labeled cells were detected using immunohistochemistry.

Immunohis tochemis try

Animals were anesthetized in a 1:500 (w/v) solution of MS 222 and fixed in 2% (w/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After rinsing in 0.1 M phosphate buffer for 12 h, the animals were transferred into 70% ethanol. Brains were removed from the skull and embedded in Histosec. The brain was cut into 20-gin-thick transverse sections. After deparaffinization, sections were rinsed in phosphate-buffered saline (PBS; 10 mM Na2HPO4, 0.3 mM KHzPO,~, 0.8% NaC1; pH 7.4), followed by denaturation of D N A in 4 N HC1 for 20 rain. The sections were then washed in 0.05 M TRIS buffer (pH 7.6) containing 0.05% (w/v) Calcium chloride and 0.2% (w/v) trypsin (according to Hayashi et al. 1988) for 10 rain. The sections were washed 4 times (20 rain total) in PBS containing 0.5% (v/v) Tween. After the sections were again washed in PBS (10 minutes), they were incubated with blocking serum for 15 min and incubated overnight at 4 ~ C with the primary antibody against BrdU (Dakopatts, Hamburg, Germany) diluted in PBS and containing 10% (w/v) bovine serum albumine (BSA) and 0.1% (w/v) NAN3. The biotin-avidin system (Vector Labs. Burlingame, Calif.) (Hsu et al. 1981) was used for the immunoreac- tion, with diaminobenzidine (DAB) as chromogen. The horseradish peroxidase (HRP) reaction product was intensified according to Adams (1981). Controls were performed by omitting the primary antibody against BrdU and by using the antibody against BrdU in an animal in which no labeling of cells with BrdU was per- formed.

Evaluation o f the BrdU-labeling

BrdU is incorporated into DNA and, therefore, labels cells that undergo mitosis during the time of application. These mitotic cells undergo further divisions. The number of divisions after the appli- cation of BrdU determines the amount of BrdU located in each labeled cell and, consequently, the staining intensity. Cells labeled during their last division are deeply stained, whereas cells that undergo further divisions after incubation in BrdU are lightly

276

stained because the amount of BrdU has been diluted. However, lightly stained elements may represent those cells that were exposed to BrdU near the end of S-phase of the cell cycle and that were thus able to incorporate only a small amount of BrdU. In the present study, the animals were thoroughly rinsed several times after the application of BrdU in order to stop incorporation of the label. However, it cannot be excluded that small amounts of free BrdU were still present within the tissue and also led to faintly stained cells. Thus, an area of labeled cells may show a gradient of staining intensity with intensely stained cells that underwent their last division during application of BrdU, together with a decrease in staining intensity in cells that underwent further divi- sions or incorporated remnants of BrdU after the application of BrdU. This gradient can be interpreted as a time gradient in cell proliferation, in that more darkly stained cells must have originated earlier than more faintly stained ones.

Results

Anatomy and lamination of the tectum

The anuran tectum is a conspicuously multilayered structure (Fig. 1 b). According to the nomenclature of Potter (1969), the adult frog tectum is divided into 9 alternating cellular and fiber layers. The superficial part of the tectum consists of layers 8 and 9 containing mi- grated nerve cells and retinal afferents, and layer 7 con- taining tectal efferents. Within the periventricular gray matter, 6 layers can be distinguished: cellular layers 6, 4 and 2, separated by deep layers of unmyelinated fibers (layers 3 and 5), and the layer of ependymal cells (layer 1).

In contrast, the salamander tectum is characterized by a thick periventricular cellular layer and an outer fiber layer (Fig. 1 a). On the basis of Golgi and HRP studies (Roth et al. 1990), the fiber layer can be divided into superficial layers 1 3 of retinal afferents and deeper layers 4-5, containing efferent fibers. The periventricular gray matter can be divided into superficial cellular layer 6, the layer of deep unmyelinated fibers (layer 7, not present in larval brains), and deep cellular layer 8. Layer 9 contains ependymal cells (Roth 1987). We use a reverse order in the numbers of different layers in the anuran and salamander tectum to make clear that the different layers in anurans and salamanders cannot necessarily be homologized.

Comparative studies on the cytoarchitecture of the tectum of frogs and salamanders (Roth et al. 1990) dem- onstrate that the deep cellular and fiber layers 1 6 of the frog tectum correspond to layers 6-9 of the salaman- der tectum. In both tecta, the upper part of the periven- tricular gray matter (layer 6 in both taxa) and the layer of tectal efferents (layer 7 in frogs, and layers 4 and 5 in salamanders) contain large projection neurons. Layers 8 and 9 in frogs correspond to layers 1-3 in sala- manders. The main gross-anatomical difference between the frog and the salamander tectum is that, in salaman- ders, the superficial layers contain few, if any, migrated cells, whereas these layers contain a large number of migrated tectal cells in frogs (20%-30% of the total number of tectal cells).

Pattern of cell proliferation and cellular migration during development

Rana temporaria: Cell proliferation during development

At early ontogenetic stages (20-24, rostral tectum), cell proliferation takes place over the whole width of the ependymal layer, but is slightly higher lateral than me- dial. At stage 24, the rostral rectum consists of one thick cellular layer; a superficial neuropil is still absent. From stage 25 until stage 30, cell proliferation in the rostral rectum increases in the medial relative to the lateral zone. At this time in the intermediate zone cell proliferation diminishes. The superficial neuropil develops, and the first migrated cells appear in this zone. In the caudal tectum, this occurs from stage 31 until stage 37. From stage 30 (rostral tectum) and stage 37 (caudal tectum) the medial and lateral proliferative zones decrease in size (Fig. 2). There is a slight increase of lateral cell pro- liferation, beginning at stage 37 in the rostral and at stage 43 in the caudal tectum.

Pattern of cellular migration as revealed by BrdU experiments

Labeling stage 31, sacrifice at stage 31. In the rostral tec- turn, labeled cells are concentrated in the medial prolifer- ation zone and to a lesser degree in the lateral prolifera- tion zone, with few labeled cells in the intermediate zone. In the central tectum, labeled cells are found in the me- dial and in the lateral proliferation zone. Only few la- beled cells are located in the periventricular layer of the intermediate zone. In the caudal tectum, labeled cells are equally distributed over almost the total thickness of the periventricular cellular layer, intermingled with less numerous unlabeled cells. This is consistent with the fact that the caudal rectum develops later than the rostral rectum. Because of the short survival time, there is only minimal cell migration.

Labeling at stage 31, survival until stage 32. In the rostral tectum, labeled cells are most numerous in the medial and lateral zone (Fig. 2d). The medial cells are found in all layers, whereas the lateral cells are restricted to the ependymal layer. In the intermediate zone, close to the medial zone, a few cells are found in layer 4, and very few in layers 8 and 9 and the upper part of layer 6. In the central rectum, cells are predominantly found within the medial zone and occur in all layers. Fewer cells occur in the lateral and intermediate rectum, where most cells are located in layers 1, 2 and 6, and a few in layers 8 and 9 (Fig. 2 e). In the caudal tectum, labeled cells are found in all zones, with a slight concentration in the medial zone, and in all layers (Fig. 2f). At this stage, the medial zone of the rectum is still thin and consists only of an ependymal layer 4 ceils thick. The lateral rectum consists mainly of a thick homogeneous periventricular layer. Little neuropil is present.

Labeling at stage 31, survival until stage 37. In the anteri- or part of the rostral rectum, labeled cells are found

277

Fig. 2. Pattern of cell proliferation during ontogeny of Rana tem- poraria based on Klfiver-Barrera staining (a-e) and as revealed by BrdU experiments (d-f). Transverse sections, a-e Shows the proliferation pattern of the central tectum during different onto- genetic stages (a stage 43; b stage 37; e stage 25). Arrows indicate proliferation zones, d-f Distribution of cells that were labeled at

stage 31 and localized at their target sites at stage 32 in different regions (d rostral, e central; f caudal) of the same tectum in Rana temporaria. The rostro-caudal gradient of cell proliferation and migration is in accordance with a temporal gradient. The caudal pattern (f) reflects the early ontogenetic pattern (e), whereas the rostral tectum (d) reflects the late pattern (a). Bar: 100 gm

in all zones of the rectum and are concentrated in layers 8 and 9 and in outer layer 6. In the posterior part of the rostral rectum and the anterior par t of the central tectum (Fig. 3 a), labeled cells are located in the medial zone, forming a vertical column that extends through all layers. There is a zone of unstained or lightly stained cells between the column of labeled cells and the medial proliferative zone. This zone consists of cells that are " b o r n " after labeling, and enlarges caudad

(Fig. 3 b) because cell proliferation is always higher in the caudal than in the rostral tectum. In the intermediate zone, a few labeled cells are found mainly in the upper part of layer 6, and layers 8 and 9, and in the ependymal layer, leaving layers 2, 4 and the lower part of layer 6 free of labeled cells. In the lateral zone, labeled cells are found only in the ependymal layer and layer 2 (Fig. 3 a). In the caudal tectum, labeled cells occupy only the lateral half of the tectum, where they are found in

278

Fig. 3a, b. Tectum of Rana temporaria. Distribution of cells that were labeled with BrdU at midlarval stage 31 and localized at their target site at stage 37. Transverse sections. In the central tectmn (a), there is a column of iabeled cells near the medial proliferative zone that extends over all cellular layers, whereas lateral to this column, labeled cells are mainly confined to superficial layers 8 and 9, upper layer 6, and deep layer 2. In the caudal tectum (b), labeled cells are found more laterally within all cellular layers. Bar: 100 gm

Fig. 4a-c. Tectum of Rana temporaria. Distribution of cells that were labeled with BrdU at late ontogenetic stage 41 and localized at their target sites 2 weeks after metamorphosis. Transverse sec- tions. Whereas labeled cells of the caudal tectum (e) still occupy all cellular layers, labeled cells of the rostral (a) and central tectum (b) are mainly found in upper layer 6, superficial layers 8 and 9, and deep layer 2. Bar: 200 gm

all layers (Fig. 3b). The medial zone consists o f un- stained and lightly stained newborn cells.

Labeling at stage 31, survival until metamorphosis. In the rostral rectum, the media l -most zone consists o f new- born unlabeled cells. Adjacent to this zone, only faintly stained cells occur ; they are found in all layers. In the intermediate zone, labeled cells consti tute a co lumn ex- tending f rom layers 4 th rough 9 with a higher density in layers 6, 8 and 9. In the lateral zone, cells are predomi- nant ly located in layers 2, and the upper par t o f layer 6, and layers 8 and 9, leaving layers 4 and the lower par t o f layer 6 free o f labeled cells (Fig. 11). In the central rectum, the medial zone consists o f newborn unlabeled cells. The intermediate zone contains faintly labeled cells in layers 4 and 6, and a few in layers 8 and 9. In the lateral rectum, labeled cells are found in all layers, but there is a concent ra t ion o f labeled cells in layers 6, 8 and 9. In the caudal tectum, the medial zone again con-

sists o f newborn unlabeled cells. The intermediate zone contains faintly labeled cells, concent ra ted in layers 4-6, with a few in layers 8 and 9. In the lateral zone, labeled cells are distributed over all layers, but are concent ra ted in layers 6, 8 and 9.

Labeling at stage 41, sacrifice at stage 41. In the rostral tectum, labeled cells are found in all layers o f the medial zone, whereas in the intermediate and lateral zone, they are confined to the ependymal layer and layer 2. In the central tectum, labeled cells are found in all layers close to the medial proliferative zone and in layers 2 and 4 o f the intermediate and lateral zone. The number o f labeled cells increases rostrocaudally. In the caudal -most tectum, labeled cells are found all over the ependymal layer.

Labeling at stage 41, survival until 2 weeks after metamor- phosis. Generally, labeled cells are found in the ependy-

279

real layer and layer 2. In the medial zone, the number of labeled cells increases in rostro-caudal direction be- cause of the proliferation gradient. In the medial-most zone of the rostral tectum, labeled cells are distributed over all layers (Fig. 4 a, 11). Lateral to this zone, labeled cells are found in the ependymal layer, layer 2, and the upper part of layer 6, and layers 8 and 9, leaving layers 4 and the lower part of layer 6 free of labeled cells. Unla- beled cells most probably arose before labeling. In the intermediate and lateral zone, cells are found in layers 2 and 4. In the central tectum (Fig. 4b, 11), labeled cells occur in all layers of the medial zone. In the intermediate and lateral zones, they are mainly confined to the epen- dymal layer, layer 2, the superficial part of layer 6 and layers 8 and 9. Few cells are found in layer 4 of the lateral rectum. In the caudal tectum (Fig. 4c), labeled cells are present in all layers.

Pleurodeles waltl : Cell proliferation during development

As in R. temporaria, cell proliferation takes place all over the ependymal layer early in development (stage 33-37, rostral rectum), with a slight concentration in the lateral part of the tectum (Fig. 5c). There is a shift in the intensity of cell proliferation at stage 38. Starting in the rostral rectum, proliferation is highest in the medial zone, whereas only a few cells proliferate in the lateral ependymal layer. In general, cell prolifera- tion is lower in the rectum mesencephali of P. waltl than in the tectum of R. temporaria. This especially affects the lateral proliferation zone (Fig. 6 a). Cell proliferation is always highest in the Caudal tectum (Fig. 6c). Cell proliferation decreases in the medial zone of the rostral tectum starting at stage 42 and in the caudal tectum starting at stage 55a. Based on evidence from nuclear staining, there is a slight increase in lateral cell prolifera- tion at late ontogenetic stages and substantial growth

of the rectum at late ontogenetic stages 53 55c. How- ever, BrdU-labeling at these stages leads to a lower number of labeled cells than at earlier stages. It is as yet unknown whether the reduced number of labeled cells occurs because of a lower proliferation rate, since late stages 55a-55c take over almost one third of the entire development, or whether these effects are the re- sult of a change in the number of subsequent mitoses of labeled cells. The superficial neuropil begins to devel- op at stage 34. The initially small numbers of migrated cells do not appear before stage 36.

Pattern of cellular migration as revealed by BrdU experiments

Labeling at stage 37, survival until stage 48. In the rostral tectum, labeled cells of the medial zone form a wedge that extends through all layers of the periventricular gray. The intermediate and lateral zone contains only few labeled cells, which are located in the ependymal layer. In the central tectum, the same situation exists as is found in the rostra1 tectum, but there is a narrow strip of unlabeled newborn cells between the wedge of labeled cells and the medial proliferative zone. In the caudal tectum, labeled cells occur in all layers of the medial, intermediate and lateral zones, again with a me- dial-most zone free of unlabeled newborn cells. The number of labeled cells in the medial and lateral zones increases from rostral to caudal.

Labeling at stage 37, survival until 5 months after meta- morphosis. In the rostral and central tectum, labeled cells form a vertical column in the intermediate zone (Figs.- 7a, 10). A column of unlabeled cells is found between

this column and the medial proliferative zone. The later- al zone contains only a few labeled cells, located in the ependymal layer and layer 8, and in the upper part of

Fig. 5. Pattern of cell proliferation in the central tec- tum of Pleurodeles waltl as revealed by light microsco- py (a late stage 55; b midlarval stage 44; c early stage 37). Transverse sections. Arrows indicate zones of cell proliferation. In b dotted lines indicate the lim- its of proliferation zones. Bar: 100 gm

280

Fig. 6a-e. Tectum of Pleurodeles waltl. Distribution of cells that were labeled with BrdU at midlarval stage 44 and localized at their target sites at stages 47. Transverse sections. The rostro-caudal (a rostral; b central; e caudal) gradient in the pattern of cell prolifera- tion reflects the temporal pattern of cell proliferation, in that cell proliferation takes place all over the ependymal layer in early stages (caudal tectum) and is confined to the medial proliferative zone and to a lesser degree to the lateral proliferative zone in later stages (rostral tectum). Bar: 100 gm

the per iven t r i cu la r g ray m a t t e r ( layer 6). In the cauda l tec tum, labe led cells are found ma in ly in the uppe r p a r t o f the g ray m a t t e r ( layer 6) in the in t e rmed ia t e and la ter- al zone (Fig. 7b) . They cover a layer o f un labe led or fa int ly labe led cells. The c a u d a l - m o s t p a r t o f the t ec tum con ta ins on ly un labe led n e w b o r n cells. The n u m b e r o f l abe led cells is h igher in the cauda l t han in the ros t ra l rectum.

Labeling at stage 38, survival until stage 41. In the ros t ra l and cent ra l tec tum, labe led cells a re concen t r a t ed in the media l zone. In the la te ra l zone, only a few cells are found in the e p e n d y m a l layer , covered by cells tha t were b o r n before labeling. In the cent ra l rectum, the same s i tua t ion exists as in the ros t ra l tec tum, bu t in the med ia l zone, an a rea o f few un labe led n e w b o r n cells is f o u n d be tween labeled cells and the med ia l p ro l i fe ra t ive zone. This zone enlarges caudad , as in frogs. In the cauda l tec tum, the media l zone is free o f l abe led cells. In the in t e rmed ia te zone, l abe led cells are found in all layers o f the g ray ma t t e r , whereas they are conf ined to deeper layers in the la te ra l zone.

>

Fig. 7a, b. Tectum of Pleurodeles waltl. Distribution of cells that were labeled with BrdU at ontogenetic stage 37 and localized at their target sites 5 months after metamorphosis. Transverse sec- tions. Labeled cells of the rostral tectum (a) constitute a column over the total thickness of the periventricular gray, whereas in the Caudal rectum (b) labeled cells are confined to the outer part of the periventricular gray. Bar: 200 ~tm

Fig. 8a-d. Tectum of Pleurodeles waltl. Distribution of cells that were labeled with BrdU at ontogenetic stage 52 and localized at their target sites 5 months after metamorphosis. Transverse sec- tions. The early developing rostral tectum (a) reveals a vertical column of labeled cells extending through the entire periventricular gray with a lateral adjacent region where cells are found only in the superficial part of the gray and in the ependymal layer. A region of unlabeled cells is found in between. In the central tectum (b), cells are distributed over the total thickness of the periventricu- lar gray. The number of cells in the deep part of the periventricular gray decreases continually caudad (e, d). Labeled cells mainly oc- cupy the outer part of the periventricular gray (d). Bar: 200 ~tm

Fig. 9a, b. Tectum of Pleurodeles waltl. Distribution of cells that were labeled with BrdU at stage 55a and visualized 5 months after metamorphosis. Transverse sections. There is only low cell prolifer- ation in the rostral rectum (a) Notable cell proliferation occurs only in the caudal-most part (b). Bar: 200 pm

281

282

Stage 37

EN

'1 5 months

etamorphosis

Stage 4 4

I ,~, ;~ )/ "

J after metamorphosis

Stage

5 months

after metamorphosis

Stage 5 5 ~ ' ~ �9

ths

after metamorphosis

Fig. 10A, B. A (left column): Schematic drawing of cell proliferation at the stage of labeling in Pleurodeles waltl. Hatched regions represent main proliferative zones. Cell proliferation strongly dimin- ishes in the intermediate zone at midlar- val and late developmental stages. B (right column): Distribution of cells that were labeled at stages 37, 44, 52 and 55 after migration

Labeling at stage 38, survival until stage 44. In the rostral and central rectum, labeled cells form a column in the medial zone, with an area of unlabeled or faintly labeled cells between them and the medial proliferative zone. In the intermediate and lateral zone, only a few labeled cells are found in the deep periventricular gray. In the caudal tectum, lateral to an area of unlabeled newborn cells in the medial zone, labeled cells are found in all layers of the gray matter in the intermediate and lateral zone with a slight concentration in the outer part of the periventricular gray (layer 6) in the caudal-most tec- turn.

Labeling at stage 44, survival until stage 47. In the rostral tectum, labeled cells occupy mostly the medial zone.

Only few cells are found in the intermediate and lateral zone, and these are confined to the ependymal layer and the adjacent deep cellular layer (Fig. 6a). In the central tectum, labeled cells are found predominantly in the medial zone where they constitute a wedge. The intermediate and lateral zones contain few labeled cells occupying the ependymal and the adjacent deep cellular layer (Fig. 6b), covered by cells that were born before the labeling. In the caudal tectum, labeled cells are found all over the tectum, with a slight concentration in the medial zone (Fig. 6 c).

Labeling at stage 44, survival until 5 months after meta- morphosis. In the medial zone of the rostral tectum, la- beled cells form a column, with some unlabeled, new-

born cells between them and the medial proliferative zone. The intermediate and lateral zones contain only few labeled cells located in the ependymal layer (Fig. 10). In the central tectum, the site of the wedge is shifted more laterally, and the medial zone contains only unla- beled cells. The lateral zone contains labeled cells only in the ependymal layer. In the caudal tectum, labeled cells are found only in the lateral zone and the outer half of the periventricular gray matter (layer 6). The deep periventricular cellular layer contains lightly la- beled and unlabeled newborn cells. The caudal-most part of the tectum contains no labeled cells.

Labeling at stage 52, survival until 5 months after meta- morphosis. In the rostral tectum, the medial zone is free of labeled cells. These cells are born after labeling. In the intermediate zone, labeled cells form a wedge, where- as more laterally, cells are found only in deep and super- ficial parts of the gray matter (Figs. 8 a, 10). There are only few labeled cells in the lateral-most zone. In the central tectum, the medial and most of the intermediate zone are free of labeled cells, whereas labeled cells form a wedge in the more lateral parts (Figs. 8b, 10). In the caudal tectum, the medial and intermediate zones are free of labeled cells. In the lateral zone, the number of cells in the deep part of the periventricular gray contin- ually decreases caudad and labeled cells are mainly con- fined to the outer part of the gray matter (layer 6) (Figs. 8c, d, 10). In the deep gray matter, there is only a minor granular staining. These cells are considered as having been born after labeling by additional mitoses of stem cells that incorporated BrdU during the labeling. The caudal-most part of the rectum contains no labeled cells.

Labeling at stage 55a and 55c, survival until 5 months after metamorphosis. In the rostral and central tectum, very few labeled cells are found in the medial zone (Fig. 10). In the caudal rectum notable cell proliferation occurs only in the caudal-most part. Labeled cells are confined to the lower half of the anterior caudal periven- tricular gray and are distributed over the whole thickness of the periventricular gray in the caudal-most part (Fig. 10).

Discussion

Our experiments show that the pattern of development of the tectum is similar in frogs and salamanders. There are quantitative rather than qualitative differences in cell proliferation and the mode of neuronal migration be- tween the two groups.

Developmental gradient

The tectum of frogs and salamanders develops along a rostro-caudal gradient. This means that, at any devel- opmental stage, the rostral tectum is more developed than the caudal tectum. Thus, by comparing transverse

283

sections along the rostro-caudal axis in the rectum at a given stage, one moves back in the time course of development, in the sense that rostral sections represent later, and caudal sections earlier, developmental stages (Figs. 2, 5, 6). This also implies that the caudal tectum from a late stage may resemble the rostral rectum of an early stage (Fig. 2). There is also a lateral-medial gra- dient with respect to tectal differentiation. Lamination and migrated cells first appear laterally and later within the medial zone.

Cell proli~eration during ontogeny

In R. temporaria, the rostro-caudal gradient in the distri- bution of cells that have been labeled with BrdU povides evidence for the following time course of development. Early in development, cells proliferate all over the epen- dymal layer, whereas at later stages, cell proliferation is confined mostly to a medial and a lateral proliferative zone, with only few proliferating cells in the intermediate zone (Fig. 2). This conclusion is in accordance with the pattern of cell proliferation observed at different onto- genetic stages by means of nuclear staining and is consis- tent with the pattern of cell proliferation described for the trout tectum (Mansour-Robaey and Pinganaud 1990). There is a slight increase of lateral cell prolifera- tion at late ontogenetic stages.

In P. waltl, we find the same rostro-caudal gradient and the same developmental pattern of cell proliferation as in Rana in that, early in development, cell proliferation takes place all over the ependymal layer and later is confined to medial and lateral zones. However, Pleuro- deles differs from Rana in that it has a lower degree of cell proliferation. This affects mainly the lateral prolif- erative zone. Reduction in lateral cell proliferation in Pleurodeles leads to suppressed lateral growth of the rec- tum and may explain differences between Pleurodeles and Rana (and differences between anurans and urodeles in general), with respect to the shape of the tectum. Whereas anurans exhibit a laterally bulging rectum, it is narrower in urodeles and resembles an anuran rectum at early ontogenetic stages.

Pattern of cell migration during development

The pattern of cell migration has been studied in diverse brain centers of many vertebrates (Angevine and Sidman 1961; Rakic 1972; Straznicky and Gaze 1972; Dann and Beazley 1988; Gona etal. 1988; Gadisseux et al. 1990; Mansour-Robaey and Pinganaud 1990). Two major pat- terns of cellular migration that differ in their time-depen- dence in the constitution of different layers have been described. In the mammalian cortex, an "inside-out" pattern of neuronal migration along radial glia has been described (Angevine and Sidman 1961; Shimada and Langman 1970; Rakic 1972). Cells born late in ontogeny migrate through layers formed by cells born previously. Thus, in the mammalian cortex, there is a time-depen- dent constitution of cellular layers. In the cerebellum

284

stage

11

EN

~ 2af::;::tamorphosis

12a Fig. 11. A (left column): Schematic drawing of cell proliferation at the stage of labeling in Rana temporaria. Hatched regions repre- sent main proliferative zones. As in Pleurodeles, cell proliferation diminishes in the intermediate zone in midlarval and late develop- mental stages. B (right column): Distribution of cells that were labeled at stages 31 and 41 after migration

Pleurodeles ~ - - Oo. . : :

I3 *~. O

12b Fig. 12. Schematic drawing of the distribution of tectal cells in Rana temporaria (a) and Pleurodeles waltl (b) that originated at early (triangles), midlarval (open squares) and late (black dots') onto- genetic stages

of frogs (Gona et al. 1988), a different pattern of cellular migration has been observed that consists of two phases. Early in development, cells proliferate throughout the ependymal layer and migrate outwards in a parallel fash- ion. Later on, granule cells originate in a medial germin- ative zone and migrate tangentially and invade mostly the superficial zones of the cerebellum, from where they migrate along Bergmann glia downward to the deep layers in an "outs ide- in" fashion.

Controversies exist over the pattern of cellular migra- tion within the anuran tectum. In Xenopus laevis, Straz- nicky and Gaze (1972) describe a simultaneous constitu- tion of all cellular layers; this occurs from rostro-ventral to caudo-medial by means of medial addition of stripes

of newborn cells that push the pre-existing tectal tissue laterally and rostrally. Kollros (1988) rejects this hypoth- esis and postulates an interpositioning of new cells into previously formed tectal regions. Constantine-Paton (1988) and Dann and Beazley (1988) propose a time- dependent constitution of tectal layers. Constantine-Pa- ton (1988) describes two different patterns of cellular migration within the tectum. The first tectal cells to orig- inate are large projection neurons characteristic of tectal layer 6. Smaller neurons are born later and are distrib- uted over all cellular layers, first in layers 4 and 6, and then in layers 8 and 9. Dann and Beazley (1988) main- tain that cells born at one ontogenetic stage are later found in all cellular layers, but that a dorso-ventral gra-

285

dient of labeled cells exists. According to these authors, cells that are born at stage 51 (stages according to Nieuwkoop and Faber 1967) invade mainly superficial layers 7-9, whereas cells born later (stage 57-58) are found in layer 6.

Our data provide evidence that simultaneous and time-dependent patterns of neuronal migration exist during the development of the tectum in R. temporaria (Figs. 11, 12). Early in ontogeny, a simultaneous consti- tution of tectal layers takes place, whereas late in onto- geny, newborn cells selectively occupy the superficial layer of migrated cells and the upper part of layer 6 and deep layer 2 (Fig. 12). In Fig. 4, the tectum of R. ternporaria contains cells labeled at stage 41 and visual- ized 2 weeks after metamorphosis. In the caudal tectum, which because of the rostro-caudal developmental gra- dient represents the most immature stage, cell prolifera- tion occurs all over the tectum, and labeled cells are found in all layers. In the central tectum, which repre- sents a later developmental stage, cell proliferation is highest in the medial proliferative zone where cells are found in all layers. However, in more lateral zones, la- beled cells occur in layer 2 on the one hand, and the upper part of layer 6, and layers 8 and 9 on the other, leaving a zone free of labeled cells in between. In the rostral tectum, representing the most mature stage of development, the situation is similar, with the exception that the superficial layers of the lateral zone contain no labeled cells.

These rostro-caudal differences in migration pattern are also present in tecta in which cells have been labeled earlier (e.g., at stage 31) (Fig. 2d-f), but now the late pattern of neuronal migration (i.e., cells mainly occupy superficial layers 8 and 9, the outer part of layer 6 and the ependymal layer) is only present in the rostral tectum and in the anterior part of the central tectum. Most of the tectum (posterior part of the central tectum and caudal tectum) is characterized by the early pattern of neuronal migration (i.e., cells are found in all cellular layers). In tecta in which cells have been labeled at the relatively late stage 41, the early pattern of migration is restricted to the caudal-most rectum. Most of the tec- turn is characterized by the late pattern. Additionally, in these late tecta, there are no unlabeled or slightly labeled cells in the medial zone, whereas many unlabeled cells are found in this zone in tecta, where cells have been labeled earlier at stage 31. We interpret these cells to be born after the labeling. This means that there is only a low degree of cell proliferation after stage 41.

Our data suggest that cells that are born throughout the ependymal layer early in ontogeny migrate towards the surface in a parallel fashion and occupy all cellular layers simultaneously. At later stages, cells born earlier are pushed laterally by cells that are born later in the medial proliferative zone. As a consequence, cells of the medial zone only carry a light labeling, caused by rem- nants of BrdU in the tissue. Interpositioning of newborn cells in previously formed layers also occurs in the lateral zone. Cells born late in ontogeny mainly invade layer 2, superficial layers 8 and 9, and outer layer 6. It seems that such cells invade these cellular layers in a parallel

fashion by a tangential migration from medial to lateral, as described for the second migration in the cerebellum (Gona et al. 1988). However, we cannot exclude that they invade the deepest and the most superficial layers from the caudal pole by migrating rostrad and again in a tangential manner. Finally, neurons could migrate radially along radial glial cells from their site of origin in the ependymal layer through pre-existing cellular layers according to the "inside-out" sequence, as de- scribed for the mammalian cortex (Angevine and Sid- man 1961 ; Shimada and Langman 1970). However, this migration pattern would leave unexplained the fact that labeled cells are found also in the deepest cellular layer.

The sequence of cellular migration in R. ternporaria, as revealed by our data, is in accordance with that de- scribed by Constantine-Paton (1988), with the exception that we have been unable to find a selective formation of layer 6 (containing large projection neurons) early in ontogeny. It may be that the formation of this layer starts before stage 31, the earliest stage that we have studied. The tecta of animals in which cells were labeled at stage 31 and which survived until metamorphosis con- tain lightly labeled cells that were concentrated in layers 4 6 medial to the column of darkly labeled cells. Under the assumption that the less differentiated medial zone represents an earlier stage, this pattern might represent an earlier migration pattern. Our experiments in P. waltl demonstrate a selective constitution of the upper part of the periventricular gray matter early in development. In the caudal tectum (which represents an early ontogen- eric stage) of animals labeled at stage 37, labeled cells are confined to the upper layer of the periventricular gray matter (Fig. 7) after metamorphosis.

Our results demonstrate that the pattern of cellular migration is similar in R. ternporaria and in P. wahl. Figure 8 shows the rostro-caudal gradient in the distri- bution of cells that were labeled at stage 52 and localized at their target sites 5 months after metamorphosis in Pleurodeles. The distribution of labeled cells is different in the rostral, medial and caudal tectum. In the late- developing caudal rectum, labeled cells occupy the outer zone of the periventricular cellular layer. This suggests that, early in development, cells migrate in a parallel fashion and mainly constitute the outer part of the peri- ventricular gray matter. This pattern is in accordance with the early constitution of layer 6 in frogs as described by Constantine-Paton (1988). In the central tectum, la- beled cells constitute a vertical column that extends over all cellular layer. This points to a parallel migration of newborn cells that occupy the total thickness of the peri- ventricular gray simultaneously. Unlabeled cells between the column of labeled cells and the medial proliferative zone suggest that, in Pleurodeles, cells that are born ear- lier are also pushed laterally by cells that are born later in the medial proliferative zone. The pattern in the early developing rostral tectum, where we find a zone lateral to the column of labeled cells, and where labeled cells are localized mainly in the outer zone as well as in the periventricular zone leaving a zone of unlabeled cells in between, suggests that the late pattern of cellular mi- gration is similar to the late pattern in R. ternporaria.

286

What are the reasons for the simple morphology of the salamander rectum ?

Frogs and salamanders differ from each other in two traits that have profound consequences on brain mor- phology, i.e., genome size and cell size. Among all ani- mals, salamanders have the second largest genome sizes (i.e., amount of DNA per nuleus, here given as pg D N A per haploid nucleus). Genome size ranges from 13.7 to 83 pg (Hally et al. 1986; Olmo 1983). Only lungfishes (Dipnoi) have larger genomes, ranging from 80 to 142 pg (Olmo 1983). The range of genome sizes from 0.9 to 19 pg in anurans (Olmo 1983) is substantially less than that in salamanders, but is higher than in other groups of vertebrates. Amniote genome sizes range from 0.9 to 5.5 pg, and those of chondrichthyans and osteich- thyans (except dipnoans) from 0.7 to 7 pg (Olmo 1983).

There is a correlation between genome size and cell size because of the nuclear/plasma ratio. Larger amounts of D N A lead to larger cells. In amphibians, this correla- tion is highly significant with respect to tectal neurons. Furthermore, there is nearly a 100% correlation between genome and neuron size on the one hand and morpho- logical complexity of the brain on the other (Roth et al. 1992; G. Roth et al., unpublished data). This means that both lungfishes and salamanders generally have brains with a simple morphology compared with frogs, whereas frogs generally have simpler brains compared with other vertebrates. Among species of these groups, there is a correlation between genome/neuron size and morpho- logical complexity of the brain (Roth et al. 1992; G. Roth et al., unpublished data).

What is the explanation for the correlation between genome size and the simplicity of the nervous system? Increased genome sizes generally lead to decreases in rates of cell metabolism, cell proliferation, and differenti- ation (Sessions and Larson 1987; Roth etal . 1990; Szarski 1976, 1983; Cavalier-Smith 1978, 1982; Horner and MacGregor 1983). These rate reductions produce brains that have fewer neurons and that have simplified morphologies characteristic of embryos or juveniles of phlyogenetic out-groups and ancestors (Gould 1977; A1- berch et al. 1979). The wide-spread influence of paedo- morphosis during salamander evolution is well-docu- mented for many morphological traits (Wake 1966), and can now also be demonstrated for the brain as well.

Paedomorphic retardation of brain development tends to affect later developmental stages more drasti- cally than earlier ones (Roth et al., in press). Thus, mor- phological structures that arise early in ontogeny have a more complex appearance and are more similar to homologous structures in non-paedomorphic out- groups than structures that appear late. These late ap- pearing structures are either strongly simplified or are not formed at all.

The results presented here are consistent with the pae- domorphosis concept. In the salamander Pleurodeles [ge- nome size: 19.7 pg, Moreschalchi (1990); tectum cell di- ameter: 9.7 ~tm, G. Roth et al., unpublished data], cell proliferation is generally lower than in the frog Rana [genome size: 4.2 pg, Olmo (1983); tectum cell diameter:

7.9 ~tm, G. Roth et al., unpublished data]. In addition, those processes that appear late in tectum ontogeny, such as the migration of cells to superficial tectal layers, are reduced in Pleurodeles. However, the general pattern of cell proliferation and cell migration is not altered. It remains an open question whether ontogenetic pro- cesses are uniformly slowed down or whether these pro- cesses are additionally affected by heterochronic pro- cesses as described for the migration of pigment cells in the white mutant axolotl (L6fberg et al. 1989). The failure of migration in the white mutant axolotl is attrib- utable to heterochrony with respect to the maturation of pigment cells and the extracellular matrix. When pig- ment cells are able to migrate into the periphery, the extracellular matrix is still immature. When the extracet- lular matrix is mature, pigment cells are no longer able to migrate. A heterochrony in tectal development could occur as follows. The slow-down of development might mainly affect cell proliferation but not the differentiation of the fiber layer. As a consequence, cells might mature later and therefore reach the ability to migrate later in ontogeny, at a moment when the neuropil might have lost its capacity to allow cells through. This hypothesis is supported by studies of Messenger and Warner (1989) who have demonstrated a heterochrony with respect to the maturat ion of glial cells and neurons in J(enopus laevis and Ambystoma mexicanum. In Xenopus, both cell types develop synchronously, whereas in Ambystoma, glial cells develop earlier than neurons.

Our findings give a clear-cut example of how changes in development in phylogenetically related taxa lead to different brain morphologies that have previously been interpreted as being qualitatively different.

Acknowledgements. We gratefully acknowledge the following for technical assistance: M. Ahlbrecht, M. Ernst, G. Kr/iger and T. Roy-Niemeyer. J. Blanke and Dr. C. Naujoks-Manteuffel gener- ously provided the light-microscopic material from Rana temporar- ia (J.B.) and Pleurodeles waltl (C. N.-M.). We thank Drs. K. Nishik- awa, S. Sessions, and M. Wullimann for helpful comments on the manuscript. This project was supported by the Deutsche For- schungsgemeinschaft (Ro 481/9-2, Schm 833/3-1).

References

Adams JC (1981) Heavy metal intensification of DAB-based HRP reaction product. J Histochem Cytochem 29:775

Alberch P, Gould SJ, Oster GF, Wake DB (1979) Size and shape in ontogeny and phylogeny. Palaeobiology 5:296-317

Angevine JB Jr, Sidman RL (1961) Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature 192:766-768

Bullock TH (1984) The future of comparative neurology. Am Zool 24:693-700

Cavalier-Smith T (1978) Nuclear volume control by nucleoskeletal DNA, selection for cell volume and ceil growth rate, and the solution of the C-value paradox. J Cell Sci 34:247 278

Cavalier-Smith T (1982) Skeletal DNA and the evolution of ge- nome size. Annu Rev Biophys Bioeng 11 : 273-278

Constantine-Paton M (1988) A neural pattern unfolding: proper- ties of retinotectal differentiation in frog tadpoles. In: Kollros JJ (ed) Developmental neurobiology of the frog. Liss, New York, pp 231-253

287

Dann JF, Beazley LD (1988) Development of the optic tecta in the frog Limnodynastes dorsalis. Dev Brain Res 44:21-35

Gadisseux JF, Kadhim H J, Bosch de Aguilar P van den, Caviness VS, Evrad P (1990) Neuron migration within the radial gliai system of the developing murine cerebellum: an electron micro- scopic autoradiographic analysis. Dev Brain Res 52 : 39-56

Gallien L, Durocher M (1957) Table chronologique du d6veloppe- merit chez Pleurodeles waltl. Michah Bull Biol 91:97-114

Gona AG, Uray NJ, Hauser KF (1988) Neurogenesis in the frog cerebellum. In: Koilros JJ (ed) Developmental neurobiology of the frog. Liss, New York, pp 255-276

Gosner KL (1960) A simplified table for staging neuron embryos and larvae with notes on identification. Herpetologica 16: 183- 190

Gouid SJ (1977) Ontogeny and phylogeny. Belknap-Harvard Uni- versity Press, Cambridge, Mass

Hally MK, Rasch EM, Mainwaring HR, Bruce RC (1986) Cyto- photometric evidence of variation in genome size of desmog- nathine salamanders. Histochemistry 85:185 192

Hayashi Y, Koike M, Matsutani M, Hoshino T (1988) Effects of fixation time and enzymatic digestion on immunohistochemi- cal demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue. J Histochem Cytochem 36:511-514

Homer HA, MacGregor H (1983) C-value and cell volume: their significance in the evolution and development of amphibians. J Celi Sci 63:135-146

Hsu S, Raine L, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a compari- son between the ABC and unlabelled antibody (PAP) proce- dures. J Histochem Cytochem 29:577-580

Kollros JJ (1988) Toward an understanding of tectal development in frogs. In: Kollros JJ (ed) Developmental neurobiology of the frog. Liss, New York, pp 207-229

L6fberg J, Perris R, Epperlein HJ (1989) Timing in the regulation of neural crest cell migration: retarded "maturation" of region- al extracellular matrix inhibits pigment celi migration in em- bryos of the white axolotl mutant. Dev Biol 131:168-181

Mansour-Robaey S, Pinganaud G (1990) Quantitative and mor- phological study of cell proliferation during morphogenesis in the trout visual system. J Hirnforsch 31:495-504

Messenger NJ, Warner A (1989) The appearance of neural and glial cell markers during early development of the nervous sys- tem in the amphibian embryo. Development 107:43-54

Morescalchi (1990) Cytogenetics and the problem of lissamphibian relationships. In: Olmo E (ed) Cytogenetics of amphibians and reptiles. Birkh/iuser, Basel, pp 1-19

Naujoks-Manteuffel C, Manteuffel G (1988) Origins of descending projections to the medulla oblongata and rostral medulla spina- lis in the urodele Salamandra salamandra (Amphibia). J Comp Neurol 273:187-206

Nieuwkoop PD, Faber J (1967) Normal table of Xenopus laevis (Daudin). North Holland, Amsterdam

Northcutt RG (1987) Lungfish neural characters and their bearing on sarcopterygian phylogeny. J Morphol 1 : 277 297

Olmo E (1983) Nucleotype and cell size in vertebrates: a review. Basic Appl Histochem 27:22%256

Potter HD (1969) Structural characteristics of cell and fiber popula- tions in the optic tectum of the frog (Rana catesbeiana). J Comp Neurol 136: 203-232

Rakic P (1972) Mode of cell migration to the superficial layers of fetal monkey neocortex. J Comp Neurol 145:61-84

Romeis B (1968) Mikroskopische Technik. Oldenbourg, Munich Romer AS (1970) The vertebrate body, 4th edn. Saunders, Philadel-

phia Roth G (1987) Visual behavior in salamanders. Springer, Berlin

Heidelberg New York Roth G, Naujoks-Manteuffel C, Grunwald W (1990) Cytoarchitec-

ture of the rectum mesencephali in salamanders: a Golgi and HRP study. J Comp Neurol 278:181-194

Roth G, Dicke U, Nishikawa K (1992) How do ontogeny, mor- phology, and physiology of sensory systems constrain and di- rect the evolution of amphibians ? Am Nat 139:$105-S124

Roth G, Nishikawa K, Naujoks-Manteuffel C, Schmidt A, Wake DB (1993) Paedomorphosis and simplification in the nervous system of salamanders. Brain Behavior Evolution (in press)

Schmidt A, Wake MH (1991) Phylogenetic changes in the morpho- logical differentiation of the caecilian rectum mesencephali. In: Elsner N, Penzlin H (eds) Synapse - transmission - modulation. Proceedings of the 19th G6ttingen Neurobiology Conference. Thieme, Stuttgart, p 335

Sessions SK, Larson A (1987) Developmental correlates of genome size in phlethodontid salamanders and their implications for genome evolution. Evolution 41:1239-1251

Shimada M, Langman J (1970) Cell proliferation, migration and differentiation in the cerebral cortex of the golden hamster. J Comp Neurol 139:227-244

Straznicky K, Gaze RM (1972) The development of the tectum in Xenopus laevis." an autoradiographic study. J Embryol Exp Morphol 28 : 87-115

Szarski H (1976) Cell size and nuclear content in vertebrates. Int Rev Cytol 44: 93-111

Szarski H (1983) Cell size and the concept of wasteful and frugal evolutionary strategies. J Theor Biol/05:201-209

Sz6kely G, L/tzfir G (1976) Cellular and synaptic architecture of the optic rectum. In: Llin/ts R, Precht W (eds) Frog neurobio- logy. Springer, Berlin Heidelberg New York, pp 407-434

Vanegas H, Ebbesson SOE, Laufer M (1984) Morphological as- pects of the teieostean optic tectum. In: Vanegas H (ed) Com- parative neurology of the optic rectum. Plenum Press, New York, pp 93-120

Wake DB (1966) Comparative osteology and evolution of the lung- less salamanders, family Plethodontidae. Mere So Calif Acad Sci 4:1 111