/. Embryol. exp. Morph., Vol. 16, 3, pp. 431-438, December 1966 431 With 1 plate Printed in Great Britain A reinvestigation of some of the tissue movements involved in the formation of the and the / system of Triturus alpestris and Xenopus laevis

ByR. S. LOWERY1 From the Department of Zoology, The University, Hull

INTRODUCTION Since the beginning of the century the generally accepted scheme of eye/lens development has been that proposed by Spemann (1901) and later confirmed by numerous workers. According to this scheme the two presumptive components of the definitive eye, the and the lens, are spatially separated at the flat stage. They later come into apposition as a result of tissue movements which occur during the formation of the neural tube; the optic vesicle then provides an inductive stimulus for the subsequent development of the presumptive lens tissue. Spemann's suggestions concerning the tissue movements involved in the early formation of the eye/lens system do not appear to have been fundamentally questioned until the publication of a number of papers by Chanturishvili (1943, 1949,1958,1959,1962), although the theory of lens induction has been modified by workers such as Liedke (1955), Jacobson (1963) and von Woellwarth (1962). Lopashov & Stroeva (1961) have reviewed the literature on eye/lens development and their paper should be consulted for further references. Chanturishvili denies the main tenet of the ' classical' scheme, namely the induction of a lens by the eyecup out of the with which it comes into contact during the closure of the neural folds. Instead, evidence is presented (Chanturishvili, 1949,1958,1959) that the presumptive eyecup anlage is from the first continuous with the presumptive lens anlage within the confines of the flat neural plate and that later these two become separated by a split within the neural tissue itself. This split is subsequently invaded by mesoderm. Thus, according to Chanturishvili, both the optic vesicle and the lens are neural in origin. Chanturishvili further suggests that this neural lens is induced by the products of cytolysis of epidermal cells which are trapped within the newly formed lens vesicle and not by the optic vesicle as stated in the classical scheme. 1 Author's address: The Department of Microbiology, The University, Birmingham 15, U.K. 27-2 432 R. S. LOWERY Text-fig. 1 is my interpretation of Chanturishvili's suggestions concerning the position of the eye/lens anlage at the flat neural plate stage in the Anura and its subsequent development. It will be seen from this figure that if the neural anlagen of the optic vesicle and of the lens are continuous in this way within the flat neural plate, the neural folds cannot be formed by simple rolling up of the neural plate as suggested by many earlier workers, such as Vogt (1929). If this were to happen the eye/lens system would be formed in the dorsal midline. Chanturishvili (1956) suggests instead that the formation of the neural folds is by some sort of building up dorsal to the anlage of the eye/lens system.

Epidermis

Split within neural tissue

Invasion of mesoderm Text-fig. 1. Chanturishvili's scheme of eye/lens development for the Anura.

The advantage which Chanturishvili sees in his main suggestion is that it re- moves the necessity to suppose that lenses can be formed in two distinct and apparently unrelated ways. According to the classical scheme the normal lens is of extraocular embryological origin and arises from the lateral ectoderm, whereas the Wolffian lens is of ocular, and so of neural, origin. If Chanturishvili is correct then the lens tissue is of neural origin in both normal development and Wolffian regeneration. The investigation here described was undertaken to see if evidence could be obtained which was irreconcilable with either the 'classical' scheme of develop- ment or that of Chanturishvili. Lens formation in Triturus 433

A. Triturus alpestris Material and Methods A cine-micrographic recording of pigmented ectodermal cell movements in the eye region of the developing T. alpestris was examined from the flat neural plate stage through to the optic vesicle stage. at the neural plate stage were stripped of their membranes in 1/10 Holtfreter solution and displayed in a paraffin-wax depression; as far as possible they were not constrained or distorted in any way. Illumination was with heat- shielded lamps which were shuttered except during exposures. The interval be- tween exposures was either 100 or 200 s and the overall magnification finally used was x 4. Filming of individual embryos was interrupted by focus changes which were necessitated by the alterations in the shape and orientation of the embryos. The movements so recorded of individual pigmented lateral ectodermal cells were traced in relation first to the developing neural folds and subsequently to the outline of the optic vesicle.

Results Eleven individual embryos were filmed but the tracings from two only will be presented (Text-fig. 2a, b) as the results were repetitive. It can be seen from the two figures that there is a dorsal movement of pigmented ectodermal cells, throughout the period studied, from a region just lateral to the flat neural plate to a region which forms part of the lateral ectoderm of the head at the early optic vesicle stage. During later stages of development the optic vesicle could be seen bulging beneath these same pigmented lateral ectodermal cells which had origi- nated outside the margin of the flat neural plate. Sections of T. alpestris embryos showed that the ectoderm is of homogeneous structure throughout the period of observation, unlike that of the Anura at a similar period of development which is clearly divided into two discrete layers of cells. Thus the movements of individual pigmented ectodermal cells would seem to be reliable markers of the movement of the ectoderm as a whole. These observations are therefore consonant with the classical scheme of eye/ lens development; they cannot easily be explained by Chanturishvili's suggestion outlined above since they indicate that in T. alpestris the origin of the presumptive lens tissue is outside the confines of the flat neural plate.

B. Xenopus laevis McKeehan (1951) designed an experiment based upon the classical scheme of eye/lens development to investigate lens induction in the chick. He used the morphogenetic movements of neural tube formation to carry strips of cellophane between the optic vesicle and the head ectoderm. This technique has been modified and here used on embryos of X. laevis to demonstrate morphogenetic tissue movements during neural tube formation. 434 R. S. LOWERY

Material and methods X. laevis embryos at the flat neural plate stage were subjected to the operation shown in Text-fig. 3. A pointed strip of Perspex, of thickness about 00064 mm, was threaded beneath the head ectoderm just laterally to the flat neural plate in the eye region. After this operation the embryos were not disturbed until the end of the experiments, when they were transferred to Smith's fixative with a wide- mouthed pipette.

(a) S3

Anterior 'Dorsal *-—Anterior Dorsal! Text-fig. 2. (a) A tracing of the surface of a Triturus alpestris embryo to show the movements of four pigmented lateral ectodermal cells (El, Fl, Gl, HI) during early neural tube formation, from positions lateral to the margin of the flat neural plate to more dorsal positions (E3, F3, G3, H3) in which they comprise part of the presumptive head ectoderm. N1, N 2, N 3: Successive positions of the edge of the neu- ral fold that was superficial in the field of view. SI, S3: Successive positions of the outline of the embryo, (b) A tracing of the surface of another T. alpestris embryo to show the movements of four pigmented lateral ectodermal cells (E1,F1,G1,H1) during late neural tube formation. The cells move anteriorly to positions (E2, F2, G2, H2) in which they form part of the presumptive head ectoderm superficial to the early optic vesicle, from part of which the lens is subsequently formed. V2, V3: Ventral margin of the neural tissue seen in outline. Other lettering as for a.

All embryos which survived to fixation were serially sectioned in paraffin wax. Sections of the Perspex were not seen in the mounted preparations but the posi- tion which it had occupied within the embryo was shown by a space in the sections. Reconstructions were made of twenty-two embryos. A reconstruction was not made if microscopic examination clearly revealed that the Perspex had been displaced during histological processing or that the embryo was badly damaged or that the strip was so deeply situated within the embryo that it had obviously been misplaced at operation. Reconstructions of two of the twenty-two embryos /. Embryol. exp. Morph., Vol. 16, Part 3 PLATE

100/<

ID

R. S. LOWERY facing p. 435 Lens formation in Triturus 435 showed in one instance that the strip had been placed too deeply and in the other instance that it had been placed too far posteriorly. These two will not be further considered.

' Plastic strip

1 Section A-B Classical lens anlage. A—r>-

\ . Classical optic vesicle anlage Text-fig. 3. Diagram to show the position of implantation of the Perspex strips described in section B. Results The data obtained from the remaining twenty reconstructions were indicative of a dorsal movement of the whole or of one end of the Perspex strip during neural tube formation. In six cases the strip was dorsal to the eye/lens system at the optic vesicle stage, an occurrence consonant with the classical scheme. Illustrations of one of these embryos are shown in Text-fig. 4a and Plate 1, figs. A, B, C. This embryo

PLATE 1 Fig. A. Xenopus embryo 31 at operation. The Perspex strip cannot be distinguished in this photograph but the heavy pigmentation lateral to the flat neural plate can be clearly seen. The damage on the left is superficial. Fig. B. Xenopus embryo 31 at the optic vesicle stage just prior to fixation. Note the Perspex strip projecting dorsally to the optic vesicle and also the heavy pigmentation of the lateral head ectoderm. Fig. C. Section A-B through embryo 31 (Text-fig. 4 a). P indicates the hole made by the Perspex strip. Fig. D. Section A-B through embryo 57 (Text-fig. 4b). Fig. E. Section A-B through embryo 5 (Text-fig. 4 c). 436 R. S. LOWERY was of particular value since it was noticed at operation that the ectoderm lateral to the flat neural plate was far more heavily pigmented than the rest of the ecto- derm (Plate 1, fig.A) . At fixation this heavy pigmentation covered the side of the head and therefore included the lens anlage (Plate 1, fig. B). This movement of the lateral ectoderm during neural tube formation exactly parallels that de- scribed earlier for T. alpestris. Thus this particular embryo provided very good evidence in support of the classical scheme, and these results do not appear to be reconcilable with Chanturishvili's suggestion.

(a)

Perspex strip

Notochord

Optic vesicle

(b) 200/i Perspex strip

Notochord Pupil Choroid fissure Optic vesicle Text-fig. 4. (a) A reconstruction of embryo 31 at the optic vesicle stage to show the Perspex strip dorsal to the eye/lens system, (b) A reconstruction of embryo 57 at the lens vesicle stage to show the Perspex strip superficial to the optic vesicle. The strip has apparently been twisted round during its movements, (c) A reconstruction of embryo 5 at the optic vesicle stage to show the position of the Perspex strip relative to the central . Note that the posterior end of the strip is near to the dorsal midline of the embryo.

None of the other embryos provided so conclusive evidence. The terminal positions of the Perspex strips in these individuals varied from superficial to the eye (ten cases) (Text-fig. 4b; Plate 1, fig.D ) to the situation shown in Text-fig. 4c and Plate 1, fig. E, in which the posterior end of the strip has been carried far dorsally during neural tube closure, while the anterior end is superficial to only Lens formation in Triturus 437 the ventral margin of the optic vesicle, or closely adjacent to it (four cases). All of these situations are, however, consonant with the classical scheme and are not explained by Chanturishvili's suggestions.

DISCUSSION The results of the cine-micrographic examination of T. alpestris provide evidence which is quite irreconcilable with Chanturishvili's scheme of eye/lens development and quite reconcilable with the classical view. The second series of experiments, using Perspex strip implantation in X. laevis, did not provide such clear-cut evidence because of the variation seen in the ultimate positions of the Perspex strips. However, those cases in which the strip eventually became lodged either dorsally or superficially to the optic vesicle are quite irreconcilable with Chanturishvili's suggestions concerning amphibian eye/lens development. The positions of the strips in the remaining cases may be due to interference between the edges of the strips and the sub- ectodermal tissues.

SUMMARY 1. A scheme of eye/lens development suggested by Chanturishvili is described and compared with the ' classical' scheme of Spemann. 2. A cine-micrographic examination of the ectodermal movements involved in neural tube formation in Triturus alpestris and an experimental embryological investigation of the process in Xenopus laevis provided data consistent with the classical scheme and irreconcilable with Chanturishvili's suggestion that the whole eye/lens system originates as a single continuous anlage within the flat neural plate of Amphibia.

RESUME Reinvestigation de certains des mouvements tissulaires lies a la formation du tube neural et du systeme ceiljcristallin chez Triturus alpestris et Xenopus laevis 1. L'auteur expose un mecanisme de developpement du systeme oeil/cristallin propose par Chanturishvili et le compare a la conception' classique' de Spemann. 2. Une etude micro-cinematographique des mouvements ectodermiques lies a la formation du tube neural chez Triturus alpestris, ainsi qu'une analyse experimentale de ce processus chez Xenopus laevis ont fourni des resultats qui sont en accord avec la conception classique. Ces memes resultats sont, par ailleurs, inconciliables avec la these de Chanturishvili selon laquelle l'ensemble du systeme oeil/cristallin proviendrait d'un ebauche unique et continue siegeant au sein de la plaque neurale des Amphibiens. 438 R. S. LOWERY

REFERENCES CHANTURiSHViLr, P. S. (1943). Materials for the new understanding of the question of the determination of Lent is Oculi. Bull. Acad. Sci. Georgian SSR 4 (5), 461-8. CHANTURiSHViLr, P. S. (1949). Towards the question of the absence of lens induction in the typical development of the eye. Bull. Acad. Sci. Georgian SSR 10 (9), 567-71. CHANTURISHVILI, P. S. (1956). Research on the Possibility of the Regeneration of a Complete Crystalline Lens in Mammals. From the Institute of Experimental and Clinical Surgery and Haematology, Tifiis. CHANTURISHVILI, P. S. (1958). The role of ectoderm in the development of the crystalline lens. Trans, ophthal. Soc. U.K. 78, 411-38. CHANTURISHVILI, P. S. (1959). Exhibition of 'A preparation showing the process of typical crystalline lens formation in some vertebrates'. Proc. XVth Int. Cong. Zoology, lxxxiii. CHANTURISHVILI, P. S. (1962). Material concerning the development of the crystalline lens of the chick from the cellular substratum of the eye. Trans. Kutais Pedagogical Inst. 24, 339—41. JACOBSON, A. G. ,(1963). Determination and positioning of the nose, lens and ear. 1. Inter- actions within ectoderm and between ectoderm and the underlying tissues. /. exp. Zool. 154, 273-83. LIEDKE, K. B. (1955). Studies on lens induction in Ambystoma maculatum. J. exp. Zool. 130, 353-79. LOPASHOV, G. V. & STROEVA, O. G. (1961). Morphogenesis of the vertebrate eye. Adv. Morphogenesis 1, 321-77. MCKEEHAN, M. S. (1951). Cytological aspects of embryonic lens induction in the chick. /. exp. Zool. 117, 31-64. SPEMANN, H. (1901). Ober Correlation in der Entwicklung des Auges. Verh. anat. Ges. 15, 61-79. VOGT, W. (1929). Gestaltunganalyse am Amphibienkeim mit ortlicher Vitalfarbung. Arch. EntwMech. Org. 120, 385-706. VON WOELLWARTH, C. (1962). Die Rolle des Neuralleistenmaterials und der Temperatur bei der Determination der Augenlinse. Embryologia 6, 219-42.

(Manuscript received 8 April 1966)