/. Embryol. exp. Morph., Vol. 16, 3, pp. 431-438, December 1966
With 1 plate
Printed in Great Britain
431
A reinvestigation of some of the
tissue movements involved in the formation of the
neural tube and the eye/lens system of Triturus
alpestris and Xenopus laevis
ByR. S. LOWERY 1
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 optic cup and the lens, are spatially separated at the flat neural plate
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 ectoderm 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 removes 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 development 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 embryo T. alpestris was examined from the flat
neural plate stage through to the optic vesicle stage.
Embryos 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 heatshielded lamps which were shuttered except during exposures. The interval between 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 originated 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 widemouthed pipette.
S3
(a)
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, F l , G l , 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 neural 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 position 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
Classical lens
anlage.
1
Section A-B
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 ectoderm (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 described 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)
Perspex strip
200/i
Notochord
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 nervous system. 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 subectodermal 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
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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.
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CHANTURISHVILI, P. S. (1959). Exhibition of 'A preparation showing the process of typical
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154, 273-83.
LIEDKE, K. B. (1955). Studies on lens induction in Ambystoma maculatum. J. exp. Zool. 130,
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LOPASHOV, G. V. & STROEVA, O. G. (1961). Morphogenesis of the vertebrate eye. Adv.
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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,
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(Manuscript received 8 April 1966)