/ . Embryol. exp. Morph. Vol. 29, 2, pp. 347-361,1973
347
Printed in Great Britain
An experimental study of eye
development in the cephalopod Loligo vulgaris:
determination and regulation during formation of
the primary optic vesicle
By H.-J. MARTHY 1
From the Laboratoire Arago, Banyuls-sur-mer, France
Dedicated to Professor Dr Adolf Portmann on his 75th birthday,
27 May 1972
SUMMARY
By a series of explantation, transplantation (yolk syncytium left intact) and incision experiments done with the eye rudiment during the stages VI-IX (Naef, 1923) it is concluded that
the yolk syncytium does not induce the differentiation of 'the outer layer of cells' from stage
VI on as suggested by Arnold (1965 b). From the explantation and transplantation experiments
the author draws the conclusion that there exists, from stage VI on, in the' outer layer of cells'
on each side of the embryo, an area which contains all factors necessary for eye formation and
which manifests itself, under experimental conditions, in regulation. The explanted eye
rudiment shows in vitro autonomous differentiation capacity only if nutritional conditions are
sufficient. The incision experiments elucidate the role of 'contractile elements' in organogenesis. Arnold's results are discussed.
INTRODUCTION
According to Arnold (19656) the Loligo embryo, at early organogenetic stages
(stage VI of Naef, 1923; stages XVI and XVII of Arnold, 1965 a), shows three
components: 'an outer layer of cells' (the actual embryo), 'an inner syncytial
epithelium' (transitory formation, usually referred to as 'yolk syncytium'),
'a central mass of yolk'.
According to this author again, the inner layer has an inductive function.
The yolk syncytium is the original egg cortex; it contains a 'morphogenetic
inductive map' determining the differentiation, from stage VI on, of the 'outer
layer of cells' (Arnold, 19656, 1968). This represents a classical induction/
reaction system.
Experiments done by the author (Marthy, 1970 a) suggest, however, that
Arnold's conclusions are not necessarily adequate to explain his very precise
1
Author's address: Laboratoire Arago, 66650 Banyuls-sur-Mer, France.
348
H.-J. MARTHY
observations. We have been able to show, by means of transplantation experiments, that the eye rudiment in stage VII has a 'differentiation tendency'
(Raven, 1938) and the capacity of autonomous retina differentiation. Thus,
determination or 'canalization' (Waddington, 1940) towards specific organ
formation appears to be irreversible. The explanted rudiment of the eye vesicle
fully integrates at its new location, as auto- or allograft at the same stage, without losing any of its elements either by mechanical or other disturbances during
the transplantation procedure or by an influence of the yolk syncytium on to
which it is grafted (which would be, to say to the least, conceivable according to
Arnold's hypothesis).
As for the donor region, it is interesting to note that the yolk syncytium,
following Arnold's concept, would not yet have entirely lost its induction activity:
the material of the ' outer layer of cells' growing over it forms one normal-sized
eye rudiment (Arnold, 19656) or two smaller-sized eye rudiments (Marthy,
1970#). This means that the formerly neighbouring material, which should be
supposed to be determined (not for retina formation) as irreversibly as the
explanted material, is in fact labile. In other words, we are facing a true regulation phenomenon. The question to be answered is then: Is the peripheral
material capable of replacing the removed material by partial autonomous
reversion of former determination (regulation) or does it only furnish cell
material of which the former determination is reversed by the inductive action
of the underlying yolk syncytium?
We have carried out our investigations in this line by a series of experiments
at the earlier stages of morphogenesis when the primary eye vesicle is formed
(stages VI-IX); the yolk syncytium was either left intact or damaged or removed.
In particular, the morphogenetic processes were analysed: (1) in the explantation
zone after complete or incomplete removal of the eye rudiment, with the yolk
syncytium left intact in all cases; (2) in the heterotopic auto- or allograft grafted
on to intact yolk syncytium; (3) in the prospective eye vesicle in situ after separation from the yolk syncytium or partial removal or destruction of the latter;
(4) in the prospective eye vesicle in situ after incision.
We should like to anticipate the conclusion to be drawn from our experiments:
An inductive influence of the yolk syncytium does not exist at stage VI or later.
Instead, an 'eye region' in the 'outer layer of cells' can be denned on either side
of the embryo: this region is definitely larger than Arnold's 'placode' (1971,
p. 297), the complete removal of this complex leads to the lack of the entire eye,
although the yolk syncytium remains intact. If this complex is, however, incompletely removed, the remaining parts regulate autonomously. The morphological appearance of this regulation (single eye or duplication) depends on the
stage at which the operation is performed. The process of duplication revealed
the presence of 'contractile elements' - their important role in the shaping of
the optic vesicle was demonstrated by a simple experiment in which only an
incision was made in the otherwise intact rudiment.
Eye development o/Loligo vulgaris
349
The eye rudiment is particularly favourable for this type of experiment
because of its large size (stage VII: 0-7/0-25 mm) and its later separation into a
pigmented part (vesicle) and unpigmented peripheral organs, which facilitate
evaluation of the various effects of operations. A discussion of Arnold's results
and conclusions is suitable as his investigations were also focused on this organ
complex.
MATERIALS AND METHODS
The eggs ofLoligo vulgaris used in the experiments were obtained by bottom
trawling off the coast of Catalonia (Western Mediterranean). The egg strings
were kept in running sea water or in large receptacles with well-aerated sea
water. For preparation, operations and culture we used the method described
earlier (Marthy, 19706) with one modification: the agar was sterilized (autoclave) and stored in 50 ml flasks and was re-liquified and distributed in Petri
dishes immediately before an experiment. After removal of the innermost egg
case (chorion), embryos were prepared for operation by several rinses in sterile
sea water. In order to diminish the pressure of the yolk mass, the yolk envelope
was perforated at the vegetative pole, with the result that a large quantity of
yolk leaked out and formed a good attachment for the embryo, preventing its
rolling over. Also, the loss of yolk pressure facilitates removal of tissue without
impeding normal development. The translucency of the agar allows operating
with illumination from below. Platinum wire (0-1 mm) and 'Wolfram' wire
were used for explantation and incision. The tip of the 'Wolfram' wire was
sharpened in an electrolyte. Grafts were implanted on to the denuded, intact
yolk syncytium in the stomodeal region or in the ventral midline between the
arm rudiments. In stages VI and VII the embryo was turned over the graft for
3-4 h in order to facilitate integration of the graft by gravity. In stages VIII and
IX the embryos were immobilized by means of glass bridges for several hours.
In general, ciliar activity of the embryo detaches the surface cells from the glass,
thus avoiding damaging of tissue by removal of the glasses.
Operations were made at room temperature (21-24 °C) in sterile sea water
to which penicillin was added (50-100 i.u./ml). The culture dishes (6 cm in
diameter, 1 cm deep) with an agar bottom (2-3 mm thick) were kept at 18-520 °C. The operated embryos were transferred to new culture dishes with fresh
sea water at 4-day intervals, since the lack of oxygen disturbs development,
slowing down its rate and eventually leading to malformations (Marthy,
1970/)). Embryos were in general fixed later than stage XVI in Halmi fixation
solution. Throughout this paper we will use the developmental stages defined
by Naef (1923), indicating the corresponding stages of Arnold (1965a), if
necessary.
For a better understanding of the processes we have investigated it will be
useful to give a brief description of the normal eye development (detailed studies
by Faussek, 1900; Naef, 1928; Sacarrao, 1954; Arnold, 19670,6, 1971).
23
E M B 29
350
H.-J. MARTHY
Fig. 1. Dorsolateral view of an embryo in stage V1I+. o, Eye rudiment.
Fig. 2. Dorsal view of an embryo in stage VIII-IX. o, Almost closed primary optic
vesicle; m, mouth (stomodeum).
Fig. 3. Optic vesicle of an embryo in stage XV.
Around stage VI the early eye rudiment can be recognized on either side of
the embryo as an ectodermal area, the ventral part of which subsequently
becomes limited by an inconspicuous semilunar ridge (Fig. 1) that progresses
dorsally so as to form a complete, closed ring of elongate shape by stage VIII.
Meanwhile mitotic activity and rearrangement of nuclei lead to a thickening of
the ectoderm within this ringfold. The latter grows over (like a closing curtain)
the central part of the rudiment until the optic vesicle is completely closed at
stage IX (Fig. 2). This primary optic vesicle, first a flattened pocket, acquires its
definitive, nearly spherical shape in subsequent stages (Fig. 3). Histological
differentiation includes thickening of the proximal part (retina) until stage IX,
pigmentation and separation into two layers (within and beyond the limit of
the membrana limitans exterior (von Lenhossek, 1894; Faussek, 1900)) of the
retina from stage XI on. Starting at stage IX, the distal part of the eye vesicle
shows differentiation into a 'lentigenic area' (Arnold, 1971) and a secondary
annular fold which forms the iris (pigmentation from stage XI on). The large
lentigenic cells are distinct before morphological separation of these two areas.
The cornea arises from a prolongation of the bases of the arms two and four
(from stage XII on); these dorsal and ventral folds of each side grow laterally,
unite behind the eye vesicle and finally close at the base of the arm crown (stage
XVIII).
It will be useful to divide the morphogenetic phase that is the particular interest
of our investigation into the following steps:
Stage V-VL Ectodermal eye rudiment monolayered, not distinguishable in
living embryos (Fig. 4); topographical extent not histologically defined.
Eye development o/Loligo vulgaris
351
Fig. 4. Dorsolateral view of an embryo in stage V-VI. x, 'Eye region'.
Fig. 5. The same embryo as in Fig. 4 in stage VIII (19 h later at 19 °C).
Fig. 6. The same embryo as in Fig. 5 in stage IX (14 h later at 19 °C). Primary optic
vesicle (o) closed, a, Arm; e, embryo; m, mantle; s, outer yolk sac; st, stomodeum;
y, yolk (leaks out).
Stage VI. Cells of eye rudiment slightly elongate, nuclei arranged on different
levels. Strong mitotic activity. Limit of primordium histologically defined only
in ventral part by an inconspicuous elevation. (About stage XVI of Arnold.)
Stage VI-V1L Peripheral elevation more distinct, extending dorsally to the
midline of the primordium.
Stage VII. This structure encloses the entire primordium as an annular
elevation, the ventral part of which becomes a distinct ridge.
Stage VII-VIII. Ridge transforms into a fold in the ventral area. Shaping of
the ridge in the dorsal part. Detached cells lie between anterior part of the eye
primordium and the yolk syncytium.
Stage VIII. Annular fold entirely surrounds prospective retina and starts
growing over it (this is not an invagination!) (Fig. 5). Optic ganglion a flat band
between anterior part of eye primordium and yolk syncytium.
Stage VIII-IX. Closure of the optic vesicle progressing.
Stage IX. Porus of the eye vesicle closed (Fig. 6). Histological differentiation
of the outer wall into a central area of lens formation and peripheral area of
iris formation. Retina thick, but not pigmented. Optic ganglion a compact mass
attached to the retina. (Stage XXI of Arnold.)
23-2
352
H.-J. MARTHY
Table 1. Schematic representation of the experiments performed on
the eye rudiment of the stages VI and VII.
15
12
Slight thickening indicates the eye rudiment, located in the 'outer layer of cells',
if not otherwise indicated in similar figures as in 10 (thickened retina) or in 18
(lack of the eye vesicle).
Yolk syncytium.
Yolk.
Optic ganglion.
1-4 ->16 Experiments from Results section 3 (a) and (b). Short lifting up of the eye rudiment
but left in situ after damaging (2), partial removal (3) or destruction (4) of the underlying yolk syncytium does not influence normal eye formation (16).
Experiments from Results section 1 (a): 1, 2 (compare Arnold, \965b, pp. 75, 76:
5-^16,
5 ->17 yolk syncytium left intact). The marginal cell material at the explantation zone
closes the wound and one normal sized eye vesicle is formed when operation is
done in stage VI, two smaller sized eye vesicles are formed when operation is done
in early stage VII.
5a ->9
The removed eye rudiment shows no differentiation in vitro (Arnold, 19656, pp.
75, 76); results confirmed by earlier experiments (Marthy 19706,c).
Experiments from Results section 2: 1, 2. The grafted eye rudiment differentiates
into a compact, later pigmented retina. (Aspect of nutrition)
Eye development o/Loligo vulgaris
353
Table 1 (cont.)
6 >16
Experiments from Results section 1 (c): 1, 2. A remaining half of an eye rudiment
forms one normal sized optic vesicle.
(7) >16 Additional experiment to 6: a remaining half of an eye rudiment also forms one
normal sized eye vesicle if the other half is explanted with its underlying yolk
syncytium and yolk.
8 ->17
Experiments from Results section 1 (d): 1. Remaining parts of a partially removed
eye rudiment develop, from stage VII on, into two optic vesicles.
11—14 Experiments leading to the complete lack of eye formation (but not of optic
ganglion formation).
11 ->18 Experiments from Results section 1 (6) (yolk syncytium left intact). After wound
closure no optic vesicle is formed (eye rudiment removed together with a circular
area surrounding the annular elevation). Optic ganglion present.
12 ->18 No formation of the optic vesicle after removal of the eye rudiment together with
some of the surrounding tissue and underlying yolk (Arnold, 19656, p. 75; result
confirmed by earlier experiments).
12a >15 The isolate differentiates in vitro into an optic vesicle and the adjacent optic
ganglion, confirming Arnold's result (19656, p. 75).
(13, 14) >18 Additional experiments to 12. No eye formation after removal of the eye
rudiment and subsequent partial removal (13) or complete destruction (14) of
the underlying yolk syncytium.
RESULTS
1. Morphogenetic behaviour of the explanation zone, with intact yolk syncytium,
after removal of eye material
Operations made at different stages were always identical: 'subtotal' (a),
total (b) and partial removal (c, d, e) of the eye rudiment without damaging the
underlying yolk syncytium. Wound closure took place without exception.
According to the amount of material removed, morphogenetic behaviour of the
explantation zone varied. The number in parentheses following the stage number gives the interpretable cases in the series of experiments.
(a) The ectodermal rudiment was removed including the annular elevation
(stage VI: estimation of dorsal part):
1. Stage VI (8). Wound closure within 20 h. Subsequently, a normal eye
vesicle was formed, which by stage IX could not be distinguished from a
normal control eye. The adjacent optic ganglion had normal size. (Table
1: 5-»16).
2. Stage VI and VII (3). Wound closure within 15-10 h. Further development resulted in formation of two optic vesicles, the common size of which
was equal to a normal vesicle. (Table 1: 5 (Fig. 7)->17 (Fig. 8).)
3. Stage VII-VIII (2). A single, small optic vesicle was formed (Fig. 9).
Duplication never occurred in the optic ganglion; it was of normal size and
structure, both in the case of vesicle duplication and with a single small vesicle.
354
H.-J. MARTHY
Fig. 7. Lateral view of an embryo in stage VII with the eye rudiment removed.
Fig. 8. Dorsal view of the head region of an embryo in stage XII. On one side two
optic vesicles are formed.
Fig. 9. Ventral view of the head region of an embryo in stage XIV-XV. On one side
a small optic vesicle is formed.
4. Stage VIII and later (6). Complete lack of optic vesicle. Growth and
development of the optic ganglion, however, were not influenced by the
operation.
(b) The ectodermal rudiment was removed together with a circular area surrounding the annular elevation (on the site of its appearance):
1. Stage VI (3). Wound closure after more than 24 h. No eye rudiment was
formed (Table 1: 11 ->18). Optic ganglion was smaller than the normal size
(Fig. 10). (This may be due to excessive removal of cell material, because
of the difficulty of locating exactly the limit of explantation.)
2. Stage VII and later (3). Wound closure after about 20 h. No eye rudiment was formed. The optic ganglion was of normal size (this may be due
to more precise explantation made possible by the greater extent of the
annular elevation). (Table 1:11 (Fig. 11)-»18 (Fig. 12).)
Eye development 0/Loligo vulgaris
355
(c) Only parts of the ectodermal rudiment were removed:
1. Stage VI (2). Wound closure according to extent of explantation, not
later than 15 h. A normal eye vesicle and a normal optic ganglion were
formed (Table 1: 6->16).
2. Stages VII and VH-VHI (3). Identical effect after removal of dorsal or
ventral part of the eye rudiment respectively. Wound closure within 10 h.
A complete optic vesicle was formed and reached normal size (Table 1:
6 (Fig. 13)->16).
3. Stage VIII-IX {\). Same effect, except for formation of iris fold, which
was inhibited.
(d) The ventral part of the ectodermal rudiment was only partially removed
leaving the ventral tip intact:
1. Stages VII (1) and VIII (1). Wound closure within 10 h. Two eye
vesicles were formed, the ventral one smaller in size than the dorsal one,
the latter smaller than normal size (Table 1: 8—>17). Both were surrounded
by a common iris fold.
2. Stage VIII-1X (2). Formation of two vesicles with differentiation of
lens, retina and pigmentation; lack of iris fold.
(e) The anterior or posterior part of the ectodermal rudiment was removed:
1. Stage VII (3). Formation of normal-sized eye vesicle and iris fold.
2. Stages VII-VIII to VIII-IX (3). Formation of normal-sized eye vesicle;
lack of iris fold.
2. The development of the eye primordium when grafted as a heterotopic auto- or
allograft on to denuded, intact yolk syncytium
The differentiation capacity of the graft depends on the stage at which transplantation is done:
1. Stage VI (3). The eye primordium grafted on to embryos at stage VII (thus
obtaining shorter time of wound closure) differentiates into a compact retina,
which becomes pigmented at later stages. No further differentiation into the
typical retina layers. No peripheral thickening (which marks beginning of vesicle
formation). (Table 1: 5<z->10.)
2. Stage VII(5). Explanted eye primordium is incorporated into the surrounding tissue and differentiates into a compact retina with pigmentation occurring
later. In contrast to our earlier description (Marthy, 1970a), differentiation into
lentigenic cells occurred in 3 of 5 cases; these cells (Fig. 14) were located in the
peripheral elevation. Further thickening at the periphery did not occur again.
3. Stage VIII (ringfold distinct) (2). Incorporation of the graft and formation
of an optic vesicle within a few hours, with normal structural and histological
differentiation. No formation of iris fold.
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12
H.-J. MARTHY
Eye development o/Loligo vulgaris
357
15
Fig. 14. Marginal region of an eye rudiment grafted in stage VII, now on stage XII.
r, Retina cells; c, lentigenic cells, x 140.
Fig. 15. Grafted eye vesicle from a stage IX in stage XVIII. /, Iris fold; c, lentigenic
cells. x280.
4. Stage IX (2). The already closed optic vesicle is incorporated and undergoes
normal differentiation including spherical shape (Fig. 15).
3. The development of the eye primordium in situ after operation of the underlying
yolk syncytium
Embryos at stage VI and at stage VII (period of reconstitution ability) were
used in these experiments in which the eye primordium was left intact, its
normal contact with the underlying yolk syncytium being interrupted. These
experiments are complementary to the others in which, on the contrary, the eye
primordium was operated on and the yolk syncytium left intact.
(a) The primordium is cut out except for a small connexion and lifted off the
yolk syncytium (Table 1: 1). Under the yolk pressure circumference of the
wound increases. After a few minutes the embryo is turned over so as to bring
FIGURES
10-13
Fig. 10. Ventral view of an embryo in stage XIV-XV. On one side lack of the optic
vesicle. Optic ganglion reduced in size, a, Arm; g, optic ganglion; w, mantle; o,
optic vesicle; s, outer yolk sac.
Fig. 11. Dorsolateral view of an embryo in stage VII with the eye rudiment removed
together with surrounding tissue; yolk syncytium left intact.
Fig. 12. Ventral view of an embryo in stage XIV-XV. On one side lack of the optic
vesicle. Optic ganglion normal sized.
Fig. 13. Ventral view of an embryo in stage XV. o, Optic vesicle formed from a half
of the eye rudiment.
358
16
H.-J. MARTHY
l
19
Fig. 16. Optic vesicle with dorsal constriction of an embryo in stage XIV.
Fig. 17. Splitted eye rudiment of an embryo in stage VIII (division into a dorsal and
a ventral half) 15 min after operation. -», Direction of the contracting forces along
the wound edges.
Fig. 18. Lateral view on the head region of an embryo in stage XVIIT. Duplication
on one side after incision in stage VIII of the eye rudiment.
Fig. 19. Eye vesicle. The two lenses (/) of the two primary optic vesicles are connected.
Aspect at hatching stage, r, Retina, typically layered; c, cornea; /, iris, x 87-5.
the eye rudiment into its original position. Wound closure is completed after
a few hours. This operation does not influence normal eye development except
for retardation which is apparent until stage XVI (Table 1: 16).
(b) Damaging (4), partial removal (2) or complete destruction (5) of the yolk
syncytium after lifting up the eye primordium has the same effect (Table 1: 2,
Eye development o/Loligo vulgaris
359
3, 4->16). The loss of yolk more or less markedly influences the wound closure
(wound contracts beyond the circumference of the primordium) and leads to
some deformation of the eye rudiment, the general development of which is
normal except for retardation as observed in the above experiment.
These experiments again demonstrate that the eye primordium from stage
VI on carries all the information necessary for further development that is
independent of the yolk syncytium. We will look in more detail into this
phenomenon by analysing the results of point (a) 2 and (d) in the Results
section 1.
4. Splitting of the eye primordium (yolk syncytium left intact or damaged)
{a) Splitting horizontally (division into a dorsal and a ventral part).
1. Stage VI (3). The dorsal and the ventral part of the eye rudiment
divided by a simple horizontal incision 'fuse' within a few hours after
operation. One normal optic vesicle was formed.
2. Stage VI-VII and earlier stage VII (2). Formation of a complete optic
vesicle showing a slight constriction of the posterior wall at the site of the
incision (Fig. 16).
3. Stage VII-VIII (5). Constriction more marked in the posterior part
becoming visible in the anterior part. The beginning of this process can be
recognized about 10 min after operation.
4. Stage VIII (6). Strong constriction along the line of incision (Fig. 17)
resulting in complete division into dorsal and ventral part, each developing
into an independent optic vesicle with a well-defined lens (Fig. 18). In four
cases the lenses of the two vesicles were connected to each other (Fig. 19).
The iris fold is always common to both vesicles, but does not form normally,
however, if the operation is done later than V1II-1X.
5. Stage IX (2). Splitting of the now closed eye vesicles does not result in
constriction and separation. Wound closure leads to complete reconstitution
of the eye vesicle; lens and iris fold form normally. Lack of pigmentation in
the retina up to hatching stage marks the site of incision.
(b) Splitting vertically (division into a posterior and anterior part):
1. Stage VI (i). No effect.
2. Stage VII (1). Constriction of the retina on the dorsal side.
3. Stage VII-VIII and later (5). Dorsal constriction, no separation into
two separate optic vesicles. The absence of the process in the earlier experiment (splitting horizontally) is apparently related to the elongate shape of
the eye rudiment.
DISCUSSION
The findings given in Results, §§ 1-3, lead us to the conclusion that the yolk
syncytium, at stage VI, does not induce the formation of the eye rudiment and,
consequently, that the eye rudiment situated in the 'outer layer of cells' does
360
H.-J. MARTHY
not have a 'reactive power' (Raven, 1938). At stage VI the 'outer layer of cells'
of the embryo has two areas that carry all factors necessary for eye formation,
providing the capacity of self-organization and autonomous differentiation
which manifests itself, under experimental conditions, in regulation. For an area
having these characteristics, we may use the term 'field' as denned by Nieuwkoop
We do not yet know how the field is established. The question is, whether
certain cells are programmed to form 'eye' or whether originally undetermined
cells receive, prior to stage VI, a morphogenetic programme from the yolk
syncytium. Our recent results from experiments prior to stage VI are in favour
of the former interpretation (Marthy, 1972).
At present, our experiments demonstrate that an eye field does exist at stage
VI. The extent of this field is shown by the experiments in the Results section
1 a and 6: It is considerably larger than the morphologically recognizable area
of the future retina and the annular elevation surrounding it. We therefore think
that Arnold's results (19656, p. 75: no yolk syncytium explanted) were due to
incomplete removal corresponding to our experiments (a) 1, 2, 3 in Results §1
(Table 1: 5).
As to the explant (cf. Table 1: 5a->9 and 12^-^15), we may add here that the
nutritive conditions in Arnold's experiments (19656: presence or absence of
yolk syncytium including yolk material!) suggest that the difference observed
in further development may be due to the presence or absence of nutritive
material, respectively. This aspect was apparently not taken into account by
Arnold, who stated that yolk material did adhere to the explanted eye primordium (19656, pp. 73, 75), but did not mention this fact in his summary,
where the supposed role of the yolk syncytium alone is denned (19656, p. 77).
Denying an inductive function of the yolk syncytium does not mean that a
morphogenetic action is not conceivable on a mechanical basis. We think that
Arnold's results (19656, p. 75: yolk syncytium explanted), which are fully
confirmed by our own experiments (Table 1: 12, 13, 14), can be interpreted as
the morphogenetic 'response' to the excessive disturbance of the material
involved. It appears that wound closure has to proceed in an 'organized' way;
the yolk syncytium serving as a ' stage' of rearrangement.
In addition to this, we note that the arrangement of the cells of a rudiment
is mechanically guided by elements that are integrated in the cell complex. Our
incision experiments (no removal of cell material) give an idea of the dynamics
of the spatial differentiation of the vesicle. The constrictions observed after
incision show the contracting force exerted by (supposedly cellular) elements.
It is uncertain at present whether the entire cell complex or a few specialized
elements take part in this process. The formation of two optic vesicles gives the
most marked evidence of this process which intensifies from stage VI-VIF
onward. The degree of morphological aberrance reflects the 'competition'
between wound closure and organogenetic contraction forces. The combined
Eye development 0/Loligo vulgaris
361
effect of these forces from stage VI-VII on in all organogenetic areas spread
out over the yolk results first in a separation of the organ rudiments and later,
from stage VIII (Naef, 1928, p. 267) on, in an increased contraction of the
entire embryo 'assembling' the organs to form a compact organism.
I thank Dr Katharina Mangold for her encouragement during the investigations presented
here and for her help with preparing the manuscript. I am also very much indebted to Dr
Sigurd von Boletzky for valuable discussions and his help during preparation of the manuscript. Mrs Leslie Rowe also kindly read the manuscript. The present investigation was
partially supported by a grant of the Swiss National Fund for the Advancement of Scientific
Research.
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{Received 2 August 1972)