/. Embryol. exp. Morph. 76, 9-25 (1983)
Printed in Great Britain © The Company of Biologists Limited
Regeneration of the eye margin in Periplaneta
americana (Insecta, Blattodea)
By P. M. J. SHELTON 1 , H.-D. PFANNENSTIEL 2 AND
E. WACHMANN2
From the Zoology Department, University of Leicester and The Institutfiir
Allgemeine Zoologie der Freien Universitdt, Berlin
SUMMARY
The regulative ability of the proliferative zone of the insect eye margin has been investigated
in larval Periplaneta americana. After sections of the eye margin are removed the eye
nevertheless recovers to form a normal shape. Using chimaeras of lavender and wild-type
animals we were able to show that the margin can regenerate from the differentiated parts of
the eye. When differentiated eye tissue is confronted with epidermis from the head capsule
adjacent to the proliferative zone (the vertex), the regenerated margin always forms from the
eye. There is no evidence that intervening levels can be intercalated between host and graft
tissues when sections of the eye margin are moved to new circumferential levels. However,
in that situation differences between tissues from non-adjacent circumferential positions lead
to the rounding up of the graft and it fails to develop normally.
INTRODUCTION
The compound eyes of hemimetabolous insects grow throughout larval
development by increase in the size of existing ommatidia and by the addition of
more ommatidia to the perimeter of the eye (Meinertzhagen, 1973; Shelton,
1976). In Periplaneta americana eye growth is due mainly to the addition of
ommatidia along the dorsal, anterior and ventral parts of the eye margin. There
is no addition of ommatidia to the posterior margin (Nowel, 1981; Stark & Mote,
1981). We have shown that cells forming new ommatidia are derived from a
proliferation zone within the eye margin (Nowel & Shelton, 1980; Nowel, 1981).
This zone has been regarded as a persistent primitive portion of the original eye
anlage in the embryo (Bodenstein, 1953). According to that view, cells within the
proliferation zone are determined by their ancestry as eye-forming cells and
removal of this anlage material should result in cessation of eye growth. There
is some evidence to support this idea. If the whole eye including the proliferation
zone is removed from a cockroach nymph, the wound becomes covered with
1
Author's address: Department of Zoology, Adrian Building, University of Leicester,
Leicester LEI 7RH, U.K.
2
Authors' address: Institut fur Allgemeine Zoologie der Freien Universitat, Konigin-LuiseStr. 1-3, D-1000 Berlin 33.
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P. M. J. SHELTON AND OTHERS
undifferentiated cuticle and eye regeneration never takes place (Nowel & Shelton, 1980). On the other hand, if the eye is only partially removed we have
noticed that the eye continues to grow and that local damage to the eye margin
in P. americana nymphs has very little effect on the size and shape of the adult
eye (Shelton, unpublished observations). This implies that damaged sections of
the proliferative zone can reform by some regulative mechanism. The purpose
of the present investigation was to provide conclusive evidence that this is so.
Having established that the eye margin can regenerate, our next objective was
to establish the source of the cells forming the regenerated eye margin. Such cells
could be derived from any of three separate sources. First, they could come from
the head epidermis adjacent to the proliferation zone (the vertex). Second, they
could be derived from the mature eye. Finally, the ablated section of the eye
margin could reform from the remaining eye margin to either side of the wound.
The present experiments show that a new eye margin including the proliferative
zone can reform from mature regions of the eye. There is some evidence that the
new eye margin forms partly by migration of cells into the wound from remaining
parts of the eye margin. We cannot exclude the possibility that in certain circumstances cells derived from the vertex can form eye margin but in our experiments
we obtained no evidence that this occurs.
MATERIALS AND METHODS
The culture conditions and operation procedure have been described
previously (Nowel & Shelton, 1980). Chimaeric eyes were generated by exchanging sections of eye margin between wild-type and lavender (Ross, Cochran
& Smyth, 1964) P. americana at stages 5 to 8 using recently moulted animals.
In some cases a second operation was performed on the eye one or two moult
stages later. Details of other types of operation are given in the results section.
Most animals were photographed at each moult following the operation and
they were fixed as adults in a paraformaldehyde/glutaraldehyde mixture (Karnovsky, 1965) and embedded in Araldite. Some specimens were fixed at larval
stages to examine the anatomy of the regenerated eye margin. Experimental
animals were photographed using a Zeiss Tessovar Photomacrographic Zoom
system. For this they were held under water following anaesthesis by cooling.
This method eliminates unwanted reflections from the eye surface. Embedded
eyes were sectioned for light microscopy at 1-0 fjm and stained with toluidine
blue in the usual way. To demonstrate the proliferation zone some animals were
injected with a 1 % colchicine solution (0-5 jul/0-1 g live weight of animal) made
up in saline (Hoyle, 1953). For the SEM, specimens were mounted on
aluminium stubs after critical-point drying. They were coated with a 1-3 nm
layer of gold using an ISI sputter-coating unit and examined with an ISI-60
SEM.
Regeneration of the eye margin in Periplaneta americana
11
RESULTS
Location of the proliferation zone within the eye margin
The anatomy of the nymphal cockroach eye margin has already been described
(Nowel, 1981). At the extreme edge of the eye there is a region of undifferentiated and dividing cells; this is the proliferation zone. It is restricted to the
dorsal, anterior and ventral parts of the circumference; new ommatidia are not
added to the posterior margin. Separating the proliferation zone from the mature
ommatidia of the differentiated eye is the maturation zone. This region contains
preommatidia in various states of differentiation with occasional dividing cells
between them (Nowel, 1981). The least differentiated ommatidia are closest to
the proliferating zone and the most differentiated ones are closest to the fully
differentiated part of the eye. In the present investigation the regions of the eye
containing the proliferation zone were identified by injecting animals (3 days
after moulting) with colchicine and fixing them for sectioning after 12 h. In
subsequent experiments to remove the eye margin this entire region was cut out
(Fig. 1A).
Regeneration of the eye after removal of a section of dorsal eye margin
To test the regenerative powers of the eye margin, strips of tissue containing
about one third of the dorsal eye margin were removed from nymphs at moult
stages 5 to 8. In different animals the tissue was removed from the anterior,
middle or the posterior regions of the dorsal eye margin (Fig. 2A, B, C). The
contralateral eye was used as an unoperated control. The results were similar in
all cases. The animals were allowed to develop to the adult stage, 5 or 8 moults
later. If no eye margin regeneration had taken place, that part of the eye normally derived from the ablated margin should have failed to form. So, for instance,
where the posterior third of the dorsal eye margin had been removed the posterior third of the eye should have ceased to grow, resulting in a large notch in
the normally smooth outline of the eye. Regular inspection of operated animals
showed that eye growth in the damaged region was temporarily interrupted for
one or two moults after the operation. The damage was visible in all animals as
a zone of undifferentiated cuticle within and at the edge of the eye. However,
after two moults a new margin had formed in the damaged area and eye growth
in that sector had resumed. Thus inspection of animals at the adult stage revealed
operated eyes of more or less normal shape and the dorsal regions of the eyes
were similar in size and shape to the contralateral controls (Figs 3A, B). These
observations provide conclusive proof that the eye margin can reform following
ablation.
Regeneration of the eye margin in \avender/wild-type chimaeric eyes
Although the previous experiment shows that the eye margin can regenerate,
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P. M. J. SHELTON AND OTHERS
Fig. 1
Regeneration of the eye margin in Periplaneta americana
B
c
13
Fig. 2. Effects of removing a third of the dorsal eye margin from a nymph (left,
central third; centre, posterior third; right, anterior third). In all cases the adult eye
has a normal appearance. A region of scar tissue (dotted) is the only detectable
consequence of the operation.
it says nothing about the origin of the cells which form the reconstituted
proliferation zone. We used chimaeras in an attempt to answer this question.
Chimaeric eyes consisting of patches of eye tissue carrying a distinctive
autonomous pigment marker were generated by exchanging dorsal eye material
between lavender and wild-type individuals. The grafts were oblong and of
variable size being one quarter to one half the length of the dorsal eye margin.
The grafts consisted of eye margin with narrow regions of adjacent vertex epidermis and mature eye to either side of it (Fig. 3C, 4A, B). In 19 cases the graft was
accepted and continued to grow. After one or two moults, a second operation
was performed to surgically remove the eye margin from the graft. The tissue
removed extended at least three rows of facets into the eye and contained a large
area of vertex material. This ensured that any vertex material implanted with the
original graft was removed and that none of the proliferation zone was left
Fig. 1. Micrographs showing sections through the eye margins in control and experimental nymphs of P. americana.
(A) Normal eye, showing the organization of the eye margin and the parts that
were removed or transplanted in the various experimental procedures. The
proliferation zone (pz) is located at the extreme edge of the eye and contains a
mitotic figure (arrow) arrested with colchicine. Dividing cells are also found in the
maturation zone (mz) which contains partly differentiated ommatidia. A well differentiated cone (c) indicates the limit of the maturation zone. In removing a margin
during ablation or transplant experiments, a region containing the zones between the
dashed lines was removed together with equal areas of mature eye and head epidermis (e) to either side.
(B) The regenerated eye margin reformed from the grafted tissue in a chimaera
from which the graft's eye margin had been removed. The major eye margin zones
have reformed and the proliferation zone (pz) contains a dividing cell (arrow). Other
labels as above.
(C) An eye margin of normal appearance which has reformed from the grafted eye
tissue after confrontation of mature eye with head epidermis. Mitotic figure arrowed.
Other labels as before. Bars represent 25jum.
P. M. J. SHELTON AND OTHERS
Fig. 3
Regeneration of the eye margin in Periplaneta americana
15
behind. The wound was repaired with an implant of vertex material from another
animal (Figs 3D, 4A, B). In one half of the animals the vertex was taken from
an animal of host phenotype, in the rest it was taken from an animal of the
phenotype of the original eye graft. Subsequent eye development was followed
over successive moults. The results for both combinations of graft were similar.
In the majority of cases (12 animals) the marked eye implant continued to grow
showing that a new eye margin had formed (Figs 3E, F; 4A, B). Regenerated
margins of selected individuals were fixed and sectioned at larval stages before
the cessation of eye growth. They had the appearance of normal eye margins with
a characteristic proliferation and maturation zone (Figs 1A, B). Ommatidia
formed by the regenerated margin were normal with the usual complement of
receptor, cone and pigment cells. Most animals were allowed to develop to the
adult stage before being fixed. Each animal was photographed at each moult
stage following the operation. Comparisons of the same animal at different
stages showed that there was a regular increase in numbers of ommatidia with
the pigmentation of the original eye graft (Figs 3E, F). These combined observations show that the regenerated margin is providing a continual supply of new
cells to the eye and that the patterns observed are not due to the redistribution
of ommatidia from the original graft. Where the implant of vertex was of host
type and the regenerated eye had the phenotype of the original eye implant this
showed that the eye itself can form a margin. In all cases where the regenerated
Fig. 3. A series of macrophotographs showing the effects of various types of operation procedure. After removal of the posterior third of the dorsal eye margin from
a nymph the resulting adult eye has an approximately normal shape (A). Comparison
of the operated (right) eye with the control shows that the eyes are both the same size
(B). The only indication of the operation is a region of scar tissue (s). Figs 3C-F
illustrate the effects of removing the eye margin from the grafted eye in a chimaera.
Chimaeras were constructed by implanting a section of donor eye margin (C). At a
later nymphal stage the eye margin in the vicinity of the graft was removed and
replaced with an implant of head epidermis (D). Two moults later the original eye
graft continues to grow indicating the regeneration of the margin (E). The resulting
adult eye is shown in (F). The continual addition of new ommatidia shows that there
is true growth of the original graft and not the redistribution of ommatidia from
within it. In some of these experimental animals the graft failed to reform a new eye
margin (G). In another type of experiment the implanted eye tissue came from the
centre of a donor eye (H). In some cases such grafts formed a margin between head
capsule and eye. Fig. 31 shows the same eye at a later stage. In a number of cases the
eye margin apparently reformed but later petered out (J). In many cases the graft
did not reform a margin but was surrounded by host-type ommatidia (K). Removal
of the eye margin to either side of the implanted mature eye leads to the formation
of a graft-type margin in all cases and a relatively much wider region of regenerated
eye margin (L). The bottom row of figures (3M-P) shows the effect of implanting a
rectangular section of vertex epidermis across the eye margin. One moult after the
graft the rectangular graft has become rounded (M). After two moults it has withdrawn from the eye (N). Later, ommatidia have reformed in the previously damaged
region (O). In the adult, the eye has a more or less normal shape and the only
evidence of the operation is scar tissue in the eye (P). Figs 3A, B, bar represents
1-0 mm. Figs 3C-P, bar represents 0-5 mm.
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P. M. J. SHELTON AND OTHERS
eye margin had the phenotype of the original eye implant, the shape of the graftderived eye had a common pattern with a constriction at the point where the eye
margin of the implant had been removed (Figs 3E, F; 4A, B). Our interpretation
Fig. 4
Regeneration of the eye margin in Periplaneta americana
17
of this observation is that the wound is repaired in part by the inward migration
of host cells from either side of the graft. The same pattern of constriction is seen
whether the vertex implant is of host or donor type. Where the vertex implant
is of the donor eye type, the inwardly moving cells can only have come from host
eye. In some cases (seven animals) implanted eye tissue failed to form a new
margin and consequently ended up isolated from the edge of the eye by
ommatidia of host phenotype (Figs 3G, 4A, B). Once again identical results were
obtained for both types of vertex implant. Certainly where the vertex was from
an animal of the same eye colour as the implanted eye (four cases), the cells
which form the regenerated margin (of host phenotype) must have come from
the host eye. Here we must assume that the inward migration of cells is the
dominant process and occurs before the implanted eye has time to regenerate its
own margin. These combined observations show that when the eye margin is
removed it can regenerate from the remaining eye tissue of the eye graft but that
it may be partly restored by the invasion of eye cells from the host to either side of
the graft. We consider the most likely source of such host cells is the eye margin.
The experiments do not exclude the possibility that in certain circumstances the
regenerated eye margin can form from vertex. We have tested the ability of the
vertex to contribute cells to the regenerated margin in later experiments.
Regeneration of the eye margin from implants of mature eye
Although we were careful to remove all of the eye margin from the graftderived eye in the previous experiment (the sections of cuticle removed were
always inspected to confirm that the underlying eye margin was attached), we
wished to eliminate the possible criticism that we had left a few cells behind. The
following experiments show that our previous conclusions were justified. Once
again we used wild-type and lavender stocks to identify graft- and host-derived
tissues. In this case we made chimaeras at nymphal stages by implanting square
grafts of mature eye tissue from the centre of donor eyes into similar shaped sites
at the dorsal margins of host eyes (Figs 3H; 4C). This type of graft confronted
mature eye with the vertex. Since the implant came from the centre of the eye
it could not possibly include eye margin cells. If our previous results represented
true regeneration of the eye margin, we predicted that a new margin should
Fig. 4. Chimaeric eyes were generated by exchanging sections of eye margin between wild-type and lavender nymphs (A, B). One or two moults later a square of
tissue containing the eye margin and adjacent head epidermis was removed and
replaced with a square of head epidermis. Some eye grafts failed to grow and became
surrounded by host ommitidia. In the majority of cases a new eye margin formed and
the graft continued to grow. In a second type of operation (C) a square of tissue from
the centre of a donor eye was implanted into a suitable site in the eye of a host so that
the graft confronted the vertex along one edge. Some of these grafts did regenerate
a narrow section of eye margin although the majority did not. The success rate was
improved to 100 % by removing host eye margin to either side of the implant at the
following moult (D).
18
P. M. J. SHELTON AND OTHERS
regenerate at the border between the graft eye tissue and the head capsule
epidermis. A number of these animals did regenerate an eye margin derived
from the graft although the success rate was rather low (3/20 animals) (Figs 31,
J; 4C). In two cases the implant became separated from the margin only after two
or three moults. In these cases it appeared that the graft had initially reformed
a small section of eye margin but that after several moults it petered out (Fig. 3J).
A similar result can occur after a control eye margin graft where the length of the
implanted margin is short (Shelton, unpublished observation). This would be
expected if cell division within the eye margin is under some sort of probabilistic
control. It argues against a population of stem cells. While this type of graft had
a low success rate, we were able to increase the success rate to 100% (nine
animals) by removing substantial sections of the host eye margin to either side
of the graft together with the dorsal edge of the implant, one moult after the
initial operation (Figs 3L; 4D). This finding provides support for the hypothesis
that cells from the host eye margin can migrate into the wounded area at the edge
of the eye. When these cells are removed, the eye margin regenerates mainly
from the implanted mature eye. Once again we sectioned eyes in the region of
the regenerated margin and found that there was a normal appearance (Fig. 1C)
and that the ommatidia derived from it had the normal complement of cells.
Implantation of marked head capsule (vertex) epidermis into the larval eye
The previous experiments show that the eye can reform a new proliferation
zone from mature eye. The following experiments were designed to test the
ability of vertex tissue to form eye margin. Once again we used exchanges of
grafts between lavender and wild-type animals so that the source of any
regenerated eye cells could be identified. In 50 % of the cases we used a lavender
host and wild-type donor; in the rest we used the opposite combination. The
results were the same for both combinations. Two types of experiment were
performed, the object of both being the same. That was to confront epidermis
from the vertex with mature eye and see if a new margin would form from vertexderived cells at the junction between the two kinds of tissue. In the first type of
experiment, a 250/im square of vertex epidermis from close to the eye margin
was implanted into the centre of the host eye at a site prepared by removing an
appropriately sized piece of mature eye tissue (Fig. 5A). In all cases (25 animals)
the epidermis was sloughed off after the first postoperative moult. We assume
that this failure of grafts to 'take' is due to the large differences in cell surface
properties of the cuboidal epidermal cells and the spindle-shaped eye cells.
In order to overcome this difficulty, a second series of experiments was devised
using lavender and wild-type tissues as before but in a configuration where the
vertex graft made contact with both the head capsule and the eye. The graft
consisted of a rectangular piece of vertex tissue. It was implanted into a rectangular site whose long axis crossed the eye/vertex border at right angles (Fig. 5B).
These grafts had a low success rate (7/30 animals) but a significant number could
Regeneration of the eye margin in Periplaneta americana
19
B
Fig. 5. The grafts illustrated here confront vertex epidermis with eye tissue. Squares
of vertex implanted into the centre of the eye are lost by the following moult (A)
leaving only a small area of scar tissue. Rectangular grafts of vertex crossing the eye
border are retained longer (B). However, the vertex material rounds up and withdraws from the eye, the eye margin regenerates from host tissue and the adult eye
has a more or less normal shape.
be followed over a number of moults. We assume the improved success rate was
due to the fact that one end of the graft was in continuity with the head capsule.
Where the grafts were successful the part of the implant within the eye was
rounded and had lost its original rectangular shape. It often formed a bump
above the smooth contour of the adjacent eye (Fig. 3M). By the second postoperative moult the graft had withdrawn completely from the eye and formed a
rounded protrusion just outside the eye in a typical case (Fig. 3N). This left a
large area of scar tissue at the edge of the eye. By the third postoperative moult
the two sides of the eye margin that had been separated previously by the graft
had become confluent (Fig. 3O). Subsequent growth resulted in an adult eye in
which the dorsal eye region had normal proportions even though a significant
part of the original eye margin had been removed and replaced with vertex (Fig.
3P). From direct observations it was clear that, in this typical case, the eye
margin reformed in the damaged area once the vertex implant had withdrawn.
In all cases ommatidia in the vicinity of the regenerated amrgin were of host
phenotype. The implant never contributed cells to the regenerated eye margin,
it must have formed from the eye tissue. These results do not prove that the
vertex cannot in other circumstances redifferentiate to form eye margin but in
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P. M. J. SHELTON AND OTHERS
this situation it is unable to do so. They do indicate that there are considerable
differences between head capsule and eye and that these differences cause an
implant to withdraw from the eye.
Effects of local reversal of the eye margin
So far we have been concerned with regulative behaviour mainly along the
radial axis of the eye. Next we wished to establish whether there is any evidence
for regulation around the circumferential axis. Where pattern regulation occurs
in postembryonic development of insects, missing intervening zones can be intercalated when non-congruent levels are combined (French, 1981; Wright &
Lawrence, 1981). In this series of experiments non-congruent parts of the eye
margin were confronted by exchanging sections of nymphal eye margin between
left and right eyes (Fig. 6C). In a parallel set of control operations, sections of
eye margin were exchanged between eyes of the same side (either left or right)
(Fig. 6A). Once again we used lavender and wild-type animals for each exchange. Exchanging grafts between left and right sides produces a local reversal
of the eye margin and consequently brings different regions of the circumference
into juxtaposition. The grafts were of a standard size and included a 250jum
length of dorsal eye margin and several rows of ommatidia. The animals were
photographed at each moult stage following the operation and they were fixed
as adults. The eyes with a section of the eye margin reversed differed from the
controls in only one detectable way. After one or two moults, tissues derived
from the reversed eye margin protruded beyond the normally smooth surface
contours of the eye to form a slight bump. As development continued this
unevenness gradually disappeared so that in the adult the eye surface was as
smooth after reversals as it always was after controls. The other feature of the
grafts that we considered was their overall shape (Figs 7C, D). For each successful reversal (8 animals) we made a direct comparison with a control operation to
the same part of the dorsal eye margin (Fig. 7A). We had access to at least 40
control operations from a previous study (Nowel, 1981) in addition to 10 of our
own. We found that the shape of the graft-derived eye after a reversal was not
detectably different from the appropriate control (Fig. 7D). If intercalation had
taken place after reversals two consequences should follow. First, the overall size
of the eye should be larger compared with the contralateral unoperated eye or
with the relevant control graft. This is because intercalation should increase the
length of the eye margin. Second, the graft-derived retina after reversals should
be larger than in controls for the same reason. Neither of these predictions was
borne out (figs 6C; 7A, C, D). We concluded that no intercalation had taken
place.
Exchanges between dorsal and anterior eye margin
As a second test for intercalation we exchanged similar 250 jum sections of eye
margin between the dorsal and anterior parts of the eye (Fig. 6D). The results
Regeneration of the eye margin in Periplaneta americana
Fig. 6. This series of controls (A, B) and circumferential exchanges (C, D) tested the
ability of confronted non-congruent levels to intercalate the normally intervening
levels. When a section of dorsal eye margin is reversed in exchanges between left and
right eyes (C) the shape of the graft-derived eye tissue and the overall shape of the
eye is the same as in the control (A). In exchanges between dorsal and anterior eye
margin the transplants round up and fail to grow as well as controls (B, D).
21
22
P. M. J. SHELTON AND OTHERS
/
\
•
f
Fig. 7. Control grafts (A, B) show the normal pattern of eye growth. Figs 7C & D
show the same experimental animal at two different stages. When sections of the eye
margin are reversed as in (C) the shape of the resulting adult graft-derived eye (D)
is indistinguishable from a control graft to the same part of the eye margin. In Fig.
7C eye tissue dorsal to the arrows has formed after grafting operation. Dotted lines
in Fig. 7D show the outlines of graft-derived eye in a comparable control. When eye
margin is grafted from anterior to dorsal regions (E) and vice versa (F, G. H) the graft
rounds up shortly afterwards (E) and forms a bumpy projection. Figs 7F, G & H show
the resulting adult eye of an animal that received dorsal eye margin in an anterior
location. A side view shows the rounding of the graft (F); seen from a dorsal position
the typical projection is visible (G). An SEM preparation (H) viewed from the
anterior shows how the graft has become isolated from the anterior edge of the eye
(lower left). Figs 7A-G, bar represents 0-5 mm. Fig. 7H, bar represents 100/an.
of these experiments were compared with control grafts at each of the two sites
(Figs 6A, B; 7A, B). The notable feature of the exchanges was the relatively low
success rate. Often there was no sign of the graft at the following moult (21
animals). Where the graft was detectable at the new site subsequent growth was
extremely poor (10 animals). In most cases the implanted graft rounded up; in
extreme cases it produced a raised projection (Figs 7E, F, G, H). It seems that
normally the host eye margin to either side of the graft outcompetes the grafted
margin, the graft becomes isolated from the edge of the eye and it ceases to grow.
The control grafts show that the eye grows much more at the dorsal than at the
anterior region (Figs 6A, B; 7A, B). The failure of the anterior grafts to grow
satisfactorily in a dorsal location could be attributed to differential growth rates.
Regeneration of the eye margin in Periplaneta americana
23
However, in the converse situation, implanted dorsal margin should be an advantage over the anterior host margin. Nevertheless, even here the implants
failed to grow properly and were outcompeted. If intercalation occurs, a graft of
the sort performed here should produce a grossly abnormally shaped eye, and the
graft-derived eye should expand with successive moults. The interpretation of
these results is that intercalation does not occur but that different parts of the eye
margin are non-equivalent in some way. Thus grafts from one region to another
fail to develop properly in the new site.
DISCUSSION
These experiments provide the first conclusive proof that the eye margin has
regulative properties. Although probably there is normally an unbroken lineage
relating the postembryonic proliferation zone of the eye to the original optic
anlage (Bodenstein, 1953), that lineage can be broken and a regenerated margin
will give rise to perfectly normal eye tissue. The results are consistent with those
of Mouze (1972) using dragonflies. However, our results are more conclusive
because we were able to use grafts carrying autonomous cellular markers. In the
case of the dragonfly experiments no markers were available and cell migration
from the remaining eye margin could have restored the ablated section.
The clearest result was that the eye itself can regenerate a proliferation zone.
Although one has to be cautious about the possible failure to completely remove
the ablated section, we took great trouble to ensure that this always occurred.
The most persuasive argument is that fully differentiated eye tissue can reform
a dividing margin when confronted with vertex epidermis. This result satisfies us
that true regeneration does occur.
The shape of the graft-derived eye tissue following removal of an implanted
eye margin tells us something of the processes of wound repair and regeneration.
A consistent feature is the constriction which occurs in the outline of the graftderived tissue at the point where the grafted eye margin was removed. In some
cases this constriction is total showing that the graft has not regenerated a margin. Where the eye and vertex implants both carry the same marker to distinguish
them from the host, the constriction can only be caused by inward movements
of host cells from either side of the eye implant. The most likely source of cells
is the host eye margin but without direct evidence we cannot exclude the possibility that the invading cells come from slightly more mature parts of the eye.
Our conclusions rest upon the assumption that the trails of graft cells extending
to the edge of the eye are the result of true growth and not the redistribution of
cells from within the graft. This conclusion is justified because sections through
the regenerated region reveal a normal eye margin with proliferation and
maturation zones. Also the number of ommatidia with the graft phenotype
increases in a regular way from moult to moult and they have the normal complement of receptor, pigment and cone cells.
24
P. M. J. SHELTON AND OTHERS
At present we do not know which class or classes of cells are respecified to form
eye margin. However, we are well aware that dividing cells are found in the
maturation zone and in mature parts of the eye (Anderson, 1978; Nowel, 1981).
In the maturation zone ommatidia already have the full complement of receptor
and cone cells so it is likely that the dividing cell population normally gives rise
to pigment cells. This is supported by the fact that such dividing cells are often
filled with pigment grains characteristic of secondary pigment cells (Shelton,
unpublished observations). During normal development, cell division within the
mature region of the eye is probably associated with the need for more secondary
pigment cells as the ommatidia increase in size (Nowel, 1981). It seems unlikely
that highly differentiated receptor cells could revert to a primitive undifferentiated state where they are capable of cell division. That is also the case for the
cone cells. However, since secondary pigment cells seem capable of cell division
it is possible that they could revert to a more primitive condition. The question
of whether the new proliferation zone is formed from the dividing cell population
or from dedifferentiation of specialized cells, requires further investigation.
The pattern-forming mechanism that allows the regeneration of a proliferation
zone also remains obscure but we see no reason why it should be the same one
that allows the regeneration of cuticular ridges (Stumpf, 1968) or even segmental
boundaries (Wright & Lawrence, 1981). This could alter the way we think of the
mechanisms for setting up patterns within the eye. While we thought of the eye
margin as a persistent primitive part of the eye anlage it was logical to assume
that it was the eye margin which acted as a progress zone and laid down the
pattern of positional values within the eye. If the eye margin reforms by the same
sort of mechanism that causes predictable cuticular features to form at particular
segment levels, then it is logical to assume that the eye margin does not generate
a sequence of positional values but that it has fixed positional value and in
consequence cells at that level form a proliferation zone.
While we could establish regulative ability in the radial axis of the eye that was
not the case for the circumferential axis. It is known that cells in different regions
of the eye margin divide at different rates (Nowel, 1981). The controlling factor
could be related to differences in positional value around the circumference. Our
experiments to shift particular circumferential levels to new locations on the
perimeter of the eye were designed with the idea that intercalation of intervening
levels would occur wherever differences in positional information are present.
Since intercalation does not occur, we concluded that the circumferential axis of
the eye may be organized in the same way as the mediolateral axis of the insect
segment. Here with only one known exception (the ecdysial line; Shelton, 1979),
transplants along this axis do not lead to intercalation. However, in the case of
the eye, transplanted sections of eye margin do not grow normally. Characteristically they round up and the neighbouring host margin cells seem to outcompete
the transplant. This tendency of grafts to round up would be consistent with the
occurrence of adhesive differences at different points on the circumference. Such
Regeneration of the eye margin in Periplaneta americana
25
adhesive differences are thought to occur in other insect systems (Nardi &
Kafatos, 1976; Nardi, 1977; Niibler-Jung, 1977) and they cannot be excluded
here. The tendency of vertex grafts to withdraw from the eye could also be
interpreted in terms of differences in adhesive properties.
Our results lead us to conclude that it is only the eye itself that can regenerate
a new margin. This would explain why it is that the Periplaneta eye never reforms
when the whole eye is removed (Nowel & Shelton, 1980) but that the eye is
capable of considerable regulation when it is only partially ablated.
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ANDERSON, H.
(Accepted 22 April 1983)