/. Embryol. exp. Morph. Vol. 46, pp. 147-170 1978
Printed in Great Britain © Company of Biologists Limited 1978
147
Postembryonic development of the visual
system of the locust, Schistocerca gregaria
II. An experimental investigation of the formation of
the retina-lamina projection
By HILARY ANDERSON 1
From the Department of Zoology, University of Leicester
SUMMARY
In the compound eye of the locust, Schistocerca gregaria, neurons from the retina project
to the lamina in a precise topographical mapping.
The formation of this projection was investigated in grafting experiments which altered
the spatial or temporal relationship between the retina and the lamina.
The results show that retina axons tend to grow along the paths of adjacent axons, with
no indication of specificity for their normal termination sites.
It is suggested that the orderly sequence of retina differentiation during normal development plays a major role in imposing pattern both upon the developing projection and,
through some form of inductive interaction between retina and lamina neurons, upon the
lamina.
INTRODUCTION
Successive layers of the nervous system are frequently connected together
in a configuration known as a topographical mapping, i.e. neurons which are
neighbours in one array form connexions with neurons which are neighbours
in another array (Gaze & Hope, 1976). The importance to the organism of this
arrangement lies in its conservation of information concerning the spatial
distribution of nervous excitation, while the formation of this precise pattern
constitutes a particularly interesting problem for the developmental biologist.
Most insects possess large compound eyes which send their photoreceptor
axons to the optic lobe of the brain, to the region known as the lamina, where
they synapse with first-order interneurons. This projection has been shown to be
a topographical mapping (Braitenberg, 1967; Horridge & Meinertzhagen,
1970; Meinertzhagen, 1976).
The visual system of the locust, Schistocerca gregaria, has been described in a
previous paper (Anderson, 1978) and consists essentially of two 2-dimensional
arrays, the retina and the lamina, of repeating structural units, the ommatidia
and cartridges, with the topographical retina-lamina projection between them.
1
Author's address: Department of Zoology, University of Oxford, South Parks Road,
Oxford, 0X1 3PS, U.K.
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H. ANDERSON
With regard to the problem of forming these connexions, we may consider the
compound eye to be organized along orthogonal axes: the dorsoventral axis
and the anteroposterior axis. The retina and lamina grow in a parallel fashion by
the addition of new ommatidia and cartridges to their respective anterior
margins (Anderson, 1978). At any one point in time, only one dorsoventral
row of new ommatidia is forming connexions with a corresponding band of
new lamina ganglion cells, thus different dorsoventral rows along the anteroposterior axis are forming connexions at different times. It is therefore possible
that the spatial and temporal aspects of the mode of growth of the two arrays
play an important role in ordering the connexions formed between them.
By observing the pattern of the retina-lamina projection as it develops
after various perturbations of the system, it is hoped that the rules by which
the nerves normally make their correct connexions may be inferred. For
example, if the retina is rotated by 180° with respect to the lamina, do the
regenerating nerves show a preference for their original termination positions
and form a 180° rotated pattern, or do they grow out in a normal pattern
suggesting that they have no regional preferences for termination sites in the
lamina?
Horridge performed this experiment on Schistocerca gregaria. He rotated
by 180° the retinae of 24 second instar nymphs and tested their visually mediated
behaviour when they were adult (Horridge, 1968). Twenty-two animals showed
normal optokinetic responses. He inferred that neurons regenerating from the
retina had made a normal pattern of new functional connexions with the underlying non-rotated lamina, i.e. they showed no preference for their original
termination sites. However, behavioural observations require histological
confirmation. Furthermore, in assessing the results no allowance was made for
growth of the retina and lamina. As pointed out by Shelton (reported in
Meinertzhagen, 1973), no conclusions can be drawn from eye rotation experiments which fail to distinguish between regeneration of axons from old (i.e.
pre-operative) ommatidia and de novo differentiation of axons from new
(i.e. post-operative) ommatidia.
I have repeated and extended the original experiments of Horridge using
direct histological observation of the pattern of nerve fibres between the operated
retina and lamina, in experiments in which ommatidia developing before and
after the operation can be distinguished, and in which the formation of the
projection can be examined in one axis at a time. The results are discussed
with respect to some simple models for the formation of topographical mappings.
MATERIALS AND METHODS
A colony of Schistocerca gregaria was reared in crowded conditions in a
room maintained on a regulated cycle of 12 h light at 32 °C and 12 h dark
at 27 °C. The insects were fed fresh wheat seedlings and bran each day.
Formation of retina-lamina projection
149
Animals were selected for operation within 12-36 h after hatching or after
moulting to the second or fourth instar. They were anaesthetized by cooling
for about 1 h in small glass vials placed on ice. They were placed on a cold
moulded bed of Plasticine and held in position with strips of Plasticine. Operations were performed under a dissecting microscope using slivers of GilletteFrancais razor blade in metal holders and fine watchmaker's forceps. Grafts
were held in place until the haemolymph from the host had clotted around the
graft.
The dorsal retina bears a patch of ommatidia with a characteristic morphology, the dorsal spot (see fig. 1, Anderson, 1978). Anterior to the retina is an
area of pale cuticle bearing trichoid sensilla whose shafts are orientated dorsoventrally (see fig. 2, Anderson, 1978). These provide useful markers and were
included in the graft wherever possible.
Operated animals were allowed to become adults and were then tested for
optokinetic behaviour.
S. gregan'a make following movements of the head when a striped pattern
is moved across the visual field (Thorson, 1964; Burtt & Rafi, 1974). This
response was used to test the presence of functional connexions between the
retina and brain following the various surgical operations.
Animals were fixed by the thorax to the centre of a rotating drum 30 cm
in diameter. The drum bore a pattern of alternating black and white stripes
2 cm in width. The head of the animal was free to move. Optomotor movements of the head in response to movements of the drum were recorded using
a light flag of paper fixed to a fine strand of straw attached to the vertex of the
head with Plasticine. Movement was measured by the flag's interruption of a
beam of light falling upon an illuminated photocell, consisting of two cells
in series with a differential output which indicates direction of movement.
The photocell was connected to a pen recorder.
To test only parts of the compound eye, the remaining eye tissue and ocelli
were covered with an opaque paint (Johnson's Matt black), which could be
peeled off easily after testing.
The eyes of adults were then dissected out, fixed in alcoholic Bouin, embedded
in paraffin, sectioned at 10 /«n and stained with reduced silver stain (Rowell,
1963). The plane of section was either vertical or horizontal. Some additional
animals were fixed immediately after the operations or as nymphs. Sections
of their eyes were prepared in the same way but stained with Mayer's Haemalum
and Eosin (Pantin, 1969). This stains up cell bodies and was used to check the
extent of damage following the operation.
Sections were photographed on Kodak Panatomic X film in a Zeiss Photomicroscope II. The film was developed in Uford Microphen and fixed in Amfix
(May & Baker).
The different types of graft performed are illustrated diagramatically in
Fig. 1 and are described in the following section.
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H. ANDERSON
Fig. .1. Diagrams of grafting operations. Dashes indicate the outline of the graft.
The dotted line indicates the position of the graft on the head of the host. (A)
Control replacement of whole second instar retina. (B) Dorsoventral inversion of
whole second instar retina. (C) 180° rotation of whole second instar retina. (D)
Removal of part of the proliferation zone of second instar retina. (E) Dorsoventral
inversion of proliferation zone of second instar retina. (F) Control replacement of
proliferation zone of second instar retina. (G) Graft of proliferation zone of first
instar retina to proliferation zone of fourth instar retina. (H) Control replacement
of part of proliferation zone of fourth instar retina.
EXPERIMENTS
Normal retina-lamina projection
Part of the retina-lamina projection of an adult Schistocerca gregaria is
shown in Fig. 2. The highly regular disposition of retinula fibres and of the
cartridges they innervate is a typical normal configuration and is shown for
comparison with those configurations obtained in experimental situations. A
more general view of the retina, lamina, and retina-lamina projection is given
in an earlier paper (Anderson, 1978).
Formation of retina-lamina projection
.
TZL
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151
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Fig. 2. Vertical section through the retina-lamina projection of an adult locust to
show the pattern of axons in the tract between the retina and lamina, and the
periodic structure of the lamina (wax/reduced silver stain). Scale, 60/im. r, Retina;
tr, trachea; Igc, lamina ganglion cells; ra, retinula axons; c, cartridges.
Whole retina grafts
The retina and surrounding head epidermis of 15 second instar nymphs
were removed and replaced in one of three orientations (Fig. 1A-C) which
altered the spatial relationship between the retina and lamina in neither axis,
in only the dorsoventral axis, or in both axes, respectively.
In all cases the grafted retina continued to grow along its anterior border,
as shown by the addition of new rows of ommatidia (Fig. 3). Four adults
showed normally orientated but reduced optokinetic responses, but this
was entirely confined to the region of the retina formed after the operation.
The remaining animals were behaviourally blind throughout the operated eye.
Histological examination showed that in all cases the lamina was either
missing or severely damaged, the retina formed before the operation failed to
regenerate any nerves, and the retina formed after the operation produced a
disordered mass of fibres (Fig. 4). These fibres frequently formed large whorls
(Fig. 4C), or tracts (Fig. 4A, C) and where they had formed cartridges, the
pattern of fibres was abnormal (Fig. 4, B).
These results show that the retina of S. gregaria is unable to regenerate
severed retinula axons. The optokinetic behaviour observed by Horridge
(1968) was therefore mediated entirely by new neurons from retina formed
after the operation and not by regenerated axons. Furthermore, the four animals
which showed normal optokinetic responses, when examined histologically,
showed a highly abnormal and disorganized pattern of new fibres. Optokinetic
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H. ANDERSON
c*
Fig. 3. Eyes of adult locusts after (A) control replacement of the retina, (B) dorsoventral inversion of the retina, and (C) 180° rotation of the retina, during the
second instar. Trichoid sensilla (ts) are shown anterior to the eye and orientated
ventrally in (A), anterior to the eye and orientated dorsally in (B), and posterior
to the eye in (C).
behaviour is clearly of limited use as a test for ordered nerve connexions.
The minimal neural input for optokinetic behaviour is not known, but an
isolated patch of 100 ommatidia can produce a normal response and it is likely
that connexions to the brain from only some of these are sufficient for the
response (Kien, 1974). On this basis alone, the conclusions of Horridge (1968)
must be considered invalid.
The lamina lies very close to the retina which is curved around it. Consequently it is impossible to prevent damage to the lamina during these operations.
Even if the lamina is not directly injured by the incision, its delicate structure
is disrupted by the forces exerted as the retinula axons are severed. In particular,
damage to the outer optic anlage will prevent further growth of the lamina.
In all three types of whole retina graft, in those patches where a retina-lamina
projection had formed at all, the pattern of retinula fibres was abnormal. A
more detailed statement about the possible organization of the projection
Formation of retina-lamina projection
153
Fig. 4. Sections through adult eyes after whole retina grafts in the second instar
(wax/reduced silver stain). (A) Vertical section through an eye after a control retina
graft. Scale, 170/tm. Note chiasma of retinula fibres between the retina and the
remaining lamina (arrow) and tract of axons running perpendicularly to the normal
pathway (double arrow). (B) Vertical section through an eye after dorsoventral
inversion of the retina. Scale, 200 /*m. (C) Horizontal section through an eye after
dorsoventral inversion of the retina. Scale, 170/*m. Only the retina formed after
the operation has formed axons (ra). Note the absence of lamina neuropil, and the
whorl of retinula fibres (arrow), r, Retina; c, cartridges; m, medulla.
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H. ANDERSON
cannot be made from this type of experiment. Therefore more controlled
operations were performed to test various classes of model which could account
for the formation of the pattern of retinula fibres in normal development.
Organization of the projection in the dorsoventral axis
Three basic classes of model which could account for organization of the
projection in the dorsoventral axis will be considered here (Fig. 5).
Neuronal Specificity
This mechanism was first proposed by Sperry (1951, 1963) for the formation
of connexions between the retina and optic tectum of amphibia and fish.
Applied to the insect eye, it supposes that the ommatidia and their axon
bundles differ intrinsically from one another, as do lamina ganglion cells; a
set of cellular properties is ordered across the retina so that each ommatidium
and its axon bundle is 'labelled' according to its position in the retina. There is a
corresponding set of 'labels' arranged dorsoventrally in the lamina, specifying
each lamina ganglion cell. Retinula fibres form an ordered projection over the
lamina by forming connexions with lamina ganglion cells possessing matching
'labels'. Each fibre grows to its target cell independently of other fibres.
Fibre Interactions
There may be no intrinsic differences between ommatidia or their axon
bundles or between lamina ganglion cells. The direction of outgrowth of
axons and their eventual termination sites could depend solely upon their
relationship with adjacent growing fibres, e.g. the fibres could repel one another.
This would ensure that the fibres did not cross over one another in the dorsoventral axis and so the spatial order of their cell bodies in the retina would be
maintained in the pattern of their termination sites in the lamina. This model
does not require a fibre to have a preferred target cell or site in the lamina.
Contact Guidance
Early neuroembryologists stressed that the paths taken by axons growing in
culture tended to reflect physical irregularities in the substrate and inferred that
similar mechanical factors guided nerve outgrowth in vivo (Harrison, 1910;
Weiss, 1939). This suggests that the temporal sequence of axon outgrowth
might be an important factor in the organization of the retina-lamina projection; new axons could be guided to the lamina by following pre-existing
fibre pathways, without the need for interaction between growing fibres or for
preferred target sites in the lamina.
To test these models, two kinds of surgical operation were performed:
(i) Partial suppression of retinula fibre input to the lamina
A small section was removed from the proliferation zone of the retina and
adjacent head epidermis of 10 second instar nymphs (Fig. ID). The operation
Formation of retina-lamina projection
155
Neuronal specificity
o
(i) c Q .
"O-
(ii)
O
Contact guidance
o—
Of-
A
Of
o^
B
Fig. 5. Three models for the formation of the retina-lamina projection in the dorsoventral axis (A), and their predicted projection patterns after suppression of part of
the retinula fibre input to the lamina (B), or after inversion of the retinula fibre input
(C). Each diagram represents a single dorsoventral row of ommatidia and corresponding lamina. Ommatidia are represented by open circles and their retinula
axons by lines. The rectangle represents the lamina. In (i), a-e, represent cytochemical 'labels'. Arrows in (ii) represent repulsion between fibres. Dashed circles
and lines in (iii) represent the adjacent row of ommatidia and their axons.
did not damage the underlying lamina (Fig. 6), but the retina did not grow
in the operated area during subsequent instars, while the retina on either side
grew normally (Fig. 7). Vertical sections through the resulting adult eyes showed
that beneath the site of operation in the retina lay an area without any retinula
fibres and a corresponding region of the lamina without any cartridges (Fig. 8).
Ommatidia on either side of the gap produced axons which grew to the underlying lamina as normal. This result may be compared with the predictions
of the three models (Fig. 5B).
The Neuronal Specificity model predicts that the axon bundles from ommatidia on either side of the operated area will grow to their normal target cells,
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H. ANDERSON
Fig. 6. Horizontal section through the eye of a second instar locust fixed immediately
after removal of part of the proliferation zone of the retina (wax/haemalum and
eosin). Scale, 160 /*m. dz, Differentiation zone of retina.
Fig. 7. The eye of an adult locust after removal of part of the proliferation zone of
the retina during the second instar. On either side of the site of operation the
retina has grown normally.
regardless of the presence of adjacent, vacant, non-matching cells. This result
is also predicted by the Contact Guidance model; fibres from one ommatidium
will follow the paths taken by pre-existing fibres from adjacent older ommatidia.
These cues are still available to the axons of the ommatidia on either side of
the gap as the anteroposterior continuity of ommatidia has not been broken.
Hence new axons should grow straight down to the subjacent lamina as normal.
The Fibre Interaction model however predicts that the repulsion between the
Formation of retina-lamina projection
157
3**^
Fig. 8. Vertical section through an adult eye after removal of part of the proliferation
zone of the retina during the second instar (wax/reduced silver stain). Scale,
255 /tm. Retinula fibres on either side of the gap (g) created by the operation,
form a normal projection to underlying cartridges (c). Beneath the gap in the
retina is an area of lamina without cartridges (inset).
EMB 46
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H. ANDERSON
y*'tFig. 9. Horizontal section through a second instar eye fixed 2 days after dorsoventral inversion of the proliferation zone of the retina (wax/haemalum and
eosin). Scale, 130 firm, gpz, Grafted proliferation zone.
fibres at the side of the gap would cause them to spread out and fill the available
space, forming a continuous sequence of cartridges across the lamina.
The observed experimental result is therefore in accord with the Neuronal
Specificity model and the Contact Guidance model but does not agree with the
predictions of the Fibre Interaction model.
(ii) Dorsoventral inversion of all or part of the retinula fibre input to the lamina
A thin strip comprising all or part of the proliferation zone of the retina
and adjacent head epidermis was removed from 65 second instar nymphs and
transplanted to a corresponding site on the other side of the head, thus inverting the dorsoventral axis of the graft (Fig. IE). Fifty-eight control grafts
were also performed in which the graft was replaced in its original position
(Fig. IF).
The transplanted proliferation zones and host tissue healed together within
2 days of operation (Fig. 9) and developed normally in subsequent instars
(Figs. 10, 11). The retina formed following inversion of the complete proliferation zone was inverted in the dorsoventral axis, as shown by the presence
of a"growing dorsal spot now in a ventral position with respect to the host.
The grafted head epidermis also retained its polarity, as shown by the trichoid
sensilla pointing dorsally with respect to the host, and the ventral position of
the ocellus (Fig. 11 A).
Histological sections of 21 experimental and 14 control grafts showed that
the grafts formed ommatidia which produced retinula fibres. However, these
fibres grew out in a mass beneath the basement membrane and often did
not approach the lamina from a direction suitable for cutting vertical sections.
In those nine cases (five experimental and four control animals) where the
retinula axons did reach the lamina from a suitable direction, they did so in a
Formation of retina-lamina projection
Fig. 10. Horizontal section through a fifth instar eye after dorsoventral inversion
of the proliferation zone of the retina during the second instar (wax/haemalum
and eosin). Scale, 130 /im. gr, Retina formed from the graft.
Fig. 11. Eyes of adult locusts following dorsoventral inversion of all (A) or part
(B) of the proliferation zone of the retina during the second instar. gr, Retina
formed from the graft; ds, dorsal spot; o, ocellus.
159
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H. ANDERSON
comparably normal pattern, with the retina forming connexions with the
underlying lamina (Figs. 12-15). In both control and experimental animals the
pattern of the retinula axons was distorted in most regions, with some fibres
taking unusually twisted and long paths between the retina and lamina (Figs.
12-15).
These results do not agree with the predictions of the Neuronal Specificity
model; if retinula axons grew to lamina ganglion cells with matching labels, a
chiasma of fibres would be observed in the dorsoventral plane (Fig. 5C).
Nor are the results fully compatible with the Fibre Interactions model, which
predicts that the retinula fibre pattern would be unaffected by inversion of the
retinal input and would be normal with the cartridges forming a continuous
sequence across the lamina (Fig. 5C). In fact cartridges were not always
formed throughout the lamina (e.g. Fig. 12") and the pattern of the retinula
axons was distorted in most regions with some fibres taking unusually twisted
and long paths between the retina and lamina (e.g. Fig. 12).
The results are compatible with the Contact Guidance model. This predicts
that the fibre pattern would not be affected by the inversion of the retinal input;
hence the patterns formed by control and experimental animals should be
comparable. This is indeed the case (compare Figs. 12 and 14 with Figs. 13
and 15). The pattern should not be normal, however, as some of the preexisting pathways are inevitably damaged by the operation and the paths of
axons subsequently formed from the graft will be correspondingly deformed.
Furthermore, these deformations should be continued through successive
rows of the graft projection. An examination of serial sections through part of
the projection illustrated in Fig. 12 shows this is the case (Fig. 16).
Organization of the projection in the anteroposterior axis
Two of the models previously considered in connexion with the dorsoventral
axis could also account for organization in the anteroposterior axis (Fig. 17).
Neuronal Specificity
Newly formed lamina ganglion cells and retinula cells could be uniquely
labelled according to their anteroposterior position in the lamina and retina,
or according to their age, cells differentiating later in development having the
equivalent of more anterior labels. Retinula fibres would form connexions with
lamina ganglion cells having the same label.
Contact Guidance
The orderly arrangement of fibres in the anteroposterior axis may originate
because of the parallel temporal sequence of development of the retina and
lamina; newly formed retinula axons could grow down the axons laid down
by the previous row of ommatidia to the region of newly formed lamina
ganglion cells, where they may form synapses with any available lamina gang-
Formation of retina-lamina projection
161
Fig. 12. Vertical section through an adult eye formed after dorsoventral inversion
of the proliferation zone of the retina during the second instar (wax/reduced silver
stain). Scale, 60 /<m. Tnset, lower power view of the same section, gr, Retina formed
from the graft; tr, trachea; c, cartridges.
lion cell. Any newly formed lamina ganglion cell may serve as a termination
site for any retinula fibre and there is no requirement for any fibre to be distinct
from its neighbour.
To test these two theories, a strip of tissue comprising part of the proliferation zone was removed from the retina of 20 fourth instar nymphs and replaced by the proliferation zone and adjacent head epidermis from 20 first
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H. ANDERSON
Fig. 13. Vertical section through an adult eye formed after control replacement
of the proliferation zone of the retina during the second instar (wax/reduced silver
stain). Scale, 100/im.gr, Retina formed from graft; tr, trachea; c, cartridges; m,
medulla; gp, distorted projection from the graft retina.
Formation of retina-lamina projection
163
gr
14
Fig. 14. Vertical section through an adult eye following dorsoventral inversion of
part of the proliferation zone of the retina during the second instar (wax/reduced
silver stain). Scale, 410 /tm. gr, Retina formed from graft; c, cartridges; gp, distorted projection from the graft retina.
Fig. 15. Vertical section through an adult eye following control replacement of part
of the proliferation zone of the retina during the second instar (wax/reduced silver
stain). Scale, 410 /tm. gr, Retina formed from graft; c, cartridges; gp, distorted
projection from graft retina.
instar nymphs (Fig. 1C). Control experiments, in which the proliferation zone
of 20 fourth instar nymphs was replaced, were also performed (Fig. 1H).
The grafted proliferation zone and host eye tissue healed together and grew
during the two remaining nymphal instars to the adult (Fig. 18). Six of the
resulting adult eyes were sectioned horizontally. In all cases the transplanted
first instar proliferation zones had developed normal ommatidia whose axons
had formed connexions with the host lamina (Fig. 19). There was a continuous
sequence of host-innervated and graft-innervated cartridges. The control
grafts showed the same results (Fig. 20).
According to a Neuronal Specificity model, those lamina ganglion cells
made available to the graft retinula axons after the operation should remain
uninnervated, as they are only able to receive innervation from fourth or fifth
instar retinula axons. Conversely, the retinula axons formed from the graft
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H. ANDERSON
Fig. 16. Serial vertical sections through the adult retina-lamina projection formed
after dorsoventral inversion of the proliferation zone of the retina during the
second instar (wax/reduced silver stain). Scale, 100 /*m. gr, Retina formed from
graft; tr, trachas; c, cartridges; ra, retinula axons.
Formation of retina-lamina projection
Neuronal specificity
a b c d e
OOOOO
a b c d
Contact guidance
a
b c
165
a b
OOOOO
e
OOOOO
OOOOO
(ii)
A
B
Fig. 17. Two models for the formation of the retina-lamina projection in the anteroposterior axis (A) and their predicted projection patterns (B) after replacing the
proliferation zone of a fourth instar retina with the proliferation zone of afirstinstar
retina. Each diagram represents a single anteroposterior row of ommatidia and
corresponding lamina in the adult. Ommatidia formed in each of the five instars
are represented by an open circle, with the youngest and more anterior ommatidia
to the right. Vertical lines represent axons and the rectangle represents the lamina,
a-e in (i) represent cytochemical 'labels'.
Fig. .18. Eye of an adult locust after replacement of part of the proliferation zone of
the retina during the fourth instar with the proliferation zone of a first instar retina.
gr, Retina formed from the graft.
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H. ANDERSON
Fig. 19. Horizontal section through an adult eye formed following replacement of
part of the proliferation zone of the retina during the fourth instar with a first instar
proliferation zone (wax/reduced silver stain). Scale, 220 /on. gr, Retina formed
from graft; gra, graft retinula axons; gc, cartidges innervated by the graft.
Fig. 20. Horizontal section through an adult eye formed after control replacement
of part of the proliferation zone during the fourth instar. Scale, 220 /tm. gr,
Retina formed from the graft; gra, graft retinula axons; gc, cartridges innervated by
the graft.
during the two instars following the operation, should not form connexions, as
the host first and second instar lamina sites are already occupied by host
axons.
The Contact Guidance hypothesis however correctly predicts that newly
formed graft retinula axons should connect with newly formed lamina ganglion
cells irrespective of their ages.
DISCUSSION
Of the classes of model considered, that of Contact Guidance fits the experimental results most satisfactorily. In all the grafting experiments described
above, axons tended to grow out in groups. These groups sometimes formed
Formation of retina-lamina projection
167
relatively normal projections (e.g. Figs. 12, 19), and sometimes abnormal
projections (Fig. 4A, B). Sometimes they formed large spherical whorls (Fig.
4C) within which the axons wound round each other. Sometimes they formed
tracts and grew considerable distances (Fig. 4A, C). It seems therefore that
retinula axons tend to grow out along other retinula axons and that abnormal
fibre patterns result from a disturbance of the relationship between pre-existing
and outgrowing fibres.
As early as 1910, attention was called to the marked tendency of nerves
in young vertebrate embryos to move along solid structures such as fibres,
blood vessels, or skeletal surfaces, and of nerves in later development to follow
the courses chosen by the earlier nerves (Harrison, 1910). The pioneer neurons
thus assume a similar guiding role as was originally played by non-nervous
structures. Similarly, in the locust embryo, pioneer sensory neurons growing
from the tip of the antenna rudiment or the limb rudiment send their axons
along the surface of the lumen of the appendage to the developing ganglion
(Bate, 1976). Observations made on postembryonic insects show that neurons
developing at these later stages grow along pioneer fibres: the axons of imaginal
neurons in the antenna of Manduca sexta join a small tract of pupal nerves
already connected to the developing brain (Sanes & Hildebrand, 1975); in
Rhodnius prolixus the axons of newly differentiated epidermal sensilla combine
with those of adjacent receptors formed earlier and thus are guided to the
nearest branch of a peripheral nerve (Wigglesworth, 1953). Ingrowing axons
which fail to find a peripheral nerve trunk become lost between the epidermis
and the basement membrane and wind around each other to form large whorls
(Wigglesworth, 1953).
Meinertzhagen has suggested that contact guidance is important in the
development of the optic tract of Diptera where larval axon bundles, probably
arising from two small larval sensory organs, penetrate the eye imaginal discs
and pass through to the larval brain to the region of the anlagen for the imaginal
optic lobe (Meinertzhagen, 1973). The first imaginal retinula axons then follow
the larval axon bundle (Meinertzhagen, 1973; Trujillo-Cenoz & Melamed, 1973).
The retina and lamina of Schistocerca gregaria grow at their anterior edges,
and innervation from the retina is required for the differentiation of the lamina
(Anderson, 1978). The orderly pattern of arrival of retinula axons delivered to
the lamina through a contact guidance mechanism could thus be imprinted
upon the developing lamina to produce a corresponding pattern of cartridges.
In the absence of any apparent organization among the ganglion cell population prior to the arrival of retinula axons, a specific recognition mechanism
between retinula axons and lamina ganglion cells would seem unnecessary.
The possibility cannot be excluded that the combination of orderly spatial
and temporal growth sequences with contact guidance, might merely ensure
that retinula axons reach the lamina in sufficiently low numbers and in sufficient
spatial order to limit the possible variety of connexions, and, once fibres have
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H. ANDERSON
entered the lamina, some form of interactive mechanism is responsible for the
final establishment of connexions. The experiments performed in this present
work are not of sufficient resolution to detect local fibre interactions within
the lamina. More subtle experiments involving grafts between eye colour
mutants are planned to help to resolve this problem.
Two observations are relevant to this argument: one from the examination
of Golgi-impregnated neurons and the other from the examination of projection
patterns from open rhabdomere ommatidia.
Retinula axons growing out from the retina possess smooth growth cones,
or are blunt-ended, and do not exhibit exploratory behaviour, as shown by
their lack of filopod activity (Sanchez, 1919c, b; Meinertzhagen, 1973). This
phase of growth could well depend upon contact guidance, by the cone of a
growing axon tracking on the path of a neighbouring bundle (Meinertzhagen,
1973). However, upon entering the outer optic anlage, the retinula axons swell,
forming expanded growth cones (Sanchez, \9l9a; Hanson, Jiang & Lee, 1972;
Meinertzhagen, 1973; Trujillo-Cenoz & Melamed, 1973; Shelton, 1976).
The function of growth cones is obscure. It is not known whether they merely
increase the surface area of the axon tip in order to make contact with available
lamina ganglion cells or whether they mediate some form of interaction between
adjacent neurons which might determine the final pattern of connexions.
Rhabdomeres are assembled in one of two ways to form the rhabdome of
the ommatidium. In the most usual arrangement, individual rhabdomeres are
fused into a central rhabdome as in the locust. The eight retinula cells share the
same visual axis (Shaw, 1967) and their axons project in a simple pattern to one
cartridge in the lamina (Meinertzhagen, 1976), so that there is a topographical
representation of visual space upon the lamina. In Diptera the arrangement is
different. Each rhabdomere is a distinct cylinder separate from the others and
has its own visual axis (Kirschfeld, 1967). Their axons project in a complex
pattern to several different cartridges in the lamina (Braitenberg, 1967) in such a
way that the axons projecting to a single cartridge are from retinula cells
with the same visual axis, although they come from different ommatidia
(Kirschfeld, 1967).
A study of the developing Dipteran projection has shown that initially
all the retinula terminals of one ommatidium grow down to a single locus
in the lamina (as in the fused-rhabdomere type of projection) and subsequent
differential lateral growth of the retinula fibres produces the final pattern
(Trujillo-Cenoz & Melamed, 1973). The model of nerve connexion formation
presented earlier accounts satisfactorily for the first stages of the projection
but is insufficient to explain the production of the final pattern.
No satisfactory alternative model has yet been provided. It has been suggested
that the different components of an ommatidium develop in response to their
position among the cluster of precursor cells (Shelton, Anderson & Eley, 1977).
The axons of open-rhabdomere ommatidia might interpret the positional
Formation of retina-lamina projection
169
information potentially available to them by autonomous growth in an intrinsically determined direction for a predetermined length of time. However,
it is difficult to see how such a precise and reliable pattern of projection could
result from autonomous growth of each axon without any regard to the axons
around them.
An alternative mechanism might be functional interaction. Rhabdomeres in
different ommatidia but with the same visual field, will receive the same visual
excitation. It is possible that after the initial phase of growth, the long retinula
axons retain their initial positions, while the growth cones of short retinula
fibres ramify extensively until they encounter the growth cone of a stable long
retinula axon having a similar pattern of excitation. The short retinula axons
may subsequently form synapses in that cartridge only. The Dipteran projection develops in the pupa and it is not clear how much patterned visual
excitation would be available to the developing retina. This form of functional
interaction could be tested simply by rearing flies in the dark. A similar mechanism in which synapses are formed between neurons deriving their excitation
from the same point in visual space, has been suggested for the intertectal nerve
connexions of amphibia (Keating, 1974).
My sincere thanks go to Dr P. M. J. Shelton for supervising this work and to the University
of Leicester for providing me with a Research Scholarship.
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(Received 6 February 1978, revised 23 March 1978)