/. Embryol. exp. Morph. Vol. 45, pp. 55-83, 1978
Printed in Great Britain © Company of Biologists Limited 1978
55
Postembryonic development of the visual system
of the locust, Schistocerca gregaria
I. Patterns of growth and developmental interactions
in the retina and optic lobe
By HILARY ANDERSON1
From the Department of Zoology, University of Leicester
SUMMARY
The visual system of the locust, Schistocerca gregaria, has a highly ordered and predictable
arrangement of neurons. The retina and the outermost layer, or lamina, of the optic lobe
are each composed of repeating units, ommatidia and cartridges respectively. Each ommatidium has eight photoreceptor cells, which send axons directly to a group of five neurons
in the lamina to form the cartridge. The importance, for the development of this precise
pattern, of the mode of growth of the two arrays and of interactions between them was
investigated.
The spatial and temporal sequences of cell proliferation, differentiation and death in the
developing retina and optic lobe were examined quantitatively under normal and experimental conditions.
The retina grows from its anterior margin by addition of new ommatidia formed from
recruited epidermal cells. The lamina also grows by addition of new neurons to its anterior
margin, but these neurons are derived from a stem cell population. The parallel pattern of
growth of the retina and lamina may be important for the formation of neuronal connexions
between them.
The retina grows and differentiates even when deprived of the underlying lamina. In
laminae deprived of the ingrowth of new axons from the retina, the production of new
neurons is also autonomous, but these neurons do not differentiate, but degenerate. A limited
amount of cell death occurs in the laminae of control insects. These two observations suggest
that a plausible mechanism for coordinating the sizes of the two arrays during normal
development might be production of lamina neurons in excess of requirements and death of
those remaining non-innervated.
INTRODUCTION
The insect visual system exhibits an extraordinary precision of geometry
both in the spatial arrangement of its constituent cells and in the pattern of its
neuronal connexions (Meinertzhagen, 1976). The retina and the lamina are
essentially two-dimensional arrays of repeating structural units, ommatidia
and cartridges respectively, connected by neurons projecting from the retina
to the lamina in a topographical mapping, so that there is an exact represen1
Author's address: Department of Zoology, University of Oxford, South Parks Road,
Oxford 0X1 3PS, U.K.
56
H. ANDERSON
tation of the primary visual field on the first-order interneurons (Braitenberg,
1967; Kirschfeld, 1967; Horridge & Meinertzhagen, 1970; Meinertzhagen,
1976). How is this precise neuroanatomical pattern generated?
In order to investigate this problem it is necessary to describe the pattern
of the finished structure, to examine the normal sequence of developmental
events leading to its production, and to experimentally perturb the developing
system and observe the consequences upon the development of the final pattern.
In this way it is hoped that the types of cellular interaction underlying pattern
formation may be inferred.
The locust, Schistocerca gregaria, was chosen for such a study because it
possesses large compound eyes which continue to develop externally on the head
throughout postembryonic life and are therefore very suitable for experimentation.
This paper provides relevant details of the structure of the compound eye
and a description of its normal development. It then describes an investigation
of the importance of interactions between the retina and lamina for their
development and maturation; the spatial and temporal sequences of cell
proliferation, differentiation and death were examined quantitatively under
normal conditions and in grafting experiments in which the developing retina
was deprived of the underlying lamina, or in which the lamina was deprived of
the ingrowth of new axons from the retina. A subsequent paper will consider
possible factors involved in the formation of the topographical retina-lamina
projection (Anderson, 1977).
MATERIALS AND METHODS
Maintenance of insect stocks
A colony of S. 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. Under these conditions the mean length of the fourth instar was 125 h, with a standard deviation
of 10 h for 60 timed animals (S. Eley, personal communication).
Histology
Animals were collected as adults and at 0, 24, 48, 72, 96 and 120 h after the
moult to the fourth instar. Their eyes were either fixed in alcoholic Bouin, embedded in paraffin, sectioned at 10 /tin and stained with Mayer's haemalum
and eosin (Pantin, 1969) or with reduced silver stain (Rowel 1, 1963) or else
fixed in buffered paraformaldehyde-glutaraldehyde mixture (Karnovsky,
1965) postfixed in 1 % osmium tetroxide, embedded in araldite, sectioned
at 1 /im using a Huxley Ultramicrotome and stained in 1 % toluidine blue
in 1 % borax solution. Sections were cut in either the vertical or horizontal
plane (Fig. 2).
Growth patterns and developmental interactions in insect eye
57
Fig. 1. Diagrams of grafting operations. (A) Transplantation of a second instar retina
to the prothorax of another second instar locust. (B) Removal of the proliferation
zone of the retina and adjacent head epidermis of a second instar locust.
Sections were photographed on Kodak Panatomic X film in a Zeiss Photomicroscope II. The film was developed in Ilford Microphen and fixed in Amfix
(May and Baker).
Investigation of cell proliferation and cell death
For a quantitative investigation of cell proliferation and cell death, the
central 60 sections of each fourth instar eye were selected and every third section
examined. This prevents any single mitosis or degenerating cell appearing on
more than one examined section. All anaphase and metaphase mitotic figures
were scored. Degenerating cells were scored according to the criterion of
Wigglesworth (1942): degenerating (pycnotic) nuclei are round, stain darkly
with Mayer's haemalum and eventually disintegrate into round chromatic
droplets of various sizes. In those few cases where several small droplets were
close together, they were scored as one degenerating cell. The number of
mitoses and degenerating cells occurring in the retina and developing optic
lobe was recorded in four animals for each of the six time points in the
instar.
58
H. ANDERSON
Grafting operations
S. gregaria nymphs were selected for operation within 12-36 h after moulting
to the second instar. The animals 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 Gillette Franc.ais 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.
Two types of operation were performed: (a) Transplantation of retina to
prothorax. The retinae and surrounding head epidermis of 20 second instar
nymphs were transplanted without the underlying optic lobe to the prothorax of
hosts of the same age (Fig. 1 a), (b) Removal of retina growth zone. A narrow
strip of the retina and adjacent head epidermis was removed from the anterior
margin of 20 second instar eyes (Fig. Ib).
In both cases the animals were allowed to pass through two moults to the
fourth instar. At 0 h or 72 h after moulting, their retinae and optic lobes were
fixed, embedded and sectioned as described above. The number of mitoses and
degenerating cells occurring in the retina and optic lobe was recorded in four
animals for both of the time points by the method described above. Some
additional animals underwent the same operations and were fixed immediately
or as adults.
RESULTS
Structure of the adult compound eye
External features
The general features of the head and compound eye of an adult locust are
illustrated in Fig. 2. In addition to the two large compound eyes there are three
ocelli; two lateral ocelli and one median ocellus on the frons. The compound
eyes provide the main organ of vision, while the function of the ocelli remains
obscure (Goodman, 1970).
The compound eyes are striped with bands of light and dark facets. Pigment
cells in the dark band are heavily loaded with dark brown pigment while those
in the light band contain only greyish-yellow pigment. There is a distinct region
of facets at the top of the eye, the dorsal spot. Ommatidia in this region differ
structurally from other ommatidia and appear to send axons to a discrete
area of the optic lobe (Fig. 5). Whether the dorsal spot has a special function
is not known but it provides a useful marker in grafting experiments described
in a later paper in this series (Anderson, 1977).
Adjacent to the anterior edge of the eye is an area of pale cuticle bearing
trichoid sensilla whose shafts are orientated dorsoventrally. They form part of
an aerodynamic organ (Weis-Fogh, 1956) and also provide a useful indicator of
graft orientation (Anderson, 1977).
Growth patterns and developmental interactions in insect eye
59
1 -4 mm
Fig. 2. Head of an adult locust. A, antenna; CE, compound eye; DS, dorsal spot;
LO, lateral ocellus; MO, median ocellus; dotted line outlines the region of epidermis bearing ventrally oriented trichoid sensilla; horizontal (H) and vertical (V)
planes of section are indicated.
The retina
The adult retina possesses some 9000 facets (Bernard, 1937; Rafi. & Burtt,
1974) beneath each of which lies one ommatidium, the structural and functional
unit of the retina. The general morphology of an ommatidium is shown in
Fig. 3. They are of the typical eucone, apposition, fused-rhabdomere type
(Goldsmith, 1964).
The corneal lens and the crystalline cone (composed of four cone cells)
direct light onto the underlying photoreceptor layer. This consists of eight
60
H. ANDERSON
Fig. 3. The arrangement of cells within an ommatidium in the fourth instar locust.
(A) Diagram and longitudinal section of an ommatidium (araldite/toluidine
blue). Scale, 26 /tm. (B-E) Transverse sections of ommatidia at various levels
(araldite/toluidine blue). Scale, 21 /*m. BM, basement membrane; BRN, basal
retinula cell nucleus; CC, crystalline cone; CCN, crystalline cone cell nucleus; CO,
cornea; LRN, long retinula cell nucleus; PPN, primary pigment cell nucleus; RA,
bundle of retinula cell axons; RH, rhabdom; SPN, secondary pigment cell nucleus.
Growth patterns and developmental interactions in insect eye
61
retinula cells arranged around an axial structure, the rhabdom. Each retinula
cell contributes a segment, or rhabdomere, to the rhabdom which is the site of
photoreception. Six of the retinula cells extend from the basement membrane
to the cone and contribute a rhabdomere to the whole length of the rhabdom.
The other two cells are restricted to the basal third of the ommatidium, and
contribute only a short rhabdomere. Each retinula cell sends out one axon, all
eight from one ommatidium passing out in a bundle through the basement
membrane.
Two large primary pigment cells containing many large pigment, grains
surround the crystalline cone, and many smaller secondary pigment cells form
a sheath around the entire ommatidium from the corneal lens to the basement
membrane. The pigment cells screen the passage of light between ommatidia.
The optic lobe
The optic lobe lies directly behind the retina and its basic morphology is
shown in the horizontal and vertical section of Figs. 4 and 5.
It consists of three ganglia which are joined by tracts of nerve fibres. They
are, from the retina inwards, the lamina, the medulla and the lobula. Each
ganglion has a central core of nerve fibres, the neuropil, and a surrounding
cortex of neuron cell bodies whose axon processes contribute to the neuropil.
The lamina is directly innervated by the bundles of retinula axons. These
axon bundles together with groups of interneurons form repeating structural
units called cartridges. The number of cartridges is equal to the number of
ommatidia and, just as the structure of ommatidia is the same throughout the
retina, so cartridge organization is isomorphic across the extent of the lamina.
Each cartridge consists of eight retinula axons and five lamina ganglion cells
(M. Nowel, unpublished electron microscope observations) and is associated
with lamina amacrine neurons, medulla tangential fibres and medulla contrifugal fibres (N. J. Strausfeld, unpublished observations). Groups of axons,
comprising two long retinula axons and the five lamina ganglion cell axons,
leave the cartridges of the lamina and pass to the medulla. In doing so they
cross over other groups in the horizontal plane, forming a chiasma, the outer
chiasma, in the tract between the lamina and the medulla.
The retina-lamina projection
Each ommatidium sends eight axons to the optic lobe where they establish
precise connexions with the two outermost neuropils. Six axons terminate in
the lamina while two longer axons from the two basal retinula cells pass
through this neuropil and terminate in the medulla. By following serial sections
through the tract from the retina to the lamina of S. gregaria, Meinertzhagen
(1976) has shown that (a) all the eight axons from one ommatidium project to
one cartridge, (b) the arrangement of retinula axons within the cartridge is
nearly always the same as the rotational sequence of their cell bodies in the
5
E M B 45
62
H. ANDERSON
Posterior
Anterior
I Posterior
Anterior
Fig 4. Horizontal sections through the left eye of an adult locust. (A) Wax section
stained by Rowell's reduced silver method to show the neuropil. Scale, 320/*m. (B)
Wax section stained with Mayer's haemalum & eosin to show the neuron cell
bodies. Scale, 320 /im. IC, inner chiasma; L, lamina; LO, lobula; M, medulla;
OC, outer chiasma; PS, perineural sheath; R, retina.
Growth patterns and developmental interactions in insect eye
Dorsal
5A
Dorsal
Ventral
63
5B1
Ventral
Fig. 5. Vertical sections through the left eye of an adult locust. (A) Wax section
stained by Rowell's reduced silver method to show the neuropil. The striated
appearance of the lamina neuropil indicates its organization into repeating structural
units, the cartridges. Scale, 420/^m. (B)Wax section stained with Mayer's haemalum
& eosin to show the neuron cell bodies. Scale, 420/*m. DS, dorsal spot; L, lamina;
LDS, discrete area of the lamina apparently innervated by retinula axons from
the dorsal spot only; LO, lobula; M, medulla; PS, perineural sheath; R, retina;
RA, bundles of retinula axons; TR, tracheae.
ommatidium, and (c) axon bundles from adjacent ommatidia project to adjacent cartridges.
The neural projection between the retina and lamina is therefore a topographical mapping. Furthermore, the retinula cells of a single ommatidium
share a common visual axis (Shaw, 1967), so that the projection provides a
point-to-point representation of visual space on the lamina.
Normal development of the nymphal compound eye
The retina
Figure 6 shows typical horizontal sections through the nymphal eye at the
beginning (0 h) and the end (120 h) of the fourth instar. At the posterior margin
of the retina, fully differentiated ommatidia abut directly onto the epidermal
cells of the head. At the anterior margin however, cells are in various stages of
5-2
64
H. ANDERSON
Posterior
Anterior
OM
Posterior
Anterior
6B ;
Fig. 6. Horizontal sections through the left eye of fourth instar locusts at 0 h (A) and
120 h (B) of the instar (wax/haemalum & eosin). Scale, 210/*m. E, epidermis; DZ,
differentiation zone; IOA, inner optic anlage; L, lamina; LO, lobula; M, medulla;
OOA, outer optic anlage; OM, fully differentiated ommatidia; PZ, proliferation
zone.
Fig. 7. Horizontal section through the growth zone of a 48 h fourth instar retina (araldite/toluidine blue). Scale, 50/*m.
DCC, developing crystalline cone; DRH, developing rhabdom; DZ, differentiation zone; E, epidermis; OM, fully
differentiated ommatidia; PP, primary pigment cell; PZ, proliferation zone; arrows indicate mitoses.
I"
Hi
66
H. ANDERSON
development from epidermal cells to fully differentiated ommatidia. Two
zones may be distinguished; a proliferation zone and a differentiation zone
(Fig. 7).
The proliferation zone is a region of high mitotic activity. The cells are
densely packed and elongated. Dividing cells are always observed at the surface
of the eye and their spindle axes are always parallel to the surface i.e. perpendicular to the long axis of the cell. Presumably this ensures that the two daughter
cells maintain contact with neighbouring cells.
Adjacent to the proliferation zone is the differentiation zone where mitoses
are relatively uncommon. The nuclei are not densely packed but become
stratified in positions suggestive of those found in fully differentiated ommatidia.
A cluster of four nuclei may be observed differentiating into the crystalline
cone (Fig. 7). Proximal to this, the retinula cells may be distinguished elaborating
the rhabdom. The primary pigment cells do not appear in their final position
with characteristic heavy pigmentation until the last stages of differentiation
(Fig. 7).
A comparison between sections through a 0 h and a 120 h retina (Fig. 6)
shows that by the end of the instar about ten new rows of ommatidia have
been formed. The high rate of cell proliferation within a confined space has
produced a bulge of cells in the developing margin of the 120 h retina and
forced the adjacent epidermal cells into a U-shaped trough. Indeed, it is possible
that it is the compression of cells that results in the hexagonal array of facets,
as this is the pattern that results when any circular objects are pressed together.
Figure 8 shows the variation in the mitotic activity in the proliferation zone,
differentiation zone and the large zone of fully differentiated ommatidia
throughout the fourth instar.
Mitoses were observed in the proliferation zone on all days of the instar.
There was no significant difference in mitotic activity between the samples
at 0, 24, 96 and 120 h but the mitotic activity at 48 and 72 h was significantly
higher than at the other time points in the instar (P < 0-05). In the other two
zones of the retina, virtually all mitotic activity was confined to the 48 h and
72 h samples, i.e. at the time when mitoses were at their peak in the proliferation zone. Mitotic figures among the fully differentiated ommatidia were
always at the surface of the retina as in the proliferation zone and differentiation
zone.
Only three degenerating nuclei were observed in the proliferation zone and
none in the other two regions of the retina in all 480 sections examined.
The optic lobe
The lamina and medulla grow by addition of cells produced by mitosis
in a common growth zone, the outer optic anlage (Fig. 6) while the cells of
the lobula arise from the inner optic anlage (Fig. 6) and will not be considered
further here.
Growth patterns and developmental interactions in insect eye
120
40
100
20
80
0
24
48 72
Time (h)
96
120
60
40
40
20
20
0
24
48 72
Time (h)
96
120
0
24
48 72
Time (h)
96
120
Fig. 8. Graphs to show the variation throughout the fourth instar, in mitotic activity
within the proliferation zone (A), differentiation zone (B), and the region of fully
differentiated ommatidia (C), of the retina. Ordinate, mean number of mitotic
figures per 60 sections. Abscissa, hours after the moult to the fourth instar. Bars
represent ± one standard error.
67
68
H. ANDERSON
B
M
D
Fig. 9. Neuron production in the outer optic anlage of the fourth instar optic lobe.
(A-C) Horizontal sections through the growing edge of the optic lobe to illustrate
the main events in the production of new neurons (wax/haemalum & eosin). Scale,
32/*m. LMM, lamina ganglion mother cell undergoing a symmetrical mitosis;
MMM, medulla ganglion mother cell undergoing a symmetrical mitosis; NM,
neuroblast undergoing an asymmetrical mitosis; single arrow, degenerating lamina
ganglion cell; double arrow, degenerating medulla ganglion cell.
(D) Semi-schematic drawing of a section through the developing optic lobe to
show the arrangement of the different cell types. C, cartridges of the lamina neuropil;
L, lamina ganglion cells; LM, lamina ganglion mother cells; M, medulla ganglion
cells; MM, medulla ganglion mother cells; N, neuroblasts; OC, outer chiasma;
PS, perineural sheath.
The outer optic anlage is a characteristically folded structure lying beneath
the growth zone of the retina and extending parallel to it along the dorsoventral extent of the optic lobe. Typical horizontal sections through the outer
optic anlage and the growing cortices of the lamina and medulla are shown
in Fig. 9. The outer optic anlage proliferates new cells from a stem cell population, the neuroblasts. These may be distinguished by their large nuclei and
Growth patterns and developmental interactions in insect eye
69
13
18
12
i
i
I
l
i
i
0
24
48
72
96
120
12
J
I
t
i
0
24
48
72
96
0
24
48
72
96
120
0
24
48 72
Time (h)
96
120
120
I8
18
12
12
I •
0
24
48 72
Time (h)
96
120
Fig. 10. Graphs to show the variation throughout the fourth instar, in the mitotic
activity of the neuroblasts (A), lamina ganglion mother cells (B), and medulla
ganglion mother cells (C), and in degeneration among the newly proliferated
lamina ganglion cells (D) and medulla ganglion cells (E) in the outer optic anlage.
Ordinate, mean number of mitotic figures, or of pycnotic nuclei, per 60 sections.
Abscissa, hours after the moult to the fourth instar. Bars represent ± one standard
error.
70
H. ANDERSON
Fig. 11. Horizontal section through the developing edge of the fourth instar optic
lobe, to show retinula axons growing to the area of newly formed lamina ganglion
cells. Scale, 100/*m. C, cartridges; GRA, growing retinula axons; L, lamina
ganglion cells; LM, lamina ganglion mother cells; N, neuroblasts; arrow indicates
the site of expanded growth cone at the tip of the retinula axon.
abundance of light-coloured cytoplasm. They divide asymmetrically to form
another neuroblast and a ganglion mother cell (Fig. 9 a). The axis of the division
spindle is always at an angle (usually perpendicular) to the surface of the
anlage. The ganglion mother cells have small densely-staining nuclei and
themselves undergo symmetrical divisions which are always parallel to the
surface of the anlage (Fig. 9b, c). Their progeny become the ganglion cells of
the lamina and medulla. As they are produced they displace older ganglion
cells from the anlage so that the distance of the ganglion cell from the anlage is
proportional to its age.
Neuroblasts and ganglion mother cells were observed undergoing mitosis
on all days of the instar (Fig. 10) and there was no significant variation in
mitotic activity between the days (P > 0-05).
Degenerating cells were rarely found among the neuroblasts or ganglion
mother cells but were frequently found among the newly proliferated ganglion
cells of both the lamina and medulla (Fig. 9b, c). The variation between days,
in the number of degenerating nuclei observed, is shown in Fig. 10 and is not
statistically significant (P > 0-05).
The retina-lamina projection
Newly formed retinula axons grow to the area of newly formed lamina
ganglion cells adjacent to the outer optic anlage (Fig. 11). Growing fibres
were observed in this position on all days of the instar.
Growth patterns and developmental interactions in insect eye
71
Fig. 12. A fourth instar locust bearing on its prothorax a retina which was grafted
on during the second instar. Scale, 2-3 mm. G, grafted retina.
Fig. .13. Horizontal section through a fourth instar retina which was grafted to the
prothorax during the second instar (wax/haemalum & eosin). Scale, 190 /*m. DZ,
differentiation zone; HE, head epidermis; OM, fully differentiated ommatidia; PE,
prothoracic epidermis; PZ, proliferation zone. The inset is a higher magnification
picture of the mitotic figure in the proliferation zone.
Development of the compound eye after grafting operations
Operation a
The transplanted retinae continued to grow during the two instars following
transplantation to the prothorax (Fig. 12) and were completely normal in
structure (Fig. 13): fully differentiated ommatidia occupied most of the retina
but at the anterior edge two zones of developing cells could be clearly distinguished, a proliferation zone and a differentiation zone, as in normal retinae.
Table 1 shows that mitotic activity in the proliferation and differentiation
zones was slightly reduced in experimental animals compared with that in
72
H. ANDERSON
Table 1. Mitotic activity at Oh and 72 h of thefourth ins tar, in the proliferation zone
and differentiation zone of control retinae or retinae transplanted to the pro thorax
Mean no. mitotic figures per
60 sections
Oh
Control
Experimental
72 h
Control
Experimental
Proliferation
zone
Differentiation
zone
9-5
8-25
0-75
0-5
27-75
2200
24-25
19-25
control animals. The number of fully differentiated ommatidia was also less in
transplanted retinae than in control retinae
Operation b
Eyes fixed immediately after removal of the anterior margin of the retina
and adjacent head epidermis showed that this operation removed all of the
proliferation zone of the retina but left most of the differentiation zone intact
and did not damage the underlying optic lobe (Fig. 14). In all 20 experimental
animals, the retina did not grow during the two instars following the operation.
Eight of the eyes were examined in detail histologically when the animals
reached the fourth instar. The cells of the retina were fully differentiated into
ommatidia except for a band at the site of operation where the cells were
considerably elongated but were not clustered and their nuclei remained near
the distal surface (Fig. 15). There was no sign of regeneration of the extirpated
proliferation zone.
The optic lobe and outer optic anlage of these fourth instar eyes were quite
normal in structure (Fig. 15) but the volume of the neuropil and cortex of the
lamina was reduced compared with control fourth instar animals. In fact, the
lamina neuropil had not increased in size in the two instars following the
operation (compare Figs. 15 & 14).
Neuroblast divisions and ganglion mother cell divisions were observed
in the outer optic anlage and degenerating nuclei were observed among the
newly proliferated lamina ganglion cells, as in control animals (Table 2).
Statistical analysis of the data summarized in Table 2 showed that there was
no significant difference in mitotic activity of neuroblasts and ganglion mother
cells between control and experimental animals (P > 0-05). Yet there was no
accumulation of new lamina ganglion cells or increase in lamina neuropil
after the operation. However, an examination of the number of degenerating
Growth patterns and developmental interactions in insect eye
73
14
Fig. 14. Horizontal section through the retina and optic lobe of a second instar locust,
fixed immediately after removal of the proliferation zone of the retina (wax/
haemalum & eosin). Scale, 160/tm. DZ, differentiation zone; OOA, outer optic
an 1 age.
Fig. 15. Horizontal section through the retina and optic lobe of a fourth instar locust
following removal of the proliferation zone during the second instar (wax/haemalum
& eosin). Scale, 160/tm. WC, elongated cells occupying the site of the wound
produced by removal of the proliferation zone.
nuclei found among new lamina ganglion cells showed that this was considerably higher in experimental animals than in control animals (Table 2 and
Fig. 16) and that this was statistically significant (P < 0-05).
DISCUSSION
Spatial aspects of compound eye development
The growth of the retina of S. gregaria was first described by Bernard (1937).
At the beginning of each of the instars he measured the size of the retina with
a micrometer eye piece. He also recorded the total number of facets and the
74
H. ANDERSON
Table 2. The numbers of dividing and dying cells of different types in the outer optic
anlage of fourth instar locusts, in the presence {control) and absence {experimental)
of innervation form the retina, at 0 h and 72 h of the instar
Mean no. mitotic figures per
60 sections
Mean no . pycnotic
nuclei per 60 sections
Neuroblasts
Lamina
ganglion
mother
Medulla
ganglion
mother
Lamina
ganglion
cells
Medulla
ganglion
cells
Oh
Control
Experimental
5-75
8-5
5-75
7-5
10-75
140
4-75
13-5
10-75
975
72 h
Control
Experimental
9-5
5-75
7-75
110
4-5
28-75
90
40
6-5
7-5
Fig. 16. Horizontal section through the outer optic anlage of a 72 h fourth instar
locust following removal of the proliferation zone of the retina during the second
instar (wax/haemalum & eosin). Scale, 23 fim. Arrows indicate pycnotic nuclei
among the newly formed lamina ganglion cells.
size of the oldest facets from isolated corneas. His findings are summarized
in Table 3. He showed that the newly hatched nymph has a well-developed
compound eye which grows during the five subsequent instars not only by a
doubling in size of the ommatidia but also by a continual addition of ommatidia
to its anterior edge.
Ran* & Burtt (1974) also examined the size and number of facets in nymphal
stages of S. gregaria. Although they used a different method, their results
(summarized in Table 3) are quite consistent with those of Bernard.
Growth patterns and developmental
interactions in insect eye
75
Table 3. Change in facet number and diameter during the postembryonic
development of the retina of Schistocerca gregaria. Measurements in jum
Instar
A
1
2
3
4
5
Adult
3850
2700
4675
3400
6480
5200
7685
7400
9400
9100
30-5
32-2
390
39-2
39-5
43-6
Facet number
a
b
2470
—
Facet diameter
a
b
25-5
200
22-5
29-3
24-9
27-1
a from Bernard, 1937.
b from Raft & Burtt, 1974.
The histological observations presented here confirm that the retina grows
by addition of new ommatidia to its anterior edge. Bernard (1937) suggested
that the new ommatidia were derived from epidermal cells. This has been
shown to be the case for several other hemimetabolous insects. In the cockroach,
Periptaneta americana, and the bug, Oncopeltus fasciatus, mutant epidermis
from anterior to the developing eye, when grafted to an equivalent position
on wild-type hosts, eventually developed into a patch of mutant ommatidia
(Hyde, 1972; Shelton & Lawrence, 1974; Shelton, Anderson & Eley, 1977). In
the dragonfly, Aeschna cyanea, small wounds made in the epidermis adjacent
to the growing eye margin were observed in later instars as scars in the eye
tissue (Lew, 1933). Mouze grafted two eye margins opposite one another in
A. cyanea and showed that the intervening epidermis and the two growth zones
were completely converted into fully differentiated ommatidia by the last
larval instar (Mouze, 1975). It is likely therefore that the locust retina also
grows by recruitment of epidermal cells and their subsequent proliferation and
differentiation into ommatidia. Attempts to demonstrate this directly, by
grafting mutant albino epidermis anterior to the growing wild-type locust eye,
have failed owing to lack of cell-autonomy of the albino mutation (Anderson,
unpublished work).
The lamina also grows by addition of cells to its anterior margin but these
neurons are derived from a stem cell population. The pattern of nerve cell
production in the optic lobe may be summarized as follows:
Neuroblast — « ^
>- Ganglion mother cell
>- Ganglion cell
(Arrows represent mitosis)
This basic pattern can be applied to the production of all neurons in the
insect central nervous system, e.g. the optic lobe of other insect species (Panov,
1960; Nordlander & Edwards, 19696); brain centres other than the optic lobe
(Nordlander & Edwards, 1970); other areas of the brain not directly associated
76
H. ANDERSON
with specific centres (Nordlander & Edwards, 1969 a); the ventral nerve ganglia
(Roonwal, 1937; Bate, 1976). However, it is still uncertain how many mitoses
precede the formation of nerve cells (Panov, 1960; Nordlander & Edwards,
1969&) and neuroblasts may divide unequally or equally during the formation
of ganglion mother cells, depending upon the insect species (Panov, 1960).
The retina and lamina show the same mode of growth, i.e. addition of
elements onto the anterior edge. This would appear to simplify the problem of
connecting the two arrays, firstly in terms of the pathways which must be
negotiated by outgrowing retinula axons since newly formed lamina immediately
underlies newly formed retina, and secondly because at any one time only a
limited number of new retinula axons will be arriving at the lamina and they
will encounter only a restricted number of new lamina ganglion cells. If the
mode of growth of the two arrays is of major importance in the formation
of the projection between them, then it should be possible to investigate disruption of the pattern of the projection following experimental alteration of
the spatial relationship between the two developing arrays. This approach will
be discussed elsewhere (Anderson, 1977).
Temporal aspects of compound eye development.
A peak of mitotic activity was observed at 48 and 72 h in all regions of the
retina. Examination of whole mounts of epidermis from the abdomen on each
day of the fourth instar showed that mitosis here was also confined almost
exclusively to the 48 and 72 h samples (unpublished data). The developing
retina of A. cyanea also shows a peak of mitotic activity but it is slightly earlier
than that observed in the abdominal epidermis (Schaller, 1964). In the proliferation zone of S. gregaria there was also a low level of mitotic activity on the
other days of the instar. Schaller (1964) also stressed the occurrence of a basal
level of mitosis throughout the instar in A. cyanea.
Cyclic mitotic activity in the epidermis is thought to be controlled by the
hormonal milieu which also varies cyclically in each instar (Novak, 1975).
It remains unclear whether cell division in the retina depends upon hormonal
influences, as suggested by the peaks of mitotic activity in all three zones of
the retina, or whether it is independent of them, as indicated by the presence of
mitosis in one zone, the proliferation zone, on all days of the instar.
Here it might be useful to know which cell types are produced in the different
zones at different times. For example, emancipation from hormonal control
may represent the first stages of a commitment of precursor cells to the production of the neural components of ommatidia, the retinula cells, while the
precursor cells of the other ommatidial components may maintain a response
characteristic of epidermal cells. Indeed, it seems unlikely that the production of
retinula cells, and hence of new sensory neurons, is cyclical since growing
retinula axons are observed in the lamina throughout the instar. Little is known
about the time or place of production of different cell types. The cells dividing
Growth patterns and developmental interactions in insect eye
11
among the mature ommatidia could be secondary pigment cells as all other
ommatidial components are fixed in number at this stage. Alternatively they
could be hair cells as the eye bears a few hairs scattered on its surface. Cells
dividing in the differentiation zone could be the precursors of pigment cells,
retinula cells or hair cells but not the cone cells which are clearly identifiable
from the earliest stages. Cells dividing in the proliferation zone could be the
precursors of any of the future components of the ommatidium. Identification
of the sites and times of production of particular ommatidial cells would require
a detailed study of the development of individually labelled cells. The use of
[3H-]thy.midine pulse-labelling in Drosophila has shown that in this insect
ommatidium production occurs in two phases (Campos-Ortega & Gateff, 1976;
Ready, Hanson & Benzer, 1976). Mitosis among recruited epidermal cells
produces a pool of cells from which ' preclusters' (Ready, Hanson & Benzer,
1976) are assembled. 'Preclusters' consist of retinula cells 2, 3, 4, 5 and 8 and
these cells undergo no further divisions. Other cells surrounding the preclusters
then divide to produce the remaining retinula cells 1, 6 and 7 and the other cells
needed to complete an ommatidium. A comparable study has not yet been made
on a hemimetabolous insect.
Mitoses were observed on all days of the instar among the neuroblasts
and ganglion mother cells of the outer optic anlage. Statistical analysis of the
data showed that there was no significant variation between the days. Neuron
production in the optic lobe of S. gregaria therefore appears to be independent
of the cyclic aspects of the hormonal environment. This is also the case for the
butterfly, Danaus plexippus, (Nordlander & Edwards, 19696) and A. cyanea
(Schaller, 1964).
Developmental interactions between the retina and lamina
The results of the experiment in which the retina was transplanted to the
prothorax show that the retina of S. gregaria can grow and differentiate independently of any neuronal connexion with the optic lobe. Similar experiments
(retina transplantation, optic lobe extirpation or section of the connexions
between the retina and optic lobe) have been performed on many insect species.
In most cases, retina development is reported as having proceeded normally
(Kopec, 1922; Pflugfelder, 1936-7; Chevais, 1937; Steinberg, 1941; Vogt, 1946;
Pflugfelder, 1947; Bodenstein, 1953; Schoeller, 1964; Wolbarsht, Wagner &
Bodenstein, 1966; Eichenbaum & Goldsmith, 1968; Mouze, 1974; TrujilloCenoz & Melamed, 1975), but in some cases the retina developed abnormally
(Plagge, 1936; Wolsky, 1938; Drescher, 1960; Wolsky & Wolsky, 1971).
Meinertzhagen (1973) has suggested a plausible explanation for these conflicting results in terms of the stage of eye development at the time of operation.
All experiments involved severing the nerves from ommatidia that were already
differentiated at the time of operation. This can result in degenerative changes
in the ommatidia (Mouze, 1974). If most of the ommatidia were already developed
6
EM B
45
78
H. ANDERSON
at the time of the operation then most of the resulting retina would be abnormal.
Hence there has been no unequivocal demonstration of the dependence of
retinal development on an influence from the optic lobe.
I have observed ommatidial degeneration in S. gregaria only following
damage to the retina tissue and basement membrane itself; whole retinae
transplanted to the prothorax, for example, remain quite normal (Fig. 13).
Probably in those recorded cases where ommatidia degenerated after nerve
section, there was additional damage to the basement membrane.
The results of the experiment involving witholding retinula innervation from
the developing lamina show that the production of new lamina ganglion cells
also proceeds autonomously, by the mitotic activity of neuroblasts and ganglion
mother cells in the outer optic anlage. However, in the absence of innervation
from a growing retina these cells do not appear to differentiate but rather to
degenerate.
Several^other authors have attempted to test the influence of the retina on
the development of the optic lobe but considerable difficulties arise when
trying to interpret their results. Most authors did not record the stage of
development of the retina and optic lobe at the time of the operation. Further,
most observations of the results were made on the adult and usually considered
only the presence or absence of the optic ganglia, as indicated by the presence
or absence of neuropil. They therefore dealt only with the end product of
development and ignored the problem of which of the component sequences
of cell proliferation, differentiation and death had been influenced by the
experimental procedure.
Evidence suggesting that lamina development, in a general sense, does
depend upon an influence from the retina was provided by the early experiments
of Alverdes (1924) and Stein (1954). After cautery of the compound eye of
larval Agrion, Cloeon, and Notonecta (Alverdes, 1924) and Libella (Stein, 1954),
the adult optic lobe showed varying degrees of deficiency. However, this was
almost certainly due not to a failure of one of the normal developmental
processes (cell proliferation, differentiation or death) in the absence of the
developing retina, but to degenerative changes following the deeply penetrating
effects of the cautery (Pflugelder, 1936-7). These experiments therefore are not
conclusive. More compelling evidence has been provided by experiments using
surgery. Following removal of the larval eye anlage (Kopec, 1922) or after
transplantation of the larval brain into the abdomen (Schrader, 1938), the
lamina of the adult optic lobe was both reduced in size and abnormal in structure.
Further evidence for an interaction between the retina and the optic lobe
comes from studies on Drosophila. Mutants of Drosophila possessing abnormally
few ommatidia, or no ommatidia at all, show a proportional reduction in the
volume of the lamina (Richards & Furrow, 1925; Power, 1943; Hinke, 1961).
Similarly, several insects which are naturally eyeless or have considerably
Growth patterns and developmental interactions in insect eye
79
reduced eyes, for example those living underground or as parasites, also
have reduced laminae or no laminae at all (Holmgren, 1909; Schimmer, 1909;
Werringloer, 1932; Pflugfelder, 1936-7; Bernard, 1937; Pflugfelder, 1947).
There are also exceptions in which the lamina is present in an eyeless animal
(Jorschke, 1914).
Mouze (1974) witheld retinula innervation from the optic lobe of Aeschna
larvae either by removing the growth zone of the retina, or by implanting a
barrier beneath the growth zone of the retina which deflected outgrowing
retinula axons from the optic lobe. The outer optic anlage was normal in
volume following the first operation, and only slightly reduced in volume
beneath the barrier in the second operation. In both cases the outer optic
anlage showed a normal or only slightly reduced level of mitotic activity.
Embryonic optic lobes of Carausius morosus transplanted into larvae or adults
showed continued mitotic activity in the outer optic anlage in the absence of
interaction with the retina (Pflugfelder, 1947). These results confirm the observation on S. gregaria that cell proliferation in the outer optic anlage proceeds
autonomously in the absence of retinula innervation.
During normal development, lamina differentiation (elaboration of neuropil)
is usually preceeded by, or occurs at the same time as, retinula axon outgrowth
(Panov, 1960; Richard &Gaudin, 1960; Hanson, 1972; unpublished observations
of Meinertzhagen, 1973). This indicates that lamina differentiation might be
dependent upon retinula innervation. Further evidence from the experiments
discussed earlier support this view. Isolated embryonic optic lobes of Carausius
cultured in vivo proliferated masses of ganglion cells which accumulated without differentiating (Pflugfelder, 1947). After witholding retinula innervation
from the larval optic lobe of Aeschna, the volume of the lamina neuropil was
reduced by 13% compared with controls (Mouze, 1974).
In the latter work, the volume of the lamina cortex was also considerably
reduced, by about 3 1 % compared with controls (Mouze, 1974). The newly
formed lamina cortex was thin, and had reduced numbers of lamina ganglion
cells and was frequently separated from the outer optic anlage (Mouze, 1974).
Mouze suggested this might result from a band of cell death in the area of
newly proliferated lamina ganglion cells (Mouze, 1974). Cell death of the
undifferentiated ganglion cells was not recorded in Carausius (Pflugfelder, 1947).
The results presented in this paper show that, in the locust eye, new lamina
ganglion cells are continuously produced by mitosis in the outer optic anlage.
The high rate of cell death and the absence of increase in lamina neuropil
volume observed after witholding innervation from the retina suggest that the
uninnervated lamina ganglion cells die. This observation suggests an important
role for cell death during normal development, since the site of degenerating
cells in the uninnervated lamina is the site to which newly-formed retinula
axons grow during normal development. Furthermore, some cell death is
observed at this site (i.e. among newly-proliferated lamina ganglion cells)
6-2
80
H. ANDERSON
in normal development but not in other regions of the outer optic anlage nor in
the retina.
The first observation of death among optic ganglion cells was in the normal
development of the butterfly, Danaus plexippus (Nordlander & Edwards, 1968).
In this insect, cell death was observed among cells produced 3-6 days earlier
and which appeared to be in the first stages of differentiating an axon, as
indicated by the appearance of an axon hillock (Nordlander & Edwards,
1968). These authors proposed that cell death occurred in response to a failure
of the ganglion cells to make their own connexions. The observations made on
S. gregaria did not indicate that the degenerating cells had developed axons.
In this case, rather than lamina ganglion cells competing for a limited number
of retinula axon terminals and dying if unsuccessful, it seems more likely that
lamina ganglion cells do not differentiate at all, and eventually degenerate,
unless they are actively contacted by ingrowing retinula axons. Direct observation of the interaction between the growth cone of the retinula axon and a
lamina ganglion cell in the crustacean Daphnia, has shown that the lamina
ganglion cell differentiates its own axon only after a sequence of extended
surface contact with the ingrowing retinula axon (LoPresti, Macagno &
Levinthal, 1973) and that gap junctions form between the lamina ganglion cell
and retinula axon during this wrapping phase (LoPresti, Macagno & Levinthal,
1974). Production of lamina ganglion cells in excess of requirements, coupled
with programmed death of those cells not utilized for cartridge formation by
incoming retinula axons would provide a plausible mechanism for regulating
the relative size of the retina and lamina arrays in the locust eye.
I thank Dr P. M. J. Shelton for his advice and encouragement throughout this work which
was supported by a Research Scholarship awarded by the University of Leicester.
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