/. Embryo!, exp. Morph. Vol. 36, 2, pp. 409-423, 1976
Printed in Great Britain
409
Cell junctions in the developing
compound eye of the
desert locust Schistocerca gregaria
By S. ELEY 1 AND P. M. J. SHELTON 1
From the Department of Zoology, University of Leicester
SUMMARY
Intercellular junctions in the developing retina of the locust Schistocerca gregaria have
been examined by electron microscopy. Different types of junction appear in a well-defined
sequence during development. Five stages of ommatidial development are described. Close
junctions and punctate junctions are present throughout development. Gap junctions appear
transiently amongst the undifferentiated cells, before clearly defined preommatidia can be
distinguished. The subsequent disappearance of gap junctions may be correlated with cell
determination. Lanthanum studies confirm these findings.
The later sequential appearance of adhesive junction types is described. These include septate
desmosomes and two types of desmosomes. In the fully differentiated ommatidium only
two types of junction remain, these are: desmosomes and rhabdomeric junctions.
INTRODUCTION
In neural ontogeny cell junctions have been implicated in at least three
functional processes. Firstly, they may be involved in determination or specification, where intercellular communication is of paramount importance
(Dixon & Cronly-Dillon, 1972; Decker & Friend, 1974; Lopresti, Macagno &
Levinthal, 1974). Determination probably involves intercellular coupling of
some kind: transfer of ions or metabolites, transfer of 'inducer' substances,
or the transfer of a 'morphogen' or gradient substance. Secondly, cell junctions
may play an important part in governing cellular adhesion; here they are
concerned with morphogenetic movements (Lentz & Trinkaus, 1971) and so
have an important function in the grouping of cells to form their final pattern.
Thirdly, cell junctions may be implicated in the transfer of electrical signals
between cells, both during development and at maturity (Loewenstein, 1966;
Sheridan, 1966, 1971; Furschpan & Potter, 1968). Various workers have
provided scattered data on cell junctions in mature ommatidia (Horridge,
1966; Lasansky, 1967; Perrelet & Baumann, 1969; Perrelet, 1970; Brammer,
1970; Boschek, 1971; Meyer-Rochow, 1972), but nothing is known of their
1
Authors'' address: Department of Zoology, School of Biological Sciences, Adrian
Building, University of Leicester, Leicester LEI 7RH.
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S. ELEY AND P. M. J. SHELTON
occurrence in ommatidial development. This communication is concerned with
the development of the locust eye and, in particular, with the identification
and possible roles of specialized cell junctions at different stages of eye
development.
Development of the insect retina has been described for a large number
of species (see Meinertzhagen, 1975), and although there are various species
specific variations, certain generalizations can be made. Retinal development
proceeds from a differentiation centre at the postero-dorsal or posterior edge
of the eye field. This centre induces cell division in the adjacent head epidermis
which spreads in a front across the prospective eye region. Following this
proliferative phase, cells are organized into clusters to form distinct nascent
ommatidia (Waddington & Perry, 1961). These cell groups have been called
preommatidia by Imberski (1967), and it is at this stage that cell determination
must occur. It was thought that within such a developing ommatidium all the
cells were clonally derived by a lineage mechanism from a single epidermal
mother cell (Bernard, 1937). From the study of genetic mosaics this hypothesis
now seems unlikely both for open rhabdom (Shelton & Lawrence, 1974) and
fused rhabdom (Shelton, 1976) eyes. Apparently cell determination is dependent
upon cell position and "cellular interaction within the developing ommatidium
and not upon cell lineage (Shelton & Larewnce, 1974).
Determination by an interactive mechanism requires intercellular communication; this probably demands specialized junctional complexes. In addition,
surface adhesive forces are almost certainly important in eye development,
initially in cluster formation and later in the grouping of cells in the mature
ommatidium. In an attempt to relate cell junction structure to developmental
function, ommatidia have been examined at all stages of eye differentiation
in the locust Schistocerca gregaria. There is a clearly defined sequence of
junctional types whose postulated functions agree with the requirements of
the developmental stages involved.
MATERIALS AND METHODS
A colony of Schistocerca gregaria was maintained on a diet of wheat and
bran in a regulated day/night cycle (12 h light, 32 °C; 12 h dark, 27 °C).
Animals were removed as they moulted on the 4th instar and placed in small
standard-sized cages, five animals per cage. They were kept under continuous
illumination at a constant temperature of 30 °C. Exactly 3 days later the
animals were decapitated and the eyes were removed for fixation.
For conventional electron microscopy the eyes were fixed in phosphate
buffered (pH 7-4) 4 % glutaraldehyde. After 4 h, the material was washed
in buffer and post-fixed for 90 min in phosphate buffered 2 % osmium tetroxide.
The eyes were dehydrated through an acetone series and embedded in Araldite,
with an intermediate propylene oxide stage.
Locust cell junctions
411
For lanthanum impregnation, eyes were placed in 2-5 % glutaraldehyde in
0-05 M cacodylate buffer (pH 7-2), containing 1 % lanthanum nitrate, for 2 h.
They were then washed in several changes of 0-1 M cacodylate buffer ( + 1 %
lanthanum nitrate) and postfixed for 90 min in cacodylate buffered 2 % osmium
tetroxide, again containing 1 % lanthanum nitrate. They were then dehydrated
and embedded as described above.
The material was orientated for cutting sections in the plane at right angles
to the long axis of the ommatidium. Sections for electron microscopy were
cut using a Huxley ultramicrotome set at 0-05 /*m. Silver/grey sections were
collected on uncoated 200 mesh copper grids. They were stained with uranyl
acetate and lead citrate. The material was examined using an AEI-EM-802
electron microscope.
RESULTS
The 4th instar of S. gregan'a consists of approximately 6200 mature ommatidia. Each ommatidium lies beneath a superficial hexagonal corneal facet and
contains about 20 cells. These can be divided into three functional groups:
(i) the dioptric apparatus, consisting of the cornea and four cone cells with
processes extending down the length of the ommatidium, (ii) the eight receptor
or retinula cells, and (iii) the pigment cells. The latter are of two types; two
large primary pigment cells surrounding the base of the cone cells and an
undetermined number of secondary pigment cells isolating each ommatidium
from its neighbours (Horridge, 1966).
Post-embryonic growth of the eye is restricted to the anterior margin where
recruitment of epidermal cells and cell division occurs (Fig. 1). Approximately
1200 new ommatidia develop between moults and this represents 10 rows of
ommatidia added to the width of the eye (measured at the equator). At the
beginning of the new intermoult period (days 1 and 2) the developing edge is
packed with dividing cells, which group to form the clusters of preommatidia
on day 3. By day 4 these have differentiated into almost mature ommatidia,
whose pigmentation is not yet complete. The new ommatidia fill this region
of the eye so that the cuticle directly above them becomes distended (Fig. 2).
After 5 or 6 days the larva moults.
In this study ommatidial differentiation was examined on the third day
of the 4th instar, where five stages of development can be identified in the
growing zone. These stages are arranged in an ascending sequence of maturity.
The least differentiated components (the ungrouped cells) are found most
anteriorly and adjacent to the basement membrane. Moving in from the eye
margin the other stages are arranged in the following predictable order: early
cell clusters, late cell clusters, the developing rhabdom stage and the fully
differentiated stage. They are considered in detail below.
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S. ELEY AND P. M. J. SHELTON
Locust cell junctions
413
Ungrouped cells
The youngest elements in the developing retina are the ungrouped cells
formed by recruitment and division of prospective eye cells from the epidermis
(White, 1963; Shelton & Lawrence, 1974). They form a sheet of elongate cells
lying parallel to the basement membrane, stretching from the corneal surface
to the point where the retinula axons leave the retina. The cells are spindleshaped (Fig. 1), have undifferentiated cytoplasm and cross-sectional diameters
of between 1-7 and 6-9/tm (Fig. 3). In cross-section they can be seen to be
very closely packed together (Fig. 3). The membranes of adjacent cells are
separated by a distance of approximately 10-15 nm. The membranes are
sufficiently regularly apposed for these structures to correspond to close
junctions (Fawcett, 1966) (Fig. 4A). They are freely permeable to lanthanum
as would be expected of close junctions (Fig. 4B). In the mature ommatidium
the intercellular spaces between all classes of cell increase to a width of up
to 35 nm which is wide enough to be considered as non-junctional.
At this stage gap junctions are present between some of the cells of this
region (Figs. 5 and 6A). The adjacent membranes are strictly parallel to
each other for relatively long distances (up to 1 fim) and they are separated
by a cleft of 3-5 nm. The intercellular material is of medium electron density
and the pentalaminar nature of these junctions is quite clear. Lanthanum freely
permeates the intercellular space of these gap junctions which is confirmed
at 3-5 nm. Using lanthanum enhances contrast in electron densities such
that a septilaminar substructure is visible (Fig. 6B).
The initial stages of septate desmosome formation are also visible at this
stage. They are represented by small numbers of irregularly spaced septae
joining the two membranes which are separated by an intercellular cleft of
about 20 nm. Septate junctions are not fully differentiated until the late cell
cluster stage.
Structures which we interpret as punctate tight junctions are also found
FIGURES
1-4
Fig. 1. A longitudinal section through the anterior edge of the retina of a newly
moulted 5th instar locust, showing a mitotic figure (arrow), differentiating bundles
(b), near to the basement membrane {bm), and young ommatidia (yo).
Fig. 2. A longitudinal section to illustrate the developing retinal margin on the
fourth day of the 4th instar. The newly forming ommatidia (arrow) distort the
cuticle anterior to the differentiated eye.
Fig. 3. A transverse section through the developing edge on the third day of the
4th instar. The cells are ungrouped and unspecialized at this stage and packed
close to the basement membrane (bm) and to each other.
Fig. 4A. A close junction of the type found between most cells in the region
illustrated in Fig. 3. The two membranes are about 15 nm apart. A punctate tight
junction is also visible on this section (arrow).
Fig. 4B. Close junction as in Fig. 4 A, with lanthanum in the intercellular space.
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S. ELEY AND P. M. J. SHELTON
Locust cell junctions
415
amongst these ungrouped cells (Figs. 4A and 7). They appear as points of
contact between two cell membranes. The membranes seem to be in very
close association when examined by conventional electron microscopy and
the lanthanum studies confirm this, for the lanthanum does not penetrate
between the membranes at the point of contact.
Early cell clusters
The first recognizable stage in the development of ommatidia is the grouping
of cells to form distinctive clusters (the preommatidia) (Fig. 8). There are
usually 12 cells clearly grouped in the centre of each bundle, although it is
sometimes difficult to assign the surrounding cells to particular bundles;
consequently numbers of cells in a well-defined cluster may vary between
11 and 15. Of the 12 cells in the centre of a bundle four are presumptive cone
cells and eight are presumptive retinula cells (Fig. 9).
There is little cytoplasmic differentiation at this stage and all the cells in
a cluster have a similar appearance. They are significantly smaller in crosssectional diameter (between 0-6 and 3-3 jam), due to the cellular elongation,
which is apparent in longitudinal sections.
At this stage the junctions between the cells are of three types, septate
desmosomes, punctate tight junctions and close junctions. The septate desmosomes are still immature and the punctate tight junctions and the close
junctions have the same structures and characteristics as described previously.
Gap junctions are totally absent from these young bundles.
Late cell clusters
These have the same gross appearance as the early cell clusters with crosssectional diameters of between 0-6 and 3-8 jam (Fig. 10A). As the clusters
of mature septate desmosomes become more prominent (Fig. 10B). They are
FIGURES
5-8
Fig. 5. A gap junction found between ungrouped cells in the developing edge of
the retina.
Fig. 6 A. The same gap junction under higher power, showing the pentalaminar
structure, characteristic of these junctions when they are block stained with uranyl
acetate and then stained with lead citrate.
Fig. 6B. Another gap junction in which the intercellular space is impregnated
with lanthanum, demonstrating the width of the gap (3 nm). The structure here
is clearer than in the conventionally stained material and the junction now has
a septilaminar appearance.
Fig. 7. A punctate tight junction (uranyl acetate/lead citrate stained), found
amongst ungrouped cells, old and young bundles. At the junction (arrow) the
two cell membranes are apposed. (See also Fig. 4A.)
Fig. 8. Transverse section further into the developing edge of the retina, showing
four young bundles which are cytologically undifferentiated.
27-2
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S. ELEY AND P. M. J. SHELTON
Locust cell junctions
All
particularly apparent between developing cone cells and between the cone
cell processes and the retinula cells, but they are also present between adjacent
developing retinula cells. The two cell membranes are separated by an intercellular cleft of between 20 and 30 nm and periodically bridged by septae of
electron-dense material. Both the junctional cell membranes and the septae
are very electron dense. The distances between the septae at this stage may
be anything between 5 and 20 nm. However, as the cluster develops the septae
become more regularly arranged with constant interseptal distances of 5 nm
in the oldest clusters. Septate junctions can extend over distances of up to
1 jam at this stage.
Punctate tight junctions are found much less frequently at this stage and
gap junctions are still absent. Close junctions are still present.
The developing rhabdom stage
As the rhabdom develops, so the final organization of the ommatidium
becomes apparent. Eight retinula cells are present, grouped into a rosette-like
configuration with a centrally placed developing rhabdom (Fig. 11). This
developing rhabdom is formed from microvilli emanating from the retinula
cells. The formation of the rhabdom has not been investigated in detail but
both intra- and inter-cellular junctions are found between the membranes of
the retinula microvilli. At least at this stage lanthanum can penetrate these
rhabdomeric junctions.
FIGURES
9-12
Fig. 9. Section through a young bundle to show the characteristic grouping of
.12 cells, which are numbered. There is very little cytoplasmic differentiation at this
stage. The surrounding cells are probably presumptive secondary pigment cells;
they are not joined by the close junctions (arrows) which characterize the young
bundle cells. The presumptive secondary pigment cells are separated by normal
intercellular spaces of at least 25 nm.
Fig. 10A. Transverse section through an older bundle where cytoplasmic differentiation is beginning. Cells 9-12 are relatively free of cytoplasmic inclusions
and are the future cone cell processes. Cells 1-8 are presumptive retinula cells,
septate junctions join the cells of the bundles at intervals (arrows).
Fig. .10B. Detail of the septate junction, cell 6, arrow, between adjacent
presumptive retinula cells, showing the typical bar-like structure of arthropod
septate junctions.
Fig. 11. Transverse section through an immature ommatidium. The rhabdom (R)
is differentiating from seven of the eight retinula cells, each of which is contributing
microvilli (m) to the developing structure. The eighth retinula cell (i?8-arbitrary
numbering) is not yet contributing to the developing rhabdom at this level of
section. External to the differentiating rhabdom desmosomes (d) join the adjacent
retinula cells. Septate junctions persist between the cells of the young ommatidium
(arrows).
Fig. 12. A higher power electron micrograph of a desmosome (d) and a septate
desmosome (sd), between adjacent retinula cells. The desmosome has cytoplasmic
filaments (tonofilaments-0 associated with it, electron-dense material associated
with the membranes and a relatively dense intercellular space.
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S. ELEY AND P. M. J. SHELTON
13
Fig. 13. Transverse section through the mature rhabdom of a 4th instar locust. The
rhabdom (R) is now fully formed with the eight retinula cells (/?I-JR8) grouped
around it.
Fig. 14. A higher power micrograph of the mature rhabdom. The membranes of
the microvilli can be seen to be closely apposed, forming the rhabdomeric junctions
(rj). The desmosomes (arrows) are less dense structures than those observed in
earlier stages of development (see Fig. 11).
The young rhabdom stage is also characterized by the appearance of very
obvious desmosomes between adjacent retinula cells near to the point when
the rhabdomeres are forming (Fig. 12). The desmosomes are separated by an
intercellular cleft of 25 nm. On each side of the junctional membranes there
is a very obvious electron-dense cytoplasmic plaque, associated with cytoplasmic
filaments (tonofilaments). In cross-section these junctions extend radially
from the rhabdom up to 045 /an and form a 300 /im strip running the entire
length of the young retinula cells. Septate junctions are particularly obvious
at this stage (Fig. 12), although they disappear as the ommatidia mature. They
are present between retinula cells and cone cell processes and between adjacent
retinula cells. At this stage the septae are regularly spaced with interseptal
distances of 5 nm. Otherwise they have the same characteristics as those
described in the late cell clusters. Close junctions are present in the early
stages of maturation but they disappear with the formation of the rhabdom.
Locust cell junctions
419
The fully differentiated stage
In the mature rhabdom (Fig. 13), only two sorts of junction remain:
desmosomes and rhabdomeric junctions. The desmosomes change in nature
at this stage, although they maintain the same position within the ommatidium.
They become smaller and less dense (Fig. 14), they have fewer tonofilaments
associated with them and they have smaller cytoplasmic plaques. Their
dimensions also change, with a wider intercellular cleft of 30-35 nm. Radially
within the ommatidium, these junctions extend for shorter distances (up to
0-4 /*m) but at the same time they still extend the whole length of the retinula
cells (up to 300 fim).
We have not examined the rhabdomeric junctions in detail but they could
be either gap or tight junctions. In all experiments lanthanum never penetrated
the rhabdomeric junctions but the lanthanum did not penetrate the mature
retina very well and further experiments are needed.
Septate junctions and close junctions are absent in the mature ommatidia.
DISCUSSION
A critical phase in the development of the insect retina is the organization
of cells into clusters to form preommatidia (Waddington & Perry, 1961;
Imberski, 1967; Meinertzhagen, 1975). From the evidence which suggests
ommatidial cell determination by an interactive process (Shelton & Lawrence,
1974), it is almost certain that there is an exchange of information between
cells at this stage. In invertebrates three types of junction have been implicated
in either electrical coupling (Loewenstein, 1966; Sheridan, 1966, 1971;
Furschpan & Potter, 1968; Shaw, 1969; Warner & Lawrence, 1973; Caveney.
1974), or the transfer of small molecules between cells (Satir & Gilula, 1973).
These are tight junctions, gap junctions and septate desmosomes. In the
present study all three classes of specialized contact were found at early stages
of development of the ommatidia. In amphibia Dixon & Cronly-Dillon (1972)
showed that gap junctions were present in the retina up to the time of specification of the retinal axes. The disappearance of gap junctions at the exact
time of neuronal specification led them to suggest a causal relationship. In the
locust retina there is a similar phenomenon, gap junctions appear transiently
amongst the ungrouped cells, i.e. prior to cluster formation and presumably
prior to specification. The disappearance of these gap junctions just before
cluster formation may mean that they have some special function in cell
determination.
It now seems unlikely that septate desmosomes have any role in intercellular
communication; it had been suggested that they were involved in coupling
processes (Gilula, Branton & Satir, 1970). However, recently gap junctions
have been found in nearly all the tissues where ionic coupling had been
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S. ELEY AND P. M. J. SHELTON
correlated with the presence of septate desmosomes (Hudspeth & Revel, 1971;
Hand & Gobel, 1972). It now seems certain that intercellular communication
in these cases is a function of gap junctions rather than the septate desmosomes.
In any case, in the insect eye the appearance and full maturation of septate
junctions occurs too late to implicate them in cell determination. They begin
to differentiate in the ungrouped cells but are not fully developed until the
late cell cluster stage, just prior to rhabdom formation. Also in Drosophila
they are absent between developing retinula cells, although present between
other classes of cells within the ommatidium (Perry, 1968). It is possible
that they selectively restrict the movement of molecules in the extracellular
space, although this is unlikely since we have found that lanthanum penetrates
these junctions at all developmental stages. In addition, in the perineurium
of the insect abdominal nerve cord ionic lanthanum moves freely across the
septae in the extracellular compartment (Treherne, Schofield & Lane, 1973).
Probably their main function in the developing retina is one of cellular
adhesion.
However, lanthanum is incapable of penetrating the punctate tight junctions,
and it is possible that these junctions may restrict movements of certain
molecules in the intercellular space at particular times during development.
They are found only in stages up to and including the older cell clusters, but
in no developmental stage after this. Such an occluding role has been postulated
for tight junctions found in the peripheral nervous system of insects (Lane,
Leslie & Swales, 1975). It has also been shown that tight junctions are the last
junctional unit to break down under stress (Lentz & Trinkaus, 1971), so it
may be that here they have some adhesive role.
The formation of preommatidial clusters certainly involves surface adhesive
factors. Kuroda (1970) showed that dissociated eye disc cells of Drosophila
sort themselves out from antennal cells and reaggregate to form clusters
reminiscent of preommatidia. Many types of cell junction have had adhesive
functions attributed to them, even those primarily involved in other roles.
Tight junctions may be adhesive as already mentioned, and gap junctions
which are primarily concerned with intercellular communication may also be
adhesive. However, the two junctional types in this tissue known to be concerned
with cellular adhesion are septate junctions (or septate desmosomes) and
desmosomes.
In the differentiating cell cluster there is a progressive elaboration of septate
desmosomes, which gradually disappear later. In the cell cluster, cells are
being organized into their final locations and it seems likely that the septate
desmosomes have some mechanical function. As the rhabdom begins to
form, desmosomes appear close to the rhabdom between adjacent retinula
cells. It is accepted that such junctions are not involved in intercellular communication and that their main function is adhesive (Lentz & Trinkaus, 1971).
They have very dense cytoplasmic modifications and many tonofilaments
Locust cell junctions
421
associated with them, which it has been postulated (Lentz & Trinkaus, 1971)
are normally arranged along the lines of stress in a cell. They almost certainly
provide strong binding forces to bring and keep together the newly formed
rhabdom in its highly regular configuration. They are not usually continuous
over long distances but the compound eye is clearly an exception where they
extend, in a strip, from the top to the bottom of the retinula cells (up to 300 /im).
At maturity the desmosomes become smaller, less dense structures, losing some
of their tonofilaments. We can only speculate with regard to the relative
adhesive properties of the desmosomes at these two developmental stages.
It is almost certain that there are greater stresses between cells during differentiation, when the cells are being regrouped within clusters, to give the rhabdomeric
configuration, than at maturity when they simply have to maintain this final
position. The desmosomes in the young ommatidium may provide greater
adhesion than those in the mature ommaditidium since the former have more
dense plaques and greater numbers of tonofilaments.
Junctions found within the rhabdom certainly have an adhesive function
but their main role is probably concerned with retinal physiology. It is now
well established that retinula cells are electrically coupled within an ommatidium
(Shaw, 1969). Depolarization of retinula cells associated with the generator
potential probably results from the flow of current across the rhabdomeric
microvilli (Perrelet & Baumann, 1969). Perrelet & Baumann (1969) found in
drone bees that lanthanum penetrated the rhabdom, indicating that the
junctions are of the gap type. As yet in locusts, we have found that lanthanum
penetrates the rhabdomeric junctions only at the early stages, when the rhabdom
is in the process of development. However, this is quite possibly because the
lanthanum never even reaches the rhabdom of the mature retina and further
studies are needed.
In the mature ommatidium two sorts of junction remain. Firstly, the
desmosomes between the retinula cells presumably maintain the rosette-like
juxtaposition of these cells. Secondly, there are the rhabdomeric junctions.
Clearly experimental studies are necessary to clarify the roles of different
types of cell junction in development. In the case of junctions with adhesive
roles there is a well-defined sequence of types: (i) septate desmosomes alone appear to bind cells together within the differentiating clusters; (ii) desmosomes are
formed as soon as the rhabdom begins to differentiate and they bring about
the rosette-like configuration of retinula cells around the differentiating rhabdom;
(iii) less dense desmosomes maintain this organization in the mature structure.
The role of cell junctions in intercellular coupling and cell determination
is far less clear and the only landmark in development is the disappearance of
gap junctions immediately prior to cell clustering. The disappearance of gap
junctions in the developing amphibian retina has been correlated previously
with cell determination (Dixon & Cronly-Dillon, 1972). It seems that the
same phenomenon may be occurring in the insect retina.
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S. ELEY AND P. M. J. SHELTON
We wish to thank the S.R.C. for financial support - S. Eley for a 3-year studentship and
P. M. J. Shelton for a research grant.
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{Received 28 June 1976)