Download Survival of photoreceptor neurons in the compound

355
Development 114, 355-366 (1992)
Printed in Great Britain © The Company of Biologsts Limited 1992
Survival of photoreceptor neurons in the compound eye of Drosophila
depends on connections with the optic ganglia
ANA REGINA CAMPOS1, KARL-FRIEDRICH FISCHBACH2 and HERMANN STELLER1
^Howard Hughes Medical Institute, Department of Brain and Cognitive Sciences and Department of Biology, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA
2
Institutftir Biologie III, SchOnzlestr. 1, D-7800 Freiburg, Germany
Summary
The importance of retinal innervation for the normal
development of the optic ganglia in Drosophila is well
documented. However, little is known about retrograde
effects of the optic lobe on the adult photoreceptor cells
(R-cells). We addressed this question by examining the
survival of R-cells in mutant flies where R-cells do not
connect to the brain. Although imaginal R-cells develop
normally in the absence of connections to the optic lobes,
we find that their continued survival requires these
connections. Genetic mosaic studies with the discon-
nected (disco) mutation demonstrate that survival of Rcells does not depend on the genotype of the eye, but is
correlated with the presence of connections to the optic
ganglia. These results suggest the existence of retrograde
interactions in the Drosophila visual system reminiscent
of trophic interactions found in vertebrates.
Introduction
been demonstrated that, in Drosophila, the birth of
lamina neurons requires retinal innervation (Selleck
and Steller, 1991).
In contrast to vertebrates, no example of retrograde
dependence during development has been described in
invertebrates. It has been shown that sensory organs as
well as motoneurons can differentiate in the absence of
interaction with their targets (e.g. Sanes et al., 1976;
Whitington et al., 1982; Anderson, 1985; Costello and
Wyman, 1986). The insect retina is particularly well
suited to studies of retrograde dependence given its
accessibility and the fact that it is not essential for
viability under laboratory conditions. Experiments
involving retina transplantation, optic lobe ablation and
sectioning of the optic stalk have been carried out in
various insects (e.g. Kopec, 1922; Chevais, 1937;
Wolski and Wolski, 1971; Mouze, 1978; Anderson,
1978a) and have demonstrated that the retina is able to
develop autonomously from the underlying optic
ganglia.
In Drosophila the best evidence for autonomy of
retinal development stems from analysis of supernumerary eyes present in flies carrying mutations in the
extra-eye (ee) gene. These structures send projections
that never reach the optic ganglia (Marcey and Stark,
1985). Morphological analyses of these extra eyes at the
electron microscopy level showed that they develop
appropriately, displaying the characteristic arrangement of photoreceptor cells seen in normal compound
eyes (Marcey and Stark, 1985). However, these studies
Certain neurons are known to degenerate as a consequence of ablation of their target cells, or after
deafferentation. These phenomema, known respectively as retrograde and anterograde transneuronal
degeneration (Cowan, 1970; Purves and Lichtman,
1985), reveal the importance of cell-cell interactions for
neuronal survival. A number of factors have been
shown to mediate target-dependent survival in the
nervous system (reviewed by Levi-Montalcini, 1987;
Oppenheim, 1989; Barde, 1989).
In invertebrates, various instances of anterograde
dependence during neural development have been
described (e.g. Anderson, 1978b; Macagno, 1979;
Schneiderman et al., 1982). The fruitfly Drosophila
melanogaster provides a classical example of such an
interaction in the development of the imaginal visual
system (reviewed by Meinertzhagen, 1973). When eyes
are reduced, due to surgical procedures or in certain
mutant flies, hypoplasia of the underlying optic ganglia
is observed (Power, 1943; Schoeller, 1964). Subsequent
mosaic analysis, using eye mutants, demonstrated that
innervation has an inductive effect on the development
of the optic ganglia (Meyerowitz and Kankel, 1978). In
fact, retinal innervation is required not only for the
generation of the second order neurons of the first optic
ganglia but also indirectly for the maintenance of higher
order neurons in the optic lobes (Fischbach, 1983;
Fischbach and Technau, 1984). More recently, it has
Key words: trophic interactions, visual system, cell death,
retinal degeneration, Drosophila.
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A. R. Campos, K.-F. Fischbach and H. Steller
did not address the importance of retrograde trophic
input for the maintenance of the fully differentiated
retina. Here, we investigate this problem by using
recessive mutations which prevent the connections of
the photoreceptor cells with their target cells in the
optic lobes of the fruitfly Drosophila melanogaster. We
examined the maintenance of the photoreceptor cells in
flies mutant for either the disconnected {disco) gene or
the ee gene.
Previous experiments have revealed that in disco
mutants the imaginal R-cells form a relatively normal
retina but generally fail to reach their target cells; the
optic lobes are extremely reduced in these cases (Steller
et al., 1987). Mosaic analysis demonstrated that this
"unconnected" phenotype occurs independently of the
genotype of the compound eye. Here we show that Rcells degenerate after eclosion in these "unconnected"
disco flies. Using genetic mosaics, we demonstrate that
the genotype of the retina does not affect R-cell
degeneration, which is strictly correlated with the
presence of connections to the optic ganglia. In ee flies,
while the normal eye retains its morphology with age,
R-cells in the supernumerary eyes progressively degenerate. We conclude that, while retinal differentiation proceeds in the absence of connections with the
brain, R-cell survival after eclosion requires interactions with the underlying optic ganglia. We propose the
existence of trophic interactions between the optic
ganglia and the photoreceptor cells in the retina.
Materials and methods
Fly stocks
All flies were raised on standard cornmeal-sugar-agar-yeast
medium supplemented with fresh yeast. Drosophila cultures
were grown at 19°C or 25°C and 60-65% humidity. A mutation
in the gene disconnected {disco') was used (Steller et al.,
1987). This chromosome carried the visible mutations for the
genes white {w) and forked if) unless otherwise stated. These
mutations confer white eyes and abnormal bristle morphology
respectively which can be easily scored under the dissecting
microscope. The extra-eye {ee) mutation was kindly provided
by William Stark. The wild-type control used carried
mutations for the genes yellow (y) and white (w).
Generation of Mosaics
Gynandromorphs were constructed using the unstable ring X
chromosome {R(l)wvC, Hall et al., 1979). R(l)wvC/Binsn
females were crossed to w disco1 f/Y males. The mutant
patches in the mosaic progeny were recognized with the aid of
visible markers {w and /).
Histology
Fly heads were fixed for 1 hour in 1% glutaraldehyde, 2%
paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4.
The heads were postfixed in 1% osmium tetroxide in the same
buffer, dehydrated and embedded in standard SPURRS
medium (Spurrs, 1969). Semithin sections (0.5 to 1 /an) were
mounted in Permount and inspected under phase-contrast
microscopy. Cryostat sections of adult heads were prepared
for mAb24B10 staining essentially as described in Steller et
al., 1987. /3-galactosidase activity staining on cryostat sections
was performed as described by Mismer and Rubin (1987).
Electroretinograms
ERGs were recorded extracellularly essentially as described
by Rendahl et al., 1991. For these experiments, flies less than
a day old were used.
Results
Photoreceptors in unconnected disco flies progressively
degenerate after eclosion
Previous studies have established that the development
of the photoreceptor cells of the Drosophila retina
proceeds independently from that of the underlying
target cells (Chevais, 1937; Marcey and Stark, 1985). In
the present study, we examined the role of the target
cells for the proper maintenance of the photoreceptor
cells after development is completed. In order to
address this question, we assessed the integrity of
retinula cells of adult flies which do not innervate the
underlying optic ganglia. In flies mutant for the Xlinked disco gene, the imaginal photoreceptor axons fail
to reach their target cells during the third larval instar,
which consequently disrupts optic lobe development
(Steller et al., 1987).
The phenotype of disco flies at various developmental stages has been described and discussed elsewhere
(Steller et al., 1987). Briefly adult flies carrying
mutations for the disco gene typically show only a
rudiment of the optic lobes ("unconnected" phenotype). No structure resembling the first optic ganglia
(lamina) is ever detected in unconnected flies (Steller et
al., 1987). Instead the photoreceptor cell axons frequently terminate in a mass of muscle tissue which often
but not always replaces the optic ganglia (Fig. IB). In
other cases the optic lobe in disco is replaced by
hemolymph or non-neuronal cells of unknown identity.
Externally these flies can be recognized by the deformity of their compound eyes. In some flies, during the
third larval instar, the retinular axons of one or both eye
imaginal discs are able to establish connections with the
developing optic lobes ("connected" phenotype). In
these cases, adult flies have optic ganglia of almost
normal size, however, their structure is abnormal and
and clearly distinct from wild type (Fig. 1C and D). The
variability in the morphology of the lamina in connected discofliesis illustrated by the two examples shown
in Fig. 1C and D. In a homozygous disco1 stock,
approximately 90-95% of theflieshave visual systems of
the unconnected type and 5-10% have the connected
type.
Adult flies hemizygous for the disco mutant allele
were aged at 25°C. Their heads were embedded in
plastic and semithin sections were inspected under
phase-contrast microscopy. The specimens were sectioned in a horizontal plane from dorsal to ventral such
that the most superficial sections represent transverse
sections of the most dorsal ommatidia. In all specimens,
deeper sections were also obtained such that the
morphology of the ipsilateral optic ganglia could be
Trophic interactions in the Drosophila eye
357
me
lo
J&X
IP
la
me
me
D
unambiguously assessed for each of the sectioned
compound eyes (as shown in Fig. IB, C and D).
Fig. 2 shows cross sections of ommatidia from a newly
eclosed (panel B) and from a 15-day-old disco1 fly
(panel C); both flies exhibited the unconnected phenotype (Fig. IB). Examination of ommatidia in newly
eclosed flies indicates that development of the retina is
not significantly impaired by absence of interaction with
the underlying optic ganglia (Fig. 2B). The normal
number of R-cells per ommatidium is present, and
these cells appear to have terminally differentiated, as
judged by the expression of photoreceptor-specific
Fig. 1. Optic lobe defects in
disco flies. All of the
photomicrographs in this figure
show horizontal semithin
plastic sections of adult fly
heads under phase-contrast
microscopy. They are
representative of the various
types of optic lobes underlying
the compound eyes analyzed in
this study. (A) Wild-type optic
lobe showing the organized
neuropil centers of the
Drosophila visual system.
(B) Optic lobe area from a
disco mutant displaying the
unconnected phenotype. In
these flies, the space normally
occupied by the optic ganglia
is replaced by non-neuronal
tissue. The R-cell axons do not
establish any connections with
their normal target cells.
(C and D) Optic lobes from
disco mutant flies displaying
the less severe connected
phenotype. Although the optic
lobe is almost normal size, its
structure is clearly
disorganized. In connected
disco flies, the pattern of
projection from the R-cells is
almost normal (Steller et al.,
1987 and Fig. 4).
Abbreviations: la, lamina; me,
medulla; lo, lobula; lp, lobula
plate. Scale bar is 20 j/m.
genes (like chaoptin and rhodopsins, Figs 4, 8 and data
not shown), and the fact that newly eclosed unconnected discofliesare able to be depolarized in response
to a light stimulus (see Fig. 6D).
In 15-day-oldfliesonly a few R-cells are found in each
ommatidium (Fig. 2C). The presence of pigment
granules in a pattern resembling the one found in young
discofliessuggests that, at this stage in the degenerative
process, the pigment cells surrounding the R-cells have
largely maintained their integrity.
These observations confirm and extend previous
results that retina development proceeds largely inde-
358
A. R. Campos, K.-F. Fischbach and H. Steller
Fig. 2. Photoreceptor cell degeneration in disco mutants. The photomicrographs in this figure show cross sections of the
most dorsal area of adult compound eyes viewed under phase-contrast microscopy. (A) Ommatidia from a wild-type fly
carrying the eye color mutation white (w); ommatidia in all of the subsequent panels are from flies carrying the wild-type
w+ gene. Each ommatidium contains 20 cells, 8 of which are photoreceptor neurons (R-cells). The rhabdomeres of these
R-cells are seen as dark spots and are arranged in a trapezoidal pattern. Because the inner R-cells, R7 and R8, are stacked
on top of each other, only 7 rhabdomeres can be seen in any given plane of section. The boundary of each ommatidial unit
is outlined by the membranes of the pigment cells. Deeper sections were always obtained in addition to these shown here
in order to assess the presence of connections to the optic ganglia. (B) Ommatidia from a disco mutant that had eclosed
approximately two hours prior to fixation. At this stage, R-cells are present and the typical number of rhabdomeres can be
identified in spite of the slight irregularity of the ommatidial units. In this and the following specimens no connections to
the optic ganglia could be found. (C) Ommatidia from an unconnected disco fly which was aged for 15 days at 25°C before
sectioning. Only a few rhabdomeres can be found in most of the ommatidia. In this field, only one ommatidium still
contains five rhabdomeres (arrow). The remaining rhabdomeres seem to correspond to both outer and inner R-cells. (D)
Ommatidia from a connected disco mutant which was aged for 15 days at 25°C. Note that the rhabdomeres in each
ommatidium are found in the correct number and are properly positioned. Scale bar is 5 /an.
pendently from interactions with the optic ganglia.
However, they suggest that survival of R-cells is
dependent on interactions with the optic ganglia.
Genetic mosaic analysis of photoreceptor cell survival
in disco flies
Previous mosaic analyses demonstrated that the unconnected phenotype in disco' flies is independent from the
genotype of the compound eye regarding disco' mutant
alleles (Steller et al., 1987). However, these studies did
not specifically address the requirement of disco1 gene
function for R-cell survival. It remained possible that
the R-cell degeneration in discofliesof the unconnected
phenotype is not due to lack of connections with the
optic ganglia but is rather due to the requirement of
disco gene function in the retina. In order to distinguish
between these two alternatives, we examined the
retinas of gynandromorphs generated by the unstable
ring X chromosome system (Hall et al., 1976).
Mosaic flies were aged at 25°C for at least 10 days.
The mutant disco chromosome carried a mutated copy
of the white (w) eye color marker in order to assess the
genotype of the cells in the eye. Adult heads of genetic
mosaic animals were embedded in plastic as described
in Materials and methods and horizontal semithin
sections were inspected under phase-contrast microscopy. The presence of connections between R-cells
and the optic lobe was assessed as described in the
previous section.
Fig. 3A shows a section through a compound eye
which was entirely mutant for disco but whose R-cells
projected to a phenotypically wild-type optic lobe. In
such cases, no degeneration of R-cells could be
detected. This was in sharp contrast to the massive
retinal degeneration seen in compound eyes of mosaics
Trophic interactions in the Drosophila eye
359
-fe&£iS*s" i>
Fig. 3. Degeneration of R-cells in disco mosaics. All of the panels in this figure show cross sections of the most dorsal area
of adult compound eyes viewed under phase-contrast microscopy. All specimens shown in this figure have been aged for at
least ten days at 25°C prior to fixation. The mutant patches were recognized by the absence of pigmentation; the disco
chromosome used in these experiments carries a mutation in the white gene. (A) Ommatidia from a compound eye entirely
mutant for the disco gene; this eye is associated with an ipsilateral optic lobe indistinguishable from wild type. No
degeneration can be seen. (B) Ommatidia from a compound eye entirely mutant for the disco gene; the ipsilateral optic
ganglia in this fly is of the unconnected phenotype. Massive degeneration can be observed (compare to Fig. 2C).
(C) Ommatidia from a compound eye entirely wild type for the disco gene. The ipsilateral optic ganglion in this fly is of
the unconnected phenotype. Despite the presence of disco* function in the entire retina, the degeneration observed is
indistinguishable from that found in compound eyes that are entirely mutant for the disco gene (compare to Fig. 3B).
(D) Ommatidia from a compound eye of mixed genotype. The ipsilateral optic ganglion in this fly is indistinguishable from
wild type. No degeneration can be detected in this specimen. The arrowhead points to an ommatidium entirely mutant for
the disco gene. The arrow indicates an ommatidium of mixed genotype. Scale bar is 5 /an.
where the underlying optic lobe displayed the unconnected phenotype (Fig. 3B, C). In these cases, R-cells
degenerated irrespective of whether cells in the compound eye were mutant (Fig. 3B) or wild type (Fig. 3C)
for disco function. Finally, no retinal degeneration was
detected in compound eyes of mixed genotype as long
as R-cells had established connections with the underlying optic lobe (Fig. 3D). Clones of disco cells in
otherwise wild-type and connected eyes showed completely normal morphology in aged flies. In contrast,
the contralateral eye of the fly shown in Fig. 3D did not
connect to the optic ganglia and had suffered retinal
degeneration (Fig. 3B). This demonstrates that R-cell
degeneration in disco mutants does not depend on the
genotype of cells in the eye, and is strictly correlated
with the failure of R-cells to project to the optic ganglia.
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A. R. Campos, K.-F. Fischbach and H. Steller
We conclude that R-cells depend on interaction with
the optic ganglia for survival after eclosion but do not
autonomously require disco gene function for survival.
Retinal degeneration in disco flies is rescued by the
establishment of connections with disorganized optic
lobes
The results presented above strongly indicate that the
R-cell degeneration observed in disco flies results from
the lack of connections with the optic lobe. In about 510% of disco flies R-axons project to an optic lobe
which is almost normal size but is significantly disorganized (the connected phenotype; Steller et al., 1987).
The connections between the eye and the optic ganglia
can be visualized in cryostat sections which have been
stained with an antibody specific for the photoreceptor
cells. Fig. 4 shows horizontal sections through heads of
wild-type, disco connected and disco unconnected flies
stained with the monoclonal antibody against the
photoreceptor-specific protein chaoptin (Zipurski et
al., 1985; Fig. 4 panel A, C and D respectively). A
neuropil pattern resembling that of lamina and medulla
of wild-type flies is found in connected disco flies (Fig.
4B,C). However in connected flies the overall morphology is significantly different from wild type as
opposed to the virtual lack of optic ganglia in the
unconnected phenotype (Fig. 4D). We were interested
in determining whether the connections found in
connected disco flies are able to prevent retinal
degeneration.
Newly eclosed disco' flies of the connected type were
selected based on the presence of a deep pseudopupil
(Franceschini, 1972). These flies were kept at 25°C for
at least two weeks after which the deep pseudopupil
phenotype was reassessed. In 139 out of 145 eyes
examined (96%) the deep pseudopupil was retained,
suggesting that no degeneration had occurred. This
conclusion was confirmed by semithin sections of these
specimens. Connected compound eyes from disco flies
did not display any signs of degeneration at that level of
resolution. Fig. 2D shows a cross section through the
compound eye of a discoflywhich has been aged for 15
days. The morphology of R-cells and their rhabdomeres, and ommatidial structure in general is indistinguishable from wild type. In such cases, we often
found that the contralateral eye displayed the uncon-
Fig. 4. Retinal innervation defects in disco mutants. Horizontal sections of adult heads stained with the R-cell specific
MAb24B10 which binds to the membranes of all R-cells. (A) Wild-type pattern of R-cell projections into the optic ganglia.
The outer R-cell axons (R1-R6) synapse in the lamina, while the inner R-cell axons (R7 and R8) synapse in the medulla.
(B) The two disco phenotypes, connected and unconnected, can occur in the same fly. This disco head has the left eye
displaying the unconnected phenotype and the right eye the connected. (C) Connected phenotype of disco flies, showing an
almost normal projection pattern. (D) In unconnected disco flies, the R-cell axons terminate in a mass of disorganized nonneuronal tissue. No regular projection pattern is discerned in these specimens. Abbreviations: re, retina; la, lamina; me,
medulla. Scale bar in A (valid for C and D) is 50 fim. Scale bar in B is 100 /m\.
Trophic interactions in the Drosophila eye
nected phenotype and had suffered severe retinal
degeneration (data not shown).
A
361
B
Retina-lamina connections in disco connected eyes
The results described in the previous section demonstrated that R-cells can survive in disco mutants if they
project to the optic ganglia (connected phenotype).
Although the overall morphology of the optic ganglia is
distorted in connected disco mutants, thesefliespossess
a lamina which is organized into cartridges that appear
relatively normal (Fig. 5A and 5B). However, we could
not determine from this analysis if all elements of the
lamina are present in connected discoflies,and whether
these elements are still functional.
In order to investigate if R-cells can establish
functional synaptic connections with the lamina in
connected disco, we performed electroretinograms
(ERG). ERGs measure the mass electrical response of
the eye consisting of the total extracellular potential
produced by retinula cells and by postsynaptic neurons
Fig. 6. Eletroretinogram recordings from disco flies. (A) A
typical eletroretinogram (ERG) waveform of -a wild-type
fly consists of a positive on-transient, a negative sustained
potential and a negative off-transient (arrow heads). These
transient potentials are observed at the beginning and end
of stimulation. Newly eclosed disco flies of the connected
phenotype as judged by the presence of a deep
pseudopupil were used to record ERGs. A total of 12
ERGs were recorded. A fraction of these (n=4) had both
transients (panel B) while the rest had only the OFF or a
very small ON transient (n=3), (panel C) or neither one of
the transient potentials (n=4) (not shown). In all cases the
negative sustained potential was present. The scale bar in
panel A is valid for panels B and C as well. (D) A typical
ERG recording from a young unconnected disco fly. The
arrows indicate when the light was turned on and off
respectively. A total of three specimens were recorded. In
all samples no transient potentials were observed. The
presence of a negative sustained potential, albeit
considerably smaller than that of wild-type flies,
demonstrates that the R-cells in unconnected disco flies are
able to respond to a light stimulus.
Fig. 5. Lamina structure in disco adults of the connected
phenotype. Semithin horizontal section through the lamina
ganglion of a wild type (A) and connected disco (B) fly. In
connected disco flies, the lamina cartridges can be readily
identified. The overall structure of lamina cartridges in
connected disco flies is very similar to wild type at that
level of resolution. Scale bar is 10 /an.
in the lamina. The ERG waveform of a wild-type fly
consists of a positive on-transient, a negative sustained
potential and a negative off-transient (Fig. 6A) (Pak,
1975). The transient components of an ERG tracing
reflect primarily a response of lamina neurons to R-cell
depolarization (Coombe, 1986). Young discofliesof the
connected phenotype were used to record ERGs. Two
typical tracings are shown in Fig. 6 panels B and C.
Often, ERG tracings were obtained which were very
similar to wild type and had clearly visible on and off
transients (Fig. 6A and B), thus indicating that, in these
cases, functional connections were established. However, in about 65% of all cases the on and/or off-
362
A. R. Campos, K.-F. Fischbach and H. Steller
transients were either very small or not detectable. This
variability is in contrast to the survival of R-cells in the
vast majority (96%) of the connected disco flies
examined. It is possible that the abnormal morphology
of the optic ganglia impairs the recording of the lamina
neuron response due to shunting. Therefore, functional
connections may be present even in these cases where
small or no transients were recorded. These results
demonstrate that functional connections between eye
and lamina can be established in at least some of the
connected disco animals.
Photoreceptors in ectopically located eyes degenerate
after eclosion
The findings described in the previous sections indicate
that the dependence of the R-cells on connections with
the optic ganglia is a general phenomenon and not
specific to disco mutant alleles. Therefore, we expected
that other mutations that prevent the interaction
between the retinal cells and the optic ganglia would
also cause degeneration.
We tested this hypothesis by analyzing extra-eye (ee)
mutants. The ee mutation causes the appearance of
supernumerary eyes, on the dorsal side of the adult
head. These structures develop properly and send
projections that never reach the optic ganglia or the
central brain and which terminate as plexuses within a
disordered tissue mass composing predominantly of
muscle cells, trachea and axon bundles (Marcey and
Stark, 1985). Fig. 7 shows cross sections of ommatidia
from supernumerary eyes of flies just after eclosion
(panel A) or 15 days after eclosion (panel B). As an
internal control, we inspected the ommatidia from the
regular compound eye (Fig. 7C) of the same fly as
shown in panel B.
As determined previously (Marcey and Stark, 1985),
the ommatidia of the supernumerary eyes from young
flies (Fig. 7A) show the usual characteristics of a fully
developed retina. There are in general seven rhabdomeres in any cross-section through each ommatidium.
They are arranged somewhat irregularly in a pattern
very similar to the one seen in eyes of young disco flies
(Fig. 2B). In contrast to young flies only a few
rhabdomeres can be found per ommatidium in ectopically located eyes of aged flies (Fig. 7B). Again, as in
disco flies, the pigment granules can be seen in a pattern
resembling that of young ee flies, suggesting that the
pigment cells surrounding the photoreceptor cells have
remained after degeneration of the R-cells. The
ommatidia of the normal compound eye in older ee flies
are indistinguishable from wild type (Fig. 7C), indicating that the R-cell degeneration observed in the
supernumerary eye is not due to lack of ee gene function
in R-cells per se.
Degeneration affects both inner and outer R-cell types
The results presented above indicated that most of the
R-cells degenerate in the absence of connections to the
optic ganglia. However, we could not determine from
these experiments whether all R-cell types, i.e. both
inner and outer R-cells, are affected. Since a few R-cells
w
*W>'~.'*'
*
Fig. 7. Retinal degeneration in ee mutants. (A) Ommatidia
from a dorsally located supernumerary eye of an ee mutant
fly which had eclosed approximately two hours prior to
fixation. Similar to the situation found in newly eclosed
disco mutants, all rhabdomeres can be identified in spite of
the slight irregularity of the ommatidial unit.
(B) Ommatidia from a dorsally located supernumerary eye
of an ee mutant fly which has been aged for 15 days at
25°C prior to fixation. R-cell degeneration very similar to
that found in disco mutants of the same age is noticeable
throughout the supernumerary eye. (C) Ommatidia from
the normal eye of the same ee mutant fly as shown in panel
B. No degeneration can be detected. Scale bar is 5 /an.
are typically present in aged unconnected eyes, it was
possible that these represent the R7 and/or R8 cell type.
A different behavior of inner versus outer R-cells would
not be entirely unexpected, because their axons
terminate in different ganglion layers of the brain. We
addressed this question by examining the expression of
rhodopsin-tacZ fusions which are specifically expressed
in different R-cell types (Mismer and Rubin, 1989;
Trophic interactions in the Drosophila eye
363
Fig. 8. Expression of Rhl and Rh4 in aged disco flies. In order to determine if the R-cell degeneration observed in
unconnected disco flies affected both inner and outer R-cells, we analyzed the expression of an Rhl-hcZ fusion gene and
an Rh4-/acZ fusion gene in aged (at least 20 days) disco flies of the connected and unconnected phenotype. In wild-type
Rhl (A, B and C) is expressed in all outer R-cells while Rh4 (D, E and F) is expressed in -30% of R7. (A) Wild type,
(B) disco connected, (C) disco unconnected, (D) Wild type, (E) disco connected, (F) disco unconnected. Expression of
either Rhl or Rh4 is not affected in connected disco flies (panels B and E respectively). Expression of Rhl and Rh4 is
equally reduced in unconnected disco flies (panels C and F, respectively). Panel F shows the mirror image of the
contralateral eye from the same fly shown in panel E. Abbreviations: re, retina; la, lamina; me, medulla. Scale bar is 50
/an.
Fortini and Rubin, 1990). A fusion between the Rhlpromoter and lacZ specifically stains outer R-cells (Rl6, Mismer and Rubin, 1989), and a Rh4-promoter-/acZ
fusion labels a subset of R7 cells in the retina (Fortini
and Rubin, 1990). These reporter gene constructs were
introduced into a disco mutant background by crossing
males which were homozygous for the rhodopsin-/acZ
fusions to disco females. The male offspring of this cross
are hemizygous for disco and heterozygous for the
element containing the rhodopsin-/acZ fusion. These
males were aged on average for 20 days at 25°C,
sectioned and stained for lacZ activity. Both unconnected and connected compound eyes were obtained in
these studies. Because we typically did not detect any
degeneration in the retina of connected disco flies (see
for example Fig. 2D), the lacZ expression in these
specimens was used as an internal control for the effect
of aging. In aged unconnected eyes, the intensity of the
lacZ staining was significantly decreased for both the
Rhl- and Rh4-/acZ fusion constructs (Fig. 8). The
reduction in the lacZ activity appears more prominent
towards the base of the retina. We conclude that
degeneration is not restricted to the Rl-6 cell type, but
also affects R7 cells. We also used northern analysis to
examine the steady state levels of mRNA from Rhl
(Zuker et al., 1985, O'Tousa et al., 1985), and another
rhodopsin gene expressed in R7 cells, Rh3 (Montell et
al., 1987). The levels of both Rhl and Rh3 message
were decreased in aged unconnected disco flies compared to young flies (data not shown). Taken together
364
A. R. Campos, K.-F. Fischbach and H. Steller
these data suggest that both outer R-cells and at least
one of the inner R-cell types, R7, degenerate in the
absence of connections to the optic ganglia.
Discussion
Many instances of retrograde transneuronal degeneration, both during and after neural differentiation is
completed, have been described in vertebrates
(reviewed by Cowan, 1970; e.g. Hamburger, 1934;
Hamburger and Levi-Montalcini, 1949; Crews and
Wigston, 1990). In contrast, only examples of anterograde dependence have been reported in invertebrates.
This study provides evidence for the existence of
retrograde transneuronal dependence in the adult
visual system of the fruitfly Drosophila melanogaster.
We determined the integrity of retinular cells in
situations where the photoreceptor cell axons do not
reach the optic ganglia. For this, we examined cross
sections of ommatidia from flies carrying mutant alleles
for either the disconnected (disco) or the extra-eye (ee)
gene. In aged flies, extensive R-cell degeneration was
found in eyes that did not make connections with the
optic ganglia. Flies that were aged in complete darkness
showed the same degree of degeneration (data not
shown). This degeneration is apparently restricted to
the photoreceptor cells since non-neuronal support
cells, e.g. cone cells (data not shown), and pigment
granules of the pigment cells, are present well after the
time when R-cells have degenerated. In most ommatidia only a few rhabdomeres are left 15 days posteclosion. This degeneration was shown to occur in all outer
R-cells (Rl-6), and in R7.
Our mosaic studies demonstrate that disco+ gene
function is not required in the retina for the survival of
R-cells. The ability of R-cells to survive is strictly
correlated with the presence of connections to the optic
ganglia. This conclusion is also supported by the
observation that in non-mosaic disco animals degeneration is only observed in flies of the unconnected
phenotype. Finally recent experiments using antibodies
against disco protein product demonstrated that the
disco gene is not expressed in the retina, and is only
detected in a relatively small number of brain cells
during the late larval, pupal and adult stages (Lee et al.,
1991; Lee and Steller, unpublished observations).
Taken together, these observations indicate that R-cells
in the compound eye of Drosophila depend on
interactions with cells outside the retina for continued
survival after eclosion.
The results discussed above also confirm previous
reports that retinal differentiation does not depend on
interactions with the optic ganglia (Kopec, 1922;
Chevais, 1937; Wolski and Wolski, 1971; Mouze, 1978;
Anderson, 1978a). Although the compound eyes of
disco individuals are not completely normal at eclosion,
displaying somewhat deranged positions of rhabdomeres and pigment cells, we have every indication to
believe that R-cells successfully complete their terminal
differentiation program in disco mutants. First, the
normal structural features of the different cell types
constituting an ommatidium are found in disco mutants,
and terminal differentiation markers, like opsins, are
properly expressed. In addition, our results indicate
that functional photoreceptor cells can develop in the
absence of connections with the optic ganglia: R-cells
from newly eclosed unconnected disco flies are able to
respond to a light stimulus, demonstrating that a
functional phototransduction machinery has been correctly assembled. We believe that the imperfect eye
geometry in disco is associated with the variable
amount of deformations previously caused by the lack
of an optic stalk (Steller et al., 1987). Due to defects in
the eye's curvature, the precise alignment of retinal
cells, most noticeably the rhabdomeres and pigment
cells, is slightly out of order.
The retinal axons of unconnected disco eyes are
sometimes seen contacting muscle tissue present in the
space otherwise occupied by the optic ganglia. It could
be argued that the degeneration observed in these cases
was caused by a cytotoxic effect from the muscle cells.
However, this explanation of our results appears very
unlikely, since only some R-cells in disco mutants
project to muscle tissue. In addition, we have seen at
least 20 cases where the optic ganglia were replaced by
either hemolymph or non-neuronal cells of unknown
identity, yet R-cells degenerated in these cases as well.
Therefore, degeneration cannot be a consequence of
ectopic contacts between retinal axons and muscle
tissue. Furthermore, the poisonous action of a putative
diffusible toxin would somehow have to be restricted to
the retina, since other neurons which are in direct
contact with the muscle tissue or hemolymph, for
example neurons of the central brain, continue to
survive and function for long times. Finally, in extra eye
mutants, which have normal optic lobes, retinal
degeneration affects only the supernumerary, unconnected eyes, but not the regular compound eyes.
Therefore, it appears that R-cell degeneration is not
due to cytotoxic effects from degenerating cells or
contact with ectopic muscle tissue, but is caused by the
lack of interactions with cells in the optic ganglia.
We have several reasons to believe that the proposed
trophic support is contributed to the retina by cells in
the optic ganglia, and not in some other tissue outside
the visual system. Since the space underlying the eye is
entirely occupied by the optic ganglia, any tissue
outside the visual system would have to produce a
factor(s) that can act over a considerable distance.
However, such long-range effects are not consistent
with the striking left/right asymmetries that we frequently observe in disco mutants or mosaics; we have
observed at least 50 cases where retinal degeneration
was restricted to only one (unconnected) eye, but did
not affect the ipsilateral (connected or wild-type) side.
This argues strongly against any long-range diffusible
factor, since it is very difficult to imagine how the action
of such a factor could always be restricted to the eye on
that side that lacks the optic ganglia. Finally, retinal
degeneration in the extra eye mutant affects only the
supernumerary eyes but not the adjacent regular
Trophic interactions in the Drosophila eye
compound eyes. We conclude that R-cell survival
depends on interactions with the optic ganglia, and not
the presence of some other structure(s) outside the
visual system.
The results described in this paper do not reveal
which cell type(s) in the optic ganglia is responsible for
survival of photoreceptor cells. In particular, we do not
know if neuronal or glial elements of the optic ganglia
provide the proposed trophic support for photoreceptor
cells. Although functional connections between R-cells
and their target neurons in the optic lobe can be found
in disco mutants, it is possible that these connections
are not essential for R-cell survival. A significant
portion of connected disco flies fail to show an optic
lobe response to a light stimulus (i.e. they lack on and
off-transients). Nevertheless, R-cells survived in the
majority of connected discofliesexamined. The caveat
for such observations is that abnormal morphology of
the optic ganglia may impair the recording of the
transient components of the ERG tracing due to
shunting. Since ERGs measure the overall electrical
response of the eye and lamina with respect to a
reference point in the body, the ability to obtain a
normal ERG waveform depends on the existence of
proper electrical barriers between the retina and the
brain, and also on barriers within the different elements
of the optic ganglia (Heisenberg, 1971, Shaw, 1977). We
believe that these barriers are severely reduced in
unconnected and also many connected disco animals,
which display significantly disorganized optic ganglia
(see Steller et al., 1987). Such a lack of proper electrical
insulation, or "shunting", may account for both the
reduced amplitude of the depolarization potential, and
also the observed lack of on and off-transients.
We have not determined the degree of degeneration
or the structure of the optic ganglia in the few
connected disco flies that lost the deep pseudopupil
after two weeks (6 out 145 animals, corresponding to
~4%). A more careful analysis of such cases may aid in
establishing a correlation between optic lobe structure
and survival of the R-cells. Mutations that cause welldefined defects in the optic lobe structure will be
particularly useful in elucidating the underlying physiological and cellular mechanisms that play a role in the
maintenance of the retina.
Glial cells are thought to play a diverse role in the
invertebrate nervous system. A supportive role for glial
cells has been reported in the crayfish where transglial
channels have been implicated in the long-term survival
of the distal stump of severed medial giant axons
(Shivers and Brightman, 1976; Shivers, 1976; Meyer
and Bittner, 1978a,b). Glial cells have also been
suggested to be mediators in the induction of glomeruli
by afferent axons in the olfactory system of the
hawkmoth Manduca sexta (reviewed by Tolbert and
Oland, 1989).
In the fly visual system, several types of glial cells
which are intimately associated with the photoreceptor
axon have been described (Trujillo-Cenoz, 1965; Saint
Marie and Carlson, 1983). A subset of these cells
establish specialized axo-glial associations called capi-
365
tate projections (Trujillo-Cenoz, 1965; Stark and Carlson, 1986; reviewed by Lane, 1981). These structures
arise from the association of the epithelial glial cell with
axons from the outer photoreceptor cells (Rl to R6),
and it has been previously proposed that they might be
involved in trophic interactions between the lamina and
photoreceptor axons from Rl-6 (Stark and Carlson,
1985, 1986). However, no direct experimental evidence
for such a function is presently available.
In conclusion, the experiments described in this
paper demonstrate the dependence of R-cells on
interactions with the optic ganglia for survival after
development is completed. These interactions are
reminiscent of trophic interactions widely studied in
vertebrates. Genes that encode molecules involved in
the proposed trophic interaction should be uncovered
by mutations that cause light-independent retinal
degeneration. These genes may be involved in the
expression of the signal or the receptor for the proposed
interaction. Alternatively, this interaction may be
mediated by a variety of molecules and cell interactions. In this case only mutations that completely
abolish the optic ganglia will cause retinal degeneration.
We thank our colleagues in the Steller lab for comments on
this manuscript and lively scientific discussions. We are
grateful to Kate Rendhal for helping with the ERGs and
William Stark for providing the ee strain. This work was
supported in part by NIH grant RO1-NS26451 to H.S..
A.R.C. is a postdoctoral associate of the Howard Hughes
Medical Institute, and H.S. is an Assistant Investigator of the
Howard Hughes Medical Institute. During the initial phase of
this work A.R.C. was supported by a postdoctoral Fellowship
from the Muscular Dystrophy Association of America.
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{Accepted 3 November 1991)