Rhodopsin patterning in central photoreceptor cells of the blowfly Calliphora vicina: cloning and characterization of Calliphora rhodopsins Rh3, Rh5 and Rh6
Institut für Zoologie, Universität Karlsruhe, Haid-und-Neu-Strasse 9, 76131 Karlsruhe, Germany
* Author for correspondence (e-mail: Armin.Huber{at}bio.uka.de)
Accepted 27 January 2005
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Summary |
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Key words: Calliphora, Drosophila, pattern formation, photoreceptor, rhodopsin, vision
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Introduction |
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The photoreceptor cells R16 and R7/R8 constitute photoreceptor
classes with distinct visual functions. Anatomical, physiological and
behavioural investigations suggested that photoreceptors R16 provide
the sensory input for high sensitivity vision, image formation and motion
detection (Hardie, 1985;
Pichaud et al., 1999
;
Wernet and Desplan, 2004
). The
R16 cells express the same rhodopsin, designated Rh1, which has been
cloned from Drosophila as well as from Calliphora
(O'Tousa et al., 1985
;
Zuker et al., 1985
;
Huber et al., 1990
). Rh1
represents over 90% of the visual pigment present in the compound eye
(Hamdorf et al., 1973
;
Hardie, 1985
;
Paulsen, 1984
;
Huber et al., 1990
;
Salcedo et al., 1999
).
The organization of the central photoreceptor cells R7 and R8 is
considerably more complex. Kirschfeld and Franceschini
(1977) first found two
populations of R7 in eye-cup preparations of the housefly Musca,
observed in transmitted light: yellow (R7y) and pale or colourless (R7p). The
yellow colour is due to the presence of a blue-absorbing, carotenoid pigment
in the rhabdomere of R7y, which occurs in 70% of the ommatidia; 30% has R7p.
The two classes of ommatidia are distributed randomly across the eye. This is
also readily recognized by epifluorescence, as the 7y rhabdomeres fluoresce
bright green under blue excitation, while the R7p rhabdomeres appear black.
Further microspectrophotometry and electrophysiological recordings
demonstrated that the R7s were accompanied by specific R8s, thus called R8y
and R8p, respectively (Hardie,
1985
).
Cloning of rhodopsins Rh3Rh6 expressed in R7 and R8 cells of the
compound eye of Drosophila provided a means of determining in which
combinations these rhodopsins are present in the various types of ommatidia
(Montell et al., 1987;
Fryxell and Meyerowitz, 1987
;
Zuker et al., 1987
;
Chou et al., 1996
;
Huber et al., 1997
;
Papatsenko et al., 1997
).
Studies using specific antibodies against Drosophila rhodopsins
revealed that the expression of rhodopsin Rh3 in an R7 cell is coupled to the
expression of Rh5 in the adjacent R8 cell, while Rh4 colocalizes with Rh6
(Chou et al., 1996
,
1999
;
Papatsenko et al., 1997
). The
Rh3/Rh5 and Rh4/Rh6 ommatidia are distributed randomly in the compound eye at
a ratio of about 29:71%, very similar to the ratio of ommatidia with R7p and
R7y in Musca (Chou et al.,
1999
). Behavioural studies indicate that the two sets of central
photoreceptors mediate colour discrimination
(Troje, 1993
;
Pichaud et al., 1999
). In the
dorsal rim region of the eye, the ommatidia of Musca have specialized
R7 and R8 photoreceptors, which are distinguishable by the larger diameter of
their rhabdomeres, which are UV sensitive. This also holds for the ommatidia
of the dorsal rim region of Drosophila, where both R7 and R8 cells
express the UV rhodopsin Rh3 (Chou et al.,
1999
; Wernet et al.,
2003
). The central photoreceptors in the dorsal rim are believed
to mediate detection of polarized light, in which the perpendicular
arrangement of the microvilli of R7 and R8 cells plays an important role
(Hardie, 1984
;
Wernet et al., 2003
). Finally,
unequivocally described for males of Musca domestica only, the R7
cells in the anterodorsal region of the compound eyes exhibit the same red
fluorescence as R16 photoreceptors, because they also express Rh1. The
R7 photoreceptors, called R7r, add their light signal to the R16
system, presumably to improve the male's chasing behaviour. The male dorsal
eye area thus is called the `love spot'
(Franceschini et al., 1981
;
Hardie et al., 1981
).
Despite the structural and functional equivalence, which is to be expected
for photoreceptor classes R16 and R7/R8, there are some differences
between the compound eyes of Drosophila, Musca and
Calliphora. For example, while the eyes of Drosophila are
composed of about 750 ommatidia, Musca has about 3000 and
Calliphora has about 5200 (Beersma
et al., 1977). The eyes of male Calliphora are much
bigger than those of the female, which results in a much higher acuity in the
dorsal area but not in a much higher number of ommatidia. The arrangement of
optical axes, and accordingly, the spatial resolution as well as temporal
photoreceptor properties are not homogeneously distributed over the ommatidial
lattice of the Calliphora compound eye
(Petrowitz et al., 2000
;
Burton et al., 2001
). Similar
properties have not yet been reported for the Drosophila eye. Also,
there are spectral differences between the central photoreceptors of
Drosophila (Salcedo et al.,
1999
) and those of Musca and Calliphora
(Hardie, 1985
), which we
further explore in this paper.
We report the cloning of three rhodopsins expressed in the central photoreceptor cells of the compound eye of Calliphora vicina. We have determined the spatial distribution of these rhodopsins and the relative abundance of the ommatidia with R7y and R7p. Our results reveal a high degree of conservation between Calliphora and Drosophila in the rhodopsin expression pattern of the central photoreceptor cells.
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Materials and methods |
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cDNA-library screening and DNA sequencing
An oligo(dT)-primed cDNA library in UniZap XR vector (Stratagene,
Amsterdam, The Netherlands) produced from poly(A)+ RNA isolated
from retinas of Calliphora vicina
(Huber et al., 1996) was used
for the isolation of cDNA clones encoding CvRh3, CvRh5 and
CvRh6. Screening of this cDNA library was carried out using a mixture
of cDNAs encoding Drosophila melanogaster rhodopsins (Rh3, Rh4, Rh5
and Rh6). The cDNAs were labelled with digoxigenin using the DIG DNA Labelling
Mix (Roche Molecular Biochemicals, Mannheim, Germany) according to the
manufacturer's instructions. The hybridization was performed in 5x SSC
(1x SSC is 0.15 mol l1 NaCl, 0.015 mol
l1 sodium citrate, pH 7.0), 0.1% laurylsarcosinate, 0.02%
SDS, 1% blocking reagent (Roche Molecular Biochemicals, Mannheim, Germany) at
55°C according to standard protocols
(Sambrook and Russell, 2001
).
Positive clones were rescreened and plasmid DNA was obtained by in
vivo excision. DNA sequencing was carried out with an Alf-Express
automated DNA sequencer (Amersham Biosciences, Freiburg, Germany) using
Cy5-labelled oligonucleotide primers and the dideoxy chain termination method
(Sanger et al., 1977
).
Different clones and subclones for each Calliphora rhodopsin were
sequenced to obtain at least twofold coverage of each sequence.
Antibodies
Polyclonal antibodies against Calliphora Rh3 and Rh5 were
generated against synthetic peptides coupled to keyhole limpet hemocyanin by
Seqlab (Göttingen, Germany). The peptides used were based on the deduced
amino acid sequence of the Calliphora rhodopsins: CNEKAPEASSTASTTG,
corresponding to amino acids 360374 of CvRh3, and CRERNYAASSSGGDNA
corresponding to amino acids 363377 of CvRh5. Monoclonal anti-DmRh6
antibodies that cross reacted with Calliphora Rh6 have been described
previously (Chou et al.,
1999).
Detection of rhabdomere autofluorescence
Autofluorescence analysis of R7y and R7p rhabdomeres was carried out by
optical neutralisation of the cornea, essentially as described by Hardie et
al. (1981).
Calliphora heads were dissected and submerged under water in a Petri
dish. Autofluorescence of central rhabdomeres was detected using a water
immersion objective (Leica 20x / 0.50) and epiillumination with blue
light (450490 nm) from a Hg lamp (HBO100W), or by confocal microscopy
after excitation with 476 nm and 488 nm laser light. To calculate the ratio of
R7y to R7p receptors, randomly chosen areas of the compound eyes of male
Calliphora were investigated for the presence (R7y) or absence (R7p)
of green fluorescence in the central rhabdomeres. Depending on the region of
the eye investigated, between 50 and 130 ommatidia were evaluated each time.
Images were obtained with a Leica DC 200 camera and were processed with
PhotoShop 6.0.
Immunocytochemistry
For immunocytochemical localization of Calliphora rhodopsins by
confocal laser scanning microscopy, dissected eyes of Calliphora were
fixed in 2% paraformaldehyde in PBS (137 mmol l1 NaCl, 3
mmol l1 KCl, 8 mmol l1
Na2HPO4, 2 mmol l1
KH2PO4, pH 7.2) for 2 h at room temperature, followed by
three washes in 10%, 25% and 50% sucrose in 0.1 mol l1
sodium phosphate buffer, and infiltration with 50% sucrose in 0.1 mol
l1 sodium phosphate buffer overnight at 4°C. The eyes
were embedded in boiled bovine liver, covered with Tissue Tek and cryofixed in
melting isopentane. Eyes were sectioned at 10 µm (cross sections) or 18
µm (longitudinal sections) in a cryostat at 25°C and placed on
coverslips pre-coated with 0.01% aqueous poly-L-lysine (Sigma,
Deisenhofen, Germany). The cryosections were fixed with 2% paraformaldehyde in
0.1 mol l1 sodium phosphate buffer for 20 min, followed by
two subsequent washes with 0.1 mol l1 sodium phosphate
buffer. For antibody staining, the sections were incubated in 0.01% Saponine
in 0.1 mol l1 sodium phosphate buffer (pH 7.2) for 2.5 h at
room temperature, washed three times with 0.1 mol l1 sodium
phosphate buffer and incubated with the primary antibody diluted 1:20 in
blocking solution (0.5% ovalbumin, 0.1% cold-water fish gelatine in 0.1 mol
l1 sodium phosphate buffer, pH 7.2) overnight at 4°C. To
block antibody reactions, diluted antibodies were preincubated with
1525 µg ml1 of the corresponding peptides for 4 h
at 4°C before they were applied to the sections. After incubation with
antibodies the sections were washed three times in 0.1 mol
l1 sodium phosphate buffer and then incubated with goat
anti-rabbit AlexaFluor 488 and goat anti-mouse AlexaFluor 660 antibodies
(Invitrogen, Karlsruhe, Germany) diluted in blocking solution. For a
concomitant staining of the photoreceptor rhabdomeres, the sections were
incubated together with the secondary antibody with 0.5 pg
µl1 rhodamine-coupled phalloidin (Sigma-Aldrich,
Taufkirchen, Germany). The sections were finally washed in 0.1 mol
l1 sodium phosphate buffer, mounted in mowiol 4.88 and
examined with a confocal laser scanning microscope (TCS-SP, Leica, Bensheim,
Germany).
To obtain isolated Calliphora rhabdoms dissected retinas were
slowly and repeatedly pipetted in 75 µl distilled water through a fine
pipette (Paulsen, 1984).
Rhabdoms were immediately transferred to coverslips pre-coated with 0.01%
aqueous poly-L-lysine. After allowing the rhabdoms to dry on the
coverslip for 30 min at 30°C, immunocytochemical detection of rhodopsins
was carried out as described above for cryosections. In addition, rhabdoms
were labelled with Oregon Green-coupled wheat germ agglutinin (Invitrogen,
Karlsruhe, Germany), which was added together with the secondary antibody at a
final concentration of 20 µg ml1.
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Results |
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Distribution of Calliphora rhodopsins in the compound eye
In larger flies, especially in Musca, the distribution of
different types of R7 and R8 photoreceptor cells across the compound eye has
been mapped precisely by evaluating the autofluorescence of the rhabdomeres
(Hardie et al., 1981;
Franceschini et al., 1981
).
Application of this method to the Calliphora chalky mutant revealed
that autofluorescence of R7y (green fluorescence) and R16 rhabdomeres
(red fluorescence) could be easily detected using water immersion objectives
combined with conventional fluorescence microscopy or confocal laser scanning
microscopy. Fig. 2shows a
fluorescence image taken from the frontal region of the compound eye, which
harbours R7y and R7p rhabdomeres. To determine the ratio of R7y to R7p
photoreceptors 20 images obtained from different eyes and eye regions were
analysed. The determined ratio, 68±5% R7y and 32±5% R7p, was
similar to the ratio previously described for Musca (70% R7y, 30% R7p;
Hardie et al., 1981
). As has
been shown for Musca, no obvious symmetry or conservation in the
distribution of R7y and R7p photoreceptors between different eyes was
observed. Contrary to the observations made for male Musca, we did
not detect any evidence for a `love spot' in the frontal dorsal region of the
male Calliphora compound eye, which would be expected to contain red
fluorescing R7 cells.
|
In order to compare the ratio of R7y to R7p photoreceptor cells with the
expression pattern of Calliphora rhodopsins we employed
immunocytochemistry. We used a monoclonal antibody directed against
Drosophila Rh6, which cross-reacted with Calliphora Rh6
rhodopsin in sections through Calliphora compound eyes. The peptide
used to make this antibody corresponds to amino acids 348362 of
Drosophila Rh6 rhodopsin, a region that is identical between
Calliphora and Drosophila Rh6 rhodopsin, except for two
amino acids (see Fig. 1). The
cross reaction of this antibody is specific for Calliphora Rh6,
because the reaction can be blocked with a peptide corresponding to the
relevant region of Calliphora Rh6
(Fig. 3). As antibodies
directed against rhodopsins Rh3 and Rh5 of Drosophila
(Chou et al., 1999) showed no
reaction with the corresponding Calliphora proteins we generated
polyclonal antibodies against peptides of the C-terminal region of these
Calliphora rhodopsins. To test the specificity of the newly generated
anti-CvRh3 and anti-CvRh5 the corresponding peptides were dot blotted and
incubated with the antibodies. Anti-Rh3 and anti-Rh5 reacted specifically with
their corresponding peptides (not shown). In addition, immunofluorescence
signals obtained with anti-Rh3 and anti-Rh5 on sections through
Calliphora eyes could efficiently be blocked with the corresponding
peptides (Fig. 3). The
anti-CvRh5 antiserum reacted also with cell nuclei. The reaction of this
antibody with nuclei was not specific for Rh5 rhodopsin as it could not be
blocked with the Rh5 peptide. With these antibodies in hand double labelling
studies of longitudinal and of cross sections through Calliphora eyes
were carried out. For identification of the rhabdomeres the actin cytoskeleton
of the rhabdomeres was also stained with rhodamin-coupled phalloidin
(Fig. 4). Longitudinal sections
probed with anti-Rh5 and anti-Rh6 demonstrated the localization of these
rhodopsins in the basal part of the retina
(Fig. 4A), suggesting that they
are expressed in R8 cells. This expression pattern is in line with the initial
characterization of these visual pigments as rhodopsins Rh5 and Rh6 on the
basis of their homology to Drosophila rhodopsins, because
Drosophila Rh5 and Rh6 are expressed in R8 cells, too
(Chou et al., 1999
). As is the
case for Drosophila Rh5 and Rh6, the corresponding
Calliphora rhodopsins are expressed in mutually exclusive sets of R8.
This can best be observed in cross sections at the R8 cell level
(Fig. 4B) which clearly reveal
that the central rhabdomere is stained either with the anti-Rh5 or with the
anti-Rh6 antibody. Double labelling experiments with antibodies directed
against Calliphora Rh3 and Rh6
(Fig. 4C,D) showed that Rh3 is
located mainly in the apical portion of the retina, suggesting its expression
in R7 cells. However, in some ommatidia anti-Rh3 labelling was observed
additionally in the basal portion of the retina, which may indicate that some
R7 cells extend into the layer of R8 cells. Analysis of cross sections at the
R7 cell level showed that the central rhabdomeres of some ommatidia are
labelled by anti-Rh3 antibody while others are not. The ommatidia not labelled
by anti-Rh3 are likely to express another rhodopsin in R7 cells, presumably
Calliphora Rh4. In addition to the rhabdomeres, the rhabdomeral
stalks are also labelled by anti-Rh3, anti-Rh5 and anti-Rh6 antibodies,
indicating that these rhodopsins are not restricted to the rhabdomeres.
However, Rh3, Rh5 and Rh6 rhodopsin are not detected in the entire plasma
membrane of the photoreceptor cells. It is likely that adherens junctions
(organized by the protein Crumbs), which separate the rhabdomeral stalk from
the basolateral membrane region of the photoreceptor cells
(Pellikka et al., 2002
;
Izaddoost et al., 2002
), form
a barrier that cannot be crossed by the rhodopsin molecules incorporated in
the rhabdomeral part of the plasma membrane.
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The Drosophila rhodopsins Rh3/Rh5 and Rh4/Rh6 are expressed in
matched pairs randomly distributed across the eye, such that 29% of the
ommatidia contain the combination Rh3/Rh5 and 71% contain Rh4/Rh6
(Chou et al., 1999
). To
determine the relative abundance of Rh5- and Rh6-expressing photoreceptor
cells in the Calliphora compound eye, we isolated rhabdoms, stained
them with anti-Rh5 and anti-Rh6 antibodies, and counted the number of rhabdoms
containing Rh5 or Rh6 (Fig. 5).
In females 28.05±1.79% of the labelled rhabdoms contained Rh5 and
71.95±1.79% contained Rh6. In males similar values (24.95±2.47%
Rh5, 75.05±2.47% Rh6) were obtained, indicating that the sexual
dimorphism that results in larger eyes in the males does not affect the
expression ratio of Rh5 and Rh6. The determined ratio is similar to that in
Drosophila, i.e. roughly 30% of the rhabdoms contain Rh5 and about
70% contain Rh6. This ratio is also in line with the relative abundance of the
two types of Calliphora ommatidia, containing either R7p/R8p or
R7y/R8y photoreceptor cells (see Fig.
2). In order to be able to count the number of rhabdoms containing
Rh3, isolated rhabdoms were incubated with anti-Rh3 antibodies and
counterstained with Oregon Green-coupled wheat germ agglutinin. In females
39.16±8.42% and in males 37.58±7.05% of the total number of
rhabdoms contained Rh3.
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Discussion |
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With respect to the spectral properties of rhodopsins, the absorption
maxima in the rhodopsin and metarhodopsin state obtained for larger flies by
microspectrophotometric measurements (summarized in
Hardie, 1985) can be compared
with those of Drosophila rhodopsins, which were determined after
ectopic expression of the R7 and R8 cell rhodopsins in R16 cells by
difference spectroscopy of extracted visual pigments and by
microspectrophotometry (Salcedo et al.,
1999
; Feiler et al.,
1992
). Since insect rhodopsins with similar spectral properties
typically fall into the same phylogenetic groups
(Briscoe and Chittka, 2001
),
the conservation of the primary structure should correlate with the spectral
properties. Table 2 shows that
Rh3 and Rh6 of Drosophila and Calliphora, which have amino
acid identity of more than 80% also display very similar spectral properties.
The less well conserved Rh5 rhodopsin shows significant differences in the
absorption maximum of the rhodopsin state. The highest differences in spectral
properties are reported for Rh4 which in its rhodopsin state has an absorption
maximum at 355 nm in Drosophila but 430 nm in the larger flies. It is
possible that this rhodopsin is the least well conserved visual pigment
between Drosophila and Calliphora which in turn could make
it difficult to isolate its cDNA by homology screening. More detailed
information on spectral tuning of fly rhodopsins came from studies in which
amino acids conserved between rhodopsins with similar spectral properties were
mutated. In a recent report it has been shown that a single amino acid
polymorphism is largely responsible for Drosophila UV vision
(Salcedo et al., 2003
). In the
second transmembrane domain the UV-absorbing Drosophila rhodopsins
Rh3 and Rh4 have a lysine (K110 in Rh3), the mutation of which results in a
large shift of the absorption maximum of Rh3 to longer wavelength. The
critical lysine residue is conserved in Calliphora Rh3, confirming
the characterization of this rhodopsin as a UV-absorbing visual pigment.
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Expression pattern of Calliphora rhodopsins
The immunocytochemical studies on the expression pattern of
Calliphora Rh3, Rh5 and Rh6 described here reveal a highly similar
expression pattern to that described for Drosophila. Calliphora Rh5
and Rh6 are detected in non-overlapping sets of R8 cells while Rh3 is
expressed in R7 cells. Furthermore, double labelling of longitudinal sections
with anti-Rh3 and anti-Rh6 showed that in most cases Rh3 and Rh6 are not
contained in the same ommatidium, that is, as in Drosophila, the
expression of Rh3 and Rh6 is usually not coupled. The quantitative evaluation
of immunolabelled isolated rhabdoms further supports the assumption that
rhodopsin patterning is highly conserved between Calliphora and
Drosophila. The ratio of Calliphora R8 cells containing Rh5
or Rh6 is not significantly different from the ratio determined for
Drosophila. Although there is a sexual dimorphism in
Calliphora resulting in larger compound eyes in the male, which is
not observed in Drosophila, this dimorphism has no significant effect
on the relative number of Rh5- and Rh6-containing R8 cells. The determined
numbers for Rh3-expressing rhabdoms are about 10% higher than the values for
Rh5-expressing rhabdoms, suggesting that some ommatidia express Rh3 but not
Rh5. Ommatidia of the dorsal rim region, which contain Rh3 in R7 and R8 cells,
may in part account for this discrepancy. In addition, besides the pairing of
Rh3-containing R7 cells with Rh5-containing R8 cells, a significant number of
Rh3-containing R7 cells may be paired with R8 cells containing Rh6. In
Drosophila, coupling of Rh3 and Rh6 was reported to occur in 6% of
ommatidia, whereas the combination Rh4/Rh5 was observed very rarely (0.3%)
(Chou et al., 1999). In
conclusion, our results suggest that the developmental mechanisms that govern
coordinated rhodopsin expression in R7 and R8 cells of the fly eye are very
well conserved between Drosophila and Calliphora. The
similar ratios of ommatidia containing R7p/R8p or R7y/R8y photoreceptors, and
of the two types of ommatidia defined by their rhodopsin expression pattern in
the Calliphora compound eye, further support the assumption that
ommatidia containing R7p/R8p express the rhodopsins Rh3/Rh5 while ommatidia
containing R7y/R8y express Rh4/Rh6. Detailed morphological studies of the
compound eye of Calliphora showed that the two major types of central
rhabdomeres not only differ in their fluorescence properties, but also in the
relative length of R7 and R8 rhabdomeres
(Smola and Meffert, 1979
).
This shows that terminal differentiation of R7 and R8 cell subtypes affects
more than just the activation of distinct rhodopsin genes.
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Acknowledgments |
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