\set{final}

\def\Author{Shim}
\def\author{shim}
\def\year{2001}
\def\vol{7}
\def\anum{30}
\def\pages{216-221}
\def\txt_title{Evidence for indirect control of phospholipase C (PLC-[beta]) by retinoids in Drosophila phototransduction}
\def\txt_authors{Kyuhwan Shim, Karen M. Zavarella, Charles F. Thomas, Randall D. Shortridge, William S. Stark}

\def\rcvd{11 June 2001}
\def\accept{14 September 2001}
\def\publ{21 September 2001}
\def\pdfsize{}
\def\PMID{}


\include{mvstyle.hsm}

\| External links

\def\NIHimage{http://rsb.info.nih.gov/nih-image/}

\| Internal defs


\article{

\title{Evidence for indirect control of phospholipase C (PLC-\beta) by
retinoids in \i{Drosophila} phototransduction}

\authors{\mailto{kshim@im.wustl.edu}{Kyuhwan Shim},\sup{1}
\mailto{karen@zavarella.com}{Karen M. Zavarella},\sup{2}
\mailto{cthomas@facstaff.wisc.edu}{Charles F. Thomas},\sup{3}
\mailto{RDS@ubvms.cc.buffalo.edu}{Randall D. Shortridge},\sup{2}
\mailto{starkws@slu.edu}{William S. Stark}\sup{1}}

\institutions{\sup{1}Department of Biology, Saint Louis University, St.
Louis, MO; \sup{2}Department of Biological Sciences, State University of
New York, Buffalo, NY; \sup{3}Laboratory for Optical and Computational
Instrumentation (LOCI), University of Wisconsin, Madison, WI}

\correspondence{William S. Stark, Ph.D., Department of Biology, 3507
Laclede Avenue, Saint Louis University, St. Louis, MO, 63103-2010;
Phone: (314) 977-7151; FAX: (314) 977-3658; email: starkws@slu.edu\br
\sp\br
Dr. Shim is now at the Howard Hughes Medical Institute, Washington
University School of Medicine, St. Louis, MO\br
Dr. Zavarella is now at IDna, Inc., Independence, OH}

\abstract

\abs_purpose{To determine how retinoids regulate the phospholipase C
(PLC) gene in the \i{Drosophila} visual system.}

\abs_methods{Western blotting, activity analyses and immunocytochemistry
were applied to \i{Drosophila} reared on various diets.}

\abs_results{Western blots and activity analyses showed that retinoid
deprivation decreases PLC, the product of the norpA gene, by
approximately 1/3 to 1/2 in \i{Drosophila}. Immunocytochemistry using
standard and confocal fluorescence microscopy confirmed the expectation
that PLC is localized to the photoreceptive rhabdomeres. Rhabdomeres of
flies that were retinoid deprived, or reared on other diets devoid of
chromophore precursors, fluoresced brightly. These observations are
consistent with earlier morphometric analyses showing that retinoid
deprivation decreases the size of rhabdomeres. In a separate control,
rhabdomeric PLC was shown to be virtually eliminated by retinoid
deprivation in transgenic \i{Drosophila} where the norpA coding sequence
was driven by the opsin promoter.}

\abs_conclusions{PLC is decreased by retinoid deprivation. Retinoid
control of PLC is indirect, as expected, since the norpA promoter is so
different from the promoter for rhodopsin's gene. PLC is not eliminated
by deprivation but decreases in proportion to the associated decrease in
rhabdomere size which, in turn, is caused by the opsin decrease. By
contrast, opsin is controlled by retinoids both translationally by
chromophore availability and transcriptionally. The fact that PLC is
eliminated by retinoid deprivation when opsin's promoter drives the PLC
gene is important evidence substantiating retinoid control via opsin's
promoter.}

\introduction

\p{In contrast to the vertebrate situation, retinoids, including
retinoic acid, are neither essential nor toxic in \i{Drosophila}. This
allows strategic manipulations of retinoid deprivation and replacement
to study photoreceptor function. Retinoids not only serve as precursors
of the light absorbing chromophore of rhodopsin, but are also involved
in the regulation of expression of the apoprotein opsin [1] at the co-
or post-translational level [2] and at the transcriptional level [3-5].
A \i{Drosophila} retinoid and fatty acid binding glycoprotein (RFABG)
also shows transcriptional regulation [6].}

\p{Do retinoids regulate other vision genes? To address this issue, we
used retinoid deprivation and replacement to examine regulation of
phospholipase C (PLC). The PLC enzymes are central to many signal
transduction systems [7]. Mutations in the \i{Drosophila} norpA gene,
encoding a homologue of mammalian PLC-\beta, are PLC deficient in heads
[8]. Mutants are named for the no receptor potential phenotype in the
compound eye [9,10] and ocelli [11]. \i{Drosophila} rhodopsin activates
a heterotrimeric G-protein, which, in turn, activates PLC [12]. PLC is
the norpA gene product [13], localized in the rhodopsin-containing
rhabdomere [14]. In the present study, we found that PLC is decreased
nearly half by retinoid deprivation (as assayed by activity analyses and
western blotting) but that the rhabdomeres still show bright emission in
immunocytochemistry. This PLC decrease can be explained by the decrease
in rhabdomere size caused by retinoid deprivation [15]. Importantly,
rhabdomere fluorescence was eliminated by retinoid deprivation in a
\i{Drosophila} stock in which opsin's promoter drives PLC, verifying the
retinoid sensitivity of opsin's promoter.}

\methods

\subsection{Flies}

\p{\i{Drosophila melanogaster} were maintained on standard medium (750
ml H\sub{2}O, 8 g agar, 100 g yellow cornmeal, 40 g brewer's yeast and
100 ml molasses, with mold inhibitors), adequate for full levels of
visual pigment [16,17] and visual function [17,18]. The lutein and
zeaxanthin in yellow cornmeal probably provide adequate carotenoids;
however, to ensure sufficient vitamin A, we supplemented our fly food
with 0.125 mg/ml \beta-carotene, the lowest dose found to maximize
visual sensitivity [17,18] and visual pigment [16,17] for retinoid
deprived flies. Flies were retinoid deprived by rearing from egg to
adult on Sang's medium without any retinoids [19]. Flies were reared on
a 12 h light / 12 h dark (L/D) cycle of fluorescent lighting in a 25
\deg C incubator at an intensity of 140 lux (calibrated with a portable
radiometer / photometer, Ealing model 27-5479, Holliston, MA).}

\p{To determine the effects of various retinoids and nutrients, flies
were reared from egg to adult on Sang's medium containing all-trans
retinoic acid (Sigma Chem. Corp., St. Louis) at 0.125 mg/ml, Beef
Brain-Heart Infusion (Difco, Chicago, IL) at 37 mg/ml or \beta-carotene
at 0.25 mg/ml. Beef Brain-Heart Infusion had been added to Sang's medium
to address the following discrepency: Huber et al. [20] found that
chromophore deprivation in blowflies (\i{Calliphora}) caused by rearing
on beef heart decreased opsin without decreasing its mRNA; our thorough
retinoid deprivation in \i{Drosophila} reared on the more defined Sang's
medium decreased both [3]. Alternatively, a yeast-glucose food was made
consisting of 100 g glucose, 100 g yeast, and 17 g agar in 1000 ml of
water with mold inhibitor. The yeast food was used to address another
discrepency: Ozaki, et al. [2] had found that a similar diet decreased
opsin, indicating that it lacked chromophore precursors, but not opsin's
mRNA; as stated above, Sang's medium eliminated both [3].}

\p{Carrot juice, especially useful for the rapid recovery it elicits
[21,22], \beta-carotene, and retinoic acid were used for "replacement
therapy" [3,5]. Retinoid replacement was with 10 ml of 20% sucrose in
water without (control) or with 0.1 ml \beta-carotene or all-trans
retinoic acid (0.125 mg/ml) dispersed in ethanol in the dark.
Replacement was achieved by transferring deprived flies into a vial with
a sliver of material from a foam stopper soaked with the fluid
[15,23,24] after the flies had been put into an empty vial for 7-9 h to
be certain that they would drink immediately.}

\p{The white-eyed mutant was used throughout, and this was especially
important in immunocytochemistry where fluorescence of eye color
pigments could have otherwise confounded the observations. For PLC
activity analyses, the w\sup{A35} allele was used in keeping with
earlier investigations [14]. The norpA\sup{P24} mutant was used as a
negative control since it is a strong allele [13,25]. Recently, a
functional norpA minigene was constructed by fusing norpA cDNA to the
ninaE promoter [14]. The ninaE (neither inactivation nor afterpotential)
gene encodes Rh1, the opsin which is expressed in R1-6 [26,27].
Transgenic flies with this ninaE-promoter norpA-coding-sequence rescued
norpA\sup{P24} mutants, but only R1-6 receptors were rescued [14]. We
used this stock to contrast retinoid regulation of PLC via the native
norpA promoter vs the ectopic ninaE promoter, the latter known to be
retinoid-sensitive.}

\p{Several ninaE mutants were tested also, including ninaE\sup{oI17}, a
large deletion [26], and ninaE\sup{ora}, a nonsense mutation in the
ninaE coding sequence [28,29]. "Ora" stands for "outer rhabdomeres
absent" [30-32]. Newly-emerged ninaE\sup{ora} flies have R1-6
rhabdomeres that diminish with age [31].}

\subsection{Western blotting}

\p{Procedures were published by Picking et al. [3] and detailed by Lee
[33]. \i{Drosophila} were frozen in liquid nitrogen. Heads were
separated from the bodies, ground and sonicated (Sonic Dismembrator 550,
Fisher Scientific, St. Louis, MO). Homogenates were centrifuged, and the
supernatant drawn off. Protein concentration was determined by BCA
(bicinchoninic acid) determination (Pierce, Rockford, IL). Proteins were
separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) [34]. Typically, 2-4 \mu g of protein were loaded per lane
onto a 4% (w/v) SDS-polyacrylamide stacking gel with a 10% (w/v)
SDS-polyacrylamide separating minigel. Electrophoresis was performed at
150 V for 30-45 min.}

\p{Proteins were transferred to Polyvinylidene difluoride (PVDF-Plus)
membranes (Micron Separations Inc.) using a Trans-Blot Semi-Dry Transfer
Cell (Bio-Rad, Hercules, CA). To block remaining adsorption sites,
membranes were incubated for 1 h at room temperature in Tris-buffered
saline containing 3% (w/v) Carnation non-fat dry milk. Membranes were
rinsed in NP-40 buffer and incubated overnight at room temperature with
the desired antiserum. To remove nonspecifically bound antibodies,
membranes were rinsed multiple times with NP-40 buffer. Membranes were
then incubated for 2 h at room temperature in NP-40 buffer containing
(\sup{125}I)-protein G (500,000 cpm/ml). To remove nonspecifically bound
(\sup{125}I)-protein G, the membrane was rinsed multiple times in
Sarkosyl buffer (pH 7.5), followed by a distilled H\sub{2}O rinse. The
membrane was dried, wrapped in Saran Wrap and exposed to Kodak X-Omat AR
film with a Cronex Lightening Plus intensifying screen at -70 \deg C.}

\p{Western blots were scanned by a Hewlett Packard Scanjet IIc using
Deskscan software. Adobe Photoshop was used to crop, label, and size the
images. An IBM compatible computer was used for all these procedures.}

\subsection{Phospholipase C activity assays}

\p{PLC activity assays were carried out by incubating a 0.1 ml volume of
50 mM Tris-Cl, pH 7.5, 10-7 M CaCl\sub{2}, 0.1 mg/ml BSA, 0.2 mM
phosphatidylionositol (Sigma), 44,000 dpm
phosphatidyl-[\sup{3}H]-inositol 4,5-bisphosphate (New England Nuclear),
and \i{Drosophila} tissue extract for five min at room temperature
essentially as described by [25]. Crude tissue extracts were prepared by
grinding tissue in a buffer of 50 mM Tris-Cl, pH 7.5, 250 mM KCl, 0.05%
sodium deoxycholate, 0.1 mM DTT, and 0.1 mM PMSF, using a teflon pestle
in a 1.5 ml microfuge tube on ice. These homogenates were then
centrifuged briefly at 12,000 x g to remove particulate matter. Protein
concentration in crude homogenates were determined using the Bradford
protein assay (Bio-Rad, Watford, Herts, England) with BSA as a standard
and an appropriate amount of extract (amount empirically determined to
yield linear results with respect to time) added to the reaction
mixture. Reactions were stopped by precipitating in 5% trichloroacetic
acid and quantifying emissions in the supernatant by liquid
scintillation.}

\subsection{Immunocytochemistry}

\p{Procedures were outlined by Shim et al. [6] and detailed by Shim
[35]. Heads were dissected and fixed in either 2.85% paraformaldehyde
and 0.15% glutaraldehyde or 2.85% paraformaldehyde alone in 0.1 M
Sorenson's phosphate buffered solution (pH 7.4) with 2% sucrose for 2.75
h. Fixed heads were washed several times in Sorenson's buffer.
Dehydration with 30%, 50%, 70%, and 90% ethanol was followed by
infiltration in L.R. White acrylic resin (London Resin Company/UK,
Reading, Berkshire, England) overnight. Samples were embedded in the
L.R. White in plastic gel capsules to avoid oxygen, then
heat-polymerized at 55 \deg C.}

\p{Sections of approximately 1 \mu m were cut with glass knives (made
with an LKB-7800 knife maker) using an Ultratome III-type 8800 (LKB
Instruments, Inc., Gaitherburg, MD) and fused to poly-L Lysine coated
glass slides (Fisher Chem. Corp., Pittsburgh, PA). A barrier on the
slide around samples was made with a hydrophobic slide marker (Research
Products International Corp, Mount Prospect, IL). Samples were treated
with sodium ethanolate for 45 min to etch the plastic, washed with
ethanol and washed with deionized water. To minimize autofluorescence,
samples were treated with 1% sodium borohydride, rinsed with deionized
water and equilibrated in PBS (pH 7.4). Blocking was with PBS with 2%
normal goat serum (Sigma) at room temperature for approximately 1 h.}

\p{Anti-PLC antibody was diluted at 1:200. To block nonspecific
staining, primary antibodies were incubated with an equal amount of
crude protein extracted from wild type fly heads after being diluted at
room temperature with antibody in PBS containing 1% goat serum for 30
min to 1 h. FITC (fluorescein isothiocyanate) conjugated goat
anti-rabbit IgG (Sigma) was diluted in PBS containing 1% goat serum at
1:50 ratio and applied. Samples were incubated at 37 \deg C for 1 h,
rinsed in PBS, and rinsed in deionized water. The samples were then
mounted in antifade mountant (2.5 g of DABCO [Sigma] dissolved in 10 ml
of 10X PBS [pH 7.4] and 90 ml of glycerol) and coverslipped. The edge of
the coverslip was sealed using clear finger nail polish (L'Oreal Super
Top Coat) which was found to have very low fluorescence. Slides were
kept in a -20 \deg C freezer.}

\p{Slides were viewed with a standard Leitz Dialux fluorescent
microscope (Leica, Inc, Deerfield, IL). Standard fluorescent images were
captured and printed using an Optronics (TEC-470, Goleta, CA) camera and
Sony RGB video printer (UP-5200MD). Images were transferred to a PC
computer with a Coreco Oculus TCX frame grabber (St. Laurent, Quebec). A
confocal microscope, Bio-Rad MRC-600 fitted with a Kr/Ar laser on a
Nikon Optiphot, was also used to obtain images. Excitation at 488 nm was
supplied by fluorecein optics and images were acquired by Bio-Rad's
COMOS software on an IBM-compatible host computer.}

\p{Image processing was performed using Image Pro Plus and Adobe
Photoshop on the Windows operating system. Images were also analyzed
using the freeware application \hot{\NIHimage}{NIH Image}, version 1.61,
running on the Macintosh OS. All the images were stored in optical disks
by the use of a magneto-optical disk drive (APS technologies, Kansas
City, MO).}

\results

\p{\figref{1} is a western blot that documents our study of the effects
of retinoids upon \i{Drosophila} PLC. The PLC band (130 kDa) [25] is
apparent in flies reared on a retinoid replete medium. Retinoid
deprivation substantially decreased but did not eliminate the band. The
stained bands at lower molecular weights are likely PLC degradative
products. This experiment was replicated four times.}

\p{Activity analysis confirmed the western blot finding in that PLC
activity was significantly reduced but not eliminated by deprivation
relative to vitamin A replete flies reared on normal medium
(\figref{2}). For comparison, data from a positive control (w\sup{A35})
and a negative control (norpA\sup{P24}) are shown.}

\p{The immunocytochemical staining using anti PLC-antibodies confirmed
the earlier demonstration [14] that PLC is localized to the R1-6 + R7
rhabdomeres (\figref{3}{A}). Staining with a different antibody that
does not label rhabdomeres demonstrated that rhabdomeres in white-eyed
\i{Drosophila} have no detectable fluorescence of their own [6].
Rhabdomeres in flies reared on Sang's medium plus \beta-carotene
(\figref{3}{B}), looked like those of flies reared on normal
cornmeal-\beta-carotene food (\figref{3}{A}). Rhabdomeres seemed to
fluoresce as brightly in deprived flies (\figref{3}{C}) as in the flies
grown on cornmeal-\beta-carotene food (\figref{3}{A}) or \beta-carotene
supplemented Sang's medium (\figref{3}{B}). Rhabdomeres from flies
reared on yeast-glucose food (\figref{3}{D}), Sang's medium supplemented
with beef brain-heart infusion (\figref{3}{E}), and Sang's medium plus
retinoic acid (\figref{3}{F}) looked like rhabdomeres of retinoid
deprived flies (\figref{3}{C}) in that they showed equally bright PLC
labeling. Recall that these latter chromophore deprivation manipulations
(\figref{3}{D-F}) had been considered useful since they eliminated opsin
but not ninaE mRNA [3].}

\p{\figref{4}{A} shows normally fluorescing rhabdomeres resulting from
vitamin A replacement, with carrot juice given to deprived flies for 3
days. Replacement with \beta-carotene yielded a similar profile,
specifically with large and bright rhabdomeres, whereas replacement with
retinoic acid resulted in flies with small but bright rhabdomeres like
those in the chromophore deprived flies (\figref{3}{C-F} and data not
shown). \figref{4}{B} shows the lack of fluorescence in the
norpA\sup{P24} mutant. In order to examine the relationship between
functional opsin and PLC expression, some ninaE mutants were tested.
\figref{4}{C,D} show ninaE\sup{oI17} and ninaE\sup{ora} respectively.
The ninaE\sup{oI17} and ninaE\sup{ora} mutants express PLC in R7 and R8
rhabdomeres. Additionally, there is fluorescence outside the center of
the rhabdomere. This may or may not be PLC-based and may be related to
the finding that newly-emerged ninaE\sup{ora} flies do have R1-6
rhabdomeres that diminish with age [31].}

\p{Vitamin A manipulations were extended to the transgenic flies rescued
by a chimeric norpA minigene driven by the ninaE gene promoter. The TI-6
allele [14] was reared on either regular or deprivational medium. Since
the ninaE promoter drives expression into R1-6, PLC labeling is only
unequivocal in R1-6 rhabdomeres in retinoid-replete flies
(\figref{4}{E}); R7/8 fluorescence is at about the level of background
(non-rhabdomeric tissue). Vitamin A deprivation (\figref{4}{F})
eliminates the PLC expression seen in vitamin A-replete TI-6. This
result is a critical piece of evidence that retinoids control
transcription at the level of the opsin gene promoter since PLC, unlike
rhodopsin, does not utilize retinoids translationally.}

\discussion

\p{Since retinoids control transcription of opsin and RFABG genes, it
was crucial to test whether other phototransduction molecules might be
regulated by retinoids. Our results support the alternative, that
rhodopsin reduction causes secondary decreases in the transduction
machinery. The PLC activity and the PLC band were reduced by retinoid
deprivation. PLC is the second molecule downstream from rhodopsin in the
phototransduction cascade. Retinoid deprivation reduces PLC activity to
63% of that of the vitamin A replete control. Furthermore, the vitamin A
replete control has an activity very close to another control
(w\sup{A35}) in an experiment that verifies the expected zero activity
in norpA\sup{P24} mutants. Although it was not quantified, the western
blot also suggests that vitamin A deprivation decreases PLC to 1/2 or
1/3. In summary, activity analyses and western blots are consistent with
deprived flies having smaller rhabdomeres [15].}

\p{Immunocytochemistry reveals that retinoid deprivation does not
eliminate PLC expression in rhabdomeres. Deprived flies (\figref{3}{C})
seemed to have rhabdomeres as brightly labeled as those of flies reared
on retinoid replete foods (\figref{3}{A,B}). Deprived flies
(\figref{3}{C}) were just like flies deprived of chromophore precursors
but not deprived of other activators of opsin and RFABG expression
(\figref{3}{D-F}).}

\p{The impression from this study's immunocytochemistry is that
rhabdomeres from retinoid and chromophore deprived flies are reduced in
size (compare \figref{3}{C-F} with retinoid replete controls,
\figref{3}{A,B}). However the present study is not optimum to prove this
point due to the low magnification of this light microscopy and the fact
that rhabdomeres taper in the distal to proximal direction; the plane of
section could not be controlled. However, thorough morphometric
comparisons from high magnification electron micrographs where the plane
of section from which measurements were taken was controlled showed that
deprived flies have smaller rhabdomeres than replete and carrot juice
replaced flies [15].}

\p{It seems likely that the reduction of PLC, as detected in activity
analyses and western blots, is secondary to lack of visual pigment. PLC
was also found in other parts of the \i{Drosophila} body, including
thorax, abdomen, and legs [25]. This suggests that PLC may be involved
in signaling pathways not associated with vision. If retinoids exert
their effects on PLC by regulating opsin, it seems unlikely that
retinoid deprivation would reduce PLC levels in these other parts of the
body, and this hypothesis could be tested.}

\p{Vitamin A manipulations on the the transgenic flies (TI-6) showed
that, when the norpA coding sequence is driven by the ninaE promoter,
the gene product is affected by retinoid deprivation. This is one of the
strongest pieces of evidence substantiating the earlier finding [3-5]
that retinoids control ninaE transcription at the promoter level. By
contrast, the results of this study suggest that retinoids do not
directly affect the transcription of the PLC gene at the level of the
PLC gene's promoter. This finding is consistent with the differences in
the norpA promoter from those of many other receptor-specific genes
[36].}

\acknowledgements

\p{We thank Dr. D. Sherry (Department of Anatomy, University of Houston)
for advice on immunocytochemistry. We thank R. D. Lee, W. D. Picking,
and W. L. Picking for assistance with the western blotting. We thank
Jonathan Grimm of Saint Louis University's Instructional Media Center
and Prof. Martin L. Katz (Department of Ophthalmology, University of
Missouri at Columbia) with assistance in producing the figures.}

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\endreferences

}

\beginfigures

\figfile{1}{
\figtitle{1}{Western blot probed with anti-phospholipase C antibodies}

\p{The norpA gene encodes for PLC (130 kDa) and is necessary for proper
phototransduction. PLC is greatly reduced in vitamin A deprived adults
as compared to controls.}

\ctr{\jpgimage{1}{112}{244}{6}}

}

\figfile{2}{
\figtitle{2}{PLC activity analysis}

\p{PLC activity analysis comparing white-eyed (w\sup{A35}) controls with
norpA\sup{P24} mutants. Vitamin A deprivation decreases PLC activity
relative to vitamin A replete controls.}

\ctr{\gifimage{2}{423}{274}{8}}

}

\figfile{3}{
\figtitle{3}{Immunocytochemistry for PLC}

\p{Immunocytochemistry for PLC in white-eyed, otherwise wild-type flies,
retinoid replete, and retinoid or chromophore deprived. \panel{A}:
standard diet, bright fluorescence in R1-7 rhabdomeres, standard
fluorescence micrograph. \panel{B}: rearing on Sang's medium
supplemented with \beta-carotene from egg to adult (one generation)
showing the same bright fluorescence as for normal diet as expected,
standard fluorescence micrograph. \panel{C}: retinoid deprived; note
that rhabdomeres fluoresce brightly but that the size of rhabdomeres may
be decreased, standard fluorescence micrograph. \panel{D}: rearing on
yeast-glucose diet, showing same features as for deprived diet, standard
fluorescence micrograph. \panel{E}: rearing on Sang's medium
supplemented with Beef Brain-Heart Infusion from egg to adult (one
generation) showing the same features as for retinoid deprived diet,
standard fluorescence micrograph. \panel{F}: rearing with retinoic acid
supplemented Sang's medium for one generation showing same features as
for deprived diet, confocal micrograph.}

\ctr{\jpgimage{3}{400}{600}{72}}

}

\figfile{4}{
\figtitle{4}{Fluorescence micrographs with PLC antibody}

\p{Fluorescence micrographs prepared with PLC antibody, repletion
manipulations, and selected mutants. \panel{A}: 3 days carrot juice
replacement of deprived flies, showing the recovery of rhabdomere size,
confocal micrograph. \panel{B}: negative control, the mutant white-eyed
norpA\sup{P24}, standard diet, standard fluorescence micrograph, no
rhabdomere fluorescence detected as expected. \panel{C}: the white-eyed
mutant ninaE\sup{oI17}, standard diet, confocal micrograph; note normal
level of PLC detected in R7 rhabdomeres. \panel{D}: the white-eyed
mutant ninaE\sup{ora}, standard diet, confocal micrograph; note normal
level of PLC detected in R7. \panel{E}: the transformant TI-6 rescued
from white-eyed norpA\sup{P24} by norpA minigene driven by ninaE
promoter, showing PLC detected in only R1-6 rhabdomeres, standard diet,
standard fluorescence micrograph. \panel{F}: the transformant TI-6
rescued from white-eyed norpA\sup{P24} by norpA minigene driven by ninaE
promoter, showing no fluorescence emission, retinoid deprived diet,
standard fluorescence micrograph.}

\ctr{\jpgimage{4}{400}{600}{87}}

}