Division of Biology, University of California, San Diego. La Jolla, CA 92093, USA
* Author for correspondence (e-mail: azelhof{at}biomail.ucsd.edu)
Accepted 5 June 2003
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SUMMARY |
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Key words: Homeodomain, Photoreceptor, Rhabdomere, Phototransduction, Terminal differentiation, Hazy, Drosophila
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INTRODUCTION |
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The Drosophila compound eye consists of an ordered array of 800
individual units known as ommatidium. Each ommatidium contains 20 cells, which
include the eight photoreceptor neurons, six outer (R1-R6) and two inner (R7,
R8) cells. The adult Drosophila eye develops from an epithelial
monolayer, the eye imaginal disc. Specification of the photoreceptor neurons
begins during the larval third instar and proceeds in a posterior to anterior
wave across the eye imaginal disc to create each individual ommatidium
(Ready et al., 1976;
Wolff and Ready, 1993
). The
early specification of photoreceptor cells has been well studied
(Heberlein and Treisman, 2000
)
and despite the obvious morphological differences between invertebrate and
vertebrate eyes, many of the necessary transcription factors required for eye
specification are conserved (Ashery-Padan
and Gruss, 2001
; Kumar and
Moses, 2001
). The best example of functional conservation is the
role of Pax6 in eye development. Mutations of PAX6 in humans
(Aniridia), mice (Small eye) and Drosophila
(eyeless and twin of eyeless) all lead to severe defects in
eye development (Czerny et al.,
1999
; Hill et al.,
1991
; Kronhamn et al.,
2002
; Quiring et al.,
1994
; Ton et al.,
1991
).
Upon specification, photoreceptor neurons immediately send axonal
projections into the optic lobe of the Drosophila brain. The outer
photoreceptor cells project into the lamina whereas the inner photoreceptor
cells (R7, R8) send axonal projections deeper into the optic lobe and
terminate in the medulla (Meinertzhagen
and Hanson, 1993; Wolff et
al., 1997
). Furthermore, the differentiation of the photoreceptor
neurons is not complete until 4 days later, at the end of metamorphosis. One
unique feature of vertebrate and Drosophila photoreceptor neurons is
the creation of a specialized light-sensing organelle on the apical cell
surface. In Drosophila, the rhabdomere is the photoreceptor
light-sensing organelle and is the functional equivalent of the outer segment
of vertebrate rod and cone cells. Each rhabdomere consists of 60,000
tightly-packed microvilli, each 50 nm in diameter and 1-2 µm in length
(Kumar and Ready, 1995
;
Leonard et al., 1992
). This
results in a tremendous increase in surface area to house the tens of millions
of rhodopsin molecules and associated signaling molecules that are responsible
for the detection of light.
As the rhabdomere develops, the signaling molecules required for the
detection and translation of light into a receptor potential are expressed and
localized to the rhabdomere. In Drosophila, the activation of
rhodopsin leads to the activation of Phospholipase C (PLC) via a coupled
heterotrimeric G protein. PLC catalyzes the breakdown of phosphatidyl
4,5-bisphosphate [PtdIns(4,5)P2] into the two
intracellular messengers inositol triphosphate
[Ins(1,4,5)P3] and diacylglycerol (DAG). This reaction
leads to the opening of light sensitive cation-selective channels (TRP, TRPL
and TRP) and the generation of a depolarizing receptor potential
(Hardie and Raghu, 2001
;
Zuker, 1996
).
We have isolated a mutation, hazy, that represents a null allele of the photoreceptor specific homeodomain gene Pph13. Pph13hazy mutants are adult viable and each ommatidium contains the appropriate set of photoreceptor and accessory cells. However, Pph13hazy mutants have a severe decrease in light sensitivity. We demonstrate Pph13 is necessary for the terminal differentiation of photoreceptor cells, in particular the morphogenesis of the photoreceptor rhabdomere as well as for the expression of phototransduction signaling proteins.
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MATERIALS AND METHODS |
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EM analysis
Electron microscopy of Drosophila eyes were carried out as
previously described (Baker et al.,
1994).
Immunofluorescence stainings
Developing whole retinas were dissected at the appropriate time and
processed as described previously (Fan and
Ready, 1997; Zelhof et al.,
2001
). Creation and processing of frozen thin sections were
performed as described by Tsunoda et al.
(Tsunoda et al., 2001
). The
primary antibodies used were rabbit anti-TRP, Rh1, TRPL, INAD, Amphiphysin
(Zelhof et al., 2001
), mouse
anti-eye Gß (Yarfitz et al.,
1991
), Chaoptin (Van Vactor et
al., 1988
), 21A6 (Zipursky et
al., 1984
), sheep anti-Bifocal
(Bahri et al., 1997
), rabbit
anti Rac1 was a gift from Dr L. Luo. If not noted, the primary antibody was
created in Dr Charles Zuker's laboratory. Rhodamine conjugated Phalloidin
(Molecular Probes) was used for the detection of Actin. FITC and
Red-X-conjugated secondary antibodies were obtained from Jackson
ImmunoResearch Laboratories.
Antibody production
The rabbit polyclonal antibody 677 was created by injecting rabbits with a
GST-fusion protein representing amino acids 219-357 of Pph13 and sera was used
at a concentration of 1:500.
Western analysis
Tissue was placed in extraction/binding buffer (EB; 100 mM KCl, 20 mM
HEPES, 5% glycerol, 10 mM EDTA, 0.1% Triton X) with the proteinase inhibitor
cocktail mix Complete (Roche Diagnostics), homogenized and sonicated. The
mixture was spun and the supernatant was collected and an equal volume of
2x sample/loading buffer was added. All extracts were resolved by
SDS-PAGE and then transferred to Immobilon-P (Millipore). Protein detection
was done as previously described (Baker et
al., 1994). Primary antibodies used in this study were rabbit
anti-Rh1, Rh4, TRP, TRPL, TRP
(Xu
et al., 2000
), NinaC (Porter
et al., 1992
), INAD, PLC, G
, G
, RdgC, NinaA,
GC
1, Arr2 and mouse anti- eye-Gß
(Yarfitz et al., 1991
). If not
noted, the primary antibody was created in Dr Charles Zuker's laboratory.
DNA constructs
The expressed sequence tag (EST) GH01528 representing the cDNA for
Pph13 was obtained from Berkeley Drosophila Genome Project
(via Research Genetics). UAS and heat shock constructs: a
BglII/SpeI fragment from GH01528 was cloned into the
BglII/XbaI sites of pUAST
(Brand and Perrimon, 1993) and
the BglII/XbaI sites of pCaSpeR-hs
(Thummel et al., 1988
) and
both were transformed into flies. pcDNA3 construct: a
BglII/XhoI fragment from the GH01528 EST was cloned into the
BamHI/XhoI sites pcDNA3 vector (Invitrogen). The p36 plasmid
(Holloway et al., 1995
) was
obtained from Dr G. Rosenfeld and contains the rat prolactin promoter inserted
into pGL2 (Promega). One copy of wild-type and mutated eye Gß
upstream regions were cloned into the NheI site. The mutated eye
Gß enhancer used in the transfection assays contained the following
sequence 5'-ggcTAATccaATCCgctAGGTgcATTAccg-3' (the uppercase
letters represent the positions of the palindromic Pph13 binding half sites).
eye Gß-GFP was constructed by placing 424 nucleotides
that are prior to the first ATG of eye Gß into the Green Pelican
vector (Barolo et al., 2000
).
GST fusion protein consisted of amino acids 1-70, representing the
homeodomain, cloned into pGEX-4T1 (Pharmacia).
EMSA assays
Electrophoretic mobility shift assays were performed as described
previously (Zelhof et al.,
1995). In vitro transcribed and translated protein was generated
by the TNT Coupled Reticulocyte Lysate System (Promega) according to
manufacturer's instructions. Upon annealing of the complimentary
oligonucleotides, the oligonucleotides were radiolabeled using a Klenow
fill-in reaction. For competition experiments both a 10- and 50-fold excess of
cold competitor were used.
Reverse transcriptase and PCR reactions
The following primer sets were used for first-strand synthesis and PCR
amplification: Arr1, 5-GAATAAATGGTAGCTCAGCGC-3,
5-CTACATGAACAGGCGTGATTT-3/5-TGTGTCTTTGCGCTTGATATC-3; InaD,
5-TAGAATCATGGTCACTACGCC-3,
5-CAGGCCAAGAACAAGTTCAAC-3/5-TGTTACATCCTGATTAACGGC-3; NinaE,
5-GGTATTCAGTGGTGTAAGGCC-3,
5-TGGCGTGGTGATCTACATATT-3/5-GACATTCATCTTCTTGGCCTG-3; Trpl,
5-AGGGAGCGCATTATATTATCA-3,
5-GAACTACGATCCGCAGATGTC-3/5-CATTTCTCGCGTGGTATGTAA-3; Eye Gß,
5-AAGTGATGCGGTTCTCGT, 5-TGTGCCCAAGATTCGATT-3/5-GGTTCATACTGGGCGATT-3. Total RNA
was isolated from cn bw and Pph13hazy homozygous
heads using Rneasy Mini Kit (Qiagen) and first strand synthesis was
accomplished using the Thermoscript RTPCR System (Invitrogen) and each mRNA
target had 35 rounds of PCR amplification.
Transfection assays
Twenty-four hours before transfection NIH 3T3 cells were seeded in 96-well
plates. Cells were grown in DMEM supplemented with calf serum. Cells were
transfected by using polyfect (Qiagen) according to manufacturer's
instructions. Transfection assay mixtures contained 20 ng of reporter with or
without 40 ng of pcDNA-Pph13 per well and assayed for luciferase
activity approximately 44 hours post transfection using the Promega Luciferase
Assay Kit and a TopCount Scintillation Counter for light detection. Sixteen to
48 wells were transfected for each DNA combination tested per experiment.
Three independent transfection experiments were performed per condition
assayed.
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RESULTS |
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To identify genes required for photoreceptor cell differentiation, we
screened for the presence or absence of the deep pseudopupil (DPP) in
Drosophila adult eyes
(Franceschini, 1972;
Franceschini and Kirschfeld,
1971
). The presence of the DPP is an indication of the overall
integrity of the photoreceptor cells and their associated rhabdomeres. Such
screens have been effective in isolating mutations that affect eye structure
and development (Baker et al.,
1992
; Banerjee et al.,
1987
; Pichaud and Desplan,
2001
). To limit our search for those mutants that affect aspects
of differentiation and not specification, we excluded any mutants that had
incorrect external morphology, particularly rough or irregular shaped eyes.
Consequently, we screened 6,000 viable second chromosome EMS mutated lines,
generated from 38,000 F3 lines (E.K. and C. S. Zuker, unpublished) for the
absence of a DPP and isolated 33 mutant stocks which represent 18
complementation groups (data not shown).
A key component of eye function is the organization of the phototransduction machinery into the rhabdomere. As import of the signaling components occurs late in photoreceptor differentiation, we reasoned that flies that lacked a DPP and could not correctly respond to light would be the best candidates for mutants defective in photoreceptor terminal differentiation. Using electroretinogram assays (ERGs) that measure the capacity of photoreceptor cells to convert light into a receptor potential, we tested our collection of 33 mutants for those that had defects in light perception. Our results indicated that among the group that had irregular ERGs, one had a severe deficiency in the detection of light. This mutant responds reproducibly only to long durations of high intensity light. The characteristic on/off transients of wild-type responses are undetectable (Fig. 1C) and by 10 days post eclosion, the mutants appear to lose all responses to light.
|
Pph13hazy encodes a paired-class homeobox gene
As a first step in understanding the molecular mechanisms leading to the
severe decrease in light sensitivity and rhabdomeric defects in hazy
mutants, we isolated the responsible gene. Recombination and deficiency
mapping placed hazy in a small genomic interval of 70 kB of DNA at
21C7-21D1 on the left arm of the second chromosome
(Fig. 2). Sequencing of
candidate genes from this interval in both the mutant and isogenic wild-type
flies revealed a single base change in the open reading frame of the
previously identified homeodomain gene Pph13
(Dessain and McGinnis, 1993).
The nucleotide change results in the introduction of a premature termination
codon at amino acid position 58 (W to Stop). The premature stop is within the
homeodomain and therefore would also effectively eliminate any DNA binding
capacity. More importantly, reintroduction of a wild-type full-length Pph13
cDNA, under the control of a heat shock promoter, by P-element mediated germ
line transformation fully rescues all Pph13hazy phenotypes
(Fig. 1C and data not shown).
Rescue is obtained without heat shock, suggesting only a basal level of
expression is required for proper photoreceptor cell morphogenesis.
|
|
If the malformed rhabdomeres were the result of improper morphogenesis, an
examination of the spatial and temporal appearance of rhabdomeric proteins
during the steps of microvilli formation would not only define the
developmental time of the defect but also identify possible Pph13
transcriptional targets. The exact mechanism of microvilli initiation and
elongation are not known but there are a few protein markers that can be used
to gauge the progression and structure of the developing rhabdomere. At 48 hrs
after pupariation (APF), when the initial microvilli folds are forming, we
find the accumulation of rhabdomeric proteins, Chaoptin
(Van Vactor et al., 1988),
Bifocal (Bahri et al., 1997
),
Amphiphysin (Zelhof et al.,
2001
), 21A6 (Zipursky et al.,
1984
), in mutants is indistinguishable from wild type (data not
shown). At 72 hrs APF, the progression of rhabdomere development and
localization of rhabdomeric proteins appears to be normal in
Pph13hazy mutants (Fig.
4). However, at 96 hours APF, the staining of F-actin clearly
reveals a severe lack of growth and elongation of the rhabdomere microvilli in
mutants (Fig. 4H). Staining for
F-actin demonstrates that the mutant rhabdomeres are not full and round when
compared with their wild-type counterparts. In agreement with our
immunofluorescent studies, EM ultrastructure analysis revealed rhabdomere
biogenesis is not proceeding correctly by 72 hours APF. The microvilli of
mutant photoreceptor cells are misaligned and loosely packed, as seen in adult
mutant rhabdomeres (Fig. 5C,D).
Whereas at 60 hours APF no discernible ultrastructure defects are observed
between wild-type and mutant photoreceptor cells
(Fig. 5A,B), suggesting Pph13
function is crucial between 60 and 72 hours APF for proper rhabdomere
formation.
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The majority of the proteins we examined are present in Pph13hazy mutant photoreceptor cells but our Western analysis did not address the question of whether these proteins are localized correctly in photoreceptor cells. To check for proper subcellular localization, we examined the expression of proteins via immunofluorescence techniques on frozen thin sections of adult eyes. Our results confirm the absence of TPRL and eye Gß in Pph13hazy mutants. Our western blot data suggested an absence of Arr1 expression but our immunofluorescent data demonstrates that Arr1 levels are only diminished and not absent in mutant photoreceptors. We can detect Arr1 in mutant R7/R8 photoreceptor cells and in the outer photoreceptor cells (R1-R6) the levels of expression are probably below our level of detection, suggesting Pph13 is only required for full expression Arr1 in all photoreceptor cells (Fig. 7). Last, molecules that show normal or reduced levels of expression, such as with INAD or TRP, show correct subcellular localization to the malformed rhabdomeres in Pph13hazy mutants (Fig. 7D). Overall, these results suggest Pph13 is downstream of the genes required for eye specification and Pph13 transcriptional targets are necessary for the proper detection of light.
Eye Gß is a transcriptional target of Pph13
Our results suggest that Pph13 exerts its affect on photoreceptor
differentiation by regulating transcription. If this is the case, we predict
that the mRNAs for the missing proteins would not be detectable. RT-PCR
reactions confirm that the transcripts for trpl and eye
Gß are absent while the transcripts for arr1 are present
(Fig. 8A). We also predict that
if trpl or eye Gß represents Pph13 transcriptional
targets, potential binding sites for Pph13 should exist in their promoter
regions. The consensus DNA binding site for a Paired class homeodomain protein
containing a glutamine at amino acid position 50 of the homeodomain is a
palindrome of TAAT separated by three nucleotides
(Fortini and Rubin, 1990;
Wilson et al., 1993
;
Wilson and Desplan, 1995
).
Scanning the transcriptional units of eye Gß and trpl
revealed one element containing strong potential binding sites for Pph13
upstream of the transcriptional start of eye Gß. Within a span
of 25 nucleotides, we find two palindromes spaced by three nucleotides and a
third overlapping palindrome separated by two nucleotides (see
Fig. 8B).
|
Based on our findings, we would predict that Pph13 acts as an activator and
not a repressor of gene transcription. To test whether Pph13 has the ability
to activate transcription we created a reporter construct containing the
eye Gß enhancer, nucleotides -323 to -105, upstream of the
minimal rat prolactin promoter controlling luciferase expression
(Holloway et al., 1995). In
transient transfection assays, upon the co-transfection of Pph13 we see an
average of a thousand fold activation of transcription specifically from the
reporter containing the eye Gß enhancer as compared to the
parental vector. Mutation of the palindromic binding sites within the response
element eliminates the transcriptional activation seen with the addition of
Pph13 (Fig. 8C), confirming the
role of Pph13 as a potential activator of photoreceptor specific gene
expression.
To further prove a direct regulation of eye Gß by Pph13, we asked whether a transgenic construct containing the eye Gß enhancer is expressed in photoreceptor cells and requires Pph13 for expression. As such, we placed GFP immediately downstream of first 424 nucleotides that are prior to the first ATG of eye Gß. As the expression of GFP is low in all of our transgenic lines and combined with the auto-fluorescence of pigmented eyes, we could not say with absolute certainty that this genomic region of eye Gß limits GFP expression only to photoreceptor cells. However, using western blot analysis, we detect GFP only in head extracts; more importantly, GFP expression is dependent on the presence of Pph13. When the transgenic construct is recombined into a Pph13hazy mutant background, GFP expression is greatly diminished (Fig. 8D).
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DISCUSSION |
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Pph13hazy mutants have two striking defects: the
ability of the photoreceptor cell to detect light and the biogenesis of the
light sensing organelle, the rhabdomere. Are the two phenotypes connected? We
cannot eliminate the possibility that the malformed rhabdomeres are
contributing to the inability of these mutants to detect light or vice versa.
However, the severity of the rhabdomere defect cannot be solely responsible.
For example, from our own screen we have isolated mutants that result in
malformed rhabdomeres equal to those seen in Pph13hazy but
have a normal ERG (data not shown). In addition, the loss of Chaoptin and
NinaC both result in a considerable loss of rhabdomeric size and rhodopsin
levels but they have a better response to light then
Pph13hazy mutants
(Matsumoto et al., 1987) (data
not shown).
In addition, our results demonstrate that Pph13 is required for the
transcription of phototransduction proteins. Clearly, trpl,
trp and Gß are not expressed in mutant photoreceptor
cells, and the absence of Pph13 affects the full expression of several other
signaling components. This is clearly observed with Arr1 expression. First,
our data demonstrates that in the inner (R7/R8) wild-type photoreceptor cells
have a considerable higher expression of Arr1 when compared with the outer
photoreceptor cells (R1-R6). Second, the loss of Pph13 does not eliminate
expression of Arr1 in photoreceptor cells. Arr1 expression can be seen in
mutant R7/R8 photoreceptor cells and the lack of signal in the outer
photoreceptors is not due to the absence of Arr1 expression but rather the
fact that these cells start out with lower levels of Arr1. Taken together,
while all of the detected protein aberrations can explain the severe loss of
light sensitivity, our results do not eliminate the possibility of a yet
unidentified molecule required for proper phototransduction. Selective rescue
and identification of any other missing components will be needed to explain
the complete molecular mechanisms responsible for the decrease in light
sensitivity.
The molecular mechanisms for rhabdomere biogenesis are for the most part unknown. Nonetheless, our data do provide a few insights into rhabdomere biogenesis.
Our results demonstrate that Pph13 is required for the generation or execution of a late acting signal necessary for the elaboration and growth of the microvilli into a rhabdomere. Immunofluorescent and EM analyses demonstrate that the defects observed in Pph13hazy mutants are the result of a developmental flaw and not of retinal degeneration. The disorganized rhabdomeres do not show any of the characteristic signs of degeneration and more significantly we detect a clear halt in rhabdomere development by 72 hours APF. In addition, by all measurements, the early events (36 to 60 hours APF) of rhabdomere biogenesis occur normally.
Our data also indicate the failure of growth is not due to the improper
localization or delivery of proteins to the rhabdomere. For example, Chaoptin,
which is required for the cross-linking of microvilli still localizes to the
developing rhabdomere before and after (data not shown) the rhabdomere has
stalled in development. In addition, the proteins composing the
phototransduction machinery, especially rhodopsin which has a role in
phototransduction and in maintaining the structural integrity of the
rhabdomere (Kumar et al.,
1997; Kumar and Ready,
1995
), are imported and stabilized within the malformed
rhabdomeres. We also do not observe the characteristic expansion of the
endoplasmic reticulum associated with defects in rhabdomeric protein cell
trafficking (Baker et al.,
1994
; Colley et al.,
1995
; Sang and Ready,
2002
).
What is responsible for the flaw in rhabdomere biogenesis? Most notably for
a transcription factor believed to be necessary for the activation and not
repression of gene transcription, we see a grossly abnormal accumulation of
Rac1 in Pph13hazy mutant photoreceptor cells. However, the
presence of Rac1 is in agreement with the fact that the terminal web does form
in Pph13hazy mutants. Given that the exact function of
Rac1 has not been resolved in rhabdomere biogenesis and that small Rho GTPases
have been implicated in mediating signals required for actin reorganization
(Etienne-Manneville and Hall,
2002; Hall, 1998
),
future experiments will address the function of Rac1 in photoreceptor terminal
differentiation and determine how the misregulation of Rac1 accumulation and
activity may be contributing to the Pph13hazy rhabdomere
phenotypes.
Homeodomain proteins and photoreceptor terminal differentiation
Our molecular cloning of Pph13hazy has identified
another homeodomain gene required for photoreceptor morphogenesis. Previous
reports have established or implicated eyeless (Pax6),
orthodenticle (otd) and Onecut homeodomain genes in
eye development (Nguyen et al.,
2000; Quiring et al.,
1994
; Vandendries et al.,
1996
). What is the relationship between these various homeodomain
transcription factors and how do they coordinate photoreceptor terminal
differentiation? Numerous possibilities exist in which each of these
transcription factors could control a unique subset of molecular mechanisms
required for a functional photoreceptor cell; alternatively, they could act in
concert on the same genes to promote differentiation. To eliminate or confirm
any one of these possibilities would be premature and further extensive
characterization of each of these genes in photoreceptor development is
necessary.
Nevertheless, our preliminary data does allow for some speculation. First,
it is clear that eyeless is required for photoreceptor cell
specification and without it a photoreceptor cell a gene like Pph13
could not function. Besides its early role in photoreceptor cell
specification, eyeless is also necessary for rhodopsin expression
(Papatsenko et al., 2001;
Sheng et al., 1997
) and
superficially, characterization of the late transcriptional targets of Eyeless
and Pph13 appear to be different. First, Pph13 is absolutely required for
trpl, trp
and Gß expression but is not necessary
for rhodopsin expression. This result would suggest that once a cell has
committed to a photoreceptor cell fate, both Eyeless and Pph13 have separate
and distinct molecular pathways that contribute to photoreceptor
differentiation.
However, comparison of otd and Pph13 mutants suggest a
more complex mode of coordination for photoreceptor differentiation. First,
the rhabdomere defects observed otduvi and
Pph13hazy mutants are similar
(Vandendries et al., 1996). In
each case, the defects appear not to be the result of degeneration but a
failure in their biogenesis. The rhabdomere terminal web does form in both
cases but the overall size and morphology are abnormal. Both Otd and Pph13 are
required in the same developmental time window for rhabdomere morphogenesis,
but neither is necessary for the expression of the other (data not shown).
Whether Otd and Pph13 represent two parallel pathways directing the expression
of the same genes or two distinct pathways with different genetic targets to
promote rhabdomere biogenesis will require further investigation. In addition,
they do not share a defect in phototransduction. otduvi
photoreceptor cells exhibit normally ERGs and we do not detect the loss of
phototransduction proteins downstream of rhodopsin as seen in Pph13 mutants
(data not shown). Clearly, Pph13 is responsible for two aspects of
photoreceptor cell differentiation: phototransduction and rhabdomere
morphogenesis.
Given that the molecular mechanisms orchestrating the differentiation of photoreceptor cells remain largely undefined, the goal of our genetic approach was to isolate genes required for photoreceptor terminal differentiation. Our work with Pph13hazy has shed some light on the regulation of this process. However, additional studies that combine the accessibility and genetic amenability of Drosophila eye development, with whole genome expression profiling techniques in both wild-type and Pph13hazy mutant photoreceptor cells, should identify additional transcriptional targets necessary for photoreceptor cells to achieve and maintain a functional state.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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Ashery-Padan, R. and Gruss, P. (2001). Pax6 lights-up the way for eye development. Curr. Opin. Cell Biol. 13,706 -714.[CrossRef][Medline]
Bahri, S. M., Yang, X. and Chia, W. (1997). The Drosophila bifocal gene encodes a novel protein which colocalizes with actin and is necessary for photoreceptor morphogenesis. Mol. Cell. Biol. 17,5521 -5529.[Abstract]
Baker, E. K., Colley, N. J. and Zuker, C. S. (1994). The cyclophilin homolog NinaA functions as a chaperone, forming a stable complex in vivo with its protein target rhodopsin. EMBO J. 13,4886 -4895.[Abstract]
Baker, N. E., Moses, K., Nakahara, D., Ellis, M. C., Carthew, R. W. and Rubin, G. M. (1992). Mutations on the second chromosome affecting the Drosophila eye. J. Neurogenet. 8,85 -100.[Medline]
Banerjee, U., Renfranz, P. J., Pollock, J. A. and Benzer, S. (1987). Molecular characterization and expression of sevenless, a gene involved in neuronal pattern formation in the Drosophila eye. Cell 49,281 -291.[Medline]
Barolo, S., Carver, L. A. and Posakony, J. W. (2000). GFP and beta-galactosidase transformation vectors for promoter/enhancer analysis in Drosophila. Biotechniques 29,726 , 728, 730, 732.[Medline]
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Chang, H. Y. and Ready, D. F. (2000). Rescue of
photoreceptor degeneration in rhodopsin-null Drosophila mutants by activated
Rac1. Science 290,1978
-1980.
Colley, N. J., Cassill, J. A., Baker, E. K. and Zuker, C. S. (1995). Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration. Proc. Natl. Acad. Sci. USA 92,3070 -3074.[Abstract]
Czerny, T., Halder, G., Kloter, U., Souabni, A., Gehring, W. J. and Busslinger, M. (1999). twin of eyeless, a second Pax-6 gene of Drosophila, acts upstream of eyeless in the control of eye development. Mol. Cell 3, 297-307.[Medline]
Dessain, S. and McGinnis, W. (1993). Drosophila homeobox genes. Adv. Dev. Biochem. 2, 1-55.
Dolph, P. J., Ranganathan, R., Colley, N. J., Hardy, R. W., Socolich, M. and Zuker, C. S. (1993). Arrestin function in inactivation of G protein-coupled receptor rhodopsin in vivo. Science 260,1910 -1916.[Medline]
Dolph, P. J., Man-Son-Hing, H., Yarfitz, S., Colley, N. J., Deer, J. R., Spencer, M., Hurley, J. B. and Zuker, C. S. (1994). An eye-specific G beta subunit essential for termination of the phototransduction cascade. Nature 370, 59-61.[CrossRef][Medline]
Etienne-Manneville, S. and Hall, A. (2002). Rho GTPases in cell biology. Nature 420,629 -635.[CrossRef][Medline]
Fan, S. S. and Ready, D. F. (1997). Glued
participates in distinct microtubule-based activities in Drosophila eye
development. Development
124,1497
-1507.
Fortini, M. E. and Rubin, G. M. (1990). Analysis of cis-acting requirements of the Rh3 and Rh4 genes reveals a bipartite organization to rhodopsin promoters in Drosophila melanogaster. Genes Dev. 4,444 -463.[Abstract]
Franceschini, N. (1972). Pupil and pseudopupil in the compound eye of Drosophila. In Information Processing in the Visual Systems of Arthropods (ed. R. Wehner), pp.75 -82. Berlin: Springer-Verlag.
Franceschini, N. and Kirschfeld, K. (1971). Pseudopupil phenomena in the Drosophila compound eye. Kybernetik 9,159 -182.[Medline]
Goriely, A., Mollereau, B., Coffinier, C. and Desplan, C. (1999). Munster, a novel paired-class homeobox gene specifically expressed in the Drosophila larval eye. Mech. Dev. 88,107 -110.[CrossRef][Medline]
Halder, G., Callaerts, P. and Gehring, W. J. (1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267,1788 -1792.[Medline]
Hall, A. (1998). Rho GTPases and the actin
cytoskeleton. Science
279,509
-514.
Hardie, R. C. and Raghu, P. (2001). Visual transduction in Drosophila. Nature 413,186 -193.[CrossRef][Medline]
Heberlein, U. and Treisman, J. E. (2000). Early retinal development in Drosophila. Results Probl. Cell Differ. 31,37 -50.[CrossRef][Medline]
Hill, R. E., Favor, J., Hogan, B. L., Ton, C. C., Saunders, G. F., Hanson, I. M., Prosser, J., Jordan, T., Hastie, N. D. and van Heyningen, V. (1991). Mouse small eye results from mutations in a paired-like homeobox-containing gene. Nature 354,522 -525.[CrossRef][Medline]
Holloway, J. M., Szeto, D. P., Scully, K. M., Glass, C. K. and Rosenfeld, M. G. (1995). Pit-1 binding to specific DNA sites as a monomer or dimer determines gene-specific use of a tyrosine-dependent synergy domain. Genes Dev. 9,1992 -2006.[Abstract]
Kronhamn, J., Frei, E., Daube, M., Jiao, R., Shi, Y., Noll, M. and Rasmuson-Lestander, A. (2002). Headless flies produced by mutations in the paralogous Pax6 genes eyeless and twin of eyeless. Development 129,1015 -1026.[Medline]
Kumar, J. P. and Ready, D. F. (1995). Rhodopsin
plays an essential structural role in Drosophila photoreceptor development.
Development 121,4359
-4370.
Kumar, J. P. and Moses, K. (2001). Eye specification in Drosophila: perspectives and implications. Semin. Cell Dev. Biol. 12,469 -474.[CrossRef][Medline]
Kumar, J. P., Bowman, J., O'Tousa, J. E. and Ready, D. F. (1997). Rhodopsin replacement rescues photoreceptor structure during a critical developmental window. Dev. Biol. 188, 43-47.[CrossRef][Medline]
Leonard, D. S., Bowman, V. D., Ready, D. F. and Pak, W. L. (1992). Degeneration of photoreceptors in rhodopsin mutants of Drosophila. J. Neurobiol. 23,605 -626.[Medline]
Matsumoto, H., Isono, K., Pye, Q. and Pak, W. L. (1987). Gene encoding cytoskeletal proteins in Drosophila rhabdomeres. Proc. Natl. Acad. Sci. USA 84,985 -989.[Abstract]
Meinertzhagen, I. A. and Hanson, T. E. (1993). The development of the optic lobe. In The Development of Drosophila melanogaster (ed. M. Bate and A. M. Arias), pp.1363 -1492. Cold Spring Harbor: Cold Spring Harbor Press.
Mollereau, B., Dominguez, M., Webel, R., Colley, N. J., Keung, B., de Celis, J. F. and Desplan, C. (2001). Two-step process for photoreceptor formation in Drosophila. Nature 412,911 -913.[CrossRef][Medline]
Nguyen, D. N., Rohrbaugh, M. and Lai, Z. (2000). The Drosophila homolog of Onecut homeodomain proteins is a neural-specific transcriptional activator with a potential role in regulating neural differentiation. Mech. Dev. 97, 57-72.[CrossRef][Medline]
Pak, W. L. (1975). Mutants affecting the vision in Drosophila melanogaster. New York, London: Plenum.
Papatsenko, D., Nazina, A. and Desplan, C. (2001). A conserved regulatory element present in all Drosophila rhodopsin genes mediates Pax6 functions and participates in the fine-tuning of cell-specific expression. Mech. Dev. 101,143 -153.[CrossRef][Medline]
Pichaud, F. and Desplan, C. (2001). A new
visualization approach for identifying mutations that affect differentiation
and organization of the Drosophila ommatidia.
Development 128,815
-826.
Pignoni, F., Hu, B., Zavitz, K. H., Xiao, J., Garrity, P. A. and Zipursky, S. L. (1997). The eye-specification proteins So and Eya form a complex and regulate multiple steps in Drosophila eye development. Cell 91,881 -891.[Medline]
Porter, J. A., Hicks, J. L., Williams, D. S. and Montell, C. (1992). Differential localizations of and requirements for the two Drosophila ninaC kinase/myosins in photoreceptor cells. J. Cell Biol. 116,683 -693.[Abstract]
Quiring, R., Walldorf, U., Kloter, U. and Gehring, W. J. (1994). Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans. Science 265,785 -789.[Medline]
Ready, D. F., Hanson, T. E. and Benzer, S. (1976). Development of the Drosophila retina, a neurocrystalline lattice. Dev Biol 53,217 -240.[Medline]
Sang, T. K. and Ready, D. F. (2002). Eyes
closed, a Drosophila p47 homolog, is essential for photoreceptor
morphogenesis. Development
129,143
-154.
Sheng, G., Thouvenot, E., Schmucker, D., Wilson, D. S. and Desplan, C. (1997). Direct regulation of rhodopsin 1 by Pax-6/eyeless in Drosophila: evidence for a conserved function in photoreceptors. Genes Dev. 11,1122 -1131.[Abstract]
Thummel, C. S., Boulet, A. M. and Lipshitz, H. D. (1988). Vectors for Drosophila P-element-mediated transformation and tissue culture transfection. Gene 74,445 -456.[CrossRef][Medline]
Ton, C. C., Hirvonen, H., Miwa, H., Weil, M. M., Monaghan, P., Jordan, T., van Heyningen, V., Hastie, N. D., Meijers-Heijboer, H., Drechsler, M. et al. (1991). Positional cloning and characterization of a paired box- and homeobox-containing gene from the aniridia region. Cell 67,1059 -1074.[Medline]
Tsunoda, S., Sun, Y., Suzuki, E. and Zuker, C.
(2001). Independent anchoring and assembly mechanisms of INAD
signaling complexes in Drosophila photoreceptors. J.
Neurosci. 21,150
-158.
Van Vactor, D., Jr, Krantz, D. E., Reinke, R. and Zipursky, S. L. (1988). Analysis of mutants in chaoptin, a photoreceptor cell-specific glycoprotein in Drosophila, reveals its role in cellular morphogenesis. Cell 52,281 -290.[Medline]
Vandendries, E. R., Johnson, D. and Reinke, R. (1996). orthodenticle is required for photoreceptor cell development in the Drosophila eye. Dev. Biol. 173,243 -255.[CrossRef][Medline]
Wilson, D., Sheng, G., Lecuit, T., Dostatni, N. and Desplan, C. (1993). Cooperative dimerization of paired class homeo domains on DNA. Genes Dev. 7,2120 -2134.[Abstract]
Wilson, D. S. and Desplan, C. (1995). Homeodomain proteins. Cooperating to be different. Curr. Biol. 5,32 -34.[Medline]
Wolff, T., Martin, K. A., Rubin, G. M. and Zipursky, S. L. (1997). The development of the Drosophila visual system. In Molecular and Cellular Approaches to Neural Development (ed. W. M. Cowan, T. M. Jessell and S. L. Zipursky), pp. 474-508. Oxford: Oxford University Press.
Wolff, T. and Ready, D. (1993). Pattern formation in the Drosophila retina. In The Development of Drosophila melanogaster (ed. M. Bate and A. M. Arias), pp.1277 -1325. Cold Spring Harbor: Cold Spring Harbor Press.
Xu, X. Z., Chien, F., Butler, A., Salkoff, L. and Montell, C. (2000). TRPgamma, a drosophila TRP-related subunit, forms a regulated cation channel with TRPL. Neuron 26,647 -657.[Medline]
Yarfitz, S., Niemi, G. A., McConnell, J. L., Fitch, C. L. and Hurley, J. B. (1991). A G beta protein in the Drosophila compound eye is different from that in the brain. Neuron 7,429 -438.[Medline]
Zelhof, A. C., Bao, H., Hardy, R. W., Razzaq, A., Zhang, B. and Doe, C. Q. (2001). Drosophila Amphiphysin is implicated in protein localization and membrane morphogenesis but not in synaptic vesicle endocytosis. Development 128,5005 -5015.[Medline]
Zelhof, A. C., Yao, T. P., Chen, J. D., Evans, R. M. and McKeown, M. (1995). Seven-up inhibits ultraspiracle-based signaling pathways in vitro and in vivo. Mol. Cell Biol. 15,6736 -6745.[Abstract]
Zipursky, S. L., Venkatesh, T. R., Teplow, D. B. and Benzer, S. (1984). Neuronal development in the Drosophila retina: monoclonal antibodies as molecular probes. Cell 36, 15-26.[Medline]
Zuker, C. S. (1996). The biology of vision of
Drosophila. Proc. Natl. Acad. Sci. USA
93,571
-576.