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First published online 31 October 2007
doi: 10.1242/dev.012781
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Department of Pediatric Ophthalmology, Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
* Author for correspondence (e-mail: tiffany.cook{at}cchmc.org)
Accepted 11 September 2007
 |
SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Prox1, Gfi1, Otx2, Opsin, Cell-specific gene expression, Photoreceptor cell
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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750 individual eye units, or
ommatidia. Initial ommatidial formation requires conserved Notch and
bHLH-dependent events important for the development of many sensory organs
(Bertrand et al., 2002
;
Frankfort and Mardon, 2002;
Lai and Orgogozo, 2004;
Pi and Chien, 2007). The first
ommatidial cell to undergo neurogenesis is called the presumptive R8 cell, the
specification of which during late larval development requires expression of
the bHLH transcription factor Atonal (Ato) and the zinc-finger transcription
factor Senseless (Sens) (Frankfort and
Mardon, 2002; Frankfort et
al., 2001; Jarman et al.,
1994; Lage et al.,
1997; Nolo et al.,
2000; White and Jarman,
2000). Next, seven additional PR neurons (R1-R7) and accessory
cells are recruited to each ommatidium through a reiterative cascade of EGF
and Notch signaling (see Doroquez and
Rebay, 2006). During late pupal development, all PRs complete
differentiation, generating several subtypes required for shape, motion, color
and polarized light perception (Fig.
1A-C) (Cook and Desplan,
2001; Wernet and Desplan,
2004). Although the events that initiate the recruitment of the
R1-R8 precursors have been much studied, far less is known regarding how these
precursors later diversify into specialized sensory neurons in the adult
eye.
During terminal differentiation, functionally distinct PRs develop
different morphologies, locations within the retina and photopigment (opsin)
expression (Cook and Desplan,
2001; Hardie,
1985). R1-R6-derived cells form adult `outer PRs' (OPRs), which
function much like vertebrate rod PRs for image formation, motion detection
and vision under dim light conditions. OPRs express the broad
wavelength-sensitive Rhodopsin (Rh) protein, Rh1 (also known as NinaE -
FlyBase), develop rhabdomeres (apical light-gathering surfaces) that extend
the full depth of the retina, and form a trapezoidal array within each
ommatidium (Fig. 1A,C). R7- and
R8-derived cells, called `inner PRs' (IPRs), are genetically distinguishable
from OPRs (Mollereau et al.,
2001) and, like vertebrate cone PRs, they discriminate color
(Cook and Desplan, 2001;
Hardie, 1985). In the adult
retina, R7s sit atop R8s in the center of the OPR trapezoid
(Fig. 1A) and they
differentiate into distinct cell populations: mature R7s express one of two
different UV-sensitive opsins, Rh3 or Rh4, whereas R8s primarily express blue
(Rh5) or green (Rh6)-sensitive opsins (Fig.
1C) (Chou et al.,
1996; Huber et al.,
1997; Montell et al.,
1987; Papatsenko et al.,
1997).
The opsins expressed within the inner PRs define three different subtypes
of adult ommatidia: pale (p), yellow (y) and dorsal rim area (DRA) (see
Wernet and Desplan, 2004). p
ommatidia couple Rh3 and Rh5 expression in the R7 and R8, respectively,
whereas y ommatidia couple Rh4 and Rh6 expression
(Fig. 1C)
(Chou et al., 1996;
Chou et al., 1999;
Papatsenko et al., 1997). p/y
subsets comprise the majority of ommatidia and are randomly distributed
throughout the retina in a 30:70 (p:y) ratio
(Bell et al., 2007;
Kirschfeld and Franceschini,
1977; Stark and Thomas,
2004). DRA ommatidia are two specialized rows of ommatidia that
express the same opsin, Rh3, in both R7 and R8 PRs, form distinct polarizing
rhabdomeres, and interpret the e-vector of polarized light
(Fig. 1B,C)
(Fortini and Rubin, 1990;
Labhart and Meyer, 1999;
Wernet et al., 2003). Thus, at
least six different adult IPRs exist: R7 and R8 cells of the p, y and DRA
subtypes.
IPRs arise through continual restriction in cell fate (for reviews, see
Bateman and McNeill, 2005;
Freeman, 2005;
Mollereau and Domingos, 2005;
Wernet et al., 2006). The
first restriction involves members of the Spalt (Sal) family of transcription
factor-encoding genes, salm and salr
(Mollereau et al., 2001).
After PR recruitment is complete during larval development, Sal genes are
specifically expressed in R7 and R8 precursors
(Domingos et al., 2004). This
expression is essential for IPR differentiation as loss of Sal gene function
causes R7 and R8 precursors to develop as OPRs
(Mollereau et al., 2001).
Downstream of the Sal genes, another transcription factor-encoding gene,
prospero (pros), is selectively activated in R7 precursors.
pros functions in R7s to block R8 characteristics such as
blue/green-opsin expression and nuclear polarity, subsequently allowing
UV-sensitive PR differentiation (Cook et
al., 2003). Thus, in the absence of pros, both
Sal-positive PRs differentiate molecularly and morphologically as R8-related
PRs in the adult retina.
|
;
Jafar-Nejad et al., 2003;
Nolo et al., 2000;
Quan et al., 2004). In the
developing Drosophila peripheral nervous system (PNS), Sens both
positively and negatively feeds back on proneural bHLH gene expression to
enhance the selection of single sensory organ precursors (SOPs)
(Acar et al., 2006;
Jafar-Nejad et al., 2003;
Jafar-Nejad et al., 2006;
Nolo et al., 2000). Similarly,
Sens is required for the specification of the eye SOP-like cell, the R8
precursor (Frankfort and Mardon,
2002). Moreover, increasing evidence suggests that sens
might participate downstream of neural selection to specify different neuronal
subtypes (Domingos et al.,
2004; Jafar-Nejad et al.,
2006; Wallis et al.,
2003). Unfortunately, owing to sens early requirements in
neural selection and in inhibiting apoptosis, such roles have been difficult
to analyze. In the eye, sens is expressed throughout R8 development
and undergoes two stages of regulation
(Cook et al., 2003;
Domingos et al., 2004;
Frankfort et al., 2001;
Frankfort et al., 2004)
(Fig. 1D). During R8
specification, sens is activated by the ato proneural gene
(Frankfort et al., 2001);
later, sens expression depends on the Sal genes. Since Sal genes are
required for IPR formation (Domingos et
al., 2004), these data suggest that sens plays two
separable roles in R8 development: specification and differentiation. This
latter function has been difficult to assess because removal of sens
during neuronal recruitment transforms the pre-R8 cell into an R2/R5 OPR
(Frankfort and Mardon, 2004;
Frankfort et al., 2001). Also,
because R7 cell recruitment requires an R8-specific signal, Boss, this loss of
R8 identity causes additional non-cell-autonomous failure in R7 recruitment
(Frankfort et al., 2001).
Here, we rescue the early sens loss-of-function (LOF) phenotype and
show that IPR development can be restored. LOF and gain-of-function (GOF)
experiments in maturing PRs reveal that sens is both necessary and
sufficient to induce R8-like characteristics and repress R7-related features
in terminally differentiating IPRs. Moreover, sens partially
recapitulates this process in vitro by differentially regulating R7 versus R8
opsin gene expression. These data reveal that IPRs require the opposing
actions of sens and pros to form functionally distinct
color-sensitive PRs. Because pros and sens are expressed in
similar, yet distinct, cell types in many developing tissues, we propose that
comparable antagonistic pros/sens-dependent regulation helps to
create cellular diversity in many developmental contexts. |
MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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13 (a white gene RNAi line)
(R. Carthew, Northwestern University, Evanston, IL),
Rh6seq56B-GAL4
(Cook et al., 2003). A mutant
Rh6 allele, Rh6[1], is present on both
FRT79D-sensE1 and sensE2 chromosomes
(data not shown). Thus, a wild-type Rh6, derived from Bloomington
Stock #93, was recombined onto the FRT79D-sensE2
chromosome similarly to previously described
(Cook et al., 2003). Two lines
were maintained, sensE2.1 and
sensE2.3. Both behaved identically to
sensE2. sensE2.1
was used for all experiments reported here. For sens full LOF
experiments, eyflp; Sp/CyO; GMRHid, cl3L,
FRT79D/TM6B flies were crossed with yw67; Sp/CyO;
sensE2.1 (or sensE1)-FRT79D/TM6B.
eyflp; Sp/CyO; GMRHid, cl3L,
FRT79D/sensE2.1-FRT79D and eyflp;
Sp/CyO; sensE2.1-FRT79D/TM6B were used for LOF and control
sections, respectively. sens lateLOF eyes were generated from
straight-winged offspring from the cross: eyflp;
UAS-sens/CyO; GMRHid, cl3L, FRT79D/TM6B x
pWIZ-w13; sca109.68-GAL4/CyO;
sensE2.1, FRT79D/TM6B.
Antibody production
Full-length sens coding sequence, kindly provided by H. Bellen,
was cloned into pET28b and transformed into BL21-CodonPlus (DE3)-RP
(Stratagene). Protein expression was induced with 0.1 mM IPTG for 4 hours.
Cells were lysed for 2 hours at room temperature (RT) in 8 M urea lysis buffer
(ULB: 100 mM NaH2PO4, 10 mM Tris-HCl pH 8.0, 10 mM
imidazole, 8 M urea, 10 mM ß-mercaptoethanol, 0.5% NP40), centrifuged 30
minutes at 16,000 g, and the supernatant mixed with Ni-NTA
beads (Qiagen) for 4 hours at RT. Beads were washed five times with ULB + 250
mM NaCl, and protein was eluted with ULB + 300 mM imidazole. This was used to
immunize rats (Cocalico Biologicals). Rt8 serum was pre-absorbed with 0- to
5-hour Drosophila embryos. Anti-Salm and anti-Otd polyclonal
antibodies were generated using the same approach as above. Salm aa410-916
were used as antigen in rabbits, whereas full-length Otd protein was used to
immunize guinea pigs. Antibody specificity was tested in the appropriate
mutant backgrounds.
Immunofluorescence, plastic sectioning and imaging
For cryosections, fly heads were embedded and frozen in OCT, sectioned (10
µm), processed and stained as previously described
(Cook et al., 2003). For
plastic sections, retinas were dissected in PBT (PBS + 0.1% Triton X-100, pH
7.2), fixed in 2% glutaraldehyde in 0.2 M PBS and post-fixed in 2%
OsO4 in PBS. Tissue was serially dehydrated with ethanol and washed
twice, 10 minutes each, with propylene oxide (Ted Pella). 1:1 propylene
oxide:Durcapan resin (Sigma) was applied overnight, and replaced with pure
Durcapan for 4 hours at RT. Resin-embedded retinas were transferred to plastic
molds, baked 70°C overnight and then sectioned on a Reichert OmU3
ultramicrotome. Sections (1 µm) were stained with 1% toluidine blue/borax
for 10 minutes, mounted in 50:50 PBS:glycerol and imaged. Whole-mounted
retinas were dissected at RT in PBT, fixed in PLP (PBS, 4% paraformaldehyde,
0.075 M lysine, 0.01 M sodium periodate, 0.05% saponin)
(McLean and Nakane, 1974), and
washed three times, 10 minutes each, with PBT. Retinas were transferred to
Signal-iT FX (Invitrogen) for 30 minutes at RT, before incubation with primary
antibodies overnight at 4°C in BNTS (PBT, 1.5 M NaCl, 0.1% BSA, 0.05%
saponin). Samples were washed three times, 20 minutes each, with PBT, and
incubated 90 minutes at RT with secondary antibodies diluted in BNTS and, when
used, Alexa Fluor 488-conjugated phalloidin (1:40) (Invitrogen). After washing
three times, 20 minutes each, with PBT, retinas were mounted in Prolong Gold
antifade-reagent (Invitrogen) and imaged 24 hours later. Antibody dilutions
were: guinea pig anti-Sens (1:800; H. Bellen) and Otd (1:750); rat anti-Sens
(1:100) and Elav (1:200; DHSB); mouse anti-Pros (1:10; DHSB), Rh3 (1:10; S.
Britt, University of Colorado, Aurora, CO) and Rh5 (1:1000; S. Britt); rabbit
anti-Salm (1:500; B. Mollereau, Ecole Normale Supérieure, Lyon, France)
or Salm (as described above, 1:150), Rh4 (1:150; C. Zuker, University of
California San Diego, La Jolla, CA), Rh6 [1:2000; C. Desplan
(Tahayato et al., 2003)], GFP
(1:500; Abcam) and ß-gal (1:1000; Cappel); chicken anti-Rh3 [1:40
(Cook et al., 2003)] and
ß-gal (1:1000; Abcam). Alexa Fluor 488, 555 and 655-conjugated secondary
antibodies (1:1500; Invitrogen) were used. Digital images were obtained with
the Apotome deconvolution system (Zeiss) and processed with Axiovision 4.5
(Zeiss) and Adobe Photoshop 7.0 software.
In vitro reporter assays
Luciferase reporter constructs were generated by subcloning minimal
promoters for Rh3 (-247 to +18), Rh4 (-159 to +85),
Rh5 (-236 to +50) and Rh6 (-555 to +121)
(Cook et al., 2003;
Papatsenko et al., 2001;
Tahayato et al., 2003) into
promoterless pGL3 (Promega). Full-length otd (E. Wimmer, University
of Göttingen, Germany), prosS [M. Mortin (NICHD/NIH, Bethesda,
MD) and C. Doe (University of Oregon, Eugene, OR)] and sens (H.
Bellen) cDNAs were subcloned into pAc5.1 (Invitrogen) (details available upon
request). pAc-LacZ (J. Culi and R. Mann, Columbia University, New York, NY)
was used for transfection controls. Drosophila S2 cells (Invitrogen)
were maintained in HyQ SFX-Insect media (Hyclone) at RT.
1x106 cells were plated in 6-well tissue culture dishes
(Corning) 48 hours prior to transfection with 3 µL Fugene HD (Roche) and
250 ng pGL3 reporter, 250 ng pAc-LacZ and 500 ng total pAc-expressing vectors
(250 ng of any one transcription factor). 48 hours post-transfection, cells
were lysed in 70 µL Passive Lysis Buffer (Promega). Luciferase activity was
measured using Luciferase Assay Reagent (Promega) and a Veritas Microplate
luminometer (Turner Biosystems). ß-galactosidase activity was measured
with ONPG substrate using a µQuant Microplate spectrophotometer (Bio-Tek).
Luciferase values were normalized to ß-galactosidase activity,
Rh-specific activity was normalized to the pGL3 control, and factor-specific
activity was normalized with the pAc control. Samples were transfected in
triplicate for each experiment, and each experiment was performed at least
three independent times. Data from single representative experiments are
shown. Statistical analysis was performed using SPSS.
Electromobility gel shift assays (EMSAs)
An EcoRI fragment encoding the four zinc fingers of Sens (amino
acids 348-541; sensZF) was subcloned into pET-28a (Novagen) and transformed
into BL21-CodonPlus-RP cells (Stratagene). Protein induction with 0.1 mM IPTG
was performed overnight at 16°C. Protein purification and EMSAs were
performed as previously described
(Gebelein et al., 2004).
Rh3, Rh4, Rh5 and Rh6 promoters were PCR-amplified from pGL3
reporters (above), gel purified (Qiagen) and end-labeled with
[
-32P]ATP. Purified His-SensZF protein (50 or 500 ng) and
approximately 30 ng probe was used for EMSAs.
Sens binding site mutagenesis and in vivo lacZ reporter assays
The core AATC Sens-binding sequence was mutated to GGTC within the
Rh3 and Rh4 promoters by PCR (details available upon
request). Sens failed to bind to these sites in vitro (data not shown). Mutant
promoters were subcloned into pGL3 or pCHABSal
(Wimmer et al., 1997). pCHAB
reporter constructs were injected into yw67 flies, and at
least three independent insertions were tested for expression. X-Gal staining
was performed as previously described
(Tahayato et al., 2003).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Maurel-Zaffran et al.,
2001). We observed sca109.68-GAL4-dependent
expression of a nuclear-localized GFP reporter (sca>nGFP)
in R8s from early specification through 55% pupation; however, by 70%
pupation, when PRs begin to terminally differentiate
(Earl and Britt, 2006;
Kumar and Ready, 1995),
reporter expression was no longer observed
(Fig. 2A). Reporter expression
was also absent in adult PRs (data not shown). Thus, we tested whether, in an
eye-specific sens mutant background (see Materials and methods),
sca109.68-GAL4 driving UAS-sens expression
(sca>sens) during PR recruitment would rescue inner PR
specification and allow subsequent sens-negative IPRs to terminally
differentiate.
|
;
Frankfort et al., 2001).
Occasional Salm-positive cells were observed and these primarily expressed the
R7-specific marker Pros (Fig.
2D,G,G', arrowheads), suggesting that few sens
mutant R8 cells persist long enough to allow R7 recruitment before being
transformed into an OPR. In contrast to sens full LOF eyes, we
observed two discrete layers of Salm-positive nuclei in sens mutants
rescued with sca>sens
(Fig. 2E,H) (henceforth called
sens late LOF). Pros is expressed in all distal-most Salm-positive
nuclei, whereas the proximal Salm-positive nuclei lack both Pros and Sens
expression. These findings indicate that transient sens expression is
able to restore IPR development.
Sens is necessary for proper R8 terminal differentiation
To address whether sens functions in R8 terminal differentiation,
we analyzed three IPR-specific features: Rhodopsin gene expression,
intracellular nuclear polarity and rhabdomere position (see
Fig. 1C). Wild-type R7s express
Rh3 and Rh4, have distal nuclei, and rhabdomeres that lie within the distal
part of the retina (Fig.
3A-B,G,I). By contrast, wild-type R8s primarily express Rh5 and
Rh6, have proximal nuclei, and have rhabdomeres that occupy the proximal third
of the retina directly beneath the R7 rhabdomere
(Fig. 3B,C,G,I). In
sens late LOF eyes, the distal-most IPRs maintained all three
characteristics of `R7-ness' (Fig.
3D,F,H,J), consistent with their maintained Pros expression
(Fig. 2H'). However,
numerous features within the R8 layer differed from that of wild-type eyes.
First, the numbers of PRs expressing R8 opsins were significantly reduced,
with only occasional Rh5- or Rh6-positive cells observed (compare
Fig. 3B,C with E,F). Instead,
the R7 opsin, Rh3, was expressed in the majority of cells within the proximal
`R8' layer (Fig. 3D,E,H,J). Rh4
was also occasionally observed proximally
(Fig. 3D,H, arrowheads).
Second, the R8 nuclear layer largely localized at the center of the retina
(compare Fig. 2C with E,
Fig. 3I with J), rather than at
the base of the retina. Finally, the R8 rhabdomeres no longer underlay R7
rhabdomeres, but instead inappropriately extended into the R7 layer
(Fig. 3H). Thin plastic
sections of these eyes confirmed the presence of two narrow rhabdomeres in the
center of many ommatidia (characteristic of IPRs) at the plane of the R7 layer
(Fig. 3L), whereas in wild-type
retina, only one IPR rhabdomere was present
(Fig. 3K). Whole-mount retinal
staining also revealed that Rh3 and Rh4 were restricted to the single R7 cell
in control eyes (Fig. 3M), but
were present in both the R7 and R8 cells within individual sens late
LOF ommatidia (Fig. 3N).
Together, these data suggest that sens mutant R8s share aspects of
both R7 and R8 PRs: the cells reside at the R7 layer of the retina, lose
R8-based opsins and acquire R7 opsin expression, yet maintain an R8-specific
proximal nuclear position.
Sens is sufficient to repress R7 features and activate R8 features in adult inner PRs
We next hypothesized that Sens misexpression in terminally differentiating
R7s would promote R8-based characteristics. Previous studies have demonstrated
that sens misexpression during early neuronal recruitment is
sufficient to convert non-R8 precursors into R8 cells
(Frankfort et al., 2001),
consistent with its requirement in R8 specification. Not surprisingly,
misexpression of sens using an early panPR GAL4 driver, GMR-GAL4,
leads to a severely disrupted retina that primarily expresses the R8 opsin,
Rh6 (B.X. and T.C., unpublished; M. Wernet, personal communication).
Misexpression of sens in maturing OPRs, by contrast, can only weakly
activate Rh6 (Domingos et al.,
2004) while other aspects of OPR differentiation appear
unaffected, even with salm co-expression (B.X., unpublished). This
suggests that differentiated OPRs are no longer sensitive to
sens-dependent R8 transformation. To specifically address whether
sens affects IPR terminal differentiation, we expressed sens
in R7 and R8 cells during late pupation. Currently, the only GAL4 drivers
restricted in expression to maturing inner PRs involve Rhodopsin promoter/GAL4
fusions. Hypothesizing that sens might repress R7-specific Rh genes,
we chose to misexpress sens using a modified Rh6-based
driver that is expressed in R7 and R8 cells during their terminal
differentiation (Cook et al.,
2003) (Fig. 4A).
Henceforth, this driver will be called `inner photoreceptor'
IP-GAL4. Note that IP-GAL4 drives high levels of gene expression in all dorsal
R7s but not in all ventral R7s (Fig.
4A), allowing us to compare wild-type and
sens-misexpressing cells in the same retina.
|
; Mikeladze-Dvali et
al., 2005; Wernet et al.,
2006), we take this as further evidence that R7s misexpressing
sens lose Rh3 and possibly other important R7-specific features. Each
sens-dependent change becomes more evident with age (data not shown),
indicating that multiple aspects of IPR differentiation remain plastic
throughout development. No changes in rhabdomere positioning were observed in
sens GOF eyes. Together, these data suggest that sens is
sufficient to mis-specify several aspects of R7 IPR differentiation into
R8-related characteristics, including opsin expression, reduction in Pros
expression and changes in nuclear position.
|
;
Zweidler-Mckay et al., 1996)
(Fig. 5A). Using a
position-weighted matrix (PWM) of Sens/Gfi binding sites
[(Sandelin et al., 2004)
jasper.genereg.net],
we found that the optimal Gfi/Sens binding site, R21
(Jafar-Nejad et al., 2003),
achieves a score of 12.2, and a bona fide Sens binding site (S-box) within the
achaete (ac) promoter
(Jafar-Nejad et al., 2003)
scores 9.0. Consistent with the idea that PWM scores correlate with binding
affinity, the purified DNA-binding domain of Sens binds with 10-fold
higher affinity to R21 than the S-box sequence in electrophoretic mobility
shift assays (EMSAs) (Jafar-Nejad et al.,
2003) (B.G., unpublished). Identical results were obtained using
purified full-length Sens protein (data not shown).
Using the Sens/Gfi1 PWM, we identified several high-scoring (>8) Sens
sites within the Rh3 and Rh4 promoters. However, only
low-scoring sites (<6.5) were found within the Rh5 and
Rh6 promoters. We next performed in vitro EMSAs using purified Sens
protein and minimal, in vivo functional, Rh promoters
(Fortini and Rubin, 1990;
Papatsenko et al., 2001;
Tahayato et al., 2003). These
showed that multiple complexes of Sens formed on the Rh3 and
Rh4 promoters (Fig.
5B,C), whereas little to no binding was observed on the
Rh5 and Rh6 promoters
(Fig. 6A,B). Individual Sens
binding sites from all four promoters were also tested using short
oligonucleotide sequences, and only sites from the Rh3 and
Rh4 promoters showed significant binding (data not shown). One site
within the Rh5 promoter (the d site in
Fig. 6A) bound Sens weakly,
consistent with the observation of a single shift complex with the full-length
Rh5 promoter (Fig. 6B,
arrow). These data suggest that R7 opsin genes are direct sens
targets, whereas R8 opsin genes are weak or indirect sens
targets.
We next measured the ability of Sens to regulate Rh promoter-luciferase
reporter expression in the non-neuronal Drosophila S2 cell line. Sens
was sufficient to repress the promoter activity of both R7 opsins,
Rh3 and Rh4, in vitro
(Fig. 5C); however, we observed
no change in R8-based Rh5 or Rh6 expression
(Fig. 5C). Mutations of site A,
the highest scoring site within the Rh3 and Rh4 promoters,
were sufficient to prevent Sens-dependent repression in vitro
(Fig. 5D, rh3A,
rh3AC, rh3ACD, rh4A, rh4AB). Mutation of the
Rh4 B site also led to a significant loss in repression. To test
whether Sens mediates direct Rh3 and/or Rh4 transcriptional
repression in vivo, we analyzed Rh3 and Rh4
Sens-binding-mutant promoter expression in adult eyes. An Rh3
reporter carrying a promoter that is unresponsive to Sens ex vivo
(rh3AC) was expanded into the majority of R8s in vivo
(Fig. 5E), whereas an in vitro
Sens-responsive rh3C promoter, rh3C, remained restricted to R7
cells (Fig. 5E). We detected
few, if any, R8 cells expressing the mutated Rh4 promoters
(Fig. 5F). This is not
surprising, as sens late LOF R8s predominantly express Rh3 and only a
few express Rh4 (Fig. 3D,H).
Lack of Rh4 expansion in R8s is also parsimonious with recent studies
indicating that Rh4 induction requires the yR7-specific factor
Spineless (Ss) in vivo, and that without Ss, Rh3 is expressed in R7s
by default (Wernet et al.,
2006) (see Discussion). Together, our in vitro and in vivo studies
demonstrate that sens is important for actively repressing R7 opsins,
particularly Rh3.
Sens activates R8 opsin promoter expression in an Otd-dependent manner
Although sens activates Rh5 and Rh6 in vivo,
sens is not sufficient to regulate these promoters in vitro
(Fig. 4H). Two likely
possibilities explain this result: (1) sens indirectly regulates R8
opsins by affecting cell fate decisions; and/or (2) Sens functions in
conjunction with other factors to regulate Rh5 and Rh6 gene
expression. One candidate for such a factor is the transcription factor
Orthodenticle (Otd; also known as Ocelliless - FlyBase). In the adult retina,
Otd is expressed in all PRs (Fig.
1G) and regulates the Rh3, Rh5 and Rh6 promoters
by binding K50 sites (Tahayato et al.,
2003). Similarly to in vivo
(Tahayato et al., 2003), we
found that Otd activates Rh3 and Rh5, but not Rh1
or Rh4, in vitro (Fig.
6C and data not shown). We also observed Otd-dependent
Rh6 activation in S2 cells (Fig.
6C), although in vivo, Otd is best characterized for Rh6
repression in OPRs. Rh6 activation in vitro required intact K50 sites
(Fig. 6E), indicating that this
Otd-dependent activation is specific.
|
0.5X), whereas a strong synergistic effect was
observed for Rh6 activation (50-100X). An Otd binding site mutation
within the Rh6 promoter significantly reduced this Otd/Sens-dependent
synergism (Fig. 6E), supporting
an essential role for Otd in mediating this activity. Together, these data
demonstrate that in vitro, Sens can recapitulate its in vivo ability to
regulate R7 and R8 opsins. Repression of R7 opsins involves direct
DNA-binding, whereas activation of R8 opsins requires cooperation with Otd.
These findings correlate with recent data showing that Sens functions as a
site-specific repressor and a DNA-binding-independent co-activator during
proneural gene regulation (Acar et al.,
2006).
Sens and Pros reciprocally regulate opsin genes in vitro
Combined, the results here and our previous results on R7 differentiation
(Cook et al., 2003) suggest
that Sal-restricted IPRs can adopt either R7 or R8 characteristics, and that
sens in R8s, or pros in R7s, is important to functionally
distinguish these PRs. In vivo, pros represses R8 opsins, but does
not affect R7-based opsins. To test whether we could recapitulate this
regulation in vitro, we also performed Rh reporter assays with Pros. As shown
in Fig. 7A, Rh promoter
activity was unaffected by pros alone. However, Pros specifically
repressed Otd-mediated Rh5 and Rh6 activation by
approximately 75% and 50%, respectively
(Fig. 7A). Pros binding sites
were required for this repression (data not shown). Together, these studies
suggest that Pros and Sens both require Otd to regulate the R8-based opsins:
Sens activates, whereas Pros represses, with Otd
(Fig. 7B). Because Otd is found
in all PRs, this combinatorial regulation is consistent with the expression of
these factors in vivo.
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Nolo et al., 2000). In
sens mutants, neuronal precursors either undergo apoptosis or, as in
the case of R8 cells, inappropriately develop into another neuronal population
(for a review, see Jafar-Nejad and Bellen,
2004). sens/Gfi1-dependent specification of distinct
neuronal populations also occurs in mouse inner ear cells and fly
mechanosensory bristles (Frankfort et al.,
2001; Jafar-Nejad et al.,
2006; Wallis et al.,
2003). However, it remains unclear what fate these neurons take in
the absence of sens/Gfi1. Here, we demonstrate that in the
Drosophila visual system, sens not only controls R8
specification and R7 recruitment, but is also essential for distinguishing
blue/green-sensitive from UV-sensitive PRs.
|
; Mikeladze-Dvali et al.,
2005; Mollereau et al.,
2001; Tahayato et al.,
2003; Wernet et al.,
2003; Wernet et al.,
2006). Once a full complement of PR neurons is recruited during
late larval development, the Sal gene complex genetically distinguishes IPRs
from OPRs (Mollereau et al.,
2001). Based on our pros and sens LOF
experiments, such Sal-specified IPRs would adopt the following
characteristics: a shortened rhabdomere that projects upwards from a
proximally localized nucleus, a cell body that resides at the R7/R8 interface,
and expression of Rh3 (Fig. 8A)
(Cook et al., 2003). From this
`generic' IPR fate, various aspects of R7 versus R8 differentiation are
promoted or repressed by the mutually exclusive expression of pros or
sens, respectively (Fig.
8B): pros functions in R7s to prevent R8 opsin expression
and to cause distal positioning of the R7 nucleus, whereas sens
functions in R8s to prevent R7 opsin expression, promote R8 opsin expression
and cause cells to develop proximally in the retina. Neither sens nor
pros seems required to suppress each other's expression
(Fig. 2)
(Cook et al., 2003). However,
misexpression of sens is sufficient to suppress pros
expression in our GOF experiments. We predict that this is owing to two
independent events: (1) sens indirectly inhibits pros
expression via its ability to alter R7 differentiation; and (2) sens
suppresses pros expression by preventing Pointed (Pnt) transcription
factor function. This latter prediction is based on previous findings that
pros is a direct transcriptional target of the MAPK-dependent Pnt
transcriptional activator (Xu et al.,
2000), and that sens suppresses Pnt-dependent
transcription during R8 selection
(Frankfort and Mardon, 2004).
Future experiments will be important to directly link cell signaling with
sens and pros antagonism during IPR development.
|
).
Interestingly, ato-dependent Sens expression occurs in all R8 cells,
but its later Sal-dependent expression is restricted to p/y ommatidia
(Fig. 1F,G and data not shown)
(Wernet et al., 2003). Since
DRA R8s are unique in expressing the typically R7-specific Rh3, absence of
sens in these cells is consistent with its importance in repressing
Rh3. Indeed, ectopic sens expression in the DRA is
sufficient to inhibit Rh3 expression and activate Rh6
expression (Fig. 4H). The
transcription factor Hth is both necessary and sufficient for DRA development:
misexpression of Hth expands Rh3 to all inner PRs and represses Sens
expression, and dominant-negative Hth leads to DRA-specific expansion of Rh6
and Sens and loss of Rh3 (Wernet et al.,
2003) (data not shown). We observe no change in Hth expression in
sens late LOF or sens GOF eyes, and Hth and Sens
co-expression causes sens-dependent Rh6 activation and
Rh3 repression (B.X., unpublished). Together, these data indicate
that Hth-dependent repression of sens is necessary for DRA
development.
Once polarization versus color decisions are made, p versus y ommatidial
subsets develop. Spineless was recently shown to be crucial for this decision
(Wernet et al., 2006), being
expressed transiently and specifically in yR7 cells to induce Rh4 (versus Rh3)
expression (Wernet et al.,
2006). That we rarely detect Rh4 in sens-negative R8s
supports the importance of R7-induction of this gene, and further indicates
that late sens LOF R8s do not fully transform into R7 cells. After p
versus y fate is established in R7 cells, Rh3-expressing pR7s induce Rh5 in
underlying R8 cells via the signaling molecule Melt
(Mikeladze-Dvali et al.,
2005). In the absence of pR8 melt induction, all R8 cells
express Rh6 (Chou et al.,
1996; Chou et al.,
1999; Papatsenko et al.,
1997). Our finding that sens more robustly activates
Rh6 than it does Rh5 both in vivo and in vitro
(Fig. 4H and
Fig. 6D) suggests that
sens is not sufficient to induce melt expression and/or
activity. Thus, current data suggest that Rh3 represents the default
opsin for all IPRs. Consistent with this, misexpression of Sal genes during
early PR development transforms OPRs into IPRs and these all express
Rh3 (Domingos et al.,
2004). Together, our ability to genetically isolate the stepwise
events of PR differentiation will allow us to uncover new mechanisms important
for achieving neuronal diversity.
Are similar restrictions important for vertebrate retinogenesis?
Interestingly, recent studies have revealed the unexpected finding that
vertebrate cone and rod PRs develop from a common precursor
(Mears et al., 2001;
Oh et al., 2007) (e.g. akin to
OPRs versus IPRs), and that medium/long wavelength-sensitive cone PRs develop
at least in part by suppressing short wavelength-sensitive cone PR development
(Applebury et al., 2007;
Deeb, 2006;
Ng et al., 2001;
Roberts et al., 2006) (e.g.
akin to R8 versus R7 decisions). Whether these developmental relationships are
indeed homologous remains unexplored. However, recent data have shown that the
thyroid hormone receptor is important in cone PR cell fate decisions, and Lim
et al. have reported that the thyroid hormone receptor associated proteins
Trap230/240 (Kohtalo/Skuld - FlyBase) are important for inducing sens
in the eye (Lim et al., 2007).
Moreover, the factors important for Drosophila PR differentiation
have vertebrate orthologs expressed in distinct mouse retinal cell populations
[e.g. Sal3 (Bpnt1), Prox1, Gfi1, Meis1/Hth and Otx2]
(Blackshaw et al., 2001;
Dyer et al., 2003;
Hisa et al., 2004;
Nishida et al., 2003;
Yang et al., 2003). Although
the interactions of these factors have not yet been explored, such studies are
likely to uncover conserved genetic cascades necessary to generate
functionally distinct PR neurons.
Gene regulation by Gfi1 and Sens
Vertebrate Gfi1 and Drosophila Sens share striking homology within
the DNA-binding four zinc-finger domains
(Jafar-Nejad and Bellen,
2004). Gfi1, but not Sens, also contains an N-terminal SNAG domain
that recruits multiple co-repressor complexes
(McGhee et al., 2003;
Zweidler-Mckay et al., 1996).
Gfi1 SNAG domain mutants behave similarly to Gfi1 DNA-binding mutants
(Grimes et al., 1996),
suggesting that Gfi1 functions primarily as a repressor. Although Sens lacks a
SNAG domain, recent studies show that, at low concentrations, Sens functions
as a site-specific transcriptional repressor of the achaete gene.
However, at high concentrations, Sens functions as a DNA-binding-independent
co-activator (Acar et al.,
2006). Both activation and repression depend on the zinc-finger
domain that is conserved with Gfi1; thus, such regulation might also be
important for Gfi1 function. Here, we similarly find that Sens represses
Rh3 and Rh4 expression through direct DNA binding, whereas
activation of Rh5 and Rh6 appears to occur through an
Otd-binding-dependent mechanism. However, we find that Sens regulation of Rh
genes is context-dependent, but not dose-dependent (B.X. and T.C.,
unpublished). Future studies aimed at comparing Ac/Sens-versus
Otd/Sens-dependent gene regulation will be important to better understand how
Sens controls such diverse aspects of PNS development.
Conserved antagonism between Sens- and Pros-related factors?
Gfi1 is best characterized as an oncoprotein in lymphoid leukemias (see
reviews, see Moroy, 2005;
Duan and Horwitz, 2005;
Jafar-Nejad and Bellen, 2004).
Gfi1 positively influences lymphoid lineage development in part by suppressing
the differentiation of the myeloid lineage. Gfi1 is also necessary for
hematopoietic stem cell maintenance, indicating an important role for this
gene in both proliferation and differentiation
(Cellot and Sauvageau, 2005;
Duan and Horwitz, 2005;
Hock et al., 2004).
Interestingly, Pros has recently been shown to repress neural stem cell
proliferation and induce differentiation, and both Pros and Prox1 have been
proposed to function as tumor suppressors
(Bello et al., 2006;
Betschinger et al., 2006;
Choksi et al., 2006;
Nagai et al., 2003;
Shimoda et al., 2006).
Although Prox1 is not associated with the hematopoiesis system under wild-type
conditions, recent studies have shown that its expression is associated with
several leukemias (Nagai et al.,
2003; Shimoda et al.,
2006). Together, we suggest that pros/Prox1 and
sens/Gfi1 factors share an evolutionarily conserved
antagonism in regulating a number of developing organ systems, ranging from
stem cell growth to neuronal and hematopoietic lineage specification.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Acar, M., Jafar-Nejad, H., Giagtzoglou, N., Yallampalli, S.,
David, G., He, Y., Delidakis, C. and Bellen, H. J. (2006).
Senseless physically interacts with proneural proteins and functions as a
transcriptional co-activator. Development
133,1979
-1989.
Applebury, M. L., Farhangfar, F., Glosmann, M., Hashimoto, K.,
Kage, K., Robbins, J. T., Shibusawa, N., Wondisford, F. E. and Zhang, H.
(2007). Transient expression of thyroid hormone nuclear receptor
TRbeta2 sets S opsin patterning during cone photoreceptor genesis.
Dev. Dyn. 236,1203
-1212.[CrossRef][Medline]
Bateman, J. M. and McNeill, H. (2005). Seeing
in color - warts and all. Dev. Cell
9, 441-442.[CrossRef][Medline]
Bell, M. L., Earl, J. B. and Britt, S. G.
(2007). Two types of Drosophila R7 photoreceptor cells are
arranged randomly: a model for stochastic cell-fate determination.
J. Comp. Neurol. 502,75
-85.[CrossRef][Medline]
Bello, B., Reichert, H. and Hirth, F. (2006).
The brain tumor gene negatively regulates neural progenitor cell proliferation
in the larval central brain of Drosophila. Development
133,2639
-2648.
Bertrand, N., Castro, D. S. and Guillemot, F.
(2002). Proneural genes and the specification of neural cell
types. Nat. Rev. Neurosci.
3, 517-530.[CrossRef][Medline]
Betschinger, J., Mechtler, K. and Knoblich, J. A.
(2006). Asymmetric segregation of the tumor suppressor brat
regulates self-renewal in Drosophila neural stem cells.
Cell 124,1241
-1253.[CrossRef][Medline]
Blackshaw, S., Fraioli, R. E., Furukawa, T. and Cepko, C. L.
(2001). Comprehensive analysis of photoreceptor gene expression
and the identification of candidate retinal disease genes.
Cell 107,579
-589.[CrossRef][Medline]
Cellot, S. and Sauvageau, G. (2005). Gfi-1:
another piece in the HSC puzzle. Trends Immunol.
26, 68-71.[CrossRef][Medline]
Choksi, S. P., Southall, T. D., Bossing, T., Edoff, K., de Wit,
E., Fischer, B. E., van Steensel, B., Micklem, G. and Brand, A. H.
(2006). Prospero acts as a binary switch between self-renewal and
differentiation in Drosophila neural stem cells. Dev.
Cell 11,775
-789.[CrossRef][Medline]
Chou, W. H., Hall, K. J., Wilson, D. B., Wideman, C. L.,
Townson, S. M., Chadwell, L. V. and Britt, S. G. (1996).
Identification of a novel Drosophila opsin reveals specific patterning of the
R7 and R8 photoreceptor cells. Neuron
17,1101
-1115.[CrossRef][Medline]
Chou, W. H., Huber, A., Bentrop, J., Schulz, S., Schwab, K.,
Chadwell, L. V., Paulsen, R. and Britt, S. G. (1999).
Patterning of the R7 and R8 photoreceptor cells of Drosophila: evidence for
induced and default cell-fate specification.
Development 126,607
-616.[Abstract]
Cook, T. and Desplan, C. (2001). Photoreceptor
subtype specification: from flies to humans. Semin. Cell Dev.
Biol. 12,509
-518.[CrossRef][Medline]
Cook, T., Pichaud, F., Sonneville, R., Papatsenko, D. and
Desplan, C. (2003). Distinction between color photoreceptor
cell fates is controlled by Prospero in Drosophila. Dev.
Cell 4,853
-864.[CrossRef][Medline]
Deeb, S. S. (2006). Genetics of variation in
human color vision and the retinal cone mosaic. Curr. Opin. Genet.
Dev. 16,301
-307.[CrossRef][Medline]
Domingos, P. M., Brown, S., Barrio, R., Ratnakumar, K.,
Frankfort, B. J., Mardon, G., Steller, H. and Mollereau, B.
(2004). Regulation of R7 and R8 differentiation by the spalt
genes. Dev. Biol. 273,121
-133.[CrossRef][Medline]
Doroquez, D. B. and Rebay, I. (2006). Signal
integration during development: mechanisms of EGFR and Notch pathway function
and cross-talk. Crit. Rev. Biochem. Mol. Biol.
41,339
-385.[CrossRef][Medline]
Duan, Z. and Horwitz, M. (2005). Gfi-1 takes
center stage in hematopoietic stem cells. Trends Mol.
Med. 11,49
-52.[CrossRef][Medline]
Dyer, M. A., Livesey, F. J., Cepko, C. L. and Oliver, G.
(2003). Prox1 function controls progenitor cell proliferation and
horizontal cell genesis in the mammalian retina. Nat.
Genet. 34,53
-58.[CrossRef][Medline]
Earl, J. B. and Britt, S. G. (2006). Expression
of Drosophila rhodopsins during photoreceptor cell differentiation: insights
into R7 and R8 cell subtype commitment. Gene Expr.
Patterns 6,687
-694.[CrossRef][Medline]
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.
Frankfort, B. J. and Mardon, G. (2002). R8
development in the Drosophila eye: a paradigm for neural selection and
differentiation. Development
129,1295
-1306.[Medline]
Frankfort, B. J. and Mardon, G. (2004).
Senseless represses nuclear transduction of Egfr pathway activation.
Development 131,563
-570.
Frankfort, B. J., Nolo, R., Zhang, Z., Bellen, H. and Mardon,
G. (2001). senseless repression of rough is required for R8
photoreceptor differentiation in the developing Drosophila eye.
Neuron 32,403
-414.[CrossRef][Medline]
Frankfort, B. J., Pepple, K. L., Mamlouk, M., Rose, M. F. and
Mardon, G. (2004). Senseless is required for pupal retinal
development in Drosophila. Genesis
38,182
-194.[CrossRef][Medline]
Freeman, M. (2005). Eye development: stable
cell fate decisions in insect colour vision. Curr.
Biol. 15,R924
-R926.[CrossRef][Medline]
Gebelein, B., McKay, D. J. and Mann, R. S.
(2004). Direct integration of Hox and segmentation gene inputs
during Drosophila development. Nature
431,653
-659.[CrossRef][Medline]
Grimes, H. L., Chan, T. O., Zweidler-McKay, P. A., Tong, B. and
Tsichlis, P. N. (1996). The Gfi-1 proto-oncoprotein contains
a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced
by interleukin-2 withdrawal. Mol. Cell. Biol.
16,6263
-6272.[Abstract]
Hardie, R. C. (1985). Functional organization
of the fly retina. In Progress in Sensory Physiology.
Vol. 5 (ed. D. Ottoson), pp.1
-79. Berlin, Heidelberg, New York, Toronto:
Springer.
Hisa, T., Spence, S. E., Rachel, R. A., Fujita, M., Nakamura,
T., Ward, J. M., Devor-Henneman, D. E., Saiki, Y., Kutsuna, H., Tessarollo, L.
et al. (2004). Hematopoietic, angiogenic and eye defects in
Meis1 mutant animals. EMBO J.
23,450
-459.[CrossRef][Medline]
Hock, H., Hamblen, M. J., Rooke, H. M., Schindler, J. W.,
Saleque, S., Fujiwara, Y. and Orkin, S. H. (2004). Gfi-1
restricts proliferation and preserves functional integrity of haematopoietic
stem cells. Nature 431,1002
-1007.[CrossRef][Medline]
Huber, A., Schulz, S., Bentrop, J., Groell, C., Wolfrum, U. and
Paulsen, R. (1997). Molecular cloning of Drosophila Rh6
rhodopsin: the visual pigment of a subset of R8 photoreceptor cells.
FEBS Lett. 406,6
-10.[CrossRef][Medline]
Jafar-Nejad, H. and Bellen, H. J. (2004).
Gfi/Pag-3/senseless zinc finger proteins: a unifying theme? Mol.
Cell. Biol. 24,8803
-8812.
Jafar-Nejad, H., Acar, M., Nolo, R., Lacin, H., Pan, H.,
Parkhurst, S. M. and Bellen, H. J. (2003). Senseless acts as
a binary switch during sensory organ precursor selection. Genes
Dev. 17,2966
-2978.
Jafar-Nejad, H., Tien, A. C., Acar, M. and Bellen, H. J.
(2006). Senseless and Daughterless confer neuronal identity to
epithelial cells in the Drosophila wing margin.
Development 133,1683
-1692.
Jarman, A. P., Grell, E. H., Ackerman, L., Jan, L. Y. and Jan,
Y. N. (1994). Atonal is the proneural gene for Drosophila
photoreceptors. Nature
369,398
-400.[CrossRef][Medline]
Kauffmann, R. C., Li, S., Gallagher, P. A., Zhang, J. and
Carthew, R. W. (1996). Ras1 signaling and transcriptional
competence in the R7 cell of Drosophila. Genes Dev.
10,2167
-2178.
Kirschfeld, K. and Franceschini, N. (1977).
Evidence for a sensitising pigment in fly photoreceptors.
Nature 269,386
-390.[CrossRef][Medline]
Kumar, J. P. and Ready, D. F. (1995). Rhodopsin
plays an essential structural role in Drosophila photoreceptor development.
Development 121,4359
-4370.[Abstract]
Labhart, T. and Meyer, E. P. (1999). Detectors
for polarized skylight in insects: a survey of ommatidial specializations in
the dorsal rim area of the compound eye. Microsc. Res.
Tech. 47,368
-379.[CrossRef][Medline]
Lage, P., Jan, Y. N. and Jarman, A. P. (1997).
Requirement for EGF receptor signalling in neural recruitment during formation
of Drosophila chordotonal sense organ clusters. Curr.
Biol. 7,166
-175.[CrossRef][Medline]
Lai, E. C. and Orgogozo, V. (2004). A hidden
program in Drosophila peripheral neurogenesis revealed: fundamental principles
underlying sensory organ diversity. Dev. Biol.
269, 1-17.[CrossRef][Medline]
Lebestky, T., Chang, T., Hartenstein, V. and Banerjee, U.
(2000). Specification of Drosophila hematopoietic lineage by
conserved transcription factors. Science
288,146
-149.
Lim, J., Lee, O. K., Hsu, Y. C., Singh, A. and Choi, K. W.
(2007). Drosophila TRAP230/240 are essential coactivators for
Atonal in retinal neurogenesis. Dev. Biol.
308,322
-330.[CrossRef][Medline]
Maurel-Zaffran, C., Suzuki, T., Gahmon, G., Treisman, J. E. and
Dickson, B. J. (2001). Cell-autonomous and -nonautonomous
functions of LAR in R7 photoreceptor axon targeting.
Neuron 32,225
-235.[CrossRef][Medline]
McGhee, L., Bryan, J., Elliott, L., Grimes, H. L., Kazanjian,
A., Davis, J. N. and Meyers, S. (2003). Gfi-1 attaches to the
nuclear matrix, associates with ETO (MTG8) and histone deacetylase proteins,
and represses transcription using a TSA-sensitive mechanism. J.
Cell. Biochem. 89,1005
-1018.[CrossRef][Medline]
McLean, I. W. and Nakane, P. K. (1974).
Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron
microscopy. J. Histochem. Cytochem.
22,1077
-1083.[Abstract]
Mears, A. J., Kondo, M., Swain, P. K., Takada, Y., Bush, R. A.,
Saunders, T. L., Sieving, P. A. and Swaroop, A. (2001). Nrl
is required for rod photoreceptor development. Nat.
Genet. 29,447
-452.[CrossRef][Medline]
Mikeladze-Dvali, T., Wernet, M. F., Pistillo, D., Mazzoni, E.
O., Teleman, A. A., Chen, Y. W., Cohen, S. and Desplan, C.
(2005). The growth regulators warts/lats and melted interact in a
bistable loop to specify opposite fates in Drosophila R8 photoreceptors.
Cell 122,775
-787.[CrossRef][Medline]
Mollereau, B. and Domingos, P. M. (2005).
Photoreceptor differentiation in Drosophila: from immature neurons to
functional photoreceptors. Dev. Dyn.
232,585
-592.[CrossRef][Medline]
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]
Montell, C., Jones, K., Zuker, C. and Rubin, G.
(1987). A second opsin gene expressed in the
ultraviolet-sensitive R7 photoreceptor cells of Drosophila melanogaster.
J. Neurosci. 7,1558
-1566.[Abstract]
Moroy, T. (2005). The zinc finger transcription
factor Growth factor independence 1 (Gfi1). Int. J. Biochem. Cell
Biol. 37,541
-546.[CrossRef][Medline]
Nagai, H., Li, Y., Hatano, S., Toshihito, O., Yuge, M., Ito, E.,
Utsumi, M., Saito, H. and Kinoshita, T. (2003). Mutations and
aberrant DNA methylation of the PROX1 gene in hematologic malignancies.
Genes Chromosomes Cancer
38, 13-21.[CrossRef][Medline]
Ng, L., Hurley, J. B., Dierks, B., Srinivas, M., Salto, C.,
Vennstrom, B., Reh, T. A. and Forrest, D. (2001). A thyroid
hormone receptor that is required for the development of green cone
photoreceptors. Nat. Genet.
27, 94-98.[Medline]
Nishida, A., Furukawa, A., Koike, C., Tano, Y., Aizawa, S.,
Matsuo, I. and Furukawa, T. (2003). Otx2 homeobox gene
controls retinal photoreceptor cell fate and pineal gland development.
Nat. Neurosci. 6,1255
-1263.[CrossRef][Medline]
Nolo, R., Abbott, L. A. and Bellen, H. J.
(2000). Senseless, a Zn finger transcription factor, is necessary
and sufficient for sensory organ development in Drosophila.
Cell 102,349
-362.[CrossRef][Medline]
Oh, E. C., Khan, N., Novelli, E., Khanna, H., Strettoi, E. and
Swaroop, A. (2007). Transformation of cone precursors to
functional rod photoreceptors by bZIP transcription factor NRL.
Proc. Natl. Acad. Sci. USA
104,1679
-1684.
Papatsenko, D., Sheng, G. and Desplan, C.
(1997). A new rhodopsin in R8 photoreceptors of Drosophila:
evidence for coordinate expression with Rh3 in R7 cells.
Development 124,1665
-1673.[Abstract]
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]
Pi, H. and Chien, C. T. (2007). Getting the
edge: neural precursor selection. J. Biomed. Sci.
14,467
-473.[CrossRef][Medline]
Quan, X. J., Denayer, T., Yan, J., Jafar-Nejad, H., Philippi,
A., Lichtarge, O., Vleminckx, K. and Hassan, B. A. (2004).
Evolution of neural precursor selection: functional divergence of proneural
proteins. Development
131,1679
-1689.
Roberts, M. R., Srinivas, M., Forrest, D., Morreale de Escobar,
G. and Reh, T. A. (2006). Making the gradient: thyroid
hormone regulates cone opsin expression in the developing mouse retina.
Proc. Natl. Acad. Sci. USA
103,6218
-6223.
Sandelin, A., Alkema, W., Engstrom, P., Wasserman, W. W. and
Lenhard, B. (2004). JASPAR: an open-access database for
eukaryotic transcription factor binding profiles. Nucleic Acids
Res. 32,D91
-D94.
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.
Shimoda, M., Takahashi, M., Yoshimoto, T., Kono, T., Ikai, I.
and Kubo, H. (2006). A homeobox protein, prox1, is involved
in the differentiation, proliferation, and prognosis in hepatocellular
carcinoma. Clin. Cancer Res.
12,6005
-6011.
Stark, W. S. and Thomas, C. F. (2004).
Microscopy of multiple visual receptor types in Drosophila. Mol.
Vis. 10,943
-955.[Medline]
Tahayato, A., Sonneville, R., Pichaud, F., Wernet, M. F.,
Papatsenko, D., Beaufils, P., Cook, T. and Desplan, C.
(2003). Otd/Crx, a dual regulator for the specification of
ommatidia subtypes in the Drosophila retina. Dev. Cell
5, 391-402.[CrossRef][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]
Wallis, D., Hamblen, M., Zhou, Y., Venken, K. J., Schumacher,
A., Grimes, H. L., Zoghbi, H. Y., Orkin, S. H. and Bellen, H. J.
(2003). The zinc finger transcription factor Gfi1, implicated in
lymphomagenesis, is required for inner ear hair cell differentiation and
survival. Development
130,221
-232.
Wernet, M. F. and Desplan, C. (2004). Building
a retinal mosaic: cell-fate decision in the fly eye. Trends Cell
Biol. 14,576
-584.[CrossRef][Medline]
Wernet, M. F., Labhart, T., Baumann, F., Mazzoni, E. O.,
Pichaud, F. and Desplan, C. (2003). Homothorax switches
function of Drosophila photoreceptors from color to polarized light sensors.
Cell 115,267
-279.[CrossRef][Medline]
Wernet, M. F., Mazzoni, E. O., Celik, A., Duncan, D. M., Duncan,
I. and Desplan, C. (2006). Stochastic spineless expression
creates the retinal mosaic for colour vision. Nature
440,174
-180.[CrossRef][Medline]
White, N. M. and Jarman, A. P. (2000).
Drosophila atonal controls photoreceptor R8-specific properties and modulates
both receptor tyrosine kinase and Hedgehog signalling.
Development 127,1681
-1689.[Abstract]
Wimmer, E. A., Cohen, S. M., Jackle, H. and Desplan, C.
(1997). buttonhead does not contribute to a combinatorial code
proposed for Drosophila head development. Development
124,1509
-1517.[Abstract]
Xu, C., Kauffmann, R. C., Zhang, J., Kladny, S. and Carthew, R.
W. (2000). Overlapping activators and repressors delimit
transcriptional response to receptor tyrosine kinase signals in the Drosophila
eye. Cell 103,87
-97.[CrossRef][Medline]
Yang, Z., Ding, K., Pan, L., Deng, M. and Gan, L.
(2003). Math5 determines the competence state of retinal ganglion
cell progenitors. Dev. Biol.
264,240
-254.[CrossRef][Medline]
Zweidler-Mckay, P. A., Grimes, H. L., Flubacher, M. M. and
Tsichlis, P. N. (1996). Gfi-1 encodes a nuclear zinc finger
protein that binds DNA and functions as a transcriptional repressor.
Mol. Cell. Biol. 16,4024
-4034.[Abstract]
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GGTC). Sites correspond to
those in A. *, P<0.05 compared with pAc alone.
(E,F) X-Gal staining of cryosections from transgenic
lacZ reporter lines carrying wild-type or Sens mutant binding sites
in the Rh3 (E) or Rh4 (F) promoters. R7 and R8 layers are
bracketed.
