1 Howard Hughes Medical Institute, Strang Laboratory of Cancer Research, The
Rockefeller University, Box 252, 1230 York Avenue, New York, NY 10021,
USA
2 Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai
School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA
* Author for correspondence (e-mail: bertrand.mollereau{at}rockefeller.edu)
Accepted 14 September 2004
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SUMMARY |
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Key words: spalt, seven-up, Drosophila, Planar cell polarity, Eye development
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Introduction |
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In the Drosophila eye, PCP is manifested by the distinct
specification of the photoreceptor (PR) fate of R3 and R4, and the rotational
movement performed by the developing ommatidia. In the adult eye, each
ommatidium contains six outer PRs (R1-R6), which are positioned in a
trapezoidal arrangement, and two inner PRs (R7 and R8), which are located in
the center of this trapezoid. The trapezoidal arrangement comes in two chiral
shapes (generated through the asymmetric positioning of R3 and R4) that form a
mirror-image symmetry on either side of the dorsoventral (DV) midline, also
called the equator (Fig. 1)
(reviewed by Tomlinson,
1988).
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The spalt (sal) gene complex encodes two related
transcription factors, spalt major (salm) and
spalt-related (salr), which are required for the
differentiation of the inner PRs (R7 and R8)
(Mollereau et al., 2001). In
sal null mutant (sal) retinas, the
morphology of the rhabdomeres (the light-sensing structure of the PR), and the
expression patterns of rhodopsins in R7 and R8, change to become
identical to those of the outer PRs
(Mollereau et al., 2001
). More
recently, we found that sal is required for R7 differentiation in the
third instar larva, as the expression of several R7 markers is lost in
sal null mutant clones (Domingos
et al., 2004
). In this last study, we found that the expression of
Enhancer of split m
0.5-lacZ
(m
0.5-lacZ a direct target of Notch signaling
in R4 and R7) (Cooper and Bray,
1999
; Cooper and Bray,
2000
) is lost in sal clones both in R4
and R7. This result was an indication that sal could also be required
for R3/R4 specification and PCP establishment.
Here, we demonstrate that sal is required for the establishment of
proper ommatidial chirality. We show that the PCP defects observed in
sal clones are due to incorrect specification of
the R3/R4 cells, as several R3/R4 markers are not correctly expressed. We find
that sal is required for R3/R4 specification upstream of
seven-up (svp), a gene that is also required for R3/R4
specification and PCP establishment (Fanto
et al., 1998; Mlodzik et al.,
1990
). Finally, we show that, posterior to row seven, svp
represses sal in R3/R4 in order to maintain R3/R4 identity and to
inhibit the transformation of these cells to an R7 cell fate.
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Materials and methods |
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Clones of mutant eye tissue were generated by the Flp/FRT technique
(Golic, 1991). Flipase
expression was induced under the control of the eyeless
(Newsome et al., 2000
) or
heat-shock promoters (larvae were heat-shocked at 37°C for 1 hour, 48
hours after egg laying).
Immunohistochemistry and histology
Third instar larval eye discs were dissected in 1xPBS, fixed in
1xPBS + 4% formaldehyde for 20 minutes at room temperature, and washed 3
times with PBX (1xPBS + 0.3% Triton X-100). Primary antibodies were
incubated in BNT (1xPBS, 1% BSA, 0.1% Tween 20, 250 mM NaCl) overnight
at 4°C. Primary antibodies were as follows: rabbit anti-Salm
(Kuhnlein et al., 1994), anti
ß-gal (Cappel), rat anti-ELAV (DSHB), mouse anti-Ro (DSHB), rabbit
anti-BarH1 (Higashijima et al.,
1992
) and mouse anti-Fmi (Usui
et al., 1999
). Samples were washed 3 times with PBX and incubated
with appropriate secondary antibodies (Cy3, Cy5, FITC from Jackson
Immuno-Research Laboratories) for 2 hours at room temperature. Samples were
mounted in Vectashield (Vector Laboratories) and analyzed on a Zeiss LSM 510
confocal microscope. Tangential sections of adult eyes were performed as
described (Tomlinson and Ready,
1987
).
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Results |
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sal is required in R3 for establishment of correct ommatidial chirality
In sal ommatidia, the rhabdomeres of both R7 and
R8 acquire the typical `large' morphology of outer PRs
(Mollereau et al., 2001). This
leads to the disruption of the normal position of rhabdomeres in each
ommatidium, thus making the evaluation of PCP defects impossible. However, on
the borders of sal clones, it is possible to detect
mosaic ommatidia with wild-type R7 and R8, allowing the analysis of PCP
defects.
To investigate the role of sal in R3/R4 specification and PCP
generation, we analyzed a large number of such mosaic
sal ommatidia and scored them for PCP defects.
Within 1391 mosaic ommatidia, we could identify 29 different mosaic
configurations with a normal number of PRs, where it was possible to score
polarity. Fig. 2A shows
examples of sal clones, containing mosaic
ommatidia, which display typical PCP defects with chirality inversions and
mis-rotations. Interestingly, this analysis of mosaic clusters reveals a
requirement of sal in R3 for PCP establishment. In the 16
configurations that always adopt the correct chiral form, R3 is always
sal+ (Fig.
2B). In mosaic ommatidia adopting the wrong chiral form, the cell
in the R4 position is invariably sal
(Fig. 2C). Presumably, in such
ommatidia, the sal precursor for R3 developed
incorrectly as an R4. In ommatidia where only the R3 precursor was
sal, we found seven cases that adopted the wrong
chirality (an example is shown in Fig.
2A, top panel), and eight cases with the correct chirality (data
not shown). These results demonstrate randomization of the R3/R4 chirality
choice when the R3 precursor is sal. We also found
15 ommatidia with symmetric R4/R4 (14) or R3/R3 (1) configurations (data not
shown). In each of these 15 symmetric ommatidia, at least one cell of the
R3/R4 pair was sal. Thus, the PCP requirement of
sal is similar to that of fz
(Tomlinson and Struhl, 1999;
Zheng et al., 1995
), and
demonstrates that sal is required in R3 for correct ommatidial
chirality and PCP establishment.
|
|
Activation of Fz/PCP signaling leads to the transient transcriptional
upregulation of Dl in R3 within approximately two to three ommatidial
rows, which can be observed by in situ hybridization
(Parks et al., 1995), or with
a Dl enhancer detector line (Fanto
and Mlodzik, 1999
). During this period, most
sal ommatidia fail to upregulate Dl in R3,
and both cells of the R3/R4 pair show a low level of expression
(Fig. 3C). We can also find
clusters where the cell in the R4 position has higher levels than R3, or where
both cells in the R3/R4 pair have increased levels of Dl expression
(Fig. 3C). These results
suggest that sal is required for the correct interpretation of the
Fz/PCP-mediated polarity signal and for the upregulation of Dl
expression in R3.
The expression of m0.5-lacZ, a marker of Notch
signaling activation in R4 (second tier), is lost in
sal ommatidia
(Fig. 3D)
(Domingos et al., 2004
).
Interestingly, in mosaic ommatidia, where only one cell of the R3/R4 pair is
sal, either R3 or R4, we observe reduced expression
of m
0.5-lacZ. This indicates that sal is
required for normal levels of m
0.5-lacZ expression,
both non-cell-autonomously in R3 (arrows in
Fig. 3D), and cell-autonomously
in R4 (arrowhead in Fig. 3D),
which is surprising given the specific genetic requirement in R3 only for
ommatidial polarity (Fig. 2). The non-cell autonomous requirement of sal in R3, for normal
m
0.5-lacZ expression in R4, is consistent with the
deficient upregulation of Dl in R3
(Fig. 3C). It is also possible
that the lack of asymmetric localization of PCP proteins, as seen in the case
of Fmi in sal
(Fig. 3B), is responsible for
the Notch activity modulation, as previously proposed
(Strutt et al., 2002
). The
autonomous requirement of sal in R4 for
m
0.5-lacZ expression either could be due to a
defective activation of Notch signaling, or sal may be required for
transcriptional activation of E(spl)m
in parallel to Notch
signaling (see also Discussion).
sal acts upstream of svp during R3/R4 specification
To further investigate the role of sal in R3/R4 specification, we
asked whether sal is required for svp expression in R3/R4.
It was previously shown that svp is also required in R3 for proper
R3/R4 specification and PCP establishment in the eye
(Fanto et al., 1998). Our data
indicate that sal genes are cell-autonomously required for the
expression of svp (svprA28; svp-lacZ)
(Hoshizaki et al., 1994
) in R3
and R4 (Fig. 3F). Conversely,
in svp (svpe22) clones, the
initiation of salm expression in R3/R4 is normal, although
salm is not repressed in more posterior rows
(Fig. 4A). As in
sal clones, in svp
clones Fmi is not properly localized in R3/R4
(Fig. 4B) and the expression of
m
0.5-lacZ is lost
(Fig. 4C). These results
suggest that sal acts upstream of svp during R3/R4
specification.
|
Repression of sal in R3/R4 by svp is required for inhibition of R7 cell fate
In svp mutants, R3, R4, R1 and R6 fail to adopt their normal fate
and are transformed into the R7 fate
(Mlodzik et al., 1990). The
PCP defects observed in svp mutant ommatidia were attributed to a
failure of the R3/R4 cells to interpret the Fz/PCP signal, because of their
transformation to R7 (Fanto et al.,
1998
). Consistent with this, we have observed that in
svp clones in the larval eye disc, both R3 and R4
express the R7 marker prospero (data not shown). In addition, in
svp clones, salm is not repressed in R3/R4
by row seven, but continues to be expressed in more posterior rows
(Fig. 4A) [sal
normally starts to be expressed in R7 by rows seven to nine, and is both
required and sufficient for R7 differentiation during larval stages
(Domingos et al., 2004
)]. Thus,
it is likely that, in svp clones, the ectopic
expression of salm in R3/R4 posterior to row seven is responsible for
their transformation into R7.
To test this hypothesis, we have analyzed the number of large (R1-R6) and
small (R7 and R8) rhabdomeres in
svp/sal double mutants
(Fig. 5). In
svp clones, most ommatidia have three to five cells
with small rhabdomeres because of the transformation of R3, R4, R1 and R6 into
R7 (Fig. 5A,D)
(Mlodzik et al., 1990). In
sal clones, most ommatidia have eight large and no
small rhabdomeres, due to the transformation of R7 and R8 to the outer PRs
subtype (Fig. 5B,D)
(Mollereau et al., 2001
).
Strikingly, svp/sal
double mutant ommatidia have the same appearance as single
sal mutant clusters
(Fig. 5C,D). This result
demonstrates that sal is required, downstream of svp
mutation, for the transformation of R3/R4 into R7. In conclusion, sal
is required upstream of svp during R3/R4 specification (rows three to
seven), but repression of sal by svp posterior to row seven
is required to avoid the transformation of these cells into R7
(Fig. 6 and Discussion).
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Discussion |
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We show that sal is also required for PCP establishment in the
Drosophila eye. The analysis of sal
mosaics reveals that sal is specifically required in R3 for
establishment of ommatidial chirality (Fig.
2). The analysis of sal clones in the
larval eye reveals that sal is required for the asymmetric
localization of Fmi in the R3/R4 precursor pair
(Fig. 3B) and upregulation of
Dl in R3 (Fig. 3C). In
a similar manner to sal, fz is required in R3 for the establishment
of ommatidial chirality (Tomlinson and
Struhl, 1999), and in fz mutants, unlike in stbm
or dgo mutants, Fmi is not localized asymmetrically in R3/R4
(Das et al., 2002
). However,
fz mutants also have a non-autonomous effect, disrupting PCP in
ommatidia located outside the mutant clone
(Zheng et al., 1995
), which is
not observed in sal mutants. Thus, sal is required for the
correct interpretation of the fz-mediated polarity signal in R3, but
not for the propagation of the polarity signal across the equatorial-polar
axis. This also indicates that the expression of Fz should not be affected in
sal clones. Finally, in
sal clones, all PCP proteins tested (Fmi, Fz, Dgo)
exhibit a defect in their asymmetric localization
(Fig. 3B and data not shown),
but their overall expression remains unaffected. A possible interpretation of
these results is that sal transcription factors induce the expression
of a yet unidentified factor, which is required for the asymmetric
localization of PCP genes.
Therefore, our results suggest that sal is required upstream or in
parallel to the Fz/PCP pathway for R3/R4 specification. Also, in support of
this model, sal expression is not affected in R3/R4, either in gain-
or loss-of-function experiments with members of the Fz/PCP and Notch signaling
pathways [fzR52 clones data not shown;
dsh1, sev-Gal4/uas-Dsh and
sev-Gal4/uas-Nicd
(Cooper and Bray, 1999);
sev-Fz and sev-N*
(Fanto and Mlodzik, 1999
);
fmi clones and sev-Fmi (Das et al.,
2000)].
We show that sal is required cell-autonomously in R4 for normal
levels of m0.5-lacZ expression
(Fig. 3D). This requirement of
sal in R4 could be due to a defect in the activation of Notch
signaling (e.g. sal may be required for the expression of
Notch or Su(H)). Alternatively, sal may be required
for transcriptional activation of E(spl)m
, in parallel to
Notch signaling. We favor the latter possibility, as the expression of a
transgenic line, where lacZ is under the regulation of 12
Suppressor of Hairless multimerized-binding sites
[12Su(H)-lacZ (Go et al.,
1998
)], is not affected when R4 is sal
(data not shown). The 12Su(H)-lacZ transgenic line is a reporter for
Su(H)-dependent Notch signaling, and thus, sal is not required for
the expression or activation of Notch, Su(H) or other components required for
signaling. In addition, exogenous expression of a constitutively activated
Notch (sev-Nact) can rescue
m
0.5-lacZ expression in
sal clones (Fig.
4E). Altogether, these results suggest that sal acts in
parallel to Notch signaling for the transcriptional activation of
E(spl)m
. Finally, although there is a reduction of
E(spl)m
expression when R4 is sal,
this does not correspond to chirality defects in mature ommatidia
(Fig. 2). This suggests that
other genes may be redundant to sal in R4 for PCP establishment.
sal and svp in R3/R4 versus R7 specification
Several pieces of evidence demonstrate that sal is required
upstream of svp for R3/R4 specification: (1) sal is required
for svp expression in R3/R4 (Fig.
3F); (2) both sal and svp are required in R3 for
the establishment of proper ommatidial chirality
(Fig. 2) (Fanto et al., 1998); (3) in
both sal and svp mutants Fmi is not asymmetrically localized
in R3/R4 (Fig. 3B,
Fig. 4B) and
m
0.5-lacZ expression is lost in R4
(Fig. 3D,
Fig. 4C); and (4) exogenous
expression of svp in R3/R4 (sev-svp) can rescue the
expression of m
0.5-lacZ in
sal clones (Fig.
4D).
In addition, we show that, posterior to row seven, svp is required
to repress sal expression in R3/R4
(Fig. 4A), and sal is
responsible for the transformation of R3/R4 into R7 in svp mutants
(Fig. 5). Based on our current
and previous results, which demonstrate that sal is both necessary
and sufficient for R7 differentiation posterior to row seven
(Domingos et al., 2004), we
propose a model for the action of sal and svp during R3/R4
specification (Fig. 6): from
rows three to seven, sal is required for svp expression in
R3/R4 and for R3/R4 specification; posterior to rows seven to nine, repression
of sal by svp in R3/R4 is necessary for the maintenance of
R3/R4 identity and the inhibition of R7 fate. This dual regulation between
sal and svp helps to understand the complex
sal phenotype in R3/R4. Strikingly, although
svp expression is lost in sal R3/R4, these
cells do not get transformed into R7, but keep an outer PR identity. Thus, in
the absence of sal, the presumptive R3/R4 remain as outer PRs with an
unspecified subtype identity.
In conclusion, our results demonstrate that sal is required in R3
to allow normal Fz/PCP signaling to specify the R3 and R4 cell fates.
Ommatidia mutant for sal show defects that are very similar to those
observed in fz and dsh mutants, as judged by the loss of
asymmetric Fmi localization at the equatorial side of the R3/R4 precursors,
and by the lack of Dl and E(spl)m upregulation within
the R3/R4 pair. In addition, sal is required upstream of svp
for normal R3/R4 specification. Finally, our results show that, posterior to
row seven, svp represses sal in R3/R4 in order to maintain
R3/R4 identity and to inhibit transformation of these cells to the R7 cell
fate.
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ACKNOWLEDGMENTS |
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