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First published online February 20, 2009
doi: 10.1242/10.1242/dev.027318
1 Program in Developmental and Stem Cell Biology, Hospital for Sick Children
Research Institute, 101 College Street, Toronto, Ontario, Canada M5G
1L7.
2 Department of Molecular Genetics, University of Toronto, 1 King's College
Circle, Toronto, Ontario, Canada M5S 1A8.
* Author for correspondence (e-mail: howard.lipshitz{at}utoronto.ca)
Accepted 11 January 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Delta, Drosophila, Hindsight (Pebbled), Notch, Cone cell, Eye
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Kadesch, 2004). Typically, one
cell expresses higher or more sustained levels of ligand. Thus, inductive
signaling occurs via unidirectional activation of Notch. The expression
patterns of Notch ligands through development are complex and contribute to
differential activation of the pathway during signaling. Although regulating
the cell-specific transcription of Notch ligands is conceptually the simplest
mode for control of the polarity and intensity of Notch signaling, we are just
beginning to understand some of the signals and transcription factors involved
(Bash et al., 1999;
Ciechanska et al., 2007;
Sasaki et al., 2002).
Additional ways of modulating ligand activity include post-translational
modification via ubiquitylation to regulate endocytosis and degradation
(Le Borgne et al., 2005;
Nichols et al., 2007),
proteolysis to affect Notch-binding affinity
(Nichols et al., 2007) and
regulation of intracellular localization by other proteins
(Bray, 2006). Controlling the
level or the activity of the Notch ligand is crucial for determining cell fate
in both the responding and, possibly, signaling cells. For example, studies in
the Drosophila ovary have established that modulated levels and
durations of Delta signaling are responsible for inducing different follicle
cell fates (Assa-Kunik et al.,
2007).
The Drosophila eye disc provides a tractable system to study how
the coordination of juxtacrine signaling pathways with cell-specific intrinsic
factors leads to differential cell-fate determination
(Doroquez and Rebay, 2006).
During Drosophila eye development, a stereotypical sequence of cell
recruitments and inductions pattern the cells comprising an ommatidial cluster
(Freeman, 1997). The first
five photoreceptor (R) precursor cells to be born – R8, -2, -5, -3 and
-4 – are patterned immediately posterior to the morphogenetic furrow. A
second wave of division gives rise to the rest of the cells that will form an
ommatidial unit. These include precursor cells for the R1, -6 and -7 neurons
as well as for the non-neuronal cone and pigment accessory cells
(Ready et al., 1976;
Wolff and Ready, 1991). In
mutants where the cone cells are not correctly specified, apical lens
secretion is defective and the structural integrity of the ommatidium is
compromised (Batterham et al.,
1996; Fu and Noll,
1997). Determination of R1, -6, -7 and the cone cells occurs in
the larval eye disc. Various cell-subtype-specific transcription factors for
these cells are regulated by the AML-1 like transcription factor, Lozenge (LZ)
(Daga et al., 1996).
Differential EGF receptor (EGFR) and Notch-Delta (N-DL) signaling among these
cells act in concert with LZ to evoke cell-specific readouts: the R1 and R6
precursor pair express the Bar and Seven up (SVP) transcription factors
(Daga et al., 1996;
Higashijima et al., 1992;
Mlodzik et al., 1990); the R7
precursor cell expresses Prospero (PROS)
(Chu-Lagraff et al., 1991;
Tomlinson and Struhl, 2001;
Xu et al., 2000) and represses
SVP (Daga et al., 1996); and
the cone cells express D-PAX2 (Shaven – FlyBase), CUT and PROS
(Blochlinger et al., 1988;
Flores et al., 2000;
Fu and Noll, 1997;
Xu et al., 2000) (see
Fig. 1).
Differential cell fate in the eye disc depends on the precise timing of
Dl transcription. The initiation of Dl transcription in the
eye disc requires the activity of two secreted factors: Hedgehog (HH) and
Decapentaplegic (DPP) at the furrow
(Greenwood and Struhl, 1999).
After neuronal determination, Dl transcription in R-precursor cells
is elevated by the EGFR pathway in the developing ommatidial clusters. Clones
lacking the EGFR ligand, Spitz (SPI), show reduced DL expression posterior to
the furrow (Tsuda et al.,
2002). Activation of EGFR relieves repression of the Dl
gene by the transcription factor Charlatan (CHN)
(Fig. 1)
(Tsuda et al., 2006).
The zinc-finger transcription factor, Hindsight (HNT) (Pebbled –
FlyBase), regulates several aspects of eye development
(Pickup et al., 2002;
Yip et al., 1997). It is
required for the assembly of the five-cell preclusters and the timing of their
neuronal determination, as well as their subsequent rotation. Later in eye
development, HNT is necessary for photoreceptor rhabdomere morphogenesis and
ommatidial integrity. Genetic screens have uncovered several potential
transcriptional targets for HNT. Loss-of-function alleles of both
charlatan and Delta are dominant modifiers of a
temperature-sensitive hnt allele, hntpeb
(Wilk et al., 2004).
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), spa-Gal4
(Jiao et al., 2001),
lz-Gal4 (Batterham et al.,
1996) and Dl05151
(Weber et al., 2000). The
pGMR-Gal4 (on the second chromosome), UAS-Dl::GFP and FLP
recombinase stock w1118; MKRS,
P[ry+t7.2=hsFLP]86E/TM6B,Tb were obtained from the
Bloomington Drosophila Stock Center. The hnt mRNAi lines 2A
and 2B (on the second and third chromosomes, respectively) were generated as
follows: the hnt RNAi construct was targeted to a 0.7 kb region of
the third exon and made by fusion of hnt genomic and cDNA fragments
(Kalidas and Smith, 2002). The
1 kb genomic fragment, including 0.3 kb of the second intron and a 0.7 kb
third exon portion downstream of the splice donor sequence, was PCR-isolated,
ligated to a 0.7 kb inverted hnt cDNA fragment corresponding to the
genomic region, and cloned into pUAST. The ligated RNAi construct was injected
into fly embryos to produce the transgenic hnt RNAi fly lines. Primer
sequences used in construction of the hnt RNAi transgenes are
available upon request. To generate the UAS-hnt transgenic line full-length hnt PCR products containing the hnt coding region and second intron, but not the 5' and 3' UTRs, were synthesized by PCR from w1118 genomic DNA. PCR products were first cloned into pCR2.1-TOPO vector (Invitrogen), then into a pUAST-egfp vector. Transgenic lines were generated by germline transformation in a w1118 background.
FLP-induced clones in the eye disc
Clones were induced in the eye as described previously
(Pickup et al., 2002).
Immunohistochemistry
Antibody staining was carried out using standard protocols
(Wolff, 2000). Primary
antibodies obtained from the Developmental Studies Hybridoma Bank were mouse
anti-HNT monoclonal (27B8 1G9, 1:10 dilution)
(Yip et al., 1997), rat
anti-ELAV (7E8A10-s, 1:20) (O'Neill et
al., 1994), mouse anti-CUT (2B10-s, 1:30)
(Blochlinger et al., 1993),
mouse anti-ROUGH (62C2AB, 1:100) and mouse anti-PROS (MR1A-s, 1:5)
(Kauffmann et al., 1996).
Other primary antibodies were rabbit anti-D-PAX2 (1:50)
(Fu and Noll, 1997), rat
anti-BAR-H1 (1:1000) (Higashijima et al.,
1992), rabbit anti-CHN (1:100)
(Tsuda et al., 2006), chicken
anti-GFP (Abcam, 1:500) and rabbit anti-β-galactosidase (Cappel, 1:1000).
All secondary antibodies were used at a dilution of 1 in 250 and were obtained
from Molecular Probes or Invitrogen. They were Alexa fluor 555-conjugated goat
anti-mouse antibody, Alexa fluor 488-conjugated goat anti-rat antibody, Alexa
fluor 555-conjugated goat anti-rabbit antibody, Alexa fluor 488-conjugated
goat anti-rabbit antibody, Alexa fluor 488-conjugated goat anti-chicken
antibody and Alexa fluor 647-conjugated goat anti-rat antibody.
To visualize two different mouse primaries simultaneously (Fig. 3C''), we used a double-labeling technique described in the Jackson ImmunoResearch catalog. Eye discs were incubated in the mouse anti-CUT antibody first, followed by an unconjugated rabbit anti-mouse Fab antibody (Jackson, dilution of 1:250), which was then visualized by a standard Alexa fluor 488-conjugated anti-rabbit tertiary antibody. The discs were then placed into the mouse anti-HNT antibody, which was detected by an Alexa fluor 555-conjugated anti-mouse secondary antibody (1:500 dilution). Although this technique was optimized, we have not achieved perfect separation of the two different primary signals and, as a result, some cross-reactivity is seen in the cone cells.
Microscopy, image capture and processing
A Zeiss Axiovert 100 microscope with LSM510 software was used for laser
confocal microscopy. Images were reconstructed using the Volocity software
(Improvision) and processed and displayed using Photoshop (Adobe). Adult eyes
were photographed using a Canon G5 camera mounted on a Leica MZ75 microscope.
Images were processed using Photoshop software (Adobe).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). To
assess the causes of this phenotype we produced transgenic flies in which
hnt expression was knocked down by RNAi
(Fig. 2). In wild-type eye
discs, HNT is expressed in all R-cell precursors posterior to the furrow but
is not found in cone-cell precursors (Fig.
2A,A'; see Fig. S1A,B in the supplementary material)
(Pickup et al., 2002). When
UAS-hntRNAi constructs were expressed using the pGMR-Gal4
driver in all cells posterior to the morphogenetic furrow, HNT protein levels
were partially reduced in the R3 and R4 cells and strongly reduced in the R1,
R6 and R7 cells (Fig.
2C-D'; see Fig. S2A-B'' in the supplementary material).
Anti-ELAV antibody staining demonstrated that neuronally determined R cells
formed, even though they lacked HNT protein
(Fig. 2B' and inset).
This result is consistent with those from a previous clonal analysis, which
showed that HNT function is not absolutely required for R cell development
but, rather, affects the timing of differentiation and the morphology of the
developing R cell clusters (Pickup et al.,
2002).
The majority of hnt RNAi flies die during late pupal stages;
however, with only one dose of the hnt RNAi transgene, adult escapers
hatched. The eyes of these escapers were glossy with mottled pigmentation and
no well-defined facets (Fig.
2E). The same phenotype was observed for another line with a
different insertion site of the hnt RNAi transgene (data not shown).
Smoothened eyes are a sign of faulty lens secretion by the underlying cone and
pigment cells (Cagan and Ready,
1989b), whereas altered patterns of pigmentation imply defective
secondary and/or tertiary pigment cells. The eyes of hnt RNAi
knockdown flies also have melanized necrotic patches on their surface,
indicative of considerable cell death (Fig.
2E, arrowheads). All aspects of this hnt knockdown
phenotype are reminiscent of the eyes of lozenge (lz)
mutants where there are demonstrable defects in the accessory cone and pigment
cells that shape the ommatidial lattice
(Batterham et al., 1996).
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).
hnt knockdown in these cells gave a gradient of loss of pigmentation
(most severe at the posterior) (Fig.
2F) in every fly, again indicating that at least a part of the
phenotype was caused by defects in the accessory cells. Although
lz-Gal4 drives expression in the cone-cell precursors, this phenotype
was unlikely to have been caused by hnt knockdown in these cells as
HNT is not expressed in cone cells. Furthermore, when the hnt RNAi
transgene was expressed in the cone cells in the larval eye disc but not in
the R cells, using the sparkling-Gal4 driver
(Jiao et al., 2001), there was
no smoothening, loss of pigmentation or necrosis
(Fig. 2G). This driver promotes
expression in at least some of the cone-cell precursors at the right time in
the larval eye disc, although it is not clear whether it is as strong a driver
as the pGMR and lz-drivers (see Fig. S3A,A' in the
supplementary material). In summary, knockdown of hnt in a subset of R-cell precursors causes defects in lens secretion and pigmentation that appear to result from a non-autonomous effect on accessory cells in the developing eye disc.
HNT function in developing R cells is necessary for cone-cell induction
The R1, R6 and R7 precursor cells arise from a basal pool of
undifferentiated cells in a second wave of division posterior to the
morphogenetic furrow. They are the last R cells to be determined in the
third-instar eye disc (Ready et al.,
1976) and play a pivotal role in cone-cell induction
(Lai, 2002). This induction
requires R-cell-precursor specification by LZ
(Daga et al., 1996), as well
as a precisely timed window of activation of the N-DL and EGFR signaling
pathways (Flores et al., 2000)
(Fig. 1).
As reduction of HNT function in just the inducing R-precursor cells
produced a glossy eye phenotype (Fig.
2F), we reasoned that part or all of this phenotype might be
caused by a cone-cell defect. To test this hypothesis we examined expression
of the cone-cell determination marker, CUT
(Blochlinger et al., 1993), in
third-instar eye discs of pGMR-Gal4; hnt RNAi flies. In control
discs, a pair of cone-cell precursor nuclei expressed CUT in a `bow-tie'
configuration by column 7. By column 10, CUT was found in two more nuclei per
ommatidium, making up a cone-cell `quartet' at the posterior of the disc
(Fig. 3A). In eye discs in
which HNT expression had been knocked down, there was a depletion of CUT
expression: CUT was found later (column 10) and at lower levels than in the
control discs at a similar stage of development (compare
Fig. 3A and 3B). No cone-cell
precursor quartets were found at the posterior of the mutant discs; at most
two cells per ommatidium expressed CUT. When pupal discs of this genotype were
examined, CUT expression was initially lower than that in a no-driver control,
but later increased to control levels (data not shown). This result suggests
that the phenotype we observe in discs where HNT expression is knocked down
may be due to a delay, rather than an outright block, in cone-cell induction.
A weaker phenotype was observed in lz-Gal4; hnt RNAi larval
eye discs in which knockdown occurred in R1, 6 and 7: CUT protein levels were
close to wild type; but again, CUT was most often expressed in only two of the
four cone-cell precursors (data not shown).
To determine whether depletion of CUT was a bona fide hypomorphic phenotype
of hnt, we tested for expression of the same marker within clones
mutant for hnt1142, an antibody-null allele
(Wilk et al., 2000)
(Fig. 3C-C''). Within
these clones we found that CUT protein was absent from ommatidia lacking HNT
expression (n=11 clones). Occasionally, a few isolated HNT-negative,
CUT-positive cells were found along the borders of clones, indicating possible
local non-autonomy. In order to establish which R cells are required to induce
CUT expression in the cone-cell precursor, we scored border ommatidia along
the edges of hnt1142 clones
(Fig. 3D,D'). For
technical reasons we are only able to examine clusters with a full complement
of R cells in a wild-type configuration. This allowed us to unequivocally
assign cone cells at the two-cell stage to a particular cluster. We examined
38 clones and found four examples of clusters where only R1/R6 (as marked by
BAR) and R7 were mutant for hnt
(Fig. 3D,D'). In all four
cases, there was no, or significantly reduced, CUT expression. We never saw
any cases of ommatidia in which all the R cells (including R1 and R6)
expressed HNT and CUT was not expressed (n=68). We were not able to
do the reciprocal experiment (where R1/R6 express HNT and all the other R
cells are mutant) as this almost always gave defective ommatidia with
incorrect R-cell specification or altered polarity
(Pickup et al., 2002). In
ommatidia in which one cell out of the R1-R6 pair was mutant for hnt,
these clusters sometimes had normal cone-cell induction (67%, n=24),
indicating that one signaling cell may sometimes be enough to induce some CUT
expression (scored at the two-cone-cell stage). A similar scenario has been
shown for R7 induction by these cells
(Tomlinson and Struhl,
2001).
|
). In
wild-type third-instar eye discs, this transcription factor is expressed in a
similar pattern to CUT and is part of the same pathway required for cone-cell
differentiation (Flores et al.,
2000; Fu and Noll,
1997) (see Fig. 1).
In the interior of the hnt-mutant patches, D-PAX2 expression was
absent (n=10 clones) (Fig.
3E-E''). Early D-PAX2 expression is required for timely
initiation of CUT expression in wild-type larval and early pupal eye discs
(Fu and Noll, 1997). Later
pupal eye discs from strong D-Pax2 mutants did express CUT,
indicating that this late phase of CUT expression is D-PAX2-independent. Thus
the effects we observed on CUT expression in the hnt RNAi mutants are
likely to have been caused by depletion of D-PAX2.
Taken together, the results shown in
Fig. 3 and the knockdown
phenotype generated by the lz-Gal4 driver
(Fig. 2F) suggest that HNT
function is required in the R1/R6-cell precursor cells during larval
development for proper cone-cell induction. As HNT is not expressed in pigment
cells, the effect of hnt RNAi on adult eye pigmentation is likely to
be a secondary effect of this cone-cell defect. Cone cells have been shown to
be necessary for proper pigment-cell determination during pupal stages
(Nagaraj and Banerjee,
2007).
In order to test whether HNT is sufficient to induce cone-cell fate we used a UAS-hnt line to over- and misexpress HNT early in all (pGMR-Gal4 driver) or a subset (lz-Gal4 driver) of the undifferentiated cells behind the furrow (see Fig. S4A-C' in the supplementary material). In this context, HNT expression is sufficient to induce ectopic anti-CUT expression in many of the basal undifferentiated cells (see Fig. S4B',C' in the supplementary material). These cells have thus taken on this aspect of cone-cell determination.
HNT is not required autonomously for R1/R6/R7 subtype specification
There are at least two possible explanations for the failure of cone-cell
precursors to adopt a normal fate in the larval eye disc. One is that the
R-cell precursor cells that usually induce their neighboring cells to initiate
the cone-cell determination pathway are not able to signal normally –
the signal is reduced or not timed correctly. A second possibility is that the
signaling cells themselves are not specified normally. We tested this latter
hypothesis by staining for the R1/R6 subtype marker, BarH1 (BAR)
(Higashijima et al., 1992), as
well as for the R7/cone precursor cell marker, Prospero (PROS)
(Kauffmann et al., 1996)
(Fig. 4). Both of these
transcription factors are necessary to determine their respective cell
subtypes and they also serve as readouts for the LZ pathway. In lz
mutants these proteins are not expressed and normal R cells fail to develop
(Daga et al., 1996).
When pGMR-Gal4; hnt RNAi knockdown flies are compared
with a non-driver control that has normal levels of HNT, expression of BAR in
the R1 and R6 precursor cells was found to be normal; BAR expression began at
the same time and was at comparable levels to BAR-positive cells in the
control eye discs (Fig. 4A,B).
Thus, the mutant phenotype we observed in hnt knockdown discs was not
a result of delayed R1/R6 determination. The R7 precursor cell was also
specified correctly. In hnt RNAi mutant discs, a single ELAV-positive
cell also strongly expressed PROS (Fig.
4D, inset). As PROS expression in the R7 precursor is in part
activated by EGFR effectors (Xu et al.,
2000), this result also indirectly indicates that there is
functional SPI ligand secreted by the R1/R6 cells (see
Fig. 1). The PROS expression
normally found in the cone-cell precursors in control eye discs
(distinguishable by their lack of ELAV in
Fig. 4C, inset) was much lower
in the cone-cell precursors in `mutant' discs (arrowheads in
Fig. 4D, inset). This
observation is consistent with the results described previously and reflects a
lack of proper cone-cell determination.
In summary, the results presented above demonstrate that wild-type HNT function is not directly required to determine R1/R6 cell identity. The R-cell precursor cells that are necessary to induce the cone cells are specified correctly after hnt knockdown. As the markers tested are induced by LZ signaling, this result also indicates that these cells have a normal LZ pathway operating in them in the knockdown scenario.
HNT is necessary for high-level DL expression and signaling
The membrane-bound N ligand, DL, is required in determined R cells for
cone-cell induction (Flores et al.,
2000). The timing of Delta expression in the R1 and R6
precursor cells is regulated by the activation of the SU(H)-EBI-SMRTR complex,
which removes the transcriptional repressor CHN from the Dl promoter
(Tsuda et al., 2006) (see
Fig. 1). We have previously
demonstrated that clones of hnt antibody-null alleles have lowered
levels of Dl-lacZ expression in their R cells
(Wilk et al., 2004). To test
whether the R1 and R6 cells in particular have less Dl expression in
a hnt RNAi mutant eye, we examined the expression of the
Dl05151 enhancer trap in a lz-Gal4; hnt
RNAi background. Control larval eye discs showed expression of this
Dl reporter gene in R cells that mimics endogenous Dl
expression in the late-developing clusters
(Weber et al., 2000):
β-galactosidase was found in the R1 and R6 precursor cells but was low in
the R7 precursor cell (Fig.
5C,C'). This asymmetrical ligand expression is thought to
account for the role of R1/R6 in inducing R7 cell fate
(Cooper and Bray, 2000;
Parks et al., 1995). In eye
discs from lz-Gal4; hnt RNAi larvae, the levels of
Dl reporter expression in the R1 and R6 cells (identified by Bar
expression) were reduced (Fig.
5D,D'). Cells that lacked HNT expression had lowered
Dl expression 37% of the time (n=165 R1/R6)
(Fig. 5B,D,D').
Apparently, this reduced level of DL is sufficient for normal R7 induction, as
the R7 precursor cell still expressed PROS and ELAV
(Fig. 4D and inset). No extra
cells expressed the R1/R6 determination marker, BAR, in the mutant eye discs
(Fig. 4B), indicating there had
been no R7 to R1/R6 fate transformation. Notably, Dl expression was
also reduced in the other R cells (where HNT was still expressed). One
possible explanation for this is that HNT-regulated signals from R1-6/7 are
necessary to maintain later Dl levels in other cells.
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).
When we drove DL-GFP in the lz-Gal4; hnt RNAi background, it
partially suppressed the knockdown phenotype (compare
Fig. 5E and 5G). The eyes of
these rescued flies were less rough and had normal pigmentation, implying that
adding DL back just to the R1, R6 and R7 cells compensated for their lack of
HNT. This effect was not caused by Gal-4 titration, as a UAS-nuclear
lacZ transgene is not able to suppress the lz-Gal4;
hnt RNAi eye phenotype (Fig.
5H). Taken together, these results suggest that, although not involved in R1 and R6 subtype specification, HNT is required in these cells for efficient DL expression in, and signaling by, these cells. The fact that DL expression is sufficient to almost completely suppress the hnt RNAi phenotype is consistent with a role for HNT protein upstream of Delta transcription and shows that HNT does not regulate any DL-independent pathways that are necessary for cone-cell induction.
HNT does not regulate DL expression in the R1-6 precursor cells via CHN
The current understanding of Dl regulation in the R1/R6 signaling
cells entails an EGFR-induced derepression of the block by CHN on Dl
transcription (Tsuda et al.,
2006) (see Fig. 1).
To determine whether HNT modulates DL levels via this pathway, we assayed CHN
expression in eye discs with lowered HNT activity. In no-driver control discs,
CHN is expressed at high levels in a band of basal nuclei around the furrow
(Tsuda et al., 2006). CHN
expression then drops and only resumes in the most posterior column of
developing clusters (Fig. 5I,
arrowhead). In eye discs where hnt expression had been knocked down
in late-developing clusters (Fig.
5J,J'), CHN expression at the furrow was unaffected. In the
posterior parts of these discs, there was still no CHN expression in the R1-
and R6-cell precursors (marked with BAR in
Fig. 5J'). This implies
that HNT does not act upstream of Chn to affect Dl
expression in these cells (Fig.
1). Notably CHN was derepressed in some of the other determined
R-cell precursors in the hnt mutant (arrows in
Fig. 5J'). This is
consistent with the non-autonomous effect on Dl expression shown in
Fig. 5D and D', and
supports the notion that there may be a HNT-dependent signal required for late
Chn repression in these precursor cells. Taken together these results
suggest that HNT affects cone-cell induction by the R1/R6 precursor cells in a
CHN-independent pathway (Fig.
1).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Earlier reports describing HNT function in the ovary show that HNT
expression is regulated by the Notch signaling pathway and controls follicle
cell proliferation and differentiation
(Sun and Deng, 2007). In this
paper we report that HNT acts upstream of Notch activation by regulating DL
ligand expression levels. These two modes of regulation are not necessarily
mutually exclusive, but we do not think that Notch activates the hnt
gene in the eye. First, HNT is expressed in all the R-cell precursors in the
eye, whereas the Notch pathway is activated at high levels only in a subset of
these precursors, as well as in the accessory cone and pigment cell
precursors, where HNT is not expressed at all
(Cooper and Bray, 2000).
Second, when Notch activity is attenuated by using the Nts
mutant (Cagan and Ready,
1989a), HNT expression in the furrow expands to all cells that now
acquire a neuronal fate (Pickup et al.,
2002). This result cannot be interpreted as a simple repression of
HNT expression by Notch activation in non-neuronal cells, as HNT expression is
not complementary to Notch activation in the eye disc
(Pickup et al., 2002). Third,
Notch activation cannot be sufficient to induce HNT expression in the eye
disc, as we do not see any expansion of HNT expression into adjacent,
non-determined cells when we ectopically express Dl early in the
cone-cell precursors (with the lz-Gal4 driver). Fourth, we have shown
here that the expression of Dl in the R-cell precursors is partly
dependent on HNT function. Others have clearly demonstrated that this late
Dl expression does not require Notch activity, as it is unaffected in
a Nts1 mutant (Tsuda
et al., 2002).
The two-signal model for R7-fate determination
The two-signal model of R7 fate hypothesizes that R7 determination requires
a strong RTK signal (achieved by the additive effects of Sevenless and EGFR
activation) together with Notch activation
(Tomlinson and Struhl, 2001).
These signals are necessary to activate pros and repress svp
expression, respectively (Kauffmann et
al., 1996; Miller et al.,
2008; Xu et al.,
2000). As the cone-cell precursor cells do not contact the
determined R8 cell at the appropriate time, they will not `see' the SEV ligand
BOSS (Van Vactor et al.,
1991). Cone cell precursors, then, will not ordinarily activate
their SEV receptors. In this model, different fates have been reinforced in
the R7/cone equivalence group by adding a second, activating ligand for EGFR
(Tomlinson and Struhl,
2001).
In this paper we suggest a further level of complexity. We have shown, by
manipulating the level of Dl in the R1/R6 signaling cells, that
activation of the key players in cone-cell determination requires high levels
of the Notch activation in the cone-cell precursor cell. Several lines of
evidence support the idea that the level of the DL ligand is translated into
cell-fate differences in a responding R precursor cell. As there is low DL
expression in the R7 precursor cell and only late expression of DL in the
cone-cell precursor cell (Flores et al.,
2000; Nagaraj and Banerjee,
2007), the adjacent R1/R6 precursor cells never activate their
Notch receptors (Cooper and Bray,
2000; Tomlinson and Struhl,
2001). Both the R7 precursor and the cone-cell precursor cells
receive their ligand signal from the R1/R6 precursor cells
(Tomlinson and Struhl, 2001;
Tsuda et al., 2002). In our
hypothesis, the R7 precursor cell requires only a low level of ligand signal
to activate the R7-like program: turning on pros and off
svp.
We suggest that the cone-cell precursor requires a high level of ligand
signal to activate the cone-cell program. Expressing a dominant-negative form
of DL in the R1/R6 signaling cells prevents cone-cell, but not R7-cell,
determination (Tsuda et al.,
2002). As both the cone and R7 precursor cells receive their DL
input from the same R1/R6 cells (Flores et
al., 2000; Tomlinson and
Struhl, 2001), it is possible that an intrinsic feature of the R7
precursor cell – possibly the high RTK activation – antagonizes N
signaling, so that D-Pax2 transcription does not occur in that cell
(Rohrbaugh et al., 2002). The
transcriptional repressor, Lola, may also be involved in this distinction, as
it is known to bias precursor cells towards R7-over cone-cell fate
(Zheng and Carthew, 2008).
A coordinated program for cone-cell induction
Although a role for Notch signaling in cone-cell induction has been shown
to be necessary for D-Pax2 expression
(Flores et al., 2000), it has
not been directly demonstrated as necessary for pros regulation in
cone cells (Xu et al., 2000).
The experiments presented here suggest that high levels of Notch signaling may
indirectly or directly be required for Pros expression in the
cone-precursor cells. This requirement is independent of the role of SU(H) in
inducing D-Pax2, as there are normal levels of PROS in the cone-cell
precursors of a D-Pax2 null mutant (A.T.P., unpublished observation).
Ectopically activating the Notch pathway in the R1/R6 precursor cells
occasionally induces ectopic PROS (but eliminates ELAV) in these cells
(Miller et al., 2008).
Although this effect on PROS expression may be a secondary result of a
cell-fate transformation, it could also be interpreted as a more direct effect
of Notch signaling on pros transcription. In a different context,
PROS expression has been shown to be affected by DL-activated Notch signaling
in a subset of glial cells in the embryonic CNS
(Thomas and van Meyel,
2007).
Why would there be two DL thresholds for different cell fates? There is
some preliminary work that suggests different mechanisms for Notch-activated
transcriptional readout in the responding cell, depending on the level of
signal received. In the cone-cell equivalence group, the cone-cell
determination pathway requires that D-PAX2 and PROS be expressed. It is
hypothesized that D-Pax2 may require a higher level of Notch
activation than Pros, which is also required for R7 determination
[Hayashi et al., unpublished data referenced by Zheng and Carthew
(Zheng and Carthew, 2008)].
Our experiments indicate that there may be coordinated regulation of both
D-Pax2 and Pros expression in the cone cells. Based on the
experiment shown in Fig. 4D, we
postulate that the mechanism of Pros-gene induction in the cone cells
is different from pros regulation in R7. By potentiating the level of
Dl gene expression in the R1/R6 signaling cells, it is possible to
overlay the cone-cell fate over the transcriptional module necessary for
R7-cell fate. This simple change has, thus, allowed for the elaboration of
very different cell fates from the same equivalence group.
|
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/136/6/975/DC1
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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