1 Genes and Development Research Group and Department of Biochemistry and
Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1,
Canada
2 Department of Biological Sciences, University of Alberta, Edmonton, Alberta
T6E 4G2, Canada
* Author for correspondence (e-mail: brook{at}ucalgary.ca)
Accepted 10 September 2002
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SUMMARY |
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Key words: Notch signalling, hnRNP I, Delta, Serrate, Polypyrimidine tract binding protein (PTB), RRM RNA-binding proteins, hephaestus
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INTRODUCTION |
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Notch signalling plays several roles in wing pattern formation.
Notch activation in cells along the DV compartment boundary of wing imaginal
discs induces expression of the Wingless (WG) morphogen conferring organizer
activity for the DV axis (de Celis et al.,
1996b; Diaz-Benjumea and
Cohen, 1995
; Neumann and
Cohen, 1996
; Neumann and
Cohen, 1997
; Rulifson and
Blair, 1995
). Later in pupal development, Notch
signalling is required in vein competent regions to distinguish between vein
and intervein boundary territories (de
Celis et al., 1997
; Huppert et
al., 1997
). In both cases, an initial broad domain of Notch
activation is refined by the inhibition of Notch activity in adjacent
ligand-expressing cells. Although the mechanisms regulating the decreases in
Notch activation are not known, it is likely that the protein levels and
activity of NICD and Notch effector genes such as the E(spl)-C
transcription factors must be tightly regulated in order for cells to change
Notch activation states.
This study shows that hephaestus (heph) is required to
attenuate Notch activity after ligand-dependent activation during
wing development. The original male sterile heph allele was
identified in a genetic screen for loci required for spermatogenesis
(Castrillon et al., 1993). We
have isolated new lethal alleles of heph that affect wing margin and
wing vein pattern formation in genetic mosaics. We report that the
Drosophila heph gene encodes the apparent homologue of mammalian
polypyrimidine tract binding protein (PTB). PTB was first identified in
vertebrates as a protein that binds to intronic polypyrimidine tracts
preceding many 3' pre-mRNA splice sites
(Garcia-Blanco et al., 1989
).
Many different functions have been identified for vertebrate PTB, including
the control of alternative exon selection
(Carstens et al., 2000
;
Chan and Black, 1997
;
Chou et al., 2000
;
Cote et al., 2001
;
Lou et al., 1999
;
Mulligan et al., 1992
;
Perez et al., 1997
;
Southby et al., 1999
;
Zhang et al., 1999
),
translational control or internal ribosome entry site (IRES) use
(Hunt and Jackson, 1999
;
Ito and Lai, 1999
;
Kim et al., 2000
;
Pilipenko et al., 2000
), mRNA
stability (Tillmar et al.,
2002
) and mRNA localization
(Cote et al., 1999
). PTB may
also act as a transcriptional activator
(Rustighi et al., 2002
). This
is the first report implicating such proteins in the regulation of the
Notch pathway.
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MATERIALS AND METHODS |
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Generation of heph genetic mosaics
To obtain a high frequency of mitotic recombination, the heat inducible
FLP/FRT recombination system was used (Xu
and Rubin, 1993). Recombination was induced using a single 1 hour
37°C heat treatment during second larval instar, unless otherwise stated.
To mark clones with yellow (y), recombination was induced in
flies of genotype y w P{hsFLP}1; P{neoFRT}82B Df(3R)G45/P{neoFRT}82B
P{y+}96E. Mitotic clones of heph03429 were
marked with pwn using a pwn+ duplication
(Dp(2;3)P32) in flies of genotype pr pwn P{hsFLP}38/+;
P{neoFRT}82B P{
M} Sb63b heph03429/P{neoFRT}82B kar
ry bx34e Dp(2;3)P32
(Heitzler et al., 1996
). For
immunohistochemistry, clones were marked by loss of a green fluorescent
protein (GFP) marker in flies of genotype y w P{hsFLP}1; P{neoFRT}82B
heph03429/P{neoFRT}82B P{hsGFP} or P{Ubi-GFP}. The
relationship between clone and twin size was measured as the number of pixels
selected using the Adobe Photoshop® magic wand tool from images of
GFP-marked heph mosaic wing discs. To mark clones with
forked, mitotic recombination was induced with a 1000 rad
-ray
dose during mid-second instar in larvae of the genotype f36a;
Df(3R)G45/bld1 P{f+}98B (f clones,
bld twins) or f36a; Df(3R)G45/P{f+}87D
RpS32 (f M+ clones). To study the
developmental profile of the heph03429 genetic mosaic
phenotype, clones were induced at 42, 54, 66, 78, 90, 102, 114 or 126±6
hours after egg lay (AEL) at 20-22°C. Developmental stages were visually
scored before each heat treatment. heph mutant clones were induced in
a Su(H) mutant background in flies of genotype y w P{hsFLP};
Su(H)2/Su(H)8; P{neoFRT}82B P{
M} Sb
heph03429/P{neoFRT}82B P{hsGFP}.
Immunohistochemistry
Mouse monoclonal antibodies for Achaete (working dilution: 1/25), Cut 2B10
(1/100), NICD C17.9C6 (1/10), NECD C458.2H (1/10) and WG 4D4 (1/10) were
obtained from the Developmental Studies Hybridoma Bank at the University of
Iowa. Monoclonal mouse anti-Distalless (1/100) was a gift from S. Cohen,
monoclonal mouse anti-DL 202.9B (1/25) was a gift from M. Muskavitch and rat
anti-SER polyclonal 2 (1/5) was kindly provided by K. Irvine. Secondary
antibodies were goat anti-mouse Alexafluor594nm (1/500; Molecular
Probes, Inc.) and goat anti-rat Cy3 (1/400; Jackson Immunoresearch).
Immunostaining of imaginal discs was performed using protocols modified from
those of Pattatucci and Kaufman
(Pattatucci and Kaufman,
1991).
Isolation, sequencing and mapping of heph cDNAs
Inverse PCR was used to confirm the insertion point of
heph03429 and to map the insertion point of
hephj11B9. Sequence from the heph2
(AQ026438), heph03429 (G00761) and
hephj11B9 (AF373596) P-element insertions were mapped to
Drosophila genomic DNA (AE003780), and ESTs were identified by
sequence homology to this genomic DNA using BLAST. The heph
P-elements mapped within a cluster of 5' and 3' ESTs that
suggested a 145 kb transcription unit including CG2094 and
CG2290. Full-length cDNA clones LD04329 (AY052367) and GH17441
(AF436844) were purchased from Research Genetics. A third cDNA sequence has
been submitted to the GenBank Nucleotide Sequence Data Library under the name
polypyrimidine tract binding protein (PTB; AF211191)
(Davis et al., 2002).
PCR-based sequencing of EMS-derived heph alleles
Single homozygous hephe1 or hephe2
first instar larvae were identified by the absence of a P{Actin-GFP}
balancer chromosome. Each heph exon and its splice sites were
amplified by PCR and sequenced. Sequence from the mutants was compared with
wild-type sequence using the same primers from the original non-mutagenized
line. When several heph exons could not be amplified by PCR from
homozygous hephe2 DNA, Southern analysis confirmed that
the hephe2 allele is a deletion of several heph
coding exons.
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RESULTS |
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The four lethal alleles and the male sterile allele of heph map to a transcription unit that is predicted to encode at least three isoforms of a protein with four RNA recognition motifs (RRMs) (Fig. 1A). The P-elements of ms(3)heph2, heph03429 and hephj11B9 are inserted in large introns. hephe2 is an EMS-induced deletion of several coding exons including the coding region for RRM1, RRM2 and part of RRM3. The temperature-sensitive hephe1 mutation is a mis-sense mutation that changes a conserved glycine (G) residue to a glutamine (Q) residue in the first predicted RRM domain of HEPH. The mapping of all five heph alleles to a single transcription unit indicates the lethality, ectopic margin, loss of vein differentiation and male sterile phenotypes are all due to loss of function in the same gene.
|
Because RRM domains are diagnostic of RNA-binding proteins and these
domains are highly conserved among HEPH, human PTB and Xenopus
Vg1RBP60 (Fig. 1B), it is
likely that the RNA-binding function of these proteins has been conserved. A
recent gene-expression report refers to this transcription unit as
PTB because of its sequence similarity to vertebrate PTB
(Davis et al., 2002). However,
in accordance with Drosophila genetic nomenclature, we will use the
name hephaestus, which has precedence
(Castrillon et al., 1993
).
Loss of heph in genetic mosaics induces ectopic wing
margin
Based on our initial observations that heph mutant clones
disrupted normal wing pattern formation, we performed several genetic mosaic
analyses with heph mutations. We find similar results for genetic
mosaics of all the strong heph alleles including heph point
and P-element insertion mutants and with Df(3R)G45, a small
deficiency that deletes heph along with a second lethal
complementation group, modulo. Clones of Df(3R)G45 and of
strong heph alleles are smaller than wild-type clones in twin spot
experiments, indicating a growth disadvantage or increased cell death in the
clone (Fig. 2A and clones
marked with GFP in Figs
4,5,6,7).
Using pixel dimensions as an estimate of clone size, heph clones
induced during mid-second instar are about 65% of the corresponding twin size
by late-third instar. When heph clones were given a growth advantage
using the Minute technique, the clone sizes increased but even clones induced
during the first instar never occupied more than a small fraction of the wing
blade. In adult wings, cell polarity and cell size (trichome density) are not
apparently affected by heph loss. Mintute+
heph clones can differentiate all wing blade structures normally with
the exception of veins.
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Mutant clones induced in larval imaginal discs are associated with ectopic wing margin (Fig. 2A-G), loss of wing margin (Fig. 2H) and loss of wing veins (described in detail below). Clones induced throughout larval development are associated with ectopic wing margin when situated within a short distance of the endogenous wing margin (Fig. 2A-G). The ectopic margin of heph genetic mosaics always conforms to the original compartment identity and resembles the adjacent endogenous margin. The autonomy of the ectopic margin was tested in experiments marking heph mitotic clones with the bristle marker yellow (y). We also marked clones with pawn (pwn) and performed twin spot experiments marking the clones and twins with forked (f) and bald (bld), which affect both bristles and trichomes. In all of these experiments, the ectopic bristles are derived almost entirely from heph+ cells immediately adjacent to the clone with the occasional ectopic bristle induced from within the heph mutant tissue (Fig. 2C). The non-autonomy of bristle induction is especially clear when the heph growth disadvantage is partially rescued by generating marked Minute+ heph clones in a Minute background. The ectopic margin is induced along the border of the marked Minute+ heph mutant tissue when that border is close to the normal margin (Fig. 2B). Outside of this domain, mutant cells do not induce ectopic wing margin. The ectopic margin is associated with small outgrowths of wing blade tissue in clones located near the junction of the wing margin and the AP boundary (Fig. 2G). Dorsal (not shown) or ventral (Fig. 2H) heph mutant clones that apparently intersect the normal margin are associated with wing margin nicks.
heph clones induce the wing margin molecular markers wingless, cut
and achaete
In vertebrates, PTB proteins regulate mRNAs involved in several cellular
processes (Valcarcel and Gebauer,
1997; Wagner and
Garcia-Blanco, 2001
). The wing margin nicks and growth
disadvantage caused by heph mutations could result from disruption of
general processes required for cell survival. However, the ectopic margin
phenotype indicates that heph plays a regulatory role in wing margin
pattern formation. In order determine what processes heph is
disrupting at the presumptive wing margin, we examined the expression of wing
margin molecular markers in heph mosaic wing imaginal discs. In
normal margin development, the WNT family member wingless
(wg) is expressed in two or three rows of cells straddling the DV
compartment boundary (Fig. 3A)
and diffuses to induce wing margin bristle fate in cells flanking the
wg expression domain (Couso et
al., 1994
; Phillips and
Whittle, 1993
). Thus, the heph ectopic margin phenotype
can be explained if heph mutant cells express wg
ectopically. Using specific antibodies, we examined the expression of
wg and cut, a second D/V boundary marker, in heph
genetic mosaic wing discs. In agreement with the distribution of ectopic
margin in adult wings, ectopic WG and CT were observed in those heph
mutant cells located within a few cell diameters of the boundary stripe of WG
and CT expression (Fig. 3A-F).
Ectopic WG or CT expression was never seen in heph mutant tissue
further away from the endogenous margin. As the expression of both wg
and cut at the boundary depends on high levels of Notch activation,
these results suggest that heph mutant cells near the endogenous
boundary are Notch activated.
|
Induction of bristles of the anterior wing margin by WG depends on downstream target genes such as the proneural gene achaete (ac). Thus, there is a strong prediction that anterior heph mutant clones should induce anterior margin-promoting genes such as ac. As predicted from the adult phenotype, we observed ectopic AC expression surrounding heph mutant clones near the DV boundary with some ectopic AC expression in the clones (Fig. 3G-I). The association of ectopic wg, cut and ac expression with heph mutant tissue suggests that ectopic margin is induced around heph clones by the same mechanisms acting during normal development.
Ser and Dl expression are reduced in heph
mutant tissue
The precise expression of WG in DV boundary cells that is present by the
late third instar evolves through interaction between the Notch and WG
signalling pathways. In mid-second instar wing discs, the Notch pathway is
activated to high levels along the boundary between dorsal and ventral cells
by Ser, which is expressed dorsally, and Dl, which is
expressed predominantly ventrally (de
Celis et al., 1996b). These ligands induce a broad domain of
Notch-activated cells at the DV interface that express wg
(de Celis and Bray, 1997
;
Diaz-Benjumea and Cohen, 1995
;
Rulifson and Blair, 1995
).
Expression of Dl and Ser is dynamic, and the initial DV
asymmetry disappears as the two ligands become expressed under the control of
WG signalling (Milan and Cohen,
2000
). By mid third instar, a broad stripe of cells along the DV
boundary express Ser, Dl and wg
(de Celis and Bray, 1997
;
Micchelli et al., 1997
;
Rulifson et al., 1996
). During
late third larval instar, this broad domain evolves into a narrow stripe of
cells expressing wg but not Ser or Dl, flanked on
either side by cells expressing Ser and Dl but not
wg (Fig. 4A). WG
secreted by the boundary cells is required to maintain high levels of
Dl and Ser expression in the flanking cells
(de Celis and Bray, 1997
;
Micchelli et al., 1997
). The
high levels of DL and SER in the flanking cells serve two roles. First, they
signal back to adjacent boundary cells to maintain the high levels of Notch
activation required for wg and cut expression. Secondly, in
the flanking cells, DL and SER autonomously inactivate Notch
signalling, which restricts Notch-dependent expression of wg
and cut to the boundary. High levels of Notch signalling in late
third instar boundary cells activates the expression of cut, which
encodes a homeodomain protein required to repress Ser and Dl
expression in the boundary cell domain.
The ectopic wing margin phenotype and association of ectopic wg,
cut and ac expression with heph mutant tissue suggests
that heph mutant cells situated near the endogenous DV boundary are
highly Notch-activated and thus behave like boundary cells and induce wing
margin fate in adjacent flanking cells. The complex interdependent signalling
network at the DV boundary (Fig.
4A) offers several possible mechanisms that could lead to Notch
activation and the ectopic margin phenotype. Ectopic boundary cell fate and
ectopic margin are induced by clones of cells mutant for dishevelled
(dsh) (Rulifson et al.,
1996), which are deficient for WG signal transduction, or by
clones mutant for both Dl and Ser
(Micchelli et al., 1997
). In
both cases, clones of cells in the flanking domains lose Dl and
Ser expression and Notch becomes activated through signalling from
the adjacent wild-type Dl- and Ser-expressing cells
(de Celis and Bray, 1997
;
Micchelli et al., 1997
). In
heph mutant cells, the levels of both DL and SER are autonomously
decreased independent of clone position within the wing disc
(Fig. 5). The decrease in DL
and SER protein levels in heph clones is sufficient to account for
the ectopic induction of wg and cut expression in cells
flanking the margin where DL and SER normally repress Notch. This reduction of
DL and SER could be the result of loss of WG signal transduction or to loss of
Dl and Ser expression. Finally, autonomous activation of
Notch signalling could result in heph mutant cells in the flanking
domains assuming a boundary fate.
heph clones are not defective for wg
signalling
Disruption of WG signal transduction is not a likely explanation for the
loss of Ser and Dl expression in heph mutant
clones. Clones mutant for heph in the antenna and leg have no pattern
phenotypes (data not shown) and heph mutations do not enhance the
phenotype of `dishevelled-weak' (data not shown), a genetic
background that is highly sensitive to dose changes in WG pathway signalling
components (Haerry et al.,
1997). Furthermore, the WG target gene acheate can be
activated in heph mutant cells
(Fig. 3G-I), and expression of
the WG target gene Distal-less is not affected in heph
clones (Fig. 3J-L). These
results suggest that clones lacking heph are able to transduce the
wg signal and that the primary effect of heph is not on
wg signalling.
Further support that Notch signalling and not wg
signalling is disrupted in heph mutants comes from a genetic
interaction observed between heph mutants and
fringeD4 (fngD4)
(Fig. 6).
fngD4 is a gain-of-function allele of fringe, a
gene encoding a Notch-modifying glycosyltransferase
(Bruckner et al., 2000;
Moloney et al., 2000
;
Munro and Freeman, 2000
). A DV
fringe expression boundary is required for maximal activation of
Notch signalling and proper induction of the DV organizer
(Fleming et al., 1997
;
Irvine and Wieschaus, 1994
;
Kim et al., 1995
;
Klein and Arias, 1998
;
Panin et al., 1997
). Ectopic
transcription of fng+ in fngD4 results
in decreased Notch activation and wg expression at the DV boundary,
causing loss of the wing margin and much of the wing blade
(de Celis and Bray, 2000
;
Irvine and Wieschaus, 1994
). A
decrease in the dose of a wg pathway component would be predicted to
enhance the fngD4 phenotype. However, flies heterozygous
for both heph and fngD4 have considerably more
wing margin and wing blade than do flies heterozygous for
fngD4 alone (Fig.
6). A similar suppression has been reported for flies heterozygous
for fngD4 and activating `Abruptex' alleles of Notch
(NAx) (de Celis and Bray,
2000
). The Abruptex phenotype probably results from an inability
to repress Notch activation (de Celis and
Bray, 2000
; de Celis et al.,
1996b
), and like heph, NAx mitotic clones are
associated with ectopic margin within a few cell diameters of the endogenous
margin (de Celis and Garcia-Bellido,
1994a
; de Celis et al.,
1996b
), and cause a cell-autonomous loss of vein differentiation
(de Celis and Garcia-Bellido,
1994a
; de Celis and
Garcia-Bellido, 1994b
).
Notch levels are increased in heph mutant cells
The ectopic margin phenotype is probably caused either by loss of
Ser and Dl expression or autonomous activation of the Notch
pathway in heph mutant cells. One consequence of Notch activation is
the cleavage of the full-length receptor, which releases the Notch
intra-cellular domain (NICD), allowing it to translocate from the membrane to
the nucleus (Gho et al., 1996;
Kidd et al., 1998
). To
determine if Notch is activated by loss of heph activity, we examined
the distribution of Notch immunoreactivity in heph genetic mosaic
wing discs. An increase in Notch immunoreactivity was found in heph
mutant cells, regardless of their position within the imaginal disc
(Fig. 7). This increase is
specific to an antibody that recognizes the intracellular domain of Notch
(NICD) and is found in the cell body away from the apical surface of the cell
(Fig. 8). As this effect was
not observed with an antibody to the extracellular domain of Notch, and the
apical levels of Notch are very similar in heph mutant and wild-type
tissue, accumulation of the full-length Notch is not apparently increased in
heph mutant cells. Consistent with this observation, comparable
changes in NICD immunostaining are found along the DV boundary, where the
Notch pathway is active and Notch target genes are expressed at high
levels (Fig. 8). As further
evidence that the changes in NICD immunoreactivity represent Notch activation,
we find that heph clones generated in Su(H) mutant discs do
not alter the levels or localization of NICD
(Fig. 9). While it is not
possible to conclude that the increased levels of NICD are localized to the
nucleus, these results are consistent with an increase in Notch activation in
heph mutant cells, and with the proposed role for SU(H) in
transporting NICD to the nucleus (Gho et
al., 1996
; Kidd et al.,
1998
). These data suggest heph acts in all wing disc
cells to repress Notch pathway function. This is consistent with a
report that heph mRNA is present uniformly in imaginal discs
(Davis et al., 2002
).
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|
Delta levels are decreased in heph mutant cells in a
Su(H)-dependent manner
We find that the level of Notch is elevated (Figs
7,
8) and the level of DL is
decreased (Fig. 5A-C) in
heph mutant cells regardless of their position in the imaginal disc.
This reciprocal relationship is typical of most tissues where Notch signalling
is acting and is generally the result of interdependent signalling causing
autonomous inhibition of Dl expression in Notch-activated cells
(Artavanis-Tsakonas et al.,
1999). DL itself is sometimes required to repress Notch activation
autonomously (Artavanis-Tsakonas et al.,
1999
; de Celis and Bray,
1997
; Doherty et al.,
1996
; Micchelli et al.,
1997
) so the observed decrease in DL levels could be either a
cause or an effect of increased Notch activation. In order to distinguish
between these possibilities, we examined Dl expression in cells
mutant for both heph and Su(H). In mature third instar
discs, Dl is expressed ubiquitously at a low level, and in elevated
levels at the DV margin, in the presumptive wing veins and in the proneural
clusters of the thorax. Discs from third instar larvae mutant for strong
alleles of Su(H) lack most of the wing pouch, because of the absence
of Notch signalling along the DV boundary, but they retain the low ubiquitous
expression of Dl. We reasoned that if Dl expression were
still reduced in heph cells in the absence of Su(H), then
heph might act directly on Dl expression. However, we
observed no change in the low levels of Dl expression in
heph clones generated in Su(H) imaginal discs
(Fig. 8A-C), indicating that
the decrease in Dl expression in heph cells depends on
Su(H). The implication of this result is that heph directly
affects Notch activity and indirectly reduces ligand expression.
heph causes a cell autonomous and Delta-dependent
loss of vein differentiation
On balance, the effects of heph on wing margin formation suggest
that heph represses Notch pathway activity. The heph
loss-of-function phenotype in the wing veins also suggests that heph
directly affects Notch signalling. Lateral inhibition involving Notch and
Epidermal Growth Factor Receptor (EGFR) signalling is required to refine
pro-vein territories in the wing blade
(Fig. 4B)
(de Celis et al., 1997;
de Celis and Garcia-Bellido,
1994b
; Huppert et al.,
1997
). The position of veins is set by the expression of
rhomboid (rho) in stripes of cells oriented perpendicular to
the DV compartment boundary in the wing pouch
(Sturtevant et al., 1993
).
RHO facilitates signalling through EGFR and EGFR activation is required for
the vein fate (Diaz-Benjumea and
Garcia-Bellido, 1990
). Loss of EGFR activity is epistatic to the
wide vein phenotype of Notch mutants, indicating that EGFR activation
induces provein regions, then Notch functions to restrict vein fate
by refining the domain of rho expression
(de Celis et al., 1997
).
Dl mutant clones that span a vein territory produce thicker veins
than normal because DL is required in the vein to activate Notch in adjacent
lateral provein cells. By contrast, heph clones covering a vein
territory cell autonomously fail to differentiate as vein
(Fig. 10A,B). Only when dorsal
and ventral clones coincide does a vein appear to be completely missing. This
phenotype is consistent with ectopic Notch activation in heph clones
as it resembles the effects of activating Notch by a variety of different
genetic manipulations (de Celis et al.,
1997
; de Celis et al.,
1996a
; de Celis and
Garcia-Bellido, 1994a
; de
Celis and Garcia-Bellido, 1994b
;
Huppert et al., 1997
;
Schweisguth and Lecourtois,
1998
; Struhl et al.,
1993
). Furthermore, despite the reduction of Dl
expression in heph clones, the heph mutant clones have a
wing vein phenotype opposite to that of Dl mutant clones
(Fig. 10A-D). This is strong
support for the interpretation that the reduction of Dl expression in
heph clones is a consequence rather than a cause of the Notch
activated heph phenotype.
|
To determine the epistatic relationship between heph and Dl, we compared the wing vein phenotypes of double mutant clones with clones lacking only heph or Dl. Clones of cells mutant for both heph and Dl cause a thick vein phenotype (Fig. 10E,F) that is indistinguishable from the effects of Dl mutant clones (Fig. 10C,D). These phenotypes indicate that heph is not required for specification of vein fate, i.e. heph is not directly required for rho expression or EGFR activity. The parsimonious interpretation that heph acts to repress Delta contradicts the loss of DL staining in heph mutant tissue, and the lack of requirement for Dl in specifying vein fate. Another interpretation is that Notch must be activated by DL before heph is required. That is, heph may attenuate the Notch signalling pathway in cells where Notch has already been activated by DL.
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DISCUSSION |
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Direct evidence of Notch activation was also observed in heph
mutant clones situated throughout the wing imaginal disc. Increased levels of
the Notch intracellular domain (NICD) were found in the cell body of normal
boundary cells and in heph mutant cells. Although we do not know that
these changes represent genuine nuclear accumulation, the altered distribution
of NICD, which is absent in Su(H) mutant tissue, is consistent with
models of Notch activation involving SU(H)-mediated translocation of NICD to
the nucleus (Gho et al., 1996;
Kidd et al., 1998
). The
position independence of this effect indicates that heph acts in all
wing disc cells to modulate Notch pathway function and is consistent with
reports that low levels of Notch pathway activation are required
throughout the developing disc for cell proliferation
(Baonza and Garcia-Bellido,
2000
; de Celis and
Garcia-Bellido, 1994b
) and for the weak general expression of
Notch target genes such as E(spl)mß
(Cooper et al., 2000
;
de Celis et al., 1996a
).
Although the effects of heph on DL, SER and NICD levels are
position independent, the margin-inducing effects of heph are
restricted to a competent region within a few cell diameters of the endogenous
wing margin. By contrast, clones of cells expressing NICD under the
control of a strong promoter are able to induce wg and cut
expression, as well as ectopic margin and outgrowths throughout the wing
blade, independent of the position of the clone
(de Celis and Bray, 1997). The
effects of heph clones, including vein loss and the restricted
location of ectopic margin, more closely resemble the effects reported for
clones expressing NICD under a weak promoter
(Diaz-Benjumea and Cohen,
1995
; Struhl and Basler,
1993
). The competent region corresponds roughly to the domain of
Dl and Ser expression in the regions flanking the boundary.
As defined by the refinement of wg expression, cells in this region
are initially Notch activated and express wg during late second and
early third instar. The flanking cells subsequently repress Notch activation
and lose wg expression in response to increased levels of DL and SER.
It is likely that the band of cells that maintains wg expression
begin with higher levels of Notch activation and that this bias is what allows
them to maintain Notch activation and wg expression
(Blair, 1997
). Even small
increases in Notch activity caused by the loss of heph activity in
the cells of the flanking domains may bias the signalling required to refine
the wg and Dl/Ser domains in favour of maintaining Notch
activity in heph clones. Thus, the competent region for
heph-induced margin formation may reflect the high levels of Notch
activation required for margin induction.
The maintenance of Notch activation in heph cells in the flanking
regions may cause them to become more like boundary cells. Thus, they maintain
wg expression and decrease Dl and Ser expression.
The reduction in Dl/Ser in heph clones would reduce Notch
inhibition to allow further increases in Notch activation by adjacent
Dl/Ser-expressing cells. This is consistent with the induction of
ectopic cut expression, which may require the highest levels of Notch
activation (Micchelli et al.,
1997), only in heph mutant cells abutting the
Dl/Ser domain. A bias toward Notch activation in heph mutant
cells may also explain the unusual shape of the boundary cell domain that is
often observed in heph mosaic discs
(Fig. 3B). Boundary Notch
activation is required to maintain the lineage restriction property of the DV
boundary and perturbing the spatial pattern of Notch activation can alter the
shape of the boundary (Micchelli and
Blair, 1999
; Rauskolb et al.,
1999
).
Notch-dependent repression of ligand expression
It has been reported that Notch activation increases Dl and
Ser expression in the wing pouch
(de Celis et al., 1996b;
Doherty et al., 1996
;
Kim et al., 1995
;
Panin et al., 1997
). However,
these effects are due to a positive feedback loop mediated indirectly through
wg (de Celis and Bray,
1997
; Micchelli et al.,
1997
). Cells mutant for heph located away from the wing
margin do not activate wg expression and do not activate Dl
and Ser expression, perhaps because of insufficient levels of Notch
activation. Instead, we observe an autonomous decrease in Dl and
Ser expression throughout heph clones. We have shown that
the decrease in Dl expression depends on Su(H), and by
implication is downstream of Notch activity in heph mutant cells.
These observations are consistent with genetic evidence that Notch
activity represses Delta activity
(de Celis and Bray, 2000
;
de Celis and Garcia-Bellido,
1994b
; Vässin et al.,
1985
), and with instances of Notch signalling that require the
progressive restriction of ligand and receptor to reciprocal cells (e.g.
during wing margin and wing vein patterning;
Fig. 4).
Is heph specific to the Notch signalling pathway?
It is unlikely that the Drosophila PTB homologue (heph)
is a dedicated Notch pathway component considering that several target RNAs
not involved in Notch signalling have been identified for vertebrate PTBs
(Valcarcel and Gebauer, 1997;
Wagner and Garcia-Blanco,
2001
). This is especially clear when considering
heph-induced wing margin nicks and the under-proliferation or lack of
survival of heph mutant cells, neither of which are Notch
gain-of-function phenotypes. Both of these defects may result from the
disruption of heph targets required for cell survival. Strong
decreases in cell survival in clones would mask the enhancement of
proliferation provided by Notch activation. Cell death in clones in the wing
margin could result in wing margin nicks through nonspecific disruption of
wg expression at the DV boundary. This may explain the greater
recovery of nicked wings resulting from clones induced prior to the formation
of the DV boundary, which are able to occupy both the dorsal and ventral
surfaces of the wing blade (Garcia-Bellido
et al., 1973
).
In addition to the heph wing phenotypes discussed above, we have
observed the effects of heph mutations on other imaginal tissues, and
clearly heph affects some but not all Notch dependent development.
For example, heph clones cause ommatidial pattern defects, whereas we
see little or no effect in the formation of leg joints or in the development
of thoracic microcheatae (data not shown). This suggests that the Notch
pathway requires modification to accommodate the diversity of processes it
regulates and that, as in the case of modifiers such as numb and
Suppressor of deltex, heph may be essential for only a limited subset
of Notch signalling events
(Busseau et al., 1994;
Cornell et al., 1999
;
Fostier et al., 1998
;
Santolini et al., 2000
). The
specificity of heph phenotypes also argues against Notch activation
in heph clones being due to the amplification of generic defects in
signal transduction or transcription as a result of the exquisite dosage
sensitivity of Notch signalling. Indeed, the lateral inhibition signalling
that regulates the spacing of adult thoracic microcheatae, perhaps the most
dosage-sensitive Notch process (Heitzler
and Simpson, 1991
), is relatively unaffected by loss of
heph activity (data not shown). Thus, although heph is
unlikely to be dedicated to the Notch signalling pathway, it is most likely to
play a specific role in the regulation of some Notch signalling events.
How does heph regulate Notch activity?
This study has linked together for the first time the PTB/hnRNPI
RNA-binding proteins and the Notch signalling pathway. Given the
strong sequence similarity shared between heph and vertebrate PTBs,
it is probable that heph regulates the processing, stability or
translation of a Notch pathway mRNA. However, the heph
mosaic wing phenotypes most closely resemble the effects of low level ectopic
Notch activation and cannot be easily correlated with an effect on any
particular known element in the Notch pathway. The phenotypes of
clones mutant for Delta and heph are most informative in
explaining where heph acts in the Notch pathway. The
epistasis of Dl over heph in double mutant clones indicates
that the Notch activation in heph clones depends on Dl. This
ligand dependency excludes the possibilities that Notch target genes
are generally de-repressed, or that the Notch receptor is constitutively
activated, in heph mutant cells. Rather, it suggests that in the
absence of heph, existing Notch activity is amplified and/or
maintained. Therefore, our favoured explanation is that heph is
required to attenuate Notch activity after ligand-dependent
activation.
The phenotypic consequences of heph are most prominent in the wing
margin cells and wing vein cells. Both of these cell types require decreases
in the levels of Notch activity during development and the
heph phenotype results from persistent Notch activity in
these cells. As described above, the wing margin cells lose Notch activation
and wg expression during the refinement of wg and
Dl/Ser expression during the late second and early third instar.
During larval development, the cells that will ultimately give rise to the
vein express low levels of Notch and Notch target genes such as
E(spl)mß (Cooper et al.,
2000; de Celis et al.,
1996a
; Fehon et al.,
1991
; Kooh et al.,
1993
), indicating that these cells have low levels of Notch
activation prior to the repression of Notch transcription in pupal vein cells.
Although it is not certain how these cells normally lose Notch activation, one
possibility is that NICD stability is tightly regulated in order for cells to
change Notch activation states and that heph+ may be
required for cells to degrade NICD following ligand activation of the Notch
receptor.
Several lines of evidence suggest that regulated degradation of NICD may be
crucial. NICD includes a PEST domain, a characteristic of proteins with very
short half-lives (Rogers et al.,
1986), and mutations of Notch that delete the PEST domain
are associated with N(gf) phenotypes
(Ramain et al., 2001
), albeit
in different Notch signalling events than those affected by heph. The
ubiquitin-proteasome pathway has been implicated in regulating the degradation
of a NICD related protein in C. elegans, where the Notch family
receptor LIN-12 is negatively regulated by sel-10
(Hubbard et al., 1997
;
Sundaram and Greenwald,
1993
). The C. elegans and mammalian SEL-10 proteins both
contain F-box motifs, and are thought to form part of a ubiquitin ligase
complex that regulates NICD protein stability
(Gupta-Rossi et al., 2001
;
Oberg et al., 2001
;
Wu et al., 2001
). In
Drosophila, the gene most similar to sel-10, archipelago
(ago), is required to destabilize Cyclin E proteins
(Moberg et al., 2001
).
However, available ago alleles do not affect the accumulation of NICD
(Moberg et al., 2001
). The
expression of a dominant-negative proteasome subunit
(Schweisguth, 1999
) stabilizes
NICD in the Drosophila wing disc, and wild-type levels of proteasome
activity are required for alternative cell fate decisions during sense organ
development. In addition, treatment of cells with chemical proteasome
inhibitors increases the accumulation of nuclear NICD
(Schweisguth, 1999
). In this
model, heph would regulate the mRNA for some element of the
proteasome-dependent degradation of NICD.
The most intriguing possibility is that heph may negatively
regulate the translation of E(spl)-C mRNAs. The E(spl)
complex bHLH genes are transcribed in response to Notch
signalling (Bailey and Posakony,
1995; Jennings et al.,
1994
; Lecourtois and
Schweisguth, 1995
) and this is counteracted by inhibition of
translation by the 3'-UTR's of E(spl)-C mRNAs
(Lai et al., 1998
;
Lai and Posakony, 1997
). This
inhibition is presumably mediated through the binding of factors to conserved
sequences found in most E(spl)-C mRNAs as well as in genes of the
Bearded family, another group of Notch mediators
(Lai et al., 2000
;
Lai et al., 1998
;
Lai and Posakony, 1997
;
Leviten et al., 1997
). In this
model, loss of heph function would increase the stability of
E(spl)-C mRNAs, resulting in amplification of the effects of
transcriptional activation by Notch signalling. Increased expression of
E(spl)-C members has been demonstrated to inhibit wing vein
differentiation (de Celis et al.,
1996a
), although the ectopic expression of individual
E(spl)-C members has not been demonstrated to induce ectopic wing
margin. However, it is possible that the stabilization of multiple
E(spl)-C mRNAs could result in more dramatic effects on the wing
margin. Furthermore, E(spl)-C members have different transcription
patterns and may have divergent roles downstream of Notch
(Cooper et al., 2000
;
de Celis et al., 1996a
;
Ligoxygakis et al., 1999
;
Nellesen et al., 1999
). If
heph were to regulate a subset of the E(spl)-C mRNAs, it
would explain the limited requirement of heph in various
Notch-mediated signalling events.
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ACKNOWLEDGMENTS |
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