1 Centro de Biología Molecular Severo Ochoa, C.S.I.C. and U.A.M.,
Cantoblanco, 28049 Madrid, Spain
2 Institute of Molecular Medicine, Department of Life Science, National Tsing
Hua University, Hsinchu, Taiwan 30034, Republic of China
Authors for correspondence (e-mail:
jmodol{at}cbm.uam.es
and
lshsu{at}life.nthu.edu.tw)
Accepted 9 September 2003
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SUMMARY |
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Key words: echinoid, Notch, EGF receptor, Cell adhesion, Signaling, Bristle patterning
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Introduction |
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In the notum territory of the imaginal wing disc, the pattern of proneural
clusters prefigures the adult pattern of chaetae
(Cubas et al., 1991;
Skeath and Carroll, 1991
).
Although many cells within a proneural cluster are competent to become SOPs,
they are prevented from doing so by the mechanism of lateral inhibition
mediated by the receptor Notch (N) and its ligand Delta (Dl) (reviewed by
Artavanis-Tsakonas et al.,
1995
; Artavanis-Tsakonas et
al., 1999
; Simpson,
1997
). According to current thinking, proneural genes activate Dl,
which upon interaction with N, triggers the proteolytic cleavage of the
extracellular domain of N by a Kuzbanian/ADAM family protease and produces
NECN (Lieber et al.,
2002
). Then, a g-secretase complex, including at least Presenilin,
Niscatrin, Aph1 and Pen2 mediates another cleavage within the transmembrane
domain to release the intracellular domain of N (NICD)
(Chung and Struhl, 2001
;
Hu et al., 2002
;
López-Schier and St Johnston,
2002
; Struhl and Greenwald,
1999
; Ye et al.,
1999
). NICD translocates to the nucleus where it
displaces Hairless (H) and acts in association with Suppressor of Hairless
[Su(H)] and Mastermind to activate transcription of the Enhancer of split
complex [E(spl)-C] (Bailey and Posakony,
1995
; Barolo et al.,
2002
; Fryer et al.,
2002
; Lecourtois and
Schweisguth, 1995
). Members of the E(spl)-C in turn prevent, in
the signal-receiving cells, self-stimulation of proneural genes, and this
leads to suppression of SOP cell fate
(Culí and Modolell,
1998
; Giagtzoglou et al.,
2003
; Heitzler et al.,
1996
). Conversely, the future SOP, which becomes insensitive to
lateral inhibition and does not express E(spl)-C genes
(Jennings et al., 1995
),
continues to accumulate AS-C proneural proteins by this self-stimulation
mechanism that involves the binding and activation of Ac, Sc and Asense to
SOP-specific enhancers of the proneural genes
(Culí and Modolell,
1998
; Giagtzoglou et al.,
2003
).
In contrast to the lateral inhibition mediated by N, which prevents SOP
cell fate, the Egfr signaling pathway favors the SOP fate lateral
stimulation by promoting the proneural gene self-stimulatory loops
(Culí et al., 2001).
Egfr signaling is mediated by the conserved Ras/Raf/MAPK signaling cassette.
Excess Egfr signaling promotes ectopic sc expression and the
production of extra SOPs, while reduced Egfr signaling results in decreased
sc expression and the loss of SOPs
(Culí et al., 2001
;
Díaz-Benjumea and
García-Bellido, 1990
). Thus, the Egfr and N pathways act
antagonistically in bristle development. Interestingly, this Egfr activity is
important for the SOPs of the notum macrochaetae, but much less so for
microchaetae or for the tergite bristles
(Culí et al., 2001
;
Díaz-Benjumea and
García-Bellido, 1990
).
echinoid (ed) encodes a cell adhesion protein with seven
Ig domains, two fibronectin type III (Fn III) domains and a transmembrane (TM)
domain, followed by a 315 amino acid intracellular domain with no identifiable
functional motif (Bai et al.,
2001). ed mutant flies exhibit extra photoreceptor and
cone cells in the eye. Conversely, overexpression of ed in the eye
leads to a decrease in photoreceptor cell number. In addition, ed
genetically interacts with several components of the Egfr pathway. These
results have suggested that ed is a negative regulator of the Egfr
pathway. Based on genetic mosaic and epistatic analyses, it has been proposed
that Ed, via homotypic interactions, activates a novel pathway that
antagonizes Egfr signaling by regulating the activity of the TTK88
transcriptional repressor, the most downstream component of the Egfr pathway
(Bai et al., 2001
). However, it
has been shown very recently that during R8 cell selection, Ed negatively
interacts with the Egfr pathway at a step upstream from the phosphorylation of
the MAP kinase (Rawlins et al.,
2003
; Spencer and Cagan,
2003
). This and other evidence obtained mostly with cell culture
assays have allowed them to propose an alternative model in which Ed
antagonizes Egfr function by direct interaction between the Ed and Egfr
molecules.
In addition to the homophilic adhesive activity, Ed also exhibits a
heterophilic trans-interaction with Neuroglian (Nrg), an L1-type CAM. L1-type
proteins are composed of six Ig domains, three to five Fn III repeats and a
cytoplasmic domain with a conserved ankyrin binding site. Co-expression of
ed and nrg in the eye exhibits a strong genetic synergy in
inhibiting Egfr signaling and this effect requires the intracellular domain of
Ed, but not that of Nrg. Together, these results suggest a model in which Ed
functions as a receptor and is activated by either its own homophilic
interaction or by an heterophilic ligand like Nrg
(Islam et al., 2003).
In addition to the eye phenotype, we noticed the presence of ectopic
bristles over the body parts of ed mutant flies. In this study, we
use the development of mesothoracic bristles macrochaetae and
microchaetae as an experimental model with which to explore the
interactions between Ed, Notch, and Egfr pathways. We show that
loss-of-function mutations at the ed locus or overexpression of a
dominant-negative form of Ed in proneural clusters promote development of
extra macrochaetae near the extant ones and increase the density of
microchaetae. These effects are due to ed mutant cells within
proneural clusters giving rise to extra SOPs. Our genetic data suggest that Ed
participates in lateral inhibition within proneural clusters and facilitates N
signaling. It also antagonizes the Egfr pathway, either by the enhancement of
N signaling or by a more direct interaction. In a parallel study, Ahmed et al.
have shown the interaction between ed and N in the embryonic
CNS, and in bristle and wing vein patterning
(Ahmed et al., 2003).
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Materials and methods |
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Molecular biology
The UAS-edintra was made by subcloning the
intracellular domain of Ed into the pUAS vector
(Brand and Perrimon, 1993).
The UAS-ed
ECD,
UAS-ed
ECD-48 and
UAS-ed
ECD-124 were generated by subcloning
either the transmembrane plus the entire intracellular domain of Ed, or
deleting the last C-terminal 48 and 124 amino acids, respectively, into the
pUAS vector.
To identify molecular lesions in ed mutants, genomic DNA, prepared from homozygous ed mutant larvae, was used as template in PCR reactions to amplify the entire ed sequence. Multiple PCR reactions were pooled and sequenced for each allele.
Mosaic analysis
To generate clones of cells mutant for ed, either yw hs-FLP122
f36a; ck Pf[+]30B FRT40/CyO or yw hs-FLP122; P[ubi-GFP]
FRT40/CyO females (stocks described in FlyBase) were crossed with w;
ed1x5 FRT40/CyO males. To generate M+
clones we either crossed w hs-FLP122; P[arm lacZ] M(2)z, FRT40/CyO
females with w; ed1x5 FRT40/CyO males or y w f36a
hs-FLP122; ed1x5 FRT40/CyO females with f36a;
M(2)z Pf[+]30B FRT40/CyO males (M(2)z stocks were from the
collection of A. García-Bellido). Recombination was induced by heat
treatment at 72-96 hours after egg laying for 1 hour at 37°C
(Xu and Rubin, 1993).
To produce germline clone embryos deficient for ed, the
FRT/FLP/DFS technique was used (Chou and
Perrimon, 1996). Maternal and zygotic mutant embryos were
identified by mating germline clone-bearing virgin females with males carrying
edlF20/Cyo, wg-lacZ, and selecting the non-lacZ
embryos.
Histochemistry
Antibody staining was performed as described [Anti-Sc, anti-Sens and
anti-ß-galactosidase (Cubas et al.,
1991); mAb22C10 (Hartenstein
and Posakony, 1990
); anti-ELAV
(Islam et al., 2003
);
anti-NICD (mAb9C6) (Parks et
al., 2000
)]. Polyclonal rabbit anti-Ed antibodies were generated
against a synthetic peptide, corresponding to the C-terminal region of Ed
(GEYSTTPNARNRRVIREIIV) and were used at a dilution of 1:200. Secondary
antibodies were from Jackson and Amersham. Hybridizations in situ to detect
E(spl)m8 mRNA were performed as described
(González-Crespo and Levine,
1993
) using an antisense DIG-labeled RNA probe. Discs from control
wild-type and overexpressing larvae were hybridized and processed in parallel
to allow comparison.
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Results |
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Within the ed1X5 clones, at positions near extant SOPs
for the notum macrochaetae, extra SOPs were detected by staining with an anti
Senseless (Sens) antibody (Nolo et al.,
2000). Often, these SOPs corresponded to homozygous
ed1X5 clones comprising just a single cell
(Fig. 1D,E). This suggests that
reaching the SOP state improved the viability of the mutant cells. Adults
bearing these clones displayed single extra macrochaeta of the mutant
phenotype (f marker;
Fig. 1F) congruent with the
very small size of the clones.
To improve the recovery of the homozygous ed1X5 cells,
we used the M+ technique
(Morata and Ripoll, 1975).
ed1X5; M+ clones were viable and they contained
extra SOPs when they included regions from where the extant SOPs arose
(Fig. 1G). No ectopic SOPs were
observed at positions far from these regions. Moreover, clones never contained
clusters of SOPs, suggesting that the mechanism of lateral inhibition was
still active in the clones. As expected from these observations, on the adult
cuticle, the ed1X5 M+ could give rise to groups
of macrochaetae (Fig. 1H) or
areas of increased density of microchaetae
(Fig. 1I,J). In both cases, the
bristles were separated by epidermal cells. In the discs, extra SOPs always
appeared within the clones, indicating that the ed phenotype was cell
autonomous. Similar results were obtained with the edlF20
allele.
Overexpression of ed
We assessed the effect of ed overexpression on bristle patterning
using two UAS-ed lines (UAS-edX and
UAS-edIII, names refer to their chromosomal positions).
UAS-edIII driven by the C253-Gal4 line, which is
expressed in proneural clusters relatively late in development (third instar
larvae and early pupa) (Culí et
al., 2001), caused a mild suppression of notum macrochaetae and
the appearance of some extra macrochaetae at the notopleural position
(Table 1). With another driver
also expressed in proneural clusters (sca-Gal4), these effects became
more pronounced (Fig. 1C). With
Gal4 lines promoting earlier and generalized expression at the notum
[C765-Gal4 (Gómez-Skarmeta
et al., 1996
) or MS1096-Gal4
(Milán et al., 1998
)]
there was little effect with UAS-edIII, but with the
ap-Gal4 driver (Calleja et al.,
1996
) at 20°C macrochaetae were removed from some positions
and extra bristles were generated in others (not shown). With the
UAS-edX line and with the generalized drivers
C765-Gal4 and MS1096-Gal4 extra macrochaetae appeared in all
notum positions (Table 1 and
not shown). Hence, the overexpression of full-length Ed can cause phenotypes
similar to those of the loss-of-function mutations of ed and suggest
that an excess of full-length Ed can act as a dominant negative.
Generation of a dominant-negative form of Ed
A form of the Ed protein with a deletion of its extracellular domain
(UAS-edECD,
Fig. 2F) was overexpressed
either early in the whole dorsal compartment of the wing disc
(ap-Gal4 driver) or late in the proneural clusters
(C253-Gal4 driver) using either one or two copies of
UAS-ed
ECD
(Table 1,
Fig. 2E). In all cases,
phenotypes similar to those of the loss-of-function ed mutant
combinations were observed, except that the PA positions seemed more
insensitive to the overexpression of
UAS-ed
ECD than to the ed
hypomorphic combinations (Table
1). More extra macrochaetae developed in the presence of two
copies of UAS-ed
ECD
(Fig. 2E) than in flies with
only one copy (Fig. 3A), while
microchaetae density was not further increased. With stronger drivers
(sca-Gal4 and MS248-Gal4)
(Cavodeassi et al., 2002
;
Sánchez et al., 1997
)
more macrochaetae or even tufts of macrochaetae developed, but always occurred
at or near the wild-type macrochaetae positions
(Fig. 3G and not shown). These
and other data indicated that Ed
ECD behaves as a
dominant-negative form of Ed. Indeed,
UAS-ed
ECD driven by ap-Gal4 and
GMR-Gal4 produce flies with extra wing veins and rough eyes,
respectively, phenotypes similar to the ed hypomorphic combinations
(not shown) (Bai et al., 2001
).
Moreover, the removal of one wild-type copy of ed increased the
number of extra macrochaetae generated by
UAS-ed
ECD (C253-Gal4 driver) (not
shown). The additional deletion of either the 48 C-terminal amino acids or the
transmembrane domain of Ed
ECD rendered the construct
ineffective (Fig. 2F),
suggesting that this terminus of the protein and its attachment to the
membrane are necessary for the dominant-negative effect.
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Extra bristles also arise from proneural clusters under conditions of
decreased N signaling (de Celis et al.,
1991a; Heitzler and Simpson,
1991
; Simpson and Carteret,
1990
). As the bHLH genes of the E(spl)-C are targets of this
signaling pathway (reviewed by
Artavanis-Tsakonas et al.,
1995
), we examined the expression of the E(spl)-m8 gene,
which is known to mediate lateral inhibition in proneural clusters
(de Celis et al., 1996
;
Jennings et al., 1995
). The
levels of E(spl)-m8 mRNA were clearly decreased in discs expressing
UAS-ed
ECD
(Fig. 2C,D). This suggested
that interference with ed function somehow reduced N signaling.
Interactions between ed and N signaling in bristle
development
Prompted by the above results, we searched for genetic interactions between
ed and members of the N signaling pathway. Halving the gene dose of
N, by using the null N55e11 allele in
heterozygous condition, had a minimal effect on notum chaetae, as it only
slightly increased the density of microchaetae (compare
Fig. 1A with
Fig. 3E). However, the
combination N55e11/+; ed1x5/edslH8
showed a strong effect, as microchaetae were almost totally suppressed
(Fig. 3F). The number of extra
macrochaetae was only slightly increased over that of the
ed1x5/edslH8 flies, but often they had double
shafts. As ed1x5/edslH8 is a relatively week
ed loss-of-function condition, we examined the phenotypes of other
genetic combinations. Expression in proneural clusters (C253-Gal4
driver) of either UAS-edECD or
UAS-NECD, a dominant-negative form of N that lacks most of
the intracellular domain (Jacobsen et al.,
1998
), had relatively mild effects
(Fig. 3A,B). By contrast,
expression of both transgenes together removed most microchaetae and either
eliminated macrochaetae or replaced them with tufts of bristles
(Fig. 3C). This is a strong
neurogenic phenotype. The tufts of bristles result from breakdown of lateral
inhibition in proneural clusters, whereas the absence of micro and
macrochaetae is normally caused by the precursors of the epidermal
constituents of the SO (basal cell and shaft) differentiating as extra neurons
because of the absence of N signaling
(Hartenstein and Posakony,
1990
). This occurred under our experimental conditions. mAb 22C10
staining of pupal nota revealed groups of neurons
(Fig. 3D) instead of the
single, well-separated neurons, each one innervating an individual chaeta,
typical of the wild-type notum (not shown)
(Hartenstein and Posakony,
1990
). As expected, groups of contiguous SOPs were detected in the
imaginal discs of these flies (Fig.
3D, inset). Synergistic interactions were also found by
overexpressing UAS-ed
ECD and
UAS-DlDN (Huppert et
al., 1997
) with the sca-Gal4 driver
(Fig. 3G-I). Moreover, the
halving of the genetic dose of Dl (Dlrev10/+) did
not affect the notum macrochaetae (not shown), but it did increase the number
of extra macrochaetae promoted by ed1x5/edslH8
(Table 2).
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Next, we examined the effect of constitutive activation of the pathway.
Overexpression of the intracellular domain of the N protein
(UAS-NICD) (Mumm and
Kopan, 2000) with the C253-Gal4 driver removed
essentially all bristles, a phenotype unmodified by reduction of ed
function (UAS-ed
ECD) (not shown). Although
this is consistent with ed acting upstream of the release of
NICD into the cytoplasm, it could also result from the strong
activation of the pathway, which might make ineffective the antagonizing
effect of the loss-of-function of ed. Thus, we examined the effect of
milder activations of the pathway. We resorted to transient activations and
administered 1.5 hour heat shocks (37°C; 0-8 hours after puparium
formation) to individuals harboring a hs-NICD transgene
(Lieber et al., 1993
;
Struhl et al., 1993
). This
eliminated most microchaetae (Fig.
4A), as their SOPs emerge during or just after the heat shock
(Rodríguez et al.,
1990
; Usui and Kimura,
1993
). By contrast, it did not prevent macrochaetae determination,
which occurred before the heat shock treatment
[Fig. 4A; note that most
macrochaetae were converted to double sockets due to the excess of N signaling
during differentiation (Schweisguth and
Posakony, 1994
)]. Under these conditions, expression of
UAS-ed
ECD (sca-Gal4 driver) gave
rise, as expected, to extra macrochaetae (extra `double sockets'), but did not
rescue the loss of microchaetae (Fig.
4B), again suggesting that ed functions previously to
NICD release into the cytoplasm. An even milder overactivation of
the N pathway was accomplished by a similar heat treatment of individuals
carrying an hs-NECN transgene
(Rebay et al., 1993
).
NECN has the NICD fragment bound to the transmembrane
domain of N and this only permits a slow release of NICD.
Heat-treated hs-NECN flies lost microchaetae on only a
relatively small region of the notum (Fig.
4C). Still, expression of the
UAS-ed
ECD could not rescue this weak
phenotype, although as expected it promoted the emergence of extra
macrochaetae (Fig. 4D). All
these data suggest that ed may interact with the N pathway in
processes previous to the release of the NICD.
|
Ed colocalizes with N at the zonula adherens of wing imaginal disc
cells
Our epistatic and clonal analyses are compatible with Ed facilitating N
signaling by acting at a step previous to the release of the NICD.
Accordingly, we tested the possibility that Ed might physically interact with
N. First, we examined the subcellular localization of both proteins in the
wing imaginal disc. Using antibodies that recognize the C terminus of Ed and
the zonula adherens marker Armadillo (Arm), we observed that Ed mainly, if not
exclusively, accumulates at the zonula adherens where it colocalizes with Arm
(Fig. 5A-C). This is in sharp
contrast to the eye disc, where Ed resides throughout the cell membrane of all
cells (Islam et al., 2003).
Using NICD-specific antibodies, we further observed that N is
mainly colocalized with Ed (Fig.
5D-F). Similar colocalization with Ed at zonula adherens can also
be detected with NECN-specific antibodies, but Ed is not present in
the NECN-containing internalized vesicles (data not shown)
(Pavlopoulos et al.,
2001
).
|
ed produces a moderate neurogenic phenotype in the
embryo
As ed promotes development of extra bristles by affecting N
signaling, we examined whether ed also affects early neural
development. Removal of N signaling causes all the neuroectodermal cells to
develop as neuroblasts (de la Concha et
al., 1988; Lehmann et al.,
1981
). Eighty percent (n=59) of
edlF20 (null) mutant germline clone-derived embryos
lacking both maternal and zygotic ed expression exhibited ventral
holes in the cuticle (Fig.
6A,B), while the rest of embryos (20%) displayed only fusion of
ventral denticle belts (data not shown). Both effects indicate a dearth of
epidermal precursors. Furthermore, we detected a moderate hyperplasia of the
embryonic nervous system, as revealed by the increase in the number of
ELAV-positive cells in stage 14 embryos
(Fig. 6C,D). Clearly,
ed embryos exhibit a N-like phenotype, although weaker than those of
mutations at the neurogenic genes (de la
Concha et al., 1988
; Lehmann
et al., 1981
).
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Discussion |
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The main steps of N signaling responsible for lateral inhibition during SO
development can be summarized as follows (reviewed by
Artavanis-Tsakonas et al.,
1995; Artavanis-Tsakonas et
al., 1999
; Mumm and Kopan,
2000
; Simpson,
1997
). (1) Activation by the proneural proteins of the Dl ligand
in the signal-emitting cell. (2) Interaction of activated Dl with N in the
receptor cell, which culminates in the intramembrane proteolytic cleavage of
the N molecule. (3) Release of the NICD, which translocates to the
nucleus and, in collaboration with Su(H) and other proteins transcriptionally
activates downstream genes. Paramount among these are the bHLH repressors of
the E(spl)-C, which interact with specific AS-C enhancers and prevent the
proneural gene self-stimulation necessary for cells to reach the SOP state
(Culí and Modolell,
1998
; Giagtzoglou et al.,
2003
). Our genetic epistasis experiments, together with the data
summarized above, suggest that ed may facilitate N signaling by
acting previously to the translocation of the NICD into the
nucleus. Hence, Ed might facilitate steps of N signaling that occur at or near
the membrane of the receptor cell, like N activation, N proteolytic processing
or its membrane release. This is consistent with the colocalization of Ed and
N at the zonula adherens and with the apparent absolute requirement for this
localization of the Ed intracellular domain to exert its dominant-negative
effect. However, at present little can be said of the molecular mechanism
underlying the Ed-N interaction. We have been unable to demonstrate, by GST
pull-down and two-hybrid assays, a direct physical interaction between the
intracellular domain of Ed and N (S.Y.W., unpublished). However, Ed might
indirectly interact with N, probably together with other proteins or through
its effects on cell adhesion, and provide an optimal environment for N
activation/processing. An excess of either the Ed full-length molecule or the
intracellular domain anchored to the membrane might disrupt this environment
by displacing other molecules necessary for effective signaling and,
therefore, exhibit dominant negative phenotypes. Hibris (Hbs), another Ig
domain-containing cell-adhesion molecule, acts as a regulator of myoblast
fusion and hbs mutant embryos show a partial block of myoblast fusion
(Artero et al., 2001
). Similar
to Ed, overexpression in the mesoderm of either full-length Hbs or of a
derivative containing the intracellular domain anchored to the membrane also
exhibits hbs loss-of-function phenotypes.
The interaction of ed with the N pathway does not appear to be limited to the process of bristle development. Wing vein determination is also affected, as the combination of loss-of-function conditions for ed and Dl results in overly enlarged veins (not shown), a characteristic of reduced N signaling. The neurogenic phenotype of the CNS of edlF20 (null) mutant germline clone-derived embryos is also consistent with reduced N function. However, clones null for ed did not disrupt formation of the wing margin (L.M.E., unpublished), another process dependent on N function.
Ed and Egfr signaling
In the eye disc, Ed functions as a receptor and elicits an independent
signaling pathway that converges into the nuclei, where it apparently acts
upstream of TTK88 to antagonize the Egfr pathway. Ed can be activated either
non-autonomously by its own homophilic interaction or autonomously by
heterophilic trans-interaction with Nrg from neighboring cells
(Bai et al., 2001;
Islam et al., 2003
). During R8
photoreceptor specification, ed also acts both autonomously and
non-autonomously to antagonize Egfr function and a model of direct
interactions between the Ed and Egfr molecules has been proposed
(Rawlins et al., 2003
;
Spencer and Cagan, 2003
).
Other work has shown that Egfr signaling is necessary for the emergence of the
SOP of the notum macrochaetae (Culí
et al., 2001
). This function, triggered by ac-sc
expression in the cells of the proneural cluster, has been denominated
`lateral cooperation', as it appears to be antagonistic to the `lateral
inhibition' promoted by N signaling. In fact, the self-stimulation of
proneural genes that occurs in the SOP and which is essential for neural
commitment (Culí and Modolell,
1998
) appears to be the target of both signals, one stimulatory
(Egfr) and the other inhibitory (Dl-N). Our present finding that ed
not only synergizes with N in lateral inhibition, but it also antagonizes Egfr
in lateral cooperation opened the possibility that the effect of ed
on either the N or the Egfr pathway might result from the action of
ed on the reciprocal pathway. However, the available data suggests
that there is an interaction during chaetae formation with the N pathway.
Indeed, evidence has been provided that N signaling downregulates Egfr
signaling by inhibiting rhomboid/veinlet mRNA accumulation in
proneural clusters, a molecule that facilitates Egfr activation
(Culí et al., 2001
). By
contrast, the activity of the N pathway, as measured by the accumulation of a
major effector of lateral inhibition, the E(spl)-m8 mRNA, seems
independent of the levels of Egfr signaling
(Fig. 7G-I). Moreover,
loss-of-function conditions for ed decreased the accumulation of
E(spl)-m8 mRNA, while that of rhomboid/veinlet mRNA was not
detectably affected (L.M.E., unpublished). The independence of the
ed-N interaction from Egfr is also supported by the
neurogenic effect of the null edlF20 allele in the embryo.
We conclude that, the reduction of N-dependent lateral inhibition concomitant
with the decrease of ed function might explain, at least in part, the
interaction of ed with the Egfr pathway. Still, a more direct
interaction between ed and this pathway, similar to that which occurs
in photoreceptor cell determination, should also be considered.
ed and fred
Recently, the presence near ed of the structurally related gene
fred has been reported (Chandra
et al., 2003). ed and fred have been considered
paralogous genes, because they have 69% identity in their extracellular
domains, although only limited similarity in their intracellular domains.
Similarly to ed, fred has been proposed to act in concert with the N
signaling pathway and the absence of either gene decreases cell viability.
However, ed and fred do not completely replace each other,
because mutations that affect only ed
(Bai et al., 2001
) (this work)
or expression of RNAi constructs specific for fred
(Chandra et al., 2003
) have
clear mutant phenotypes. The fact that these genes are not redundant may be
related to their largely different intracellular domains
(Chandra et al., 2003
). The 48
amino acid C-terminal region of Ed necessary for the activity of the
intracellular domain (this work) is absent from Fred.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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(2003). echinoid mutants exhibit neurogenic phenotypes
and show synergistic interactions with the Notch signaling pathway.
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Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J.
(1999). Notch signaling: cell fate control and signal integration
in development. Science
284,770
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