Institute für Genetik, Universitaet zu Koeln, Weyertal 121, 50931 Koeln, Germany
* Author for correspondence (e-mail: th.klein{at}uni-koeln.de)
Accepted 30 January 2003
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SUMMARY |
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Key words: Suppressor of Hairless, Enhancer of split complex, Notch-signalling, Bristle development, Presenilin, Kuzbanian, Senseless
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INTRODUCTION |
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In many developmental processes Su(H) seems to act as a repressor of the
expression of target genes in the absence of the Notch signal
(Barolo et al., 2002;
Furriols and Bray, 2000
;
Morel and Schweisguth, 2000
).
For this `default repression' it requires the Hairless protein (H), which acts
as a bridge between Su(H) and its co-repressors CtBP and Groucho
(Barolo et al., 2002
;
Morel et al., 2001
).
One intensely studied process in which the Notch pathway plays an
important role is the development of the bristle sense organ of the adult
peripheral nervous system (PNS) of Drosophila
(Modolell and Campuzano,
1998). These bristles are simple mechanosensory organs that
consist of only four cells. All four cells are generated by a single
precursor, referred to as the sensory organ precursor cell (SOP)
(Fig. 1A). The SOP is selected
from a cluster of cells that are defined by the expression patterns of the
proneural genes, such as the genes of the achaete-scute complex
(AS-C) (Fig. 1B,C).
The activity of the proneural genes enables all cells of a cluster (proneural
cluster) to develop as SOPs. The selection of the SOP in the proneural cluster
occurs through a process called lateral or mutual inhibition and is mediated
by the Notch signalling pathway. Lateral inhibition ensures that only
a defined number of cells of a proneural cluster develop as SOP, whereas the
rest switch fate and develop to epidermoblasts. During lateral inhibition, the
SOP sends an inhibitory signal via the Notch ligand encoded by Delta
(Dl) to its neighbours to activate the expression of the genes of the
Enhancer of split-complex [E(spl)-C] in these cells
(Bailey and Posakony, 1995
;
Hinz et al., 1994
;
Jennings et al., 1994
;
Lecourtois and Schweisguth,
1995
; deCelis et al.,
1996
; Lai et al.,
2000a
; Lai et al.,
2000b
). The activity of the genes of the E(spl)-C
antagonizes that of the proneural genes
(Knust et al., 1992
;
Nakao and Campos-Ortega,
1996
). As a result the neighbouring cells switch fate and develop
as epidermal precursors. The expression of the genes of the E(spl)-C
is directly activated by a transcription complex that includes Su(H) as the
DNA-binding part (Bailey and Posakony,
1995
; Hinz et al.,
1994
; Jennings et al.,
1994
; Lecourtois and
Schweisguth, 1995
; deCelis et
al., 1996
; Lai et al.,
2000a
; Lai et al.,
2000b
).
|
Once a SOP is selected through lateral inhibition, it starts to express
genes such as asense (ase), neuralized
(neur), senseless (sens; Ly
FlyBase) and hindsight (hnt; pub FlyBase).
The expression of these markers is important for the correct development of
the SOP. In particular, sens appears to be essential for the normal
development of the sensillum (Nolo et al.,
2000). It encodes a zinc-finger transcription factor and is
activated in the SOP by the proneural proteins Achaete (Ac) and Scute (Sc)
(Nolo et al., 2000
). Upon
ectopic expression, Sens is able to induce supernumerary SOPs, indicating that
it is sufficient to initiate the development of sensory organs
(Nolo et al., 2000
). At the
time when sens and neur expression is initiated, the
expression of the proneural genes is switched off in the SOP
(Cubas et al., 1991
).
The SOP subsequently divides to generate two second-order precursors, pIIa
and pIIb (Hartenstein and Posakony,
1990) (Fig. 1C).
pIIa divides once more and generates the bristle and socket cell. pIIb divides
to give rise to a pIIIb precursor cell and a glia cell
(Gho et al., 1999
)
(Fig. 1C). The glia cell
migrates away from the developing sense organ. The pIIIb divides another time
to give rise to the neurone and a sheath cell
(Fig. 1C).
The Notch pathway is required repeatedly during the further
development of the bristle sensillum
(Hartenstein and Posakony,
1990) (Fig. 1C,D): It sends an inhibitory signal from the second order precursor cell pIIb to
pIIa that prevents pIIa from choosing the pIIb fate. Later, the Notch
pathway is again required to send a signal from the bristle to the socket cell
and from the neuron to the sheath cell to prevent the receiving cells from
choosing the same fates as the sending cell
(Fig. 1C). Thus, loss of
Notch function results in the development of all cells of a proneural
cluster into SOP cells. These SOP cells then generate an excess of neurones at
the expense of the other fates (Fig.
1D). This scenario implies that loss of function mutants of all
genes that are involved in the Notch pathway should display an excess
of SOPs that subsequently generated an excess of neurones
(Fig. 1D), a phenotype named
neurogenic.
Schweisguth and Posakony (Schweisguth
and Posakony, 1992) have reported that in Su(H) mutant
wing imaginal discs not all proneural clusters can be detected with the
SOP-specific neurA101-lacZ marker
(neurA101). By contrast, cells of all clusters express
this marker in kuz mutant wing discs
(Sotillos et al., 1997
). The
differences in the mutant phenotype of kuz and Su(H) raise
the possibility that Su(H) might have a function during SOP development that
is independent from its function during Notch signalling. To test
this possibility, we compared the consequences of loss-of-function mutations
of genes that are involved in the Notch pathway on the development of
the SOP, and the differentiation of neurones. We found that in Su(H)
mutants, the development of the SOPs arrests during an early phase. This is
not observed in Psn, Notch and kuz mutants, and suggests
that Su(H) is required for SOP development in a Notch independent
fashion. We provide evidence that this arrest is caused by the loss of the
activity of the gene senseless (sens), which is crucial for
SOP development. Our results suggest that Su(H) acts as a repressor of one or
more members of the E(spl)-complex that in turn repress the
expression of sens.
We further find that Su(H) mutant cells are unable to prevent the SOP fate in normal neighbours that are located at the position of the proneural cluster, where the SOP normally forms. It appears that cells at this position are insensitive to the Dl signal. This observation suggests that the positional information within a proneural cluster is more precise than anticipated and that the position of the SOP is strongly determined.
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MATERIALS AND METHODS |
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Reporter strains were E(spl)m8-lacZ
(Lecourtois and Schweisguth,
1995; Nakao and Campos-Ortega,
1996
), E(spl)mß CD2
(de Celis et al., 1998
), SOP-E
(Culi and Modolell, 1998
) and
Gbe+Su(H) (Furriols and Bray,
2001
).
UAS stocks were UASsens (Nolo
et al., 2000), UASSu(H)
(Klein et al., 2000
), UAS
Su(H)
H (Furriols and Bray,
2000
) and UAS GFP [a gift from S. Bahri and Yeh et al.
(Yeh et al., 1995
)].
Gal4 drivers wre scaGal4 (Hinz
et al., 1994) and dppGal4 (a gift from S. Carroll).
Histochemistry
Antibody staining was performed according to standard protocols. The anti
Dl, anti Wg and anti Hnt antibodies were obtained from the Developmental
Studies Hybridoma Bank developed under the auspices of the NICHD and
maintained by the University of Iowa, Department of Biological Sciences, Iowa
City, IA 52242. The anti Sens antibody was a gift of H. Bellen
(Nolo et al., 2000). The anti
22C10 and anti Elav antibody were a gift of C. Klämbt.
Fluorochrome-conjugated antibodies were purchased from Molecular Probes.
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RESULTS |
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To find out whether there is a difference in the phenotypes of
Su(H) and mutants of other genes involved in the Notch
pathway, we have compared the development of the SOPs of the machrochaete in
wing imaginal discs that were mutant for of Su(H), Notch, Psn and
kuz. We used two other markers, besides
neurA101-lacZ, that specifically label SOPs of the wing
imaginal disc (sens and hnt). Expression of both genes is
restricted to the SOPs in the notum (Fig.
2A,E,J) (Nolo et al.,
2000; Pickup et al.,
2002
).
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During the course of our experiments, we noticed that the loss of
Psn function causes the strongest phenotype. We found that the
proneural clusters are larger than in other mutants and often fused (see
Fig. 2G,L). This observation is
in agreement with the results of the analysis of Psn mutants during
wing development, where loss of its function also causes the strongest
phenotype (Klein et al.,
2000).
Absence of neurones in Su(H) mutant wing imaginal discs
To look at the consequences of loss of the SOP markers for neural
differentiation of the progenies of Su(H) mutant SOPs, we monitored
the expression of 22C10 (futsch) and elav, which
are specific for mature neurones (Fig.
3). Although all cells of Psn and Notch mutant
proneural clusters express 22C10 and some in addition express
elav (Fig. 3A,B,D), the expression of these markers was not detectable in cells of Su(H)
mutant clusters (Fig. 3C,E).
This indicates that the loss of expression of SOP marker such as sens,
hnt and neurA101 was accompanied by a lack of neural
differentiation in Su(H) mutant wing imaginal discs.
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Forced expression of sens can re-establish expression of SOP
markers normally absent in cells of Su(H) mutant proneural
cluster
sens encodes a zinc-finger containing transcription factor that is
essential for SOP development (Nolo et
al., 2000). Thus, we wondered whether it is the loss of
sens activity that causes the arrest in SOP development in
Su(H) mutant wing imaginal discs. To test this hypothesis, we looked
to see if forced expression of sens could restore expression of
neural markers such as 22c10 and elav and SOP markers such
as hnt and the SOP-E in Su(H) mutant discs
(Fig. 6). In the first series
of experiments, we expressed UAS sens with dppGal4
(Fig. 6A-G). We found that most
cells that express Sens were able to activate the expression of the SOP-E, as
well as hnt (Fig.
6A,C,F,G). Most sens-expressing cells of the notum also
initiated 22C10 expression (Fig.
6B,C). The ability of Sens to activate these genes ectopically was
restricted to the notum.
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In summary, the results show that Sens is able to activate the expression of those genes that are normally absent in Su(H) mutant proneural clusters and suggests that the loss of sens activity causes the arrest in SOP development in Su(H) mutants. Hence, Su(H) appears to be required in the SOP to activate the expression of sens in a Notch-independent manner.
SOP development in Su(H) mutant cell clones
We also analysed the function of Su(H) during SOP development by
inducing mutant cell clones in the notum during first larval instar [24-48
hours after egg laying (ael)] (Fig.
7). Using this type of analysis, we found a spectrum of
phenotypes. In several cases, the mutant cells express early markers such as
neurA101 or the SOP-E, but fail to express hnt.
The arrowhead in Fig. 7A-E points to such an example. In this case one Su(H) positive cell lies
in the cluster. It is this cell that expresses SOP-E and hnt. By
contrast, the mutant cells of the cluster express only the SOP-E. In other
cases, we found a varying fraction of Su(H) mutant cells that express
the early marker and also hnt, suggesting that some of the mutant
cells do not arrest their development (arrows in
Fig. 7B-E;
Fig. 7F-I). However, many of
the cells of this class expressed lower levels of hnt than normal
(see Fig. 7H,I). Altogether,
these observations confirm our conclusion that Su(H) is required for the
development of the SOP.
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For the other clusters, where we have less cases examined, we found that in four out of six cases of clones, including parts of the pPA/tr2/aPA+tr1 cluster we found a fraction cells weakly expressing hnt. In one out of the four observed cases of the PSA cluster, we found weak hnt expression in one cell. In the two clones we found for the ANP/PNP cluster no expression was observed.
As expected, kuz mutant cell clones that include regions of proneural clusters contain big clusters of hnt-expressing cells, indicating that cells of kuz mutant proneural clusters can progress in their development as SOP (Fig. 7J).
The SOP is insensitive to the DI signal from its Su(H)
mutant neighbours
During the course of the clonal analysis of Su(H), we very often
found that a single enlarged and Su(H)-positive cell adjacent or
nearly surrounded by mutant cells that express the SOP-E
(Fig. 7B-E;
Fig. 8I-M). Invariantly, this
Su(H)-positive cell expressed hnt
(Fig. 7B-E) and was located at
the position of the cluster, where a SOP would normally arise
(Fig. 7B-G). This observation
contradicts the lateral inhibition model, which predicts that cells in which
Notch is least active have the highest potential to inhibit their neighbours
from adopting the SOP fate. Hence, cells in a cluster that are defective in
Notch signal reception should be very potent to inhibit their
wild-type neighbours and a Su(H)-positive cell should therefore never
adopt the SOP fate, if adjacent to Su(H) mutant cells.
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We found that Su(H) mutant cells of a proneural cluster can activate the Gbe+Su(H) construct in their Su(H)-positive neighbours. This is indicated by the upregulation of the construct in these cells (Fig. 8K-O, arrowhead in N, O). Hence, the cells of Su(H) mutant proneural cluster seem to express an active form of Dl. However, one exception was observed: Gbe+Su(H) is not activated in the hnt expressing, Su(H) positive SOPs that are located next to mutant cells (arrows in Fig. 8K,M,O). Expression of Notch itself in Su(H) mutant cells was normal (data not shown). These observations indicate that Su(H) mutant cells, although expressing active Dl, cannot activate the Notch pathway in cells that are located at positions where the SOP develops. Thus, it appears that the position of the SOP within a proneural cluster is strongly determined. Cells at this position appear to be insensitive to lateral inhibition.
The conclusion that the position of the SOP within a proneural cluster is pre-determined is further supported by another observation: We found three cases where a Su(H) mutant clone includes almost all cells of a proneural cluster. In these cluster, one or two cells of the cluster weakly express Hnt (arrows in Fig. 7B,E). These cells are located at positions, where the SOP would be expected to arise. Thus, it appears that cells at certain positions in a cluster are strongly biased towards the SOP fate.
The repressor function of Su(H) is required for expression of
sens in the SOP
The results above suggest that Su(H) is required for the proper expression
of sens in the SOP. Su(H) could activate the expression of
sens by binding directly to its promoter. Such a
Notch-independent activation of target genes by Su(H) has recently
been discovered (Barolo et al.,
2000; Klein et al.,
2000
). Alternatively, Su(H) could act as a repressor that switches
off the expression of a factor that in turn represses the expression of
sens. Default repression by Su(H) in absence of
Notch-signalling seems to be a common mechanism to silence expression
of Notch target genes in the absence of Notch activity
(Barolo et al., 2002
). We have
performed the following experiments to discriminate between the two
possibilities. First, we made use of a construct in which a VP16
transactivation domain is fused to Su(H) (Su(H)VP16)
(Kidd et al., 1998
). UAS
Su(H)VP16 acts exclusively as an transcriptional activator that
activates all Notch target genes in the embryo and wing, similar to
activated forms of Notch (UAS Nintra)
(Kidd et al., 1998
;
Klein et al., 2000
). If Su(H)
is a direct activator of sens transcription, expression of UAS
Su(H)VP16 might activate sens in cells where it is
expressed. However, if Su(H) represses the expression of a repressor, UAS
Su(H)VP16 should activate expression of this repressor and thus
sens expression should be lost. We observed that expression of UAS
Su(H)VP16 with dppGal4 did not induce expression of
sens in the notum. By contrast, Su(H)VP16 appears to
suppress its expression in most parts of the notum
(Fig. 9D,E). This result
favours the possibility that the repressor function of Su(H) is required for
sens expression. However, we cannot rule out that, in this
experiment, the loss of sens expression is caused indirectly through
the suppression of the determination of the SOP by UAS Su(H)VP16.
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We further expressed a form of Su(H) that cannot bind H because it
lacks the H-binding domain (Furriols and
Bray, 2000). Overexpression of this UAS Su(H)
H
construct by scaGal4 abolished hnt expression in
Psn mutant wing imaginal discs
(Fig. 9F). As in the H
Psn double mutants, the cells of the proneural clusters were present, as
visualized by the expression of UAS GFP driven by scaGal4. Thus,
expression of UAS Su(H)
H in cells of the proneural clusters
appears to cause an arrest of SOP development as observed in Su(H)
mutants. In summary, these results support the conclusion that Su(H) requires
H for its function in SOP development (see
Fig. 9H).
The arrest of SOP development in Su(H) mutants is caused by
one or more members of the E(spl)- complex
Su(H) is directly required for the activation of the genes of the
E(spl)-C (Bailey and Posakony,
1995; Hinz et al.,
1994
; Jennings et al.,
1994
; Lecourtois and
Schweisguth, 1995
; deCelis et
al., 1996
; Lai et al.,
2000a
; Lai et al.,
2000b
). Seven of the genes of this complex encode bHLH repressor
proteins that are required for the suppression of the SOP fate in the cells of
the proneural clusters during the process of lateral inhibition
(Knust et al., 1992
;
Nakao and Campos-Ortega, 1996
)
(Fig. 10A). Four other genes
of the complex encode members of the bearded protein family
(Lai et al., 2000a
;
Lai et al., 2000b
)
(Fig. 10A). The data raise the
possibility that the repressor of SOP development is encoded by one or more
genes of the E(spl)-C.
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We then tried to determine, which of the genes of the complex encode the
repressor. From previous work, it is known that only one of the bH1H proteins,
E(spl)m8, and two of the bearded-like proteins, M and M4, are
expressed in cells of Su(H) mutant proneural clusters
(Bailey and Posakony, 1995
;
deCelis et al., 1996
;
Lai et al., 2000a
;
Lai et al., 2000b
)
(Fig. 10A). The other members
of the complex are either not expressed in the notal region of the wing
imaginal disc, or the expression is lost in Su(H) mutant cells
(Bailey and Posakony, 1995
;
deCelis et al., 1996
;
Lai et al., 2000a
;
Lai et al., 2000b
). Hence, it
is likely that persistent expression of one of the three proteins expressed in
cells of Su(H) mutant proneural clusters causes the arrest of SOP
development. However, forced expression of UAS m4 or UAS m
in cells of
the proneural clusters with scaGal4 results in the formation of
supernumerary bristles (Lai et al.,
2000b
). This suggests that these proteins stimulate rather than
preventing SOP development.
By contrast, the expression of E(spl)m8 is switched off in the SOP
during normal development (Nolo et al.,
2000) (Fig. 5D,E).
Thus, Su(H) appears to be required to switch off the expression of
E(spl)m8. Furthermore, expression of UAS E(spl)m8 with
scaGal4 prevents SOP development in Su(H) or Psn
mutant proneural clusters (Klein et al.,
2000
) (data not shown). These facts suggest that the abnormal
persistent expression of E(spl)m8 in Su(H) mutant proneural
clusters might cause the arrest in SOP development. One prediction for this
hypothesis is that E(spl)m8 should not be abnormally expressed in
SOPs of mutants that are involved in Notch signalling, but do not
affect the formation of the Su(H)/H repressor complex. One such an example is
the mutants of Psn. Thus, we monitored the expression of
E(spl)m8 in Psn mutant wing discs. In addition we looked at
the expression of the E(spl)mß gene, which is expressed in a
broader domain and seems to include all regions of the wing imaginal discs
where Notch is active (de Celis et al.,
1996
) (Fig. 10E).
We found that E(spl)m8 is strongly expressed in cells of Psn
mutant proneural clusters in a similar way to expression in Su(H)
mutants (compare Fig. 10D with
Fig. 5C). Hence, it is unlikely
that the abnormal expression of E(spl)m8 alone causes the arrest in
SOP development observed in Su(H) mutants. As expected, expression of
E(spl)mß was reduced in both mutants in a similar manner and is
not elevated in cells of the proneural clusters
(Fig. 10E,F; data not
shown).
Altogether, the results indicate that the repressor of SOP development in Su(H) mutants is encoded by one or more genes of the E(spl)-C. At the moment, it is difficult to determine whether the repressor activity is encoded by one member of the complex or by a combination of E(spl)m8 with one or both of the beaded-like proteins.
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DISCUSSION |
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A role of Su(H) in development of the SOP is surprising, because it is a
core element of the Notch signalling pathway and the activity of this
pathway is required to prevent SOP development in cells of the proneural
clusters (Schweisguth and Posakony,
1992; de Celis et al.,
1996
; Heitzler et al.,
1996
; Nakao and Campos-Ortega,
1996
; Klein et al.,
2000
). Importantly, in this new role, Su(H) seems to function
independently of the Notch signalling pathway. This is indicated by
the finding that the Su(H) mutant phenotype is epistatic over that of
Psn mutants.
The data presented here indicate that Su(H) appears to be required to
suppress the activity of one or more members of the E(spl)-C, that in
turn suppress the expression of genes such as hnt and sens.
This conclusion is based on: (1) the failure of Su(H)VP16 to activate
sens, (2) the fact that Psn H double mutants display a
similar loss or reduction of sens expression as Su(H) and
Su(H); Psn double mutants and (3) the fact that expression of a
Su(H) construct that is unable to bind H (UAS Su(H)H)
leads to an arrest of SOP development in Psn mutant wing imaginal
discs. Several reports show that H is involved in Su(H)-related
suppression of gene expression in the absence of Notch signalling
(Furriols and Bray, 2001
;
Klein et al., 2000
; Morel and
Schweisguth, 2001; Barolo et al.,
2002
). Recently, it has been shown that H acts as a bridge between
Su(H) and the general co-repressors CtBP and Gro
(Barolo et al., 2002
; Morel and
Schweisguth, 2001). It is therefore likely, that this Su(H)/H/Gro/dCtBP
complex mediates the repressor function required during SOP development.
Repression by Su(H) is not strictly required in all proneural clusters to allow expression of sens and other late SOP markers. Examples are the clusters in the wing region, such as the clusters of the dorsal radius. However, even in these clusters, sens and hnt are not expressed in all cells that express early markers, such as neurA101 (e.g. compare wing imaginal discs in Fig. 2C with 2H,M; clonal data not shown). Therefore, it appears that the activity of Su(H) promotes SOP development also in these clusters. The clusters of the dorsal radius give rise to other types of sense organs, such as companiforme sensilla, and it is possible that there are different requirements for the activity of Su(H) for the development of the different types of sense organs
The repressor activity requires the activity of one or more genes of
the E(spl) complex
We show that the removal of one copy of the E(spl)-C is already
sufficient to relieve the block in SOP development in Su(H) mutants,
indicating that the arrest is probably caused by the abnormal expression of
one or more members of the complex. Although the complex encodes for several
well-characterized repressors of neural development, we were not able to
pinpoint the repressor function to any particular gene. Many studies by
various groups have studied the regulation of the genes of the
E(spl)-C (Bailey and Posakony,
1995; deCelis et al.,
1996
; Lai et al.,
2000a
; Lai et al.,
2000b
). From these studies, it is clear that only three genes of
the complex are expressed in the cells of Su(H) mutant proneural
clusters. All other members are either not expressed in the notal region of
the wing imaginal disc or their expression is lost in the mutant cells.
Previous studies have shown that both bearded-like proteins that are
expressed in Su(H) mutant proneural clusters promote SOP development
(Lai et al., 2000a
;
Lai et al., 2000b
). Hence, it
is unlikely that the abnormal expression of these genes causes the observed
arrest in SOP development. To our surprise, we found that the strongest
candidate, the bHLH repressor encoded by E(spl)m8, is also abnormally
expressed in Psn mutants, where SOP development proceeds and the
Su(H)/H-containing complex is intact. The observation is interesting, because
it suggests that the activity of the whole Notch pathway is required
to switch off the expression of E(spl)m8 in the SOP, but it also
indicates that abnormal expression of the gene cannot be the reason for the
arrest in SOP development in Su(H) mutant cells. Thus, the repressor
activity might not be encoded by a specific member of the
E(spl)-C.
One possibility is that the combination of the three abnormally expressed genes of the complex generates the repressing activity. An alternative is that Su(H) controls the expression of other genes that act in combination with the upregulated members of the complex to suppress SOP development. Another possibility is that more genes of the complex are de-repressed in Su(H) mutants at a level not detectable by the currently available methods. In this scenario, the weak expression of several bHLH-encoding genes will sum up to a level of repressor activity sufficient to stop SOP development. Using currently available techniques, it is very difficult to discriminate between these possibilities.
The stability of the Su(H) protein
We found that in Su(H) mutant cell clones induced during the first
larval instar stage, hnt is expressed in a fraction of cells of
specific proneural clusters, such as the scutellar cluster, but absent or
strongly reduced in other clusters. We further found that in Su(H)
mutant wing imaginal discs, expression of sens and hnt is
lost or stronger reduced than in mutant cell clones induced during the first
larval instar.
A high stability of the Su(H) protein is a possible explanation for this
discrepancy. In favour of this explanation is the observation that the
maternal component of Su(H) is sufficient to allow the development of
homozygous animals until early pupal stages
(Lecourtois and Schweisguth,
1995). Furthermore, we found that vestigial (vg), a
target gene of the Notch/Su(H) pathway during wing development
(Kim et al., 1996
) is
expressed in Su(H) mutant wing imaginal disc of the early third
larval instar stage (S.K. and T.K., unpublished). This indicates the presence
of Su(H) activity at this stage. This residual activity of Su(H) must
be provided by the maternal component. Both observations suggest that the
Su(H) protein is degraded slowly and thus persists in mutant cells for a long
time. It is therefore likely that Su(H) mutant cells, induced at the
first larval instar, contain residual amount of Su(H). This residual amount of
Su(H) might be sufficient to activate expression of late SOP marker in cells
of certain proneural clusters.
An alternative explanation for the milder phenotype observed in the Su(H) mutant clones is that it requires time to accumulate a sufficient level of activity of the repressor(s) of the E(spl)-C to stop SOP development. Hence, in the case of the clonal analysis, the loss of Su(H) activity occurs later than in homozygous mutant wing imaginal discs and lower levels of repressor activity would be present in cells of the proneural clusters of the machrochaete.
Determination of the sensory organ precursor cell
The development of the machrochaete is a paradigm for the assignment of
different fates to initially equivalent cells. Proneural genes are expressed
in clusters of cells and confer on these cells the potential to become SOPs.
Careful examination has revealed that the SOPs of the machrochaete arise at
the same positions within the proneural cluster (Cubas and Modollel, 1992),
indicating that the selection of the SOP is not random. It is thought that
other factors, such as Extramachrochaete, Pannier and Wingless introduce small
differences in proneural activity that favour cells at specific positions
within the cluster to become the SOP (Cubas and Modollel, 1991) (reviewed by
Simpson, 1997). These small
differences in proneural activity are enhanced through the activity of the
Notch pathway: a cell with high proneural activity expresses high
levels of Dl and is therefore potent to inhibit its neighbours
(Heitzler and Simpson, 1991
;
Hinz et al., 1994
). Cells with
a high activity of Notch have less proneural activity and Dl. Thus, they are
less potent to inhibit its neighbours (reviewed by
Simpson, 1997
). In this
scenario, the Notch pathway is required to amplify initially small
differences in proneural activity in cells among a cluster. This amplification
eventually results in the accumulation of high levels of activity in the SOP
and loss of activity in the neighbours. In this way, the pathway acts to
resolve a crude pre-pattern to the level of a single cell. According to this
model, cells defective in Notch signal reception should be very
potent in lateral inhibition. However, we here found the opposite: A cell that
is located at the position where the SOP arises is able to adopt the SOP fate,
even if surrounded by Su(H) mutant cells. It can do so despite the
fact that the mutant neighbours accumulate high levels of proneural activity
(indicated by the expression of the SOP-E), as well as Dl. We here show that
Dl, expressed in the Su(H) mutant cells at high level, is active and
can activate Notch signalling in wild-type cells with the exception
of the SOP. Thus, although the SOP was adjacent to cells with an extremely
high potency for lateral inhibition during the whole live of a proneural
cluster, it has succeeded in adopting the SOP fate. It appears that the SOP is
determined by positional cues that are much more precise than anticipated.
These cues render the cell at the correct position in the cluster insensitive
to lateral inhibition. This suggests that small differences in proneural
activity are not the crucial bias imposed on cells within a proneural cluster
and that lateral inhibition might not be required to resolve a crude
pre-pattern.
Nevertheless, the big clusters of SOPs observed in other neurogenic mutants, indicate that the Notch pathway has a function in preventing the SOP fate in all cells of a proneural cluster and also in cells that are located further away from the SOP.
Furthermore, we observed a ring of high expression of the Gbe+Su(H) construct around the SOP during normal development, suggesting that the SOP sends a signal that activates the Notch pathway in its immediate neighbours. This lateral inhibition is relatively late, as we observe it only around SOPs that already express hnt. It also occurs only in the cells adjacent to the SOP.
Altogether, these observations suggest that the Notch pathway
might have two separable functions during SOP development. During early phases
of a proneural cluster, the activity of the pathway keeps the cells of the
cluster undecided, perhaps by mutual repression. Owing to positional cues, one
cell becomes insensitive to the inhibitory signal and adopts the SOP fate.
Subsequently the SOP inhibits its immediate neighbours by sending an
inhibitory signal through Dl. A similar function of the Notch pathway
has recently been proposed for the segregation of the embryonic neuroblasts of
Drosophila (Seugnet et al.,
1997).
Schweisguth (Schweisguth,
1995) reported that, during michrochaete development, cells can
adopt the SOP fate, even if they are located adjacent to Su(H) mutant
cells, suggesting that, also during development of this bristle type, the
mutant cells cannot inhibit its normal neighbours. It was suggested that the
Su(H) mutant cells might loose contact with neighbouring wild-type
cells and, because Dl/Notch signalling depends on cell contact, this
could prevent the activation of the Notch pathway in the wild-type
cells. Our data suggest that this is not the case: the mutant cells can
activate the pathway in adjacent wild-type cells. Hence, the results of
Schweisguth (Schweisguth,
1995
) suggest that also during michrochaete development positional
cues might be important for the determination of the SOP.
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ACKNOWLEDGMENTS |
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