Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA
* Author for correspondence (e-mail: mlundell{at}utsa.edu)
Accepted 12 May 2003
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SUMMARY |
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Key words: Numb, Notch, Apoptosis, Serotonergic neurons, Drosophila
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INTRODUCTION |
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The segmented Drosophila nerve cord develops from stereotyped
division of 30 neuroblasts (NB) in each hemisegment
(Doe, 1992;
Goodman and Doe, 1993
). A pair
of serotonergic neurons in each hemisegment arise from NB7-3
(Lundell et al., 1996
). NBs
are stem cells that undergo several asymmetric divisions producing a specific
number of ganglion mother cells (GMCs). Each GMC divides once to form two
neuronal or glial progeny (Hartenstein et
al., 1987
). The divisions of the NB7-3 lineage have recently been
determined using a combination of molecular markers and clonal analysis
(Isshiki et al., 2001
;
Novotny et al., 2002
). NB7-3
produces three GMCs. GMC-1 produces two neurons: GW, a motoneuron, and EW1,
the more medial serotonergic neuron. GMC-2 produces EW2, the more lateral
serotonergic neuron. GMC-3 produces EW3, a neuron that synthesizes the
neuropeptide corazonin. The GW neuron projects an axon ipsilateral and
posteriorly, and the three EW interneurons all project axons anteriorly to the
posterior commissure (Fig. 1)
(Bossing et al., 1996
;
Higashijima et al., 1996
;
Dittrich et al., 1997
;
Schmid et al., 1999
).
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In this study, we examine the role of Numb/Notch signaling, in the
differentiation of the NB7-3 lineage. Numb is a membrane-associated protein
that has been shown to partition asymmetrically with one daughter cell during
binary mitotic divisions and to be responsible for establishing alternative
cell fates in the Drosophila CNS, PNS (reviewed by
Lu et al., 2000;
Cayouette and Raff, 2002
;
Skeath and Thor, 2003
) and
myogenic precursor cells (Ward and Skeath,
2000
). Numb has also been suggested to have a role in the
differentiation of CNS lineages in vertebrates (reviewed by
Lu et al., 2000
;
Cayouette and Raff, 2002
;
Shen et al., 2002
;
Sommer and Rao, 2002
). Numb
affects cell fate by inhibiting the intercellular Notch signaling pathway.
During cell division, the cell that inherits Numb will lack Notch signaling,
while the sibling cell that does not receive Numb will maintain Notch
signaling (reviewed by Lu et al.,
2000
; Cayouette and Raff,
2002
; Sommer and Rao,
2002
; Skeath and Thor,
2003
). The mechanism for how Numb inhibits Notch signaling is
still under investigation, but Numb binds to the intercellular domain of Notch
(Frise et al., 1996
) and may
regulate
-Adaptin, a protein involved in receptor-mediated endocytosis
(Berdnik et al., 2002
).
We present evidence that Numb/Notch signaling regulates apoptosis within
the NB7-3 lineage. Notch signaling has previously been associated with
apoptosis in an array of both negative and positive effects. Notch has been
shown to inhibit apoptosis in T and B lymphocytes
(Pear et al., 1996;
Deftos et al., 1998
;
Jehn et al., 1999
;
Shelly et al., 1999
;
Morimura et al., 2000
;
Jundt et al., 2002
).
Constitutive Notch activity has been associated with cell transformation and
cancer, presumably by inhibiting apoptosis (reviewed by
Artavanis-Tsakonas et al.,
1999
; Miele and Osborne,
1999
; Mumm and Kopan,
2000
). Notch has also been implicated in inhibiting apoptosis in
the brain. In the cerebellum of mice, Notch1 mediates the onset of
neurogenesis and prevents apoptosis of neuroepithelium
(Lütolf et al., 2002
). A
number of investigations have linked neurodegeneration in Alzheimer's disease
to a reduction in Notch signaling (reviewed by
Miele and Osborne, 1999
).
Presenilins, which are often mutated in individuals with Alzheimer's disease,
are also necessary for Notch processing. It has been proposed that a loss in
presenilin function may lead to a decrease in Notch signaling and a subsequent
increase in cell death. Furthermore it has been shown in Drosophila
that constitutive expression of Notch can inhibit presenilin-induced apoptosis
(Ye and Fortini, 1999
). Thus
Notch signaling may protect neurons from apoptotic cell death.
By contrast, Notch has also been shown to induce apoptosis during
development of the Drosophila retina
(Cagan and Ready, 1989;
Wolff and Ready, 1991
;
Rusconi et al., 2000
;
Yu et al., 2002
), wing
(Milan et al., 2002
) and PNS
(Orgogozo et al., 2002
).
Constitutive expression of Notch in zebrafish causes cells of the developing
retina to enter apoptosis (Scheer et al.,
2001
). Notch has also been shown to induce apoptosis in vertebrate
neural crest cells (Maynard et al.,
2000
). Recently, several studies have found a reduction in Notch
activity to be coincident with cell proliferation and cancer
(Verdi et al., 1999
;
Wakamatsu et al., 1999
;
Talora et al., 2002
),
suggesting that Notch-induced apoptosis may be an important mechanism in
regulating cell growth. In this study, we demonstrate that during terminal
mitotic divisions of the Drosophila CNS Notch signaling can induce an
apoptotic cell fate.
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MATERIALS AND METHODS |
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Immunohistochemistry
Dissected CNS were fixed in 4% paraformaldehyde and incubated with primary
and secondary antisera as previously described
(Lundell and Hirsh, 1994).
Primary antibodies used were: rabbit anti-eg (1:2000, G. Technau), guinea pig
anti-Hb (1:600, C. Doe), rabbit anti-Ddc (1:50, M. Lundell), rat anti-Ddc
(1:30, J. Hirsh), rabbit anti-corazonin (1:200, C. Doe), rabbit anti-zfh-1
(1:400, R. Lehman), rat anti-zfh-2 (1:200, A. Tomlinson), rabbit anti-pdm1
(1:1000, T. Dick), guinea pig anti-lacZ (1:1000, M. Lundell) and
rabbit anti-lacZ (1:1000, Cappel). All Texas Red, FITC and Cy5
secondary antibodies were from Jackson Laboratories and used at 1:200. Images
were obtained using a BioRad 1024 laser-scanning microscope and processed with
Adobe Photoshop.
TUNEL assay
Apoptosis was assayed by TUNEL using a TACS 2 TDT-Fluor In Situ Kit from
Trevigen. To reduce background the Strep-Fluor solution was preabsorbed
against embryonic tissue and used at a 1:400 dilution. After the TUNEL
procedure, tissue was washed with an Avidin/Biotin blocking kit from
Vector.
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RESULTS |
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Numb is necessary for development of GMC-2 and GMC-3 progeny
As the GMC divisions of NB7-3 appear to be asymmetric, we asked whether
Numb might have a role in specifying cell fates within this lineage. We found
that in the mutant allele numb1, the development of EW2
and EW3 was dramatically altered, but there was only a mild effect on the
development of EW1 (Fig. 2).
Comparison of stage 17 CNS immunoassayed for Ddc/Zfh-2/Hb from wild-type
(Fig. 2A,A') and
numb1 (Fig.
2B,B') embryos showed that the number of immunoreactive Ddc
cells decreased from two per hemisegment in a wild-type CNS to one per
hemisegment in a numb1 CNS
(Table 1C). The Ddc cells that
remained in numb1 mutants were immunoreactive for Hb (95%,
n=65), indicating they were EW1 cells
(Fig. 2B). The EW1 cell was
detectable in most hemisegments, but in 7% of the numb1
mutant hemisegments there were no detectable Ddc cells
(Fig. 2D,
Fig. 4D and
Table 1C). Comparison of stage
17 CNS immunoassayed for Ddc/Eg-lacZ/corazonin from wild-type
(Fig. 2C) and
numb1 (Fig.
2D) embryos showed that no corazonin-positive EW3 cells were
detectable in the numb1 mutant
(Table 1D). Not only were EW2
and EW3 undetectable with Ddc and corazonin in a numb1
mutant, but they also failed to express Eg-lacZ at stage 17. In a
wild-type CNS three cells per hemisegment were detected with Eg-lacZ,
whereas in a numb1 mutant CNS only one cell per
hemisegment was detected with Eg-lacZ
(Fig. 2C,D and
Table 1E). These data indicate
that in a numb1 mutant development of the EW1 neuron
proceeds normally, but the development of the EW2 and EW3 neurons is
significantly altered.
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This suggests that in a numb1 mutant, division of GMC-1 may sometimes produce two cells with the GW fate, rather than a single GW and EW1 cell. This 10% transformation in cell fate to two GW cells, may account for the 7% of hemisegments that showed no detectable EW1 cell with Ddc immunoreactivity. Taken together, these results indicate that the differentiation of GMC-1 progeny is mostly independent of Numb function, whereas Numb is essential for the differentiation of GMC-2 and GMC-3 progeny.
numb1 alters the identity of the serotonergic
cell in segment A8
In a wild-type CNS, there is a pair of serotonergic neurons within each
hemisegment, with two exceptions: in A8, the most posterior abdominal segment,
there is a single serotonergic cell; and in T1, the most anterior thoracic
segment, there is a triplet of serotonergic cells
(Fig. 4A)
(Valles and White, 1988). We
found that in a numb1 mutant there is a change in the
identity of the A8 serotonergic cell. In a wild-type animal the single cell in
A8 expressed Zfh-2, indicating that it has characteristics similar to EW2
cells (Fig. 4B). In a
numb1 mutant, there was also a single cell in A8, but it
expressed Hb similar to an EW1 cell (Fig.
4E) and similar to the more anterior segments of a
numb1 mutant (Fig.
2B).
Examination of A8 in a wild-type embryo, at stage 15, showed three Eg immunoreactive cells. Two of the cells expressed Hb and one expressed Zfh-2 (Fig. 4C). The Zfh-2 expressing cell presumably develops into the Ddc-expressing neuron. EW3 cells were not detected with corazonin in either segments 7 or 8 (Fig. 5A), suggesting that GMC-3 does not form in these segments. These results suggest that although GMC-1 and GMC-2 both produce progeny in A8, only EW2 develops into a serotonergic neuron and the development of EW1 into a serotonergic neuron is suppressed.
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Ectopic expression of Notch disrupts the differentiation of
NB7-3 progeny
As Numb has previously been shown to inhibit Notch signaling, we next asked
whether ectopic expression of Notch within the NB7-3 lineage would produce a
similar phenotype to the numb1 mutant. Ectopic expression
of Notch was achieved using the UAS/Gal4 system
(Brand and Perrimon, 1993),
where the expression of a UAS-NotchACT transgene was
driven by an eg-gal4 transgene. The extracellular domain of Notch is
absent in the NotchACT allele, thus this allele provides
constitutive Notch activity (Doherty et
al., 1996
). In UAS-NotchACT embryos and
larvae, very few Ddc- and corazonin-containing cells were detected
(Fig. 5C and
Table 1C,D). This phenotype is
extremely penetrant, most CNS show no serotonergic or corazonin-containing
cells, the example presented in Fig.
5C is an exception. Some of these flies actually eclosed as
adults, but died within several days. This phenotype is similar to
numb1 in that EW2 and EW3 are undetectable, but is
different in that EW1 is also undetectable.
The number of Eg immunoreactive cells detectable in the UAS-NotchACT allele at stage 15 was variable (Fig. 3D,K; Table 1A). Four percent of hemisegments showed four Eg-positive cells, with the normal distribution of Hb and Zfh-2 expression (Fig. 3D). Fifteen percent of hemisegments showed three Eg-positive cells, but most hemisegments showed two Eg-positive cells (Fig. 3D). This variable number of Eg immunoreactive cells suggests that the NB7-3 lineage divisions occur when Notch is ectopically expressed, but that the cells quickly follow an alternative fate that makes them undetectable, similar to the numb1 phenotype. Interestingly, 89% of the UAS-NotchACT hemisegments had two cells that were immunoreactive for Zfh-1 (Fig. 3K and Table 1B). This suggests that most EW1 cells were converted to a GW cell fate in the UAS-NotchACT genotype. This would explain the inability to detect EW1 cells with Ddc immunoreactivity (Fig. 5C). This transformation in cell fate is similar to the 10% of hemisegments in the numb1 mutant that had two Zfh-1-positive cells. Therefore in both the numb1 and UAS-NotchACT genotypes, the number of hemisegments that have no detectable Ddc immunoreactivity is proportional to the number of hemisegments that show two Zfh-1 immunoreactive cells (Table 1B-C).
These results suggest that Notch signaling must be inactivated within the NB7-3 lineage for normal development of EW1, EW2 and EW3. As the previous results show that Numb is required for the development of EW2 and EW3, Numb is most probably responsible for repressing Notch signaling in EW2 and EW3. However, as the effect of a numb1 mutation on EW1 development is minor, some additional mechanism must be primarily responsible for repressing Notch signaling in EW1.
sanpodoG104 produces ectopic Ddc cells and can
rescue the numb1 mutant phenotype
Because the ectopic expression of Notch produced a phenotype that inhibited
development of the NB7-3 lineage, we next investigated the phenotype
associated with a loss of Notch function in the NB7-3 lineage. To
produce a loss of Notch function we used a mutant allele of
sanpodo (spdo). spdo has been reported to be a
tropomodulin-like molecule (Dye et al.,
1998) but its functional capacity has not yet been shown. Previous
experiments indicate that spdo mimics loss-of-function Notch
mutations (Buescher et al.,
1998
; Dye et al.,
1998
; Park et al.,
1998
; Skeath and Doe,
1998
; Ward and Skeath,
2000
). It is advantageous to use a spdo mutation, instead
of a Notch mutation, because spdo is not involved in
Notch-mediated lateral inhibition during early neurogenesis and thus does not
have the severe morphological defects of a Notch embryo
(Salzberg et al., 1997
).
In a homozygous spdoG104 mutant embryo at stage 15, 50% of the hemisegments had one or two ectopic cells immunoreactive for Eg (Fig. 3E and Table 1A). The additional cells were also immunoreactive for Zfh-2 suggesting that they are derivatives of GMC-2 or GMC-3. After stage 16 the homozygous spdoG104 mutant CNS became quite fragile and difficult to dissect, nevertheless we were able to isolate fragments of stage 17 CNS from spdoG104 mutants. These CNS showed random ectopic expression of Ddc cells and altered morphology. In the position of the NB7-3 lineage the thoracic clusters showed numerous Ddc immunoreactive cells (Fig. 5D), and there were three to five Ddc immunoreactive cells in each abdominal cluster (Fig. 5E and Table 1C). These clusters of Ddc-expressing cells showed two cells that were immunoreactive for Hb, with the remaining cells expressing Zfh-2 (Fig. 5F). A wild-type CNS has only one Ddc cell that expresses Hb (Fig. 1D), suggesting that in the spdoG104 mutant there is a transformation in cell fate such that, GMC-1 produces two EW1 cells, rather than a single EW1 cell and the GW cell. Corazonin immunoreactivity was poorly developed in spdoG104 mutants, only 50% of the hemisegments had a corazonin-immunoreactive cell (Table 1D) and there was no evidence of ectopic corazonin-containing cells. This phenotype of ectopic Ddc cells in spdoG104 is opposite to the absence of Ddc cells observed in numb1 and UAS-NotchACT phenotypes.
We then asked whether the spdoG104 allele could rescue
the numb1 mutant phenotype. Because the
spdoG104 homozygous phenotype is so severe, we attempted
the rescue with just one copy of the spdoG104 allele. The
spdoG104 heterozygote alone showed a phenotype that was
similar to wild-type at stage 17 (Fig.
5G). When the spdoG104 allele was combined
with the homozygous numb1 mutation we found evidence that
EW2 and EW3 were partially rescued. 23% of hemisegments showed both EW1 and
EW2 Ddc neurons and 7% of hemisegments showed an EW3 corazonin-containing
neuron (Fig. 5H;
Table 2). This is a substantial
increase over the number of hemisegments in the numb1
mutant that have a wild-type pattern of EW neurons. The ability of
spdo to rescue the numb1 mutant phenotype
demonstrates that spdo is epistatic to numb, which has been
demonstrated in other lineages (Dye et
al., 1998; Skeath and Doe,
1998
; Ward and Skeath,
2000
). These results imply that the function of Numb in the NB7-3
lineage is to inactivate Notch signaling during differentiation of EW2 and EW3
neurons.
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Notch-mediated apoptosis in the NB7-3 lineage
Previous studies with the apoptosis deficient line H99 have shown
that supernumerary Eg cells were produced within the NB7-3 lineage
(Isshiki et al., 2001;
Novotny et al., 2002
). This
result suggests that there are cells within the NB7-3 lineage that undergo
apoptosis. The ectopic NB7-3 progeny we observe by inhibiting Notch signaling
with spdoG104 and UAS-Numb might be due to an
inhibition of apoptosis and the rescue of cells in the NB7-3 lineage that
normally undergo apoptosis. Conversely, the loss of NB7-3 progeny we observe
by enhancing Notch signaling with numb1 or
UAS-NotchACT, might be due to Notch-mediated
apoptosis.
To test this hypothesis, we first examined the NB7-3 lineage for apoptosis using TUNEL analysis, which measures DNA fragmentation, a hallmark of programmed cell death. In wild-type embryos we examined the occasional hemisegments that had five immunoreactive Eg cells rather than the standard four Eg cells. In these hemisegments we found that the extra Eg-positive cell undergoes apoptosis (Fig. 6A,A'). This indicates that during the mitotic divisions of the NB7-3 lineage some cells are eliminated by apoptosis. In numb1 mutant embryos, we examined the hemisegments that had three immunoreactive Eg cells and found that the cell, which we previously showed to be Zfh-2 positive (Fig. 3C), underwent apoptosis (Fig. 6B). We also found a small fraction of numb1 hemisegments where the EW1 and GW neurons were apoptotic (Fig. 6C). It is likely that these hemisegments correspond to the 7% of numb1 hemisegments that have no detectable Ddc neurons at stage 17 (Table 1C and Fig. 2D, Fig. 4D). Thus, a loss of numb function initiates novel apoptosis within the NB7-3 lineage.
|
We next addressed whether inhibition of apoptosis could produce ectopic Ddc
and corazonin-containing neurons. We first examined the apoptosis deficient
line H99 (White et al.,
1994), which had previously been shown to produce ectopic NB7-3
cells at stage 15 (Isshiki et al.,
2001
; Novotny et al.,
2002
). We found that H99 mutant embryos showed a
wild-type pattern of expression for Ddc and corazonin at stage 17 (data not
shown). Apparently, the extra cells produced at stage 15 in H99
mutants are unable to mature into Ddc- and corazonin-containing neurons.
Therefore, we tried another approach and examined the effect of ectopic
expression of the apoptosis inhibitor, p35. Ectopic expression of p35 was
achieved using a UAS-p35 transgene driven by an eg-gal4
transgene. At stage 15, UAS-p35/eg-gal4 embryos produced
ectopic Eg cells in 56% of all hemisegments
(Fig. 3G;
Table 1A). This phenotype is
similar to H99 (Isshiki et al.,
2001
; Novotny et al.,
2002
), spdoG104 and UAS-Numb embryos
(Fig. 3E-G;
Table 1A). At stage 17,
UAS-p35/eg-gal4 embryos produced a pattern of Ddc and corazonin
immunoreactivity that was mostly wild-type with occasional ectopic cells
(Fig. 7A,D; Table 1C,D). Two
corazonin-containing cells were found in 9% of the hemisegments instead of the
normal one per hemisegment (Fig.
7A). Corazonin-containing cells were also found in A7, where
corazonin is normally not expressed (data not shown). Triplets of
Ddc-expressing cells were observed in 6% of the hemisegments
(Fig. 7D). These ectopic cells
suggest that p35 can, to a limited degree, rescue cells of the NB7-3 lineage
that would normally undergo apoptosis.
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To test directly if Notch signaling induces apoptosis within the NB7-3 lineage, we examined whether inhibiting apoptosis with ectopic expression of p35 would rescue the phenotype produced by the ectopic expression of Notch. When both UAS-p35 and UAS-NotchACT were expressed simultaneously with eg-gal4, p35 was able to protect cells from Notch-induced apoptosis (Fig. 7C). However, only 18% (n=146) of larval CNS showed the rescued phenotype depicted in Fig. 7C, the majority of CNS appeared identical to the UAS-NotchACT phenotype alone (Fig. 5C). Of the larval CNS that showed rescue there were no patterns of partial expression (Table 2), all hemisegments showed the wild-type number of Ddc- and corazonin-expressing cells with occasional ectopic cells. This all-or-nothing phenotype suggests that there is some global trigger that determines whether UAS-NotchACT or UAS-p35 will have the dominant effect on development of the NB7-3 lineage. When en-gal4 was used to drive expression of both transgenes, 100% (n=14) of larval CNS showed the rescued wild-type pattern. eg expression fades during late embryogenesis, whereas en expression persists into larval stages. Therefore the extended expression of en-gal4 may maintain sufficient levels of p35 to prevent apoptosis, whereas declining expression of eg-gal4 may cause levels of p35 to drop such that Notch-induced apoptosis predominates.
Our results suggest that Notch-induced apoptosis is an essential mechanism
for regulating cell fates within the CNS. In support of this hypothesis, we
observed in heterozygous spdoG104 larval CNS, tumor-like
overproliferation of Ddc-expressing cells that protrude from the surface
(Fig. 7E,E'). These areas
of cell growth appeared in random positions throughout the CNS and varied in
size and frequency. Ten percent of the CNS showed regions of cell
proliferation as large as those in Fig.
7F and another 30% of the CNS showed smaller foci of cell
proliferation. We do not know the origin of these cells, but because we have
seen overproliferation in the brain lobes they cannot originate exclusively
from the NB7-3 lineages. They may not even originate from Ddc-expressing
lineages, as induction of Ddc expression is a characteristic of human
small-cell lung carcinomas (Bepler et al.,
1987). This observation that a reduction in Notch signaling can
promote abnormal cell growth, suggests that Notch-induced apoptosis may have
an important role in preventing overproliferation of cells during CNS
development.
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DISCUSSION |
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The A8 segment is unique in that it only has a single serotonergic neuron instead of the pair of serotonergic neurons found in the more anterior segments. In a wild-type fly, this cell appears to be a derivative of GMC-2, because it expresses Zfh-2, but in a numb1 mutant, this cell appears to be a derivative of GMC-1, because it expresses Hb. A single Hb/Ddc-expressing cell in A8 is identical to the phenotype in the more anterior segments of a numb1 mutant. This suggests that in a numb1 mutant an EW1 cell is the default developmental pathway for this lineage. One possibility is that the redundant Notch-inactivating mechanism we proposed for EW1 is only induced in the presence of a numb mutation. This would explain why the A8 EW1 cell is only seen in the numb mutant and not in a wild-type animal. If this were true, then the preservation of EW1 cells in a numb mutant would be due to the mutation itself. Until a putative redundant factor is identified it is impossible to determine whether it is expressed normally in wild-type animals or is expressed only in numb mutant animals.
Like GMC-1, most GMCs divide producing two progeny cells. However, GMC-2
and GMC-3 of the NB7-3 lineage only produce one neuron. It has previously been
suggested that the mitotic sisters of EW2 and EW3 may undergo apoptosis
(Isshiki et al., 2001;
Novotny et al., 2002
). This
idea is supported by our detection of apoptotic cells with TUNEL in the
wild-type NB7-3 lineage and our experiments with the apoptosis inhibitor p35,
which can produce ectopic Ddc and corazonin-containing cells. The origin of
the ectopic cells within NB 7-3 has not been formally determined by lineage
tracing; however, the hypothesis that they are mitotic sisters of EW2 and EW3
is supported by the observations that GMC-2 and GMC-3 progeny often appear as
mitotic pairs (Fig. 6E,F) and
that ectopic NB7-3 cells are immunoreactive for Zfh-2
(Fig. 3E-G).
During the divisions of GMC-2 and GMC-3, genetic alterations in the expression of Notch leads to a switching between a neuronal cell fate and apoptosis. A reduction of Notch signaling with either spdoG104 or UAS-Numb embryos produces ectopic NB7-3 cells that express Zfh-2. Conversely, the overexpression of Notch in either UAS-NotchACT or numb1 embryos led to an increase in TUNEL labeling of GMC-2 and GMC-3 progeny. Additionally, inhibiting apoptosis with UAS-p35 or reducing Notch activity with spdoG104 can rescue the numb1 phenotype. We hypothesize that during the divisions of GMC-2 and GMC-3, Numb partitions asymmetrically into EW2 and EW3 where it inactivates Notch signaling and leads to neuronal development. The mitotic sisters of EW2 and EW3 do not receive Numb, maintain Notch signaling and undergo apoptosis. The difficulty in detecting wild-type hemisegments that have more than four immunoreactive Eg cells, suggests that any other cells produced during divisions of the NB7-3 lineage quickly undergo apoptosis.
Ectopic Eg cells in the NB7-3 lineage can be induced at stage 15 by
H99 (Isshiki et al.,
2001; Novotny et al.,
2002
), UAS-Numb, spdoG104 and UAS-p35
(Fig. 3E-G). However, the
ability of these alleles to produce ectopic Ddc and corazonin-containing
neurons at later stages is variable. We were unable to detect significant
ectopic Ddc or corazonin-containing cells in either H99 or
UAS-Numb CNS. We have shown for UAS-Numb that the ectopic Eg
cells detected at stage 15 can undergo apoptosis. spdoG104
mutants produce only ectopic Ddc cells, but the reduction in the number of
corazonin-containing cells in general suggests that either GMC-3 does not
consistently form in these mutants or that GMC-3 progeny may convert from a
corazonin-containing cell fate to a serotonergic cell fate. UAS-p35
mutants produce both ectopic Ddc and corazonin-containing cells at low
frequency, but the allele is much more efficient at rescuing the EW neurons in
numb1 and UAS-Notch mutants. Therefore, apoptosis
is harder to reverse in cells that normally undergo apoptosis, than in the
cells genetically induced to undergo apoptosis. The ability of these various
alleles to produce ectopic Ddc- and corazonin-containing cells could be
influenced by mutant affects they cause outside the NB7-3 lineage or may
reflect different roles they have in the apoptotic pathway. The mechanism by
which Notch induces apoptosis in the NB7-3 lineage remains to be determined,
but the apoptotic genes reaper, grim and hid may be involved
because all three of these genes are deleted in the H99 allele
(White et al., 1994
).
Notch-induced apoptosis in the NB7-3 lineage will probably be regulated by
other factors in addition to Numb. The Ras signaling pathway has been shown to
inhibit Notch-induced apoptosis in the Drosophila pupal retina
(Cagan and Ready, 1989).
Wingless has been shown to mediate Notch signaling
(Axelrod et al., 1996
;
Brennan et al., 1999
;
Wesley, 1999
;
Lawrence et al., 2001
;
Ramain et al., 2001
) and
mutations in the Wingless pathway can lead to ectopic serotonergic cells
(Patel et al., 1989
). It will
be a challenge to determine how these different signaling pathways interact to
specify apoptosis within the NB7-3 lineage.
The tumor-like expansion of Ddc-expressing cells we observe in heterozygous
spdoG104 larvae suggests that Notch-induced apoptosis may
be essential for regulating cell proliferation. This spdo phenotype
is reminiscent of three tumor-suppressor genes; discs large
(dlg), lethal giant larvae (lgl) and
scribble (scrib), which produce tumors in the CNS and
imaginal disks (Gateff, 1978;
Manfruelli et al., 1996
;
Woods et al., 1996
;
Bilder et al., 2000
).
Interestingly, these three genes work in a common pathway that regulates cell
polarity, and lgl and dlg have been shown to be essential
for the distribution of Numb and other asymmetric determinants
(Ohshiro et al., 2000
;
Peng et al., 2000
). Further
investigation will be necessary to determine if spdo is part of this
same mechanism and exactly how spdo mutants inhibit Notch signaling.
Spdo expression is ubiquitous throughout embryogenesis and persists through
the larval stages and into adults (Dye et
al., 1998
). If a spdo mutation can alter the response of
the Notch receptor to environmental cues that induce apoptosis, one would
expect to see overproliferation in additional tissues.
Finally, although not discussed explicitly, our figures demonstrate that
Numb/Notch signaling also affects development of the midline dopaminergic
cells. The expression of Ddc is essential to the biosynthesis of both
serotonin and dopamine. In the images presented, anti-Ddc antibody detects not
only the serotonergic neurons, but also midline dopamine neurons. As a
consequence of using Ddc as a marker for the serotonin lineage, a number of
observations could be made about the development of midline dopamine cells. We
found that in a numb1 mutant very few midline dopamine
cells were detectable with Ddc (Fig.
2, Fig. 4D,
Fig. 5B).
spdoG104 mutants produce ectopic dopamine cells (data not
shown) and can rescue dopamine cells in the numb1 mutant
phenotype (Fig. 5H). Thus,
Numb/Notch signaling also has a role in the development of midline dopamine
cells, but further investigation into the significance and whether apoptosis
is involved in this lineage will require lineage analysis to determine the
origin of the midline dopamine cells. Neither eg
(Fig. 2C) nor en
(Lundell et al., 1996) is
expressed in midline dopamine cells. As the UAS alleles in this paper were
induced with either eg-gal4 or en-gal4, the phenotype of
dopamine cells in these experiments was not affected by these genetic
backgrounds.
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ACKNOWLEDGMENTS |
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