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First published online 17 September 2008
doi: 10.1242/dev.023986
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Institute of Genetics, University of Mainz, 55099 Mainz, Germany.
* Author for correspondence (e-mail: technau{at}uni-mainz.de)
Accepted 1 September 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Programmed cell death, Motoneurons, Segment specificity, Hox genes, CNS, Drosophila
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
McGinnis and Krumlauf, 1992).
In Drosophila larvae, Hox genes have also been suggested to
orchestrate the assembly of neuromuscular networks that control
region-specific peristaltic movements in crawling
(Dixit et al., 2008).
Drosophila Hox genes are grouped into two complexes that specify the
different segment identities: Antennapedia-Complex genes that specify segments
in the head and anterior thorax, and Bithorax-Complex genes that specify
segments of the posterior thorax and abdomen
(Kaufman et al., 1990;
Lewis, 1978). In addition to
their role as regulators of segment identity, Hox genes can directly affect
expression of the so-called `realizator' genes, which regulate cell behavior,
e.g. cell adhesion, migration, proliferation and apoptosis
(Garcia-Bellido, 1975;
Hombria and Lovegrove, 2003;
Pearson et al., 2005). Within
the last decade, several reports have emerged describing the involvement of
Hox genes in regulating developmental programmed cell death
(Economides et al., 2003;
Liu et al., 2006;
Lohmann et al., 2002). They
also regulate apoptosis in the developing Drosophila central nervous
system (CNS), where abdominal A (abdA) is required to induce
apoptosis of larval abdominal neuroblasts, thus limiting neuronal numbers in
abdominal segments (Bello et al.,
2003), and where Abdominal B (AbdB) expression
ensures survival of differentiated pioneer neurons in the posterior segments
of the embryo (Miguel-Aliaga and Thor,
2004). These studies also underscore the importance of apoptosis
in patterning the nervous system, as it enables the selective removal of cells
that might otherwise disturb the highly refined circuitry required for control
of locomotion.
Generally, apoptotic cell death is abundant in the Drosophila
embryonic CNS and occurs both in apparently random cells and in a regular,
segmentally repeated pattern (Abrams et
al., 1993; Rogulja-Ortmann et
al., 2007) that implies strict spatio-temporal regulation. The
mechanisms of this regulation are largely unknown. The identification of the
developmental signals involved is crucial to understanding how apoptosis is
integrated into tissue and organ patterning during development. We report here
that the Hox genes Ultrabithorax (Ubx) and
Antennapedia (Antp) have antagonistic functions in
motoneuron survival in the embryo. We show that Ubx expression in the
CNS is strongly upregulated at a late point in development, when most cells
have begun to differentiate. This upregulation shortly precedes
Ubx-dependent segment-specific apoptosis of two differentiated
motoneurons. In addition, the proapoptotic gene reaper (rpr)
(White et al., 1996) is
transcriptionally activated following Ubx upregulation in these
motoneurons. Furthermore, we demonstrate that Antp is required for
the survival of these cells. In segments where these two Hox genes are
coexpressed, Ubx counteracts the anti-apoptotic function of
Antp, resulting in cell death. Taken together, our results
demonstrate that Hox genes contribute to segment diversity at the cellular
level by regulating cell numbers in a segment-specific manner, and that they
do so by exerting opposing effects on the survival of individual cells. They
may thus contribute to the diversification of neural circuits along the
anteroposterior body axis.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
Def(3R)109/TM6,abdA- lacZ (Lewis,
1978), abdAMX1/TM3,Sb,Kr-Gal4,UAS-GFP
(Sanchez-Herrero et al.,
1985), hthP2/TM6B,abdA-lacZ
(Sun et al., 1995),
exdB108/FM7,ftz-lacZ
(Rauskolb et al., 1993),
numb1/CyO,wg-lacZ
(Uemura et al., 1989),
zfh15/TM3,Kr-Gal4,UAS-GFP (kindly
provided by Z. C. Lai, Pennsylvania State University, University Park, PA),
Ubx1/TM6B,Tb,Sb,Dfd-lacZ, UAS-Ubx (both
kindly provided by L. Shashidara, Centre for Cellular and Molecular Biology,
Hyderabad, India), eagle-Gal4 (Mz-360)
(Dittrich et al., 1997),
engrailed-Gal4 (Tabata et al.,
1995), poxN-Gal4,UAS-GFP (Pox neuro -
FlyBase) (Boll and Noll, 2002),
UAS-hb (second and third chromosomal insertion)
(Wimmer et al., 2000) and
hs-Ubx/TM6B,Tb,Sb,Dfd-lacZ (Mann
and Hogness, 1990).
Immunohistochemistry and in situ hybridization
Antibody stainings were performed as described
(Rogulja-Ortmann et al.,
2007). Primary antibodies used were: mouse anti-GFP (1:250,
Promega), rabbit anti-human activated caspase 3 (1:50, Cell Signaling
Technology), mouse anti-Eg (1:100, C. Doe, University of Oregon, Eugene, OR),
rabbit anti-Eg (1:500) (Dittrich et al.,
1997), guinea pig anti-Hb (1:500, J. Urban, University of Mainz,
Mainz, Germany), rabbit anti-β-gal (1:2000, Cappel), mouse
anti-β-gal (1:750, Promega), mouse anti-Ubx (1:20, Developmental Studies
Hybridoma Bank), mouse anti-Antp (1:20, Developmental Studies Hybridoma Bank),
guinea pig anti-Zfh1 (1:500, J. Skeath, Washington University School of
Medicine, St Louis, MO), rabbit anti-Hth (1:500)
(Kurant et al., 1998) and
mouse anti-Exd (1:5) (Aspland and White,
1997). The secondary antibodies used were: Cy3-conjugated
anti-mouse and anti-rabbit, Cy5-conjugated anti-guinea pig and FITC-conjugated
anti-rabbit (1:500, all from donkey, all Jackson ImmunoResearch) and donkey
Alexa488-conjugated anti-mouse (1:500, Molecular Probes).
For fluorescent in situ hybridization, digoxigenin-labeled RNA probes for
rpr and hid were made from cDNAs obtained from H. Steller
(Rockefeller University, New York, NY) and J. Abrams (University of Texas
Southwestern Medical Center, Dallas, TX), and for grim from cDNA
RE28551 (Stapleton et al.,
2002), using the DIG-RNA Labeling Kit (Roche Applied Science). In
situ hybridizations were performed according to standard procedures
(Tautz and Pfeifle, 1989),
using a 40% formamide hybridization solution. A Leica TCS SPII confocal
microscope was used for fluorescent imaging, and the images were processed
using Leica Confocal software and Adobe Photoshop.
Heat-shock procedure
For the 3.5-hour heat-shock (HS) experiment, 30-minute collections were
made. At 3.5 hours after egg laying (AEL), embryos were dechorionated in 6%
bleach and rinsed into a mesh. The HS was administered by placing the mesh
into a beaker of PBS in a 35°C water bath for 30 minutes. For the 12-hour
HS, 60-minute collections were made. At 12 hours AEL, embryos were
dechorionated as above and the HS performed for 60 minutes at 35°C. In
both experiments, after the HS embryos were allowed to develop at 25°C
until late stage 16, when they were fixed as described by Rogulja-Ortmann et
al. (Rogulja-Ortmann et al.,
2007).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), were analyzed. These two lineages, along with NB3-3 and
NB6-4, express the transcription factor eagle (eg)
(Dittrich et al., 1997;
Higashijima et al., 1996),
which we used as an identification marker. In NB7-3 and NB2-4, eg is
expressed in all progeny cells throughout development, although expression in
NB2-4 is weaker. NB7-3 comprises three interneurons (EW1-3) and one motoneuron
(GW) (Fig. 1A)
(Bossing et al., 1996;
Dittrich et al., 1997;
Higashijima et al., 1996;
Novotny et al., 2002;
Schmid et al., 1999). In the
anterior thoracic segments (T1 and T2), the cluster often comprises five cells
in late embryos, the fifth cell probably being one of the progeny that dies
immediately after birth in abdominal segments
(Lundell et al., 2003). This
cell also undergoes apoptosis in T1 and T2, but this takes place at the end of
embryogenesis (A.R.-O., unpublished). The individual NB7-3 neurons can be
identified based on marker gene expression and their position in the cluster.
The GW motoneuron and the fifth cell in T1 and T2 are positive for the
transcription factor zinc-finger homeodomain protein 1
(zfh1) (Isshiki et al.,
2001; Novotny et al.,
2002). In addition, GW and EW1 both express the transcription
factor hunchback (hb), but are easily distinguished from one
another based on their size and position: GW is smaller and lies posterior to
the three interneurons (Bossing et al.,
1996; Dittrich et al.,
1997; Higashijima et al.,
1996; Isshiki et al.,
2001; Novotny et al.,
2002; Schmid et al.,
1999). We used anti-Hb and anti-Zfh1 antibodies to identify the GW
motoneuron.
As we reported previously, GW undergoes Caspase-dependent cell death
specifically in segments T3 to A8 (Fig.
1B) (Rogulja-Ortmann et al.,
2007). We restricted further analyses to the thorax and abdominal
segments A1-A7, as NB7-3 appears to develop differently in A8 and comprises
only two to three cells. At late stage 16, GW was either positive for
activated Caspase 3 (a marker for apoptosis activation) or had already been
removed from the CNS in 55.6% of analyzed T3 hemisegments (hs) (n=27)
and 81.2% of analyzed abdominal hs (A1-A7, n=69)
(Table 1). The considerable
difference in the percentage of apoptotic GWs between T3 and the abdomen
appears to be a consequence of differential apoptosis timing, as wild-type
embryos at mid stage 16 showed Caspase 3-positive GWs in 12.5% of analyzed
A1-A7 hs (n=112) and we did not observe dying T3 GWs at this stage
(n=16). In addition, Caspase 3 activation did not occur
simultaneously in the GWs of all abdominal segments: late stage 16 embryos
showed hemisegments where GW appeared intact, others where GW showed Caspase 3
activation, and a third group where GW could no longer be visualized with the
Eg marker. These observations are indicative of the dynamics of apoptosis, and
show that the analyzed specimens can only be regarded as snapshots taken at
the time of embryo fixation. In addition, they suggest that GW apoptosis in T3
is initiated slightly later than in the abdomen. TUNEL staining on late stage
16 embryos revealed TUNEL-positive GWs, confirming GW apoptosis. By the time
of hatching (L1), GW had been cleared from the ventral nerve cord in
practically all T3 and A1-A7 hs examined (see Fig. S1C in the supplementary
material).
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;
Schmidt et al., 1997). Later
in development, these two motoneurons lie in a dorsomedial position, separate
from the other NB2-4 neurons. In the abdomen, the motoneurons lie slightly
apart from one another, whereas in the thorax they occupy the same
dorsoventral position, with one lying anterior to the other
(Fig. 1D,E). In segment T3, and
only in this segment, the anterior NB2-4t motoneuron (from here on referred to
as MNa) underwent Caspase-dependent apoptosis at late stage 16
(Fig. 1E). As eg
expression is low in the NB2-4 lineage, we used the eg-Gal4 driver
combined with UAS-mCD8::GFP for easier identification of NB2-4 cells.
In 64.7% of analyzed T3 hs (n=17), MNa either showed Caspase 3
activation or had already been removed from the CNS
(Table 1). Apoptosis of this
motoneuron was confirmed by TUNEL staining (see Fig. S1 in the supplementary
material).
We also examined which of the three Drosophila proapoptotic genes
is involved in GW and MNa apoptosis. Fluorescent in situ hybridization
revealed transcriptional activation of rpr
(White et al., 1996) in both
GW (Fig. 1C) and MNa
(Fig. 1F), but not of
head involution defective (hid; Wrinkled -
FlyBase) (Abbott and Lengyel,
1991; Grether et al.,
1995) or grim (Chen
et al., 1996) (data not shown).
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; Carroll et al.,
1988; White and Wilcox,
1985). We therefore examined the CNS expression of Ubx
more closely, and found that it was present in the NB7-3 lineage from segments
T2 to A7 (Fig. 2A and see Fig.
S2 in the supplementary material). In T2, the early NB7-3 neuroblast did not
express Ubx. Its progeny showed no, or very low, Ubx
expression until stage 15, when Ubx was strongly upregulated, but
only in the EW interneurons. High Ubx levels were then maintained in
the EWs at least until early stage 17, while the GW remained devoid of
Ubx. In segments T3 to A7, Ubx was expressed already in the
newly delaminated NB7-3 neuroblast (Fig.
2A and see Fig. S2 in the supplementary material) and showed weak
expression in all progeny cells until stage 15. At stage 15, Ubx was
strongly upregulated in all cells of the NB7-3 cluster. At late stage 16,
Ubx was expressed at high levels in all NB7-3 cells of segments T3 to
A7 (Fig. 2A,
Fig. 3A). Thus, the pattern of
Ubx expression in GW coincides with the timing and pattern of
apoptosis. Because apoptosis in NB2-4t occurs in segment T3, where Ubx levels are highest, we examined Ubx expression in this lineage and found that it is expressed from segments T3 to A8 (Fig. 2B and see Fig. S2 in the supplementary material). In T3, the early NB2-4t neuroblast did not express Ubx. The first Ubx-positive cells in NB2-4t of T3 probably appeared around stage 12. However, cells of NB2-4t and of the Eg-positive NB3-3 intermingle, making it difficult to distinguish between their progeny. From stage 14 onwards, the NB2-4t motoneurons are identifiable from their dorsal position, and both showed weak Ubx expression. At stage 15, Ubx was strongly upregulated in MNa, whereas the posterior motoneuron showed low Ubx levels. This difference was maintained at least until late stage 16 (Fig. 2B, Fig. 3D), when the motoneuron exhibiting high Ubx levels underwent apoptosis. In segments A1 to A7, the young NB2-4 neuroblasts were all Ubx-positive (Fig. 2B, see Fig. S2 in the supplementary material). The progeny cells all appeared to show weak Ubx expression (although they could not be distinguished from progeny of NB3-3). At stage 14, when the motoneurons can be identified based on their position, they still maintained weak Ubx expression. After this stage, and in contrast to segment T3, Ubx was strongly upregulated in both motoneurons in segments A1 to A6 (Fig. 2B and see Fig. 2 in the supplementary material). In A7, the pattern of Ubx expression in these cells resembled that in segment T3: it was strong in the anterior cell and weak in the posterior cell. By late stage 16, Ubx levels were reduced only in the posterior cell of all abdominal segments, whereas MNa maintained high Ubx expression but did not undergo apoptosis (Fig. 2B, Fig. 3D). The pattern of Ubx upregulation in MNa of T3 thus correlates with apoptosis induction, although its counterparts in other segments also upregulate Ubx but do not undergo apoptosis.
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In poxN-Gal4,UAS-GFP/UAS-Ubx embryos, MNa underwent apoptosis in 75% of analyzed T3 hs (n=16). In addition, segments T1 and T2 exhibited apoptotic MNas in 68.8% of cases (n=32) (Fig. 3F, Table 1). Interestingly, this motoneuron seemed less responsive to Ubx in T1 than in T2, as we observed apoptosis in 43.8% (n=16) and 93.8% (n=16) of cases, respectively. Thus, Ubx is necessary and sufficient to induce apoptosis of the GW and of the anterior NB2-4t motoneuron.
Ubx-dependent apoptosis is a late function of this gene
Considering that both en-Gal4 and
poxN-Gal4,UAS-GFP drive Ubx expression from early
stages of CNS development through to the end of embryogenesis, there are two
ways to explain our results. First, Ubx might be required only in the
early neuroblast, specifying its segmental identity and developmental program.
As such, the apoptotic fate of the progeny would be predetermined, either
intrinsically or in response to an extrinsic apoptotic signal, and would not
require Ubx at a later point in development. In a second scenario,
Ubx would be required specifically at a later developmental stage to
induce the apoptotic program. In support of the second hypothesis,
Ubx is strongly upregulated at developmental stage 15 in both
motoneurons, a few hours before detectable activation of Caspase 3.
To test which of the above possibilities is true, we expressed Ubx under the control of a heat-shock promoter. Since the early NB7-3 and NB2-4t neuroblasts and the GW and NB2-4t motoneurons show no Ubx expression in wild-type T1 and T2, we reasoned that providing Ubx in these cells at relevant developmental timepoints should clarify whether it is required early (at the time of neuroblast determination) or late (in the differentiated neurons) to induce apoptosis. The heat shock (HS) was administered at two different timepoints in separate experiments: at 3.5 hours after egg laying (AEL) (prior to NB7-3 delamination) and at 12 hours AEL (when Ubx is normally upregulated in T3 to A7). In the first experiment (early HS), Ubx was present in segments T1 and T2 at the time of neuroblast formation, but was degraded by the time it is required to induce apoptosis. This early Ubx expression in T1 and T2 might transform the NB7-3 lineage into a T3 identity, as the NB7-3 clusters in T1 and T2 in 26/27 cases contained only four cells instead of five. In the wild type, four cells were observed in 29/36 cases. In addition, we observed a transformation of the thoracic NB6-4 lineage (which contains neurons and glia) into an abdominal lineage (glia only), which indicates that ectopic Ubx expression can transform the tagma-specific identity of the neuroblasts. Following early HS, we did not observe apoptotic GWs in T1 and T2 (n=27), which suggests that early Ubx expression is not sufficient to induce apoptosis in the NB7-3 lineage (Fig. 3G, Table 1). This conclusion is supported by the fact that the percentage of apoptotic GWs in the abdomen of these embryos (82.4%, n=85) did not differ from that of wild type (81.2%, n=69) (Table 1). In T3, however, we did find an increase in apoptotic GWs (100%, n=12) as compared with the wild type (55.6%, n=27) (Table 1), indicating that in this segment, Ubx might be required both early in order to specify an apoptosis-susceptible progeny fate, and late to initiate apoptosis itself.
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We were not able to analyze NB2-4t in these experiments because expression of the marker protein Eg was not strong enough to permit reliable evaluation of the data. However, we suggest that Ubx is required late in this lineage as well, as it is not expressed in the early NB2-4t neuroblast in T3 and thus probably does not participate in providing this neuroblast with its segment-specific identity. Moreover, early ectopic Ubx expression using the poxN-Gal4,UAS-GFP line was unable to transform the thoracic NB2-4 lineage into the abdominal lineage, but it did induce apoptosis of the MNa in all thoracic segments (Fig. 3F).
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;
Hirth et al., 1998), with
strongest expression from the posterior half of T1 to the anterior half of T3.
At the end of embryogenesis, Antp levels were high in all NB7-3
progeny cells in T1 (Fig.
5A,B). In T2, the EWs had low Antp levels, but GW was
strongly Antp-positive (Fig.
5A,C). In T3 and in the abdomen of late stage 16 embryos, NB7-3
progeny cells exhibited variable, low-level Antp expression
(Fig. 5A,
Fig. 6A). Considering that
Antp expression is high in surviving GWs of T1 and T2, and is low in
GWs of T3 to A7, which undergo apoptosis, we wondered whether Antp
might be required for GW survival in T1 and T2. Indeed, in
AntpW10 mutant embryos, we found that GWs underwent cell
death in T1 and T2 in 35% of analyzed hs (n=20)
(Fig. 4A,
Table 1). Additionally, in T3,
the frequency of GW apoptosis increased from 55.6% (n=27) in wild
type to 90% (n=10) in AntpW10. In the abdominal
segments, the frequency of GW apoptosis was slightly lower than in the wild
type (Table 1). We conclude
that Antp is necessary for the survival of GW in T1 and T2, and might
also be responsible for the slightly later onset of GW apoptosis in T3. In order to test whether Antp can also rescue GWs from apoptosis, we overexpressed Antp using the en-Gal4 driver. In segments T1 and T2 of en-Gal4/UAS-Antp embryos, no apoptotic GWs were observed (Table 1). In T3, only 30.8% of hs showed apoptotic GWs (n=13) as compared with 55.6% in the wild type (n=27). In abdominal segments, only 34.7% of GWs were apoptotic (n=95), significantly fewer than in the wild type (81.2%, n=69) (Fig. 4B, Table 1). In addition, driving Antp expression with poxN-Gal4 reduced the percentage of apoptotic NB2-4t MNas in T3 (Fig. 6B). This suggests that Ubx might counteract Antp in NB2-4t, in a similar way as in NB7-3, to prevent it from promoting survival of this motoneuron. Taken together, our results suggest that Antp is necessary and sufficient for GW motoneuron survival and that it can counteract the proapoptotic effect of Ubx.
Ubx opposes Antp in promoting survival of the GW motoneuron
To examine how Antp counteracts the proapoptotic function of
Ubx, we looked at Ubx and Antp expression in
Antp and Ubx loss- and gain-of-function mutants,
respectively. In AntpW10 mutants at late stage 16,
Ubx expression was unaffected. In en-Gal4/UAS-Antp
embryos of the same stage, Ubx expression in the NB7-3 cells
occasionally appeared somewhat reduced compared with the wild type (summarized
in Fig. 6A). This suggests that
Antp overexpression reduces GW apoptosis, either by downregulating
Ubx expression or by sustaining cell survival, or both. In the wild
type, however, Antp is probably unable to suppress Ubx, as
Antp mutants showed no change in Ubx expression
(Fig. 6A) and no increase in
abdominal GW apoptosis (Table
1). In Ubx1 mutants, high Antp
expression levels extended to the anterior half of segment A1, as has been
shown previously (Carroll et al.,
1986), and in all NB7-3 cells Antp expression was equally
strong from T1 to T3 (Fig.
5D-F). In the abdomen, by contrast, Antp levels in GWs
did not differ between Ubx1 mutants and wild type in most
segments (Fig. 6A). In
en-Gal4/+;UAS-Ubx/+ embryos, Antp was downregulated
in the whole NB7-3 cluster, in both thoracic and abdominal segments
(Fig. 5G-I). These results show
that Ubx overexpression represses Antp, which contributes to
an increase in thoracic and abdominal GW apoptosis. In
Ubx1 mutant embryos, however, NB7-3 Antp
expression increased only in T2 and T3, and not in the abdominal segments
(Fig. 6A), although apoptosis
was abolished in both tagmata (Table
1). This suggests that, in the wild type, high Antp levels are
required to prevent activation of apoptosis in GWs. In the third thoracic
segment and in abdominal segments, low Antp levels were not sufficient to
counteract the proapoptotic effect of Ubx. Accordingly, Antp
overexpression was able to significantly reduce GW apoptosis in all segments
(Table 1), without affecting
Ubx levels (Fig. 6A). Thus,
Antp seems to be required to prevent activation of GW apoptosis, and
Ubx appears to oppose this function of Antp.
To confirm the opposing roles of Antp and Ubx in regulating GW survival, we examined AntpW10,Ubx1 double mutants and found that GW apoptosis was now partly restored, with 19.6% of GWs being apoptotic in segments A1 to A7 (n=112) (Fig. 5J, Table 1). In segment T3, 23.5% of counted hs (n=17) contained a cell that was triple-stained for Eg, activated Caspase 3 and Hb. However, thoracic segments of these mutants often showed a disordered pattern of Eg staining, making it difficult to unambiguously identify the GW motoneuron. We also attempted coexpressing Antp and Ubx using the en-Gal4 driver. The NB7-3 cluster was severely affected in these embryos: it was completely missing in 20% of T3 segments (n=10) and in 31.4% of abdominal segments (n=70), and in most other cases only one to two cells remained at stage 15, making it impossible to analyze GW fate. We therefore examined embryos in which the function of one or both of these genes was partially or completely removed. We made use of the observation that in Ubx1/+ heterozygous embryos, GW apoptosis is reduced in T3 from 55.6% (in wild type, n=27) to 7.1% (n=14), and in the abdomen from 81.2% (in wild type, n=69) to 21.7% (n=97) (Table 1), which confirms that Ubx is required for GW apoptosis and that this effect is dose dependent. Removing one copy of the Antp gene in these embryos (AntpW10,Ubx1/+) resulted in an increase in GW apoptosis to 16.7% (n=18) in T3 and 41.3% (n=126) in the abdomen. Moreover, completely removing Antp (AntpW10,Ubx1/AntpW10) further increased the proportion of dying GWs to 36.4% (n=22) in T3 and to 52.2% (n=90) in abdomen (Table 1).
Taken together, our results demonstrate that, in abdominal segments, Ubx induces apoptosis of the GW motoneuron late in development, and it does so by counteracting the positive effect of Antp on cell survival. In anterior thoracic segments, where Ubx expression is repressed by an unknown factor, Antp protects these neurons from apoptosis.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Regulation of CNS cell number is achieved through control
of cell proliferation and death, and both of these processes have been shown
to be orchestrated by Hox genes, in vertebrates and invertebrates
(Bello et al., 2003;
Economides et al., 2003;
Liu et al., 2006;
Miguel-Aliaga and Thor, 2004;
Peterson et al., 2002;
Prokop et al., 1998). It has
recently also been reported that mutations in Hox genes affect locomotor
behavior in larval crawling (Dixit et al.,
2008), suggesting that they provide positional information for
neurons and thus regulate the formation of neuromuscular networks that control
region-specific peristaltic locomotion. Our studies reveal that the
Drosophila Hox genes Ubx and Antp regulate
segment-specific survival of two differentiated motoneurons, and may thus
pattern neuromuscular networks by regulating the segment-specific presence of
individual motoneurons. We show that Ubx is necessary and sufficient
to induce apoptosis, and that it does so by impeding the positive effect of
Antp on the survival of these cells.
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);
and (2) a late role in inducing apoptosis of the motoneurons. Our heat-shock
experiments confirm that, at least in NB7-3, it is this late upregulation of
Ubx that leads to apoptosis. NB2-4t could not be tested for the
proapoptotic function of Ubx in the heat-shock experiments. However,
we believe that it plays a late role in this lineage as well: as Ubx
does not play an early role in specifying the T3 identity of the NB2-4t
neuroblast, this suggests that the proapoptotic function of Ubx must
be executed at a later point in development. Also, in
poxN-Gal4,UAS-GFP/UAS-Ubx embryos, the NB2-4t
lineage does not appear to be transformed into its abdominal counterpart, as
we observe two closely positioned dorsal motoneurons in thoracic segments. In
these embryos, Ubx is still capable of inducing apoptosis of the
anterior motoneuron in all thoracic segments. Taken together, these data
indicate that, also in this lineage, Ubx is needed at a late
developmental stage to induce apoptosis. Dual requirements for Hox genes have
also been described in determining thoracic bristle patterns
(Akam, 1987;
Rozowski and Akam, 2002) and
in cardiac tube organogenesis (Perrin et
al., 2004). It is not clear how the late expression of
Ubx is regulated. It has been suggested that this might depend on
genes that define the differences between cell types
(Akam, 1987), and it will be
interesting to see whether this is also the case for the NB7-3 neurons.
The ability of Ubx to induce apoptosis is context dependent
Interestingly, the late Ubx expression in NB7-3 extends to the
second thoracic segment, but in a cell-specific manner: the GW motoneuron does
not activate Ubx expression, whereas the EW interneurons do. These
findings evoke the following questions: (1) why is it that the EWs in T2 to A7
do not undergo apoptosis although they also upregulate Ubx? and (2)
what represses Ubx expression in the T2 GW?
Regarding the first question, it is likely that the differentiation program of the EWs creates a different cellular context in which Ubx is unable to induce apoptosis. The results of our ectopic Ubx expression experiments support this assumption: whereas GW undergoes apoptosis, the EWs do so very rarely, although they express Ubx at equally high levels. In addition, GW seems to acquire the competence to undergo Ubx-dependent apoptosis rather late, as en-Gal drives expression strongly from earlier stages but apoptosis does not occur until stage 15. This would suggest that the susceptibility to apoptosis of at least some motoneurons is coupled to differentiation. The context-dependent ability of Ubx to activate apoptosis also holds true for the NB2-4t lineage: the anterior motoneuron is susceptible to Ubx-induced apoptosis, whereas the posterior one is not, even when Ubx is overexpressed.
Our preliminary attempts to determine at least some of the factors
contributing to apoptosis-susceptibility were not successful. For the NB7-3
lineage, we examined abdA expression and found that, at the onset of
GW apoptosis, it is weakly expressed only in the EWs from A1 to A7, but not in
GW. However, the survival of the EWs is not impaired in abdA mutants
(data not shown). We also tested the cofactors homothorax
(hth) (Pai et al.,
1998; Rieckhof et al.,
1997) and extradenticle (exd)
(Peifer and Wieschaus, 1990;
Rauskolb et al., 1993), which
are known to be required for some functions of homeotic genes, but obtained no
compelling evidence for their involvement in the apoptosis of these cells.
Mutants of other factors that are differentially expressed in GW and EWs
[numb (Uemura et al.,
1989), zfh1 (Isshiki
et al., 2001)] were also examined, and no indication was found
that any of these is involved in the differential effect of Ubx on
cell survival (data not shown).
Regarding the question of differential Ubx expression in NB7-3
cells of T2, this is an intriguing observation that might prove to be key in
determining the developmental signal that upregulates Ubx late in
development. One candidate for repressing Ubx expression in GW is the
gap gene hb (White and Lehmann,
1986). It is known to repress Ubx in early embryonic
development, and hb overexpression can suppress Ubx in the
NB7-3 lineage (A.R.-O., unpublished). However, hb is also necessary
to activate Antp expression and thereby specify the second thoracic
segment (Wu et al., 2001). In
hb mutants, T2 is not present and we were thus unable to test whether
this is the factor repressing Ubx expression in GW. Other obvious
candidates are the Polycomb group (PcG) proteins, well-known repressors of Hox
genes (Ringrose and Paro,
2007). It has recently been shown that, contrary to what had been
believed for a long time, target gene repression by these proteins is not
necessarily maintained throughout development, but can be reversed in certain
developmental contexts (Chen et al.,
2005). It is conceivable that repression by PcG proteins could be
lifted in some cells (e.g. EWs in T2) and not in others (e.g. GW). However,
the question would still remain as to how the difference between the GW and EW
neurons is established specifically in this segment. Alternatively,
differential Ubx regulation might be effected via micro RNAs
(Pearson et al., 2005) or
non-coding RNAs (Petruk et al.,
2006).
Ubx counteracts Antp to induce programmed cell death
We also show that Antp is required for GW survival in all
segments, and that Ubx counteracts Antp in T3 to A7 to
induce apoptosis. Although the lower percentage of dying abdominal GWs in
Antp mutants (69%) as compared with wild type (81.2%)
(Table 1) might indicate a
proapoptotic function of Antp in the abdomen, we believe that this is
not the case because Antp overexpression actually reduces the amount
of abdominal GW apoptosis more than twofold (see
Table 1, en>Antp).
Moreover, removing Antp function in a Ubx heterozygous
background increases GW apoptosis (both in T3 and in abdomen) in a
dose-dependent manner, and removing both Ubx and Antp
results in a recurrence of GW apoptosis, albeit with low penetrance
(Table 1), lending further
support to a pro-survival function of Antp.
It is not clear at which level these two factors interact. GW apoptosis is
inhibited both in T3 and in abdominal segments of Ubx mutants.
However, Antp expression in Ubx mutants is upregulated only
in T3, and remains low in abdominal segments, suggesting that here
Ubx does not induce apoptosis through Antp repression. In
addition, the pattern and levels of Ubx expression do not change at
all in Antp mutants, indicating that in this context Antp
does not promote survival via Ubx regulation. We therefore propose
that in the wild type, Antp and Ubx might compete for a co-factor or for a
target enhancer, rather than cross-regulating each other. The proapoptotic
gene rpr (White et al.,
1996), which is transcriptionally activated in both GW and MNa
motoneurons, is a candidate target. In fact, we found several binding sites
for both Ubx and Antp in the enhancer of the rpr gene. It will be
interesting to see whether Antp can prevent activation of the
apoptotic machinery by affecting an upstream factor or through direct
repression of rpr. The presence of several Antp binding sites in the
rpr enhancer permits such speculation, and although it awaits
experimental validation, this does suggest a model for the antagonistic
effects of Antp and Ubx on cell survival. According to this model, Ubx and
Antp compete for sites in the rpr enhancer. In cells that express
Antp at high level, this would repress rpr transcription. Ubx would
overcome repression by Antp and activate rpr transcription to induce
apoptosis. Such positive Hox regulation of rpr has already been
demonstrated in shaping segment borders in Drosophila embryos, where
Deformed directly activates rpr transcription
(Lohmann et al., 2002). In
addition, antagonistic transcriptional regulation of the P2 Antp
promoter in the embryonic ventral nerve cord has been demonstrated for Antp
and Ubx (Appel and Sakonju,
1993). In this case, Antp positively autoregulates its own P2
promoter in the thoracic segments, and Ubx competes with Antp for the same
binding sites and thus prevents high-level expression of Antp in the
more posterior segments.
Hox-dependent apoptosis as a mechanism for CNS patterning
A requirement for Hox genes in segment-specific cell survival has already
been shown for the MP2 and MP1 pioneer neurons
(Miguel-Aliaga and Thor,
2004), where AbdB expression is necessary for survival of
these neurons in the three most-posterior abdominal segments. In the more
anterior segments, the dMP2 and MP1 motoneurons undergo apoptosis at the end
of embryonic development, after they have completed their role in pioneering
axonal tracts. The surviving dMP2 neurons innervate the hindgut and
differentiate into insulinergic neurons
(Miguel-Aliaga et al., 2008).
The exact function and targets of the GW and the anterior NB2-4t motoneurons
in the first and second thoracic segment are unclear
(Bossing et al., 1996;
Schmid et al., 1999;
Schmidt et al., 1997), as is
the reason for their removal in the relevant segments. The surviving GW and
MNa might exert a region-specific neurosecretory function and thus modulate
neuronal or muscle activity, as has been described for neurosecretory cells in
the larval brain, the processes of which arborize on the wall of the anterior
aorta adjacent to the ring gland (Johnson
et al., 2003). Alternatively, the elimination of certain
outward-projecting neurons in the posterior thoracic and/or abdominal segments
might be related to the pattern of muscle fibers, which differs considerably
between the thorax and the abdomen (Bate,
1993). We suggest that Hox-regulated segment-specific motoneuron
survival is a part of the patterning process that enables formation of
region-specific functional neuromuscular networks.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/20/3435/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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