Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA
Author for correspondence (e-mail:
steth{at}ifm.liu.se)
Accepted 6 October 2004
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SUMMARY |
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Key words: Drosophila, Abd-B, reaper, grim, dMP2, MP1
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Introduction |
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Amongst such regulators are Hox genes, whose expression in specific domains
along the antero-posterior (AP) axis is translated into axial morphological
differences through their action on a number of biological processes
(Mann and Morata, 2000;
McGinnis and Krumlauf, 1992
;
Warren and Carroll, 1995
),
including apoptosis (Alonso,
2002
). Indeed, increased or reduced cell death has been observed
in mouse, Drosophila and nematode Hox gene mutants
(Economides et al., 2003
;
Lohmann et al., 2002
;
Sommer et al., 1998
;
Tiret et al., 1998
).
Furthermore, a direct, in vivo modulation of the apoptotic machinery by a Hox
gene was recently reported; in Drosophila, the Hox gene
Deformed (Dfd) promotes localized PCD in the embryonic head
lobes by activating the transcription of the RHG-motif gene reaper
(rpr) (Lohmann et al.,
2002
). Together, these results point to a link between Hox
function and apoptosis, and suggest that the effect of Hox function on cell
death is dependent on cellular context.
The nervous system is also regionally specified, and Hox genes have been
shown to control its segmentation
(Capecchi, 1997) and to
specify neuronal identity along its anteroposterior (AP) axis
(Dasen et al., 2003
;
Jungbluth et al., 1999
;
Liu et al., 2001
). In the
vertebrate spinal cord, axial differences in the size of embryonic motor
neuron pools exist long before they become dependent upon target-derived
signals (Brown, 1981
).
Similarly, in the developing Drosophila ventral nerve cord (VNC),
segmental differences in the numbers of neurons and glia arise from an
initially equivalent set of 30 neuroblasts in each hemisegment
(Prokop and Technau, 1994
;
Schmid et al., 1999
;
Udolph et al., 1993
). These
differences are likely to result from differential Hox gene modulation of both
proliferation and apoptosis along the AP axis
(Prokop et al., 1998
). Indeed,
the recent finding that the Drosophila Hox gene abdominal A
(abd-A) activates apoptosis of postembryonic neuroblasts, thereby
regulating the final number of neurons in the adult fly, provided an elegant
example of how Hox genes can act on neuronal precursors to generate axial
diversity (Bello et al., 2003
).
However, differences in neuronal architecture along the AP axis could
alternatively emerge from selective elimination of mature neurons in specific
axial domains. In this model, an identical neuronal profile would initially be
generated along the AP axis. Subsequently, patterning genes would act in their
specific expression domains to differentially trigger or prevent apoptosis of
mature cells.
In this study, we demonstrate the first example of apoptosis of
differentiated neurons in an invertebrate embryo by showing that the
Drosophila dMP2 and MP1 pioneer neurons undergo segment-specific
apoptosis at a late developmental stage. These neurons are initially generated
throughout the VNC (Bossing and Technau,
1994; Doe, 1992
;
Schmid et al., 1999
;
Schmidt et al., 1997
), extend
their axons and perform a critical pioneering role in guiding follower axons
(Hidalgo and Brand, 1997
;
Lin et al., 1995
). We have
found that at a later stage, dMP2 and MP1 neurons undergo apoptosis only in
anterior segments. While neither the generation nor the initial function of
these neurons depends on Hox function, their late, segment-specific survival
is under homeotic control; the Hox gene Abdominal B (Abd-B)
acts in posterior segments in a cell-autonomous and postmitotic fashion to
prevent apoptosis of dMP2 and MP1 neurons by repressing two RHG-motif cell
death activators, rpr and grim. Our findings provide clear
evidence for a cell-autonomous and anti-apoptotic function of a Hox gene in
vivo. Furthermore, they identify a novel mechanism linking Hox positional
information to axial differences in neuronal architecture by the selective
elimination of mature neurons.
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Materials and methods |
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Immunohistochemistry and DiI injections
Immunolabeling was carried out as previously described
(Allan et al., 2003).
Antibodies used were: mouse
-myc mAb 9E10 (1:25), mouse
-Antp
mAb 4C3 (1:50), concentrated mouse
-Abd-B 1A2E9 (1:5) (all from
Developmental Studies Hybridoma Bank); rabbit
-Proctolin (1:2000)
(Taylor et al., 2004
), rabbit
-Odd (1:1000) (Spana et al.,
1995
), rabbit
-GFP (Molecular Probes, 1:1000), mouse
-Ubx mAb FP3.38 (1:20) (White and
Wilcox, 1984
), mouse
-Abd-A mAb DMabdA.1 subclone 6A8.12
(1:400) (Kellerman et al.,
1990
). FITC-, Rhodamine-Red-X- and Cy5-conjugated secondary
antibodies were obtained from Jackson Immunolabs and used at 1:200 (1:100 for
the Cy5-conjugated antibody). Lipophilic DiI injections were done as
previously described (Landgraf et al.,
1997
). Phalloidin-TX (Molecular Probes) was used at 1:1000. Where
appropriate, images were false-colored for clarity, and red was converted to
magenta for the benefit of color-blind readers.
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Results |
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Because the dMP2 neurons extend axons posteriorly to pioneer the
longitudinal tract (Thomas et al.,
1984), they had been previously characterized as interneurons.
However, analysis of their axonal projections using a membrane-targeted
reporter (dMP2-GAL4/UAS-myc-EGFPF), as well as lipophilic
dye (DiI) backfills from the hindgut, allowed us to determine that posterior
dMP2 `interneurons' actually exit the VNC and innervate the hindgut
(Fig. 1J-L). Given that most,
if not all, Drosophila motor neurons show signs of activated BMP
signaling (Marques et al.,
2002
), this observation is in accord with our previous finding
that the dMP2 neurons (or Vaps) express phosphorylated Mad (pMad)
(Allan et al., 2003
). The
Drosophila hindgut is innervated by axons positive for the
invertebrate myomodulator Proctolin
(Anderson et al., 1988
), and we
found that the dMP2s indeed express this neuropeptide
(Fig. 1I). Thus, the
Drosophila dMP2 `interneurons' are, in fact, Proctolin-expressing
peptidergic motor neurons that innervate the hindgut
(Fig. 1M).
dMP2 neurons undergo segment-specific apoptosis at a late embryonic stage
The absence of anterior (S3, T1 to T3 and A1 to A5) dMP2 neurons in larvae
(but not in stage 16 embryos) could result from specific downregulation of
dMP2-GAL4 expression anteriorly. Alternatively, it could reflect the
selective death of anterior dMP2 neurons at a late embryonic stage. To resolve
this issue, we used dMP2-GAL4 to examine these neurons at embryonic
stage 17. Surprisingly, the dMP2 neurons displayed several of the
morphological hallmarks of apoptosis; they appeared pyknotic (shrunken and
densely stained) and their axons were fragmented
(Fig. 1B-E). Pyknotic corpses
seemed to be transported to the dorsal surface of the VNC
(Fig. 1D, arrow), where dying
cells have been reported to be engulfed by macrophages
(Sears et al., 2003;
Sonnenfeld and Jacobs, 1995
).
By late embryogenesis (air-filled trachea stage), all 18 anterior dMP2 neurons
were lost (0% survival; Fig.
7B). In contrast, the six dMP2 neurons in segments A6 to A8
(subsequently referred to as posterior dMP2s) survived and were maintained
throughout the four-day larval period (Fig.
1F; not shown). We further examined the larval expression of Odd,
as well as that of other previously described dMP2 markers namely,
15J2-GAL4 (Hidalgo and Brand,
1997
) and AJ96-lacZ
(Spana et al., 1995
)
and found them to be absent from anterior segments (see below; not shown),
further suggesting that anterior dMP2 neurons die at a late embryonic
stage.
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To rule out the possibility that the persistence of anterior dMP2 neurons
in the aforementioned mutant scenarios resulted from blocking cell death in a
non-autonomous fashion, we used the dMP2-GAL4 line to express the
baculoviral anti-apoptotic caspase inhibitor P35
(Hay et al., 1994)
postmitotically in all dMP2 neurons. This resulted in the survival of anterior
dMP2 neurons in all VNC segments (Fig.
2D), with accompanying expression of Odd and, in some cases,
Proctolin (Fig. 2F,H). This
further supports that the death of anterior dMP2 neurons is apoptotic in
nature.
Cell death of anterior dMP2s could be achieved by selective activation of rpr transcription anteriorly. Alternatively, rpr could be transcribed in both anterior and posterior dMP2s, with post-transcriptional mechanisms or regulation of other genes downstream of rpr accounting for the differential survival of anterior and posterior dMP2s. This issue was difficult to address since the embryonic expression of rpr is highly dynamic and there are no antibodies available with which to detect Rpr protein. However, a GAL4 P element transposon line inserted 125 base pairs upstream of the predicted rpr transcriptional start site was recently generated (subsequently referred to as rprGAL4; see Materials and methods). Since this insertion line allows for expression analysis with single-cell resolution, we used rprGAL4 as a probable readout of rpr expression. Its expression in the VNC is dynamic and commences at stage 15 (Fig. 2I). Double labeling for rprGAL4 and Odd indicates that anterior, but not posterior, dMP2s express rprGAL4 at the time when they begin to die (Fig. 2J). This suggests that differential transcriptional activation of rpr in anterior dMP2s underlies their death. Anterior expression is variable and may reflect the fact that Odd expression is sometimes already lost by the time that rprGAL4 levels become high enough to be detected. In fact, we often observed cells with strong rprGAL4 expression in positions where Odd-positive dMP2s would have been expected (not shown).
As expected from its insertion site, rprGAL4 is an
allele of rpr; 32% of anterior dMP2s survive in
rprGAL4/H99 embryos
(Fig. 7B). The difference in
dMP2 survival between the rprGAL4 allele and the
XR38 deletion (32% versus 98% over H99) may indicate that
rprGAL4 is not a null rpr allele. Alternatively,
or additionally, XR38 may remove the recently identified RHG-motif
gene sickle, located close to rpr but outside the
H99 region (Christich et al.,
2002; Srinivasula et al.,
2002
; Wing et al.,
2002
) (Fig. 7A). If
that is the case, XR38/H99 would be heterozygous for skl,
which might contribute to anterior dMP2 survival.
Based upon (1) the loss of Odd, 15J2-GAL4 and AJ96-lacZ expression in anterior dMP2 neurons at stage 17, (2) the apoptotic appearance of pyknotic dMP2 cell bodies and their fragmented axons as visualized by dMP2-GAL4/UAS-myc-EGFPF at this stage, (3) the analysis of Df(3L)H99/XR38 and rprGAL4/H99 mutants, (4) the cell-autonomous rescue of anterior dMP2 obtained with dMP2-GAL4/UAS-p35 and (5) the expression of rprGAL4, we conclude that the Drosophila dMP2 neurons are generated in all VNC segments, that they differentiate and extend axons at stage 12, and that at stage 17 they activate rpr and undergo rpr- (and partly grim-) mediated programmed cell death in all VNC segments anterior to A6.
The expression of Abdominal B, but not of other homeotic genes, parallels the segment-specific survival of posterior dMP2 neurons
Why would only anterior dMP2s undergo apoptosis at stage 17? Since dMP2
neurons are motor neurons that exit the VNC, we initially speculated that they
perhaps depend upon a target-derived signal. Anterior dMP2s would fail to exit
the VNC on time to receive this signal and would therefore die. Although such
survival signals have not been described in invertebrates, recent studies have
revealed that both Drosophila motor neurons and peptidergic neurons
depend upon target-derived BMP signals for both proper differentiation and
maturation (Aberle et al.,
2002; Allan et al.,
2003
; Marques et al.,
2002
; Marques et al.,
2003
). To test this idea, we carried out a number of mutant and
tissue-ablation experiments (details available upon request). However, they
all failed to lend support to this model.
We then hypothesized that the mechanism preventing posterior dMP2 death may
be cell-autonomous. Recently, two studies have uncovered a pro-apoptotic
function for Hox genes in Drosophila
(Bello et al., 2003;
Lohmann et al., 2002
). While
Hox genes often appear to be broadly expressed within their AP domain
(Hirth et al., 1998
), their
precise expression patterns at the time when neurons and glia are generated
have not been well characterized in Drosophila. For these reasons, we
sought to examine in detail the expression of the Hox genes normally expressed
in the Drosophila VNC, and to determine whether it may be of
relevance to the segment specificity of dMP2 death.
When anterior dMP2s degenerate, the vast majority of embryonic neurons and glial cells have differentiated. We observed that at this stage, the Hox proteins Antennapedia (Antp), Ultrabithorax (Ubx), Abd-A and Abd-B were not ubiquitously expressed within their expression domains. For example, some postmitotic neurons appeared not to express specific Hox proteins (Fig. 3A-C, arrowheads). Furthermore, Hox expression was largely absent both from lateral and midline glia (Fig. 3E-L), although Abd-A and Abd-B were expressed in a small subset of glia (Fig. 3L, arrowhead; not shown). We noticed that Hox expression boundaries were relatively imprecise, especially posteriorly; Hox expression domains did not end in well-defined lines across the cord, and some neurons lost Hox expression before others. The limits of Hox expression were also variable depending on the specific embryonic stage (Fig. 3Q; not shown). In the dMP2 neurons, Hox gene expression was particularly restricted; Ubx was absent from them in all segments (Fig. 3N,Q) and Antp was only expressed in the T2 dMP2 (Fig. 3M,Q). Expression of Abd-A spanned A5-A7 (Fig. 3O,Q) and, importantly, Abd-B was specifically expressed in the A6-A8 dMP2s (Fig. 3P,Q). Thus, Hox gene expression in the Drosophila VNC is dynamic and complex, and Abd-B is the only Hox gene whose expression profile in dMP2 neurons parallels the segment specificity of their survival (Fig. 3Q).
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dMP2 neurons require Abdominal B for their survival, but not for their generation
The specific expression of Abd-B in posterior dMP2 neurons, as well as its
ability to fully suppress anterior dMP2 cell death when misexpressed,
suggested that Abd-B is normally involved in preventing apoptosis in posterior
dMP2 neurons. In wild-type embryos, the two different Abd-B proteins are
expressed in distinct domains; while Abd-Bm expression is apparent in segments
A5 to A8, Abd-Br is confined to A9 (Boulet
et al., 1991; Delorenzi and
Bienz, 1990
; Kuziora and
McGinnis, 1988
;
Sanchez-Herrero and Crosby,
1988
) and should therefore not be expressed in posterior dMP2
neurons at the time of their death. To confirm this, and to determine whether
Abd-Bm has an anti-apoptotic function in posterior dMP2 neurons, we
analyzed the development of dMP2 neurons in Abd-Bm mutant embryos. In
Abd-Bm mutants, dMP2 neurons were generated throughout the cord
(including posterior segments) in normal numbers
(Fig. 5A,B) and appeared
properly specified, as evidenced by their expression of dMP2-GAL4 and
Odd (Fig. 5F). As expected, at
stage 16, Abd-B staining (corresponding to Abd-Br expression) was
confined to the posterior tip of the VNC and was absent from posterior dMP2
neurons, including the posterior-most pair
(Fig. 5E). At stage 17, all
dMP2 neurons appeared to undergo apoptosis, and no posterior dMP2 neurons were
detected at late embryonic stages using dMP2-GAL4 and Odd
(Fig. 5D; Fig. 6D), strongly suggesting
that Abd-Bm is normally required in posterior dMP2 neurons to prevent
apoptosis. In contrast, and as would be expected from the Hox expression data
(Fig. 3Q), the pattern of dMP2
survival/death was unaffected in mutant embryos lacking other Hox genes (not
shown). To rule out a possible early contribution of Abd-Br to the
generation of posterior dMP2s, we additionally analyzed Abd-B mutant
embryos in which neither Abd-Bm nor Abd-Br is expressed, and
obtained identical results to those described for Abd-Bm mutants (not
shown). Thus, Abd-B is not required for the generation of posterior
dMP2 neurons.
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The absence of rprGAL4 expression from posterior dMP2
neurons suggested that the segment specificity of dMP2 survival is achieved,
at least in part, by the transcriptional repression of the rpr gene.
Since Abd-B has been shown to activate rpr transcription in
the periphery (Lohmann et al.,
2002), it is conceivable that Abd-B can also act to
repress rpr in certain cellular contexts. If this were the case in
the dMP2 lineage, we would expect rprGAL4 to be expressed
in posterior cells in Abd-B mutant embryos. To test this idea, we
examined rprGAL4 expression in an Abd-Bm mutant
background. While in control embryos no rprGAL4 expression
was apparent in posterior dMP2 neurons
(Fig. 2J), it was often
observed in the posterior dMP2 neurons of Abd-Bm mutants
(Fig. 5G).
Thus, in spite of its broad posterior expression, Abd-B is not necessary for the generation of the posterior dMP2 neurons. However, Abd-B is absolutely required cell-autonomously and postmitotically for the survival of posterior dMP2 neurons. Furthermore, the activation of rprGAL4 expression in posterior dMP2 neurons in Abd-B mutant embryos strongly suggests that Abd-B prevents dMP2 apoptosis, at least in part, by repressing the transcription of rpr.
Abdominal B prevents grim-mediated apoptosis of MP1 pioneer neurons in posterior segments
In invertebrate embryos, developmental apoptosis typically takes place
shortly after cells are generated. dMP2 cell death is unusual in this respect,
in that it occurs at a later developmental stage when these neurons have
differentiated and extended their axons, thus resembling apoptosis of
developing vertebrate neurons. We wondered whether there were other cases of
apoptosis of differentiated neurons in the VNC of the Drosophila
embryo, and whether Hox genes may be involved in regulating its segmental
specificity.
Odd is expressed in both the dMP2 neurons and the smaller, more medial and
more dorsal MP1 pioneer neurons (Fig.
1G,H). Previous studies have shown that the MP1 neuron is
generated in all VNC segments and extends an ipsilateral projection that
bifurcates both posteriorly and anteriorly
(Bossing and Technau, 1994;
Schmid et al., 1999
). While
using Odd as an additional marker for dMP2 neurons, we confirmed that MP1
neurons were present throughout the VNC at stage 16. However, we observed that
Odd expression was conspicuously absent from anterior MP1 neurons in late
embryos, and only A5-A8 MP1 neurons were apparent
(Fig. 6A). Compared to dMP2
expression, Odd expression in MP1 neurons was lost at a slightly later stage
(late stage 17, before tracheal inflation). Conceivably, this could have been
a consequence of downregulation of Odd expression in anterior MP1s. However,
while using Odd as a marker to identify dMP2 neurons in cell death mutants, we
observed that its expression in anterior MP1 neurons was maintained in late
embryos and larvae (H99/XR38; Fig.
6B); only two out of the four Odd-positive cells apparent in
anterior segments were dMP2 neurons (Fig.
6H). This confirms that anterior MP1 neurons also undergo
apoptosis at a late developmental stage.
Having identified an additional neuronal subtype that undergoes segment-specific apoptosis after differentiation, we sought to determine whether MP1 cell death was effected by the same molecular mechanisms involved in anterior dMP2 death. Similar to the complexity observed for dMP2 apoptosis, we found that several deletion combinations resulted in the survival of anterior MP1 neurons (Fig. 7B). Like dMP2 cell death, anterior MP1 death is mediated by both rpr and grim, while hid appears to play a very limited role. This is revealed by the almost complete rescue observed in XR38/H99 and X25/H99 mutants, but not in X14/H99 or X14/X14 mutants (Fig. 6C; Fig. 7B). However, there is an important difference between dMP2 and MP1 death. In XR38/XR38 mutant embryos, a significant number of anterior dMP2 neurons survive but no MP1 neurons are rescued, indicating that loss of rpr alone cannot affect MP1 survival. By contrast, no anterior dMP2 neurons survive in X25/X25 (but not X14/X14) mutant embryos, whereas most MP1 neurons are rescued, implying that loss of grim alone is sufficient to prevent MP1 apoptosis (Fig. 6C). There is, however, some contribution of rpr to MP1 apoptosis since, in the absence of one copy of grim, loss of rpr is sufficient to rescue most MP1 neurons (XR38/H99, Fig. 6C). Thus, we conclude that grim and rpr effect apoptosis of both dMP2 and MP1 neurons. However, grim levels are critical in MP1 neurons, while dMP2 apoptosis depends more on rpr. The fact that we can readily detect rprGAL4 expression in anterior dMP2s, but only in nine anterior MP1 neurons out of 30 VNCs examined, supports this notion (Fig. 2J; Fig. 6G).
We next sought to determine whether Abd-B function underlies the posterior survival of MP1 neurons. We were initially surprised to find that in wild-type embryos MP1 neurons always survived one segment more anteriorly than dMP2 neurons (A5-A8; Fig. 6A). However, when we analyzed the expression of Abd-B in MP1 neurons, we found that not only was Abd-B expressed in posterior MP1 neurons, but its expression in this neuronal type extended into A5 (Fig. 6F). Abd-B expression thus parallels the segmental survival of MP1 neurons. To test whether Abd-B functions in posterior MP1 neurons to prevent their death, we examined Abd-Bm mutants and found that MP1 neurons were initially generated throughout the VNC (Fig. 5F, asterisk). However, in late embryos, both anterior and posterior MP1 neurons were lost (Fig. 6D). Thus, Abd-B is required for the survival of posterior MP1 neurons, but not for their generation. Conversely (and as in the case of dMP2 neurons), anterior MP1 cell death could be completely suppressed by pan-neuronal expression of Abd-B (elavGAL4/UAS-Abd-Bm; Fig. 6E).
Abdominal B prevents apoptosis by repressing the function of more than one RHG-motif gene
The finding that the death of both anterior dMP2 and MP1 neurons is
completely prevented by anterior expression of Abd-B cannot solely be
explained by a repressive function of Abd-B on the rpr gene;
loss of rpr does not result in survival of anterior MP1 neurons
(XR38/XR38; Fig. 7B).
Instead, MP1 survival requires an at least partial loss of grim
(X25/X25 and XR38/H99;
Fig. 7B). Furthermore, in dMP2
neurons, loss of rpr alone is not sufficient to prevent all anterior
cell death; complete anterior rescue is only observed in mutants with
additionally reduced grim gene dosage (XR38/XR38 versus
XR38/H99; Fig. 7B).
This implies either that Abd-B represses rpr and grim, or
that it somehow acts downstream of both rpr and grim to
prevent apoptosis in both lineages. To resolve this issue, we generated flies
mutant for both the H99 region and Abd-Bm. In these
double-mutants, dMP2 and MP1 neurons were properly specified throughout the
cord, but they all failed to undergo apoptosis in late embryos
(Fig. 8A,B). This indicates
that loss of pro-apoptotic gene function is epistatic to Abd-B
function and probably places Abd-B upstream of the RHG-motif genes
removed by the H99 deletion (Fig.
8C).
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Discussion |
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Specification of neuronal numbers and identity along the AP axis
In the developing vertebrate neural tube, a number of studies have shown
that Hox genes are critical for AP organization and for proper neuronal
specification (Capecchi, 1997;
Carpenter, 2002
;
Lumsden and Krumlauf, 1996
).
Although their action may be largely confined to progenitor cells, recent
studies have revealed that Hox genes can also act to control the identity of
early postmitotic neurons (Dasen et al.,
2003
). In the light of our findings, it will be of interest to
determine if selective, Hox-dependent elimination of mature neurons gives rise
to differences in motor neuron numbers along the AP axis of the vertebrate
spinal cord. Increased apoptosis of postmitotic motor neurons has been
observed in mouse mutants lacking Hoxc-8, one of the vertebrate
homologues of abd-A (Tiret et
al., 1998
). This may be the result of the aberrant connectivity
pattern of Hoxc-8-deficient motor neurons, which would restrict their
access to target-derived neurotrophic factors. However, this increase in cell
death is also consistent with the possibility that Hoxc-8 normally
acts to prevent apoptosis of postmitotic neurons in its expression domain.
Specificity and context dependence of Hox genes
Our results contrast with the previous finding that Abd-B appears
to activate rpr transcription to regulate segment boundary formation
in the posterior region of early Drosophila embryos
(Lohmann et al., 2002).
Decreased apoptosis has also been observed in mouse mutants lacking
Hoxb13, one of the vertebrate homologues of Abd-B
(Economides et al., 2003
). It
has previously been shown that the target functions of Hox genes are highly
dependent on cellular context, and the regulation of apoptosis appears to be
no exception. This context dependence may not be unique to the Abd-B
gene. abd-A has been previously reported to activate apoptosis in
post-embryonic neuroblasts during normal development. When Antp and
Ubx were misexpressed in these neuroblasts, they too were able to
trigger apoptosis (Bello et al.,
2003
). In contrast, none of these genes acted in a pro-apoptotic
manner in our study. It is, therefore, conceivable that the pro-apoptotic
function of Hox genes is confined to progenitors, at least in the nervous
system. Alternatively, or additionally, availability of certain cofactors may
determine whether a Hox gene activates or represses transcription of
pro-apoptotic genes in a specific cell.
In addition to their dependence on cellular context, specific Hox proteins
may control pro-apoptotic genes differently. Abd-B and its vertebrate
homologues share several properties that distinguish them from other Hox
proteins, such as the absence of a hexapeptide motif and a preference for a
different DNA core sequence (Pradel and
White, 1998). Together, these differences may confer unique
transcriptional properties on proteins of the Abd-B family, and may explain
why Abd-B is the only Hox protein capable of fully rescuing anterior pioneer
neurons. The finding that Abd-B is the only Hox gene that was unable
to rescue the embryonic brain phenotypes of Drosophila mutants for
the Hox gene labial is consistent with this idea
(Hirth et al., 2001
).
Is the cellular control of Hox gene expression functionally relevant?
Our results show that while Hox genes are broadly expressed within their
domains, they are largely absent from certain cell populations; at stage 16,
few glial cells express Hox genes in the VNC. Since many Drosophila
neuroblasts give rise to both neurons and glia
(Schmid et al., 1999;
Schmidt et al., 1997
), it is
possible that Hox gene expression is actively suppressed by factors promoting
glial fate. Alternatively, an initial wave of Hox expression in progenitors
could be followed by a second, neuron-specific re-activation of Hox
expression. In any case, it will be of interest to identify the molecular
mechanism by which Hox gene expression is confined to specific populations of
postmitotic cells in the nervous system.
While cellular context may determine whether a Hox gene acts in a pro- or
anti-apoptotic manner, apoptosis of specific cells within a Hox expression
domain may also be achieved by differential Hox gene expression. For example,
while Abd-A is broadly expressed in abdominal segments during larval stages,
it is absent from post-embryonic neuroblasts. However, at the last larval
instar, a neuroblast-specific pulse of abd-A results in the
activation of the cell death program in these cells
(Bello et al., 2003).
Similarly, and given the novel role for Hox proteins in the apoptosis (this
study) and differentiation of postmitotic neurons
(Dasen et al., 2003
), the
expression of Hox genes in specific postmitotic neurons is likely to be of
functional significance. Together, these findings are not consistent with the
view that Hox genes solely function as `segment identity' factors specifying
global properties of the segments in which they are active. Instead, they lend
functional support to the proposal that Hox genes are required for a number of
decisions taken at the cellular level
(Castelli-Gair, 1998
;
Hombria and Lovegrove,
2003
).
The developmental regulation of apoptosis
The combined activity of RHG-motif genes is critical to the initiation of
all cell death in the Drosophila embryo
(White et al., 1994). Both our
findings and previous work indicate that these genes act in an additive manner
(Zhou et al., 1997
). However,
not all cell death activators are simultaneously expressed in every cell fated
to die, and their specific expression patterns do not always overlap
(Chen et al., 1996
;
Grether et al., 1995
;
Robinow et al., 1997
;
White et al., 1994
).
Therefore, it is likely that they are differentially regulated by specific
developmental signals. While Abd-B acts to repress rpr and
grim function in posterior pioneer neurons, the developmental
stimulus activating their expression in these neurons throughout the cord is
currently unknown. Three developmental signals are known to regulate the
function of RHG-motif genes in the Drosophila nervous system. The
insect hormone ecdysone appears to be important for blocking cell death of
certain peptidergic neurons during metamorphosis
(Draizen et al., 1999
).
However, the ecdysone-receptor complex has also been shown to promote cell
death by activating rpr transcription in other tissues during
Drosophila metamorphosis (Jiang
et al., 2000
). While an embryonic ecdysone pulse occurs around the
time when pioneer neurons die, our preliminary experiments have failed to lend
any support to an ecdysone-dependent activation of apoptosis in these neurons
(not shown). The EGF-receptor/Ras/MAPK pathway has been shown to phosphorylate
Hid protein, thereby preventing apoptosis of midline glial cells
(Bergmann et al., 2002
).
However, neither Rpr nor Grim appear to be regulated in this fashion, and this
model would not address the specific transcriptional activation of these genes
in pioneer neurons. Lastly, Notch signaling has been described as resulting in
both activation and inhibition of apoptosis
(Miele and Osborne, 1999
). In
Drosophila, recent studies have revealed that Notch can act
cell-autonomously to induce apoptosis during final mitotic divisions both in
the central and peripheral nervous systems
(Lundell et al., 2003
;
Orgogozo et al., 2002
).
Although this Notch-induced developmental apoptosis is prevented in
H99 mutant embryos, the molecular mechanisms by which activated Notch
signaling results in the activation of IAP inhibitors are still unknown.
Nevertheless, Notch signaling is unlikely to be relevant to dMP2 death, since
it is not active in dMP2 neurons (Spana
and Doe, 1996
; Spana et al.,
1995
). It is, therefore, likely that an as yet unidentified factor
is responsible for the activation of the apoptotic machinery in pioneer
neurons. This factor could be Odd, given its specific expression in dMP2 and
MP1 neurons. Because of the early role of odd in embryonic patterning
(Nusslein-Volhard and Wieschaus,
1980
), its possible postmitotic function in these neurons cannot
be addressed using the currently available odd mutants.
Developmental apoptosis in invertebrate embryos typically occurs shortly
after cells are generated. In Drosophila, this has often precluded
the identification of dying cells until apoptosis has been genetically
prevented. Consequently, progress in identification of the mechanisms
controlling apoptosis has been relatively slow, and little is known about the
upstream pathways that initiate cell death in specific tissues or lineages.
Furthermore, in the Drosophila VNC, studies have shown that apoptotic
corpses are engulfed by glia, transported to the dorsal surface of the VNC and
transferred to macrophages for final destruction
(Freeman et al., 2003;
Sears et al., 2003
;
Sonnenfeld and Jacobs, 1995
).
The molecular genetic mechanisms underlying this intriguing series of events
are only just beginning to be unraveled
(Baehrecke, 2002
). The
identification of a late apoptotic event in two of the best-studied and least
complex lineages in the Drosophila CNS, as well as the
characterization of the dMP2-GAL4 line, should contribute to the
elucidation of the mechanisms involved in both the developmental initiation
and execution of apoptosis.
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ACKNOWLEDGMENTS |
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Footnotes |
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