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First published online 7 February 2007
doi: 10.1242/dev.02793
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Biozentrum, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
* Author for correspondence (e-mail: Bruno.Bello{at}unibas.ch)
Accepted 28 December 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Polycomb group, Drosophila, Neurogenesis, Hox genes, Neuroblasts
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Ringrose and Paro, 2004;
Simon and Tamkun, 2002). They
thus contribute to a cell memory system that prevents changes in cell identity
by maintaining the transcription patterns, which are set early in
embryogenesis, during subsequent development and in adult life. PcG genes were
originally discovered in Drosophila as repressors of the homeotic
(Hox) genes. Through PcG gene action, spatial patterns of Hox gene expression,
which are initially set in early embryogenesis by segmentation genes, are
maintained; when the segmentation proteins decay, PcG proteins assume Hox
expression control for the remainder of development
(Gould, 1997;
Kennison, 1995;
Pirrotta, 1998;
Schumacher and Magnuson,
1997).
In Drosophila, approximately 15 PcG proteins are thought to
participate in two separate multiprotein complexes. These are the Polycomb
repressive complex 1 (PRC1), which contains most of the characterized PcG
proteins such as those encoded by Polycomb (Pc),
Posterior sex combs (Psc), polyhomeotic
(ph) and Sex combs extra (Sce), and Polycomb
repressive complex 2 (PRC2), which contains proteins such as those encoded by
extra sex combs (esc), Enhancer of zeste
(E(z)) and Suppressor of zeste 12 (Su(z)12). PRC2
is thought to be involved in the initiation of gene silencing, whereas PRC1 is
implicated in stable maintenance of gene repression. The expression of PcG
genes is widespread and nearly ubiquitous throughout fly development, and
notably includes expression in the embryonic and larval CNS (e.g.
Martin and Adler, 1993;
DeCamillis and Brock, 1994;
Carrington and Jones, 1996;
Bornemann et al., 1998).
Homologs of the fly PcG genes have been identified in mammals, and in some
cases these gene homologs have also been implicated in developmental Hox gene
regulation (Gould, 1997).
Indeed, deregulation of Hox gene expression is one of the hallmarks of both
Drosophila and mammalian PcG phenotypes. However, as general
chromatin regulators, PcG genes in Drosophila and mammals also
control a diverse set of other target genes that include those involved in
cell cycle regulation (reviewed by Gould,
1997; Orlando,
2003; Simon,
1995).
Recent work on mouse models indicates that PcG genes are involved in stem
cell fate and proliferation control (reviewed by
Valk-Lingbeek et al., 2004).
Direct evidence for this comes from studies of the murine Bmi1 gene,
which is the homolog of the two closely related fly genes Psc and
Suppressor of zeste 2 (Su(z)2). The analysis of
Bmi1-deficient mice, which suffer from progressive loss of neuronal
and hematopoietic cells, implicates Bmi1 in the renewal of multiple
stem cell types (Lessard and Sauvageau,
2003; Molofsky et al.,
2003; Park et al.,
2003). In the developing nervous system, Bmi1 is required
for the self-renewal of neural stem cells in the telencephalon and for the
proliferation of cerebellar granule cell progenitors
(Leung et al., 2004;
Molofsky et al., 2003).
Bmi1 has been shown to regulate three Hox genes, Hoxd8,
Hoxd9 and Hoxc9, in neural stem cells
(Molofsky et al., 2003). Since
loss of Bmi1 leads to upregulation of these Hox genes in vitro, it is
conceivable that Bmi1 loss could affect neural stem cells in vivo
through aberrant reactivation of Hox gene expression. This possibility has not
been addressed experimentally. Although many aspects of PcG gene structure and
function are conserved from flies to mammals, little is known about the roles
of PcG genes in neuronal developmental processes in Drosophila. In
ph mutants, global misrouting of CNS axons and ecdysone-dependent
loss of neuronal subtype identity during metamorphosis have been reported
(Smouse and Perrimon, 1990;
Wang et al., 2006). However,
there is currently little evidence for a role of PcG genes in neural stem cell
fate or neuronal proliferation in Drosophila.
The neuroblasts of Drosophila are similar to mammalian neural stem
cells in that they self renew and have the potential to generate different
types of neurons and glia. Neuroblasts divide repeatedly in an asymmetric mode
which is self-renewing and generates smaller ganglion mother cells (GMCs),
which usually divide once to produce two postmitotic progeny.
Drosophila neuroblasts generate the larval CNS during embryonic
development. Following a period of quiescence, most neuroblasts resume their
asymmetric mode of proliferation and generate the bulk of the adult CNS during
postembryonic development (Prokop and
Technau, 1991; Truman and
Bate, 1988; Truman et al.,
1993). Mechanisms involved in asymmetric neuroblast division and
neural proliferation during embryogenesis have been characterized in detail
(Betschinger and Knoblich,
2004; Chia and Yang,
2002; Jan and Jan,
2001; Pearson and Doe,
2003; Pearson and Doe,
2004; Skeath and Thor,
2003). By contrast, the mechanisms that control the number of
cells generated by larval neuroblasts during postembryonic development are not
yet well understood (Maurange and Gould,
2005). However, proliferation and self-renewal of larval
neuroblasts are currently subjects of intense investigation, given that these
processes may have implications for stem cell biology (reviewed by
Yu et al., 2006;
Bello et al., 2006;
Betschinger et al., 2006;
Lee et al., 2006a;
Lee et al., 2006b;
Bowman et al., 2006. Recently,
it has been shown that reactivation of specific Hox genes is involved in
terminating neuroblast proliferation during larval stages
(Bello et al., 2003). During
the last larval instar, a brief pulse of Hox gene expression occurs in
abdominal neuroblasts, which activates proapoptotic genes and results in
neuroblast death. Thus, the Hox axial patterning system is directly linked to
neuronal proliferation and stem cell fate during postembryonic development of
the Drosophila CNS. Given that PcG genes are involved in maintaining
spatial patterns of Hox gene expression, this suggests that PcG genes might
also play a role in controlling neuronal proliferation in
Drosophila.
Here we investigate the role of several PcG genes in postembryonic development of the Drosophila CNS. We use mosaic-based MARCM techniques to carry out a clonal analysis of these genes in the persistent larval neuroblasts and their progeny ('neuroblast clones'). Our findings show that neuroblast clones fail to proliferate normally during larval development in the absence of the PcG genes and that the observed lack of proliferation in the PcG mutants is due to the loss of the neuroblasts through apoptosis. This proliferation defect can be rescued by blocking apoptosis in the neuroblast of mutant clones, indicating that PcG gene action is required to prevent neuroblast death and, thereby, ensure normal neural proliferation. Significantly, ectopic expression of posterior Hox genes, which in wild type leads to neuroblast death, is observed in the PcG mutant clones, as well as in the PcG mutant neuroblasts rescued from apoptosis-block. These results suggest that loss of PcG genes leads to aberrant derepression of posterior Hox gene expression in postembryonic neuroblasts, which causes neuroblast death and termination of proliferation in the mutant clones. Taken together, our findings demonstrate for the first time that PcG genes are essential for normal neuroblast survival and proliferation in postembryonic CNS development of Drosophila. Moreover, together with studies on mammalian PcG genes, these studies imply that repression of aberrant reactivation of Hox gene expression may be a general and evolutionarily conserved role for PcG genes in nervous system development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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);
FRT2A, Su(z)121; FRT42D, Su(z)21.B8;
FRT2A, PcXT109; FRT82B, Sce1;
FRT82B, ScmD1 (Beuchle
et al., 2001). Additional recombinants were generated for this
study: FRT82B, ScmD1, UAS-p35BH2; FRT82B,
Sce1, UAS-p35BH2; UAS-mCD8::GFPLL5,
UAS-nlslacZ20b, UAS-p35BH1. Recombinants not
described are found in Bello et al. (Bello
et al., 2003). Further details can be found at Flybase:
http://flybase.bio.indiana.edu. Combinations of chromosomes were assembled using standard genetics to generate the following MARCM clones:
wt control (Fig. 1): y,w, hsFLP/+; UAS-mCD8::GFPLL5, UAS-nlslacZ20b/tubP-GAL4; FRT82B/FRT82B, tubP-GAL80LL3.
wt control (Figs 2 and 4): y,w, hsFLP/+; UAS-mCD8::GFPLL5, UAS-nlslacZ20b/tubP-GAL4; FRT2A/FRT2A, tubP-GAL80LL9.
Sce: y,w, hsFLP/+; UAS-mCD8::GFPLL5, UAS-nlslacZ20b/tubP-GAL4; FRT82B, Sce1/FRT82B, tubP-GAL80LL3.
Sce+P35: y,w, hsFLP/+; UAS-mCD8::GFPLL5, UAS-nlslacZ20b/tubP-GAL4; FRT82B, Sce1, UAS-p35BH2/FRT82B, tubP-GAL80LL3.
Scm: y,w, hsFLP/+; UAS-mCD8::GFPLL5, UAS-nlslacZ20b/tubP-GAL4; FRT82B, ScmD1/FRT82B, tubP-GAL80LL3.
Scm+P35: y,w, hsFLP/+; UAS-mCD8::GFPLL5, UAS-nlslacZ20b/tubP-GAL4; FRT82B, Sce1, UAS-p35BH2/FRT82B, tubP-GAL80LL3.
Pc: y,w, hsFLP/+; UAS-mCD8::GFPLL5, UAS-nlslacZ20b/tubP-GAL4; FRT2A, PcXT109/FRT2A, tubP-GAL80LL9.
Pc+P35: y,w, hsFLP/+; tubP-GAL4/UAS-mCD8::GFPLL5, UAS-nlslacZ20b, UAS-p35BH1/tubP-GAL4; FRT2A, PcXT109/FRT2A, tubP-GAL80LL9.
Ez: y,w, hsFLP/+; tubP-GAL4, UAS-mCD8::GFPLL5/+; FRT2A, E(z)731/FRT2A, tubP-GAL80LL9.
Su(z)12: y,w, hsFLP/+; tubP-GAL4, UAS-mCD8::GFPLL5/+; FRT2A, Su(z)121/FRT2A, tubP-GAL80LL9.
Psc-Su(z)2: y,w, hsFLP/+; FRT42D, Su(z)21.68/FRT42D, tubP-GAL80; tubP-GAL4LL7, UAS-mCD8::GFPLL6/+.
UAS-abdA: y,w, hsFLP/+; UAS-mCD8::GFPLL5, UAS-nlslacZ20b/tubP-GAL4; FRT82B, UAS-abdAH3/FRT82B, tubP-GAL80LL3.
UAS-abdA+UAS-P35: y,w, hsFLP/+; UAS-mCD8::GFPLL5, UAS-nlslacZ20b, UAS-p35BH1/tubP-GAL4; FRT82B, UAS-abdAH3/FRT82B, tubP-GAL80LL3.
For MARCM experiments, embryos of the appropriate genotype were collected
on yeasted grape juice agar plates over a 4- to 8-hour time-window and raised
at 25°C for 24-28 hours before heat shock treatment. Heat shock induction
of FLP in the newly hatched larvae was performed by immersing the plates at
37°C in a water bath for 1 hour. Larvae were then collected and plated at
low density on standard cornmeal-yeast-agar medium supplemented with live
yeast. GFP-labeled MARCM clones were examined in nervous systems dissected out
of wandering third-instar larvae (time of dissection was 
96 hours after
heat shock). For the time course experiment shown in
Fig. 3, batches of newly
hatched larvae were raised in vials on standard medium and heat shocked at
24-hour intervals. Clones were examined on 10-20 nervous systems dissected out
at the wandering stage 24, 48 and 72 hours after heat induction.
Immunostaining and antibodies
CNSs were dissected in PBS, fixed in 2% paraformaldehyde in PBL (75 mM
lysine-HCl in sodium phosphate buffer, pH 7.4) for 1 hour at room temperature
(RT), washed three times for 10 minutes in PBS containing 0.5% Triton X-100
(PBT), blocked for 1 hour at RT in PBT containing 10% normal goat serum, and
incubated with primary antibodies in blocking solution overnight at 4°C.
Samples were washed three times for 10 minutes each in PBT at RT, and
secondary antibodies were applied in blocking solution for 2 hours at RT.
After washing three times for 10 minutes each in PBS, samples were mounted in
Vectashield (Vector Labs). The following antibodies were used: rabbit anti-Grh
(1:200) (Bello et al., 2006),
rabbit anti-PH3 (1:400; Upstate Biotechnology), mouse anti-Mira Mab81 (1:50;
gift of P. Overton, VASTox, Abingdon, UK), mouse anti-Pros MaMR1A (1:10;
DSHB); rat anti-Elav Mab7E8A10 (1:30; DSHB), rat anti-Abd-A (1:200; gift of J.
Casanova, CSIC, Barcelona, Spain), mouse anti-UBX (1:20; gift of R. White,
Cambridge University, UK), mouse anti-CycE (Cyclin E2) (1:50; mAb8B10, gift
from H. Richardson, Peter MacCallum Cancer Center, East Melbourne, Victoria,
Australia), rabbit anti-Caspase-3 (1:75; Cell Signaling Technology). Alexa
Fluor-conjugated secondary antibodies (Molecular Probes) were used at
1:200.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The extent of ongoing proliferation in the wild-type third larval instar
CNS was determined by studying expression of the mitotic marker phosphorylated
Histone H3 (PH3), and of the cell cycle marker Cyclin E
(Fig. 1A). Highest levels of
proliferation were seen in the developing optic lobes where dense zones of
mitotically active cells characterized the outer and inner proliferation
centers. This proliferation in the optic lobes was not studied further in this
report. Clearly delimited from the optic lobes were the scattered
proliferative clusters in the developing central brain and thoracic ganglia.
Consistent with previous findings, only limited proliferation was seen in the
abdominal ganglia (Awad and Truman,
1997). To characterize the proliferating clusters of the wild-type
central brain and thoracic ganglia in more detail, we examined expression of
the PH3 marker in MARCM neuroblast clones that were positively labeled with
nuclear ß-galactosidase. Each of these clones was composed of a large
neuroblast and its associated progeny. Mitotic activity assayed with the PH3
marker was observed in the neuroblast, in the associated smaller GMC, or in
both cell types (Fig. 1B-E).
Approximately half of the labeled MARCM clones in late third-instar larvae
showed mitotic activity as judged by expression of PH3.
We next examined labeled MARCM clones that were mutant in the PcG genes Sce, Scm, Pc, E(z), Su(z)12 or Psc-Su(z)2. An analysis of the proliferating clusters in the central brain and thoracic ganglia of these PcG mutants revealed a striking phenotype in five of the six cases examined. The overall size of labeled clones was dramatically reduced in Sce, Scm, Pc, E(z) and Su(z)12 mutants as compared with wild-type control clones (Fig. 1F-I). For example, whereas 76% of the wild-type clones contained more than 50 nuclei (n=25), 81% of Sce clones (n=75) and 88% of Pc clones (n=56) contained less than 20 nuclei. This marked reduction in clone size was observed throughout the central brain and thoracic ganglia. In contrast to the other PcG genes studied, mutant clones for Psc-Su(z)2 in the CNS appeared morphologically normal.
The small size of the labeled clones in the five PcG mutants was associated with a dramatic reduction in mitotic activity as assayed with the marker PH3 (Fig. 1J). Thus, in contrast to wild-type control clones, only 6% of the E(z) clones showed PH3 immunoreactivity (n=66), and none of the 50 Sce clones contained mitotic cells. Mutant clones for Su(z)12, Pc and Scm showed a comparable reduction in clone size associated with a low mitotic index in the same range. In accordance with their normal overall size, mutant clones for Psc-Su(z)2 in the CNS had a mitotic index similar to that of wild-type controls. These findings indicate that there is a marked deficit in neural proliferation in the brain and thoracic ganglia of Sce, Scm, Pc, E(z) and Su(z)12 mutants.
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Supporting this notion were differences in the spatial organization of labeled clones in wild type versus mutants. In the wild type, the large Grh-expressing neuroblasts of each clone were located in the outermost cortical layer and their labeled progeny extended in a continuous, columnar fashion from the neuroblast into inner cortical layers (Fig. 2C). As expected for postmitotic neurons, these progeny expressed the neuron-specific label Elav and projected neuronal processes towards the neuropile. By contrast, the small, labeled MARCM cell clones of the Sce, Scm, Pc, E(z) and Su(z)12 mutants were generally located in the inner cortical layers and were rarely associated with a Grh-expressing precursor cell of any size (Fig. 2D). These small clones were composed of cells that expressed Elav and extended neurites, indicating that the cells are postmitotic neurons.
Further evidence for the lack of neural precursors in the mutant clones was
provided by studying the expression of Miranda (Mira), which is expressed at
the cell cortex in both neuroblasts and GMCs
(Ikeshima-Kataoka et al.,
1997). As expected, the neuroblasts of labeled MARCM wild-type
clones always expressed Mira (Fig.
2E). By contrast, the small clones of Sce, Scm, Pc, E(z)
and Su(z)12 mutants were generally Mira-negative and only rarely
contained large cells (Fig.
2F,G). Unlike wild-type and Psc-Su(z)2 mutants clones
that always showed Grh- or Mira-expressing cells, the PcG mutants Sce,
Scm, Pc, E(z) and Su(z)12 had Grh- or Mira-expressing cells in
less than 10% of the clones (Fig.
2G). Taken together with the data on clone size and mitotic
activity, these results suggest that the deficit in proliferation observed in
Sce, Scm, Pc, E(z) and Su(z)12 mutants is causally related
to the absence of neural precursor cells in the brain and thoracic
ganglia.
We next investigated if the neuroblasts in PcG mutant clones might be
eliminated by programmed cell death. Most of the PcG mutant clones induced
after larval hatching and examined 96 hours later at the late third larval
instar stage no longer contained neuroblasts. To generate mutant clones that
still had neuroblasts, induction was timed to occur 24 hours, 48 hours, and 72
hours before examination at the late third larval instar stage. The percentage
of Pc mutant clones that contained neuroblasts in each of these
conditions is shown in Fig. 3A.
Neuroblasts were present in 88% of Pc clones examined 24 hours after
induction, indicating that significant elimination had not yet occurred during
this short time interval. By contrast, neuroblasts were absent in 89% of the
mutant clones induced 72 hours before examination, implying that the
elimination process had largely taken place during this long time interval.
However, in mutant clones induced 48 hours before the late third larval
instar, neuroblasts were present in approximately half of the cases (59%),
suggesting that the neuroblast elimination process might still be ongoing at
this intermediate time interval. To investigate this possibility, these clones
were immunostained with an antibody against the activated form of the human
Caspase-3 protein, shown previously to recognize apoptotic cells of
Drosophila (Brennecke et al.,
2003). Forty-eight percent of the neuroblasts present in
Pc mutant clones expressed activated Caspase-3 at this stage and
were, therefore, undergoing programmed cell death
(Fig. 3B). This result supports
the notion that neuroblasts in PcG mutant clones are eliminated by apoptosis.
Moreover, given the marked proliferative activity of most neuroblasts during
the third larval instar, this finding also suggests that loss of PcG gene
function can induce neuroblast death even if these precursors are in an
actively proliferating state.
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By contrast, in the markedly reduced PcG mutant clones, ectopic expression of Hox genes was regularly observed anterior to their endogenous expression domains. Many of the cells in these PcG mutant clones, which lacked neuroblasts and were much smaller than wild-type clones, showed aberrant expression of the posterior Hox genes (Fig 4D-F). This ectopic Hox gene expression was more prevalent in the PcG mutant clones for abd-A and Abd-B, in that the number of clones with ectopic Hox gene expression, as well as the number of Hox gene-expressing cells in a given clone, were larger for abd-A and Abd-B than for Ubx. This prevalence of ectopic abd-A and Abd-B expression was not only seen in the ventral ganglia, but was also characteristic of mutant clones in the central brain (Fig. 4G-I). It should be noted that ectopic Hox gene expression was only seen in Sce, Scm, Pc, E(z) and Su(z)12 mutant clones, and not in Psc-Su(z)2 mutant clones. These observations imply that the ectopic expression of posterior Hox genes is a cell-autonomous consequence of the loss of specific PcG genes in postembryonic neuroblast lineages.
Targeted misexpression of posterior Hox genes mimics the PcG mutant phenotype
Previous work has shown that experimentally induced misexpression of
posterior Hox genes such as abd-A in postembryonic neuroblasts causes
the death of these precuror cells (Bello et
al., 2003). As a consequence of induced neuroblast death, the
clonal lineages observed in these experiments were small and consisted of a
limited number of postmitotic neurons. The size and cell composition of the
lineages obtained in clones that are mutant for the PcG gene Sce, are
very similar to the size and cell composition of the lineages that result
following targeted misexpression of the Hox gene abd-A
(Fig. 5A-C). In both cases, the
clone size is severely reduced as compared with wild type and comprises only
small cells; neuroblasts are lacking and clones ectopically express
abd-A.
The clonal phenotypes observed following misexpression of abd-A in postembryonic neuroblasts can be rescued by blocking cell death. Fig. 5D,E shows that blocking cell death in abd-A misexpression experiments through co-misexpression of the caspase inhibitor P35 fully restored both clone size and neuroblast survival such that wild-type-like clones were obtained. Thus, rescued lineages consisted of a large neuroblast and a normal number of neuronal progeny that extended in a continuous, columnar fashion from the neuroblast into inner cortical layers. This restoration of wild-type-like lineages occurred in spite of the elevated level of abd-A in the neuroblast of the labeled clones. Thus, targeted misexpression of abd-A leads to a phenotype that mimics the PcG mutant phenotype, and that can be fully rescued by blocking apoptosis in the neuroblast clones.
Restoration of wild-type-like neurogenesis by apoptosis-block in PcG mutant clones
If derepression of Hox genes in PcG mutant lineages restricts lineage size
by promoting neuroblast apoptosis in a manner comparable to that caused by the
targeted misexpression of Hox genes, then blocking apoptosis should be able to
rescue the PcG mutant phenotype. To test this, we expressed P35 in mutant
clones for the PcG genes Pc, Sce and Scm. In all three cases
we recovered wild-type-like clones characterized by the presence of a large
neuroblast and the normal number of neuronal progeny.
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A quantification of the obtained rescue efficiency is given in Fig. 6G. For all three PcG genes investigated, over 80% of the labeled clones contained neuronal precursor cells based on expression of Mira or Grh. Moreover, approximately half of these clones showed signs of mitotic activity as assayed with PH3 expression; this value corresponds to that seen in wild-type lineages. Taken together, these results demonstrate that apoptosis-block in PcG mutant lineages is sufficient to fully restore wild-type-like properties to CNS neuroblast clones. We therefore posit that PcG mutant lineages are capable of normal neurogenesis if the consequence of homeotic deregulation in neuronal precursors is bypassed through prevention of cell death.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PcG genes are required for postembryonic neuronal proliferation in Drosophila
Numerous genes are required for the continued mitotic activity of
neuroblasts during postembryonic life
(Maurange and Gould, 2005).
Our findings provide the first demonstration that PcG genes are essential for
neuroblast survival and proliferation in the postembryonic CNS. Previous work
on PcG gene action during embryonic neurogenesis has been carried out by
Prokop and Technau (Prokop and Technau,
1994). This demonstrated that the derepression of posterior Hox
genes in PcG mutants leads to a change in the segmental determination of
neuroblasts and their lineage, but not to their mitotic arrest and death.
Thus, the effects of PcG gene loss on neurogenesis are context-dependent and
differ during embryonic development as compared with postembryonic
development. This is underscored in recent work which indicates that the PcG
gene ph is essential for maintaining neuronal identity and diversity
during metamorphosis (Wang et al.,
2006).
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). In the
ventral ganglia, a neuroblast-specific pulse of abd-A during the
third instar provides the cue for cell death, which limits the number of
progeny produced per neuroblast. Our data indicate that this mechanism, which
in the wild type relates Hox gene expression to the clone size of neural stem
cells, also operates in PcG mutants and is responsible for the PcG mutant
phenotypes. Indeed, a general function of PcG genes in postembryonic
neurogenesis may be to prevent the premature and widespread operation of this
mechanism for temporal regulation of neurogenesis through termination of
neuroblast life. It is noteworthy that the Hox gene-dependent activation of
apoptosis within the CNS is selective for the neuroblast and does not occur
when Hox genes are derepressed in neurons, either during normal development or
in misexpression experiments (Bello et al.,
2003). This explains why the neurons in PcG mutant clones, which
were generated before the induction of neuroblast cell death, continue to
survive despite the presence of ectopic Hox gene derepression.
Our data indicate that loss of specific PcG genes in larval neuroblasts
leads to ectopic Hox gene expression that is sufficient to cause neuroblast
cell death. However, the PcG proteins may also contribute to neuroblast
survival by repressing other unidentified target genes which, when
derepressed, might result in premature death of postembryonic neuroblasts.
Indeed, although deregulation of Hox gene expression is one of the hallmarks
of PcG phenotypes in Drosophila, a diverse set of other target genes,
including genes involved in cell cycle regulation, are controlled by PcG genes
(see Gould, 1997;
Orlando, 2003;
Ringrose and Paro, 2004).
PcG genes act in postembryonic neuronal proliferation in flies and mammals
Interesting parallels to our findings on the role of PcG genes in neural
proliferation come from studies of mammalian PcG genes, specifically of the
Bmi1 gene (reviewed by Shakhova
et al., 2005; Valk-Lingbeek et
al., 2004). Bmi1 mutant mice develop ataxia, seizures and
tremors in early postnatal life, and display a significant reduction in
overall brain size, which is particularly severe in the granular and molecular
layers of the cerebellum. Strikingly, Bmi1-deficent mice become
depleted of cerebellar neural stem cells postnataly, indicating an in vivo
requirement for Bmi1 in neural stem cell renewal. Bmi1
deficiency leads to increased expression of the cell cycle regulators
p16Ink-4a and p19Arf (both now known as Cdkn2a - Mouse Genome Informatics),
and the neurogenesis defect in the mutant mice can be partially rescued by
further deleting p16Ink4a (reviewed by
Molofsky et al., 2004). This
suggests that one way in which Bmi1 promotes the maintenance of adult
stem cells is by repressing the p16Ink4a pathway. However, it is also likely
that Hox gene repression through Bmi1 is involved in this process,
given that loss of Bmi1 has been shown to cause a deregulation of
posterior Hox gene expression in neural stem cells in vitro. Moreover, a
direct molecular link between Bmi1 and Hox gene regulation has
recently been discovered in mammalian development, in that the promyelocytic
leukemia zinc finger (Plzf; Zbtb16 - Mouse Genome Informatics) protein
directly binds Bmi1 and recruits PcG proteins in the HoxD cluster
(Barna et al., 2002).
In Drosophila, the homologs of the mammalian Bmi1 gene
are the PcG genes Psc and Su(z)2. Psc and Su(z)2
encode very similar proteins and are partially redundant in function, but both
genes are eliminated in a deletion in the Psc-Su(z)2 line; see Soto
et al. (Soto et al., 1995).
Rather surprisingly, mutational loss of Psc-Su(z)2 does not lead to
ectopic Hox gene derepression and, in consequence, does not appear to affect
neuronal proliferation in the postembryonic CNS of Drosophila. This
is in stark contrast to the other five PcG genes investigated, which do play
important roles in proliferation control by preventing ectopic Hox gene
expression and cell death in postembryonic neuroblasts. The discrepancy
between murine Bmi1 and Drosophila Psc-Su(z)2 function in
neuronal proliferation suggests that although a general role of PcG genes in
neuronal proliferation control may be conserved between mammals and flies,
conservation of gene action may not always be retained at the level of
individual PcG homologs.
In terms of overall development, it is clear that one and the same PcG gene
can have very different functions depending on the developmental context in
which it acts. For example, as mentioned above, during embryonic neurogenesis
the Drosophila Pc gene acts in tagmata-specific differentiation of
neuroblasts, in contrast to its role in postembryonic neurogenesis
(Prokop and Technau, 1994)
(this report). Moreover, in postembryonic development of imaginal discs,
deletions in the Drosophila Psc-Su(z)2 genes have been shown to
result in cellular hyperproliferation, which contrasts with the
lack-of-proliferation phenotype of Psc-Su(z)2 mutants in
postembryonic development of the CNS
(Beuchle et al., 2001) (this
report). Similarly, in the mouse, the Bmi1 gene has been implicated
in tumor progression in mantle cell lymphoma, colorectal cancer, liver
carcinomas and non-small cell lung cancer, in addition to its role in nervous
system development. Nevertheless, in all of the Drosophila and
mammalian phenotypes mentioned, deregulation of Hox gene expression appears to
be one of the conserved and thus unifying features of PcG gene functional
loss.
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ACKNOWLEDGMENTS |
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