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First published online 14 June 2006
doi: 10.1242/dev.02429
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Biozentrum, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
* Author for correspondence (e-mail: frank.hirth{at}unibas.ch)
Accepted 5 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, Brain tumor, Prospero, Stem cell, Progenitor cell, Proliferation, Tumour
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the embryonic neurogenic period, individual neuroblasts
delaminate in a stereotyped pattern from the neuroectoderm and divide
repeatedly and asymmetrically to generate a new neuroblast and a smaller
daughter cell, called a ganglion mother cell (GMC)
(Campos-Ortega, 1997;
Edenfeld et al., 2002;
Skeath and Thor, 2003;
Technau et al., 2006). Each
GMC is a transient intermediate progenitor cell that generally divides once to
produce two lineage-specific postmitotic ganglion cells (GCs), which
subsequently initiate their differentiation processes. Towards the end of
embryogenesis, most neuroblasts stop proliferating and enter a period of
quiescence. During the second, larval neurogenic period, most neuroblasts
resume proliferation and generate the majority of the cells that comprise the
central brain and ventral ganglia of the adult
(White and Kankel, 1978;
Truman and Bate, 1988;
Prokop and Technau, 1991;
Ito and Hotta, 1992;
Truman et al., 2004).
In contrast to embryonic neurogenesis, less is currently known about the
genetic systems which control and limit the activity of neural progenitor
cells during postembryonic development, notably in the developing brain, where
over 90% of the neurons in the adult central nervous system are generated. A
candidate gene for proliferation control in the fly brain is the
tumour-suppressor gene brain tumor (brat) which encodes a
member of the conserved NHL family of proteins that regulate growth and
differentiation in a variety of organisms
(Arama et al., 2000). Recessive
mutations in brat lead to neoplastic overgrowth, characterized by a
dramatic enlargement of the larval brain hemispheres and the ability of pieces
of brain tissue to metastasize and form secondary tumours when injected into
the abdomen of a wild-type host fly
(Kurzik-Dumke et al., 1992;
Woodhouse et al., 1998;
Arama et al., 2000). However,
the functional role of the brat gene in this process is not clear
since the neural phenotype has not been characterized in detail and initial
findings are contradictory. Thus, the brain overproliferation phenotype of
brat mutants is thought to be primarily due to expansion of optic
lobe neuroblasts (Kurzik-Dumke et al.,
1992; Gateff et al.,
1993), yet the major sites of brat expression in the CNS
throughout development are not the optic lobes, but rather the central brain
and ventral nerve cord (Arama et al.,
2000). In addition to its involvement in brain development, the
brat gene acts in translational repression during early embryogenesis
(Sonoda and Wharton, 2001) and
negatively regulates cell growth and ribosomal RNA synthesis in imaginal discs
(Frank et al., 2002); whether
brat acts by similar molecular mechanisms in the brain is not
known.
In this report, we use molecular markers and mosaic analysis to study the role of brat in neural proliferation control and tumour suppression in the Drosophila brain. Our findings indicate that brat mutation leads to loss of proliferation control in the central brain. Mosaic analysis with a repressible cell marker (MARCM) analysis demonstrates a larval requirement for brat since induction of mutant clones in the first larval instar is sufficient to cause massive overproliferation in the central brain that continues into adulthood. Molecular analysis of brat MARCM mutant clones demonstrates that loss of brat function leads to loss of nuclear Prospero (Pros) expression. Both brat and pros mutant MARCM clones comprise supernumerary cells, which do not express neuron-specific markers such as Embryonic lethal abnormal vision (Elav). By contrast, the vast majority of cells in mutant clones strongly express molecular markers and cell cycle control genes which characterize neural progenitor cells of the wild-type postembryonic brain. Detailed clonal analysis indicates that loss of brat function, and loss of pros function, cell-autonomously affect the differentiation of ganglion cells. Targeted expression of wild-type brat or wild-type pros in brat mutant clones restores cell cycle exit and differentiation of brat mutant cells, which in turn abrogates central brain tumour formation. Taken together, our results demonstrate that the tumour suppressor brat negatively regulates cell proliferation during larval central brain development of Drosophila and identify prospero as a downstream effector of brat in cell fate specification and proliferation control.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (a gift from D. Frank); FRT82B,
pros17 (Reddy and
Rodrigues, 1999) (a gift from V. Rodrigues);
UAS-bratFL1B (Sonoda
and Wharton, 2001) (a gift from R. Wharton);
UAS-prosL(wt)9D (a gift from C. Doe). The following
recombinant chromosomes other than described in Bello et al.
(Bello et al., 2003), were
generated for this study: brat11, GAL4NP3262;
FRT40A, UAS-mCD8::GFPLL5, UAS-nlslacZ20b; FRT40A,
UAS-mCD8::GFPLL5; FRT40A, brat11,
UAS-mCD8::GFPLL5, UAS-nlslacZ20b. GFP labelling of
wild-type and zygotic brat mutant larval nervous systems was achieved by
crossing brat11, GAL4NP3261/CyO,
actGFPJMR1 to FRT40A; UAS-mCD8::GFPLL6,
UAS-nlslacZJ312 or FRT40A, brat11/CyO,
actGFPJMR1; UAS-mCD8::GFPLL6,
UAS-nlslacZJ312. Heterozygous wild-type and homozygous
brat mutant larvae lacking CyO, actGFPJMR1 were
selected under the fluorescence microscope. The detailed MARCM genotypes
indicated in the figure legends were obtained from various stocks assembled
using standard genetics. For MARCM experiments, embryos of the appropriate genotype were collected on yeasted grape juice-agar plates over a 4- to 6-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 60 minutes. Larvae were then collected and plated at low density on standard cornmeal/yeast/agar medium supplemented with live yeast. GFP-labelled MARCM clones were examined in mid (66-72 hours) to late (96-120 hours) third instar larval central nervous systems (CNSs).
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). To
generate a neural progenitor cell-specific anti-grainy head (Grh) antibody, a
synthetic peptide covering 19AA of Grh [aa 798-816 of the O-isoform
(Uv et al., 1997)] was used
for immunization of rabbits (carried out by Neosystems). Polyclonal rabbit
anti-Grh 711 was used at a 1:200 dilution. Other primary antibodies used were:
rat anti-Brat (Sonoda and Wharton,
2001) 1:200; rabbit anti-ß-galactosidase (ß-gal; Cappel)
1:7000; mouse anti-ß-gal (Promega) 1:1000; rabbit anti-PH3 (Upstate
Biotechnology) 1:400; mouse anti-Mira [mAb81
(Matsuzaki et al., 1992); a
gift from P. Overton] 1:50; mouse anti-Pros (mAbMR1A, Developmental Studies
Hybridoma Bank, DSHB, Iowa University) 1:5; mouse anti-cyclin B (CycB)
(mAbF2F4, DSHB) 1:10, mouse anti-CycE (mAb8B10; a gift from H. Richardson)
1:50; rat anti-Elav (mAb7E8A10, DSHB) 1:30; and mouse anti-Grh (BF1; a gift
from S. Bray) 1:3. All fluorescent images were captured using a Leica TCS SP
scanning confocal microscope. Optical sections ranged from 0.5 to 2 µm,
recorded in line average mode with picture size of 512x512 pixels.
Captured images from optical sections were arranged and processed using IMARIS
(Bitplane) or ImageJ software. Figures were arranged and labelled using Adobe
Photoshop. |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
overgrowth of brain hemispheres is gradual during larval development and
becomes prominent in whole mounts of late third larval instars
(Fig. 1A,B). At earlier larval
stages, brain hemispheres in brat mutants still have the same overall
size as in wild type; in both cases, the larval brain hemispheres are
characterized by a central brain region and laterally located developing optic
lobes (Fig. 1C,D). Examination
of cell morphology in the wild-type central brain reveals a limited number of
large cells, which are the postembryonic neuroblasts, associated with smaller
cells, which are their progeny (Fig.
1E). By contrast, the brat mutant central brain is
characterized by dense cellular masses of numerous small and pleiomorphic
cells in addition to a limited number of large cells
(Fig. 1F).
brat mutation affects postembryonic cell proliferation in a cell-autonomous manner
In order to gain insights into the origin and genetic mechanisms of tumour
formation caused by loss of brat function, we used MARCM clonal
analysis to label individual brat mutant neural lineages in a
wild-type heterozygous background (Lee and
Luo, 1999; Lee and Luo,
2001). Positively marked somatic clones mutant for
brat11, a strong loss-of-function allele of brat,
were generated and examined 4 days later in late third instar larvae.
Wild-type clones were induced in parallel as control, hence for each genotype
(wild type and brat) we examined more than 300 specimens carrying
5-20 clones in each brain hemispheres. Our analysis indicated that
brat mutant clones in the larval optic lobes are indistinguishable
from wild-type clones. By contrast, brat mutant clones in the larval
central brain populated large areas of the brain hemispheres
(Fig. 2C), as compared to
wild-type central brain clones (Fig.
2A). Whereas wild-type clones are composed of 50-100 cells in
average (n>100), the majority of late third instar brat
mutant clones (n>100) were up to ten times larger than control
clones. Thus, less than 20% of brat mutant clones comprised <100
cells, whereas more than 80% of the clones comprised 200-1000 cells.
Examination of mutant clones in early third instar larvae revealed clearly
distinguishable and separated brat mutant clones (not shown), whereas
in late third instars, we frequently observed hemispheres within which the
central brain was almost entirely labelled with GFP
(Fig. 2C). This suggested that
multiple brat mutant clones had already merged into an
indistinguishable labelled cell mass overgrowing the whole central brain.
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). Yet,
brat mutant clones of mushroom body neuroblast lineages did not
result in enlarged clones (data not shown).
Proliferation in wild-type neural lineages of the larval central brain
To investigate the nature of the cells that overproliferate in
brat mutant lineages, we first defined a set of molecular markers
that enabled us to characterize the cell types in wild-type neural lineages.
Wild-type MARCM clones examined in late third instar animals typically contain
a single large neuroblast (
10 µm in size) associated with a discrete
packet of smaller cells that corresponds to its entire progeny generated
during larval development (Fig.
3A) (Bello et al.,
2003). The latter population is mostly composed of immature
adult-specific ganglion cells which extend neurites towards the inner
neuropile. Neuronal extensions associate in a common bundle known as cell body
fibre tract (CBT) (Fig.
3A).
Neuroblasts express the coiled-coiled protein Miranda (Mira) at the cell
cortex (Ceron et al., 2001;
Akong et al., 2002). Cortical
Mira becomes polarized during mitosis forming a crescent that segregates into
the budding GMC (Fig. 3C). The
transcription factor Grainy head (Grh) also labels the large neuroblast
(Fig. 3H). The mitotic profile
of the neuroblast is revealed by expression of the cell cycle markers
phosphorylated histone H3 (PH3) (Fig.
3B,D) and CycE (Fig.
3D,E). Careful inspection of mitosis using the nuclear
localisation of PH3 and visualisation of centrosomes further indicates that
CycE protein is detectable in neuroblasts throughout the cell cycle.
Although GMCs are not distinguishable morphologically from the surrounding
postmitotic ganglion cells, their identification in close proximity to the
parental neuroblast is defined in wild-type clones by expression of the cell
cycle markers CycE (Fig. 3D,E)
and/or PH3 (Fig. 3E-G), as well
as nuclear Grh expression (Fig.
3H) (Cenci and Gould,
2005; Almeida and Bray,
2005) and association of Mira at the cellular cortex
(Fig. 3F,I). Beside their
difference in size, two molecular characteristics make it possible to
distinguish neuroblasts and GMCs. First, expression of CycE is detectable in
GMCs only at interphase (PH3-negative nucleus,
Fig. 3D) but not during mitosis
(PH3-positive nucleus, Fig.
3E). Second, localisation of Mira at the cellular cortex is never
polarized during GMC mitosis (PH3-positive nucleus in
Fig. 3F).
GMCs are also characterized by nuclear localization of Pros, which is clearly detectable by co-staining with PH3 (Fig. 3G) or Grh (Fig. 3H). In addition, nuclear Pros expression is also detectable in postmitotic ganglion cells (Fig. 3G,H), which also express the neuronal marker Elav (Fig. 3I). These data show that in wild-type neural lineages of the larval central brain, only neuroblasts and GMCs are actively engaged in the cell cycle, and that each cell type can be unambiguously identified by a unique combination of markers (Table 1).
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). To
determine whether the observed increase in cell number reflects changes in
cell cycle regulation, we analyzed the distribution of cell cycle markers in
brat11 clones using antibodies against CycE, CycB and PH3.
In sharp contrast to wild-type neuroblast lineages, most brat mutant
cells within a labelled mass of clones expressed CycE
(Fig. 4A) and CycB (not shown)
and a large number of cells appeared engaged in mitosis as indicated by PH3
staining of numerous nuclei (Fig.
4B,E).
With few exceptions, all mutant cells within the clones lacked
immunoreactivity for nuclear Pros (Fig.
4C,C') and nuclear Elav
(Fig. 4D), unlike the
surrounding heterozygous wild-type cells devoid of GFP labelling. Moreover, we
never observed any cellular extensions comparable to axon fascicles,
suggesting an invariable lack of axonal processes. Since MARCM clones allow
unambiguous identification of neuronal processes in both, wild-type and
various mutant lineages, due to stable GFP labelling
(Lee and Luo, 1999;
Lee and Luo, 2001), these data
suggest that brat mutant cells are impaired in differentiation. In
support of this notion, we observed that the vast majority of cells in
brat mutant clones showed both nuclear Grh
(Fig. 4C,C') and cortical
Mira (Fig. 4E). Close
inspection of clones revealed that a few large cells scattered at the surface
showed polarized Mira distribution in a cortical crescent. As in wild-type
neuroblasts, this asymmetric distribution of Mira was always associated with
PH3 immunoreactivity, suggesting the presence of several asymmetrically
dividing neuroblasts because of the merger of individual GFP-labelled
brat mutant clones (see also Fig.
2C). In contrast to these large cells displaying Mira in a
cortical crescent, the majority of mutant cells showed uniform cortical
distribution of Mira even during mitosis
(Fig. 4E). These data indicate
that brat mutant clones comprise an excessive number of neural
progenitor cells that are unable to exit the cell cycle but continue to
proliferate.
brat mutant cells in the central brain continue to proliferate into adulthood
Previous studies have shown that cessation of proliferation in the
developing Drosophila brain occurs during puparium formation and
metamorphosis (Ito and Hotta,
1992). We, therefore, wondered whether brat mutant clones
cease proliferation or whether they are able to escape cell cycle stop signals
during later stages of development. MARCM brat11 clones
were induced in first instar larvae and immunostained for PH3 in adult brains
at 1 day, 2 weeks and 3 weeks after eclosion. Control wild-type clones induced
in parallel and examined in the brain of adult flies comprised a limited
number of cells that never showed PH3 immunoreactivity
(Fig. 5A,B), consistent with
the general notion that the adult brain is composed of postmitotic cells. In
contrast, brat mutant clones appeared dramatically enlarged in size
and cell number in the adult brain (Fig.
5C). Moreover, a large number of mutant cells displayed PH3
immunoreactivity even in 3-week-old adult flies, suggesting that these mutant
cells were still mitotically active (Fig.
5D). These data indicate that loss of larval brat
function results in mutant cells that are unable to respond to cell cycle
termination signals during metamorphosis.
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).
Loss of Pros function results in continued mitotic activity that correlates
with de-repressed, ectopic transcription of cell cycle genes
(Li and Vaessin, 2000). This
prompted us to investigate whether loss of nuclear Pros expression in
brat mutant clones could be causally related to the overproliferation
phenotype observed. We therefore generated wild-type MARCM clones and clones mutant for the loss-of-function allele pros17. When assayed in third instar larvae, nuclear ß-gal or GFP-labelled pros17 clones appeared to have increased in size and be populating large areas of the central brain (Fig. 6B) when compared to wild-type control clones (Fig. 6A). The majority of pros mutant central brain clones were five to ten times larger than wild-type control clones (n>100). Thus, less than 5% of pros mutant clones comprised less than 100 cells, whereas more than 90% of these clones comprised 200-1000 cells. Occasionally we also observed brain hemispheres in which the central brain was almost entirely labelled with nuclear ß-gal or GFP, suggesting that multiple pros mutant clones had already merged to an indistinguishable labelled cell mass. Close inspection of such a nuclear ß-gal-labelled cell mass revealed the presence of numerous pleiomorphic cells including large neuroblast-like cells (Fig. 6C).
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).
Importantly, we did not observe any difference in anti-Brat immunoreactivity
between GFP-labelled pros mutant cells and surrounding wild-type
heterozygous cells (Fig.
6C,C'). These data suggest that Brat expression is
unaffected in larval central brain pros mutant clones. Next, we utilized the molecular markers used to characterize wild-type and brat mutant lineages to further analyze pros mutant clones. Almost all cells in the clones were found to express CycE (Fig. 6D,D'), CycB (Fig. 6E), and numerous cells displayed PH3 immunoreactivity (Fig. 6D''). Co-labelling with PH3 and CycE showed that CycE is present at significant levels in most mitotic cells, regardless of morphology, size or position within these clones (Fig. 6D',D''). GFP-labelled pros mutant clones did not show any cellular extensions comparable to axon fascicles, suggesting an invariable lack of axonal processes. Moreover, pros mutant clones lacked Elav immunoreactivity (Fig. 6F,F'), suggesting the lack of differentiated ganglion cells. By contrast, most pros mutant cells showed nuclear Grh (Fig. 6G,G') and cortical Mira (Fig. 6F-G'). As for wild-type neuroblasts, few large cells scattered at the surface of clones showed polarized Mira distribution in a cortical crescent (Fig. 6F',G', arrows), suggesting the presence of several asymmetrically dividing neuroblasts. However, the vast majority of pros mutant cells showed uniform cortical distribution of Mira (Fig. 6F',G'). These findings indicate that pros mutant clones phenocopy larval central brain brat mutant clones.
pros can promote cell cycle exit and differentiation of brat mutant cells
The remarkable similarities between brat and pros mutant
clones suggested that both genes might act in the same pathway regulating
proliferation in the larval central brain. Hence, we used MARCM to express
full-length wild-type pros specifically in brat mutant
clones. As a control we tested the ability of a full length wild-type
brat cDNA (Sonoda and Wharton,
2001) to rescue the brat mutant brain phenotype. Targeted
expression of wild-type brat in brat mutant clones
(n>50) resulted in a significant reduction of clone size in the
central brain of third instar larvae (Fig.
7A-B'). Thus, more than 90% of late third instar central
brain clones comprised <100 cells, whereas less than 10% of these clones
comprised 100-500 cells. We frequently observed that individual clones merged
to form a nest of GFP-labelled cells (Fig.
7A,B), however clones were still distinguishable because only a
few superficially located cells showed nuclear Grh
(Fig. 7A-B') and cortical
Mira (data not shown). Moreover, most cells within clones expressed Elav
(Fig. 7A,A') and Pros
(Fig. 7B,B') in their
nuclei and they formed axonal processes contributing to individual fibre
tracts (Fig. 7A,B), indicating
the presence and proper differentiation of postmitotic ganglion cells. These
data suggest that UAS-driven expression of the brat transgene in
brat mutant clones results in clones that are comparable in size,
number and marker gene expression to wild-type controls. In addition, even
when examined in the adult brain, these clones appeared similar to wild-type
control clones and lacked PH3 staining (data not shown).
Targeted expression of wild-type pros in brat mutant clones (n>50) resulted in a very similar phenotype (Fig. 7C-D'). More than 90% of late third instar central brain clones comprised <100 cells, whereas less than 10% of these clones comprised 100-500 cells. Similar to the brat rescue, a significant decrease in mitotic activity occurred, as exemplified by clone size and by PH3-immunoreactivity restricted to a few large cells and to a limited number of smaller associated cells, both of which expressed cortical Mira and nuclear Grh (Fig. 7C-D'). The presence and proper differentiation of postmitotic ganglion cells could be assessed by the detection of Pros (Fig. 7D,D') and Elav (not shown) in the vast majority of cells within the clones. Moreover, most cells formed axonal processes assembled in wild-type-like fibre tracts (Fig. 7C,D). Thus, as for brat, targeted expression of pros in brat mutant clones results in clones that are comparable to wild-type lineages in size, number, and marker gene expression. These results demonstrate that pros can promote cell cycle exit and differentiation of brat mutant cells in a wild-type-like pattern, which in turn abrogates central brain tumour formation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Gateff et al., 1993;
Woodhouse et al., 1998;
Arama et al., 2000). By
contrast, our analysis showed that the brat overproliferation
phenotype is due to loss of proliferation control in the larval central brain;
the optic lobes initially appear wild-type-like but subsequently are overgrown
by neoplastic central brain brat mutant tissue. This conclusion is
further supported by MARCM clonal analysis which demonstrated that loss of
brat function causes overproliferation in the larval central brain
only.
|
) and
larva (our study, Fig. 6A-C),
induction of brat mutant clones in the first larval instar is
sufficient to cause massive overproliferation in the central brain but not the
ventral ganglia. This may suggest that either unknown compensatory mechanisms
actively suppress a brat mutant phenotype in the larval ventral
ganglia, or that this reflects region-specific differences in cell cycle
control. Indeed, transcriptional activity of the mitotic regulator
string/Cdc25 is regulated by a plethora of cis-acting elements, most
of which are devoted to differential control of cell proliferation during
embryonic and larval neurogenesis (Lehmann et al., 1999).
During postembryonic neurogenesis, intense proliferation takes place in the
brain (Truman and Bate, 1988;
Ito and Hotta, 1992). Our
analysis shows that central brain brat mutant clones display
sustained cell cycle marker expression, indicating that mutant cells are
unable to withdraw from the cell cycle. This is further supported by the
presence of enormous brat mutant clones with pronounced proliferative
activity even in 3-week-old adult brains, an observation that contrasts with
the postmitotic adult wild-type brain. Previous studies have shown that
cessation of proliferation in the developing Drosophila brain occurs
during metamorphosis (Ito and Hotta,
1992), although the underlying genetic mechanisms are currently
unknown. The elevated and aberrant cell cycle activity of central brain
brat mutant cells suggests that these cells are either able to escape
or that they lack cell cycle termination signals
(Peterson et al., 2002;
Bello et al., 2003).
brat negatively regulates progenitor cell proliferation by acting on pros
Our mosaic analysis demonstrates that enlarged brat mutant clones
comprise cells that display sustained expression of neural progenitor cell
markers, and simultaneously lack marker gene expression specific for
differentiating ganglion cells. Indeed, lack of axonal processes suggests that
brat mutant clones comprise an excessive number of mutant cells that
are unable to exit the cell cycle and hence do not differentiate into ganglion
cells but rather continue to proliferate. These data indicate that
brat mutation impairs proliferation control of neural progenitor
cells, namely either neuroblasts and GMCs or only one of these progenitors,
since in the wild-type central brain only these two cell types are actively
engaged in the cell cycle. Based on our analysis we cannot distinguish
unambiguously between the two possibilities and the underlying mechanisms. We
do, however, exclude the possibility that differentiating ganglion cells
de-differentiate due to brat mutation, because we consistently
observed lack of differentiation right after clone induction and also at any
later stages of mutant clone development. This was especially exemplified by
the lack of nuclear Pros expression, which in the wild type is unambiguously
detectable in differentiating progeny of larval neuroblast lineages, namely
GMCs as well as ganglion cells (see Fig.
3) (see also Ceron et al.,
2001; Akong et al.,
2002).
Moreover, our loss-of-function analysis indicates that brat mutant
MARCM clones lack Pros and also phenocopy pros mutant clones. Thus,
enlarged pros mutant clones consist of cells that are devoid of Elav
expression, that lack axonal processes but display sustained expression of Grh
and Mira as well as cell cycle markers such as CycE, CycB and PH3. These data
suggest that mutant clones are essentially devoid of terminally
differentiating postmitotic ganglion cells, indicating that Pros functions
like Brat in terminating neural progenitor cell proliferation and inducing
ganglion cell differentiation. In the embryonic CNS, Pros functions to
terminate cell proliferation by repression of cell-cycle activators and
simultaneously to induce a differentiation program, effectively coupling the
two events. This Pros function appears to be warranted by its localization in
the basal cortex of asymmetrically dividing neuroblasts and hence its
distribution to only one daughter cell, the GMC. Upon completion of mitosis,
Pros translocates from cytoplasm into the nucleus where it executes its
transcriptional program ensuring both terminal division of the GMC and cell
differentiation of its progeny (for a review, see
Prokopenko and Chia, 2005). In
the larval CNS we observe nuclear localisation of Pros in GMCs and ganglion
cells but not in the neuroblast, suggesting that Pros has comparable
functional features in larval central brain neurogenesis.
In addition, our results provide evidence that Pros acts downstream of Brat
in neural proliferation control. The following points support this notion: (1)
brat mutant clones lack nuclear Pros; (2) brat and
pros mutant clones are indistinguishable both at the morphological
and at the molecular level; (3) Brat expression is unaltered in pros
mutant clones, which together with point no. 1 strongly suggests that Brat is
epistatic over Pros; and (4) trans-activation of wild-type pros in
brat mutant clones is sufficient to promote both cell cycle exit and
differentiation. Our experiments, however, do not provide any evidence about
the direct or indirect nature of their interaction. Since overexpressed Pros
is detected specifically in brat mutant clones in a wild-type-like
pattern (Fig. 7D'), we
exclude the possibility that brat acts as a translational repressor
of Pros, comparable to its role in hunchback repression
during embryonic abdominal segmentation
(Sonoda and Wharton, 2001). In
addition, brat mutation apparently does not affect pros
transcription, since pros RNA in situ hybridization in zygotic
brat mutants produced a pattern indistinguishable from wild-type
controls (data not shown). Thus, Brat and Pros may act indirectly in the same
pathway, regulating progenitor cell proliferation control in the brain.
Alternatively, Brat may act in a process required to cargo Pros, comparable to
the function of its mammalian homolog BERP
(Yan et al., 2005).
Somatic mutation of brat and pros can cause clonally derived brain tumours
Our in vivo mosaic analysis demonstrates that a single mutation in either
brat or pros is sufficient to cause brain tumour formation
in a cell-autonomous manner, suggesting that indefinite proliferation of
brat and pros mutant cells is a cell intrinsic property
(Caussinus and Gonzalez, 2005).
GFP-labelled MARCM cells each derive from a common precursor cell, implying
that brat and pros mutant cells all descend from individual
tumour cells of origin and hence lead to brain tumour formation in a clonally
related manner. Moreover, our data indicate that pros and
brat mutant clones in the larval central brain are composed of an
excessive number of mutant progenitor cells that are unable to differentiate
into ganglion cells but rather continue to proliferate (summarized in
Fig. 8). In this sense our
results provide in vivo support for the notion that the initiating event in
the formation of a malignant tumour is an error in the process of normal
differentiation (Harris,
2005).
In addition, the unlimited capacity to generate undifferentiated,
proliferating progeny suggests that cells mutant for brat or
pros retain self-renewing capacities. In human, brain cancers are
thought to arise either from normal stem cells or from progenitor cells in
which self-renewal pathways have become activated (reviewed in
Al-Hajj and Clarke, 2004;
Oliver and Wechsler-Reya,
2004), however the underlying mechanisms are elusive. Our results
in Drosophila may therefore provide a rationale and genetic model for
the origin of brain cancer stem cells. Although parallels to human tumour
formation are speculative, it is noteworthy that TRIM3, a human
homolog of brat is located on chromosome 11p15
(El-Husseini et al., 2001), a
region frequently deleted in brain tumours
(Fults et al., 1992;
Sonoda et al., 1995;
Schiebe et al., 2001).
Moreover, functional studies have shown that the pros homologue
Prox1 regulates proliferation and differentiation of neural
progenitor cells in the mammalian retina
(Dyer et al., 2003). These
data may indicate that brat and pros function in cell
differentiation and tumour suppression in an evolutionarily conserved
manner.
|
) and
Lee et al. (Lee et al., 2006)
reported comparable findings on the function of brat in larval
central brain tumour suppression. |
ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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