MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
* Authors for correspondence (e-mail: mb2{at}mrc-lmb.cam.ac.uk and kkhanson{at}mrc-lmb.cam.ac.uk)
Accepted 24 August 2005
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SUMMARY |
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Key words: Chromosomal passenger complex, Mitotic spindle, Polyploidy, Borealin/Dasra, Tissue repair
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Introduction |
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Borealin/Dasra was identified in human cell lines and in Xenopus
extracts, respectively, and found to colocalise with other CPC proteins
throughout mitosis (Gassmann et al.,
2004; Sampath et al.,
2004
). The correct localisation of human Borealin in mitotic cells
depends on the function of the other CPC components; conversely, RNAi-mediated
depletion of Borealin in HeLa cells causes mislocalisation of Aurora B, Incenp
and Survivin (Gassmann et al.,
2004
). Human Borealin binds directly to Incenp and Survivin in
vitro, and forms a complex with the other CPC components in vivo. Its loss of
function, like that of other CPC components, causes multiple mitotic defects,
including failures in chromosome attachment to the spindle, multifocal
spindles and uneven chromosome segregation. This typically results in
multinucleate cells, aneuploidy and polyploidy, as well as, ultimately,
apoptosis (Gassmann et al.,
2004
; Sampath et al.,
2004
). However, cells that lack CPC function can also occasionally
escape apoptosis as they appear to be defective for their spindle attachment
checkpoint (Lens and Medema,
2003
; Yang et al.,
2004
).
Little is known about the role of the CPC during development, except for
its function in the early C. elegans embryo
(Kaitna et al., 2000;
Kaitna et al., 2002
;
Romano et al., 2003
). Here, we
present the first detailed characterisation of a CPC mutation in
Drosophila, using a loss-of-function allele of borealin.
This gene was identified independently in a recent RNAi screen for cytokinesis
defects in cultured Drosophila cells, and was named borr
(borealin-related) (Eggert
et al., 2004
). We provide evidence, based on its subcellular
localisation and function during the cell cycle, that Borr is the functional
counterpart of vertebrate Borealin/Dasra. We show that borr is an
essential gene, and that loss of borr function causes mitotic
defects, including multipolar spindles that result in large polyploid cells
and often in delayed apoptosis. The developmental consequences of these
defects include striking cell-autonomous and non-autonomous defects in
cell-type specification and tissue architecture.
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Materials and methods |
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Plasmids, cell culture and transfection
Borr was tagged N-terminally with green fluorescent protein (GFP) by
Gateway cloning (Invitrogen), using the borr cDNA from clone LD36125
and the pAGW vector (Terence Murphy, Carnegie Institution of Washington). The
resulting construct was confirmed by sequencing.
Kc167 cells were cultured at 25°C in Schneider's medium supplemented with 10% heat-inactivated foetal bovine serum and antibiotics. DmD8 cells were obtained from the Drosophila Genome Resource Center, and cultured similarly, with 10 µg/ml insulin (Sigma) added to the medium. They were transfected with the FuGene transfection reagent (Roche) according to the manufacturers instructions, with a ratio of 4 µg DNA:1 µl FuGene. Cells were processed for analysis 24 hours after transfection.
RNA interference
To identify the gene responsible for the embryonic phenotype of E133, an
RNA interference screen of candidate open reading frames within the genomic
region 30A was performed, as follows. Genomic DNA was isolated from
yw flies, and amplified by PCR with primer pairs containing a T7
promoter sequence at the 5' end designed to amplify a large
uninterrupted stretch of coding DNA. PCR products were used as templates in
transcription reactions using the MegaScript RNAi kit (Ambion), which
resulted, in the case of CG4454, in a dsRNA of 264 bp. The predicted size of
the dsRNA products was verified by agarose gel electrophoresis, and their
concentrations were determined by comparison with a known standard.
Injection of dsRNA into embryos was carried out as described
(Desbordes and Sanson, 2003),
except that the dsRNA was delivered in water. All preparation and injection
steps were carried out at room temperature, and the embryos were aged for
24 hours at 18°C before fixation.
RNAi of Kc167 cells was carried out basically as described
(Clemens et al., 2000), except
that 500 µl of cells were plated at a concentration of 106 per
well of a 24-well plate. Control cells were treated identically, but without
dsRNA.
Estimation of nuclear volumes and mitotic indices
To estimate nuclear volumes, individual wild-type and borr mutant
ventral nerve cord (VNC) nuclei stained with Hoechst were outlined using
ImageJ, and their maximal circumference was measured. From these measurements,
the volumes of the corresponding spheres were calculated, providing estimates
of nuclear volumes. This modelling of nuclear volumes by spheres was validated
as a best approximation by 3D reconstructions of individual nuclei. To
estimate mitotic indices, the mitotic cells were identified on the basis of
chromatin morphology, Hoechst and serine-10 phosphorylated histone H3 (P-H3)
staining, and their numbers were determined per hemineuromere for abdominal
segments 4, 5 and 6 (see also Results and
Fig. 4 legend).
Clonal analysis
FRT/FLP mediated recombination (Xu and
Rubin, 1993) was used to induce homozygous mutant borr
clones. Flies of the genotypes borrE133 FRT40A/SM6a-TM6b
and yw hsflp; Ub-NLS-GFP FRT40A/CyO or f yw hsflp; ck,
f+FRT40A/Cyo (kindly provided by K. Basler) were crossed. Embryos were
collected for 24 hours, aged at 25°C, and heat-shocked after a further 36
or 84 hours. Mutant phenotypes were analysed in dissected larval imaginal
discs, dissected pupal wings or in adult tissues.
Antibody staining and fluorescence microscopy
Embryos were immunostained as previously described
(Cliffe et al., 2003). Imaginal
discs and pupal wings were stained using standard methods. Briefly, tissues
were fixed with 4% formaldehyde (30 minutes at room temperature for imaginal
discs, overnight at 4°C for pupal wings), washed, blocked and incubated
overnight at 4°C with primary antibodies in PBS+0.1% Triton-X-100+1% BSA
(BBT). Tissues were then washed several times in BBT and incubated with
secondary antibodies (Molecular Probes) for 2-3 hours at room temperature. The
following primary antibodies were used: mouse E7 anti-ß-tubulin (1:100;
Developmental Studies Hybridoma Bank, DHSB); rabbit anti-P-H3 (1:500; Abcam);
rabbit anti-activated human caspase 3 (1:700; BD Biosciences), which has been
shown to cross-react with the Drosophila ortholog
(Yu et al., 2002
); mouse
anti-Wg (1:100; DHSB); mouse anti-Cut (1:100; DHSB); rabbit anti-GFP (1:2000;
gift from R. Arkowitz); guinea pig anti-Senseless (1:1000)
(Barbosa et al., 2000
); rabbit
anti-Aurora B (Giet and Glover,
2001
) (1:200); and rabbit anti-Incenp
(Adams et al., 2001
) (1:500).
DNA was stained with Hoechst dye or DAPI
(Fig. 5). Images were collected
on a BioRad 1024 confocal microscope or a Zeiss Axiovert 200M
(Fig. 5).
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Results |
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borr is an essential gene required for embryonic mitoses
Zygotic homozygosity for the borr mutation results in late
embryonic lethality, but the mutant embryos lack overt morphological defects,
probably owing to rescue by maternal gene product. Consistent with this,
borr is ubiquitously expressed in the early Drosophila
embryo, although it appears to be restricted to the VNC and brain during later
embryonic stages (Berkeley Drosophila Genome Project in situ data).
Given its high expression levels in the embryonic nervous system, we
scrutinised this tissue more carefully after staining embryos with Hoechst
dye. Indeed, by stage 12, we detected cells in the VNC and brain with
abnormally large nuclei (Fig.
1B,C). We estimate that the volumes of the borr mutant
VNC nuclei are on average 3 times larger than those of wild-type VNC
nuclei (Fig. 1D). This implies
an increased DNA content (>2N) of the mutant cells, and suggests that
borr loss affects the divisions of VNC cells. We also observed
similarly oversized nuclei in other tissues (in addition to severe
morphological defects such as failure of germ band retraction), after
injection of borr dsRNA into wild-type embryos, which potentially
also depletes maternal gene product (not shown). Thus, borr loss
appears to affect many, if not all, dividing cells in the embryo.
To monitor the mitotic events that are affected in the borr mutant
embryos, we stained these embryos with an antibody against serine 10
phosphorylated histone H3 (P-H3), a histone modification specifically found in
mitotic cells that has been ascribed to Aurora B kinase activity in several
organisms, including Drosophila (see below)
(Giet and Glover, 2001;
Hsu et al., 2000
). Counting
the mitotic cells per hemi-neuromere in wild-type and borr mutant
embryos, we found that these numbers were reduced significantly in the
mutants, to
50% of the wild type at stage 12, and to
20% at stage 14
(Fig. 2A; see also
Fig. 4). Our estimates suggest
that, in mutant embryos, the overall number of cells per hemi-neuromere is
also lower than normal (although it is technically difficult to obtain
accurate counts of total cell numbers). Nevertheless, these counts suggest
that the fraction of mitotic cells (i.e. the mitotic index) in the VNC of
borr mutant embryos may be reduced compared with the wild type.
To see whether the borr mutation affects a specific mitotic stage, we classified each P-H3-positive cell as one of four different mitotic stages (based on the shapes of their chromatin masses; see below), and we determined the frequencies of these stages as a percentage of the total of mitotic cells. This revealed that the percentages of prophase and prometaphase cells were higher in borr mutants compared with the wild type, whereas anaphases and telophases were underrepresented in the mutants (Fig. 2B,C). This profile shift of the mitotic stages appears to be progressive during embryonic development, and becomes more pronounced by stage 14 when telophases have become exceedingly rare (Fig. 2C), maybe as a result of cumulative defects during consecutive abnormal cell divisions. This profile shift suggests that borr loss causes a severe attenuation, or block, prior to metaphase.
Two further features were noticeable in the P-H3 staining patterns of the
borr mutant VNC cells. First, many of the rare anaphases detected at
stage 12 appeared abnormal, showing evidence of uneven segregation of
chromatin (Fig. 2D; see also
Figs 3,
5). Second, the P-H3 staining
intensity was reduced markedly, which is particularly noticeable during
metaphase, but also during telophase when P-H3 staining normally fades away
(Fig. 2D; see also
Fig. 4). These observations are
consistent with the profile shift of the mitotic stages in borr
mutant embryos (Fig. 2B,C), and
they underscore the notion that the first major defect during the mutant cell
cycle occurs prior to metaphase. A similar prometaphase block has been
reported for human Borealin (Gassmann et
al., 2004) and for other CPC components in Drosophila
cells (Adams et al., 2001
;
Giet and Glover, 2001
).
Borr colocalises with CPC components
In order to observe the subcellular localisation of Borr,
Drosophila DmD8 cells were transfected with a construct encoding
GFP-tagged full-length Borr. As expected, GFP-Borr is associated with
chromatin during prometaphase (Eggert et
al., 2004) (not shown), and is subsequently concentrated at the
central spindle midbody and at the cell cortex in the cleavage furrow during
telophase and cytokinesis (Fig.
3A-C,E,F). We shall refer to this pattern as `localisation to the
mitotic spindle'. Significantly, GFP-Borr colocalises with both endogenous
Aurora B and Incenp (Fig.
3B-G), in agreement with the results by Eggert et al.
(Eggert et al., 2004
), who
also observed co-localisation of Borr and Aurora B throughout mitosis. These
results are consistent with Borr being a CPC component, like its vertebrate
counterparts.
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|
Despite the strong reduction of the P-H3 levels in mitotic VNC cells of borr mutant embryos, these cells display only a slight undercondensation of their chromatin (Fig. 4I, arrow, compare with mitotic cell in F), although the degree of undercondensation is somewhat variable from cell to cell (Fig. 4I, and not shown). These results suggest that borr may not be essential for chromatin condensation.
borr is required for the localisation of Aurora B and Incenp to mitotic spindles
To examine the effects of borr loss on actively dividing
epithelial cells, we used FRT-FLP-mediated recombination
(Xu and Rubin, 1993) to
generate borr mutant clones in imaginal discs whose cells undergo
cell divisions throughout larval development. If borr mutant clones
are induced during early larval stages and examined in fully grown larval
discs, these clones are rare and are much smaller than the corresponding
wild-type twin spots, suggesting that a large fraction of the mutant cells die
(see below). Hoechst staining revealed that many of the surviving
borr mutant cells are large, with giant but well-formed nuclei that
appear healthy, and well integrated into the epithelial tissue (see Movie 1 in
the supplementary material).
We stained imaginal discs bearing borr mutant clones with antibodies against Incenp and Aurora B, to assess the effect of borr loss on these CPC components during mitosis. Wild-type cells in metaphase show characteristic well-ordered mitotic spindles, with distinct staining of Aurora B and Incenp at specific sites along condensed chromatin (Fig. 5A,F,B'-J'). By contrast, borr mutant cells invariably show abnormal mitotic spindles, including multipolar ones (Fig. 5A-J). Most of these mutant spindles do not show any chromatin-associated Incenp or Aurora B staining (Fig. 5C,H), although occasionally patches of Incenp staining can still be observed, but they do not seem to be associated with any of the spindle components (not shown). These staining patterns suggest that these CPC components fail to localise properly to mitotic spindles in the absence of borr (and their levels may also be reduced, though the low frequency of surviving borr mutant cells does not allow us to assess this quantitatively). Therefore, as in mammalian cells, the correct localisation of Incenp and Aurora B to mitotic spindles of dividing imaginal disc cells depends on Borr. This is further evidence that Borr is a CPC protein, and that it interacts functionally with other known CPC components.
|
Closer examination of the borr mutant cells revealed essentially two distinct phenotypes: large cells with giant well-formed nuclei, as described above (Fig. 6B,C, grey arrow), and cells that appear to be undergoing apoptosis. The clearest examples of the latter show compacted almost perfectly spherical nuclei that are found at the basal-most level of the disc epithelium, well separated from the healthy nuclei of the wing pouch (Fig. 6C, white arrow). We also observed borr mutant cells that may be at an earlier step in the apoptotic process: their nuclei are less compacted, and they are just beginning to drop basally within the epithelium (Fig. 6D, red arrow). Antibody staining against active caspase 3 confirmed that the borr mutant cells with compacted DNA are indeed undergoing apoptosis (Fig. 6E, white arrows), in contrast to the borr mutant cells that are well-integrated into the epithelium and display only background levels of active caspase 3 staining (Fig. 6E, grey arrow). Cells with low caspase staining can also be observed (Fig. 6E, red arrow): these show apparently fragmented but not yet compacted DNA, and may thus represent an intermediate stage similar to that shown in Fig. 6C.
These results, together with our observations in Borr-depleted embryos and tissue culture cells, suggest that borr mutant cells can undergo several consecutive abnormal mitoses, which results in large polyploid cells that eventually undergo apoptosis. Apoptotic cells appear to be cleared by basal extrusion from the epithelium.
Early borr mutant clones have non-autonomous effects on tissue architecture
To assess the consequences of Borr loss on the development of the imaginal
discs, we induced borr mutant clones in first or early second instar
larvae, and we examined the resulting adult flies. The most common defects in
these flies are abnormal legs and rough eyes (see Fig. S1 in the supplementary
material). In addition, they often show other striking defects in tissue
architecture, e.g. large wing nicks (Fig.
7A,B). In all these cases, a twin spot is apparent (e.g.
Fig. 7C, outlined in white),
but no mutant tissue is detectable. This indicates that, by the adult stage,
each of these early-induced borr mutant cells has undergone
apoptosis. The nature and extent of the adult defects also suggests that they
may be due partly to non-autonomous effects of the borr mutant clones
on their neighbouring wild-type tissue.
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We also observe clear non-autonomous effects of borr mutant cells
if we examine the expression of cut and senseless, two of
the ultimate target genes responding to the Wg morphogen in the marginal
region (Neumann and Cohen,
1996; Parker et al.,
2002
). For example, a single surviving giant borr mutant
cell expressing high levels of Cut can cause suppression of Cut and Senseless
expression in neighbouring wild-type cells
(Fig. 7H-J). A similarly
striking example is the introduction of a V shape into the patterns of Cut and
Senseless expression caused by a borr mutant clone
(Fig. 7K-M). The presence of a
large twin spot associated with this abnormality indicates that the causative
borr mutant clone arose early when the disc contained only a small
number of cells. Again, the borr mutant cells have disappeared in
this case, most likely through apoptosis (see above). The kink introduced into
the expression domains of both proteins appears to coincide with a
rearrangement of cells in this region (Fig.
7L, arrow). Indeed, it appears that a single giant borr
mutant cell (see Movie 1 in the supplementary material), in the process of
basal displacement, might drag along normal epithelial cells. Thus, apoptosis
and basal extrusion of a giant cell may exert sufficient disruption of the
epithelium to induce compensatory cell rearrangements aimed at repairing
epithelial integrity, which in the event compromise patterning.
Late borr mutant clones are viable, but affect external sensory organ development
If borr mutant clones are induced late (from the early third
larval instar onwards), the resulting flies are viable and display no gross
patterning defects. Indeed, analysis of marked clones and twin spots in adult
wings suggests that all borr mutant clones are fully viable, given
that they occupy roughly the same amount of territory as their twin spots
(Fig. 8A). This is somewhat
unexpected in the light of our results with earlier-induced clones whose
survival was severely compromised (Figs
6,
7) owing to abnormal mitoses
(Fig. 5). Indeed, the size of
the late-induced borr mutant clone in
Fig. 8A indicates that the
mutant cells have survived three or four consecutive (abnormal) mitoses
without entering the apoptotic pathway.
Closer examination of the flies bearing late-induced borr mutant clones revealed that their wing blades contain clusters of hairs (trichomes) surrounded by large clearings, rather than the usual regularly spaced single hairs (Fig. 8A). The number of hairs per cluster varies, with the largest cluster observed consisting of 12 hairs. All these hair clusters are produced by borr mutant cells (as judged by their trichome marker), so this phenotype is strictly cell-autonomous. The borr mutant clones do not significantly affect the planar polarity in the wing blade as mutant and surrounding wild-type hairs appear normally oriented (Fig. 8A).
Examination of borr mutant clones in pupal wing discs supports our
notion that all late-induced borr mutant clones occupy roughly the
same amount of territory as their twin spots, confirming that the mutant cells
are fully viable at this stage (Fig.
8B-D). In support of this, we did not observe any nuclei with
compacted DNA (that would indicate imminent apoptosis; see
Fig. 6E). As in the larval
discs, the surviving borr mutant cells in the pupal discs are much
larger than their neighbours, often with giant nuclei
(Fig. 8B, arrows), indicating a
high degree of ploidy. These giant borr mutant cells appear healthy
and are well integrated within the epithelial tissue
(Fig. 8B-D). Their large size
provides an explanation for the observed adult phenotype, and are consistent
with a single borr mutant cell producing multiple hairs: other
conditions that produce large cells for example, cdc2, UltA
or UltB mutant clones, or wounding result in similar
cell-autonomous clusters of trichomes, albeit in some cases with fewer hairs
per cluster (Adler et al.,
2000; Weigmann et al.,
1997
) (data not shown).
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Bristles are part of sensory organs, which are composed of four cells
the trichogen (bristle-producing cell), tormogen (socket-producing
cell), neuron and thecogen (sheath cell); these are the progeny of a single
sensory organ precursor cell produced by consecutive invariant lineage
divisions (Lai and Orgogozo,
2004). Evidently, loss of borr compromises the
lineage-generating divisions, and the single polyploid mutant cell seems to
develop invariably as a trichogen at the expense of the tormogen and,
possibly, of the other two sensory organ cells.
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Discussion |
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However, in borr mutant embryos, the condensation of the
chromosomes in mitotic VNC cells is barely affected, yet their P-H3 staining
is often strongly reduced (Fig.
2D; Fig. 4D,J).
This argues that H3 phosphorylation occurs in parallel or subsequent to
chromosome condensation, rather than driving it. Consistent with this, others
have also reported a lack of correlation between chromosome condensation and
P-H3 levels (Adams et al.,
2001), including Yu et al. (Yu et al., 2004) who have observed
normal levels of P-H3 on undercondensed chromosomes in greatwall
mutants of Drosophila. Indeed, it has been suggested that P-H3 may be
a sort of licensing factor, namely a mark placed on mitotic chromosomes to
indicate their readiness to undergo separation during the subsequent stages of
the cell cycle (Hans and Dimitrov,
2001
).
The striking reduction of the P-H3 levels in borr mutant embryonic
cells, and in Borr-depleted cultured Drosophila cells
(Eggert et al., 2004), is in
contrast to the situation in HeLa cells in which RNAi-mediated depletion of
Borealin did not affect their P-H3 levels
(Gassmann et al., 2004
). These
authors suggested that, in these cells, H3 phosphorylation may be mediated by
a Borealin-independent subcomplex of Aurora B and Incenp
(Gassmann et al., 2004
). More
work is required to determine whether this apparent discrepancy between human
and Drosophila Borealin function in mediating phosphorylation of H3
is genuine and cell type- or species-specific, or whether it is simply due to
methodological differences in the analyses.
borr loss causes polyploidy and delayed apoptosis in developing tissues
We have shown that borr is an essential gene in
Drosophila, and that borr loss results in multiple
successive defects during mitosis, including a reduction of P-H3, a severe
attenuation prior to metaphase, multipolar spindles and uneven chromosome
segregation. These defects may all reflect a function of Borr in the
attachment of kinetochores to the mitotic spindle, given that this process
often fails in Borealin-depleted HeLa cells
(Gassmann et al., 2004).
However, it is also possible that they reflect additional underlying
activities of the CPC during the progression of mitosis. However, all of these
mitotic defects are probably due, ultimately, to the observed failure of other
CPC components such as Aurora B to localise correctly to the mitotic spindle
(Fig. 5).
Multifocal spindles as observed in borr mutant cells
(Fig. 2D;
Fig. 3J; Fig. 5D,I) are expected to
cause aneuploidy, and may trigger checkpoint function. They should thus be
cleared from the developing tissue by apoptosis. Our observation of apoptotic
borr mutant cells in larval imaginal discs
(Fig. 6E) provide direct
support that cell death is often the ultimate consequence of borr
loss at the cellular level. However, borr mutant cells can also
clearly evade apoptosis, and can undergo several consecutive abnormal
divisions, given that the surviving (and dying) borr mutant cells in
imaginal disc epithelia are typically large, with giant nuclei and greatly
increased ploidy. Consistent with this, mammalian cells lacking CPC function
appear to be defective for their spindle attachment checkpoint and can thus
escape apoptosis (Lens and Medema,
2003; Yang et al.,
2004
). A similar defect in the checkpoint function of
borr mutant epithelial cells would explain why these cells can
survive multiple abnormal mitoses, instead of entering apoptosis in response
to the uneven chromosome segregation of a single abnormal mitosis. However,
the survival capacity of the mutant cells is clearly limited, and most of them
die ultimately except in late larval and pupal discs in which they
survive, possibly because of the slowing down of mitotic activity and/or
growth at these stages, which perhaps provides a more permissive environment
for the abnormally dividing borr mutant cells.
|
We propose that a similar situation arises in the case of borr
mutant imaginal disc cells: given that these can survive multiple abnormal
divisions, they may be doomed i.e. on a suspended apoptosis path
for an extended period of time and thus mimic some characteristics of
`undead' cells. Like the latter
(Perez-Garijo et al., 2004;
Ryoo et al., 2004
), doomed
borr mutant cells may induce a burst of compensatory responses in
their neighbours by stimulating the expression of extracellular signals such
as Wg. This is suggested by our analysis of larval discs bearing early-induced
mutant clones (Fig. 7), which
revealed examples of overexpressed Wg in giant borr mutant cells, and
also lateral expansion of Wg in twin spot areas whose associated borr
mutant cells have died. Doomed borr mutant cells may also affect
signalling by other pathways, e.g. the Notch pathway, given some of the
borr mutant phenotypes (Fig.
7H-J; see Fig. S1 in the supplementary material) (e.g.
Neumann and Cohen, 1996
), but
we have not examined this directly.
|
Polyploidy caused by borr loss may be instructive for bristle development
We have shown that borr loss also affects the lineage divisions of
the external sensory organs: our evidence from late-induced borr
mutant clones indicates that surviving giant borr mutant cells
develop large bristles without sockets
(Fig. 8). This phenotype
suggests a defect or block in the division of the pIIa precursor cell that
normally gives rise to the trichogen and tormogen
(Lai and Orgogozo, 2004). It
is less likely that the division of pI (the initial sensory organ precursor
cell) is blocked by borr loss in these instances, as evidence from
the analysis of embryonic sensory organs suggests that blockage of the first
lineage division should result in the precursor cell adopting a neural fate
(Hartenstein and Posakony,
1990
).
Why a borr mutant cell should adopt the bristle fate at the
expense of the socket fate is not immediately obvious. One possibility is that
the determining factor is its increased DNA content and large size. Notably,
the trichogen cells that produce the stout bristles of the wing margin undergo
at least one round of endoreplication during their differentiation
(Hartenstein and Posakony,
1989; Hartenstein and
Posakony, 1990
) (though in other external sensory organs the
tormogen does as well) (Lai and Orgogozo,
2004
). Thus, borr loss could mimic an aspect of normal
trichogen development, and could actively promote the acquisition of the
bristle fate. It is thus conceivable that endoreplication is instructive
during the process of sensory organ development.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/21/4777/DC1
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