Medical Research Council, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
* Author for correspondence (e-mail: agould{at}nimr.mrc.ac.uk)
Accepted 8 June 2005
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SUMMARY |
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Key words: Grainyhead, Neuroblasts, Apoptosis, Mitotic activity, Neural progenitors, Drosophila
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Introduction |
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Recent studies in the developing central nervous system (CNS) of
Drosophila have identified a striking example of an expression
sequence of transcription factors that modulates neural progenitor potential
with time (reviewed by Pearson and Doe,
2004). The progenitors involved, neuroblasts, share two properties
with mammalian neural stem cells: they are multipotent and they undergo
self-renewing divisions (reviewed by Doe
and Bowerman, 2001
; Jan and
Jan, 2001
; Chia and Yang,
2002
; Betschinger and Knoblich,
2004
). Each asymmetric neuroblast division generates a smaller
progenitor, termed a ganglion mother cell (GMC), which usually divides only
once to produce two neurons or glia. In the Drosophila embryo,
dividing neuroblasts express four transcription factors in a characteristic
sequence Hunchback
Kruppel
Pdm1
Castor, providing each GMC
with a temporal label (Kambadur et al.,
1998
; Brody and Odenwald,
2000
; Isshiki et al.,
2001
; Novotny et al.,
2002
; Pearson and Doe,
2003
). When combined with the anteroposterior and dorsoventral
positional information defining neuroblast type (reviewed by
Skeath and Thor, 2003
), this
temporal tag confers a unique cell identity to each GMC and its progeny.
Interestingly, there is also evidence that two members of the temporal
transcription factor series may regulate the overall number of divisions that
a neuroblast undergoes. For example, neuroblast 7-3 normally stops dividing in
the embryo after it has switched to Pdm1 (Nub FlyBase)-positive status
but it can be forced to produce a much larger lineage than normal by
persistently expressing either one of the `early' factors, Hunchback or
Kruppel (Isshiki et al.,
2001
).
The majority of neuroblasts appear to undergo the Pdm1 to Castor transition
in the embryo (Kambadur et al.,
1998), and go on to divide numerous times during postembryonic
stages, generating large numbers of neurons that will function in the CNS of
the adult fly (reviewed by Truman et al.,
1993
). This raises the issue of whether there are additional
temporal transcription factors expressed in neuroblasts during postembryonic
stages. In vivo and cell culture studies indicate that the expression of
another nuclear protein, Grainyhead (Grh), is first switched on in neuroblasts
towards the end of embryonic neurogenesis and thus may follow on from Castor
(Bray et al., 1989
;
Uv et al., 1997
;
Brody and Odenwald, 2000
). Grh
is a sequence-specific DNA-binding protein that defines a family of
transcription factors conserved from Drosophila to mammals
(Bray et al., 1989
; Dynlacht,
1989; Bray and Kafatos, 1991
;
Attardi and Tjian, 1993
;
Uv et al., 1994
;
Uv et al., 1997
; Wilanowski,
2002; Venkatesan et al.,
2003
). In Drosophila, Grh is required for several
non-neural developmental processes (Bray
and Kafatos, 1991
; Huang, 1995; Liaw, 1995;
Ostrowski et al., 2002
;
Hemphala et al., 2003
;
Lee and Adler, 2004
). Although
it is known that most neuroblasts continue to express a neural-specific
isoform of Grh during postembryonic stages
(Bray et al., 1989
;
Uv et al., 1997
;
Bello et al., 2003
), a specific
function in this late context has yet to be identified. Therefore, although
Grh has been proposed to be a late member of the temporal transcription factor
series (Brody and Odenwald,
2000
; Brody and Odenwald,
2002
), any supporting evidence that it temporally regulates
neuronal fate and/or number has been lacking.
The developmental stage at which a postembryonic neuroblast ceases dividing
is one of several crucial control points determining the overall number of
progeny that it generates (reviewed by
Maurange and Gould, 2005).
This endpoint is highly region specific, with neuroblast divisions stopping
two days later in the thorax than in the abdomen, correlating with average
lineage sizes of
100 cells for the former but only
5 cells for the
latter. Such anteroposterior differences strongly implicate the conserved
axial patterning system encoded by Hox homeodomain proteins
(Lewis, 1978
;
Wakimoto and Kaufman, 1981
;
McGinnis and Krumlauf, 1992
;
Mann and Morata, 2000
).
Although mechanisms linking Hox proteins to neuroblast activity have yet to be
identified in most regions, in the abdomen it is known that programmed cell
death plays a crucial role. In brief, neuroblasts stop dividing soon after
they upregulate Abdominal-A (AbdA), which activates one or more of the three
H99 proapoptotic genes grim, head involution defective
(hid; Wrinkled FlyBase) and reaper (rpr),
and thus triggers their death (White et
al., 1994
; Bello et al.,
2003
). Like other Hox proteins, AbdA acts in a highly
context-dependent manner (Lohmann and
McGinnis, 2002
). However, the factors that restrict the competence
to undergo this particular AbdA output, apoptosis, to neuroblasts rather than
neurons and to late rather than early neuroblasts have yet to be
identified.
Here, we analyse the roles of Grh during postembryonic stages and find that it regulates late region-specific patterns of neural proliferation. Characterisation of neural-specific mutations and marked neural clones lacking Grh activity demonstrate that Grh regulates the mitotic activity and apoptosis of neuroblasts. We show that the differential regulation of these neuroblast properties in the thorax and abdomen accounts for the very different proliferation endpoints in these two regions. Genetic analysis reveals that Grh plays two separable roles within abdominal neuroblasts, regulating the duration of the late-phase of AbdA expression and also the competence to respond appropriately to it. Epistasis tests show that the latter competence function of Grh in late-stage neuroblasts is essential for terminating neural proliferation in the abdomen. This study reveals, for the first time to our knowledge, a proliferation stop mechanism linking a late stage-specific neuroblast factor to the Hox axial patterning system.
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Materials and methods |
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Rearing and staging of larvae
Newly hatched larvae (0 hour) were collected during a 4- or 6-hour time
window and raised at 25°C at low density on standard cornmeal/yeast/agar
medium supplemented with live yeast. Hence, developmental stages referred to
as 48 hour, 63 hour, 72 hour and 96 hour correspond to larval age intervals of
46-50 hours, 60-66 hours, 70-74 hours and 94-98 hours, respectively. For the
104-hour time point, animals were morphologically selected at the white
prepupal stage. BrdU-incorporation studies were performed as described
(Truman and Bate, 1988), using
0.2 mg/ml BrdU for continuous labelling and 1 mg/ml for pulse labelling with
no live yeast supplementation of the medium. As the rate of development of
grh370/Df2RPcl7B larvae is more variable than
that of wild type, both age and morphological criteria were used for staging
(Bodenstein, 1994
). For MARCM
experiments, embryos of the appropriate genotype were collected on yeasted
grape juice-agar plates over a 4-hour window. Heat-shock induction of FLP was
at 37°C for 90 minutes, using larvae of 4-8 hours in age. For the
experiments using hs-abdA, grh370/Df2RPcl7B, a
1-hour heatshock at 37°C was used and the control larval genotypes were y
w; hs-abdA/CyO and
grh370/Df2RPcl7B.
Immunolabelling
Larval tissues were fixed and immunostained, as previously described
(Bello et al., 2003). For BrdU
staining, larval tissue was treated with 2N HCl as described
(Truman and Bate, 1988
), or
with 50 Units/ml of RQ1 RNase-free DNase (Promega) for 30 minutes at 37°C.
The primary antibodies used were: rabbit anti-ß-galactosidase (ßgal,
Cappel) 1:7000, mouse anti-ßgal (Promega) 1:1000, rabbit anti-Cas (gift
of W. F. Odenwald) 1:2000, rabbit anti-H3p (Upstate Biotechnology) 1:400, rat
anti-AbdA (gift of J. Casanova) 1:500, mouse anti-Ubx (FP.3.38, gift of R.
White) 1:20, mouse anti-Mira (Mab81, gift of F. Matsuzaki) 1:50, mouse
anti-BrdU (G3G4, Developmental Studies Hybridoma Bank) 1:200, mouse anti-Grh
(BF1, gift of S. Bray) 1:3. All fluorescent images were taken using a Leica
TCS SP scanning confocal microscope with a pinhole of 1 and represent
projections of multiple sections unless otherwise indicated in the figure
legend. Clone/lineage sizes were determined from confocal z stacks of
sections, spaced by
1 µm. Using ImageJ, cells were counted
section-by-section and marked to avoid double counting. Sample sizes, means
and standard deviations for all histograms are indicated in the text and
figure legends. For other data points, only illustrated by photographic
panels, n
4.
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Results |
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We first sought to identify all of the postembryonic neural cell types that
express Grh and thus have the potential to mediate grh functions
during late neurogenesis. Individual wild-type neuroblast clones were labelled
in the thorax from 0 to 96 hours using the MARCM technique
(Lee and Luo, 1999).
Consistent with previous studies (Uv et
al., 1997
; Bello et al.,
2003
), we observed that Grh protein is expressed by postembryonic
neuroblasts. Importantly, labelling single postembryonic clones shows clearly
that Grh is never detected in postmitotic adult-specific neurons, although,
surprisingly, we do observe it within the nucleus of new-born GMCs
(Fig. 1A-C). The expression
analysis thus indicates that the potential sites-of-action for Grh are within
neural progenitors rather than their postmitotic progeny.
Neural proliferation is regulated by Grh in a segment-specific manner
To determine the function of Grh during postembryonic neurogenesis, we made
use of the larval-viable grh370 mutation. This introduces
a premature stop codon into an alternatively spliced exon such that, although
it is not a null allele, it selectively inactivates the Grh protein isoforms
that are CNS specific (Uv et al.,
1997). We used larvae transheterozygous for
grh370 and a chromosomal deficiency for grh
(hereafter termed grh370 hemizygotes), and compared these
with balanced sibling controls. Phosphorylated Histone-H3 (H3p) labelling at
mid-L3 (78 hours) indicates that the frequency of M-phase within the CNS is
reduced within the thorax by
40% but, within the central abdomen, we
observe a very different effect, with mitoses continuing past 72 hours, the
time at which they would normally cease
(Fig. 2A-C and data not
shown).
|
In the abdominal CNS, BrdU-pulse labelling showed that cells in
grh370 hemizygotes remain actively engaged in the cell
cycle after the 72-hour time point when they would normally stop dividing
(Fig. 2D,E). This observation
is consistent with the previous H3p staining. We then labelled all neural
cells born during the postembryonic phase using continuous BrdU treatment from
0 hours onwards. At 96 hours, grh370 hemizygous larvae
display three abdominal rows of labelled lineages corresponding to the
positions of the ventromedial (vm), ventrolateral (vl) and dorsolateral (dl)
wild-type neuroblast lineages (Truman and
Bate, 1988). Combined cell counts of the vm and vl lineages show
that both are larger than the wild-type average count of four cells (72 hours:
n=19 lineages, mean=3.9, s.d.=1.5; 96 hours: n=18, mean=4.2,
s.d.=1.5), containing an average of 7.4 cells by 72 hours (n=14,
s.d.=2.4) and 13.7 cells by 96 hours (n=18, s.d.=2.4,
Fig. 3A-F; see Fig. S1 in the
supplementary material). Although part of this 3.5-fold increase in vm/vl
lineage size is likely to involve increased cell cycle speed, this is
difficult to quantitate as the corresponding neuroblasts in
grh370 hemizygotes also prematurely exit from the period
of non-proliferation (quiescence) that separates embryonic from postembryonic
neurogenesis (data not shown). Interestingly, grh370
hemizygotes also generate some supernumerary postembryonic lineages that
occupy ectopic positions outside the vm, vl and dl rows, often located close
to the midline (Fig. 3B, see
Discussion). Together, these abdominal labelling studies demonstrate that
grh is required for timely cessation of postembryonic neurogenesis.
In its absence, mitotic activity persists for at least 24 hours longer than
normal and, for the vm/vl lineages, we showed that this is associated with a
3.5-fold increase in cell number.
|
Grh maintains the mitotic activity of thoracic neuroblasts
We next identified which type of neural progenitor is affected by the
pro-proliferative function of Grh in the thorax. grh loss-of-function
MARCM clones were induced at 0 hours and, to ensure that no residual zygotic
Grh activity remained, we used the grhB37 null allele that
disrupts all isoforms of Grh (Bray and
Kafatos, 1991; Uv et al.,
1997
). Consistent with the previous analysis of
grh370 hemizygous larvae, thoracic
grhB37 clones display a reduction in clone size that is
already apparent at 72 hours (Fig.
4A,B). By 96 hours, the mean size of grhB37
clones is 28.6 cells (n=43 clones, s.d.=12.7), approximately half the
corresponding value of 57.3 cells for wild-type clones (n=61,
s.d.=15.7, Fig. 4C,D). At 72
hours,
40% of grhB37 mutant clones contain a single
large cell that expresses the neuroblast marker Miranda (Mira, data not
shown), but, whereas 37% of wild-type neuroblasts (n=56) are
H3p-positive, none of the grhB37 neuroblasts analysed
(n=51) were observed in M phase
(Fig. 4A,B). As this
grhB37 null phenotype is stronger than the reduced
neuroblast number and frequency of mitoses observed in 72-78 hour
grh370 larvae, it is likely that the
grh370 allele retains a low-level of neural Grh activity.
Importantly, the large but inactive neuroblast observed in
grhB37 clones at 72 hours is completely absent by 96 hours
(n=43 clones, Fig.
4C). Together, the grh370 and
grhB37 analyses indicate that the pro-proliferative role
of Grh in the thorax is mediated, at least in part, by maintaining the
neuroblast in a mitotically active state.
|
|
|
Abdominal neuroblasts remain Grh-positive until their last asymmetric division
We next addressed the mechanism-of-action of Grh in abdominal neural
lineages. The previous finding that Grh may promote survival of neuroblasts in
the thorax, raises the possibility that neuroblast death in the abdomen might
involve a transition from Grh-positive to Grh-negative status at mid-L3. In
apparent support of this notion, we previously observed that Grh expression
ceases to be detectable in all three abdominal lineages just prior to the
appearance of TUNEL labelling (Bello et
al., 2003). To resolve whether abdominal neuroblasts switch off
Grh expression prior to apoptosis, we generated abdominal neuroblast clones
deficient for H99 proapoptotic genes. These continue to divide for at
least 24 hours after they would normally have died
(Bello et al., 2003
) and we now
find that, during this `extra time', they remain Grh-positive
(Fig. 7A). This indicates that
levels of Grh protein become downregulated only as a consequence of activation
of the cell death pathway. Importantly, these results rule out the possibility
that a late switch from Grh-positive to Grh-negative status provides the
trigger for initiating H99 proapoptotic gene activity and subsequent
neuroblast apoptosis.
|
Grh is required for maintenance, but not initial activation, of AbdA neuroblast expression
Persistence of mitotically active abdominal neuroblasts in
grh370 hemizygotes after 72 hours not only mimics the
H99 phenotype but also that associated with loss of function of
abdA (Bello et al.,
2003). This raises the possibility that grh might
regulate the L3 phase of AbdA expression that is required to trigger
neuroblast apoptosis (Bello et al.,
2003
). We first determined the temporal relationship between
wild-type AbdA expression and the onset of neuroblast apoptosis. Abdominal
neuroblasts become TUNEL-positive at
72 hours
(Bello et al., 2003
), and, at
this stage, we find that they still continue to express AbdA despite Mira
expression now being weak and punctate
(Fig. 7D). This abnormal Mira
pattern suggests that AbdA expression continues until after the first stages
of neuroblast apoptosis have been initiated. We estimate that AbdA expression
is maintained for
9 hours after the previously described expression at 63
hours (Bello et al., 2003
),
but, given the method of larval staging (see Materials and methods), the
precise duration could lie anywhere within a 4- to 14-hour window. Blocking
cell death in H99 clones further supports the notion that there is no
downregulation of AbdA prior to apoptosis, as, in this artificial context,
many abdominal neuroblasts continue expressing AbdA at 96 hours
(Fig. 7E). AbdA expression is
also observed in a subset of neurons positioned close to the neuroblast,
consistent with expression being maintained in the late subset of progeny
generated after the neuroblast first becomes AbdA positive. These results
reveal that wild-type AbdA expression, once switched on in neuroblasts, is not
downregulated prior to the onset of apoptosis.
We next analysed the time course of AbdA expression in grh370 hemizygotes. Mira/AbdA double labelling reveals that AbdA is expressed in interphase neuroblasts at 63 hours, as normal (Fig. 7F). Also similar to in wild type, AbdA is expressed in M-phase (H3p-positive) neuroblasts at this time (Fig. 7G). However, whereas AbdA expression is maintained until 96 hours in H99 neuroblasts, this is not the case for grh370 hemizygous larvae at 96 hours (Fig. 7H). We conclude that grh is not essential for initiating the late larval phase of AbdA expression in neuroblasts, but that it is required to maintain it.
Grh provides the competence for neuroblasts to undergo AbdA-dependent apoptosis
To test whether rescuing the AbdA maintenance deficit in
grh370 hemizygotes would be sufficient to restore
neuroblast apoptosis, we made use of an hs-abdA transgene. In the
first set of experiments, hs-abdA activity was transiently induced at
the 63-hour time point to prolong the endogenous phase of AbdA expression in
grh370 hemizygotes. However, we failed to observe any
abdominal neuroblast apoptosis (data not shown). In a second series of
experiments, hs-abdA expression was induced at 48 hours, a protocol
known to provide sufficient AbdA activity to stop all abdominal neuroblast
divisions prematurely (Bello et al.,
2003). In a wild-type background, we find that this
early-induction protocol leads to sustained ectopic AbdA expression and the
associated elimination of all Mira-positive neuroblasts by 72 hours
(Fig. 8A,B). In sharp contrast,
neuroblasts in a grh370 hemizygous background are
resistant to the induction of apoptosis by hs-abdA
(Fig. 8C). Strikingly, they
continue to express AbdA for at least 24 hours after heat-shock induction, yet
are not eliminated by programmed cell death
(Fig. 8D). Together, the
abdominal experiments demonstrate that Grh is a terminal neuroblast factor
that restricts postembryonic lineage size by promoting the proapoptotic
subfunction of AbdA in two distinct ways. First, it regulates the duration of
the AbdA burst, and, second, it installs the competence to undergo an
apoptotic response to it. The grh370, hs-abdA epistasis
tests clearly separate these two Grh activities by showing that, although AbdA
maintenance may be necessary, it is not sufficient and, importantly, that AbdA
competence is essential for neuroblast apoptosis.
|
![]() |
Discussion |
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|
We provided four lines of evidence that the underlying basis for premature
cessation of thoracic proliferation in grh mutant clones is reduced
mitotic activity of the neuroblast, most probably followed by Hox-independent
apoptosis. First, although grh mutant neuroblasts are present at 72
hours they are mitotically inactive. Second, by 96 hours, no recognisable
grh mutant neuroblasts remain. Third, inhibiting cell-death effector
caspases by misexpressing P35 rescues the loss of grh mutant
neuroblasts. And fourth, although misexpression of Hox proteins in thoracic
neuroblasts induces apoptosis (Bello et
al., 2003), Ubx, the resident Hox protein of the posterior thorax,
remains excluded from grh mutant neuroblasts at 72 hours.
Importantly, the role of Grh in maintaining mitotically-active neuroblasts is
not a general `housekeeping' function but is specific for their age. Thus,
wild-type neuroblasts in the early embryo are Grh-negative yet viable and
actively dividing. This observation suggests that the late switch to
Grh-dependency involves additional factors. These could be intrinsic to the
neuroblast or provided by a glial-cell niche (reviewed by
Maurange and Gould, 2005
).
Consistent with the niche idea, neuroblast divisions within the postembryonic
brain require DE-cadherin-dependent interactions between glia and neural cells
(Dumstrei et al., 2003
).
Grh is required to stop abdominal neuroblast divisions
In the central abdomen, we previously found that, at 72 hours, many
neuroblasts downregulate Grh and become TUNEL positive
(Bello et al., 2003). In the
present study, when the neuroblast death pathway was blocked in H99
clones, Grh expression continued in mitotically active neuroblasts long after
the 72-hour stage. This indicates that abdominal neuroblasts remain in
Grh-positive mode during their final division and that Grh is only
downregulated after the onset of apoptosis. Moreover, we showed that loss of
Grh activity leads to the failure of neuroblasts to undergo apoptosis. As
these persistent neuroblasts not only survive but also remain actively engaged
in the cell cycle, they generate a 3.5-fold excess of cells within each
abdominal neuroblast lineage. Together, these findings identify Grh as a
terminal neuroblast factor that is an essential component of the
abdomen-specific `stop' programme.
Grh installs the competence for AbdA-dependent neuroblast apoptosis
Two different interactions with the Hox gene AbdA underlie the dramatic
reversal of Grh function from pro-proliferative in the thorax to
anti-proliferative in the abdomen. First, Grh acts upstream of AbdA to
maintain its late phase of expression, and, second, it functions in parallel
with AbdA to activate apoptosis. Although the functional significance of
grh-dependent AbdA maintenance is not clear, it may be that efficient
neuroblast apoptosis requires AbdA levels to remain high for a significant
proportion of the interval separating initial AbdA upregulation and the
TUNEL-positive stage. More definitively, we used epistasis tests to show that
Grh, acting in parallel with AbdA activity, is essential for abdominal
neuroblast apoptosis. Thus, when the AbdA-maintenance deficit was rescued
using hs-AbdA, neuroblast death remained blocked. As AbdA is not
required to activate neuroblast Grh expression, Grh and AbdA must work in
parallel to activate apoptosis. Together with the finding that AbdA is
required to activate H99 gene activity
(Bello et al., 2003), our study
demonstrates that inputs from Grh and AbdA are both essential to activate
proapoptotic genes and thus trigger neuroblast apoptosis. Whereas the late
upregulation of AbdA provides a timing cue to schedule the onset of apoptosis,
the much broader phase of Grh expression defines the period of neuroblast
competence to respond appropriately to it.
The restricted temporal pattern of Grh expression ensures that competence
to undergo AbdA-dependent apoptosis, rather than some other AbdA-dependent
output, is only installed at late stages. Consistent with this, neuroblasts in
the early embryo that are AbdA positive but Grh negative go on to generate
substantial embryonic lineages. Low levels of expression from UAS-grh
transgenes (data not shown) make it difficult to test whether Grh is
sufficient to confer apoptotic competence to these early embryonic
neuroblasts. In the late embryo, however, neuroblasts have already switched on
Grh, and, within the central abdomen, all but three undergo
abdA-dependent death (Truman and
Bate, 1988; Bray et al.,
1989
; White et al.,
1994
; Prokop et al.,
1998
). Our observation that reduced neural grh function
leads to supernumerary postembryonic neuroblasts positioned outside the vm, vl
and dl rows, raises the possibility that Grh is required for all
developmentally programmed neuroblast apoptosis.
![]() |
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/132/17/3835/DC1
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