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First published online 23 October 2008
doi: 10.1242/dev.025189
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1 Center for Frontier Research, National Institute of Genetics, 1111 Yata,
Mishima, Shizuoka 411-8540, Japan.
2 Department of Genetics, SOKENDAI, 1111 Yata, Mishima, Shizuoka 411-8540,
Japan.
Author for correspondence (e-mail:
tisshiki{at}lab.nig.ac.jp)
Accepted 3 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Drosophila, Nab, Neuroblast, Quiescence, Temporal
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Zhao et al., 2008). Quiescent
adult neural stem cells in the mouse forebrain can generate multiple cell
types over time, in response to niche-derived sonic hedgehog activity
(Ahn and Joyner, 2005).
Although such extrinsic controls of stem cell proliferation have been
reported, the mechanisms by which neural stem cells/progenitors enter and exit
quiescence remain largely unknown.
During development of Drosophila CNS, neural stem cells
(neuroblasts; NBs) proliferate in the embryo to generate the neurons that
drive larval behaviors. Once embryogenesis is completed, most abdominal NBs
are eliminated through programmed cell death
(Abrams et al., 1993;
Peterson et al., 2002;
White et al., 1994), whereas
most of the cephalic and thoracic NBs enter mitotic quiescence at the
embryo-larval transition. After a long period of quiescence in the larval
stage, the quiescent NBs that transformed into larval types receive extrinsic
mitogenic signals, such as Hedgehog, in a niche-dependent fashion, and resume
cell division to produce the huge number of neurons needed to control the
highly developed cephalic and thoracic segments of the adult fly
(Barrett et al., 2008;
Britton and Edgar, 1998;
Datta, 1995;
Ebens et al., 1993;
Park et al., 2003;
Prokop and Technau, 1991;
Truman and Bate, 1988).
One advantage of studying neural stem cell quiescence in
Drosophila is the ability to trace identified NBs and their lineages.
NBs divide asymmetrically to bud off a series of daughter cells (ganglion
mother cells; GMCs), each of which typically makes two postmitotic neurons.
Each Drosophila hemisegment contains a set of 
30 NBs that can be
individually identified and named according to their position within the
hemisegment (Broadus et al.,
1995; Doe, 1992).
Each NB has a unique cell lineage (Bossing
et al., 1996; Schmid et al.,
1999; Schmidt et al.,
1997). NBs with identical positions in a segment, but located in
different segments, share many gene expression and developmental features but
generate slightly different segment-specific neuronal clones. For example,
abdominal NB3-3 (NB3-3A) generates 11 Even-skipped (Eve)-positive neurons,
whereas the thoracic NB3-3 (NB3-3T) generates just six Eve-positive neurons,
yet the axon projections of these interneurons are virtually indistinguishable
(Schmid et al., 1999;
Schmidt et al., 1997).
A single NB can produce diverse neuronal cell types in an invariant order
by changing its property over time. In the earliest stages of lineage
development, NBs sequentially express a series of transcription factors:
Hunchback (Hb), Seven-up (Svp), Krüppel (Kr), Pdm1/Pdm2 (Pdm) and Castor
(Cas), which control the temporal change of NBs and temporal cell fate
specification of their progeny
(Grosskortenhaus et al., 2006;
Isshiki et al., 2001;
Kambadur et al., 1998;
Kanai et al., 2005;
Novotny et al., 2002;
Pearson and Doe, 2003). As
these transcription factors mutually regulate each other's expression in a
network of feed-forward loops, the temporal change of NBs proceeds in a
cell-intrinsic manner (Grosskortenhaus et
al., 2005).
To ensure that a sufficient variety of neurons are generated before quiescence, NB entry into quiescence must be temporally regulated. However, the timing of entry into quiescence and whether it is controlled extrinsically or intrinsically in NBs is currently unknown. How NBs re-adopt the appropriate cell lineage upon re-entry to the cell cycle after quiescence is also unclear. Such issues have been difficult to address because markers and tools for analyzing the later stages of embryonic NB lineages have been lacking.
In this study, we first searched for transcription factors that are expressed temporally at the later stages of most, if not all, NB lineages, and characterized the model NB lineage NB3-3. This new model lineage allowed us to observe reproducibly the entire process of lineage development from embryogenesis to larval stages at the level of single cell divisions. We report that the sequence of temporal changes in NBs, defined by the switching of temporal transcription factor expression, runs continuously from embryonic to larval stages, and that quiescence suspends - but does not disrupt - the switching of temporal gene expression. Moreover, we report that NB entry into quiescence is regulated intrinsically and cooperatively by two independent controls: spatial control by Hox genes and temporal control by known temporal transcription factors in conjunction with the transcription co-factor Nab.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), worniu-GAL4
(Lee et al., 2006),
pdmED773 and UAS-pdm
(Grosskortenhaus et al.,
2006), nabSH143 and UAS-nab
(Terriente Felix et al.,
2007), nabG26
(Clements et al., 2003),
sqzie (Allan et al.,
2003), sqz02102, Antp25, UAS-abd-A,
UAS-mCD8GFP, UAS-LacZ and scabrous-GAL4 (Bloomington Drosophila
Stock Center). The cas20-1 null mutant line was generated
by imprecise P-element excision from the 1530 line
(Cui and Doe, 1992).
UAS-sqz was generated by inserting the cDNA fragment from RE47124
(BDGP) into pUAST. eagle-GAL427-1 was generated by
inserting an eagle enhancer 2.0 kb fragment into pGAWB. To visualize
NB3-3 and NB2-4 lineage clones, we crossed either
eagle-GAL427-1 or MZ360-eagle-GAL4 to
UAS-mCD8GFP or UAS-LacZ. To misexpress UAS-nab, UAS-pdm,
UAS-sqz or UAS-abd-A, we crossed worniu-GAL4 or
scabrous-GAL4 to the UAS lines at 29°C.
Antibodies and immunostaining
Antibody staining for embryos or larval CNS was performed according to
standard protocols. Primary antibodies used were mouse and rat anti-BrdU
(1:200, BD Biosciences, Abcam), rabbit and mouse anti-GFP (1:500, Invitrogen,
1:200, Roche), mouse anti-b-galactosidase (1:300, Promega), guinea pig
anti-Kr, guinea pig anti-Castor, guinea pig and rat anti-DmLin29, rat
anti-Seven-up, guinea pig anti-Nab, rabbit anti-Even-skipped, guinea pig
anti-Miranda (1:500), rat anti-Squeeze (1:750), rat anti-β-galactosidase
(1:1000), rabbit and mouse anti-Miranda, (1:2000, 1:50)
(Matsuzaki et al., 1998;
Ohshiro et al., 2000), rabbit
anti-Grainyhead (1:300) (Bello et al.,
2006), rabbit anti-Nab (1:500)
(Terriente Felix et al.,
2007), rat anti-Pdm2 (1:10)
(Grosskortenhaus et al.,
2006), rabbit anti-Eagle (1:1000)
(Karcavich and Doe, 2005),
rabbit anti-Castor (1:2000) (Kambadur et
al., 1998) and mouse anti-Even-skipped monoclonal 2B8 (1:50,
DSHB). Detailed information about primary antibodies can be supplied upon
request. Images were obtained using Zeiss LSM510 META or LSM5 LIVE confocal
microscopes. Images and counted data were from T3 or A2-A6 segments unless
otherwise noted.
Microarray analysis
We isolated the CNS from cas20-1 homozygous and
heterozygous embryos 12.5-13.5 hours after egg laying (AEL) (stages late 15 to
early 16) in PBS. We used Drosophila Genome 2.0 Array (Affymetrix).
Total RNA was extracted from the CNS and purified. Experiments were performed
using 1 µg total RNA following the Affymetrix manual.
BrdU labeling
A 1-hour collection of embryos was dechorionated, permeabilized in octane
for 4 minutes, and incubated in BrdU solution (BrdU 1 mg/ml in Schneider's
medium) for 20 minutes. Immediately after labeling, embryos were fixed. As for
larvae, a 5-hour collection of newly hatched larvae was transferred to
BrdU-containing medium (BrdU 0.1 mg/ml), and grown for the specified time. To
prepare 0 to 6 hour after larval hatching (ALH) larvae, each isolated CNS was
incubated in BrdU-containing Schneider's medium (BrdU 1 mg/ml) for 20
minutes.
In vitro NB culture
In vitro NB culture was performed according to Grosskortenhaus et al.
(Grosskortenhaus et al.,
2005), except that NB cultures were made from 5 to 7 hour AEL
embryos.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Higashijima et al., 1996). To
examine when NB3-3 stops entering a new S phase, we observed the incorporation
of BrdU into NB3-3 during transient labeling
(Fig. 1). When we labeled
embryos at stages late 13 to 14 (10-11 hours AEL), 76% of thoracic NB3-3
(NB3-3T) and 92% of abdominal NB3-3 (NB3-3A) incorporated BrdU
(Fig. 1A-C). Labeling just 1
hour later [stages late 14 to 15 (11-12 hours AEL)] resulted in a significant
reduction of BrdU incorporation in NB3-3T cells, and after 12-hour AEL (stage
15) we rarely observed BrdU-positive NB3-3T
(Fig. 1A,C). Thus, NB3-3T
enters quiescence at stages late 14 to 15. By contrast, more than 70% of
NB3-3A incorporated BrdU even at stage 16
(Fig. 1B,C), showing that
NB3-3A proliferates. Despite the different number of GMCs made in each
lineage, both lineages generate neurons with similar axonal projections
(Schmid et al., 1999;
Schmidt et al., 1997).
We also found that, upon entering quiescence, NB3-3T underwent marked
changes in shape. We used Miranda, a cargo-binding protein involved in NB
asymmetric cell division (Ikeshima-Kataoka
et al., 1997), as a marker for visualizing NB shape. Whereas
mitotic NBs were round, NB3-3T began to extend a thin protrusion around the
time it entered quiescence (stages late 14 to 15)
(Fig. 1A). This non-spherical
shape persisted until at least stage 16, well after NB3-3T entered quiescence
(Fig. 1A). At stage 16, many
such elongated Miranda-positive cells were detected in thoracic, but not
abdominal, segments. These cells never incorporated BrdU (data not shown).
These observations strongly suggest that the elongated Miranda-positive cells
are indeed quiescent NBs. Individual NBs underwent this shape change in a
stereotypical order, rather than simultaneously at a specific time of
development (Fig. 1E).
Furthermore, isolated NBs cultured in vitro often exhibited quiescent NB-like
features (see Fig. S1 in the supplementary material).
Next, we evaluated the timing of NB3-3T exit from quiescence in larvae by continuously feeding larvae with BrdU after hatching. NB3-3T could first be labeled with BrdU at late first-instar to early second-instar stages (20-25 hours ALH) and assumed a round shape at this point (Fig. 1D). We conclude that NB3-3T exits quiescence by late first-instar to early second-instar.
These observations revealed the period of quiescence in the model NB (NB3-3T), and that quiescent NBs have distinct morphological features that may reflect their unique cell physiology. Furthermore, the fact that NBs change shape asynchronously and in vitro suggests that an intrinsic property in each NB controls the timing of quiescence.
Relationship of sequential expression of temporal transcription factors and the timing of quiescence in NB3-3T and NB3-3A
NBs sequentially express a series of temporal identity factors that
intrinsically regulate sequential neuronal identity
(Grosskortenhaus et al., 2006;
Isshiki et al., 2001;
Kanai et al., 2005). To
determine whether any correlation exists between sequential expression of
these factors and quiescence, we analyzed the timing of expression of the
known temporal transcription factors Hb, Kr, Pdm, Cas, Svp and Grainyhead
(Grh) in NB3-3T (Almeida and Bray,
2005; Cenci and Gould,
2005; Maurange et al.,
2008) (Fig. 2).
NB3-3T was not Hb positive at birth but sequentially expressed Kr, Pdm, Cas,
Grh and Grh/Cas (Fig. 2A,F).
Two distinct windows of Cas expression have previously been noted
(Cleary and Doe, 2006) but
never analyzed in an identified NB. In NB3-3T, the second Cas expression phase
began at stage 15, when NB3-3T started to change shape (i.e. enter
quiescence), and Cas levels were maintained in the quiescent NB
(Fig. 2A,F). Within the NB3-3T
lineage, the quiescent NB was the only Cas-positive cell at late first-instar
to early second-instar (20-25 hours ALH)
(Fig. 2C,D); at this point the
NB had increased Cas levels, returned to a round shape, began to incorporate
BrdU and exited quiescence (Fig.
2C,D; Fig. 1D). By
early- to mid-second instar (30-35 hours ALH), Cas expression decreased in
NB3-3T, and the NB expressed the temporal factor Seven-up (Svp), which
transiently overlapped with the Cas expression window
(Fig. 2D). Similar to NB3-3T,
most thoracic NBs showed transient late Svp expression in larval stages but
not in embryos (see Fig. S2 in the supplementary material).
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We conclude that the model NB3-3 lineage allows us to track the sequence of temporal identity gene expression in abdominal segments (where it occurred rapidly and was completed during embryogenesis) and in thoracic segments (where it is interrupted by a period of quiescence and completed only in larval stages) (Fig. 2E,F). The fact that a period of quiescence delays but does not disrupt the sequence of temporal identity factor switching suggests that the NB can `remember' its temporal identity during quiescence.
Hox proteins regulate segment-specific entry into NB quiescence
In embryos, NB3-3T becomes quiescent, whereas NB3-3A continuously
proliferates. Among the Hox proteins, Antennapedia (Antp) and Abdominal-A
(Abd-A) are expressed in NBs in thoracic T1-T3 segments and in abdominal A1-A7
segments, respectively (Carroll et al.,
1986; Carroll et al.,
1988; Hirth et al.,
1998; Prokop et al.,
1998). We therefore investigated whether these two Hox proteins
are involved in the regulation of quiescence in NB3-3A and NB3-3T
(Fig. 3).
In Antp mutant embryos, NB3-3T incorporated BrdU until late in embryogenesis, and maintained a round shape (Fig. 3J). Thus, Antp is required for NB3-3T to enter quiescence during mid-embryogenesis. Moreover, Antp mutants occasionally showed precocious Svp expression in NB3-3T during embryogenesis (13%, n=23) (Fig. 3A,C,G). Corresponding to the change in mitotic behavior, Antp mutant NB3-3T generated an increased number of GMCs and neurons. Wild-type NB3-3T in the T3 segment typically produced seven GMCs that generated six Eve-positive lateral interneurons (EL neurons) (Fig. 2F; Fig. 3B), whereas Antp mutants generated as many as nine EL neurons (average of 7.8 cells, n=41) (Fig. 3D). This is an increase in number but is still fewer than the number of EL neurons produced by NB3-3A: at least 12 GMCs generated 11 EL neurons (Fig. 2F, Fig. 3H; see Fig. S3 in the supplementary material) (see also sections below).
We further characterized the NB3-3 lineage using a marker: the protein encoded by the gene CG2052. This protein is a Krüppel-type zinc-finger protein related to Caenorhabditis elegans LIN-29, which we named DmLin29. DmLin29 was expressed in most or all late-born neurons (data not shown). We thus consider DmLin29 as a marker for late-born neurons. In the NB3-3T lineage, DmLin29 was not expressed in any of the six EL neurons but was expressed after quiescence in larval stages (Fig. 2F, Fig. 3B,I; see Fig. S4 in the supplementary material). Not surprisingly, Antp mutants that had a delay in NB3-3T quiescence showed up to three DmLin29-positive EL neurons (average of 2.3 cells, n=18), born after the first six DmLin29-negative EL neurons (Fig. 3D). This was similar to the NB3-3A lineage, which does not enter quiescence at mid-embryogenesis and generated five DmLin29-positive late-born EL neurons during embryogenesis (Fig. 2F, Fig. 3H,I; see Fig. S3 in the supplementary material). We can thus conclude that in Antp mutants NB3-3T fails to enter quiescence during mid-embryogenesis, leading to the precocious expression of Svp and DmLin29 during embryogenesis.
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; Isshiki et al.,
2001; Novotny et al.,
2002). Thus, we tested whether any of these factors also regulate
the timing of entry into quiescence (Fig.
4). In pdm mutants, many thoracic NBs prematurely changed
to an elongated cell shape and appeared to enter quiescence
(Fig. 4C,G). By contrast, in
cas mutants most thoracic NBs retained the round shape characteristic
of proliferating NBs (Fig.
4B,F). When we assayed the model NB3-3 lineage, we found that
pdm mutants showed precocious quiescence of NB3-3T and generated
fewer thoracic EL neurons (average of 4.2 cells, n=20)
(Fig. 4K,K',W,Z).
pdm mutant NB3-3T lost its ability to incorporate BrdU 3 hours
earlier than in the wild type (Fig.
4Y, compare with wild type in
Fig. 1C). By contrast,
cas mutants showed prolonged proliferation of NB3-3T and generated
extra thoracic EL neurons (average of 6.8 cells, n=15)
(Fig. 4J,J',V,Z). We
conclude that Pdm delays quiescence in NBs, whereas Cas induces
quiescence.
How do Pdm and Cas oppositely regulate quiescence? cas mutants
show prolonged Pdm expression in NBs
(Grosskortenhaus et al.,
2006), suggesting that the cas mutant phenotype (delayed
quiescence) may simply be due to prolonged Pdm expression. To test this
hypothesis, we assayed pdm cas double mutants. In pdm cas
double mutants, we found that most NBs showed precocious quiescence similar to
pdm mutants (Fig.
4D,H,L,L',S,T,X). Furthermore, forced expression of Pdm
delayed quiescence, regardless of Cas (see Fig. S6 in the supplementary
material). Pdm thus acts downstream of Cas to inhibit NB quiescence. Because
Pdm expression normally disappeared 3-4 hours before NB3-3T entered
quiescence, Pdm probably controls expression of other genes to regulate the
timing.
nab is a Pdm-regulated gene required for triggering NB quiescence
To identify unknown Pdm-regulated factors involved in triggering NB
quiescence, we performed microarray analyses to compare wild-type embryos with
cas mutant embryos, in which entry into quiescence is inhibited due
to prolonged Pdm. We selected genes that show significant differences in
expression at the time when most NBs enter quiescence. Genes that are
upregulated in cas mutant (Pdm overexpression) embryos might be
involved in inhibiting quiescence, and conversely downregulated genes are good
candidates for promoting quiescence. Among the candidate genes that showed
severe reductions at the transcript level in cas mutant embryos was
nab, which encodes a transcriptional cofactor of the NGFI-A-binding
protein family (Clements et al.,
2003; Terriente Felix et al.,
2007). Although it has previously been reported that Nab is
temporally expressed in many NB lineages, and that cas mutants lose
Nab expression from all but a medial NB
(Clements et al., 2003), Nab
function in NBs has not been investigated.
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To determine whether Nab is actually involved in triggering NB quiescence, we assayed the NB3-3T lineage using BrdU pulse-labeling (Fig. 6). In nab mutant embryos, NB3-3T continued to incorporate BrdU well beyond its normal time of quiescence (Fig. 6A); consequently, NB3-3T generated additional EL neurons (as many as eight cells, an average of 7.1 cells, n=23) (Fig. 7B). This effect was not limited to NB3-3T; nab mutant first instar larvae showed delayed quiescence and prolonged proliferation of many thoracic NBs, based on their round morphology and ability to incorporate BrdU, which was not seen in wild type (Fig. 6B). We conclude that nab is a Pdm-regulated gene that is required for triggering NB entry into quiescence (Fig. 6E).
Squeeze, a putative Nab partner, is required for timely triggering of NB quiescence
Nab function requires the presence of a co-factor. In Drosophila,
two transcription factors, Squeeze (Sqz) and Rotund, can associate with Nab
(Terriente Felix et al.,
2007). Because sqz was transcribed in a temporal pattern
within the CNS, we focused our attention on Sqz. As with Nab, Sqz protein
level was controlled by temporal factors (see Fig. S7 in the supplementary
material). In NB3-3T, we detected Sqz protein slightly prior to Nab
(Fig. 2F;
Fig. 5A), with Sqz detected in
the fifth-born EL neuron (Fig.
2F; Fig. 5A,C,E),
one cell division prior to Nab (Nab is detected in the sixth-born EL neuron;
see above).
Consistent with the idea that Nab acts as a co-factor of Sqz, Sqz and Nab were co-expressed at high levels when NB3-3T entered quiescence (Fig. 2F; Fig. 5A). Sqz expression declined over time in proliferating larval stages. By contrast, Nab expression continued.
To investigate Sqz function, we examined two genotypes: a null sqzie mutant and a strong hypomorphic sqz mutant (in which Sqz protein can barely be detected) (Figs 6 and 7). We found that the loss or reduction of Sqz function led to a delay in NB3-3T entering quiescence, and a corresponding increase in the number of EL neurons (as many as eight cells, an average of 7.1 n=31) (Fig. 6A,E; Fig. 7D,E). Despite the delay, NB3-3T eventually entered quiescence, as we found no proliferating NBs in the thorax of newly hatched sqz mutant larvae (Fig. 6B). As expected, the extra seventh-born EL neurons in sqz mutants were occasionally DmLin29 positive (Fig. 7D,E). Sqz is unlikely to regulate NB quiescence by controlling the expression of Nab, because sqz mutants usually showed normal timing of Nab expression in the sixth-born EL neuron (79%, n=29, data not shown).
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Nab and other downstream mediators of Pdm cooperate to regulate NB quiescence
Despite the ability of Nab/Sqz to bypass the requirement of Cas to induce
NB quiescence, several lines of evidence suggest that additional factors
downstream of Pdm are involved in determining the timing of entry into
quiescence. First, precocious expression of Nab alone and precocious
co-expression of Nab and either Sqz or Rotund were all insufficient to
accelerate the entry into quiescence (Fig.
6C and data not shown). Second, when co-misexpression of Nab and
Sqz rescued the NB quiescence phenotype of the cas mutant, this
manipulation also rescued the prolongation of Pdm expression that occurs in
cas mutant (Fig. 6F;
see Fig. S8 in the supplementary material). This was unexpected because the
loss of function of either nab or sqz does not affect Pdm
expression (see Fig. S9 in the supplementary material). Thus, co-misexpression
of Nab and Sqz might have resulted in proper expression of other genes
downstream of Pdm, through an artificial downregulation of Pdm. Taken
together, these results suggest that the pathway that controls NB quiescence
branches at Pdm, and that Nab/Sqz and another yet unidentified factor
cooperate to regulate the timing of quiescence (see Fig. S10 in the
supplementary material).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Spatial regulation of NB entry into quiescence
In the Antp mutant and following ectopic expression of Abd-A there
was a lack of NB quiescence, and consequently we observed what appeared to be
a precocious generation of larval neurons during embryogenesis. This strongly
supports the notion that temporal changes in NBs actually continue in sequence
before and after quiescence, i.e. through embryogenesis and larval stages, and
in the absence of quiescence the changes occur precociously. In addition, this
indicates that spatial and temporal factors control NB quiescence through
independent routes (see Fig. S10 in the supplementary material).
Antp mutants did not exhibit NB3-3T quiescence in all thoracic
T1-T3 segments. In Antp mutants, epidermis in T2 and T3 segments
transform into that in the T1 segment, and some thoracic NB lineages retain
thoracic-specific features (Berger et al.,
2005; Martinez-Arias,
1986; Wakimoto and Kaufman,
1981). These facts indicate that the inhibition of NB3-3T
quiescence by Antp mutation is not just a consequence of global
transformation of thoracic-to-abdominal segments but rather results from
specific effects on individual NBs. NB-specific misexpression of Abd-A also
repressed Antp and inhibited NB3-3T quiescence
(Fig. 3K and data not shown).
This also provides evidence that the effect is specific to NBs. Furthermore,
because the effect could be observed even when Abd-A was induced after several
divisions of the NB, thoracic NBs probably maintain plasticity of their
identities during lineage formation.
Temporal regulation of NB entry into quiescence
We showed that the temporal transcription factors/co-factor Pdm, Cas, Sqz
and Nab play a role in triggering NB quiescence intrinsically in NBs (Figs
4 and
6; see Fig. S10 in the
supplementary material). All of these factors also controlled temporal
specification within late lineages of embryonic NBs in both thoracic and
abdominal segments. We confirmed this by further examining the relationships
of the temporal factors (Figs
4,
6 and
7; see Fig. S7 and Fig. S9 in
the supplementary material). For example, the loss of Pdm function in NB3-3T
resulted in precocious transcription factor switching and precocious
quiescence. Conversely, in cas mutant embryos, in which Pdm
expression was de-repressed, quiescence was inhibited and expression of
late-stage-specific temporal factors disappeared. Similar to Pdm upregulation,
loss of nab function resulted in loss of both transcription factor
switching and quiescence.
Although Nab and Sqz can form a complex, nab and sqz mutants displayed very different phenotypes. Both mutants showed de-repression of Kr expression; however, sqz mutants showed no other abnormality in transcription factor switching, whereas nab mutants showed the above-mentioned defects in transcription factor switching and timing of quiescence. These mutant phenotypes revealed that regulation of late temporal events is distributed into multiple pathways. Pdm probably coordinately regulates all factors involved in the timing of NB quiescence, because the loss of Pdm alone is sufficient to cause precocious entry into quiescence.
Function of Nab and Sqz in lineage formation
We showed that Nab and Sqz work for NB quiescence in NBs. The
Nab/Sqz-mediated repression of Kr may be controlled in NBs due to changes in
NB temporal identity, or in both NBs and their neurons. Nab might inhibit
transcription by recruiting the nucleosome remodeling and deacetylase
chromatin remodeling complex as mammalian Nab does
(Srinivasan et al., 2006).
Mammalian Nab acts with EGR-1, EGR-2 to determine the fate of cells in
hematopoiesis (Laslo et al.,
2006; Svaren et al.,
1996), but whether it can act with the mammalian homolog of
LIN-29/Sqz has not been reported. Loss of lin-29, a C.
elegans homolog of sqz, causes a heterochronic phenotype in
which adulthood is not reached and molting is repeated
(Ambros and Horvitz, 1984;
Rougvie and Ambros, 1995).
C. elegans has a nab homolog gene, mab-10, that
acts with lin-29 in a heterochronic genetic cascade (D. Harris and H.
R. Horvitz, personal communication).
Quiescent NBs memorize temporal identity during quiescence
It is unclear what molecular mechanisms enable NBs to suspend the switching
of transcription factor expression and maintain temporal identity during
quiescence. We know that the mechanisms for switching expression of early
temporal transcription factors can be either cell division dependent or
independent (Grosskortenhaus et al.,
2005; Mettler et al.,
2006). Irrespective of the mechanism used, a NB can `memorize' its
temporal state before quiescence and resume the intrinsic temporal changes
once cell cycle progression is reactivated. Embryonic stem cells may maintain
multipotency during a slow proliferation state by staying in S phase
(Andang et al., 2008). When
quiescent NBs re-entered the cell cycle, their initial progeny incorporated
BrdU fed since hatching (Fig.
2C), indicating that quiescent NBs stay either prior to S phase or
early in S phase. It will be important to identify the point in the cell cycle
at which NB enters quiescence.
Evolution of temporal lineage development
Another well-established mechanism that governs temporal aspects of lineage
formation is the heterochronic gene cascade in C. elegans. This
cascade contains one each of the hunchback homolog and
lin-29 genes and generates five distinct temporal cell identities
within a single cell lineage (Moss,
2007). Drosophila NB lineage formation uses two types of
Zn-finger proteins, namely the Hb/Cas class [Cas shares DNA-binding
characteristics with Hb (Kambadur et al.,
1998)] and the Kr/LIN-29 class. These pairs are expressed three
times in NB lineages in the following order: (1) Hb and Kr
(2) Cas, Kr
and Sqz(3) Cas and DmLin-29end of lineage. This sequence seems to
produce at least ten distinct temporal identities within an NB lineage. The
repetitive use of these temporal transcription factors in three distinct
phases appears to have made the NB lineage longer and more diverse. Lack of Hb
also generates NB lineage variety; the NB3-3 and NB2-1 lineages lack Hb and
initiate their lineage with Kr. Although the model NB employed in this study
lacks Hb, the sequence and entry into quiescence we described here are common
to many typical NB lineages that start with Hb. Interesting questions from the
perspective of evolution are how do the three phases combine to form a single
lineage and how has NB quiescence evolved in the middle of the NB
lineages?
Neural stem cells in the mouse cerebral cortex go through 11 divisions
and some enter quiescence in late embryo
(Merkle et al., 2004;
Takahashi et al., 1995;
Ventura and Goldman, 2007). We
thus consider the possibility that mammalian neural stem cell and
Drosophila NB share a similar intrinsic mechanism that induces
quiescence.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/23/3859/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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