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First published online 7 March 2007
doi: 10.1242/dev.02810
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1 Department of Molecular Biology, University of Texas Southwestern Medical
Center at Dallas, Dallas, TX 75390-9148, USA.
2 Graduate School of Biomedical Sciences, University of Texas Southwestern
Medical Center at Dallas, Dallas, TX 75390-9148, USA.
* Author for correspondence (e-mail: Dennis.McKearin{at}utsouthwestern.edu)
Accepted 17 January 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Stem cells, Epigenetics, Drosophila, Oogenesis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The germline stem cells (GSCs) of the Drosophila ovary have
emerged as a valuable model for studying stem cell behavior and its
regulation. In contrast to stem cells in many other tissues, the ovarian GSCs
are easily identified by location and by molecular markers. Two or three
ovarian GSCs reside at the anterior of each ovariole, closely apposed to the
somatic cap cells of the germarium. Mutational analysis in Drosophila
has revealed a suite of genes that is necessary for GSC maintenance
(Gilboa and Lehmann, 2004a
;
Spradling et al., 2001). These
studies have demonstrated that a microenvironment, or niche, composed of
multiple cell types influences GSC self-renewal. Factors intrinsic to the stem
cell, as well as signals from the somatic cells of the niche, are important
for GSC maintenance.
The emerging view of GSC maintenance in Drosophila indicates that
GSCs are maintained largely by preventing their differentiation
(Kai et al., 2005;
Li and Xie, 2005;
Wang and Lin, 2004). Stromal
cells produce Dpp locally to prevent GSC differentiation by initiating a Smad
signaling cascade in GSCs that silences transcription of the key
differentiation factor bag of marbles (bam)
(Chen and McKearin, 2003a;
Song et al., 2004;
Xie and Spradling, 1998).
Adherens junctions anchor GSCs to cap cells and are essential for GSC
maintenance and for Smad signaling (Song
et al., 2002). Other factors required in the cap cells, such as
piwi (Cox et al.,
2000) and fs(1)Yb
(King and Lin, 1999), are also
essential GSC maintenance factors, but their mode of action remains
unknown.
Gene products that are required intrinsically in stem cells for their
maintenance also function to prevent germline differentiation. For example,
the translational inhibitors Nanos (Nos) and Pumilio (Pum) are required in
stem cells (Forbes and Lehmann,
1998; Lin and Spradling,
1997; Wang and Lin,
2004). The Nos-Pum complex is thought to repress the translation
of key differentiation factors to prevent GSC differentiation, but the targets
of Nos and Pum in GSCs are unknown. A recent report has also suggested that
chromatin state influences GSC maintenance, because Iswi, a key
chromatin-remodeling factor, is required in GSCs for their maintenance
(Xi and Xie, 2005).
Here, we present evidence that the DNA-associated protein Stonewall (Stwl) is required in GSCs, and that it promotes their maintenance by repressing germ cell differentiation. Stwl is a nuclear factor that has protein domains that suggest an interaction with histone-modifying enzymes. We demonstrate that stwl mutants act as dominant suppressors of variegation, indicating a requirement for stwl in heterochromatin assembly or maintenance. We order the requirement for stwl with respect to other GSC maintenance factors and find that stwl is not essential for bam silencing, but rather that it acts in opposition to bam activity to prevent germ cell differentiation. We propose that Stwl represses the expression of many genes, including those targeted by Nos-Pum translational inhibition. We identify a group of genes that are specifically upregulated in the absence of stwl and, among those genes, find candidates for Nos-Pum translational inhibition.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Other
mutations included nosRC
(Gavis and Lehmann, 1992),
Df(nos) Df(3R)DL-FX1 (Sonoda and
Wharton, 1999) (gift from R. Wharton), pumET9
(Barker et al., 1992),
bam
86 (McKearin
and Ohlstein, 1995), Su(var)3-91
(Reuter et al., 1986) and
EY00986 upstream of CG3919 (stwl neighbor) from Bellen
(Bellen et al., 2004).
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),
P{hsp70-stwl} (Clark and
McKearin, 1996), P{FRT 79D} and P{hsFLP} from Bloomington Stock
Center, P{nosP-Gal4:VP16} (a gift from R. Lehmann)
(Van Doren et al., 1998), DX1
P{w+} (a gift from S. Henikoff)
(Dorer and Henikoff, 1994),
P{Nos-myc} (a gift from R. Wharton)
(Verrotti and Wharton, 2000).
Histone-GFP fusion flies were a gift from J. Duffy (Indiana University,
Bloomington, IN). Ectoptic Stwl was produced by combining the stwlEP allele, which allows misexpression of stwl+ when a source of Gal4 transactivator is introduced, with the germline-specific Nos-Gal4:VP16. Flies were transferred to wet yeast and shifted to 29°C for a minimum of 18 hours prior to ovary dissection. For each genotype, a minimum of 30 ovarioles were scored. {HS-stwl} produced a similar, but more variable, stem cell expansion phenotype.
For clonal analysis, flies of the genotype {hsFLP}; {histoneGFP} {FRT79D} were crossed to stwl95 {FRT 79D} flies. To make clones of wild-type alleles, such as for Lola expression experiments, mitotic recombination was induced in P{hsFLP}/+; {histoneGFP} {FRT 79D}/{FRT 79D} females. Adult females were subjected to heat-shock (37°C) for 1 hour, three times per day for 2 days. All fly stocks were raised on standard cornmeal molasses agar at 25°C, unless noted otherwise. Stem cell maintenance was assayed by retention of mutant stem cells over time. At 5, 7, 9, 11 and 13 days post-clone-induction, ovaries were examined and scored for the number of GSC clones/ovariole. For each time-point and genotype, a minimum of 30 ovarioles were scored.
stwl bam double-mutant analysis used stwl95 bam86 and stwlZ1 bam86 or stwlEY bam86 chromosomes. All three stwl alleles acted as strong loss-of-function alleles and behaved similarly in combination with bam86.
For eye pigment extraction, ten heads from male flies aged for 3 days were homogenized in 700 µl of methanol with 0.1% HCl and were then incubated overnight at 4°C. Following incubation, the extracts were cleared by centrifugation and measured for optical density at 488 nm. Each genotype was assayed in triplicate.
Immunohistochemistry
Ovaries were prepared for reaction with antibodies as described previously
(Christerson and McKearin,
1994). The antibodies and dilutions used were: rabbit anti-GFP
(Invitrogen) (1:10,000 dilution); mouse anti-
-Hts (1B1)
(Zaccai and Lipshitz, 1996)
(1:750); mouse anti-BamC (A7) (McKearin
and Ohlstein, 1995) (1:10); rat anti-Pum 1637
(Macdonald, 1992) (1:500);
rabbit anti-Vasa (Lasko and Ashburner,
1990) (1:1000); rabbit anti-Bru
(Sugimura and Lilly, 2006)
(1:3000), rabbit anti-Lola (Giniger et
al., 1994) (1:100); rat anti-Stwl F6 (K. Clark, PhD thesis,
University of Texas Southwestern Medical Center, 1996) (1:100); and mouse
anti-Myc (9E10, DSHB) (1:1000). Alexa Fluor secondary antibodies (Molecular
Probes) were used at a 1:500 dilution.
Phenotypic analysis and genetic interactions
Ovaries isolated from 3-day-old well-fed flies were incubated with anti-Hts
and anti-GFP antibodies. Images were collected on a Zeiss LSM 510 Meta and
projected to count the number of spherical spectrosomes/fusomes, the number of
bam-GFP-negative cells and to identify differentiated cysts. Round
spectrosome-containing cells that were negative for bam-GFP were
scored as GSCs. GSC number was determined by scoring a minimum of 20 germaria
per genotype.
Microarray analysis
Microarray analyses for stwl bam versus bam ovaries were
performed at the UT Southwestern Microarray Core Facility using Affymetrix
Drosophila Genome 2.0 chips, representing 
18,500 transcripts.
Ovaries from each genotype were hand-dissected and placed immediately into a
Trizol RNA isolation reagent (Invitrogen). Total RNA was used to probe
microarray chips. The expression of over 18,500 transcripts represented on the
Affymetrix Drosophila Genome 2.0 microarray was examined in ovarian
tissue samples obtained from stwl bam and bam mutant
females. Triplicate hybridizations were performed for each sample. Overall,
9000 transcripts were expressed in the tissues of each genotype.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The microenvironment or niche that regulates GSC maintenance includes somatic cells of the cap and terminal filament, which produce survival and differentiation factors that are crucial for GSC maintenance. Stwl is expressed in both germ cells and in somatic cells (K. Clark, PhD thesis, University of Texas Southwestern Medical Center, 1996); thus, stwl may be required in somatic cells to produce the microenvironment necessary for GSC maintenance and/or in the stem cell to respond to that microenvironment. To differentiate between these possibilities, we used mitotic recombination to eliminate stwl specifically in germ cells. Under conditions that produced GFP-negative GSCs (hereafter referred to as `clones') in approximately 35-40% of ovarioles (Fig. 1D,G), wild-type GSC clones were maintained for over 2 weeks, whereas most stwl- GSC clones were rapidly depleted (Fig. 1E-G). TUNEL labeling in both wild-type and stwl- ovaries failed to reveal any germ cell apoptosis in the more than 100 germaria that were examined (data not shown).
To distinguish further between stem cell loss by germ cell death or by differentiation, we followed cyst progression in mosaic ovaries. stwl- clones were induced by heat-shock and animals were aged for a week or more to ensure that persisting clonal germ cells were derived from a mutant stem cell. Careful analysis of ovarioles containing stwl- germline clones revealed germaria containing clonally related mutant germ cell clusters without a corresponding stem cell, indicating that the stem cell that gave rise to these germ cell clusters had been lost (Fig. 1D-F). Together with the lack of apoptotic germ cells, these studies suggested that stem cell loss in stwl mutants was not a consequence of cell death, but rather that stwl- stem cells were lost by differentiation.
In addition to its requirement for GSC maintenance, stwl was also
essential as a cell-autonomous factor for proper oocyte maturation. Developing
egg chambers derived from stwl- GSCs displayed several
defects previously described in ovaries from stwl- females
(Clark and McKearin, 1996),
including a failure of oocyte differentiation, as evidenced by abnormal oocyte
karyosome formation and by a failure to concentrate Orb within a single cell
(data not shown). Likewise, pycnotic nuclei and widespread TUNEL labeling in
cystocytes of stage-4 egg chambers revealed that stwl-
germline clones eventually became apoptotic, as was described for homozygous
stwl ovaries (Clark and McKearin,
1996). By contrast, egg chambers containing
stwl- follicle cells but wild-type germ cells developed
normally (data not shown), suggesting that, like GSC loss, stwl acts
in germ cells rather than in soma to ensure oocyte development.
Ectopic Stonewall expression delays cystoblast differentiation
A corollary to the observation that stwl inactivation caused
premature differentiation is that excess stwl expression might be
sufficient to delay or block cystoblast differentiation. Although ovaries
expressing ectopic Stwl in germ cells were morphologically similar to wild
type, they contained extra GSC-like cells in the germarium, as measured by the
presence of round fusomes (spectrosomes) and by the quiescence of a
bam transcriptional reporter in germ cells at the anterior end of
germarium (compare Fig. 2A with
2B). Ectopic Stwl resulted in two to three extra stem-cell-like
cells per germarium, on average, compared with Gal4 control ovaries
(Fig. 2C).
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Stwl acts as a regulator of chromatin structure
Our data suggested that stwl acted autonomously in the stem cell
to promote GSC maintenance or to repress cystoblast differentiation. Because
the Stwl protein contains modified SANT domains, which have been implicated in
chromatin regulation (Boyer et al.,
2002), we considered the possibility that stwl may
influence gene expression via the modification of chromatin structure or
function. We noticed that Stwl was expressed in eye discs (data not shown),
and took advantage of a well-described assay for chromatin-mediated gene
repression to test for stwl-dependent chromatin modification
(Dorer and Henikoff, 1994).
Position-effect variegation (PEV) occurs when euchromatic genes are placed in,
or adjacent to, heterochromatin, at which point they become subject to
heterochromatic gene silencing. This silencing is often mosaic, or
variegating, and can be relieved by reducing the concentration of
heterochromatin-binding proteins or other associated transcriptional
repressors (Karpen, 1994).
The DX1 chromosome contains a `heterochromatinized'
white+ (w+) gene array that normally
produces weak w+ expression
(Dorer and Henikoff, 1994),
resulting in mostly white eyes with a few spots of red eye pigment
(Fig. 3A). Mutant alleles of
known chromatin-regulatory genes, such as Su(var)3-91,
which encodes a histone methyltransferase required for heterochromatin
structure (Rea et al., 2000),
suppressed the variegated phenotype by producing more-uniform expression of
w+ (Fig.
3D). Surprisingly, inactivating stwl alleles acted as
strong dominant Su(var) mutations
(Fig. 3B,C). We quantified the
degree of suppression by extracting A488-absorbing eye pigment from
fly heads of each genotype and found that a reduction in stwl gene
dose resulted in a two- to three-fold increase in eye pigment, a reflection of
w+ expression level
(Fig. 2E). stwl
mutants showed a similar dominant Su(var) affect when tested with the
variegated wm4 and bwD alleles (data
not shown).
Stwl in the hierarchy of GSC maintenance genes
One attractive candidate for a target silenced by stwl was the
bam gene, because previous studies had shown that bam
silencing was required for GSC maintenance
(Ohlstein and McKearin, 1997).
If bam transcription were de-repressed in stwl mutants, GSCs
would be lost because bam expression would cause GSC-to-cystoblast
differentiation (Ohlstein and McKearin,
1997). We therefore scored bam expression in
stwl- ovaries using both a Bam-specific antibody and a
bam transcriptional reporter. We found, however, that bam
transcriptional silencing was properly maintained in stwl-
ovaries (Fig. 4A), indicating
that Stwl is not required for bam silencing.
Mutations that cause GSC loss without disrupting bam silencing,
such as those in pum and pelo, have been described
previously (Chen and McKearin,
2005; Szakmary et al.,
2005; Xi et al.,
2005). Tests of genetic epistasis have shown that double mutants
of bam and the GSC-loss gene (pum bam or pelo; bam)
can form differentiating germ cell cysts, indicating that bam
function is dispensable when either pum or pelo is also
absent (Chen and McKearin,
2005; Szakmary et al.,
2005; Xi et al.,
2005). We similarly constructed stwl95
bam86 double-mutant animals to determine whether
stwl and bam also constituted an antagonistic gene pair. In
approximately 80% of stwl bam germaria, we found germ cells
interconnected by branched fusomes, indicating that cystoblasts had formed and
initiated cyst differentiation (Fig.
4D). On average, 30% of germ cells in these ovaries were in
clusters penetrated by branched fusomes. Taken together, these findings
indicated that stwl antagonizes bam action by a mechanism
that is independent of bam transcriptional silencing.
Identifying targets of Stwl repression
To identify Stwl targets whose transcriptional silencing is required for
GSC maintenance, we carried out microarray analysis of undifferentiated germ
cells that lack bam and compared them to stwl bam mutants.
Both of these genetic backgrounds provided a nearly homogeneous population of
cell types, because bam mutant cells failed to differentiate into
cystoblasts and stwl bam germ cells arrested as partially formed
cysts. The homogeneity and early arrest of the mutant germ cells improved the
quality of microarray data by eliminating late-stage egg chambers that produce
complex and abundant populations of mRNA, which can distort microarray
analysis.
We identified 501 genes that were differentially expressed twofold or more (P<0.05) in stwl bam mutants relative to bam mutant ovaries. Of these candidate transcripts, 235 were upregulated in stwl bam versus bam mutants. Differentially expressed transcripts designated as having functions in mRNA processing, transcription or other roles are listed in Table 1, and those with the largest differential change in expression are highlighted by bold. Transcripts that appeared as downregulated are listed in Table S1 in the supplementary material.
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Bruno (also known as Arrest - FlyBase), a translational repressor required
for cyst formation (Parisi et al.,
2001), appeared in greater abundance in stwl bam
profiles. Bruno accumulation did not change significantly in
stwl+/- versus stwl-/- germline clones
(Fig. 5A,B). Bruno accumulation
did increase, however, throughout stwl bam germaria
(Fig. 5E,F) but not in
bam mutant germaria (Fig.
5C,D), correlating with cyst differentiation. Thus, Bruno was not
a direct Stwl target gene, but bruno mRNA was present in greater
abundance in the stwl bam transcript profile due to cyst
differentiation.
Transcripts from lola, a gene implicated in chromatin organization
(Zhang et al., 2003), also
appeared in greater abundance in stwl bam profiles. Likewise, Lola
protein was detected in 48% of the cells in stwl bam ovaries, in
contrast to in 12% of bam cells and in 18% of early germ cells (GSCs
to eight-cell cysts) in stwl+/- ovaries. Lola is a
dynamically expressed protein (Giniger et
al., 1994), and its apparent increase in transcript profiles and
in ovaries could be explained if a greater percentage of stwl bam
cells was trapped at Lola-expressing stages. We therefore examined Lola
expression more closely in stwl mutant and stwl mosaic
backgrounds, in which cystocytes do not arrest differentiation until much
later in oogenesis. Unlike Bruno, Lola protein was expressed in much greater
abundance in germ cells of stwl homozygous mutant flies compared with
stwl+/- (data not shown). Analysis of stwl mosaic
germlines provided the clearest demonstration of the dependence of Lola
expression on stwl activity. As shown in
Fig. 5G,H, almost all
stwl-/- cells in mosaic germaria expressed Lola, whereas
most stwl+/- germ cells were Lola-negative. Lola was
detected in 84% of germ cells lacking stwl (54/64
stwl-/- germ cell clones) and in only 26% of germ cells
expressing stwl+ (15/52 stwl+/+ germ
cell clones). We concluded, therefore, that induction of lola mRNA
reflected in the transcript profiling experiments was due to upregulation of
lola transcription in the absence of Stwl, suggesting that Stwl
directly repressed lola transcription.
As previously noted, genetic interactions had suggested a link between Stwl
and Nos-Pum action. We examined the expression of Nos and Pum in stwl
mutant ovaries, but found no change in the levels or localization of Nos or
Pum in the absence of stwl (see Fig. S1 in the supplementary
material). Because these proteins act in different cellular compartments
(nucleus versus cytoplasm), we suspected that an overlapping set of targets
might account for the genetic interactions. To identify Stwl-repressed mRNAs
that might also be targets of Nos-Pum translational repression, we searched
the 235 genes upregulated in the absence of stwl for those that
contained consensus Pum-binding sites (termed Nanos response element or NRE:
AUUGUA) within their 3'-UTRs (Murata
and Wharton, 1995; Sonoda and
Wharton, 1999). We identified 25 transcripts with one or more NRE
consensus sites within their 3'-UTR, including six transcripts with two
NREs and five transcripts with three NREs
(Table 2). Although the number
of transcripts containing one NRE in their 3'-UTR sequences was not
different from random occurrence, the frequency of 3'-UTRs with multiple
NREs was significantly higher in the induced group of transcripts than in a
group whose levels did not change in the absence of stwl (4.7% versus
2%). Intriguingly, several of the multiple lola transcripts carried
two predicted NREs within their 3'-UTR, suggesting that lola
may be a candidate for both Nos-Pum translational regulation and stwl
transcriptional regulation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Wang and Lin,
2004; Wong et al.,
2005). Dpp signaling within the GSC niche silences Bam expression
(Chen and McKearin, 2003a;
Li and Xie, 2005;
Song et al., 2004), and the
translational repressors Nos and Pum prevent translation of putative
cystoblast-promoting mRNAs (Gilboa and
Lehmann, 2004b; Wang and Lin,
2004). Our finding that GSCs prominently express a marker of
transcriptional quiescence is another indication that the stem cell state
depends heavily on mechanisms that restrict gene expression. Similar
conclusions have been made for several stem cell populations in mammals. For
example, the transcriptional repressor Plzf (Zbtb16) is required for
spermatogonial stem cell maintenance in the mouse
(Buaas et al., 2004;
Costoya et al., 2004). Plzf
associates with the Polycomb family member bmi-1 (bmi1) and
has been implicated in chromatin-mediated repression
(Barna et al., 2002). Other
adult stem cells are also likely to be regulated by chromatin state, as
bmi-1 is required for the self-renewal of hematopoetic and neural
stem cells in the mouse (Lessard and
Sauvageau, 2003; Molofsky et
al., 2003; Park et al.,
2003).
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; Cutler et al.,
1998). SANT-domain proteins in humans and yeast are most
frequently associated with chromatin-remodeling and histone-modifying
activities (Boyer et al.,
2002). In many instances, the SANT domains are essential for
histone binding. Peterson (Boyer et al.,
2004) has advanced the hypothesis that SANT domains bind histone
tails in histone-modifying complexes, such as histone acetyl transferases
(HAT) and histone de-acetylation complexes (HDACs). On the other hand, SANT
domains in Myb, Adf1 and Dip3 have been shown to act as sequence-specific
DNA-binding motifs (Bhaskar and Courey,
2002; Cutler et al.,
1998; Gabrielsen et al.,
1991). Although we cannot exclude the possibility that Stwl acts
as a sequence-specific DNA-binding protein, we favor the idea that it acts as
a histone-interacting protein, because the Su(var) phenotype has been
remarkably predictive of proteins involved in histone modification and
chromatin remodeling (Ebert et al.,
2004). For instance, Su(var)3-7 is a Drosophila
protein with a modified SANT domain. This domain interacts with a major
heterochromatin protein, HP-1 [Su(var)205]
(Delattre et al., 2000), and
contributes to heterochromatic gene silencing
(Delattre et al., 2004).
Chromatin silencing factors and GSC maintenance
The Su(var) activity and stem-cell-loss phenotype of stwl
led us to propose that Stwl is a component of a chromatin-modifying complex
and that chromatin state may influence GSC fate. Indeed, the chromatin
remodeler iswi also plays an essential role in maintaining GSCs
(Xi and Xie, 2005), indicating
a significant function for chromatin remodeling in GSC chromosomes. Our
finding that stwl acts as a potent Su(var) and is required
for GSC maintenance suggests that domains of chromatin silencing are
established and maintained in these stem cells to regulate
differentiation.
Remarkably, loss- and gain-of-function experiments documented that Stwl was
both necessary and sufficient for GSC fate
(Akiyama, 2002; this paper).
Our data indicates that Stwl represses the expression of
differentiation-promoting genes, probably by chromatin-mediated
transcriptional silencing. Strikingly, imposing Stwl-mediated silencing in
germ cells caused more of them to persist as GSC-like cells. These ectopic
GSCs retained properties of stem cells, including bam silencing.
Eventually, these supernumerary GSCs differentiated, probably when they exited
the crucial niche region.
Only a few genes that can expand the number of GSCs have been identified
and, in each case, these genes occupy crucial positions in the genetic
circuits maintaining GSCs. Constitutive activation of the Dpp signaling
pathway can block germ cell differentiation and maintain all germ cells in a
GSC-like state (Casanueva and Ferguson,
2004; Xie and Spradling,
1998). Prolonged somatic expression of piwi can expand
the number of GSC-like cells, but only within the anterior-most region of the
GSC niche (Cox et al., 2000;
Szakmary et al., 2005).
Ourselves and others have proposed that the region defined by the limited
expansion of GSCs represents a domain of graded Dpp signaling
(Casanueva and Ferguson, 2004;
Chen and McKearin, 2005;
Szakmary et al., 2005).
Overexpression of factors that enhance Dpp signaling, such as piwi
(Chen and McKearin, 2005;
Szakmary et al., 2005), expand
the niche by `flattening' the gradient. We propose that Stwl represses GSC
differentiation cell-autonomously, delaying germ cell differentiation and
expanding GSC number within the limits of the niche. Our finding that an
intrinsic transcriptional repressor can expand GSC number might have important
implications in systems that require stem cell number to expand (for example,
during growth), or in cases of cancer metastasis when cancer stem cell numbers
might amplify.
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). For example, the Polycomb proteins are bound to a suite of
promoters in embryonic stem (ES) cells and the corresponding genes become
activated when ES cells begin to differentiate
(Boyer et al., 2006;
Lee et al., 2006). A separate
study identified another PcG protein, Ezh2, as necessary for maintenance of
mouse hematopoietic stem cells (Kamminga
et al., 2006).
The current paradigm for the mechanisms that regulate GSCs predicts that
stem cells are maintained by blocking their differentiation. GSCs are subject
to repressing activities by niche signaling
(Chen and McKearin, 2003a;
Song et al., 2004) and
translational control (Gilboa and Lehmann,
2004b; Wang and Lin,
2004). Here, we propose that Stwl preserves GSCs by imposing
epigenetic transcriptional quiescence that acts downstream of
bam-promoter silencing and depends on full nos+
and pum+ function for the preservation of GSC fate. We
note that previous studies on transcriptional silencing in pole cells in
Drosophila and germ cells in C. elegans have implicated Nos
and its orthologous proteins in regulating epigenetic modifications
(Schaner et al., 2003). In
flies, nos+ was required to delay the accumulation in pole
cell genomes of lysine 4 methylation of histone H3 (H3meK4) that would signal
broad transcriptional activation (Schaner
et al., 2003).
The fact that Stwl action depended on the genetic dosage of
nos+ can be explained if Stwl silences the transcription
of genes whose transcripts are also translationally inhibited by Nos-Pum.
Alternatively, Stwl-dependent and Nos-dependent epigenetic mechanisms might
collaborate to provide full-genome transcriptional silencing. Because
transcripts bearing multiple NREs were over-represented in the class of
Stwl-repressed genes, we favor the initial hypothesis, and present a model for
Stwl function in Fig. 6. We
propose that Stwl represses the transcription of a cohort of genes, including
key differentiation factors also targeted by Nos-Pum translational repression.
It is also possible that nos and stwl are both involved in
regulating chromatin state in the stem cell. In pole cells, Nos activity has
been shown to influence chromatin state, probably by repressing the
translation of chromatin modifiers
(Schaner et al., 2003). The
possibility of dual mechanisms for silencing the expression of this group of
genes might suggest that they can promote key steps in GSC differentiation and
may require overlapping mechanisms to block their expression. Perhaps it will
be possible to produce a chart of the earliest steps in differentiation from a
stem cell to a differentiated daughter by noting genes that are common targets
of multiple repressors.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/8/1471/DC1
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ACKNOWLEDGMENTS |
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