Department of Genetics, Washington University School of Medicine, Saint Louis, MO 63110, USA
* Author for correspondence (e-mail: ts{at}genetics.wustl.edu)
Accepted 13 October 2003
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SUMMARY |
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Key words: Germline development, Stem cells, Proliferation, Tumor, Meiotic entry, Notch signaling, gld-1, nos-3, glp-1
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Introduction |
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The Caenorhabditis elegans germline is an excellent system for
studying the balance between proliferation and differentiation in a stem cell
population because cells can be found in all stages of development in a linear
spatial pattern (Schedl,
1997). The most distal end of the adult gonad contains a stem cell
population that covers a region of approximately 20 cell diameters in length
(Fig. 1A) (Crittenden et al., 1994
;
Hansen et al., 2004
). Cells
immediately proximal to the stem cells, in the transition zone, have entered
meiotic prophase and continue to progress through meiosis as they move
proximally.
|
While no direct transcriptional targets of GLP-1 signaling have yet been
characterized in the germline, genetic evidence indicates that gld-1
and gld-2 function in redundant pathways downstream of GLP-1/Notch
signaling to promote meiotic development and/or inhibit proliferation
(Fig. 1B)
(Francis et al., 1995b;
Kadyk and Kimble, 1998
). GLD-1
is a KH domain-containing RNA binding protein
(Jones and Schedl, 1995
), and
GLD-2 is the catalytic portion of a poly(A) polymerase
(Wang et al., 2002
). The gene
for either of these is sufficient to promote meiotic entry since in either
gld-1 or gld-2 single null mutant animals, germ cells enter
meiosis normally (Francis et al.,
1995a
; Kadyk and Kimble,
1998
; Hansen et al.,
2004
). However, in animals that lack both gld-1 and
gld-2 activity, a germline tumor is formed that is similar to that of
glp-1(gf) mutants (Kadyk and
Kimble, 1998
). This tumorous phenotype is epistatic to
glp-1 null indicating that gld-1 and gld-2 function
downstream of GLP-1/Notch signaling (Kadyk
and Kimble, 1998
). Therefore GLP-1 signaling promotes
proliferation, at least in part, by turning off the activities of
gld-1 and gld-2. It is not known how alteration of GLP-1
signaling in the distal germline changes gld-1 and gld-2
activities there, or how gld-1 and gld-2 become active more
proximally. The mechanism appears to involve spatial regulation of GLD-1
protein accumulation. GLD-1 is at the lowest level at the very distal end and
increases until reaching maximum levels approximately 20 cell diameters from
the distal tip (Jones et al.,
1996
) (Fig. 1C,D).
Since gld-1 promotes meiotic entry, the low levels of GLD-1 protein
in the distal end may be necessary to maintain the stem cell population.
Likewise, the high levels of GLD-1 protein achieved at the approximate
location of meiotic entry may be important for meiotic entry to occur.
Recently, FBF, a homolog of Drosophila Pumilio that is the product
of two nearly identical adjacent genes, fbf-1 and fbf-2
(Zhang et al., 1997), has been
shown to inhibit GLD-1 accumulation in the distal end of the germline
(Crittenden et al., 2002
). FBF
is also necessary for germ cell proliferation in late larvae and adults; loss
of FBF activity results in premature entry into meiotic prophase and a
depletion of the stem cell population in the late fourth larval stage
(Crittenden et al., 2002
). FBF
is a post-transcriptional repressor of gld-1 and Crittenden et al.
have proposed that FBF promotes proliferation by keeping GLD-1 levels low in
the distal most germline (Crittenden et
al., 2002
).
We show that a major mechanism by which GLP-1/Notch signaling maintains the stem cell population is by inhibiting GLD-1 protein accumulation in the distal end of the germline, thereby restricting its activity to more proximal regions. We further show that not only does low GLD-1 allow proliferation, but that high GLD-1 promotes meiosis. We also show that the position of the rise in GLD-1 levels determines the size of the stem cell population and the location where germ cells begin meiotic development. We find that nos-3, whose role we identified in a mutant screen, functions redundantly with gld-2 to promote the rise in GLD-1 that is necessary for entry into meiosis. Genetic experiments indicate that repression of GLD-1 accumulation by FBF is acting through nos-3, while regulation of gld-2 in this processes is likely by something other than, or in addition to, FBF. Our data suggest a model in which GLP-1 signaling regulates the size of the stem cell population by regulating GLD-1 levels, at least in part, through antagonism between the repressive activity of fbf and the positive activities of nos-3 and gld-2.
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Materials and methods |
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Nematode strains and culture
Standard procedures for culture and genetic manipulation of C.
elegans strains were followed with growth at 20°C unless otherwise
noted (Sulston and Hodgkin,
1988). Descriptions of genes, alleles and phenotypes related to
this study are in Hodgkin and Martinelli
(Hodgkin and Martinelli,
1999
).
Measurement of distal GLD-1 accumulation pattern
Eleven wild-type (N2) gonad arms from animals grown at 20°C and
dissected one day past L4 were stained with anti-GLD-1-specific antibodies
(see below) and analyzed using a Leica TCS SP2 confocal microscope. Images
were collected well below saturation. For the distal end of each arm, images
were obtained as 1 µm serial sections and then flattened into one image.
Pixel intensity was determined on a Macintosh computer using the public domain
NIH Image program (developed at the US National Institutes of Health and
available on the Internet at
http://rsb.info.nih.gov/nih-image/).
In short, the program divided the arm into a grid 20 units in height and 150
units in length, which corresponds to approximately 24 cell diameters. The
pixel intensity was measured for each location on the grid and each of the 150
columns was averaged (20 spots per column). These 150 values were then
averaged with the 150 values of the remaining 10 gonad arms and plotted on a
graph (Fig. 1D).
Antibody staining and RNA in situ hybridization
Antibody staining of dissected gonads has been described previously
(Jones et al., 1996). In
short, animals were dissected and fixed with either 3% formaldehyde/0.1 M
K2HPO4 (pH 7.2) for 1 hour at room temperature (RT)
followed by 5 minutes incubation with 100% methanol at 20°C (this
fixative was used when not using GLD-1 antibodies), or 3%
formaldehyde/0.5x PBS/75% methanol for 5 minutes at 20°C
(this fixative used when GLD-1 antibodies were used). The use of nucleoplasmic
REC-8 staining to identify proliferative germ cells is described elsewhere
(Hansen et al., 2004
).
Fluorescent images were captured with a Zeiss Axioskop microscope equipped
with a Hamamatsu digital CCD camera (Hamamatsu Photonics). For all strains
stained with GLD-1, wild-type control animals were dissected in the same dish,
co-stained, mounted on the same slide and images were captured with the same
camera settings. In many cases, both the N2 and mutant gonads were captured in
the same field (Fig. 6C). In
order to confirm that the low GLD-1 levels seen in gld-2(q497);
nos-3(oz231) animals was not due to the germlines being masculinized, we
also stained gld-2(q497) fog-3(q443); nos-3(oz231); unc-32(e189)
animals and found that GLD-1 levels were still low (data not shown).
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Results |
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We first examined animals that have constitutively active, ligand
independent, GLP-1 signaling (Berry et al.,
1997), predicting that if GLP-1/Notch signaling inhibits GLD-1
protein accumulation, then constitutively active signaling would result in
lower GLD-1 levels. Animals with one copy of the gain-of-function allele
glp-1(oz112) and one copy of the glp-1(q175) null allele
have a late onset tumorous phenotype where the distal proliferative zone
increases in size over time, reflecting constitutive GLP-1 activity
(Berry et al., 1997
). In these
animals, low GLD-1 levels extend much further proximally than in wild-type
(Fig. 2). The maximum level,
however, still coincides with the transition of germ cells from proliferation
to early meiotic prophase as judged by nuclear non-chromosomal axis REC-8
staining (Pasierbek et al.,
2001
), which under our fixation conditions stains proliferating
germ cells (Hansen et al.,
2004
). In animals homozygous for glp-1(oz112gf), and
carrying an extra copy of glp-1(+) on a free duplication, GLD-1
levels do not increase (Fig.
2). These animals have completely tumorous germlines with no
evidence of entry into meiosis (Berry et
al., 1997
; Hansen et al.,
2004
). Therefore, GLP-1/Notch signaling activity leads to low
GLD-1 levels, suggesting that in wild-type animals, GLP-1/Notch signaling
inhibits GLD-1 accumulation in the distal end.
|
|
To determine if GLP-1/Notch signaling inhibits GLD-1 accumulation at the
level of transcription, we looked at gld-1 mRNA levels by in situ
hybridization in gld-2(q497) gld-1(q361) and gld-2(q497)
gld-1(q361); glp-1(q175) animals. Previous studies suggested that
gld-1 mRNA accumulation is only modestly regulated along the distal
proximal axis in wild-type hermaphrodites
(Jones et al., 1996). We did
not see an increase in gld-1 mRNA levels in gld-2(q497)
gld-1(q361); glp-1(q175) animals, but approximately the same spatial
patterning as in gld-2(q497) gld-1(q361) animals
(Fig. 3). Therefore the lack of
GLD-1 accumulation in glp-1(gf) tumorous germlines
(Fig. 2) and in the distal-most
region of wild-type and gld-2(q497) gld-1(q361)
(Fig. 3A,B), probably reflects
the inhibition of GLD-1 accumulation by GLP-1 signaling at a post
transcriptional level, possibly through inhibiting translation or promoting
protein degradation.
Increased GLD-1 accumulation in the distal most end results in germ cells entering meiosis more distally
We have shown that GLP-1/Notch signaling represses GLD-1 accumulation in
the distal end of the gonad. To determine if this repression of GLD-1 is
functionally important in maintaining the stem cell population, we sought to
determine the effect of ectopically increasing GLD-1 levels in the distal-most
end. Since gld-1 has previously been shown to inhibit proliferation
and/or promote meiotic entry, this would imply that glp-1-mediated
repression of GLD-1 accumulation in the distal end allows for proliferation in
this region (see also Crittenden et al.,
2002). In order to test this further we utilized
gld-1(oz10gf) animals, which have increased GLD-1 accumulation in the
distal-most end (Jones et al.,
1996
). gld-1(oz10gf) animals display a semi-dominant Mog
phenotype (masculinization of the germline),
with both heterozygous and homozygous hermaphrodites having increased sperm at
the expense of oocytes. This Mog phenotype results from GLD-1's role in
regulating germline sex determination, a function that is separate from its
function in regulating meiotic entry
(Francis et al., 1995a
).
We measured the size of the proliferative zone in gld-1(oz10gf)
homozygotes following staining for proliferative and meiotic prophase nuclei
using anti-REC-8 and HIM-3 antibodies respectively
(Pasierbek et al., 2001;
Zetka et al., 1999
;
Hansen et al., 2004
).
gld-1(oz10gf) homozygotes have a proliferative zone 13 cell diameters
in length as compared with 19 in wild-type animals of the same age
(Fig. 4A).
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Screen to identify genes that function in the GLD-1 pathway
gld-1 and gld-2 function redundantly to regulate the
switch of germ cells from the mitotic proliferative state to meiotic
development (Francis et al.,
1995b; Kadyk and Kimble,
1998
) (Fig. 1B). In
the absence of gld-1 or gld-2 activity, cells are able to
enter meiosis properly, however, if the activities of both gld-1 and
gld-2 are absent, cells fail to enter meiosis properly and a germline
tumor results (Kadyk and Kimble,
1998
). In order to identify genes that function with
gld-1 either to promote entry into meiosis and/or inhibit
proliferation, we screened for recessive mutations that, when in combination
with a gld-2 null mutant, form a germline tumor (a synthetic tumorous
phenotype, Syt). The genetic screen we employed
(Fig. 1E) involved mutagenizing
animals that were homozygous for gld-2(q497) but that carried the
gaDp1 free duplication, which contains a copy of
gld-2(+).
The screen yielded new alleles of gld-1 (three), as well as
mutations that define three other loci. We describe the locus initially called
syt-1 in which five alleles were identified. The reference allele,
oz231, mapped between let-241 and unc-4, although
closer to unc-4 (4/16 Unc non Let recombinants carried the
oz231 allele), approximately 300 kb from unc-4 on the
physical map
(http://www.wormbase.org,
release WS100, May 2003). An examination of genes in the region identified
nos-3, which encodes a putative translational regulator, as a likely
candidate to encode syt-1. NOS-3 was previously identified from its
similarity to Drosophila Nanos
(Subramaniam and Seydoux,
1999), as well as for its ability to bind FBF-1 and FBF-2
(Kraemer et al., 1999
). FBF-1
and FBF-2 are products of two nearly identical genes, fbf-1 and
fbf-2 (Zhang et al.,
1997
), which are members of a larger family of Pumilio-related
`puf' genes (Pumilio and FBF) (Wickens et
al., 2002
). FBF can bind to the 3'UTR of the mRNA of the sex
determining gene fem-3 (Zhang et
al., 1997
), and working with NOS-3, is thought to repress FEM-3
translation to allow the switch from spermatogenesis to oogenesis in the L4
hermaphrodite.
Four pieces of evidence confirm that oz231 and the other four
mutations are alleles of the nos-3 gene. First, reducing the activity
of nos-3 by RNAi in a strain lacking gld-2 mimics the
gld-2; oz231 double mutant phenotype in that they have tumorous
germlines (data not shown). Second, sequencing genomic DNA of all five alleles
revealed lesions in the nos-3 gene with each lesion predicted to
result in a truncation of the protein prior to the zinc finger motifs
(Fig. 5A). Third, staining of
animals carrying one of the alleles (oz231) with the NOS-3 antibody
(Kraemer et al., 1999) fails
to detect a signal, confirming that oz231 is an allele of
nos-3, and probably a null (data not shown). Fourth, double mutant
animals for gld-2(q497) and nos-3(q650), a previously
identified allele of nos-3
(Kraemer et al., 1999
), form a
germline tumor in hermaphrodites and males similar to that formed in
gld-2(q497) nos-3(oz231) animals (data not shown). Therefore we
conclude that syt-1 is nos-3.
|
NOS-3 and GLD-2 function redundantly to promote GLD-1 accumulation
Since gld-1 and nos-3 function in the same pathway for
entry into meiosis (see above), we next wanted to determine their regulatory
relationship. As both proteins are thought to be translational regulators, we
looked at the level of protein accumulation. GLD-1 accumulation in
nos-3 mutants was very similar to the accumulation in wild type
(Fig. 5D), as was NOS-3
accumulation in gld-1 mutants (data not shown). This suggests that
neither GLD-1 nor NOS-3 is solely responsible for promoting the expression or
stability of the other. However, we already knew through genetic analysis that
nos-3 functions redundantly with gld-2 in regulating entry
into meiosis, therefore we looked at protein accumulation in gld-2;
nos-3 double mutants and found that GLD-1 accumulation is greatly reduced
or absent (Fig. 5E). Since
GLD-1 accumulates at wild-type levels in gld-2 single mutant
(Fig. 5C), we infer that
nos-3 and gld-2 function redundantly to promote GLD-1
accumulation.
To determine the relationship between GLP-1/Notch signaling and the redundant activities of gld-2 and nos-3 in regulating GLD-1 accumulation, we assayed GLD-1 levels in gld-2; nos-3; glp-1 triple mutants and found that GLD-1 levels were low (Fig. 5F). This suggests that the high level of GLD-1 found in the absence of GLP-1/Notch signaling requires nos-3 and gld-2 activity, and that nos-3 and gld-2 function downstream of GLP-1/Notch signaling in regulating GLD-1 accumulation. Furthermore, RNA in situ hybridization of gld-2(q497); nos-3(oz231) animals (data not shown) shows gld-1 mRNA levels similar to gld-2(q497) gld-1(q361) animals, which express GLD-1 protein at near wild-type levels. This suggests that gld-2 and nos-3 are promoting GLD-1 accumulation at the level of translation or protein stability.
fbf-1 fbf-2 proliferation/meiosis phenotype depends on nos-3 activity
Animals lacking FBF activity have germ cells entering meiotic prophase
prematurely resulting in a depletion of the proliferative germ cells
(Crittenden et al., 2002;
Zhang et al., 1997
). This
depletion is suggested to be due to high levels of GLD-1 in the distal end
(Crittenden et al., 2002
). FBF
is a negative regulator of GLD-1 accumulation and binds to the 3'UTR of
gld-1 mRNA in the region deleted by the oz10gf allele
(Crittenden et al., 2002
). FBF
and NOS-3 physically interact in vitro and in a yeast 2-hybrid assay
(Kraemer et al., 1999
), and
are thought to function together in repressing fem-3 translation
relating to germline sex determination. This is apparently analogous to the
canonical Puf/Nanos interaction where Drosophila Pumilio and Nanos
form a ternary complex with hunchback RNA to prevent its translation
(Sonoda and Wharton, 1999
). It
is, therefore, interesting that FBF and NOS-3 function in opposite
directions to regulate meiotic entry. FBF promotes proliferation and/or
inhibits meiotic entry (Crittenden et al.,
2002
), while NOS-3 inhibits proliferation and/or promotes meiotic
entry (this work), both accomplishing these functions, at least in part, by
regulating GLD-1 accumulation.
To determine the epistatic relationship between nos-3 and fbf for entry into meiosis, we compared the size of the proliferative zone and pachytene region of fbf-1(ok91) fbf-2(q704) double null mutants with fbf-1 fbf-2 nos-3(oz231) triple null mutants, in young adults (Fig. 6A). While all fbf-1 fbf-2 germlines lacked a proliferative zone, and all but one lacked any pachytene cells, all fbf-1 fbf-2 nos-3 germlines have extensive proliferative zones and pachytene regions, although somewhat smaller than those of wild type (Fig. 6A). Therefore the lack of nos-3 activity suppresses the fbf-1 fbf-2 null late-onset Glp lf phenotype, suggesting that nos-3 functions downstream or parallel to fbf in regulating meiotic entry.
We next analyzed GLD-1 levels in fbf-1 fbf-2 nos-3 animals. The
rise in GLD-1 protein accumulation in the distal germline is similar in
wild-type males and hermaphrodites (female), but the magnitude of the rise is
much lower in the male germline (Jones et
al., 1996). Since fbf-1 fbf-2 nos-3 animals have a
masculinized germline, and to allow a comparison of the GLD-1 accumulation
pattern with other strains in this study, we feminized fbf-1 fbf-2
nos-3 animals with fog-3(q443), which did not affect the
suppression of the fbf-1 fbf-2 mutant Glp phenotype by nos-3
null. In these animals the pattern of GLD-1 accumulation is very similar to
that of wild type, with low levels at the very distal end and increasing to a
high level as germ cells enter meiosis, although overall levels appear to be
slightly lower (Fig. 6B). Thus,
NOS-3 activity is required for the higher distal GLD-1 levels thought to occur
in fbf mutants.
We next examined the relationship of gld-2 to fbf to test whether the fbf-1 fbf-2 Glp phenotype requires gld-2 activity. We examined the germlines of gld-2(q497); fbf-1 fbf-2 triple null adult hermaphrodites and found that they lacked a distal proliferative region (n=24), although the total number of germ cells appears to be slightly higher (data not shown). Thus, in contrast to nos-3, the activity of gld-2 is not required for the fbf-1 fbf-2 double mutant Glp phenotype.
GLD-1 levels rise as germ cells enter meiosis in the feminized fbf-1 fbf-2 nos-3 triple null mutants (Fig. 6B). Removal of gld-2 activity (in feminized gld-2(q497); fbf-1(ok91) fbf-2(q704) nos-3(oz231) quadruple mutants), results in GLD-1 levels that are very low or absent (Fig. 6C). This result supports the view that GLD-2 is sufficient to promote high levels of GLD-1. However, since there is a proliferative region and low levels of GLD-1 in the very distal end of feminized fbf-1 fbf-2 nos-3 triple mutants (Fig. 6B), GLD-2 must be inactive in the very distal end, even in the absence of fbf. Taken together, these results suggest that GLD-2 is sufficient to promote high levels of GLD-1 and that its activity in the most distal end of a wild-type germline is inhibited by something other than, or in addition to, FBF.
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Discussion |
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GLP-1/Notch signaling controls spatial accumulation of GLD-1
The spatial pattern of GLD-1 accumulation is important for regulating the
balance between proliferation and meiotic entry
(Crittenden et al., 2002). The
extended low GLD-1 levels in the larger than normal proliferative zone of
glp-1(oz112gf)/glp-1(null) hemizygotes, as well as the low or absent
GLD-1 levels in glp-1(oz112gf)/glp-1(oz112gf)/glp-1(+) animals
(Fig. 2), supports the
hypothesis that GLD-1 levels in the most distal end of wild-type animals must
be low in order to enable the stem cell population to be maintained.
Conversely, the correlation of increased GLD-1 levels with meiotic entry in
glp-1(oz112gf)/glp-1(null) hemizygotes
(Fig. 2) and of increased GLD-1
levels in the distal end resulting in more distal meiotic entry
(Fig. 4), indicates that the
wild-type rise in GLD-1 levels causes germ cells to enter meiotic prophase. It
is currently unknown, however, what level of GLD-1 is necessary to promote
meiotic entry. Cells may commit to enter meiotic prophase when GLD-1 levels
are near their highest, or it is possible that cells commit to enter meiotic
prophase more distally, where GLD-1 levels are still increasing.
GLP-1/Notch signaling, activated by a ligand produced by the DTC, is the
initial spatial polarizing cue in regulating the proliferation versus entry
into meiosis decision (Seydoux and Schedl,
2001). The rise in GLD-1 accumulation as cells move proximally is
probably due to a lowering of GLP-1/Notch signaling. Inhibition of distal
GLD-1 accumulation is probably achieved post-transcriptionally because when
GLP-1/Notch signaling is absent, gld-1 mRNA levels do not increase
(Fig. 3E), even though there is
a dramatic increase in protein levels (Fig.
3C). However, since the culminating third component of the core
Notch signaling pathway is a CSL transcription factor [LAG-1 bound to
GLP-1(INTRA)], gld-1 is unlikely to be directly regulated by this
complex. Instead a factor(s), whose transcription is regulated by
LAG-1/GLP-1(INTRA), may control GLD-1 protein levels. None of the genes known
to regulate GLD-1 levels, and that have known expression patterns (NOS-3,
GLD-2 and FBF-1), have significant changes in accumulation in the region where
GLD-1 protein levels increase (Crittenden
et al., 2002
; Kraemer et al.,
1999
; Wang et al.,
2002
), therefore they probably are not transcriptional targets of
LAG-1/GLP-1(INTRA).
Even though GLP-1/Notch signaling inhibits GLD-1 accumulation, it is
interesting that GLP-1 protein levels are still high at the same location
where GLD-1 levels are high (20 cell diameters from the DTC)
(Crittenden et al., 1994
;
Jones et al., 1996
). This
suggests that the level of GLP-1 visible on the membrane does not,
necessarily, reflect the level of signaling that is occurring.
GLD-2 and NOS-3 promote GLD-1 accumulation
We have shown that GLP-1/Notch signaling inhibits GLD-1 accumulation, while
NOS-3 and GLD-2 function redundantly to promote GLD-1 accumulation
(Fig. 7A). Therefore both
positive and negative influences shape the pattern of GLD-1 accumulation,
allowing a spatially controlled balance between proliferation and
differentiation to be maintained. One possible model for how these opposing
factors regulate GLD-1 accumulation is that GLP-1/Notch signaling could
inhibit the activities of GLD-2 and NOS-3 in the most distal end of the
germline (Fig. 7B). As germ
cells move proximally, away from the DTC-bound LAG-2 ligand, GLP-1 signaling
is reduced, allowing for NOS-3 and GLD-2 to promote the accumulation of GLD-1.
Supporting this model are the low GLD-1 accumulation and tumorous germline
phenotypes in gld-2; nos-3; glp-1 triple mutants, indicating that
gld-2 and nos-3 are epistatic to glp-1 with respect
to GLD-1 accumulation. As mentioned above, however, NOS-3 and GLD-2 are
unlikely to be direct targets of GLP-1/Notch signaling. Current data do not
rule out an alternate model where GLP-1/Notch signaling, nos-3 and
gld-2 each function independently on GLD-1 accumulation and that the
sum of their positive and negative regulation determines GLD-1 levels
(Fig. 7C). In this model NOS-3
and GLD-2 may continually promote GLD-1 accumulation, but only when the
inhibiting influence of GLP-1 signaling is reduced by distance from the DTC,
are high GLD-1 levels achieved.
|
NOS-3 is an RNA binding protein similar to Drosophila Nanos
(Kraemer et al., 1999). It is
currently unclear how nos-3 functions redundantly with gld-2
in promoting GLD-1 accumulation. One possibility is that gld-2 and
nos-3 (or genes that they regulate) accomplish similar biochemical
functions that are mutually compensatory. Alternatively, each may be involved
in promoting the translation of GLD-1 through independent means and only when
both activities are reduced is a threshold crossed where a dramatic decrease
in GLD-1 levels is realized. Since nos-3 and gld-2 activity
are each sufficient to achieve the normal pattern of GLD-1 accumulation, both
genes must be negatively regulated in the distal-most germline to keep GLD-1
levels low and allow proliferation.
Antagonistic relationship between FBF and NOS-3
FBF probably functions downstream of GLP-1/Notch signaling in inhibiting
GLD-1 accumulation (Fig. 7D),
because loss of FBF and GLP-1/Notch signaling have similar germline
phenotypes, and because FBF appears to directly inhibit GLD-1 translation. FBF
binds the gld-1 3'UTR, and there are putative binding sites in
the UTR that are removed in the gld-1(oz10gf) deletion
(Crittenden et al., 2002). In
gld-1(oz10gf) mutants, distal GLD-1 levels are increased
(Jones et al., 1996
) and
meiotic entry occurs more distally than normal (see Results). Therefore, FBF
probably functions directly to translationally inhibit GLD-1 accumulation.
Furthermore, since GLP-1 signaling also inhibits GLD-1 accumulation,
GLP-1/Notch signaling probably positively regulates FBF. It should be noted
that GLD-1 accumulation reaches a high level at
20 cell diameters from
the DTC (Jones et al., 1996
),
where FBF-1 levels are high (Crittenden et
al., 2002
), therefore the spatial patterning of FBF-1 does not
explain the distribution of GLD-1 in the distal arm.
Since FBF inhibits GLD-1 accumulation, it functions in opposition to NOS-3,
which promotes GLD-1 accumulation. We have shown that nos-3 mutants
suppress the Glp lf phenotype of fbf-1 fbf-2 mutants, and that
fbf-1fbf-2 nos-3 triple mutants display near wild-type distal GLD-1
patterning. This suggests that nos-3 functions genetically downstream
of fbf (Fig. 7D), or
parallel to it. The antagonistic relationship between FBF and NOS-3 contrasts
with their relationship in hermaphrodite germline sex determination where they
are thought to work together to inhibit fem-3 translation
(Kraemer et al., 1999) and is
at odds with their Drosophila homologues, Nanos and Pumilio, which
function together to repress translation
(Sonoda and Wharton,
1999
).
There are a number of possibilities to explain this unique antagonistic
relationship between Nanos and Pumilio homologues. First, although both FBF
and NOS-3 regulate entry into meiosis, they may not partner in this process.
Instead, FBF may partner with one of the other two NOS homologues
(Kraemer et al., 1999;
Subramaniam and Seydoux,
1999
), and NOS-3 may partner with one of the other ten PUF
proteins (Wickens et al.,
2002
). The genetic epistasis of fbf and nos-3
suggests that the FBF/NOS-X complex could function upstream and inhibit the
PUF-X/NOS-3 complex. However, this model is unlikely to be correct since FBF
directly binds to the gld-1 3'UTR in vitro
(Crittenden et al., 2002
).
Also, nos-3 cannot be a direct target of translational inhibition
because NOS-3 protein accumulation is uniform throughout the gonad
(Kraemer et al., 1999
),
although its partner PUF protein could be a target. Furthermore, FBF can bind
NOS-3, but not NOS-1 or NOS-2 in a two-hybrid assay or as GST-fusion proteins
in vitro (Kraemer et al.,
1999
). The possibility still remains, however, that binding
between FBF and NOS-1 or NOS-2 is dependent upon the presence of the target
RNA, as is the case with Drosophila Pumilio and Nanos
(Sonoda and Wharton,
1999
).
A second possible reason why FBF and NOS-3 have an antagonistic
relationship, unlike Nanos and Pumilio, could have to do the divergence of the
Nanos and NOS-3 proteins. Nanos is 401 amino acids in length while NOS-3 is
over twice that size at 871. Most similarity between the proteins exists in
the putative zinc finger domains, and even there they are only 26% identical
over 57 amino acids (Kraemer et al.,
1999; Subramaniam and Seydoux,
1999
). Furthermore, while Nanos and Pumilio are unable to
interact, except in the presence of target RNA
(Sonoda and Wharton, 1999
),
interaction of NOS-3 and FBF-1 is not RNA dependent
(Kraemer et al., 1999
). Nanos
appears to require its zinc finger motifs to complex with Pumilio and the
hunchback RNA (Sonoda and
Wharton, 1999
), while the NOS-3 zinc fingers are dispensable for
binding to FBF-1 (Kraemer et al.,
1999
). Perhaps the extensive differences between Nanos and NOS-3
reflect different molecular functions, and the relationship between NOS-3 and
FBF may not be completely analogous to Nanos and Pumilio, allowing an
inhibitory relationship to exist between NOS-3 and FBF.
Repression of GLD-2 activity in the proliferative zone
gld-2 and nos-3 are each sufficient to promote high
levels of GLD-1 since only in the double mutant are levels of GLD-1
dramatically reduced (Fig. 5,
Fig. 6C). Therefore, in the
most distal end of a wild-type germline, where GLD-1 levels are low, the
activities of GLD-2 and NOS-3 must each be repressed
(Fig. 7D). FBF probably
represses NOS-3 activity since nos-3 lf mutants suppress the
premature entry into meiosis phenotype of fbf-1 fbf-2, and since
fbf-1 fbf-2 nos-3 triple mutants have low GLD-1 levels in the distal
end (see above), and higher GLD-1 levels at 20 cell diameters away,
probably as a result of GLD-2 activity
(Fig. 6C). However, if
repression of GLD-2 was solely accomplished through FBF activity, then in
fbf-1 fbf-2 nos-3 triple mutant animals, the repression of
gld-2 would be relieved and wild-type gld-2 would be
sufficient to promote not just proximal (
20 cell diameters), but also
distal GLD-1 accumulation. Since distal GLD-1 accumulation is low in fbf-1
fbf-2 nos-3 triple mutants, GLD-2 activity must be repressed in the most
distal end by something (X) other than (or in addition to) FBF
(Fig. 7D).
Furthermore, since meiotic entry is normal in both gld-1 and
gld-2 single mutants (Francis et
al., 1995a; Kadyk and Kimble,
1998
), the activities of either gld-1 or gld-2
are sufficient for the switch from proliferation to meiotic prophase to occur.
The premature meiotic entry phenotype of fbf-1 fbf-2 double mutants
is probably primarily due to increased gld-1 activity, and not
gld-2 activity, because reducing the amount of gld-1 by half
(gld-1/+; fbf-1 fbf-2), suppresses the fbf-1 fbf-2 premature
meiotic entry phenotype (Crittenden et al.,
2002
). Since gld-2 activity is sufficient to cause the
switch from proliferation to meiotic prophase, if fbf-1 fbf-2
inhibits gld-2 activity in the most distal end then in gld-1/+;
fbf-1 fbf-2 mutants, the gld-2 suppression would be relieved and
cause premature meiotic entry. However, this is not seen, and therefore we
suggest that something other than fbf, or in addition to
fbf, inhibits gld-2 activity in the most distal end of the
germline (Fig. 7E). We note
that the lack of gld-2 activity does seem to weakly repress the
fbf-1 fbf-2 Glp phenotype, with gld-2 fbf-1 fbf-2 having
slightly larger germlines than fbf-1 fbf-2 double mutants. This
repression is minimal compared to that observed in fbf-1 fbf-2 nos-3
mutants, and could be caused by the lack of gld-2 activity slightly
reducing GLD-1 levels, since GLD-2 is a positive regulator of GLD-1
accumulation. Alternatively, FBF may function redundantly with the activity of
another factor(s) in repressing GLD-2 activity, therefore only minimal
repression of the fbf-1 fbf-2 Glp phenotype is observed when
gld-2 activity is removed.
gld-2 must have another role(s) in regulating entry into meiosis
in addition to promoting GLD-1 accumulation
(Fig. 7E). If gld-2
only promoted GLD-1 accumulation, then a gld-2 gld-1 double mutant
would have a similar phenotype to a gld-1 single mutant, however,
this is not the case. gld-2 gld-1 double mutants have a germline
tumor because of a defect in entry into meiosis
(Kadyk and Kimble, 1998),
while germ cells in a gld-1 single mutant enter meiosis normally
(Francis et al., 1995a
). In
addition, gld-2 gld-1 double mutant males have tumorous germlines
(Kadyk and Kimble, 1998
),
while gld-1 single mutant males have wild-type germlines
(Francis et al., 1995b
).
Therefore, there must be another downstream target(s) of gld-2
activity in regulating entry into meiosis. GLD-2, in part, may be a positive
regulator of meiosis-specific genes since it is thought to lengthen poly(A)
tails of target mRNAs (Wang et al.,
2002
), thereby promoting translation. Conversely, GLD-1 functions
as an inhibitor of translation (Clifford et
al., 2000
; Jan et al.,
1999
; Lee and Schedl,
2001
), and therefore may, in part, represses
proliferation-specific gene products.
Maintenance of a stem cell population
The balance between proliferation and differentiation must be tightly
controlled in order for a stem cell population to be maintained and for
required tissues to be generated. In order to understand the behavior of stem
cells, and thereby harness their therapeutic potential, it is important that
we understand the mechanisms involved in regulating the proliferation versus
differentiation decision. In the C. elegans germline, we have shown
that this decision relies on the spatial pattern of GLD-1 levels. The genetic
hierarchy controlling this spatial pattern, beginning with the restriction of
GLP-1/Notch signaling to the most distal end of the gonad and culminating in
the promoting influence of gld-2 and nos-3, provides an
excellent example of how tight control of protein levels can set the boundary
for a niche, within which stem cell proliferation can occur.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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