|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 11 March 2009
doi: 10.1242/dev.030619
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Division of Developmental Biology, Cincinnati Children's Hospital Research
Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
2 Molecular and Developmental Biology Graduate Program, Cincinnati Children's
Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229,
USA.
3 Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2
1QR, UK.
* Author for correspondence (e-mail: Christopher.Wylie{at}cchmc.org)
Accepted 9 February 2009
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Primordial germ cell, Steel factor, Stem cell niche, Mouse
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Saitou et al., 2002;
Tanaka and Matsui, 2002).
Within this group of epiblast cells, about six cells activate the expression
of Blimp1 (Prdm1 - Mouse Genome Informatics), a marker of lineage-restricted
PGC precursors, whilst neighboring cells acquire a somatic fate. The
Blimp1-positive PGC precursors increase in number as they move to the
posterior primitive streak and allantois, where they become specified at E7.25
to form exclusively PGCs, and express markers of specified PGCs, such as
alkaline phosphatase (AP) and Stella (Dppa3 - Mouse Genome Informatics)
(Hayashi et al., 2007;
Ohinata et al., 2005). The
details of PGC behavior between E7.25 and E8.5 are not clear. However, by
E8.5, PGCs are localized around the hindgut diverticulum, and by E9.0 they are
incorporated into the hindgut epithelium
(Anderson et al., 2000). PGCs
emigrate from the dorsal aspect of the hindgut between E9.0 and E9.5, separate
into left and right streams of individual cells, and migrate laterally across
the dorsal body wall. At E10.5, PGCs close to the genital ridges continue to
migrate, singly or in clusters, into the genital ridges to form the primary
sex cords, while PGCs remaining in the midline structures die by apoptosis
(Molyneaux et al., 2001).
Steel factor (also known as stem cell factor, kit ligand or mast cell
growth factor) is the product of the Steel locus and a member of the
short-chain helical cytokine family. It has been shown by many studies to be
an essential survival factor for PGCs (De
Felici and Pesce, 1994; Dolci
et al., 1991; Godin et al.,
1991; Matsui et al., 1991). There are two forms of Steel protein,
generated by alternative splicing: soluble Steel factor and membrane-bound
Steel factor. The membrane-bound form lacks an extracellular domain containing
a proteolytic cleavage site, which normally causes release of the
extracellular region of the protein
(Flanagan et al., 1991;
Huang et al., 1992). The
receptor for Steel factor is the product of the W locus, c-Kit, a
tyrosine-kinase receptor of the PDGFRB superfamily that is expressed in PGCs
throughout migration from E7.25 (Loveland
and Schlatt, 1997; Yabuta et
al., 2006). In the absence of either ligand or receptor, mice are
sterile, and reduced numbers of PGCs are seen during migration. In addition,
surviving PGCs in Steel-/- embryos are often described as
being retarded in their migration, clumped, and in ectopic locations
(Bennett, 1956;
Mahakali Zama et al., 2005;
McCoshen and McCallion, 1975;
Mintz and Russell, 1957).
Recent work from our laboratory has revealed novel aspects of Steel factor
function in regulating migratory PGC behavior
(Runyan et al., 2006). At
E9.5, Steel factor is expressed in midline structures as well as in the
genital ridges. At E10.5, Steel factor expression is lost from midline
structures, but is enriched in the genital ridges. The change in location of
Steel factor expression causes PGCs in the midline to die through the
intrinsic apoptotic pathway, but the survival of PGCs closest to the genital
ridges is maintained. Steel factor functions at close range, as the genital
ridges are only about 100 µm away from the midline at E10.5. Analysis of
embryos in which PGC apoptosis was blocked showed that, in addition to its
role in PGC survival, Steel factor is also required between E9.0 and E10.5 for
PGC proliferation and migration (Runyan et
al., 2006). These data suggest the existence of a `traveling
niche' of Steel factor-expressing cells that control many aspects of PGC
behavior.
Little is known about when the association between PGCs and Steel
factor-expressing cells begins, or whether it exists throughout PGC migration.
Classical studies of W and Steel mutants have reported that PGCs only become
Steel factor-dependent in the hindgut
(Bennett, 1956;
Mahakali Zama et al., 2005;
McCoshen and McCallion, 1975;
Mintz and Russell, 1957).
However, the inability to visualize PGCs in living embryos before gut
colonization has made this period of their development inaccessible to study.
In this paper, a novel reporter mouse line expressing GFP under the
Stella promoter (Payer et al.,
2006) has been used to visualize PGCs from the time of their first
appearance in the embryo. We show that PGCs are surrounded by Steel
factor-expressing cells, from the time they first turn on expression of
Stella in the allantois to the time they colonize the genital ridges.
Fewer PGCs are found in the allantois in Steel-null mutant embryos
when compared with their wild-type littermates, indicating that Steel factor
is required to maintain PGC numbers as early as E7.5. Moreover, PGCs in
Steel-/- embryos show reduced motility and increased clump
formation, but the direction of PGC migration is not randomized. Although germ
cells are known to express E-cadherin
(Okamura et al., 2003),
clumping is not due to an upregulation of this adhesion protein. The results
were further confirmed by the acute loss of Steel signaling following the use
of an Ack2 antibody, an antibody that blocks the c-Kit receptor. Analyses of
PGC migration in E9.0 embryos revealed that Steel factor is also required for
normal PGC motility at this stage, in addition to its role in regulating PGC
survival and proliferation. These data show that a wave of Steel factor
expression moves with the PGCs, from the time of their specification to their
arrival in the genital ridges, thereby forming a traveling niche for this
important population of migrating pluripotential cells.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
) on a B6/CBA background with 6- to 8-week old CD1
females (Charles River). Embryonic day 0.5 (E0.5) was assumed to be noon of
the morning a vaginal plug was observed. Stella-GFP mice were crossed
with KitlSl heterozygotes (Jackson Laboratories, Stock
number 000693) to obtain mice that were Stella-GFP+,
Steel+/-. These were interbred to yield
Stella-GFP+, Steel-/- embryos. To generate
Steel/Bax double mutants, Stella-GFP+, Steel+/-
mice were bred with Bax heterozygotes
(Knudson et al., 1995). For
E9.0 studies, Oct4
PE:GFP transgenic mice
(Anderson et al., 2000) were
used instead of Stella-GFP mice. Genomic DNA was isolated from tails
snips (adults), embryo halves (E7.5 embryos) and heads (E9.0 embryos), and
genotypes were determined by PCR. Genotyping primers used were as follows:
Stella-GFP, F-5'-TGCATCGGTAACCCACAGTA-3',
R-5'-GAACTTCAGGGTCAGCTTGC-3'; KitlSl,
Common-F-5'-CGGGGTTTATGAGGGTAGGA-3',
WT-R-5'-TTGGGCCTGTGTGACAAACT-3',
DEL-R-5'-ACTTCCTAGGGCTGGAGAGATG-3'; Bax
(Deckwerth et al., 1996),
EX5-F-5'-GAGCTGATCAGAACCATCATG-3',
IN5-R-5'-GTTGACCAGAGTGGCGTAGG-3',
NeoPGK-R-5'-CCGCTTCCATTGCTCAGCGG-3';
Oct4PE:GFP (Yeom
et al., 1996), F-5'-GGAGAGGTGAAACCGTCCCTAGG-3',
R-5'-GCATCGCCCTCGCCCTCGC-3'. Stella-GFP expression was
determined by the presence of a 289-bp fragment. For
KitlSl, wild-type and deleted alleles were determined by
the presence of 294- and 646-bp fragments, respectively. For genotyping of
Bax, wild-type and mutant alleles were determined by the presence of
304- and 507-bp fragments, respectively. Oct4PE:GFP
transgene expression was determined by the presence of a 250-bp fragment.
Embryo slice culture
E7.25 and E7.5 embryos were cut into halves along the sagittal axis using a
scalpel. One half of each embryo was placed onto a millicell CM organ culture
insert (Millipore) pre-coated with collagen IV (BD) and the other half was
used for genotyping. For E9.0 embryos, the caudal halves were cultured on the
inserts as described previously (Molyneaux
et al., 2001). The millicell organ inserts were then placed into a
metal stage that contained glass-bottom chambers, and incubated in 600 µl
Hepes-buffered DMEM/F-12 (Gibco) medium with 0.04% lipid-free BSA and 100 U/ml
penicillin/streptomycin (Gibco). To generate an acute loss of Steel signaling,
10 µg/ml c-Kit blocking antibody, Ack2 (a kind gift from Dr Fred Finkelman,
CCHMC), was added to the slice culture medium. Mouse IgG (10 µg/ml, Jackson
ImmunoResearch) was used as a control. Embryo slices were maintained at
37°C by placing the metal stage in a temperature-controlled stage (Zeiss)
and maintaining humidity with wet paper towels placed in a 100 mm culture dish
fastened over the organ culture chambers.
Time-lapse analysis of migrating PGCs
Slices were filmed with a Zeiss LSM510 confocal system attached to a Zeiss
axiovert microscope. Pictures were acquired every 5 minutes for 6 to 10 hours,
and movies were analyzed using NIH image as described
(Molyneaux et al., 2001).
Briefly, all cells that remained in focus for the duration of the filming were
traced, and the average velocity, maximum velocity and displacement were
measured for each of these cells using two cell-tracking macros written for
the NIH ImageJ software by Kathy Molyneaux or by Erik Meijering. Their
directionality was also recorded and the net trajectory for all cells in each
embryo was plotted on a windrose diagram. Experiments were repeated at least
three times, and three to eight embryos were analyzed per group. For
statistical comparisons, the data were analyzed using an unpaired, two-tailed
Student's t-test with equal variances.
RT-PCR
For RT-PCR analysis, allantoides from E7.5 embryos, and genital ridges from
E10.5 embryos were dissected, and RNA extracted from dissected tissues using
the RNeasy Kit (Qiagen). RNA (10 ng) was reverse transcribed by using
Superscript III First-Strand Synthesis Systems (Invitrogen), following the
manufacturer's recommendations. PCR reactions were performed using Redmix Plus
(GeneChoice). Primers used were as follows: membrane-bound Steel factor,
F-5'-TCCCGAGAAAGGGAAAGC-3',
R-5'-CTGCCCTTGTAAGACTTGACTG-3' (predicted fragment length, 149
bp); soluble Steel factor, F-5'-TTATGTTACCCCCTGTTGCAG-3',
R-5'-CTGCCCTTGTAAGACTTGACTG-3' (predicted fragment length, 195
bp).
Immunofluorescence analysis on whole-mount embryos or frozen sections
Embryos were fixed in 4% paraformaldehyde (PFA). For whole-mount staining,
embryos were then washed in 0.5% NP-40 (2x10 minutes), blocked in PBSST
(PBS/0.3% Triton X-100 with 5% goat or donkey sera, 2x1 hour washes) and
incubated overnight at 4°C in PBSST with primary antibody. The following
day, the embryos were washed in PBST (PBS/0.3% Triton X-100, 2x15
minutes, then 3x1 hour) at 4°C, incubated overnight at 4°C in
PBSST with Cy3- or Cy5-conjugated secondary antibodies (Jackson
ImmunoResearch), washed in PBST (2x15 minutes, then 3x1 hour) and
cleared in 50%, then 90%, glycerol for imaging. Embryos to be sectioned were
dehydrated in sucrose and mounted in OCT compound (Tissue-Tek) for
cryosectioning. Sectioned-embryos were rehydrated with PBST (10 minutes),
blocked with PBSST (1 hour at room temperature), and incubated with primary
antibody overnight at 4°C. The next day, slides were washed with PBST
(3x15 minutes), incubated in PBSST with secondary antibody (2 hours),
washed with PBST (2x15 minutes) and mounted with DABCO (Sigma) for
imaging.
Primary antibodies were used at the following dilutions: 1:50 for anti-Steel factor (R&D Systems), 1:200 for anti-cleaved-PARP (Cell Signaling), 1:2000 for anti-phospho-histone H3 (Upstate), 1:250 for anti-E-cadherin (a kind gift from Dr Masatoshi Takeichi, RIKEN Center for Developmental Biology, Japan). Secondary antibodies (Jackson ImmunoResearch) were used at the following dilutions: 1:300 for Cy5 donkey anti-goat; 1:300 for Cy5 goat anti-rabbit; 1:300 for Cy5 goat anti-rat; 1:300 for Cy3 goat anti-mouse. Images were captured using a Zeiss LSM 510 confocal system.
|
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
), which
showed that Steel factor was expressed in PGCs at E7.25, but was downregulated
within 24 hours. Somatic cells immediately adjacent to PGCs also showed high
levels of Steel factor staining on their cell surfaces at E7.5
(Fig. 1A-C). Diffuse staining
was seen in other regions. We interpret this to be soluble Steel factor, on
the basis that the Steel-/- embryos completely lacked this
diffuse staining (Fig. 1I).
Whole-mount staining of E8.5 embryos showed that Steel protein was strongly
expressed in cells of the visceral endoderm, as well as the adjacent hindgut
diverticulum containing the PGCs (Fig.
1D). Transverse sections through the hindgut diverticulum at E8.5
showed that PGCs occupied only the ventral aspect of the hindgut epithelium,
which was stained strongly for Steel factor, but not the dorsal part, which
lacked Steel factor expression (Fig.
1E,F). At E9.0, Steel factor was expressed by all cells of the
hindgut, and PGCs now occupied the dorsal, as well as the ventral, hindgut
(Fig. 1G). At E10.0, Steel
factor staining was reduced or absent in the hindgut epithelium, and was
enriched in tissues surrounding the PGCs in the midline dorsal body wall and
the coelomic angles (Fig. 1H).
As published previously (Runyan et al.,
2006), Steel factor expression is subsequently lost in midline
structures at E10.75 and becomes restricted bilaterally to the genital ridges.
The data presented here, together with the previously published results, show
that PGCs are immediately surrounded by cells expressing Steel factor from the
time they first appear in the allantois to the time they colonize the genital
ridges. As shown in Fig. 1B,C, cell membrane-localized Steel factor was seen in the allantois at E7.5. To confirm the presence of membrane-bound Steel factor in the allantois, we fixed E7.5 embryos with 2% trichloroacetic acid (TCA), which gave a better cross-reaction with the anti-Steel factor antibody. TCA fixation causes loss of the GFP signal, so PGCs could not be identified. However, a group of cells in the core of the allantois showed strong membrane staining of Steel factor (Fig. 1J, arrow). We also dissected allantoides from E7.5 embryos, and performed RT-PCR using primers that distinguish membrane-bound and soluble Steel factors. The results of the RT-PCR confirmed the presence of both soluble and membrane-bound Steel factor in the allantois at E7.5 (Fig. 1K).
PGCs first express Stella in the extraembryonic allantois, and migrate proximally into the posterior epiblast
It has been reported that PGCs are specified in the allantois around E7.25
(Hayashi et al., 2007;
Ohinata et al., 2005), but
little is known about the behavior of PGCs after their specification. To study
PGC behavior before they colonize the hindgut, we developed a method of
culturing bisected Stella-GFP embryos at E7.25 and E7.5, and made
time-lapse movies during these time periods.
Fig. 2A shows a sequence of
frames from one movie started at E7.25 (see also Movie 1 in the supplementary
material). During the time period of this movie (2.5 hours), PGCs turned on
Stella expression in the extraembryonic mesenchyme of the allantois,
and migrated proximally towards the embryo
(Fig. 2A). Seven out of the
eight movies started at E7.25 showed PGC migration in this direction.
Fig. 2B shows a movie started
at E7.5 (Movie 2 in the supplementary material). More PGCs were seen at the
beginning of movies started at this stage. During the culture period, PGCs
moved from the allantois (AL) into the posterior epiblast (EP) of the embryo.
They came to occupy a region that included the proximal epiblast and proximal
allantois. Once in this region, they migrated in random directions within it.
PGCs that migrated to the edges of the region turned back again, indicating a
mechanism for retention of the PGCs in this posterior region of the embryo. In
eight out of 10 movies started at E7.5, we observed the PGC behavior shown in
Fig. 2B.
|
|
;
Buehr et al., 1993; Matsui et
al., 1991) and survival (Godin et al.,
1991; Dolci et al.,
1991; Matsui et al., 1991; De
Felici et al., 1999; Stallock
et al., 2003). To distinguish between effects on proliferation and
apoptosis of PGCs in the absence of Steel factor, whole-mount embryos at E7.5
were stained using the phospho-histone H3 antibody to detect mitosis and the
cleaved-PARP antibody to detect apoptosis. We observed that a few PGCs were
positively stained for phospho-histone H3 or cleaved-PARP in the embryos (data
not shown). However, the low PGC numbers present at this stage made it
difficult to perform statistical analysis.
Recent work from our laboratory showed that loss of Steel factor leads to
PGC apoptosis beginning on or before E9.0, which can be rescued by removal of
the pro-apoptotic protein Bax (Runyan et
al., 2006). We therefore examined embryos from
Steel/Bax crosses at E7.5. In five Bax+/-,
Steel-/- embryos examined, loss of one allele of Bax
rescued the decrease of PGC number in Steel-/- embryos at
E7.5 (Fig. 3A). In
Steel-/- embryos, PGC numbers increased from 15±4.3
to 29±9.3 (P=0.041) in the absence of one allele of
Bax. Sample embryos from which these counts were made are shown in
Fig. 3B. These data show that
Steel factor is an essential survival factor for PGCs even before they enter
the hindgut, and that PGCs die through the Bax-dependent apoptotic pathway at
E7.5 when they lack Steel factor. The data also exclude the possibility that
Steel factor is required for initial PGC specification, as the number of PGCs
increased in Steel-null embryos when apoptosis was inhibited.
Steel factor is required for normal PGC migration before entry into the hindgut
Time-lapse movies were made using sagittally bisected E7.5 Steel
null embryos and their littermates. Examples of movie frames from embryos of
different genotypes are shown at time=0 and time=413 minutes
(Fig. 4A,B; from Movies 3-5,
see supplementary material). We manually traced PGCs in time-lapse movies,
obtained trajectories of all the PGCs whose migratory routes remained in the
confocal plane throughout the movie (Fig.
4C,D), and calculated the velocities and displacements of PGC
movement (Fig. 4E). PGCs in
Steel+/+ embryos were highly motile, with a maximum
velocity of 51.4±3.1 µm/hour, an average velocity of 24.7±1.2
µm/hour and an average total displacement during the time of filming of
83.1±8.9 µm. The maximum velocity (50.1±3.3 µm/hour),
average velocity (23.6±1.3 µm/hour) and displacement
(78.9±10.8 µm) of PGCs in Steel+/- embryos were
not significantly changed from those in Steel+/+ embryos
(P=0.58, 0.23 and 0.57, respectively). However, PGCs in
Steel-/- embryos had significantly decreased maximum
velocities (37.4±2.9 µm/hour), average velocities (17.5±0.8
µm/hour) and displacements (44.9±7.7 µm) compared with PGCs in
Steel+/+ (P=1.0x10-6,
3.8x10-10 and 1.2x10-7, respectively) and
Steel+/- littermates
(P=1.8x10-7, 7.4x10-12 and
2.2x10-6, respectively). These data indicate that Steel
factor is necessary for active PGC motility before entry into the hindgut.
|
One result of decreased PGC motility was that fewer PGCs reached the posterior epiblast. To show this, the positions of individual PGCs at the end of the movies were scored. PGCs that had migrated across the boundary (white line, Fig. 4C) between the extraembryonic region (EEM) and the posterior end of the embryo (PEM) were scored as `enter embryo'. Only 18.8±12.3% of PGCs entered the posterior region of the embryo in the absence of Steel factor (Fig. 4G), which was a significant reduction when compared with Steel+/+ (66.8±5.4%, P=3.7x10-5) and heterozygous (51.4±4.0%, P=0.0001) littermates. This result confirmed the hypothesis that Steel factor is required for normal PGC migration before they enter into the hindgut.
|
|
;
Bendel-Stenzel et al., 2000).
To test whether PGC clumping is due to precocious expression of E-cadherin, we
stained wild-type and Steel-/- embryos at E7.5 as whole
mounts with the ECCD2 antibody against E-cadherin
(Shirayoshi et al., 1986).
E-cadherin was not upregulated by PGCs in Steel-/- embryos at this
stage (Fig. 5A). It is possible
that the expression of other adhesion molecules is responsible for clumping
and for the failed motility. However, other adhesion molecules have not yet
been characterized in early migrating germ cells. To exclude the possibility that the failure of PGC motility was due to apoptosis, we analyzed maximum and average velocities, and total displacements of PGCs in E7.5 Steel-/-, Bax+/- embryos. The loss of one allele of Bax rescues germ cell numbers (Fig. 3), but does not rescue germ cell motility (Fig. 6).
To exclude the possibility that the defects of PGC migration observed in
Steel-/- embryos were consequences of a previous
requirement for Steel factor before migration in the allantois, we carried out
an `acute' blockade to Steel factor signaling by culturing bisected E7.5
Steel+/+ embryos in the presence of Ack2 antibody, which
has been shown to effectively block Steel signaling and Steel factor function
in PGCs (Nishikawa et al.,
1991; Runyan et al.,
2006; Stallock et al.,
2003). As shown in Fig.
7, both the velocity and displacement of PGCs were significantly
decreased by treatment with 10 µg/ml Ack2 for 6 hours when compared with
those in control embryos (P<0.01;
Fig. 7A). In addition, the
proportion of PGCs entering the posterior embryo was significantly decreased
by Ack2 treatment (25.5±9.2% compared with 53.4±6.9%,
P=0.003; Fig. 7B).
PGCs also clumped more in the presence of Ack2 (42.3±7.5% of PGCs in
clusters) than did those in control embryos (15.3±5.6%,
P=0.002; Fig. 7C).
Movies of E7.5 Ack2-treated embryos and control embryos incubated in the same
concentration of a non-immune IgG, which did not affect PGC behavior can be
found in the supplementary material (see Movies 6 and 7). Treatment with the
Ack2 antibody did not alter PGC numbers, probably because of the short
time-period (6 hours) of the experiment. The results of this acute blockade of
Steel factor signaling confirm that it is required for PGC migration at E7.5,
and that the defects seen at this stage in Steel-/-
embryos were not due to a previous requirement for Steel factor (for example,
in PGC specification).
Steel factor is required for PGC motility in the hindgut
We reported previously that the rescue of PGC apoptosis in
Steel-/- embryos by the removal of Bax revealed roles for
Steel factor in both proliferation and migration between E9.0 and E10.5
(Runyan et al., 2006). The
defects in later migration in Steel/Bax mutant embryos could have
been due to either a failure of motility or directionality. In this study, we
have shown that at an earlier stage, before colonization of the hindgut, Steel
factor is required for motility but not directionality of the PGCs
(Fig. 4). To test whether the
same is true once PGCs have colonized the hindgut, time-lapse movies were made
using E9.0 Oct4PE:GFP embryos from Steel/Bax
crosses. No differences in migration were observed between
Bax-/- and Bax+/- PGCs, so the data
from these two groups were combined and the embryos were grouped based upon
their Steel genotype. Frames from the time-lapse movies are shown at time=0
(Fig. 8A) and time=420 minutes
(Fig. 8B); the migration
directions of individual PGCs were plotted using the method described above
(Fig. 8C).
Fig. 8D shows that PGCs in
Steel-/- embryos had significantly decreased displacement
(22.6±5.22 µm) compared with those in Steel+/+
(56.8±5.8 µm, P=6.93x10-8) and
Steel+/- (65.1±8.1 µm,
P=1.89x10-9) littermates. They also showed a
decreased maximum velocity (11.8±1.4 µm/hour), compared with
Steel+/+ (22.2±1.4 µm/hour,
P=2.49x10-10) and Steel+/-
(20.9±1.7 µm/hour, P=1.00x10-9), and a
decreased average velocity (5.0±0.5 µm/hour) compared with
Steel+/+ (9.6±0.6 µm/hour,
P=3.48x10-12) and Steel+/-
(10.1±0.8 µm/hour, P=1.24x10-12). Windrose
assays (Fig. 8E) showed that
all of the PGCs migrated in a net dorsal and slightly rostral direction (right
panel, Fig. 8E), regardless of
the Steel gene dosage. These results suggest that Steel factor plays
an essential role in PGC motility, both in the allantois and in the hindgut,
in addition to its role in PGC survival.
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Godin et al.,
1991; Matsui et al., 1991). Early studies reported that PGC
numbers in Steel mutants are similar to those in wild-type embryos during the
period E8.0 to E9.0, suggesting that PGCs are independent of Steel factor
until colonization of the hindgut (Bennett,
1956; Mahakali Zama et al.,
2005; McCoshen and McCallion,
1975; Mintz and Russell,
1957). However, recently published data suggest the need for a
reappraisal of this. First, PGC numbers are already dramatically reduced at
E9.0 in Steel-mutated embryos
(Runyan et al., 2006). Second,
Steel expression in the allantois, and c-Kit expression by PGCs, have recently
been found to commence at E7.25, the time of PGC appearance
(Yabuta et al., 2006). In this
paper, we have shown that PGCs are surrounded by Steel factor-expressing cells
from the time that they first express Stella, and that normal PGC
behavior is controlled by Steel factor from the time of their first appearance
in the embryo. Together with previous work, this shows that PGCs require Steel
factor throughout their migration, from the time of formation to the time of
colonization of the gonads.
In a previous paper, we showed that PGCs are surrounded by Steel factor at
E9.5, when emigrating from the hindgut, and at E10.5, when in the genital
ridges (Runyan et al., 2006).
Here, we show that Steel factor expression surrounds PGCs at all earlier
stages. First, in the allantois, where Stella expression is first
activated in PGCs, Steel factor is expressed in a group of cells in the
mesodermal core. PGCs also initially express Steel factor at this time,
consistent with RT-PCR results from a previous study
(Yabuta et al., 2006). By
E8.5, PGCs occupy the ventral aspect of the hindgut epithelium, which also
stains strongly for Steel factor. At E9.0, both the ventral and the dorsal gut
epithelium express Steel factor, and as described before
(Molyneaux et al., 2001), PGCs
have now moved dorsally in the hindgut. At E10.0, when PGCs are concentrated
in the dorsal midline, Steel factor is enriched in the midline, but has been
lost from the hindgut. These data show that migratory PGCs are in a Steel
factor-enriched microenvironment from the time of their first appearance to
the time they colonize the gonads.
There are two forms of Steel protein, generated by alternative splicing of
Steel precursor RNA. The transmembrane form lacks an extracellular domain
containing a proteolytic cleavage site, which normally causes release of the
extracellular region of the protein
(Flanagan et al., 1991;
Huang et al., 1992). The
Steel-dickie (Steeld/d) mutation, in which only soluble
Steel factor is made, is sterile (Brannan
et al., 1991), suggesting that the membrane-bound form is
essential at some stage of PGC differentiation. Moreover, PGC numbers are
already significantly reduced in Steeld/d embryos at E9.5
(Mahakali Zama et al., 2005),
indicating a requirement for membrane-bound Steel factor earlier than this. In
our study, the staining pattern of Steel factor and the RT-PCR analyses at
E7.5 show that PGCs become surrounded by cells expressing membrane-bound Steel
factor when they are in the allantois, suggesting that PGC behaviors may be
dependent on this close-range signaling as they first form in the embryo. It
is possible that the membrane-bound Steel protein controls different aspects
of PGC behaviors from the soluble Steel factor. It is also possible that the
higher local concentration of membrane-bound Steel makes it indispensable for
PGC development. Future work will focus on trying to distinguish between these
possibilities.
In the Steel-/- embryos examined, PGC numbers were
already reduced (
40% of control numbers) at E7.5, suggesting that PGC
numbers are controlled by Steel factor as soon as they appear in the embryo.
This is almost certainly a direct interaction, as only PGCs express c-Kit in
the allantois at this stage (Yabuta et
al., 2006). The reduction of PGC numbers in the absence of Steel
factor could be caused in three ways: increased apoptosis, decreased
proliferation, or defects in PGC specification. The low PGC numbers at this
stage make statistical analysis of cleaved-PARP staining (cell death) or
phospho-histone H3 staining (mitosis) extremely difficult. However, in the
five embryos examined so far that were Steel-/- and
Bax+/-, PGC numbers were dramatically increased by the
removal of one allele of Bax, implying that Steel factor is not
required for PGC specification, but is required for their survival. This
result does not exclude the possibility that PGCs also require Steel factor
for their proliferation at this stage. Further investigations will be
performed in a Bax-null background to study the role of Steel factor
on PGC proliferation.
Steel factor has been considered an essential factor for PGC survival and proliferation in previous studies. However, the precise role played by Steel factor in PGC migration has not been clear. Here, we show that germ cells are actively migratory before hindgut colonization, and that Steel factor is required for their motility, but not their directionality, at this stage. Tracing individual PGCs on movie frames starting at E7.5 revealed that both the velocities and the displacements of PGCs were significantly decreased in Steel-null embryos. PGCs also failed to move away from each other in Steel-/- embryos, and instead formed clusters in the proximal region of the extraembryonic allantois. It is not clear why PGCs should adhere to each other without Steel factor. It may be that germ cells become specified as a group, and decreased motility causes them to fail to move away from the group. Alternatively, Steel factor may inhibit the expression of adhesion proteins. Acute blockade of Steel factor signaling by culturing E7.5 bisected embryos in the presence of Ack2 antibody confirmed the effects of Steel factor on PGC motility at this stage, demonstrating that the motility defects of PGCs are not consequences of a previous requirement for Steel factor.
|
). Because of these small numbers, the Bax-null
mutation was bred into Steel mutants in order to reduce PGC apoptosis
and generate enough PGCs for motility analyses. The defects in PGC migration
in the hindgut in the absence of Steel factor were the same as those found in
the allantois earlier. Although PGCs in all genotypes migrated in a net dorsal
and slightly rostral direction in the hindgut at E9.0, both the velocities and
the displacements of PGCs were significantly decreased in the absence of Steel
factor. These data may explain why clumps of PGCs are found in structures
ventral to the gut in both Steel and W mutant embryos after
E9.0 (Buehr et al., 1993;
Mahakali Zama et al., 2005;
Runyan et al., 2006).
Previously published data show that Steel factor is also required for later
stages of PGC migration. In E10.5 Steel/Bax double-null embryos, germ
cells were not found in their normal site, the dorsal body wall, but instead
were found clumped around the hindgut
(Runyan et al., 2006).
In vitro, using modified Boyden chambers, it has been reported that Steel
factor has a chemotactic function (Farini
et al., 2007). However, the in vivo experiments described here at
two stages, before and after colonization of the hindgut, show that Steel
factor is essential for PGC motility, but that the direction of movement is
not randomized in the absence of Steel factor. This discrepancy could be due
to other guidance cues being available in the absence of Steel factor, or to a
requirement for Steel factor for guidance later in germ cell migration, which
was not tested here. At E7.5, during migration from the allantois to the
posterior epiblast, the directionality of PGCs in Steel-null embryos
is altered a little. This could be because the germ cells migrate more slowly
in these embryos, and are therefore affected more by other morphogenetic
movements taking place at this time, although this possibility could not be
tested.
An essential feature of PGC specification is the turning off of genes that
initiate somatic cell fate, including Evx1, Hox genes and brachyury,
and the maintenance (or switching on) of genes that maintain pluripotency in
the germ cell lineage, including Stella, fragilis (Ifitm3 -
Mouse Genome Informatics), Oct4, Sox2, Nanos3 and Nanog
(reviewed by Saitou et al.,
2005). This process takes place in the allantois, and both the
sources and the natures of the signals that control it are poorly understood.
In well-characterized stem cell populations, such as male and female stem
cells in the gonad, epidermal stem cells and hemopoietic stem cells, signals
that control pluripotency and behavior are provided by the `niche' in which
the stem cells reside. However, germ cells are migratory and have no obvious
niche, although the maintenance of all aspects of their behavior, including
proliferation rate, migration and survival, are all continuously controlled
(Kunwar et al., 2006;
Runyan et al., 2006;
Surani et al., 2007). Our
results, together with previously published data, show that PGCs are
surrounded by cells releasing Steel factor throughout their migration, from
the time they first form in the allantois to the time they colonize the
gonads, and that normal PGC behaviors, including survival, proliferation and
motility, are all controlled by this close-range signaling. This suggests the
existence of a `traveling niche' in which the Steel factor-expressing cells
provide a spatio-temporal environment along the migratory route to retain the
normal properties of PGCs as they occupy different regions of the embryo. How
this Steel factor niche is established and maintained, and whether the Steel
factor-expressing cells also release other signaling ligands that control PGC
behavior, are interesting questions to answer in future studies.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/8/1295/DC1
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Anderson, R., Copeland, T. K., Scholer, H., Heasman, J. and
Wylie, C. (2000). The onset of germ cell migration in the
mouse embryo. Mech. Dev.
91, 61-68.[CrossRef][Medline]
Bendel-Stenzel, M. R., Gomperts, M., Anderson, R., Heasman, J.
and Wylie, C. (2000). The role of cadherins during primordial
germ cell migration and early gonad formation in the mouse. Mech.
Dev. 91,143
-152.[CrossRef][Medline]
Bennett, D. (1956). Developmental analysis of a
mutation with pleiotropic effects in the mouse. J.
Morphol. 98,57
-70.
Brannan, C. I., Lyman, S. D., Williams, D. E., Eisenman, J.,
Anderson, D. M., Cosman, D., Bedell, M. A., Jenkins, N. A. and Copeland, N.
G. (1991). Steel-Dickie mutation encodes a c-kit ligand
lacking transmembrane and cytoplasmic domains. Proc. Natl. Acad.
Sci. USA 88,4671
-4674.
Buehr, M., McLaren, A., Bartley, A. and Darling, S.
(1993). Proliferation and migration of primordial germ cells in
We/We mouse embryos. Dev. Dyn.
198,182
-189.[Medline]
Deckwerth, T. L., Elliott, J. L., Knudson, C. M., Johnson, E.
M., Jr, Snider, W. D. and Korsmeyer, S. J. (1996). BAX is
required for neuronal death after trophic factor deprivation and during
development. Neuron 17,401
-411.[CrossRef][Medline]
De Felici, M. and Pesce, M. (1994). Growth
factors in mouse primordial germ cell migration and proliferation.
Prog. Growth Factor Res.
5, 135-143.[CrossRef][Medline]
De Felici, M., Carlo, A. D., Pesce, M., Iona, S., Farrace, M. G.
and Piacentini, M. (1999). Bcl-2 and Bax regulation of
apoptosis in germ cells during prenatal oogenesis in the mouse embryo.
Cell Death Differ. 6,908
-915.[CrossRef][Medline]
Dolci, S., Williams, D. E., Ernst, M. K., Resnick, J. L.,
Brannan, C. I., Lock, L. F., Lyman, S. D., Boswell, H. S. and Donovan, P.
J. (1991). Requirement for mast cell growth factor for
primordial germ cell survival in culture. Nature
352,809
-811.[CrossRef][Medline]
Farini, D., La Sala, G., Tedesco, M. and De Felici, M.
(2007). Chemoattractant action and molecular signaling pathways
of Kit ligand on mouse primordial germ cells. Dev.
Biol. 306,572
-583.[CrossRef][Medline]
Flanagan, J. G., Chan, D. C. and Leder, P.
(1991). Transmembrane form of the kit ligand growth factor is
determined by alternative splicing and is missing in the Sld mutant.
Cell 64,1025
-1035.[CrossRef][Medline]
Godin, I., Deed, R., Cooke, J., Zsebo, K., Dexter, M. and Wylie,
C. C. (1991). Effects of the steel gene product on mouse
primordial germ cells in culture. Nature
352,807
-809.[CrossRef][Medline]
Hayashi, K., de Sousa Lopes, S. M. and Surani, M. A.
(2007). Germ cell specification in mice.
Science 316,394
-396.
Huang, E. J., Nocka, K. H., Buck, J. and Besmer, P.
(1992). Differential expression and processing of two cell
associated forms of the kit-ligand: KL-1 and KL-2. Mol. Biol.
Cell 3,349
-362.[Abstract]
Knudson, C. M., Tung, K. S. K., Tourtellotte, W. G., Brown, G.
A. J. and Korsmeyer, S. J. (1995). Bax-deficient mice with
lymphoid hyperplasia and male germ cell death. Science
270, 96-99.
Kunwar, P. S., Siekhaus, D. E. and Lehmann, R.
(2006). In vivo migration: a germ cell perspective.
Annu. Rev. Cell Dev. Biol.
22,237
-265.[CrossRef][Medline]
Lawson, K. A. and Hage, W. J. (1994). Clonal
analysis of the origin of primordial germ cells in the mouse. Ciba
Found. Symp. 182,68
-84; discussion 84-91.[Medline]
Loveland, K. L. and Schlatt, S. (1997). Stem
cell factor and c-kit in the mammalian testis: lessons originating from Mother
Nature's gene knockouts. J. Endocrinol.
153,337
-344.
Mahakali Zama, A., Hudson, F. P., 3rd and Bedell, M. A.
(2005). Analysis of hypomorphic KitlSl mutants suggests different
requirements for KITL in proliferation and migration of mouse primordial germ
cells. Biol. Reprod. 73,639
-647.
Matsui, Y., Toksoz, D., Nishikawa, S., Nishikawa, S.-I.,
Williams, D., Zsebo, K. and Hogan, B. L. M. (1991a). Effect
of Steel factor and leukaemia inhibitory factor on murine primordial germ
cells in culture. Nature
353,750
-752.[CrossRef][Medline]
McCoshen, J. A. and McCallion, D. J. (1975). A
study of the primordial germ cells during their migratory phase in Steel
mutant mice. Experientia
31,589
-590.[CrossRef][Medline]
Mintz, B. and Russell, E. S. (1957).
Gene-induced embryological modifications of primordial germ cells in the
mouse. J. Exp. Zool.
134,207
-237.[CrossRef][Medline]
Molyneaux, K. A., Stallock, J., Schaible, K. and Wylie, C.
(2001). Time-lapse analysis of living mouse germ cell migration.
Dev. Biol. 240,488
-498.[CrossRef][Medline]
Nishikawa, S., Kusakabe, M., Yoshinaga, K., Ogawa, M., Hayashi,
S., Kunisada, T., Era, T., Sakakura, T. and Nishikawa, S.
(1991). In utero manipulation of coat color formation by a
monoclonal anti-c-kit antibody: two distinct waves of c-kit-dependency during
melanocyte development. EMBO J.
10,2111
-2118.[Medline]
Ohinata, Y., Payer, B., O'Carroll, D., Ancelin, K., Ono, Y.,
Sano, M., Barton, S. C., Obukhanych, T., Nussenzweig, M., Tarakhovsky, A. et
al. (2005). Blimp1 is a critical determinant of the germ cell
lineage in mice. Nature
436,207
-213.[CrossRef][Medline]
Okamura, D., Kimura, T., Nakano, T. and Matsui, Y.
(2003). Cadherin-mediated cell interaction regulates germ cell
determination in mice. Development
130,6423
-6430.
Payer, B., Chuva de Sousa Lopes, S. M., Barton, S. C., Lee, C.,
Saitou, M. and Surani, M. A. (2006). Generation of stella-GFP
transgenic mice: a novel tool to study germ cell development.
Genesis 44,75
-83.[CrossRef][Medline]
Runyan, C., Schaible, K., Molyneaux, K., Wang, Z., Levin, L. and
Wylie, C. (2006). Steel factor controls midline cell death of
primordial germ cells and is essential for their normal proliferation and
migration. Development
133,4861
-4869.
Saitou, M., Barton, S. C. and Surani, M. A.
(2002). A molecular programme for the specification of germ cell
fate in mice. Nature
418,293
-300.[CrossRef][Medline]
Saitou, M., Payer, B., O'Carroll, D., Ohinata, Y. and Surani, M.
A. (2005). Blimp1 and the emergence of the germ line during
development in the mouse. Cell Cycle
4,1736
-1740.[Medline]
Shirayoshi, Y., Nose, A., Iwasaki, K. and Takeichi, M.
(1986). N-linked oligosaccharides are not involved in the
function of a cell-cell binding glycoprotein E-cadherin. Cell
Struct. Funct. 11,245
-252.[Medline]
Stallock, J., Molyneaux, K., Schaible, K., Knudson, C. M. and
Wylie, C. (2003). The pro-apoptotic gene Bax is required for
the death of ectopic primordial germ cells during their migration in the mouse
embryo. Development 130,6589
-6597.
Surani, M. A., Hayashi, K. and Hajkova, P.
(2007). Genetic and epigenetic regulators of pluripotency.
Cell 128,747
-762.[CrossRef][Medline]
Tanaka, S. S. and Matsui, Y. (2002).
Developmentally regulated expression of mil-1 and mil-2, mouse
interferon-induced transmembrane protein like genes, during formation and
differentiation of primordial germ cells. Mech. Dev.
119 Suppl. 1,S261
-S267.[CrossRef][Medline]
Yabuta, Y., Kurimoto, K., Ohinata, Y., Seki, Y. and Saitou,
M. (2006). Gene expression dynamics during germline
specification in mice identified by quantitative single-cell gene expression
profiling. Biol. Reprod.
75,705
-716.
Yeom, Y. I., Fuhrmann, G., Ovitt, C. E., Brehm, A., Ohbo, K.,
Gross, M., Hubner, K. and Scholer, H. R. (1996). Germline
regulatory element of Oct-4 specific for the totipotent cycle of embryonal
cells. Development 122,881
-894.[Abstract]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Mithraprabhu and K. L Loveland Control of KIT signalling in male germ cells: what can we learn from other systems? Reproduction, November 1, 2009; 138(5): 743 - 757. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
