Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK
Author for correspondence (e-mail:
c.stern{at}ucl.ac.uk)
Accepted 19 March 2004
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SUMMARY |
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Key words: Gastrulation, Embryonic axis, Regulative development, Vg1, FGF, Chordin, Nodal
![]() |
Introduction |
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By contrast, amniote embryos do not appear irreversibly to fix their
polarity until much later, when gastrulation begins and the embryo may already
contain many thousand cells. In the mouse, the inner cell mass can be
dissociated and reaggregated, or made up from cells derived from different
embryos, yet it will develop into a single normal embryo, although the extent
to which the axis of polarity is specified by maternal cues or by the point of
sperm entry has recently generated considerable controversy (reviewed by
Gardner, 2002;
Piotrowska and Zernicka-Goetz,
2002
; Tam, 2002
;
Zernicka-Goetz, 2002
;
Gardner and Davies, 2003
;
Johnson, 2003
). A more extreme
case in mammals is seen in the armadillo, which normally produces four embryos
from a single blastoderm (Enders,
2002
). Likewise, chick embryos until the time of primitive streak
formation (when they contain as many as 50,000 cells), can be cut into four
pie-shaped fragments, all of which can spontaneously give rise to a complete,
miniature embryo (Lutz, 1949
;
Spratt and Haas, 1960
).
Furthermore, composite embryos made up of equivalent pie-slices (for example
the most anterior quadrant) derived from four or even more donor embryos will
develop only a single axis, arising randomly from a margin of any one of these
slices. Although this finding does not rule out a role for maternally
inherited, differentially distributed components in biasing the initial
polarity of amniote embryos, it certainly does rule out the concept that these
components are true `determinants' of cell fate, as no quadrant of the embryo,
even at this advanced stage, possesses a unique ability for polarity
determination or cells committed to particular fates, not shared by any other
quadrant.
Surprisingly, the remarkable ability of the chick embryo to regulate has received little attention, as it is an excellent experimental system in which to study the molecular mechanisms underlying polarity determination in an extremely regulative system. The finding that any fragment of the chick embryo right up to the beginning of primitive streak formation can form a complete axis when isolated, while none of these regions other than the posterior part (containing Koller's sickle and the marginal zone) do so in intact embryos, strongly suggests that the normal site of axis formation actively inhibits other regions from initiating the same process.
We provide evidence for an inhibitor operating before primitive streak formation. It is induced by Vg1 misexpression, travels across the embryo (3 mm distance) at a speed of at least 500 µm/hour, and acts upstream of, or in parallel with, the signalling factors Nodal and Chordin. We argue that this inhibitor is distinct from the Nodal antagonist Cerberus, which is produced by the hypoblast. We also reveal a role for the FGF signalling pathway in initiation of the embryonic axis and suggest that this acts in cooperation with Nodal and Chordin.
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Materials and methods |
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FGF gain and loss of function
Heparin acrylic beads (Sigma) were incubated in 50 µg/ml FGF4 or FGF8b
(R&D Systems) in PBS for 2 hours at 4°C. AG1X2-formate beads (100
µm) were coated with 250 µM SU5402 (Calbiochem) for 1-2 hours at room
temperature. The beads were then washed in PBS before grafting.
Vg1, Wnt1, Chordin and Nodal misexpression
COS-7 cells were cultured in DMEM with 10% fetal calf serum. BVg1
(Shah et al., 1997),
Chordin (Streit et al.,
1998
) or Nodal
(Bertocchini and Stern, 2002
)
were transfected using Lipofectamine Plus (Gibco-BRL). After 24 hours, 1000 or
500 cells were allowed to aggregate into pellets in 20 µl hanging drops of
medium. Mock-transfected cells were used as control. For experiments involving
Vg1 grafted with SU5402-coated beads, 500-cell pellets were prepared each
containing 250 Vg1-transfected cells and 250 mock-transfected cells. In all
other experiments, 1000 cell pellets were used. For Wnt1 misexpression, we
used a stable cell line (rat B1; a kind gift from Dr Jan Kitajewsky) as
previously described (Joubin and Stern,
1999
; Skromne and Stern,
2001
; Skromne and Stern,
2002
).
Assays
The experiments described here, like those of previous work in this field,
rely on misexpression of secreted factors delivered by grafts of transfected
cells. Although it is possible to deliver factors by electroporation of
expression constructs, we have not yet succeeded in doing this at pre-streak
stages. Grafts of expressing cells have the advantage, however, that it is
possible to titrate the number of cells grafted and thus adjust the levels of
factor supplied. Although it is not possible to compare the absolute protein
levels available to the embryo after such grafts, this method allows for
internal comparison.
It is also worth pointing out that in this study, as in all other previous studies on embryos at these early stages of development, the results are `statistical' even those manipulations that give the strongest effects (such as Vg1 or Vg1+Wnt misexpression) produce ectopic streaks in only about 70% cases. The reasons for this are unknown, but one reason may be that there are many endogenous factors regulating axis development, as the present study reveals.
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Results |
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To confirm at a molecular level the earlier findings
(Lutz, 1949;
Spratt and Haas, 1960
) that
embryos can regulate, we divided stage X-XIII
(Eyal-Giladi and Kochav, 1976
)
chick embryos into anterior and posterior halves, and each half was grown
separately in modified New culture (Fig.
1A) (New, 1955
;
Stern and Ireland, 1981
). The
posterior half (where the streak normally forms) develops an axis in 93/98
(95%) cases, within the same time course as normal uncut embryos. At each
stage analysed, the anterior half forms an axis in almost 60% of cases [20/27
(74%) at stage X; 9/23 (39%) at stage XI; 17/28 (61%) at stage XII; and 11/20
(55%) at stage XIII). The forming axis expresses markers of mesoderm (chick
Brachyury) and organizer (Chordin)
(Fig. 1B,C), and develops a
lower layer composed of hypoblast (Crescent and FGF8
expression) and endoblast (Fig.
1D,J). Primitive streak formation in the anterior half is
initiated with a delay of at least 8 hours compared to the posterior half and
always arises from the most posterior part of the anterior half. A primitive
streak (and Brachyury expression) is first visible after 15-18 hours.
These findings confirm the observations of
(Spratt and Haas, 1960
) that
isolated fragments of the early chick embryo can initiate primitive streak
formation following a posterior-to-anterior gradient of `embryo-forming
potentiality'.
|
Vg1 induces an inhibitor that prevents multiple streak formation
The above experiments confirm the finding that any region of the
pre-primitive streak stage embryo has the ability to form an axis
(Spratt and Haas, 1960). The
fact that the anterior half of intact embryos never does so suggests the
existence of endogenous inhibitory factors, from whose influence the anterior
half is released upon cutting. For such a mechanism to work effectively to
prevent multiple axes in normal development, the inhibitor should travel
quickly across the embryo, which measures some 3 mm in diameter.
The existence of such an inhibitor can also be revealed by ectopic
expression of Vg1 at various positions in the marginal zone. When misexpressed
anteriorly, Vg1 induces an ectopic streak that co-exists with the endogenous
axis, in more than 60% of cases (Seleiro
et al., 1996; Shah et al.,
1997
; Skromne and Stern,
2001
; Skromne and Stern,
2002
). By contrast, when misexpressed in the lateral marginal zone
at 90° from the posterior part (Fig.
2A), the newly induced primitive streak often inhibits the
formation of the original axis [31/55 (56%) with only one axis arising from
the Vg1 source; 5/55 (9%) with two axes, 10/55 (18%) with one axis arising
half-way between the original site and the Vg1 pellet, 7/55 (13%) with only
the original axis and 2/55 (4%) with no axis]
(Fig. 2B). This result suggests
that Vg1 induces an inhibitor that prevents the development of additional
axes.
|
Recently, we reported that one mechanism contributing to ensure the
formation of a single axis during normal development relies on the expression
of the Nodal antagonist Cerberus by the hypoblast, which is then removed just
prior to primitive streak formation by the appearance of the endoblast (which
does not express Cerberus)
(Bertocchini and Stern, 2002).
Could Cerberus, or another molecule produced by the hypoblast, be the
inhibitor revealed by the above experiments? To answer this, we analysed the
expression pattern of hypoblast markers including Cerberus and
Hex in time-course in embryos grafted with Vg1 pellets. We did not
observe any increase in Cerberus expression around the second pellet
(0/9 embryos). The hypoblast, marked by both Cerberus and
Hex, is displaced from both sites as in the normal embryo when two
streaks form (0-6 hours; Fig.
2G). When the pellets are grafted 6 hours apart, in no case did
the hypoblast arising from the first pellet reach the second pellet after 6
hours (Fig. 2H). This finding
suggests that Cerberus is not the inhibitory molecule induced by Vg1.
Moreover, as hypoblast formation and movements similar to those of the normal
embryo are seen in isolated anterior halves
(Fig. 1D,J), this suggests that
the inhibitor from which the anterior half is released by cutting is also
distinct from Cerberus.
The primitive streak emits an inhibitor
In both experimental paradigms that reveal the existence of an inhibitor
(isolated half-embryos and ectopic Vg1), additional axes can no longer be
induced after the normal primitive streak appears (stage 2-3)
(Spratt and Haas, 1960;
Seleiro et al., 1996
;
Shah et al., 1997
). Two
different situations can explain this: either cells in the anterior marginal
zone lose their competence to Vg1 at stage 2-3, or these cells are still
competent but somehow inhibited. To distinguish between these possibilities,
we grafted a Vg1 pellet in the anterior marginal zone of stage 2-3+
embryos, cultured either intact or after removal of the half of the embryo
containing the primitive streak (Fig.
3A). In no case did an ectopic streak form in intact embryos (0/21
this study) (see also Shah et al.,
1997
); likewise, in no case did an axis develop when
mock-transfected cells were grafted into anterior halves isolated at stage 2-3
(0/14). By contrast, 31% of isolated anterior halves implanted with a source
of Vg1 developed a primitive streak arising from the pellet (8/26;
P<0.01; Fig. 3B).
However the capacity to respond to Vg1 is lost at still later stages: anterior
halves from older streak stage embryos (4, 4,
4+), never form a streak after a Vg1 graft (0/22).
|
The inhibitor acts downstream of Vg1 but upstream of Nodal and Chordin
Vg1 has been shown to require Wnt signalling to induce a primitive streak
(Joubin and Stern, 1999;
Skromne and Stern, 2001
),
raising the possibility that the inhibitor might work by antagonising Wnt
signalling. To test this, we misexpressed Vg1 on the lateral margin, followed
6 hours later on the opposite side by another pellet of Vg1 together with a
pellet of Wnt1-expressing cells. Only 3/34 (9%) embryos produced a primitive
streak from the second implantation site, not significantly different from
when a second Vg1 pellet was implanted alone (see above).
Vg1 misexpression in the anterior part of a pre-streak embryo induces a
cascade of gene activation: Nodal is induced after 6 hours, followed
by Chordin after 9 hours (Skromne
and Stern, 2002). As both Nodal and Chordin have been implicated
in primitive streak formation (Streit et
al., 1998
; Streit and Stern,
1999b
; Bertocchini and Stern,
2002
), it is important to determine whether the inhibitor acts by
antagonising these factors. To this end, we grafted a Vg1 pellet on one side
(stage X-XII), followed 6 hours later by either Vg1 together with Nodal or Vg1
with Chordin on the opposite side (Fig.
4A). If the inhibition takes place downstream of Chordin
activation, co-expression of Chordin should not be sufficient to induce an
ectopic axis. If the inhibitor works between Nodal and Chordin, or upstream of
Nodal, misexpression of Chordin in the first case, or either Chordin or Nodal
in the second case, should be enough to bypass the inhibitory step. Grafting a
second Vg1 pellet together with either factor induces an ectopic primitive
streak [18/27 (67%) with Vg1+Chordin; 14/29 (48%) with Vg1+Nodal]
(Fig. 4B,C), suggesting that
the inhibition takes place downstream of Vg1 but upstream of Nodal and
Chordin. However, when either Nodal or Chordin is grafted alone 6 hours after
the first Vg1 pellet on the opposite side, no induction takes place (0/33 with
Nodal, 1/34 with Chordin). This latter result suggests that in addition to
Nodal and Chordin, Vg1 must induce another factor necessary to bypass the
inhibitory step and allow an ectopic axis to form.
|
|
Does FGF signalling overcome the Vg1-induced inhibitor? We grafted Vg1 in the lateral marginal zone and FGF4 in the opposite side 6 hours later (Fig. 6A); 14/26 (54%) embryos developed a double axis, one from each side (Fig. 6B), and 4/26 (15%) developed a streak only from the FGF4 side. This indicates that the inhibition initiated by Vg1 does not affect the response to FGF. The same result was obtained with FGF8b [which again was less potent; 4/40 (10%) with two axes; not shown].
|
Does FGF synergise with Vg1, or with one or both of the downstream
components Nodal and Chordin? We addressed this using both loss of FGF
function (SU5402) and gain-of-function (misexpression) approaches. For the
former, we misexpressed Vg1 in the anterior marginal zone, together with a
SU5402-soaked bead and either Chordin or Nodal in the adjacent area pellucida.
An ectopic primitive streak developed in 18/39 (46%) embryos when Nodal was
used and in 27/44 (61%) with Chordin, while 4/17 (24%) embryos grafted with
Vg1+SU5402+mock-transfected cells developed a primitive streak
(P<0.05 for Chordin, P0.2 for Nodal). This result
suggests that Chordin can induce an axis in the absence of FGF signalling, and
Nodal has a weaker effect.
As a second approach, we misexpressed FGF8b together with either Chordin
(Fig. 6G) or Nodal. For
Chordin, 16/27 embryos (59%) grafted with FGF8b+Chordin in the area pellucida
developed an ectopic axis (Fig.
6H), more frequently than either FGF8b [1/12 (8%)] or Chordin
alone [3/15 (20%)]. When FGF8b+Chordin were misexpressed in the lateral side 6
hours after a Vg1 pellet in the opposite side
(Fig. 6E), a second streak
developed in 9/39 (24%) cases (Fig.
6F), more frequently than with either Chordin alone [1/34 (3%)] or
FGF8b alone [4/40 (10%)]. For Nodal, however, the results were different.
FGF8b+Nodal yielded an axis in 26/80 embryos (33%)
(Fig. 4L), when compared with
0/20 with Nodal alone (Bertocchini and
Stern, 2002) and 1/12 (8%) with FGF8b alone (P
0.1);
and misexpression of Nodal+FGF8b 6 hours after a Vg1 graft generated an
ectopic axis in 6/39 embryos (15%), when compared with 0/33 with Nodal alone
and 4/40 (10%; P>0.05) with FGF8b alone.
We suspected that the weak effect of Nodal in these experiments might be
due to the expression of the Nodal antagonist Cerberus by the
underlying hypoblast at the time of the graft
(Bertocchini and Stern, 2002).
Consistent with this, the same experiment performed after hypoblast removal
led to an ectopic streak in a much higher proportion of cases (9/21; 43%),
when compared with 0/24 for Nodal alone (P<0.001). Taken together,
these results suggest that FGF signalling is required for primitive streak
formation and that it cooperates with Nodal and Chordin in this process.
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Discussion |
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Other recent work revealed that the hypoblast (an extraembryonic tissue
that underlies the embryonic epiblast, from which the embryo proper arises)
emits Cerberus, which acts as an antagonist of Nodal and prevents premature
and ectopic primitive streak formation
(Bertocchini and Stern, 2002).
It was proposed that just prior to primitive streak formation, the hypoblast
(along with Cerberus expression) is displaced away from the posterior
edge, which allows Nodal signalling to initiate the process of streak
formation (Bertocchini and Stern,
2002
). Comparable findings were made in mouse embryos lacking both
cerberus and another Nodal antagonist, Leftb: in the absence
of both antagonists, multiple primitive streaks develop
(Perea-Gómez et al.,
2002
), confirming that release from an inhibitor of Nodal
signalling is a conserved mechanism to ensure that only a single primitive
streak forms in amniotes.
The inhibitor whose properties are revealed by the present experiments does not appear to be Cerberus, and is not associated with hypoblast movements. Although misexpression of Vg1 does coordinate the formation of a new lower layer (including both the coalescence of hypoblast cells into a layer and the induction of endoblast) from a new site, as revealed by the expression of hypoblast markers (goosecoid, Hex, Cerberus, crescent, HNF3ß), the appearance of this layer does not correlate with the spreading of the fast travelling inhibitor described.
Other candidate inhibitors of primitive streak formation
Our experiments in the chick
(Bertocchini and Stern, 2002)
(this paper) are consistent with work from zebrafish
(Erter et al., 1998
;
Feldman et al., 1998
;
Rebagliati et al., 1998
;
Feldman et al., 2000
;
Shimizu et al., 2000
;
Sirotkin et al., 2000
;
Chen and Schier, 2001
;
Chen and Schier, 2002
;
Schier, 2003
),
Xenopus (Jones et al.,
1995
; Smith et al.,
1995
; Lustig et al.,
1996
; Agius et al.,
2000
; Kodjabachian,
2001
) and mouse (Zhou et al.,
1993
; Conlon et al.,
1994
; Varlet et al.,
1997
; Perea-Gómez et
al., 2002
), implicating Nodal as an essential inducer of axial
mesoderm and required for gastrulation. In addition to Cerberus, two
antagonists of Nodal signalling have been identified in zebrafish,
Xenopus and mouse: Leftb (Lefty1) and Ebaf (Lefty2)
(Meno et al., 1996
;
Meno et al., 1997
;
Meno et al., 1999
;
Chen and Schier, 2002
).
Could Lefty proteins be the early primitive streak inhibitors? This
possibility is made likely by the finding that Lefty proteins act as
long-range antagonists of Nodal (Squint) signalling without affecting short
range Nodal (Cyclops) signals (Chen and
Schier, 2002). These properties are exactly what would be required
for an inhibitor emitted by the site of axis formation, as they would allow
local Nodal signalling (primitive streak formation) while inhibiting this
process remotely.
To date, only a single Lefty gene has been identified in the chick, named
Lefty1 (Ishimaru et al.,
2000). However, this is more likely to be the orthologue of mouse
Ebaf (previously Lefty2), as it is expressed in the
primitive streak itself but not at earlier stages
(Meno et al., 1997
;
Meno et al., 1998
;
Meno et al., 1999
;
Ishimaru et al., 2000
;
Meno et al., 2001
;
Bertocchini and Stern, 2002
).
Despite several attempts, we have been unable to isolate another Lefty gene
from the chick to test this. If a true orthologue of mouse Leftb does
exist in the chick, its expression should reveal whether it fulfills the
criteria for being the inhibitor described here. In the mouse, Ebaf
is expressed in the anterior visceral endoderm (AVE), which is equivalent to
the chick hypoblast. If the putative chick orthologue is expressed only in the
hypoblast, it could also not be the inhibitor whose identity we seek, because,
as described above, the spreading of the hypoblast layer does not correlate in
space or time with the inhibitor induced by Vg1 misexpression and is also too
slow to ensure the formation of a single axis in bisected embryos.
Therefore, there appear to be at least two distinct inhibitors of primitive
streak formation in the chick: an early, fast-travelling wave initiated by Vg1
or one of its targets in the marginal zone (for which the missing orthologue
of mouse Leftb is a candidate); and a later antagonist of Nodal signalling
(Cerberus), which accompanies the spreading of the hypoblast layer
(Bertocchini and Stern, 2002).
At later stages, we have also argued
(Bertocchini and Stern, 2002
)
for a role of chick Lefty1 (or the orthologue of mouse Ebaf) from the
primitive streak as a third mechanism to ensure that only a single primitive
streak develops.
Koller's sickle, the posterior marginal zone and the amniote `Nieuwkoop centre'
A previous study identified the posterior marginal zone (PMZ) of the avian
embryo, the region that expresses Vg1, as the functional equivalent
of the Nieuwkoop centre of amphibians
(Bachvarova et al., 1998). This
was based on the observation that grafts of the marginal zone that exclude the
neighbouring Koller's sickle can induce the formation of a complete embryonic
axis without making a cellular contribution to the induced axis, the
properties that initially defined the Nieuwkoop centre (see
Nieuwkoop, 1969a
;
Nieuwkoop, 1969b
;
Lemaire et al., 1995
;
Harland and Gerhart, 1997
).
However, others have argued that Koller's sickle is indispensable for axial
development, and that this rather than the PMZ is the equivalent of the
Nieuwkoop centre (Callebaut and Van Nueten,
1994
). Our present results argue strongly in favour of the former
interpretation: both in embryo fragments and when an ectopic axis is initiated
by misexpression of Vg1, the primitive streak appears without any sign of
prior induction of a Koller's sickle-like region. Furthermore, molecular
markers for the sickle (Chordin, FGF8, goosecoid) are relatively late
responses to either manipulation, and are not expressed until after the
primitive streak can be seen both morphologically and by expression of the
mesodermal marker brachyury (this study)
(Skromne and Stern, 2002
).
Koller's sickle has also been claimed to be the region through which the
endoblast forms (Azar and Eyal-Giladi,
1979; Azar and Eyal-Giladi,
1983
; Callebaut and Van Nueten,
1994
), which led to the original name of the latter as `sickle
endoblast' (Vakaet, 1970
;
Callebaut and Van Nueten,
1994
). In our experiments, an endoblast layer forms both in cut
embryos and after misexpression of Vg1, but in the absence of a sickle. This
finding is more consistent with the alternative explanation that the endoblast
arises not from the sickle but from the adjacent yolky germ wall margin
(Stern and Ireland, 1981
;
Stern, 1990
). In conclusion,
the present experiments suggest that Koller's sickle is not essential either
for the formation of the embryonic axis or for the development of endoblast in
the lower layer.
A role for FGF signalling in axis formation
A large body of evidence has implicated Nodal signalling in
mesoderm/endoderm induction (see above). Other experiments have established
that the induction of mesoderm/endoderm by TGFß signals related to Nodal
requires activation of the FGF pathway
(Kimmelman and Kirschner,
1987; Cornell and Kimmelman,
1994
; LaBonne and Whitman,
1994
; Cornell et al.,
1995
). In the chick embryo, similar to early findings in
amphibians, it was reported that FGF can act as a sufficient mesoderm inducer
(Mitrani et al., 1990
). The
present results are consistent with the now widely accepted notion that FGFs
act in synergy with Nodal-related signals in mesendoderm induction. Of all the
factors implicated in primitive streak initiation to date (Vg1, Wnt8C, Nodal,
Chordin, FGFs), only the latter can by itself overcome the effects of the
inhibitor induced by Vg1, consistent with the idea that FGFs act through a
parallel pathway with TGFßs, and synergise with them. We also show that,
as in amphibians, FGF signalling is required for normal primitive streak
formation and for induction of a streak by Vg1 and its targets, as both
processes are completely blocked in the presence of the FGF inhibitor
SU5402.
A cascade of genes regulating axis development in the chick embryo
Our findings, together with previous studies on the molecular bases of
initiation of primitive streak formation in the chick embryo, allow us to
propose a model (Fig. 7) for
how the embryo initiates primitive streak formation and how it ensures that
only a single axis forms despite the widespread potential of any region of the
embryo to do so. The earliest known molecular asymmetry preceding axis
formation is the localised expression of chick Vg1 in the posterior
marginal zone, in a region overlapping with expression of chick
Wnt8C. It has been shown that Wnt activity is required for Vg1 to
induce an axis, and that chick Wnt8C is expressed all around the
margin of the embryo, suggesting that this factor defines the entire marginal
zone as a unique region (Skromne and
Stern, 2001). The convergence of Vg1 and Wnt activity then induces
Nodal in the adjacent epiblast of the area pellucida
(Bertocchini and Stern, 2002
;
Skromne and Stern, 2002
).
However, Nodal cannot act because it is covered by the hypoblast, which
secretes Cerberus (Bertocchini and Stern,
2002
): primitive streak formation is repressed until the removal
of Cerberus that results from displacement of the hypoblast by endoblast
tissue (which does not express Cerberus). We now suggest that FGF8
(which is expressed both by the hypoblast and by Koller's sickle cells) and/or
other members of the FGF family synergise with Nodal to initiate primitive
streak formation at this time.
|
At the same time, an antagonist of primitive streak formation other than Cerberus further ensures the formation of a single primitive streak by rapidly conveying to remote parts of the embryo the information that primitive streak development is already underway elsewhere. A crucial feature of this inhibitor, which remains to be identified, must therefore be either rapid propagation through the embryo without affecting cells near its site of production (therefore Lefty1 may be a candidate; see above). Our results also suggest that it is induced by Vg1 and acts between Vg1 and Nodal in the genetic cascade.
An additional mechanism that remains to be understood is how the embryo
positions Vg1 expression at the site where primitive streak formation
will begin. Chick Vg1 is the earliest known gene to be expressed in
anterior half-embryos in a manner that predicts where the axis will form and
is also the only known marker for the PMZ. Anterior half-embryos, which only
generate a single axis, positioned randomly on the left or right of the
fragment, also possess only a single randomly located site of chick
Vg1 expression. Whatever is responsible for this must therefore also
travel rapidly across the embryo (and therefore may be identical to the fast
inhibitor described), or be present as a pre-existing gradient (for which
Wnt8C is a candidate) (Skromne and Stern,
2001), which might bias Vg1 expression to one side.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
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---|
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