Developmental Genetics Group, Graduate School of Frontier Biosciences,
Osaka University, and CREST, Japan Science and Technology Corporation (JST),
1-3 Yamada-oka, Suita, Osaka 565-0871, Japan
* Present address: E. Kennedy Shriver Center, Division of Developmental
Neuroscience, 200 Trapelo Rd, Waltham, MA 02254, USA
Author for correspondence (e-mail:
hamada{at}fbs.osaka-u.ac.jp)
Accepted 13 January 2003
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SUMMARY |
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Key words: Foxh1, Left-right asymmetry, Midline, Nodal, Mouse
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INTRODUCTION |
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Nodal and Lefty (Ebaf Mouse Genome Informatics), both of which are
members of the transforming growth factor ß (TGFß) family of
proteins, play important roles in several embryonic patterning events
(Schier and Shen, 2000;
Brennan et al., 2001
;
Juan and Hamada, 2001
). Lefty
antagonizes Nodal signaling by acting as a feedback inhibitor
(Meno et al., 1999
;
Cheng et al., 2000
;
Sakuma et al., 2002
). In LR
patterning, genetic evidence suggests that Nodal expressed on the left side of
the lateral plate acts as a left-side determinant and induces left
side-specific morphogenesis of visceral organs
(Oh and Li, 1997
;
Yan et al., 1999
;
Lowe et al., 2001
), whereas
Lefty2 (Leftb Mouse Genome Informatics), which is induced by Nodal in
the left lateral plate, restricts the timing and the region of Nodal activity
(Meno et al., 2001
). Nodal and
Lefty have similar roles among vertebrates from the zebrafish to mouse
(Sampath et al., 1997
;
Rebagliati et al., 1998
;
Bisgrove et al., 1999
;
Thisse and Thisse, 1999
).
Despite the recent progress in our understanding of LR patterning, many
important questions remain unanswered. One such question concerns the
mechanism by which symmetry is broken in the first place. Breaking of symmetry
in mammals appears to involve nodal flow, the leftward flow of extra-embryonic
fluid in the node generated by the vortical movement of nodal cilia
(Nonaka et al., 1998). Thus,
nodal flow is impaired in mutant mice in which LR patterning is randomized
(Okada et al., 1999
). Indeed,
many of the genes whose mutation results in LR patterning defects encode
proteins required for the formation or motility of cilia. Furthermore,
imposition of an artificial flow was able to direct LR patterning in early
mouse embryos (Nonaka et al.,
2002
). The mechanism by which Nodal flow achieves this effect,
however, has remained unclear. It is possible that the flow transports a LR
determination factor toward the left side, but the identity of such a factor
is unknown.
The mechanism of signal transfer from the node to the lateral plate is also unknown. Both the identity of the signal (or signals) transported from the node to the lateral plate mesoderm (LPM) and whether the signal is transferred directly from the node to the lateral plate or is first relayed to an intermediate region such as the paraxial mesoderm remain to be determined.
Another important and related question concerns the mechanism by which the
expression of Nodal is initiated in left LPM. Although an
autoregulatory mechanism involving signaling by Nodal and the transcription
factor Foxh1 (previously known as Fast2) is responsible for amplification of
Nodal expression in left LPM
(Saijoh et al., 2000;
Norris et al., 2002
), it is
not known how Nodal expression is initiated. Ectopic expression of
Nodal in right LPM of chick embryos was not able to induce Nodal
expression (M. Levin, PhD thesis, Harvard University, 1996), suggesting that
an unknown factor other than Nodal initiates Nodal expression in left
LPM. Bone morphogenetic protein (BMP) signaling has been proposed to regulate
Nodal expression negatively in the chick, and a BMP antagonist such
as Caronte may initiate Nodal expression by inhibiting BMP activity
on the left side (Rodriguez Esteban et
al., 1999
; Yokouchi et al.,
1999
). However, recent evidence has suggested that BMP signaling
positively regulates Nodal expression by inducing an EGF-CFC factor
in LPM (Schlange et al., 2001
;
Schlange et al., 2002
;
Piedra and Ros, 2002
). The
factor responsible for the initiation of asymmetric Nodal expression
in LPM thus remains elusive. Finally, the midline structures serve as a
barrier that prevents the diffusion of asymmetric signals
(Danos and Yost, 1996
;
Lohr et al., 1997
;
Meno et al., 1998
), but it is
unclear how the midline barrier is established and precisely how it functions.
Analysis of Lefty1 mutant mice has shown that Lefty1, a Nodal
antagonist expressed on the left side of the prospective floor plate (PFP),
contributes to midline barrier function
(Meno et al., 1998
). However,
it is unknown how Lefty1 expression is induced at the midline.
We have now studied the role of Nodal-Foxh1 signaling in LR patterning by analyzing Foxh1 conditional mutant mice. We have also examined Nodal function by developing transplantation and electroporation systems for use with mouse embryos and applying these systems to the Foxh1 mutant mice. Unexpectedly, Nodal-Foxh1 signaling was shown to be able to initiate Nodal expression in LPM and to induce Lefty1 expression at the midline. Our results indicate that the leftsided expression of Nodal in LPM is initiated by Nodal produced in the node, and that Lefty1 expression at the midline is induced by Nodal produced in left LPM. We propose that Nodal activity travels from the node to left LPM, and from left LPM to the midline.
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MATERIALS AND METHODS |
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In situ hybridization and histology
Mouse embryos were staged on the basis of their morphology
(Downs and Davies, 1993).
Whole-mount in situ hybridization was performed according to standard
procedures (Wilkinson, 1992
).
Wild-type and mutant embryos were processed in the same tube. Embryos were
genotyped by PCR analysis of yolk sac DNA.
Transplantation of LPM
Fragments of tissue (containing 20 cells) were isolated for
transplantation from the left LPM of mouse embryos at the four-somite stage.
For use as recipients, mouse embryos were recovered at the two-somite stage,
dissected free of decidual tissues and Reichert's membrane, and maintained in
culture until manipulation. Cell clumps from donor embryos were grafted to the
right LPM, left paraxial mesoderm or left LPM of the host embryos with the use
of tungsten needles. The transplanted embryos were cultured for 3 hours at
37°C in 35 mm disposable dishes containing 4 ml of 50% Dulbecco's modified
Eagle's medium supplemented with 50% rat serum
(Lawson et al., 1986
); this
volume of medium was sufficient for culture of eight embryos
(Sturm and Tam, 1993
). A
transplant remained as a mass after the culture, showed distinct density and
thickness, and was easily distinguished from the host tissues.
Electroporation of a Nodal expression vector into LPM
The full-length cDNAs of mouse Nodal, constitutive active human ALK4
(caALK4), lacZ and EGFP were subcloned into the eucaryotic expression
vectors pEF-BOS (Mizushima and Nagata,
1990), pcDNA3, pEF and pCX, respectively. Each plasmid was
suspended in phosphate-buffered saline at a concentration of 5 mg/ml. Mouse
embryos at the two-somite stage were dissected free of decidual tissues and
Reichert's membrane and maintained in culture until electroporation.
Electroporation was performed with a CUY21 electroporator and a pulse monitor
(BTX, Tokyo). Platinum electrodes were used as an anode and a cathode and were
positioned near the posterior and anterior regions of the embryo,
respectively. Electric pulses were applied (14 V for 129 ms, five times) while
the DNA solution (1 µl) was injected into the anterior region of left or
right LPM. The electroporated embryos were cultured for 6 hours in a 50 ml
disposable tube containing 4 ml of 50% Dulbecco's modified Eagle's medium
supplemented with 50% rat serum (Lawrence
and Struhl, 1996
), a volume sufficient for the culture of eight
embryos (Sturm and Tam, 1993
).
The tubes were rotated at 30 rpm on a roller apparatus placed in a 37°C
incubator containing 5% CO2. This culture condition is optimized
for normal LR development so that left-sided Nodal expression in LPM
is preserved in >95% of the cultured embryos. A Nodal or caALK4
expression vector was introduced together with an EGFP expression vector by
electroporation, and regions that received the vectors were confirmed by the
presence of EGFP fluorescence.
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RESULTS |
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To confirm the expression pattern of the Cre transgene in 77b
mice, we crossed the animals with Cre-reporter mice that express lacZ
in response to Cre activity (Sakai and
Miyazaki, 1997). Embryos harboring both Lefty2-3.0 Cre
and the Cre-reporter lacZ transgene were recovered at various stages
and stained for ß-galactosidase activity with the substrate X-gal.
Staining was first evident at embryonic day 6.75 (E6.75) in the nascent
mesoderm. At E7.5, most of the embryonic region of the mesoderm exhibited
staining, whereas the primitive streak, ectoderm and endoderm were negative
(Fig. 1A). Mesoderm-specific
staining remained evident at E8.2; however, the midline structures, including
the axial mesoderm, lacked Cre activity
(Fig. 1B). At this stage, the
pattern of Cre activity differed between the anterior and posterior regions of
the embryo (Fig. 1C). In the
anterior region, X-gal staining was complete in the LPM, paraxial mesoderm,
definitive endoderm and heart. In the region posterior to the node, however,
Cre activity was detected only in the most lateral region of LPM. About 30 to
50% of the extra-embryonic mesoderm cells in the yolk sac and amnion were also
positive for X-gal staining. At E9.5, mesoderm-derived cells in the anterior
region were positive whereas the mesoderm in the posterior region was negative
(data not shown). Thus, at stages later than the early somite stage, Cre
activity was mostly restricted to the mesoderm of the anterior region of the
embryo.
|
Right isomerism in Foxh1 conditional mutant embryos
We first examined LR patterning in the Foxh1c/- embryos
by analyzing the transcription of asymmetrically expressed genes such as
Nodal, Lefty1, Lefty2 and Pitx2. In wild-type embryos,
Nodal is expressed in two domains at the early somite stage: the node
and left LPM (Fig. 2A,B). In
most Foxh1c/- embryos examined (31/37, 84%), however,
left-sided expression of Nodal in left LPM was absent
(Fig. 2C); in the remaining
embryos (6/37, 16%), a low level of Nodal expression was detected in
a small region of left LPM adjacent to the node
(Fig. 2D,E), probably because a
Nodal-positive loop was operative in this region before deletion of
Foxh1 was complete. Nodal expression in the node was
maintained in all (37/37) Foxh1c/- embryos (insets in
Fig. 2C,D). The expression of
Lefty2 apparent in left LPM of wild-type embryos
(Fig. 2F) was abolished in all
(16/16) Foxh1c/- embryos examined
(Fig. 2G). The expression of
Lefty1 observed in the PFP of wild-type embryos
(Fig. 2F) was also lost in all
(16/16) Foxh1c/- embryos
(Fig. 2G) (in some
Foxh1c/- embryos, Lefty1 expression was detected
in a small region of PFP adjacent to the node at the two-somite stage but this
expression disappeared at four-somite stage). This latter effect was
unexpected given that Foxh1 is preserved in midline structures
including the PFP (Fig. 1B).
The expression of Lefty1 apparent in the node of wild-type embryos
was maintained in the mutant embryos (inset of
Fig. 2G). The asymmetric
expression of Pitx2 apparent in wild-type embryos
(Fig. 2H,I,N-P) was abolished
in two-thirds (29/43) of Foxh1c/- embryos at E8.2
(Fig. 2J) and in 70%
(18/26) of the mutant embryos between E9.0 and E10.5
(Fig. 2K,Q-S), whereas
bilateral Pitx2 expression in the branchial arch was maintained in
all Foxh1c/- embryos. In the remaining mutant embryos, a
reduced level of left-sided Pitx2 expression was detected both in LPM
at E8.2 (Fig. 2L) and in
several organs and other structures, including the common atrial chamber, lung
bud, sinus venosus, vitelline vein, common cardinal vein and gut, at later
stages (Fig. 2M,T-V).
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Initiation by Nodal of Nodal expression in the lateral
plate
Our results suggested that an unknown factor derived from left LPM is able
to initiate Nodal expression in right LPM. An obvious candidate for
this factor was Nodal itself present in left LPM. To test this possibility, we
introduced a Nodal expression vector and an EGFP expression vector
into embryos at the early somite stage by electroporation. The embryos were
first examined for fluorescence to locate the cells that received the vectors
(Fig. 5A), and were subjected
to in situ hybridization. Introduction of the Nodal vector into right
LPM of wild-type embryos resulted in the induction of endogenous
Nodal expression (Fig.
5B,C). Nodal expression induced in right LPM expanded
along the anteroposterior axis and extended throughout the entire region of
the right LPM (25/25) (arrowheads in Fig.
5C). Electroporation of an EGFP expression vector alone into right
LPM of wild-type embryos did not give rise to such expanded Nodal
expression except in one (1/20) case. By contrast, introduction of the
Nodal expression vector into the left or right LPM of
Foxh1c/- embryos failed to induce Nodal
(Fig. 5F). Thus, ectopic Nodal
is able to induce Nodal expression in right LPM, but this induction
requires the presence of Foxh1 in LPM.
|
Lefty1 is expressed in the PFP on the left side of wild-type
embryos (Meno et al., 1997). A
piece of left LPM transplanted to the right LPM of wild-type embryos was able
to induce not only Nodal in the right LPM but also Lefty1 in
the right PFP (3/3) (Fig. 4C). Thus, Lefty1 expression in the PFP became bilateral only at the
levels where Nodal was ectopically expressed in right LPM
(Fig. 4D,E), supporting the
idea that Nodal produced in LPM induces Lefty1 expression in the PFP.
Similar experiments were performed with Foxh1c/- embryos,
which retain Foxh1 in the PFP but lack Lefty1 expression in
this region. Transplantation of left LPM from wild-type embryos to the left
LPM of Foxh1c/- embryos did not result in the induction of
Lefty1 expression in the PFP (0/10)
(Fig. 4F,G). However,
transplantation of the left LPM to the paraxial mesoderm, a site closer to the
PFP, resulted in the induction of Lefty1 (3/4)
(Fig. 4H,J). Furthermore, left
LPM obtained from Lefty2
ASE/
ASE embryos, in
which Nodal activity is increased as a result of the lack of Lefty2
(Meno et al., 2001
), induced
Lefty1 expression in the PFP even when transplanted to the left LPM
of Foxh1c/- embryos (4/5)
(Fig. 4I,K).
Introduction of the Nodal expression vector into the right LPM of wild-type embryos also induced Lefty1 expression in the PFP (18/25) (Fig. 5D,E). The spatial level of ectopic Lefty1 expression along the anteroposterior axis of the PFP corresponded to that of ectopic Nodal expression in the right LPM. In most instances, Lefty1 expression was bilateral throughout the entire PFP, while ectopically induced Nodal expression extended throughout the entire region of right LPM (Fig. 5E). Furthermore, introduction of the Nodal expression vector into the left LPM of Foxh1c/- embryos also induced Lefty1 expression in the PFP (6/8) (Fig. 5G,H), making it unlikely that Lefty1 was induced by secondary signals produced by the Nodal-Foxh1 pathway. In these various experiments, Nodal expression was never induced in the PFP either by the transplanted left LPM or by introduction of the Nodal expression vector into right LPM (Fig. 4B, Fig. 5B).
These results suggest that Lefty1 expression in PFP is directly induced by Nodal produced in LPM but do not exclude an alternative possibility that it is induced by secondary factor(s) produced in LPM by a Nodal-dependent yet Foxh1-independent pathway. To test the latter possibility, we examined the effects of constitutive active ALK4 (caALK4). As expected, caALK4 was able to induce Nodal in the right LPM of the wild-type embryos (5/5) (Fig. 5I). However, introduction of the caALK4 expression vector into the left LPM of Foxh1c/- embryos failed to induce Lefty1 expression in the PFP (11/11) (Fig. 5J). These results now demonstrate that Nodal activity produced in LPM directly induces Lefty1 expression in PFP.
Nodal activity travels from left LPM to the PFP
Our results suggest that Nodal ectopically produced in LPM may diffuse over
the relatively long distance to the PFP and there induce Lefty1
expression. We next examined whether the PFP indeed receives Nodal signals
from left LPM with the use of a lacZ transgene whose expression is
strictly dependent on Nodal signaling. This transgene, (n2)7-lacZ,
contains seven tandem repeats of a Foxh1 binding site and its expression is
induced by the Nodal-Foxh1 pathway (Saijoh
et al., 2000; Sakuma et al.,
2002
). X-gal staining of transgenic embryos harboring
(n2)7-lacZ revealed lacZ expression in the PFP predominantly
on the left side as well as in left LPM
(Fig. 6A,B). Given that
Nodal is not expressed in the PFP, Nodal produced elsewhere (either
in left LPM or the node) must have traveled to the PFP. By contrast, X-gal
staining of Foxh1c/- embryos harboring the
(n2)7-lacZ transgene revealed that lacZ expression was
abolished in the left LPM and PFP, although staining in the allantois and at
the base of the allantois remained (10/10)
(Fig. 6C,D). Similarly, another
Nodal-responsive lacZ reporter gene, Lefty2 ASE-lacZ, that
contains the asymmetric enhancer (ASE) of Lefty2 also gave rise to
X-gal staining in the PFP in addition to the left LPM of wild-type embryos
(Saijoh et al., 1999
);
however, this transgene was inactive in the PFP of
Foxh1c/- embryos (11/11) (data not shown).
Foxh1c/- mice lack Nodal expression in left LPM
but that in the node is unaffected (Fig.
2C-E). Furthermore, they retain Foxh1 in the PFP
(Fig. 1B,C). Finally,
introduction of the Nodal expression vector into left LPM of
Foxh1c/- embryos harboring the (n2)7-lacZ (7/7)
(Fig. 6E,F) or Lefty2
ASE-lacZ (2/2) (data not shown) transgene resulted in expression of
lacZ in the PFP. Together, these results suggest that Nodal
synthesized in left LPM, not Nodal produced in the node, travels to the PFP
and activates the Nodal-responsive lacZ transgenes.
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DISCUSSION |
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Foxh1 is expressed bilaterally in LPM at the early somite stage when
Nodal is expressed in left LPM
(Saijoh et al., 2000), and may
function in both the initiation and amplification of Nodal expression
in left LPM. Although it is difficult to distinguish these two processes
experimentally, Foxh1 is implicated in both by our observation that the
transplantation of left LPM to Foxh1c/- embryos
failed to induce Nodal expression even in the cells adjacent to the
transplant site.
If Nodal synthesized in the node acts on left LPM, what might prevent Nodal
activity from traveling toward the right side? Nodal flow, the leftward flow
of extra-embryonic fluid in the node generated by vortical movement of the
cilia (Nonaka et al., 1998),
may transport Nodal preferentially toward the left side. Indeed, the role of
nodal flow in LR patterning was recently demonstrated by testing the effects
of artificial fluid flow in embryos
(Nonaka et al., 2002
).
Although ciliated cells can be found in the organizer region of non-mammals,
including the chick (Essner et al.,
2002
), fluid flow may not be generated there. Coincidentally,
ectopic introduction of Nodal into the right LPM failed to induce endogenous
Nodal expression (M. Levin, PhD thesis, Harvard University, 1996).
Thus, a different mechanism may operate in the chick for the transfer of
asymmetric signals from the node to left LPM.
Nodal protein produced in left LPM induces Lefty1 expression
at the midline
The midline structures, including the floor plate and notochord, are
required to separate the two sides of the embryo
(Danos and Yost, 1996), with
Lefty1 being critical for midline barrier function
(Meno et al., 1998
). Our
observations now suggest that Lefty1 expression in the PFP is induced
by Nodal produced in left LPM. First, Fixhlc/-
embryos, which lack Nodal expression in left LPM but retain it in the
node, fail to express Lefty1 in the PFP, suggesting that Nodal
produced in the node is unable to induce Lefty1 expression in the
PFP. Second, and more importantly, transplanted left LPM or a Nodal
expression vector introduced into right LPM induced Lefty1 expression
in the PFP of wild-type embryos but not in that of
Foxh1c/- embryos. Third, introduction of
constitutively active ALK4 into the left LPM of
Foxhlc/- embryos was unable to induce
Lefty1 expression in PFP, excluding a possibility that an unknown
factor produced by a Nodal-dependent yet Foxh1-independent pathway induces
Lefty1. The idea that Lefty1 expression is induced by
LPM-derived Nodal is also consistent with previous observations. Comparison of
the kinetics of Lefty1 and Nodal expression thus revealed
that Lefty1 expression in the PFP is preceded by Nodal
expression in left LPM (C. M. et al., unpublished data). Furthermore,
Nodal is not expressed in the PFP. Finally, mutant mice lacking a
component of the Nodal signaling pathway, such as the coreceptor Cryptic, fail
to express Lefty1 in the PFP as well as Nodal in left LPM
(Yan et al., 1999
).
After Nodal produced in left LPM travels to the midline and induces
Lefty1 expression, Lefty1, which is also able to travel over long
distances (Sakuma et al.,
2002), might then be expected to diffuse toward the LPM. Lefty1
that reaches the right LPM would render it incompetent for Nodal signaling and
prevent Nodal expression there. Lefty1 that reaches left LPM,
together with Lefty2 produced in the left LPM, may contribute to rapid
repression of Nodal expression in this region. Midline barrier
function is abolished in mutant mice that lack Lefty1, resulting in bilateral
expression of Nodal and Lefty2
(Meno et al., 1998
). We
previously suggested that, in the absence of Lefty1, an unknown left-side
determinant travels across the midline and reaches the right LPM, where it
induces the expression of Nodal and Lefty2
(Meno et al., 1998
). Our data
now suggest that this left-side determinant is most likely Nodal.
Although our results indicate that Lefty1 expression at the
midline is induced by Nodal produced in left LPM, it is not clear whether this
expression depends on Foxh1. Lefty1 expression is lost even in the
least severe type of Foxh1-null mutant
(Yamamoto et al., 2001).
However, this effect may be secondary to misspecification of the midline cells
in the absence of Foxh1 (Hoodless et al.,
2001
; Yamamoto et al.,
2001
). Our previous analysis of the transcriptional regulatory
elements of Lefty1 by transgenic approaches
(Saijoh et al., 1999
)
suggested that the 1.2 kb region immediately upstream of Lefty1 is
sufficient for its asymmetric expression in the PFP. Although this 1.2 kb
region contains three potential binding sites for Foxh1, mutation of these
sequences did not impair the PFP-specific expression of Lefty1 (Y.S.
and H.H., unpublished). Nodal signaling that induces Lefty1 in the
PFP therefore may not involve Foxh1.
Overall, our results obtained with Foxh1c/- mice suggest that Nodal activity travels from the node to left LPM, and from left LPM to the midline. A direct test of this conclusion will require visualization of the behavior of Nodal in mouse embryos.
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ACKNOWLEDGMENTS |
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