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Vol. 14, No. 9, pp. 1146-1155, May 1, 2000
signaling network during axis formation
1 Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138 USA
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ABSTRACT |
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 Top
 Abstract
 Introduction
Results
Discussion
Materials and methods
References
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In vertebrates, specification of anteroposterior (A/P)
and left-right (L/R) axes depends on TGF
-related
signals, including Nodal, Lefty, and BMPs. Endoproteolytic maturation
of these proteins is probably mediated by the proprotein convertase
SPC1/Furin. In addition, precursor processing may be
regulated by related activities such as SPC4 (also known as PACE4).
Here, we show that a proportion of embryos lacking SPC4 develop situs
ambiguus combined with left pulmonary isomerism or complex craniofacial
malformations including cyclopia, or both. Gene expression analysis
during early somite stages indicates that spc4 is genetically upstream
of nodal, pitx2, lefty1, and lefty2 and perhaps maintains the
balance between Nodal and BMP signaling in the lateral plate that is critical for L/R axis formation. Furthermore, genetic interactions between nodal and spc4, together with our analysis of chimeric
embryos, strongly suggest that during A/P axis formation, SPC4
acts primarily in the foregut. These findings establish an important
role for SPC4 in patterning the early mouse embryo.
[Key Words:
Axis formation; mouse embryo; TGF signals; maturation; regulation]
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Introduction |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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In the mouse, Nodal activities in the epiblast
and overlying visceral endoderm are required for anteroposterior
(A/P) axis formation (Conlon et al. 1994
; Varlet et al.
1997), whereas at a later stage asymmetric nodal expression in
the node and the left lateral plate mesoderm mediates positional
information specifying the left side of the embryo (Collignon et al.
1996; Lowe et al. 1996). Asymmetric, left-sided expression has also
been documented for the TGF-related molecules Lefty1 and Lefty2
(Meno et al. 1996, 1997). The unique structure and the activity of
Lefty proteins indicate they may act as antagonists of TGF-like
signaling molecules including Nodal or BMPs (Meno et al. 1997; Thisse
and Thisse 1999). Moreover, ectopic expression of Nodal, Lefty1, or
Lefty2 in chick right lateral plate mesoderm induces the bicoid-related
homeobox gene pitx2 that normally is confined to the left
side, suggesting Pitx2 acts downstream of Nodal and/or
Lefty in the left-right (L/R) signaling pathway (Logan
et al. 1998; Piedra et al. 1998; Ryan et al. 1998; Yoshioka et al.
1998). Mutations perturbing the asymmetric expression of these genes in
mice may reverse or randomize the body situs and/or
result in visceral organ isomerisms (Collignon et al. 1996; Lowe et al.
1996; Meno et al. 1996; Ryan et al. 1998; Meyers and Martin 1999).
TGF-related activities are controlled by multiple regulatory
mechanisms. A critical step involves the maturation of inactive precursor proteins via endoproteolytic cleavage, which is thought to
occur within the trans-Golgi network prior to secretion (Sha et al. 1989). Recently, recombinant soluble forms of several
subtilisin-like proprotein convertases (SPCs), including
SPC1/Furin, SPC4/PACE4, SPC6B, and SPC7,
have been shown to cleave purified BMP4 precursor in vitro (Cui et al.
1998). However, results obtained under these experimental conditions
need to be interpreted with caution because they may not necessarily
reflect a physiological enzyme-substrate interaction. In
Xenopus embryos, the serpin-like polypeptide 
1-PDX that
has been reported to selectively inhibit SPC1 and SPC6 activities (Jean
et al. 1998) efficiently blocks the ventralizing activity of BMP4,
suggesting that proteases other than SPC1 and SPC6 may be unable to
activate BMP4 in vivo (Cui et al. 1998). Likewise in transfected tissue
culture cells, we found that SPC7 and SPC6B activities are
post-translationally regulated and fail to enhance BMP4 processing
(D.B. Constam, unpubl.; Constam and Robertson 1999). Thus, of the known
convertases, SPC1 and possibly SPC6A appeared to be solely responsible
for BMP4 cleavage.
Recently, we analyzed a loss-of-function mutation of SPC1 in the mouse:
SPC1-deficient embryos fail to undergo turning and develop severe
ventral closure and heart morphogenesis defects (Roebroek et al. 1998).
A nearly identical phenotype has been reported for embryos lacking
Smad5, a transcription factor mediating BMP signal transduction (Chang
et al. 1999), consistent with the idea that SPC1 is required for
efficient maturation of BMP activities. However, BMP4- and
BMP2-deficient embryos both develop more severe defects (Winnier et al.
1995; Zhang and Bradley 1996), suggesting that processing of these BMPs
does not solely depend on SPC1. During gastrulation, both the epiblast
and cardiogenic mesoderm transiently express spc6 mRNA, and
SPC6A dramatically enhances BMP4 precursor cleavage in cell
transfection assays (D.B. Constam and E.J. Robertson, unpubl.; Constam
et al. 1996). Thus, SPC1 and SPC6A probably act in concert to ensure
optimal activation of BMP ligands.
In Xenopus, inhibition of SPC activities by 1-PDX
expression failed to uncover a physiological role for SPC4. However,
recombinant SPC4 has been reported to process activin (Cui et al.
1998), and overexpression of SPC4 in tissue culture cells also promotes
Nodal maturation (Constam and Robertson 1999). To assess the
physiological function of SPC4 in the embryo, and to test its potential
regulatory role in the activation of Activin or Nodal signals, we have
now generated a loss-of-function mutation of spc4. In the
absence of SPC4, a proportion of embryos develop situs defects
and/or display complex craniofacial malformations.
Analysis of the expression patterns of nodal and putative
Nodal target genes suggests that SPC4 may regulate both Nodal
processing and the activation of BMPs that normally confine
nodal expression to the left side. To identify the tissues
where spc4 expression is primarily required, we also studied
the distribution of spc4 mRNA by whole-mount in situ
hybridization and analyzed chimeric embryos composed of wild-type and
SPC4-deficient cells. From these experiments we conclude that SPC4
expression in the foregut is critical for anterior CNS development. In
addition, SPC4 activities are probably required in the adjacent splanchnic mesoderm to maintain the balance between mutually
antagonistic TGF signaling pathways.
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Results |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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L/R axis defects in SPC4-deficient embryos
To determine the role of SPC4 during mouse development, a
loss-of-function mutation was generated by deleting coding sequence essential for SPC4 activity (Fig. 1A).
spc4+/
mice appeared normal and
were fertile. Irrespective of the genetic background, heterozygous
intercrosses yielded homozygous mutant viable offspring at nonmendelian
frequencies (Fig. 1B; Table 1). To confirm that we
had generated a null allele, total RNA from adult brains was analyzed
by RNase protection. No wild-type spc4 mRNA and only small
quantities of a 309-nucleotide product corresponding to a
nonfunctional, truncated spc4 transcript were detected in homozygous mutants, indicating that the spc4 locus is
inactivated (Fig. 1C).
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Genotyping of progeny from heterozygous intercrosses suggested that between 24% and 28% of the homozygous mutant embryos die prenatally. Analysis of 13 litters from homozygous mutant intercrosses collected between embryonic day (E)13.5 and E15.5 confirmed that by E15.5, a corresponding proportion (25%, n = 19 of 77) of mutant embryos had died (Table 2). Embryonic failure was associated with severe cardiac malformations, including the formation of double outlet right ventricles in combination with ventricular septal defects and in some instances dextrocardia. Common atria were also observed either on the left side or, in association with dextrocardia, on the right side (Fig. 2). By E13.5, the deteriorating hearts of three resorbing embryos displayed persistent truncus arteriosus and had formed a single ventricular chamber (Table 2; data not shown), implicating cardiovascular insufficiency as a probable cause of death. Moreover, a proportion of embryos displayed left pulmonary isomerism (Fig. 2D,E). In ~50% of these animals (n = 6 of 11; Table 2), the stomach, spleen, and pancreas were abnormally positioned on the right side of the midline (Fig. 2F,G). Specific laterality defects are also detected at E9.5, including reversal of the direction of heart looping (n = 2 of 35) or embryo turning (n = 4 of 35), or both (n = 4 of 35) in a corresponding proportion (28%) of homozygous mutants (Fig. 2H,K). These results establish a function for SPC4 during L/R axis formation.
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Dual role for SPC4 in the regulation of asymmetric nodal, pitx2, and lefty expression patterns
As might be expected, the laterality defects of spc4
mutants are preceded by specific alterations in the expression patterns of nodal, lefty, and/or the
bicoid-related homeobox gene pitx2, a putative target of Nodal
signaling. At early somite stages, 71% (n = 29 of 41) of
the mutant embryos express nodal mRNA in both the left and
right lateral plate (Fig. 3; Table
3). Similarly, pitx2 and
lefty mRNAs are bilaterally expressed, although in a smaller
proportion of mutant embryos [21% (n = 5 of 24) and 15%, (n = 3 of 20), respectively]. This disturbance in
asymmetric gene expression probably accounts for left pulmonary
isomerism, because both lefty1 and pitx2 impose
left-sided pattern on the lung primordia (Meno et al. 1998; Ryan et al.
1998; Gage et al. 1999; Lin et al. 1999; Lu et al. 1999). On the other
hand, a proportion of embryos failed to express either lefty1
(n = 2 of 20) or lefty2 (n = 3 of 20),
or both (n = 1 of 20) in the prospective floor plate or
lateral plate mesoderm, respectively (Table 3). We conclude that
asymmetric nodal, lefty, and pitx2 gene
expression patterns are regulated by SPC4-dependent activities.
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The nodal locus contains two FAST transcription factor binding
sites that are necessary and sufficient to mediate tissue-specific nodal expression in the lateral plate (Saijoh et al. 2000).
Xenopus FAST1 and its mouse homolog, FAST2, cooperate with
specific Smad transcription factors to transduce TGF, Activin, and
Nodal signals, raising the possibility that nodal regulates
its own expression via a positive feedback mechanism (Saijoh et al.
2000). If Nodal is required to induce or maintain its own expression in
the lateral plate, a failure to appropriately process Nodal precursor
might be expected to result in loss of nodal expression. To
test this prediction, we reduced the gene dosage of nodal by
crossing a loss-of-function nodallacZ reporter
allele into the spc4 null background (Collignon et al. 1996).
Interestingly, only 29% of the
nodallacZ/+;spc4/
embryos examined at the 4- to 8-somite stage express lacZ
bilaterally, whereas 47% show normal left-sided expression, and 24%
fail to activate nodal on either side, even though
lacZ expression in the node is unperturbed (Table 3). These
findings are consistent with the idea that SPC4 contributes to
establish a threshold concentration of Nodal protein necessary to
maintain nodal expression in the lateral plate.
SPC4 regulates anterior patterning
A proportion of spc4 mutants analyzed between E13.5 and
E15.5 also displayed complex craniofacial abnormalities, including cyclopia, and anterior truncations marked by the absence of the telencephalon, nasal capsule, and upper and lower jaws (Fig. 4A,B; Table 2). The maxillary and mandibular components of
the first branchial arch are often fused (Fig. 4F,K). Midline fusion of the eye anlagen and anterior patterning defects of the CNS are common
in embryos lacking a functional prechordal plate (Chiang et al. 1996;
Izraeli et al. 1999). This most anterior aspect of the axial midline
forms the roof of the rostral foregut and is responsible for inducing
the overlying neurectoderm to acquire anterior character (Dale et al.
1997; Foley et al. 1997). Interestingly, in spc4 mutants
showing anterior malformations, the axial midline marked by
hnf3 expression is severely truncated compared
with stage-matched control embryos (Fig. 4C,D). This early defect
presages the loss of ventral forebrain structures expressing
nkx2.1 (Fig. 4E,F) and vax1 (Fig. 4G-K). We conclude
that SPC4 regulates activities contributed by anterior mesendoderm that
are necessary for specifying the ventral forebrain.
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SPC4 expression is required in the foregut but not in extraembryonic ectoderm
The establishment of anterior pattern depends on signals from both
axial mesendoderm and anterior visceral endoderm (Perea-Gomez et al.
1999; Shawlot et al. 1999). To determine whether spc4 may act
in extraembryonic tissues, we examined its expression pattern by
whole-mount in situ hybridization. spc4 mRNA is abundant in the extraembryonic ectoderm before and during gastrulation, initially in cell populations lining the exocoelomic cavity (E5.5-E7.5) and
subsequently throughout the forming chorion (Fig.
5A-C). In contrast, no spc4 mRNA is
detectable in the visceral endoderm. Within the embryo, spc4
transcripts are first detected during early somite stages in the
definitive foregut endoderm and adjacent splanchnic mesoderm and at low
levels in cephalic mesenchyme (Fig. 5D,E). It seemed likely, therefore,
that the L/R and anterior CNS defects of spc4
mutants are both due to the loss of SPC4 activity in the embryo itself.
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To test this possibility, ES cells heterozygous for the
nodallacZ allele (Collignon et al. 1996), but wild
type with respect to the spc4 locus, were injected into
blastocysts derived from spc4/
intercrosses to generate chimeric embryos lacking SPC4 specifically in
extraembryonic tissues (Beddington and Robertson 1989). Of 34 chimeric
embryos recovered at 4- to 8-somite stages, 31 were efficiently
colonized by ES cell derivatives as judged by strong lacZ
staining of the node and left lateral plate mesoderm. None of these
chimeras displayed anatomical abnormalities or ectopic lacZ
expression (data not shown). Thus, spc4 expression in epiblast derivatives is sufficient for normal development.
Anterior truncations similar to those seen in spc4 mutants
were observed previously in a proportion of embryos
trans-heterozygous for nodal and
hnf3 (Collignon et al. 1996) or nodal
and smad2 (Nomura and Li 1998) and in nodal
heterozygotes lacking ActR-IIA, a putative Nodal receptor (Song et al.
1999). To test for genetic interactions between nodal and
spc4, we similarly examined whether loss of one copy of
nodal exacerbates the phenotype of spc4 mutants. Interestingly, the notochord of nodallacZ/+;
spc4/ embryos displayed large
gaps in the expression of shh and
hnf3, and the foregut was truncated anteriorly
(n = 4 of 6; Fig. 6; data not
shown), demonstrating that nodal and spc4
synergize to promote the formation or maintenance of axial mesendoderm.
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Discussion |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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Here, we have shown that the proprotein convertase SPC4 has a
regulatory function during anterior CNS patterning and
L/R axis formation. Thus, SPC4-deficient embryos
frequently exhibit anterior truncations and/or develop
specific laterality defects. Within the embryo itself, spc4
mRNA expression is first detectable throughout the foregut, the rostral
roof of which constitutes the endodermal component of the prechordal
plate (Seifert et al. 1993). Moreover, severe perturbations of anterior
notochord and foregut are observed in nodallacZ/+;
spc4/ embryos. Thus, in
spc4 mutants, defective anterior patterning may reflect a
specific requirement for SPC4 activity within the endoderm-derived
prechordal plate or in maintaining prechordal mesoderm function. Tissue
explant recombination assays have shown that during early somite stages
the prechordal plate and anterior paraxial mesoderm both contribute
diffusible activities sensitizing the neurectoderm to SHH signals that
impart anterior character. This activity can be mimicked by recombinant
BMP7 and BMP4, whereas preincubation with anti-BMP7 antibodies blocks
activity at least in prechordal plate explants, strongly suggesting BMP
signals cooperate with SHH to pattern the anterior neural plate (Dale et al. 1997). Impaired BMP processing therefore seems most likely to
account for the forebrain defects observed in spc4 mutants.
During L/R axis formation, SPC4 also activates a
signaling pathway that inhibits nodal expression in the right
lateral plate. In the chick, repression of nodal on the right
side is mediated by BMP activities that are themselves antagonized on
the left side by Caronte, a novel member of the DAN family of secreted BMP antagonists (Rodriguez Esteban et al. 1999; Yokouchi et al. 1999).
Also in the mouse, nodal apparently is repressed in the right
lateral plate by BMP signals because loss of the BMP effector molecule
Smad5 results in bilateral nodal expression (Chang et al.
2000). Repression of nodal and lefty2 in the right
lateral plate also requires lefty1 expression (Meno et al.
1998), possibly to prevent diffusion of a Caronte homolog across the
midline (Yokouchi et al. 1999). However, because lefty mRNA
was abundant in the midline of spc4 mutants showing bilateral
expression of lefty2, ectopic induction of lefty2 or
nodal unlikely reflects simply a lack of lefty1
expression. Instead, we propose that in spc4 mutants
derepression of nodal on the right side is caused by impaired BMP processing in the lateral plate mesoderm or adjacent gut endoderm. Consistent with this, several BMPs are expressed in definitive endoderm, including BMP3, BMP4, BMP6, and BMP7, whereas BMP4, BMP5, and
BMP7 are coexpressed in the lateral plate (Dudley and Robertson 1997;
Solloway and Robertson 1999).
In marked contrast to the frequent incidence of bilateral
nodal expression seen in homozygous spc4 single
mutants, spc4/;
nodallacZ/+ embryos carrying only one functional copy of
nodal often fail to induce nodal expression on either side.
This is never observed in spc4+/+ or
spc4+/ embryos, suggesting SPC4
promotes nodal feedback signaling. We propose this novel
finding reflects a direct role for SPC4 in establishing the threshold
concentration of mature Nodal necessary to ensure nodal
autoinduction. In normal embryos, Nodal also induces expression of
pitx2, as well as Lefty proteins thought to inhibit Nodal
feedback signaling (Logan et al. 1998; Meno et al. 1998, 1999; Piedra
et al. 1998; Ryan et al. 1998; Yoshioka et al. 1998; Bisgrove et al.
1999). In keeping with this view, a proportion of spc4 mutants
also display bilateral pitx2 and lefty expression. However, in a majority of
spc4/ embryos, bilateral
nodal expression is not sufficient to ectopically induce
lefty2 or pitx2 in the right lateral plate.
Furthermore, a significant proportion of spc4 mutants lack
lefty2 expression even in the left lateral plate. This is
expected if SPC4 is required for efficient maturation of Nodal
activities. On the other hand, because Lefty2 is a Nodal antagonist,
lack of its expression or inhibition of its own maturation should
further reduce the threshold concentration of Nodal that is required on
the left side to propagate a signal of "leftness," thus
facilitating normal development of embryos unable to optimally process
Nodal. We propose that in addition to regulating BMP activities, SPC4
also ensures efficient Nodal processing and thus plays an important
role in establishing the balance between mutually antagonistic TGF
signaling pathways that is critical for L/R axis
formation (Fig. 7). This simple model is in keeping
with results obtained in cell transfection experiments showing
efficient cleavage of both BMP4 and Nodal in the presence of SPC4
(Constam and Robertson 1999). However, it does not preclude the likely
possiblity that nodal, pitx2, and lefty
genes may differentially respond to slight changes in the
concentrations of either Nodal or BMP proteins and, perhaps, are
differentially modulated by additional signaling pathways.
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In Xenopus embryos, BMP4 maturation is abolished by
1-PDX, an SPC inhibitor reported to selectively block SPC1 and
SPC6 (Cui et al. 1998; Jean et al. 1998). These data argue that in vivo SPC4 has a unique substrate specificity (Cui et al. 1998). However, another possible explanation for the inability of SPC4 to compensate for SPC1 and/or SPC6 in the presence of 1-PDX
instead may simply be its poor expression levels during early
gastrulation stages. Consistent with this idea, the specificities of
SPC1 and SPC4 for TGF-related substrates in vitro have been shown
to overlap (Cui et al. 1998; Constam and Robertson 1999). Another
possibility is that 1-PDX has a higher affinity for
Xenopus SPC4 in comparison with its human homolog and thus may
also impair SPC4 function in the embryo. 1-PDX recently has also
been shown to form SDS-stable complexes with SPC4 and to block its
activity (Benjannet et al. 1997; Tsuji et al. 1999). The present
experiments clearly demonstrate an important role for SPC4 during early
mouse embryogenesis and strongly suggest that BMP activities are
regulated by this convertase.
In previous experiments, we have analyzed chimeric embryos composed of
wild-type and SPC1/Furin-deficient cells. In contrast to
SPC4, SPC1 is required for embryo turning and ventral closure. However,
SPC1 is also required to ensure repression of pitx2 and lefty2 in the right lateral plate, implicating SPC1 as a
likely regulator of BMP and/or Lefty1 activities (Constam
and Robertson 2000). In contrast, it remains unclear whether Nodal
signaling depends on SPC1 in vivo. In particular, we failed to observe
alterations in nodal mRNA expression patterns in SPC1 mutants
(D.B. Constam and E.J. Robertson, unpubl.; Roebroek et al. 1998). Thus,
in SPC1 mutants, pitx2 and lefty2 can apparently be
derepressed on the right side even if nodal expression is
unperturbed, consistent with the assumption of our model that both
Nodal and BMPs provide direct positive and negative regulatory input,
respectively, into these common target genes (Fig. 7). Based on these
considerations, we propose that the fine balance between Nodal and
antagonists of nodal expression in the lateral plate is
established by several SPCs, including SPC1 and SPC4, which partially
overlap in their functions. Functional overlap among these proteases
may also account for the incomplete penetrance of the spc4
mutant phenotype, and/or explain why spc4
expression in the extraembryonic ectoderm is nonessential. SPC1 and
SPC4 mRNAs are coexpressed at high levels both in this extraembryonic
tissue and in definitive endoderm and lateral plate mesoderm (Roebroek
et al. 1998). Future analysis of SPC1/SPC4 double
mutants, therefore, will probably provide further insight into the
partially overlapping functions of these proteases during gastrulation.
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Materials and methods |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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Generation of spc4/ mice
The SPC4 gene targeting vector designed to delete a 280-bp
XbaI-NcoI fragment, including the coding region for
the catalytic serine of SPC4, was derived from a 129SvJ genomic phage
library. Restriction map analysis of the SPC4 locus revealed multiple
polymorphisms among 129SvJ and 129/SvEv alleles.
Therefore, homologous recombination was performed in R1 ES cells
derived from an (129/SvJ × 129/Sv) F1 hybrid (gift of Dr. J. Rossant, Mount Sinai Hospital,
Toronto, Canada). In this cell line, homologous recombination occurred at a frequency of 1 of 30, although exclusively in the SvJ allele (data
not shown). Eleven correctly targeted ES cell lines were injected into
C57BI/6J blastocysts, one of which gave rise to germ line
chimeras that were backcrossed to C57BI/6J or
129/SvEv females to obtain spc4 heterozygous
offspring. For Southern blot analysis, tail DNA samples were digested
with BamHI and probed with an external 3' genomic
fragement detecting 9.5-kb or 6.2-kb fragments corresponding to the
wild-type and mutant alleles, respectively. Embryos were genotyped by
PCR analysis of yolk sac DNA samples using the intron-specific primer
5'-GGCAGCCGAGGCAGACCAATACAGAG-3', in combination with 5'
primers complementary to neomycin cDNA (5'-CCTGCTTGCCGAATATCATGGTGGAAAA-3'), and to exon sequences
deleted in the mutant allele (5'-CGCTGCACCGACGGCCA CACT-3'). To
confirm that the mutation abolished spc4 expression, total RNA
(20 µg) from adult brains was subjected to RNase protection
analysis using an antisense probe complementary to nucleotides
751-1332 of the murine spc4 cDNA (Hosaka et al. 1994),
spanning the deleted region and the entire adjacent upstream exon.
Whole-mount in situ hybridization
Protocol and probes used for whole-mount mRNA in situ hybridization
have been described (Varlet et al. 1997; Constam and Robertson 2000).
In addition, an antisense RNA probe hybridizing to both lefty1
and lefty2 transcripts was synthesized using a lefty1
cDNA template (Meno et al. 1996). Nodal transcripts were detected using full-length or exon 2-specific probes. A Vax1-specific probe (Hallonet et al. 1998) was kindly provided by Dr. P. Gruss (Max-Planck Institute, Göttingen, Germany). A 2.1-kb nkx2.1 cDNA probe was provided by
Dr. J. Rubenstein (UCSF).
Analysis of chimeric embryos
Chimeric embryos lacking SPC4 specifically in extraembryonic
lineages were generated by injecting
spc4/ blastocysts obtained
from homozygous mutant intercrosses with ES cells carrying one copy of
a nodallacZ reporter allele (Collignon et al. 1996).
At stage E8.5, the resulting chimeras were stained for lacZ
expression as described (Hogan et al. 1994).
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Acknowledgments |
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We thank Elizabeth Bikoff, Dominic Norris, and Georgy Koentges for critical review of the manuscript and Patricia Lewko and Joseph Rocca for animal care. This work was supported by a grant from the NIH.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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Received February 23, 2000; revised version accepted March 22, 2000.
2 Present address: Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges, Switzerland.
3 Corresponding author.
E-MAIL ejrobert{at}fas.harvard.edu; FAX (617) 496-6770.
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References |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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and Lim1 interact in the visceral endoderm to regulate primitive streak formation and anterior-posterior polarity in the mouse embryo.
Development
126:
4499-4511[Abstract].1-antitrypsin portland (1-PDX), a blocker of proteolytic activation of bone morphogenetic protein during embryogenesis: Evidence that PACE4 is able to form an SDS-stable acyl intermediate with 1-PDX.
J. Biochem.
126:
591-603
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