Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge MA 02138, USA
* Author for correspondence (e-mail: ejrobert{at}fas.harvard.edu)
Accepted 7 January 2004
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SUMMARY |
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Key words: Mesoderm induction, Smad2/3, Axis patterning, Nodal, Mouse
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Introduction |
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Previous studies have described dose-dependent functions for the TGFß
ligand Nodal and Smad2 (Madh2 Mouse Genome Informatics), its cognate
downstream effector, in coordinating cell fate specification and cell
movements during early vertebrate development
(Nomura and Li, 1998;
Norris et al., 2002
;
Schier and Shen, 2000
;
Vincent et al., 2003
;
Whitman, 2001
). In mouse,
Nodal is expressed throughout the epiblast prior to gastrulation and
then becomes rapidly confined to the posterior side of the embryo, marking the
site of primitive streak formation (Conlon
et al., 1994
; Varlet et al.,
1997
). Genetic studies have shown that Nodal expression
in the epiblast is essential for maintaining reciprocal tissue interactions
that assign early embryonic polarity. Nodal signals to the overlying visceral
endoderm (VE) where it activates Smad2 and promotes formation of the anterior
visceral endoderm (AVE). This specialized signaling center first appears
within a distal patch of VE and establishes proximodistal (PD) polarity within
the early epiblast, and then emerges via directed cell movements as the early
anterior organizer, converting initial PD polarity into the definitive
embryonic anteroposterior (AP) axis. At the same time, Smad2-independent Nodal
signaling within the epiblast is required to induce posterior markers
(Brennan et al., 2001
). Thus,
Nodal mutant embryos arrest at the egg cylinder stage, failing to
form either the AVE or primitive streak and therefore entirely lacking AP
identity (Brennan et al., 2001
;
Conlon et al., 1991
;
Conlon et al., 1994
).
The primitive streak normally becomes visible at embryonic day (E) 6.5 when
cells on the posterior side of the epiblast ingress and emerge as mesoderm and
definitive endoderm. Cells entering the streak adopt different fates depending
on their position relative to the PD axis. Proximal epiblast cells give rise
to extra-embryonic mesoderm. Cells at intermediate levels give rise to heart,
lateral plate and paraxial mesoderm precursors, while more distal populations
generate the axial mesendoderm (AME) and the node (reviewed by
Lawson, 1999). At early to
mid-streak stages, a discrete subpopulation of epiblast cells that exhibits
classical `organizer' activity, i.e. the ability to induce a secondary axis
following heterotopic transplantation (reviewed by
Camus and Tam, 1999
), gives
rise to the anteriormost AME, which comprises the prechordal plate (PCP) and
anterior definitive endoderm (ADE) (Camus
et al., 2000
; Lawson,
1999
). During gastrulation, the progenitors of the PCP and ADE
displace the VE and migrate anteriorly to underlie the neural plate, where
they produce secondary inductive cues that reinforce the initial anterior
identity established by the AVE
(Rubenstein et al., 1998
;
Stern, 2001
). Graded Nodal
signals within the epiblast and the primitive streak have been shown to govern
specification of the AME. For example, recent studies have shown that reducing
Nodal expression within the mouse epiblast with partial
loss-of-function Nodal alleles selectively disrupts specification of
the anterior AME and consequently mutant embryos display varying degrees of
anterior truncations (Lowe et al.,
2001
; Norris et al.,
2002
; Vincent et al.,
2003
).
TGFß cell-surface receptors activate downstream intracellular
effectors, the so termed R-Smads, that in turn associate with the co-mediator
Smad4. This heteromeric complex translocates into the nucleus to regulate cell
type-specific target gene expression (reviewed by
Massagué and Wotton,
2000). Smad1, Smad5 and Smad8 function downstream of BMP
sub-family ligands, whereas TGFß/Activin/Nodal receptors activate the
closely related Smad2 and Smad3 proteins
(Moustakas et al., 2001
;
Shi and Massagué,
2003
). Nodal signaling via the Alk4 or Alk7 type I receptor in
association with either the ActRIIA or ActRIIB type II receptor has been shown
to activate Smad2 (Kumar et al.,
2001
; Reissmann et al.,
2001
; Whitman,
2001
). Interestingly, the closely related intracellular effector
molecule Smad3 (Madh3 Mouse Genome Informatics) shares an overall 92%
amino acid identity with Smad2, and both Smad2 and Smad3 induce expression of
dorsal mesodermal markers in Xenopus explant assays
(Baker and Harland, 1996
;
Chen et al., 1997
;
Graff et al., 1996
). Moreover,
both Smad2 and Smad3 efficiently interact with the transcription factor Foxh1
(FAST) in vitro (Labbé et al.,
1998
; Yeo et al.,
1999
). These findings suggest that for a broad spectrum of in vivo
functions, Smad2 and Smad3 are functionally interchangeable and that Smad3
also functions downstream of the Nodal receptor complex.
However, the N-terminal Smad2 MH1 domain contains a distinctive thirty
amino acid insert predicted to impose steric constraints that selectively
disrupt DNA binding (Shi et al.,
1998). Consistent with this DNA-binding difference, Smad2 and
Smad3 exhibit distinctive functional activities in a variety of
transcriptional reporter assays (reviewed by
Liu, 2003
;
Moustakas et al., 2001
;
Shi and Massagué,
2003
). For example, Smad3 suppresses whereas Smad2 activates the
Xenopus goosecoid promoter
(Labbé et al., 1998
).
These findings suggest that Smad2 and Smad3 also play unique roles in
directing cell type-specific responses. Consistent with this, Smad2
and Smad3 mutant mice exhibit strikingly different phenotypes.
Smad3-deficient mice develop to term and exhibit only subtle
developmental abnormalities (Datto et al.,
1999
; Yang et al.,
1999
; Zhu et al.,
1998
). We have previously shown that Smad2 and
Smad3 expression domains mostly overlap at early embryonic stages
(Tremblay et al., 2000
).
However Smad3 is not expressed in the VE, where Smad2
functions uniquely to specify the AVE. Thus, Smad2 mutant embryos
fail to form the AVE, display early patterning defects, and consequently
become highly disorganized by E6.5 (Brennan
et al., 2001
; Heyer et al.,
1999
; Waldrip et al.,
1998
).
To further dissect shared and/or unique roles provided by Smad2 and Smad3,
we have manipulated their expression ratios in vivo. We find that
Smad2;Smad3 double heterozygous animals are born at the
expected Mendelian ratio and are fully viable and fertile. However, loss of
Smad3 in the context of one wild-type copy of Smad2 results
in embryonic lethality around E9.5. As for conditional loss of Smad2
from the epiblast (Vincent et al.,
2003), these
Smad2+/;Smad3/
mutant embryos display impaired production of anterior AME during
gastrulation. Surprisingly, selective removal of both Smad2 and
Smad3 from the epiblast disrupts specification of axial and paraxial
mesodermal derivatives. Finally, we demonstrate that Smad2;Smad3
double homozygous mutants entirely lack mesoderm and fail to gastrulate. The
present work reveals for the first time that Smad3 expression
contributes essential signals at early post-implantation stages of mouse
development.
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Materials and methods |
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Whole-mount in situ hybridization and histology
Individually genotyped or groups of embryos were processed for whole-mount
in situ hybridization as described (Nagy
et al., 2003) with the following probes: Foxa2, Shh, T, Sox2,
Meox1, Bmp4, Oct4 (Pou5f1 Mouse Genome Informatics),
Otx2, Six3, Fgf8, Krox20 (Egr2 Mouse Genome
Informatics), Chrd, Nog, Cer1, Hex, or Smad3 (IMAGE clone
45861). For histology, embryos or decidua were fixed in 4% paraformaldehyde,
dehydrated through an ethanol series, and embedded in paraffin wax.
Hematoxylin and eosin staining was performed according to standard
protocols.
Protein purification and western blotting
Protein extracts (50 µg) derived from CCE ES cells, KT15
Smad2Robm1 homozygous ES cells, STO fibroblasts, or thymi,
spleen and livers of adult mice were mixed with an equal volume of
2xLaemmli buffer and then separated on a 10% SDS-PAGE gel followed by
transfer to nitrocellulose (Protran). Blots were incubated with either mouse
anti-Smad2/3 (Transduction) or rabbit anti-Smad3 (Zymed) primary antibodies
(Abs) and then with either anti-mouse or anti-rabbit IgG HRP-conjugated
secondary Abs (Amersham). Blots were developed by chemiluminescence using ECL
(Amersham).
Plasmids and transfections
N-terminally FLAG-tagged human Smad2 and Smad3 cDNA
cassettes (Yagi et al., 1999)
were subcloned into pCAGGS (Niwa et al.,
1991
). Mv1Lu mink lung cells (ATCC) were grown overnight prior to
transfection with a total of 1.5 µg DNA complexed with Lipofectamine 2000
(Invitrogen). The following plasmids (0.25 µg/well) were used in
transfections: (n2)7-luc, caALK4, pCR3-FAST2, pcDNA3-Smad4myc,
pCAGGS-FLAG-hSmad2 and pCAGGS-FLAG-hSmad3, and the renilla luciferase
expression vector pRL-CMV (Promega). Cell lysates were analyzed after 40 hours
using the Dual-Luciferase Reporter Assay System (Promega). Mean values of each
set of triplicates were plotted as fold induction compared to activation of
the reporter by caALK4 alone. FLAG-hSmad2 or FLAG-hSmad 3 protein expression
was verified by western analysis with the M5 anti-FLAG mouse monoclonal.
Supernatant from the 9e10 hybridoma (ATCC) was used to detect Smad4myc.
Anti-mouse IgG HRP (Amersham) was used to detect bound primaries.
RT-PCR and ribonuclease protection assays
Total RNA from CCE ES cells, thymus or wild-type embryos was prepared using
the Trizol® method (Invitrogen). Ribonuclease protection assays were
performed on 10 µg total RNA (RPA IIITM kit, Ambion). The
Smad2 probe corresponds to a 3' 221 bp
BglII-BamHI fragment
(Waldrip et al., 1998), and
the Smad3 probe represents a 3' 237 bp HincII
fragment.
Blastocyst RNA was isolated with the Absolutely RNA MicroprepTM Kit
(Stratagene). Random-primed total RNA was reverse transcribed with
SuperScriptTM reverse transcriptase (Invitrogen), and resulting cDNA
assayed by RT-PCR for Smad2 and Hprt
(Oxburgh and Robertson, 2002),
Smad3 (Rosendahl et al.,
2001
) and Smad4 expression in the presence of 1 µCi
P32-dCTP. Smad4 RT-PCR reactions use forward primer
5'-GCCATTGGTTTTCACTGCCTTC-3' and reverse primer 5'-GGGTGTTGG
ATGGTTGAATCG-3'), and yield a 632 bp product.
RPA and RT-PCR products were separated on 5% PAGE gels, exposed to film and quantitated by phosphoimager.
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Results |
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Smad3 expression domains were further analyzed by whole-mount in
situ hybridization. Consistent with previous results, Smad2 is
broadly expressed throughout all tissues and at all stages analyzed
(de Sousa Lopes et al., 2003;
Tremblay et al., 2000
;
Waldrip et al., 1998
). As
expected, the visceral endoderm lacks Smad3 transcripts, but
Smad3 is expressed throughout the epiblast
(Fig. 1G) (Tremblay et al., 2000
). At
early gastrulation stages, Smad3 transcripts are most abundant in the
extra-embryonic ectoderm and later in its derivative the chorion
(Fig. 1G,H). Highest levels of
Smad3 expression are seen along the ventral midline and in the
somites by E8.5 (Fig. 1I).
Both Smad2 and Smad3 activate the Nodal ASE
Both Smad2 and Smad3 associate with the activated Alk4 receptor and can
propagate Nodal signaling in P19 embryonal carcinoma cells
(Kumar et al., 2001;
Lebrun et al., 1999
). However,
it remains unknown whether Smad2 and Smad3 both transduce Nodal/Alk4 signals
in early mouse embryos. A Foxh1-dependent autoregulatory enhancer, termed the
ASE, directs Nodal expression in the early epiblast, VE and left
lateral plate mesoderm (Adachi et al.,
1999
; Norris et al.,
2002
; Norris and Robertson,
1999
), and interestingly an ASE-lacZ reporter transgene
is activated appropriately in the epiblast of Smad2-deficient embryos
(Brennan et al., 2001
). This
observation suggests that Smad3 functionally compensates for the loss of Smad2
in this genetic context. To test this possibility directly, we compared the
abilities of Smad2 and Smad3 to activate the (n2)7-luc
reporter construct containing seven tandem repeats of a Foxh1-responsive 24 bp
oligonucleotide from the mouse Nodal ASE. This reporter is activated
by Foxh1 in a TGFß-dependent manner in Mv1Lu cells
(Saijoh et al., 2000
). As
shown in Fig. 2A, in the
presence of a constitutively active Alk4 receptor, both Smad2 and Smad3 give
robust amplification of the transcriptional response. These results
demonstrate that both Smad2 and Smad3 mediate Foxh1-dependent activation of
the Nodal ASE.
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|
We used a panel of molecular markers to evaluate AME specification in Smad2+/;Smad3/ mutant embryos. The winged helix transcription factor Foxa2 (Hnf3b) is first expressed in the anterior primitive streak and AVE, then slightly later in the patent node, AME, notochord and floorplate (Fig. 4A,C,E). Smad2+/;Smad3/ embryos show Foxa2 expression within the AVE and node, but very little to no expression is detected in the region of the AME that normally extends rostrally from the node (Fig. 4B,D,F). Shh is also expressed in the node, AME and notochord (Fig. 4G,I,K), but in mutant embryos Shh transcripts are limited to the node and notochord, with only occasional Shh-positive AME cells being found in the presumptive anterior midline (Fig. 4H,J,L). Thus, we conclude that the AME fails to form in Smad2+/;Smad3/ mutant embryos.
|
The definitive endoderm emerges either side of the midline as a thin,
superficial layer of cells that displaces VE proximally, and is marked by
expression of the Nodal/Wnt antagonist cerberus (Cer1) and
Hhex. Expression levels of these two diagnostic markers are greatly
diminished but not completely lost in the distal region of mutant embryos
(Fig. 4Q,R), allowing us to
conclude that ADE formation is significantly impaired. At E8.5, Hhex,
which normally marks invaginating cells of the foregut pocket, is lost
(Fig. 4T), and Shh
expression in the hindgut pocket is significantly reduced compared with wild
type (Fig. 4L'). At E9.5,
we observe a rudimentary gut tube in
Smad2+/;Smad3/
mutants (Fig. 3E,F,H). The
tissue occupying this region probably originates from extra-embryonic endoderm
that fails to be displaced, as was previously shown in embryos that develop
from an epiblast lacking Smad2
(Vincent et al., 2003).
Similar to other reports describing defects in the specification or
maintenance of the AME,
Smad2+/;Smad3/
mutant embryos are morphologically recognizable at the late headfold stage due
to the development of a thickened anterior neuroepithelium
(Fig. 4N,P) (Hallonet et al., 2002;
Martinez Barbera et al., 2000
;
Shawlot et al., 1999
). At
E8.5, these embryos display a flattened neural region, with anterior
truncations and fusions and persistent lack of neural groove
(Fig. 4L,V,X,Z). Forebrain
(Six3 and Otx2), midbrain (Fgf8 and Otx2)
and hindbrain (Krox20) markers are induced
(Fig. 4L,U-Z). Among the
markers analyzed, only Fgf8 expression in the anterior neural ridge,
a forebrain organizing center within the anteriormost neural plate
(Rubenstein et al., 1998
), is
consistently absent (Fig. 4Z),
confirming loss of forebrain structures. Taken together, these results show
that in
Smad2+/;Smad3/
mutants specification of the neural plate occurs normally, but the loss of
anterior AME compromises its subsequent growth and patterning. Collectively,
these experiments demonstrate that Smad2 and Smad3 signals cooperatively
specify anterior streak fates.
Smad2 and Smad3 co-expression within the epiblast is essential for mesodermal patterning
To further assess Smad3 contributions during gastrulation and
mesoderm patterning, the Smad3 null allele was introduced into the
Smad2CA conditional background
(Vincent et al., 2003). In
embryos with Smad2-deficient epiblast but expressing one wild-type
copy of Smad3
(Sox2Cre;Smad2Robm1/CA;Smad3null/+),
all derivatives of the anterior primitive streak, including PCP, ADE,
notochord, node and posterior endoderm are eliminated
(Fig. 5B). The loss of axial
structures is confirmed by the complete absence of Shh expression
(Fig. 5D); similar results were
obtained with Foxa2 (Vincent et
al., 2003
). Somites, a derivative of paraxial mesoderm, are fused
across the midline, a feature associated with the loss of the node and
notochord (Fig. 5B,B').
Anterior development is compromised, and in some embryos the allantois is
enlarged (Fig. 5B,D).
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Homozygous Smad2/;Smad3/ double mutant embryos entirely lack mesodermal tissues
As shown above (Fig. 1), we
observe Smad2 and Smad3 co-expression from the blastocyst
stage onwards. Notably, by E5.5 Smad2 and Smad3 are both
strongly expressed in the extra-embryonic ectoderm
(Tremblay et al., 2000). To
further evaluate their respective functional contributions,
Smad2Robm1/+;Smad3null/+
double heterozygous mice were intercrossed. Four different phenotypic classes
of embryos were recovered at late primitive streak stages, corresponding to:
(1) overtly wild-type embryos, including
Smad2+/;Smad3+/+,
Smad2+/;Smad3+/ and
Smad2+/;Smad3/,
which are unremarkable at this stage; (2)
Smad2/;Smad3+/+ mutant
embryos with defects described previously
(Waldrip et al., 1998
); (3)
Smad2/;Smad3+/
embryos; and (4)
Smad2/;Smad3/
double mutants.
Smad2/;Smad3+/+
embryos fail to establish anterior pattern, and the epiblast gives rise
exclusively to mesoderm that proliferates to form extra-embryonic structures
including allantois and blood islands, as well as germ cells
(Tremblay et al., 2001;
Waldrip et al., 1998
). We find
that
Smad2/;Smad3+/
mutant embryos are more severely compromised and develop a distinct thickened
rind of corrugated, prominently vacuolated columnar endoderm
(Fig. 6C). Although cavitation
occurs, we fail to observe a distinct boundary between extra-embryonic and
embryonic regions (Fig. 6G).
Rather the extra-embryonic ectoderm and epiblast are often folded and
distorted, and only limited mesoderm formation is observed. Loss of
pluripotential epiblast in these embryos was confirmed by analyzing expression
of Oct4, which is normally robustly expressed at this stage of
development (Fig. 6K,I). The
identity of the presumptive extra-embryonic ectoderm and mesoderm populations
was confirmed using Sox2 and Bmp4
(Fig. 6O,S).
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Discussion |
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Similar to Smad2, Smad3 elicits formation of dorsal mesoderm in
Xenopus explant assays (Chen et
al., 1997). Moreover, a variety of reporter assays implicates
Smad2 and Smad3 functional redundancy. These findings raise the possibility
that Smad3 also acts downstream of TGFß/activin/Nodal signaling in the
embryo. However, Smad3 mutant mice develop normally and exhibit only
subtle phenotypic abnormalities (Datto et
al., 1999
; Yang et al.,
1999
; Zhu et al.,
1998
). By contrast, Smad2 mutant embryos develop early
patterning defects because of a failure to establish the AVE
(Brennan et al., 2001
;
Heyer et al., 1999
;
Waldrip et al., 1998
). The
simplest explanation to account for these distinct phenotypes is that Smad2
acts alone as the key transducer of TGFß/activin/Nodal-related signals
during early development. However, this is not the case. The present work
demonstrates that (1) both Smad2 and Smad3 cooperate with the transcription
factor Foxh1 to regulate the Foxh1-dependent autoregulatory enhancer present
in the Nodal locus; (2) Smad2 and Smad3 as well as
Smad4 transcripts are expressed from the blastocyst stage onwards,
with Smad3 expression domains appearing more tightly regulated in
comparison to widespread Smad2 expression; (3) Smad2 and
Smad3 expression ratios are independently regulated; and, finally,
(4) combinatorial Smad2 and Smad3 activities indeed regulate mesoderm
formation and patterning in the developing mouse embryo.
It has previously been shown that Smad2 function in the epiblast
is not required for establishment of the AVE, primitive streak formation or
gastrulation movements (Tremblay et al.,
2000; Waldrip et al.,
1998
). Rather selective loss of Smad2 in the epiblast
results in a failure to correctly specify progenitors of the AME
(Vincent et al., 2003
).
Similarly, the AVE forms normally in
Smad2+/;Smad3/
embryos, and the anterior epiblast is endowed with neural pre-pattern.
However, in this genetic context, loss of Smad3 combined with
decreased Smad2 expression disrupts the production of anterior AME
during gastrulation. Thus, we conclude that formation of the AME not only
requires Smad2 but is also governed by the closely related molecule Smad3.
In the absence of anterior AME production during gastrulation,
Smad2+/;Smad3/
mutant embryos probably recruit extra-embryonic endoderm that fails to be
displaced into a gut-like structure
(Vincent et al., 2003).
Consequently, the crucial refining signals that normally emanate from the
anterior AME and orchestrate the continued patterning and morphogenesis of the
anterior neuroectoderm are lost, and mutant embryos develop anterior
truncations. The common origin, intimate morphogenesis and final topological
proximity of the AME component tissues, the prechordal mesoderm and ADE, make
it difficult to distinguish patterning activities contributed by these
distinct cell populations. The divergent homeobox gene Hhex is
specifically expressed in ADE, and chimeric embryos in which the ADE is
largely Hhex-deficient show forebrain abnormalities
(Martinez Barbera et al.,
2000
). By contrast, conditional disruption of Foxa2
activity with nestin:Cre yields embryos that initially form, but fail
to maintain the ADE and lack all other AME including prechordal mesoderm
(Hallonet et al., 2002
).
Similar to
Smad2+/;Smad3/
mutants, conditional loss of Foxa2 also results in severe anterior
truncations and heart defects. Therefore, disrupting the morphogenesis or
signaling by any single component of the anterior AME seems to impact the
patterning activity of the remaining, tightly apposed cell lineage. Consistent
with this idea, experiments in mice and chick have strongly implicated planar
signaling within anterior midline tissues
(Camus et al., 2000
;
Dale et al., 1997
).
We further demonstrate that reduction in Smad2/3 gene dose
specifically within the epiblast sequentially eliminates anterior streak
derivatives, first impacting the anterior AME
(Smad2Robm1/CA), then the node and remaining axial
mesoderm
(Smad2Robm1/CA;Smad3+/), and
finally affecting tissues that normally originate from the mid-streak
including paraxial and lateral mesoderm
(Smad2Robm1/CA;Smad3/).
These experiments thus reveal a previously unappreciated role for
Nodal-Smad2/3 signals in patterning middle primitive streak derivatives.
Considering that Foxh1-null mutants display defects confined to the
anterior streak (Hoodless et al.,
2001; Yamamoto et al.,
2001
), we conclude these activities are mediated via
Foxh1-independent pathways. Smad2 and Smad3 may therefore govern target gene
expression by associating with other, yet to be described DNA-binding
partners. Smad2/3 effectors may also regulate cellular responses by modulating
intracellular components downstream of other signaling molecules. Indeed,
considerable data demonstrate an interplay between TGFß signaling
pathways and those controlled by ERK/MAPK and EGF-Ras (reviewed by
Derynck and Zhang, 2003
;
Shi and Massagué,
2003
). Interestingly, disruption of FGF signaling leads to
enhanced formation of axial mesoderm
(Yamaguchi et al., 1994
). A
fine balance of signaling by TGFß family members, FGFs and other growth
factors seems to precisely control cell fate allocation in the primitive
streak.
Smad2/ embryos fail to form the AVE
(Heyer et al., 1999;
Waldrip et al., 1998
).
Consequently, anterior fates are not imposed on the epiblast. Rather,
posteriorizing signals such as Bmp4 predominate and convert this tissue into
nascent mesoderm that ultimately becomes specified to form extra-embryonic
mesoderm and primordial germ cells (Lawson
et al., 1999
). We show that progressive reduction of
Smad3 expression in the context of Smad2-deficient embryos
causes more severe phenotypes. Thus, mesoderm formation is greatly diminished
in Smad2/;
Smad3+/ mutants, presumably as a result of highly
reduced Nodal signaling within the epiblast
(Brennan et al., 2001
;
Norris et al., 2002
).
Nonetheless, low levels of Nodal appear to promote maintenance of the adjacent
extra-embryonic ectoderm, and this tissue as well as the visceral endoderm
appears to proliferate normally (Fig.
5).
Smad2/;Smad3/
embryos are even more severely compromised. We observe minimal elaboration of
the embryonic and extra-embryonic cell populations, and mesoderm induction is
entirely absent. Interestingly, these double mutant embryos closely resemble
Smad4-deficient or ActRIIA/IIB-doubly deficient
embryos, and are consistently smaller than Nodal mutant embryos
(Sirard et al., 1998;
Song et al., 1999
;
Yang et al., 1998
). It is
tempting to speculate that TGFß-family members secreted by the maternal
decidual tissue and acting upstream of Smad2/3, such as activin, collaborate
with Nodal to promote growth and organization of the early embryonic and
extra-embryonic lineages.
The genotypes and corresponding tissue disturbances generated by
intercrossing Smad2Robm1/+ and
Smad3null/+ mutant mice are summarized in
Fig. 7. The striking phenotypic
similarity shared between
Smad2+/;Smad3/
embryos and those selectively lacking Smad2 activity in the epiblast
(Smad2Robm1/CA;Sox2Cre) suggests that
functions mediated by one copy of Smad2 are roughly equivalent to
those provided by wild-type levels of Smad3. If the combined levels
of Smad2 and Smad3 activities within cells of the epiblast and primitive
streak fall below a crucial threshold level, formation of anterior AME is
eliminated. The fact that Smad2;Smad3 double heterozygous
animals develop normally defines the upper limit of this threshold.
Progressive loss of Smad3 in the context of a
Smad2-deficient epiblast eventually disrupts node and axial mesoderm
formation. Further reduction of Smad2/3 signals compromises paraxial and
lateral mesoderm formation (Fig.
7). The simplest explanation to account for the relatively strong
Smad2 gene dose effects is that Smad2 is more abundant in the early
embryo. RPA experiments indeed reveal approximately twofold higher levels of
Smad2 transcripts in early gastrulation stage embryos
(Fig. 1), which is consistent
with the less robust expression of Smad3 in zebrafish and
Xenopus gastrula-stage embryos
(Dick et al., 2000;
Howell et al., 2001
).
Interestingly surveys of Smad2 and Smad3 protein distribution using
cross-reactive antibodies reveals that Smad2 is more broadly expressed,
whereas Smad3 shows a more restricted pattern in embryos, tissues and cell
lines (Flanders et al., 2001
)
(N.R.D., S.D.V., L.O., E.J.R. and E.K.B., unpublished). Smad3 may therefore
amplify Smad2 signals in selected cell types.
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ACKNOWLEDGMENTS |
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REFERENCES |
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