1 Department of Molecular and Cellular Biology, Harvard University, Cambridge,
MA 02138, USA
2 Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115,
USA
* Author for correspondence (e-mail: ejrobert{at}fas.harvard.edu)
Accepted 27 April 2004
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SUMMARY |
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Key words: Smad4, TGFß, Anterior primitive streak, Mesoderm patterning, Mouse
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Introduction |
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The receptor complex is composed of two distinct transmembrane
serine-threonine kinases termed type I and type II receptors. Upon ligand
binding, the activated receptor complex phosphorylates members of the Smad
family. The receptor-associated Smads (R-Smads) function as both signal
transduction mediators and transcription factors essential for TGFß
signaling and can be subdivided into two groups. While Smad1, Smad5 and Smad8
are phosphorylated in response to Bmp signals, Smad2 and Smad3 are
phosphorylated in response to activation of the TGFß, activin and Nodal
pathways. Phosphorylation of the R-Smads allows their association with the
common-mediator Smad, Smad4, resulting in nuclear translocation and formation
of higher order transcriptional complexes. Current models emphasize the
central requirement of Smad4 for the regulation of TGFß target genes. By
itself or in combination with R-Smads, Smad4 weakly binds DNA and only
moderately activates transcription. For robust gene activation or suppression,
heteromeric Smad4-containing complexes must associate with additional nuclear
co-factors or tissue-specific transcription factors such as FoxH1 or OAZ
(reviewed by Attisano and Wrana,
2000; Moustakas et al.,
2001
).
During early vertebrate development, the Nodal signaling pathway plays a
conserved role in the establishment of the anteroposterior (AP) axis,
gastrulation and left-right patterning (reviewed by
Schier, 2003;
Whitman, 2001
). In the early
mouse embryo, Nodal signals from the epiblast are transduced in the overlying
visceral endoderm (VE) to establish the early anterior organizer known as the
anterior visceral endoderm (AVE) (Lu et
al., 2001
). The AVE acts prior to gastrulation to impart early
anterior character in the adjacent epiblast. Nodal mutants fail to
pattern the AVE and arrest prior to gastrulation
(Brennan et al., 2001
).
Similarly, null mutations within the Nodal receptor complex, either the type I
receptor ALK4 or the type II receptors ActRIIA and ActRIIB, result in embryos
that do not gastrulate and overtly resemble Nodal mutants
(Gu et al., 1998
;
Song et al., 1999
). Continued
Nodal signaling during gastrulation instructs epiblast cells passing through
the anterior primitive streak to become definitive endoderm (DE), prechordal
plate, node and notochord. Downstream of Nodal signaling, loss of
Smad2 in the epiblast or reducing both Smad2 and
Smad3 gene dose yield phenotypes indistinguishable from
Nodal hypomorphic mutations (Dunn
et al., 2004
; Lowe et al.,
2001
; Norris et al.,
2002
; Vincent et al.,
2003
). Similarly, loss of Foxh1, which encodes a forkhead
transcription factor that associates with Smad2/3 and Smad4, yields phenotypes
that closely resemble those resulting from disruption of the Nodal signaling
pathway (Schier, 2003
). Taken
together, these biochemical and genetic results underscore the significance of
the Smad pathway in transducing Nodal signals during early embryogenesis.
Signaling via the Bmp pathway is also crucial in early embryonic
development, but loss-of-function phenotypes are much more varied (reviewed by
Zhao, 2003). Mutations in the
type I receptor Bmpr1a/Alk3 and type II receptor Bmpr2 lead
to a block prior to gastrulation. Inactivation of Bmp4 results in a
reduction in extra-embryonic mesoderm characterized by a lack of an allantois
and loss of primordial germ cells (PGCs)
(Lawson et al., 1999
). Loss of
Bmp2 and Bmp8b also affect allantois development and PGC
number (Loebel et al., 2003
).
Mutations in the Bmp effectors Smad1 and Smad5 result in
allantois, PGC and yolk sac defects as well as impaired angiogenesis. In
addition, genetic analysis has implicated a number of Bmps in heart
development including Bmp2, Bmp4, Bmp5, Bmp6 and Bmp7.
Compared with the early gastrulation defects associated with Bmp receptor
mutations, the phenotypes resulting from loss of individual Bmp ligands or
their corresponding downstream Smads are more moderate, perhaps reflecting
functional redundancy among Bmp or Smad members.
Consistent with the abundant biochemical data that suggest a central role
for Smad4 in the transduction of all TGFß-related signals, mouse embryos
homozygous for a Smad4 null allele arrest prior to gastrulation
(Sirard et al., 1998;
Yang et al., 1998
). Mutant
embryos show disorganization by embryonic day (E) 6.5 and do not form
mesoderm, a phenotype more severe than that of any individual R-Smad mutation.
Limited analysis of Smad4-null embryos indicates that the initial
requirement for Smad4 activity resides in the extra-embryonic lineages:
aggregation of Smad4-deficient embryonic stem (ES) cells with tetraploid
wild-type embryos gives rise to chimeric embryos that express the mesodermal
marker T and show limited formation of mesodermal derivatives such as
somites (Sirard et al., 1998
).
However, the specific roles of Smad4 within the developing embryo proper have
not been well characterized.
We have selectively manipulated Smad4 gene activity in the epiblast and its derivatives in order to address the combined roles of TGFß-related signaling during gastrulation and specification of the definitive embryonic lineages. We generated a Smad4 conditional allele that undergoes efficient Cre-mediated DNA recombination within the epiblast in response to the robust Sox2Cre transgene. Loss of Smad4 does not prevent the establishment of the primary AP axis and gastrulation is initiated normally. However, we observe a highly focal defect in patterning of the primitive streak as gastrulation proceeds. Mutant embryos fail to form derivatives of the anterior primitive streak (APS), such as the node, notochord and definitive endoderm, and thus share many phenotypic similarities associated with downregulation of the Nodal/Smad2/3/Foxh1 pathway. Surprisingly, the transduction of extra-embryonic Bmp signals within the epiblast is only moderately impaired in the absence of Smad4. Posterior streak derivatives probably patterned in response to extra-embryonic Bmp signals are induced normally, and mutant embryos consistently form a rudimentary heart tube, yolk sac mesoderm and allantois. By contrast, specification of the germline is Smad4-dependent as PGC formation is dramatically diminished. Taken together, these studies indicate that Smad4 is an essential component of the Nodal signaling pathway that specifies the APS, but is required to transduce only select Bmp signals during early development.
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Materials and methods |
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Mouse strains
Smad4CA/CA was maintained on a (129xC57BL/6)
hybrid background. Smad4N/+ and
Smad4CA/CA;ROSA26R/R were maintained
on a partially outbred CD1 background.
Sox2Cre/+;Smad4N/+ mice were generated from
crosses between Smad4N/+ and Sox2Cre/+ parents
(Hayashi et al., 2002b) and
maintained on a CD1 background.
Whole-mount in situ hybridization, X-gal staining, histology and PGC staining
Whole-mount in situ hybridization was performed according to standard
procedures (Nagy et al.,
2003), with the following commonly available probes Shh, T,
Nodal, Foxa2, Gsc, Hex, Cer1, Otx2, Fgf8, Krox20, Bmp4, Eomes and
Oct4 (Brennan et al.,
2001
; Vincent et al.,
2003
). Additional probes used for this study: Lim1
(Barnes et al., 1994
),
Mml (Pearce and Evans,
1999
), and Nkx2.5 and cardiac alpha-actin
(Norris et al., 2002
). X-gal
staining was performed as described by Nagy et al.
(Nagy et al., 2003
). For
Hematoxylin and Eosin histology, embryos were processed, sectioned and stained
using standard procedures. Histochemical staining of PGCs and mounting of
specimens were performed essentially as described
(Lawson et al., 1999
).
Western blots
ES cells were sonicated in 2x sample buffer containing protease
inhibitor cocktail (Roche). Protein extract (40 µg per cell line) was
separated on a 10% SDS-PAGE gel and electroblotted onto nitrocellulose
(Schleicher & Schuell). The membrane was then hybridized overnight at
4°C with a mouse monoclonal antibody showing affinity for the linker
and/or MH2 domain of Smad4 (B-8; Santa Cruz) diluted 1:1000, followed by
HRP-conjugated sheep anti-mouse IgG and ECL (Amersham).
Embryoid body differentiation and RT-PCR
Embryoid bodies were generated from ES cell lines
(Robertson, 1987), harvested
at day 5 of differentiation and then every 2 to 3 days until day 12. RNA was
isolated using Absolutely RNA Miniprep Kit (Stratagene). RNA (10 µg) was
reverse transcribed using Superscript II RNase H-reverse transcriptase
(Invitrogen). For all genes analyzed, cDNA reverse transcribed from 0.1 µg
of RNA was used for PCR amplification. PCR amplification was carried out using
1 µCi [
-32P] dCTP per reaction and performed for 22-26
cycles, maintaining linear amplification. The PCR products were separated on a
6% nondenaturing polyacrylamide gel, vacuum dried and subjected to
autoradiography. Primer sequences used were as follows: G3PDHF,
5'-ACCACAGTCCATGCCATCAC-3'; G3PDHR,
5'-TCCACCACCCTGTTGCTGTA-3'; Msx1F,
5'-AACCCCTTGCTACACACTTCCTCC-3'; Msx1R,
5'-GGACCACGGATAAATCTCTTGGC-3'; Msx2F,
5'-GGAGCACCGTGGATACAGGAG-3'; Msx2R,
5'-GCACAGGTCTATGGAAGGGGTAG-3'; HnfF,
5'-ATGCCTGCCTCAAAGCCATC-3'; HnfR
5'-CCACTCACACATCTGTCCATTGC-3; TransferrinF,
5'-GCCATCCCATCACAACAAGGTATC-3'; TransferrinR,
5'-CTGCTTCAGATTCTTAGCCCATTC-3'
Isolation of primary embryonic fibroblasts
In order to obtain Smad4 null murine embryonic fibroblasts (MEFs),
we crossed the tamoxifen-inducible Cre transgene CAGGCre-ERTM
(Hayashi and McMahon, 2002)
onto the Smad4N/+ background. Litters of
CAGGCre-ERTM;Smad4N/+xSmad4CA/CA
embryos were harvested at E13.5. Embryos were genotyped and fibroblasts were
isolated using standard procedures (Nagy
et al., 2003
). Fibroblasts from
CAGGCre-ERTM;Smad4CA/N embryos were pooled as were
Smad4CA/+ littermates. Cre recombinase expression was
induced by addition of 1 µM 4-OH-Tamoxifen to the culture medium at passage
2. Cells were passaged twice in the presence of 4-OH-Tamoxifen, and seeded for
transfection at passage 5. Excision of Smad4 was verified by PCR
genotyping.
Transcriptional reporter assays
Cells were seeded at 3x105 cells/well in 12-well dishes
prior to transfection with Lipofectamine 2000 (Invitrogen) according to the
manufacturer's instructions. The following plasmids (0.75 µg) were used for
the 3TP-lux assay: 3TP-lux (Wrana et al.,
1992) and pCMV5B-MADR4
(Macías-Silva et al.,
1996
). For the Msx2-lux assay, 0.5 µg of the following plasmids
were used: Msx2-lux (Sirard et al.,
2000
), pCMV5B-ALK3/HAQD
(Hoodless et al., 1996
) and
pCMV5B-MADR4. Each well was supplemented with 0.25 µg pRL-CMV (Promega) and
an empty CMV expression vector was added to bring the total amount of DNA to
1.75 µg/well. Fifteen hours after transfection, growth medium was replaced
with serum-free medium with or without 1 nM TGFß1 (R&D systems). Cell
lysates were harvested 30 hours after addition of ligand, and analyzed using
the Dual-Luciferase Reporter Assay System (Promega). Mean values of each set
of triplicates were plotted as fold induction compared with activation of the
reporter in the absence of ligand or constitutively active receptor.
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Results |
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As alternate splicing plays a significant role in Smad4 processing
and activity (Pierreux et al.,
2000), it was important to confirm that deletion of exon 1
generates a null allele. We generated both Smad4N/N ES
cells by retargeting the remaining wild-type allele in
Smad4N/+ ES cells and Smad4N/N MEFs
using a tamoxifen-inducible Cre transgene
(Hayashi and McMahon, 2002
)
and performed functional assays (see Fig. S1 at
http://dev.biologists.org/supplemental).
Western blot analysis shows that the predominant wild-type 64 kDa
Smad4 protein present in control ES and MEFs is absent in
Smad4N/N cells. Embryoid bodies (EBs) made from
Smad4N/N ES cells were found to have marked downregulation
of the visceral endoderm markers Hnf4 and transferrin and the
Bmp-responsive genes Msx1 and Msx2, as described for the
previously characterized null Smad4 allele
(Sirard et al., 1998
;
Sirard et al., 2000
).
Similarly, transfection assays confirmed that Smad4N/N
MEFs are unresponsive.
Finally, we examined the phenotype of embryos resulting from
Smad4N/+ intercrosses. Mutant embryos were grossly
identifiable at E6.5 and characterized by a shortened proximodistal axis and
thickened endoderm (Fig. 2A,B).
Histological analysis showed disorganization of the epiblast and overlying
visceral endoderm, with no evidence of mesoderm formation
(Fig. 2C,D). This phenotype is
indistinguishable from the independently generated
Smad4tm1Ari and Smad4ex8 null
mutations (Sirard et al.,
1998; Yang et al.,
1998
). Moreover, aged Smad4N/+ heterozygous
mice developed gastric tumors, similar to those previously reported (data not
shown) (Takaku et al.,
1999
).
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Gastrulation occurs in the absence of Smad4 in the epiblast
The failure of Smad4-deficient embryos to gastrulate prevents an
analysis of the later roles of Smad4 during embryonic development. To
characterize further the requirement of Smad4 during early
gastrulation, we used the Sox2Cre deleter strain to specifically
inactivate Smad4 in the epiblast
(Hayashi et al., 2002b;
Vincent et al., 2003
). The
Sox2Cre transgene exhibits activation in blastocyst outgrowths and is
strongly expressed in the early epiblast prior to E5.75 (data not shown).
Sox2Cre was first introduced into the Smad4N/+
line of mice, and Sox2Cre/+;Smad4N/+ and
Smad4CA/CA animals were subsequently intercrossed. PCR
analysis of tissue fragments confirms that rearrangement of the Smad4
conditional allele efficiently occurs in a Cre- and cell lineage-specific
manner (Fig. 3P).
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Embryos deficient in Smad4 in the epiblast lack anterior primitive streak and axial mesendoderm
To examine axis formation and patterning in more detail, Smad4
mutants were examined histologically and by whole-mount in situ hybridization.
At E7.5, the primitive streak is evident both in cross section and by
T expression, but is somewhat broadened
(Fig. 3J,K,
Fig. 4G-J). In wild-type
embryos, mesoderm emerges from the primitive streak and migrates
anterolaterally between the epiblast and VE. The anterior primitive streak
gives rise to a specialized single layer of cells termed the axial mesendoderm
(AME) that occupies the ventral midline and marks the point where axial
mesoderm and DE converge (Fig.
3H). In Smad4 mutants, migrating mesoderm traverses the
midline without interruption, suggesting loss of AME
(Fig. 3I). At E8.5, transverse
sections show no evidence of notochord. The neural plate is also flattened and
fails to form a tube at any point along the AP axis
(Fig. 3M-O).
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We next evaluated whether failure to pattern the anterior primitive streak in Smad4 mutants results in complete loss of its derivatives, the AME, the node and the notochord. At E7.5 the patent node is readily identified by the expression of Nodal. In Smad4 mutant embryos, Nodal expression is not observed (Fig. 4M,N). Consistent with this result, T and Shh transcripts, which also identify the node and anteriorly extending AME, were not detected (Fig. 4I,J,O,P). At E8.5 T and Shh are additionally expressed in the notochord, but no evidence of a notochord structure is apparent in mutants, although scattered Shh-expressing cells are occasionally observed (Fig. 4K,L,Q,R). From these analyses, we conclude that Smad4 in the epiblast functions to specify the APS, and in its absence, midline structures are completely lost.
Smad4 is required for formation of the definitive endoderm
Cell lineage experiments have shown that some of the epiblast cells
entering the APS during gastrulation exits and intercalates into the overlying
visceral endoderm. These nascent definitive endoderm cells form a sheet that
displaces the visceral endoderm proximally and, with the exception of a few
residual VE cells, eventually gives rise to the entire embryonic gut
(Dufort et al., 1998;
Lawson et al., 1991
). Chimera
studies have uncovered an important requirement for Foxh1 and
Smad2 in specification of DE progenitors
(Hoodless et al., 2001
;
Tremblay et al., 2000
). To
investigate whether Smad4 collaborates with these known components of
the Nodal signaling pathway in DE specification, we analyzed expression of
Hex, which identifies the AVE as well as the earliest population of
anterior definite endoderm (ADE) to emerge from the APS
(Martinez Barbera et al.,
2000
; Thomas et al.,
1998
). At E6.5, the ADE domain of Hex expression is
absent in Smad4 mutants (Fig.
5A,B). Consistent with result, at E7.5 Smad4 mutants fail
to activate a Hex-lacZ transgene that identifies ADE adjacent to the
midline (Fig. 5C-F)
(Rodriguez et al., 2001
).
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To distinguish whether loss of DE markers reflects loss of gene expression
or a failure to produce the cells themselves, we performed an in vivo cell
lineage assay. The ROSA26R conditional allele
(Soriano, 1999) was introduced
into the Smad4CA background. The Sox2Cre
transgene robustly activates the ROSA26R reporter allele
in the epiblast. At early headfold stages, the three embryonic lineages -
ectoderm, mesoderm and definitive endoderm - comprise exclusively
lacZ-positive cells, while extra-embryonic tissues are lacZ
negative (Fig. 5M,N). By
contrast, in Smad4 mutants the superficial endoderm layer is
completely devoid of lacZ activity, indicating that these cells are
extra-embryonic in origin (Fig.
5O,P). This result conclusively shows that Smad4-deficient
epiblast cells do not form DE, and consequently the overlying visceral
endoderm layer is not displaced.
Anterior neural development in Smad4 mutant embryos
Anterior identity in the mouse is initially specified by the AVE but
subsequently reinforced by APS derivatives such as the ADE and prechordal
plate. The AVE is normally patterned in Smad4 mutants and the AP axis
is correctly specified. However, mutant embryos fail to form classic headfolds
at E7.5 and form a pair of bulbous structures suggestive of rostral CNS at
E8.5. We therefore characterized the degree of neural pattern in
Smad4 mutants. Otx2 is initially expressed in the epiblast
and visceral endoderm, but by E7.5 transcripts are restricted to the anterior
neurectoderm in both control and mutant embryos
(Fig. 6A,B). At E8.5
Otx2 expression normally resolves to demarcate the forebrain and
midbrain. By contrast, expression is confined to the most apical portion of
the headfolds in Smad4 mutants
(Fig. 6C,D). A similar
reduction was seen in the expression of the forebrain marker Six3
(Fig. 6E,F). To evaluate
anterior CNS defects further, we analyzed Fgf8 expression, which
identifies the anterior neural ridge and the midbrain-hindbrain junction (or
isthmus). In Smad4 mutants, while Fgf8 is weakly expressed
in a region that probably corresponds to the isthmus, the most rostral domain
of expression is lost (Fig.
6G,H). Hindbrain patterning was examined using Krox20
expression, which delineates rhombomeres 3 and 5. Mutant embryos variably
exhibit one or two stripes of expression that frequently span the midline
(Fig. 6I,J). Collectively,
these results suggest that Smad4 mutants exhibit limited neural
patterning, probably imparted by the early activity of the AVE. In the absence
of secondary refining signals provided by the anterior AME, the growth and
patterning of the neural plate is disrupted.
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PGCs can first be identified by their high levels of endogenous alkaline phosphatase (AP) activity around E7.5 and localization in a cluster at the base of the incipient allantois (Fig. 7J). Smad4 mutants at this stage show disorganized extra-embryonic development, with the collapsing extra-embryonic ectoderm shifted toward the posterior visceral yolk sac. Rudimentary allantois formation is observed in all mutant embryos. However, in the majority of mutants (seven out of nine) no cells resembling PGCs with darkly stained cell membranes or a coarse spot are detected either at the base of the allantois or ectopically within the neighboring visceral yolk sac (Fig. 7K).
To address the possibility that PGC formation is delayed in the absence of
Smad4, PGC number was assayed one day later at E8.5. In wild-type
embryos at this stage, AP-positive PGCs (100) have dispersed from the
base of the allantois and intercalated into the hindgut epithelium
(Fig. 7L). All Smad4
mutants (21 out of 21) show an allantois-like outgrowth that varies in size
and that in the majority of cases (70%) associates or fuses with the
stalk-like chorionic ectoderm (Fig.
3O, Fig. 7H,I). In
the remaining embryos, the allantois terminates blindly within the exocoelomic
cavity (Fig. 7F). Despite
relatively normal formation of the allantois, the majority of E8.5 mutant
embryos (16 out of 21) lack AP-positive PGCs in the posterior extra-embryonic
region, including the base of the allantois and visceral yolk sac, or in the
posterior embryo proper, which lacks a recognizable hindgut
(Fig. 7M). The remaining
mutants have fewer than ten PGCs widely scattered in the disorganized
posterior region (data not shown). Taken together, these data suggest that
Smad4 is required in the epiblast for specification of the PGC
precursor population.
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Discussion |
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Smad4 and formation of the anterior primitive streak
After the onset of gastrulation, inductive events occurring within the APS
are responsible for formation of the node and its derivatives, as well as
definitive endoderm. In Smad4 mutants, these midline structures fail
to form, resulting in an embryo with fused somites and anterior defects. The
midline defects reflect the inability of the embryo to correctly pattern the
APS as seen by failure to activate Gsc, Lim1 and Foxa2. The
promoter regions of Gsc and Lim1 have been shown to contain
activin response elements occupied by a complex containing Smad4, Smad2 and
Foxh1 (Labbé et al.,
1998; Watanabe et al.,
2002
), and failure to activate Gsc and Lim1
expression in the APS domain is thus consistent with a essential role for
Smad4 in the regulation of these genes.
The definitive endoderm arises from cells within the epiblast that ingress through the APS, displacing the visceral endoderm layer. Our lineage studies and marker analysis indicate that DE is not specified in Smad4 mutants. As chimeric analysis and conditional gene ablation have also identified a role for Smad2 and Foxh1 in endoderm specification, our finding suggests that Smad4 complexes with Smad2/Foxh1 to mediate formation of DE.
Overall, removal of Smad4 from the epiblast leads to a phenotype
that closely resembles that of several other mutants. Embryos in which
extra-embryonic lineages are wild-type while the epiblast lacks Foxa2
fail to form a node and notochord, have fused somites, and establish
rudimentary neural patterning (Dufort et
al., 1998; Hallonet et al.,
2002
). Foxh1 mutant embryos also lack APS derivatives,
and show somite fusion (Hoodless et al.,
2001
; Yamamoto et al.,
2001
). Removal of Smad2 alone or in the context of
Smad3 deficiency also leads to similar phenotypes (see below).
Collectively, these findings provide genetic evidence that Smad4,
Smad2/3 and Foxh1 lie in the same pathway and support previously
characterized biochemical interactions. These mutant contexts each result in
downregulation of Foxa2, probably accounting for the convergent
phenotype. Curiously, no Foxh1/Smad-binding sites have been documented in the
Foxa2 gene, and regulation by Foxh1/Smad proteins may be indirect
(Nishizaki et al., 2001
).
Smad4 and mesoderm patterning
The central role of Smad4 in transducing TGFß signals was in part
initially characterized by gain-of-function experiments in Xenopus
embryos. Overexpression of Smad1 in animal caps simulated the ability
of Bmp2 and Bmp4 to induce ventral mesoderm, while Smad2 induced
dorsal mesoderm similar to activin stimulation. As expected, Smad4
injection produced both dorsal and ventral mesoderm
(Lagna et al., 1996;
Zhang et al., 1997
). These
results suggest that Smad4 contributes essential activities in the
events of gastrulation and mesoderm formation. That Smad4 homozygous
null embryos fail to gastrulate was unsurprising, especially in light of
similar phenotypes observed in activin/TGFß and Bmp receptor mutants.
However, when wild-type extra-embryonic lineages are provided either by
aggregation of Smad4-deficient ES cells with tetraploid wild-type
embryos or selective loss of Smad4 gene activity with
Sox2Cre, gastrulation commences and mesoderm forms. In
Sox2Cre;Smad4CA/N mutant embryos, we observe numerous
mesodermal derivatives, including somites, heart, allantois and lateral plate
mesoderm. We also find that teratomas made from Smad4N/N
ES cells form mesoderm (G.C.C. and E.J.R., unpublished). These results suggest
that the primary defect in Smad4 homozygous null embryos is
restricted to the extra-embryonic lineages. When this requirement is bypassed,
Smad4-independent gastrulation and mesoderm formation proceeds. Of
particular interest, the Smad4 mutant shows a phenotype less severe
than a Smad2;Smad3 doubly-deficient epiblast
(Dunn et al., 2004). Stepwise
removal of Smad2 and Smad3 from the embryo results in a
progressive loss of primitive streak lineages, first impacting APS derivatives
such as the AME, node and notochord followed by middle primitive streak
lineages such as somites and LPM. The more extensive mesoderm formation seen
in Smad4 mutants therefore suggests that Smad2 and
Smad3 function in a Smad4-independent manner in eliciting
Nodal-mediated induction of middle streak derivatives. Thus, in the
mouse embryo, as in Xenopus, Smad4 appears to potentiate TGFß
signaling to allow the correct patterning of the mesoderm lineage, but
Smad4 activities are unnecessary for mesoderm formation per se.
Initial heart specification does not require Smad4
The role of Bmp-dependent signals in heart formation is evolutionarily
conserved. In Drosophila, Dpp initiates signaling involving the
R-Smad Mad, the Smad4 homolog Medea and
Tinman, the homolog of Nkx2.5, to specify the cardioblast
fate (Zaffran et al., 2002).
In chick, a homologous pathway has been described in which Bmp2 or Bmp4
induces expression of Nkx2.5 in non-cardiogenic mesoderm and directs
cardiac differentiation (Schlange et al.,
2000
; Schultheiss et al.,
1997
). Intriguingly, we found that removal of Smad4 in
the epiblast affected neither early cardiac development nor correct induction
of Nkx2.5. Previous promoter analysis identified multiple Smad
binding elements in the AR2 enhancer of Nkx2.5 that governs
expression within the cardiac crescent. Mutation of a specific Smad4 binding
site leads to complete loss of transgene activity in cardiogenic mesoderm
(Lien et al., 2002
). Our
results therefore suggest that Smad4 is not essential for activation
of the AR2 enhancer and that R-Smad binding and association with other
requisite cofactors are sufficient to promote early Nkx2.5
expression.
Requirement of Smad4 in allantois and PGC development
The Bmp signaling pathway is essential for the normal specification of the
mammalian germline (McLaren,
2003). Bmp4 and Bmp8b secreted by the extra-embryonic ectoderm
predispose a small number of progenitor cells within the extreme proximal
epiblast towards the germ cell fate. Importantly, these precursor cells are
multipotent in that they give rise not only to committed PGCs but also to
descendants in the extra-embryonic mesoderm, particularly the allantois
(Lawson and Hage, 1994
).
Bmp4 homozygous null embryos entirely lack PGCs and allantois,
whereas heterozygotes show reduced PGC numbers and normal allantois formation
(Lawson et al., 1999
). Similar
gene dose effects are observed in Bmp8b mutant embryos
(Loebel et al., 2003
). These
observations suggest that allocation to the germ-cell lineage is more
sensitive to the levels of Bmps than is allocation to the allantois.
Furthermore, fate mapping studies reveal that the allantois is derived from a
much broader region of the proximal epiblast, where Bmp concentrations are
expected to be lower, than the narrow belt at the junction with the
extra-embryonic ectoderm in which the common PGC/allantois progenitors reside
and which is presumably exposed to the highest local Bmp concentration. The
loss of PGCs with persistent formation of allantois in Smad4 mutant
embryos is therefore consistent with the simple model in which Smad4 is
required for maximal Bmp signaling within the proximal epiblast, with
intermediate signaling reliant upon the available Bmp R-Smads Smad1/5/8 within
the epiblast and sufficient for formation of allantois
(Hayashi et al., 2002a
).
Is Smad4 an obligate member of TGFß signaling?
The widely held conclusion that Smad4 occupies a central role in
transduction of TGFß signals comes from multiple lines of biochemical and
genetic evidence (reviewed by
Massagué, 1998).
Smad4 participates in both activin and Bmp pathways as demonstrated
in Xenopus overexpression assays and in dominant-negative
experiments. In reconstitution experiments, cell lines that lack
Smad4 fail to respond to TGFß signals; transfection of wild-type
Smad4 restores the signaling capabilities of these cells
(de Caestecker et al., 1997
;
Lagna et al., 1996
;
Zhang et al., 1997
). Moreover,
in Drosophila, Medea/Smad4 is necessary for Dpp function,
and elimination of maternal and zygotic Medea in the embryo results
in dorsoventral patterning defects identical to that of null Dpp
mutants, indicating that Medea is required for Dpp-dependent
signaling (Das et al., 1998
;
Hudson et al., 1998
).
By contrast, our data suggest that in the early embryo, Smad4 is
required for certain TGFß/Bmp signaling pathways, but not obligate for
others. A growing body of data corroborates our observations. For example, in
Smad4-deficient MEFs as well as several Smad4-deficient
human tumor cell lines, TGFß addition still results in classical growth
inhibition (reviewed by Derynck and Zhang,
2003; Wakefield and Roberts,
2002
). In Drosophila, oogenesis is more severely
disrupted by the loss of the R-Smad Mad than by Medea.
Interestingly, in wing imaginal discs, loss of Medea most severely
affects regions receiving low Dpp signal
(Wisotzkey et al., 1998
). One
possible explanation is that Medea normally potentiates weak
Dpp signals and that in the absence of Medea, R-Smads
regulate target genes only when the Dpp signal strength is high.
R-Smads may substitute for or bypass the requirement for Smad4, resulting in
unpotentiated levels of downstream signal
(Yeo et al., 1999
).
Additionally, alternate TGFß pathways have been described, including the
Ras/Mapk, Pp2a/S6 kinase, Rhoa and PI3K-Akt pathways, some of which are
entirely Smad independent and others that involve crosstalk with Smad signals.
Finally, an independently-derived Smad4 conditional allele was
recently employed to eliminate Smad4 function during CNS and mammary
gland development and the resulting phenotypes were surprisingly mild
(Li et al., 2003
;
Zhou et al., 2003
). These
findings in combination with our genetic results emphasize the versatility in
the intracellular transduction of TGFß-related signals, and encourage a
more careful consideration of the current canonical model of TGFß
signaling that places Smad4 as a central effector molecule.
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ACKNOWLEDGMENTS |
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Footnotes |
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