|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 24 October 2007
doi: 10.1242/dev.010645
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Zoology,
University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
2 Interdisciplinary Research Institute and Institut de Biologie de Lille, 1 rue
du professeur Calmette, BP447, 59021 Lille Cedex, France.
Author for correspondence (e-mail:
jim{at}gurdon.cam.ac.uk)
Accepted 12 September 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Xenopus, Smads, Activin, Nodal-related proteins, Bimolecular fluorescence complementation, Midblastula transition
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). TGFß family members signal by binding to type II
serine-threonine kinase receptors, which then associate with, and
phosphorylate, type I receptors. The activated type I receptors go on to
phosphorylate receptor-regulated Smad proteins (R-Smads, including Smad1,
Smad2, Smad3, Smad5 and Smad8) and these in turn associate with Smad4,
accumulate in the nucleus, and regulate the expression of downstream target
genes, usually by interacting with a specific cofactor.
Activin and the Xenopus Nodal-related proteins (Xnrs) induce
formation of the mesendoderm during early Xenopus development
(Heasman, 2006). Although one
can visualise the dynamic spatial and temporal regulation of Activin/Xnr
signalling by use of an antibody that recognises the phosphorylated form of
Smad2 (Schohl and Fagotto,
2002), this technique involves the use of serial cryosections of
embryos at different stages of development and does not permit true real-time
analysis. To address this point, and to allow one to monitor Smad signalling
in other cells and tissues, we have investigated whether bimolecular
fluorescence complementation (BiFC) (Hu et
al., 2002) might provide an alternative approach to monitor Smad
signalling. BiFC takes advantage of the fact that when the two non-fluorescent
amino- and carboxyl-terminal fragments of yellow fluorescent protein (YFP) are
brought into close apposition they interact to form a fluorescent protein. We
have optimised this technique for use in the highly autofluorescent
Xenopus embryo by using mutated versions of VENUS, and thereby
succeeded in detecting homomeric interactions between two Smad2 and two Smad4,
as well as heteromeric complex formation between Smad2 and Smad4. Importantly,
our approach is at least semi-quantitative, and will allow us to monitor Smad
signalling during the normal development of Xenopus. Interestingly,
we note that during mitosis Smad complexes associate with chromatin; this may
provide a mechanism for the equal distribution of Smad proteins at cell
division.
Finally, we note that the nuclear accumulation of Smad2-Smad4 complexes in response to induction only occurs after the midblastula transition, irrespective of the stage at which cells are exposed to Activin. This observation may help shed light on the complex problem of developmental timing in the early Xenopus embryo.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). They were staged according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1994).
Animal cap cells were dissociated in Ca2+- and Mg2+-free
medium (75 mM Tris pH 7.5, 880 mM NaCl, 10 mM KCl, 24 mM NaHCO3)
for 30-45 minutes at room temperature before treating with Activin.
Western blotting
Fertilized eggs were injected with the relevant RNA at the one-cell stage
(see figure legends for details) and animal caps were dissected at blastula
stages 8 to 9. Protein extracts were prepared from the animal caps as
described previously (Saka and Smith,
2004) and the equivalent of 10 animal caps was applied to each
lane of a SDS polyacrylamide gel before blotting onto Hybond P membrane
(Amersham Bioscience). Antibodies used were anti-Smad2/3 (rabbit IgG, from
Cell Signaling, 1:1000) or anti-phospho-Smad2 (monoclonal rabbit IgG from Cell
Signaling, 1:1000). HRP-conjugated secondary antibodies were anti-rabbit IgG
(whole antibody, Amersham, 1:1000; or Santa Cruz, 1:4000).
BiFC constructs, mRNA injection and transfection of mammalian cells
The N-terminal and C-terminal halves of VENUS
(Nagai et al., 2002) were
amplified by PCR and cloned into pCS2+. VENUS fragments were fused to the N
terminus of human SMAD4 with an 11 amino acids linker (TGGGGGGGGGG) or to the
N terminus of Xenopus Smad2 with a four amino acids linker (TGSS).
For the constructs VC155-Smad3 and VC155-Smad2
exon3, Smad2 was replaced
either by Xenopus Smad3 (a gift from Caroline Hill, Cancer Research
UK London Research Institute, London, UK) or Xenopus
Smad2exon3 (a gift from Oliver Nentwich, Gurdon Institute, Cambridge,
UK). Site-directed mutagenesis to create modified forms of VENUS was performed
as described previously (Sawano and
Miyawaki, 2000). VENUS, mRFP1, ECFP, ECFP-GPI, ECFP-NLS, histone
H2B-ECFP and Emerin-ECFP were all cloned into the expression vector pCS2+ and
checked by sequencing. GFP-emerin was a kind gift from Chris Hutchison (Durham
University, Durham, UK), YFP-histone H2B, a kind gift from Andrea Brand
(Gurdon Institute, Cambridge, UK) and the NLS sequence a kind gift from Steve
Jackson (Gurdon Institute, Cambridge, UK).
|
Real-time PCR
Total RNA was prepared from 15 animal caps using the Trizol reagent
(Invitrogen), followed by isopropanol precipitation. RNA was dissolved in
water and used as template for real-time RT-PCR. Real-time RT-PCR with the
LightCycler (Roche) was carried out using the manufacturer's RNA amplification
kit. All determinations included a negative control. Primers specific for
Xbra, goosecoid (gsc), chordin and ornithine decarboxylase
were as described previously (Piepenburg
et al., 2004). All values are normalised to the level of ornithine
decarboxylase in each sample.
Imaging
Animal caps were dissociated in Ca2+- and Mg2+-free
medium (CMFM) at the indicated stages. Dissociated cells were seeded onto a
fibronectin-coated glass bottom dish (MatTek) in 0.75% normal amphibian medium
(NAM) with 0.2% BSA and with or without 16 U/ml Activin
(Cooke et al., 1987). The image
in Fig. 1 was taken using a
Leica MZ FLIII dissecting microscope equipped with RFP and YFP filters. The
image in Fig. 3 was taken using
a Leica DM IRB inverted fluorescent microscope with a Hamamatsu ORCA ER camera
and analysed with Open Lab software. Fig.
3C, Figs 4,
5,
6 and Fig. S1 (see Fig. S1 in
the supplementary material) images were taken using a Perkin Elmer Spinning
Disk confocal microscope with a Zeiss Axiovert 200M inverted microscope and a
Hamamatsu ORCA ER camera. CFP protein was excited at 440 nm and VENUS BiFC at
514 nm. Pictures were taken with Ultra View ERS software. All confocal
pictures are taken with a 40x objective and represent a merged
z-stack comprising 15 slices of 1 µm each.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), to VENUS,
a variant of enhanced yellow fluorescent protein (EYFP) which is less
sensitive to pH and chloride ion concentration than EYFP and
which-matures more rapidly
(Nagai et al., 2002). We chose
two positions, both located within a loop between two ß-sheets, to divide
VENUS into two fragments: immediately after N144 or immediately after A154.
The latter position was used in the original BiFC experiments using unmodified
YFP (Hu et al., 2002); the
former was used in the development of a fluorescent reagent to monitor
Ca2+ levels in HeLa cells
(Nagai et al., 2001). We refer
to the two types of VENUS N-terminal fragments as VN144 and VN154 and the two
types of C-terminal fragments as VC145 and VC155.
In contrast to previous work using COS-1 cells
(Shyu et al., 2006), we found
that Xenopus embryos injected with as little as 50 pg of RNAs
encoding either pair of complementary VN and VC fragments became fluorescent
(Fig. 1A,D). No fluorescence
was observed when the VN or VC fragments were expressed alone (data not
shown). This apparent self-assembly of VENUS, which would significantly
compromise its utility in BiFC experiments, may derive from the mutations
introduced into EYFP that were designed to improve its folding and maturation
(Nagai et al., 2002). To
address this problem, mutations were introduced back into VENUS. Point
mutation T153M is located at the junction between one of the ß-strands
and a loop in the crystal structure; it is likely to affect protein folding.
The other mutations (L46F, L64F and Y203H) affect maturation of the
chromophore (Rekas et al.,
2002). Different combinations of N-terminal and C-terminal VENUS
fragments carrying different combinations of mutations were tested for
self-assembly by injecting RNAs into Xenopus embryos
(Table 1). Out of 30
combinations, ten produced little fluorescence
(Fig. 1B,E and data not shown).
Of these, VNm9, a VN154 fragment carrying the point mutation T153M, did not
fluoresce in combination with VC155, and this combination of VENUS fragments
was used in further studies. We note that constructs tagged with VN154 and
VC155 tend to be more fluorescent than those tagged with VN144 and VC145
(compare Fig. 1A with
Fig. 1D, and data not
shown).
|
; Smith et al.,
1991) and then for the elongation or inversion movements that
presage differentiation as mesoderm (Armes
and Smith, 1997; Symes and
Smith, 1987). Animal pole regions derived from uninjected embryos or embryos injected with mRNAs encoding individual tagged Smad constructs expressed neither Xbra nor gsc at the early gastrula stage. However, strong expression of these genes was observed in caps derived from embryos injected with a combination of either HA- or VENUS-tagged forms of Smad2 and Smad4 (Fig. 2B), at levels comparable to those induced by injection of 5 pg Activin RNA (Fig. 2B). Such animal caps also underwent elongation (data not shown). These results indicate that Smad constructs carrying VNm9 or VC155 tags at their N termini are able to interact with each other to induce the expression of their target genes. The inability of the individual tagged proteins to induce target gene expression at these low levels of injected RNA is likely to be due to limiting amounts of the complementary endogenous Smad protein.
Activin-induced nuclear accumulation of Smad complexes
To investigate whether tagged Smad constructs can reveal the ligand-induced
association of Smad2 and Smad4 and thus signalling by members of the TGFß
family, such as Activin, Xenopus eggs were injected with RNA encoding
VNm9-tagged Smad2 or Smad4, together with VC155-tagged Smad2 and, as a lineage
tracer, mRFP1 (Campbell et al.,
2002). Animal pole regions containing mRFP1 fluorescence were
dissected at midblastula stage 8, and cells were dissociated and cultured to
the equivalent of early gastrula stages 10-11 on a fibronectin-coated surface
in the presence or absence of Activin. As a control for non-specific
fluorescence we also injected a VC155-Smad2 construct that lacks the MH2 and
linker regions of Smad2 as well as part of the MH1 domain
(VC155-Smad2C; Fig. 3A).
This protein should not interact with Smad4
(Hata et al., 1997).
Preliminary western blot analyses using VN154-Smad2 (which differs from
VNm9-Smad2 by just one amino acid) confirmed that the truncated constructs are
expressed at levels similar to those of the full-length proteins (data not
shown) and experiments described below
(Fig. 6E) indicate that levels
of VN154-Smad2 resemble those of endogenous Smad2.
In the absence of Activin, we noted cytoplasmic fluorescence associated with the formation of Smad2-Smad2, Smad2-Smad4 and Smad4-Smad4 complexes (Fig. 3Ba,e,i and see Fig. 4A). Activin treatment of these animal cap cells caused detectable nuclear accumulation of Smad2-Smad2 complexes (Fig. 3Bb) and strong accumulation of Smad2-Smad4 complexes compared to levels of cytoplasmic fluorescence (Fig. 3Bf).
|
As we discuss below, it is possible that interactions between Smad2 and
Smad4 are stabilised by the BiFC technique. However, prolonged culture of
Activin-treated animal pole blastomeres reveals that levels of BiFC
fluorescence in the nucleus have decreased by stage 13
(Fig. 3C), by which stage,
levels of nuclear GFP-Smad2 and phospho-Smad2 have also declined
(Faure et al., 2000;
Grimm and Gurdon, 2002;
Lee et al., 2001).
Subcellular localisation of Smad complexes
The subcellular localisation of Smad complexes as revealed by BiFC was
further analysed by use of spinning disk confocal microscopy, which provides
an improved signal-to-noise ratio. These experiments revealed that in the
absence of ligand, Smad2-Smad4 fluorescence is associated with the cytoplasm
and is enriched at the nuclear membrane
(Fig. 4A). The distribution of
Smad2-Smad4 complexes at the nuclear membrane was confirmed in experiments in
which embryos were co-injected with RNA encoding ECFP-tagged emerin
(Fig. 4B), an integral membrane
protein that interacts with lamins A/C on the inner nuclear membrane
(Bengtsson, 2007;
Gruenbaum et al., 2005).
Following Activin treatment the nucleus becomes particularly bright
(Fig. 4C). There appears to be
no consistent sub-nuclear distribution of BiFC fluorescence, although in some
cells distinct spots can be made out (Fig.
4C arrows).
Spinning disk confocal microscopy revealed that all combinations of Smad
dimers that have been tested, including Smad2-Smad2, Smad2-Smad4, Smad4-Smad4,
Smad2exon3-Smad4 and Smad3-Smad4 (see Materials and methods) associate
with chromosomes during mitosis, both in the presence and in the absence of
ligand (Fig. 4D,E). This
observation contrasts with data obtained using an antibody directed against
Smad2, indicating that (presumably monomeric) Smad2 associates with the
mitotic spindle (Batut et al.,
2007; Dong et al.,
2000). Both these phenomena may ensure the equal partition of Smad
proteins and complexes between daughter cells.
The localisation of Smad-BiFC complexes to nuclear membrane and chromosomes appears to be specific; injection of 200 pg RNA encoding the complementary VENUS fragments VC155 and VNm9 (representing a 20-fold molar excess over the levels used elsewhere in this investigation) does not cause fluorescence to appear in these structures (Fig. 4F; arrow points to mitotic chromatin). Rather, fluorescence is present at the periphery of cells, perhaps because it is displaced elsewhere in the cell by yolk platelets.
BiFC can reveal Smad signalling during normal development
In an effort to ask whether BiFC can be used to assess quantitative aspects
of signalling by members of the TGF-ß family, we first investigated the
extent to which increasing extracellular concentrations of Activin cause
increased nuclear accumulation of Smad2-Smad4 complexes. Different
concentrations of Activin were therefore added to cultures of dissociated
animal cap cells derived from embryos that had been injected with RNA encoding
VC155-Smad2 and VNm9-Smad4 BiFC constructs and, to allow normalisation, RNA
encoding ECFP (see Materials and methods). The fluorescence intensity in the
nucleus after 3 hours of incubation was then quantified with Volocity software
and plotted as a ratio of VENUS fluorescence to ECFP
(Fig. 5A,B). Our results showed
that fluorescence deriving from the accumulation of Smad2-Smad4 complexes
became more intense as the extracellular concentration of Activin
increased.
These observations suggest that BiFC might be used to monitor levels of
Smad signalling during development. To test this idea, Xenopus eggs
were injected with RNA encoding VNm9-tagged Smad4 together with VC155-tagged
Smad2. The large size and yolkiness of Xenopus blastomeres precluded
direct observation of BiFC fluorescence in the intact embryo, so we dissected
early gastrulae into animal and vegetal halves and studied the two
half-embryos. In such embryos, marginal zone cells show nuclear accumulation
of Smad2-Smad4 BiFC in a pattern resembling that observed with
immunohistochemistry for phosphorylated Smad2
(Schohl and Fagotto, 2002),
whereas in the animal hemisphere, as in cells derived from isolated animal
pole regions, nuclei are only weakly fluorescent (compare
Fig. 5C with nuclear
membrane-associated fluorescence in Fig.
4A).
|
). Thus, addition of Activin to dissociated animal cap cells
after the MBT caused detectable accumulation of Smad2-Smad4 complexes within
10 minutes (Fig. 6Af), whereas
treatment at any time before the MBT does not result in nuclear accumulation
of BiFC signal until control embryos reach stage 8.5
(Fig. 6Aa-d).
|
), phosphorylation of VC155-Smad2 occurs within 30 minutes of
treatment with Activin (Fig.
6E). In this respect we note that the effects of Activin differ
from those of the Xnr proteins, which cannot cause phosphorylation of Smad2
until after the MBT (Lee et al.,
2001). Together, these observations suggest that although Activin can cause the phosphorylation of Smad2 as early as stage 7, Smad2-Smad4 complexes are prevented from accumulating in the nucleus until after the MBT. We discuss this phenomenon below.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Hu and Kerppola, 2003), and
which does not require the sophisticated equipment needed for techniques such
as fluorescence resonance energy transfer (FRET). In this paper we use this
approach to study the TGFß signal transduction pathway, and in particular
the association of Smad2 and Smad4 with themselves and with each other. Our
results show that our Smad BiFC constructs can be used to reveal
homodimerisation and heterodimerisation of Smad molecules during early
amphibian development.
Formation and subcellular distribution of Smad complexes
We have shown that in the absence of Activin, BiFC reveals homomeric
interactions between Smads2 and Smads4 and heteromeric interactions between
Smad2 and Smad4 in the cytoplasm of Xenopus animal pole blastomeres.
Although gel filtration experiments do show that Smad2 is present as part of a
high molecular mass complex in untreated HaCaT cells
(Jayaraman and Massague,
2000), our results contrast with previous reports that Smad2-Smad4
interaction can only be detected after Activin stimulation. However, we note
that these conclusions were based on co-immunoprecipitation assays, and they
cannot exclude the possibility of transient interactions, which may be
stabilised to some extent by the BiFC technique
(Kerppola, 2006). Indeed, we
note that Smad2 and Smad4 can form homo- or hetero-oligomers in yeast
two-hybrid assays (Hata et al.,
1997), where there is no ligand-induced phosphorylation at the
C-terminus of Smad2. Weak fluorescence, associated with the formation of
complexes containing Smad2 and Smad4, was detectable in the nuclei of cells
that had not been treated with Activin. This fluorescence was present in
nuclear membranes that were marked using ECFP-tagged emerin
(Fig. 4B), although its
significance is not completely clear, because we cannot exclude the
possibility that these cells are experiencing very low doses of endogenous
TGFß signalling that are not sufficient to induce detectable target gene
expression in our assays (see Fig.
2B: `caps'). We also note that similar perinuclear accumulation of
Smad3 was observed in an in vitro nuclear import assay when no active
cytosolic factors for nuclear import were added
(Kurisaki et al., 2001).
|
Our conclusion that homomeric interactions occur between Smad2 and Smad4 is
consistent with the observation that TGFß can stimulate the formation of
a complex comprising two Smad2s, one Smad4 and one FoxH1
(Inman and Hill, 2002), and
with structural analyses showing that the Smad4 MH2 domain can form a
homo-trimer (Qin et al.,
1999). However, our results
(Fig. 2C) also indicate that in
the presence of Activin, Smad2-Smad4 interactions are preferred in the nucleus
over Smad4-Smad4 interactions, whereas in the cytoplasm of untreated cells
Smad4-Smad4 interactions may predominate. Homomeric interactions between
Smad4s may therefore play a significant role in the regulation of TGF-ß
signalling by limiting the availability of Smad4 for heteromer formation with
Smad2.
|
; Dong et al.,
2000).
Use of Smad BiFC in the analysis of early development
In this investigation we have used BiFC to follow the temporal aspects of
Smad signalling in response to the mesoderm-inducing factor Activin. We have
shown that the technique is sensitive, quantitative, and, importantly, rapid:
complexes containing Smad2 and Smad4 accumulate in the nucleus just 10 minutes
after Activin treatment (Fig.
6A). This delay is shorter than that observed with GFP-Smad2
(Bourillot et al., 2002),
perhaps because our approach provides a better signal-to-noise ratio.
Remarkably, however, the earlier cells are treated with Activin, the longer it
takes for BiFC to occur, and we conclude that accumulation in the nucleus can
only occur after the MBT (Fig.
6). This delay in the translocation to the nucleus of Smad2-Smad4
complexes is not any consequence of the sensitivity of our assay, because the
BiFC constructs are expressed and cytoplasmic Smad2-Smad4 fluorescence can
readily be observed as early as stage 7
(Fig. 6C,E). It is also not due
to any failure of the animal pole blastomeres to phosphorylate our tagged
Smad2; as with the endogenous protein, phosphorylation is clearly detectable
within 30 minutes of Activin treatment
(Fig. 6D,E).
Our observation may be relevant to the fact that the time of gene
activation, or even of morphogenetic movements
(Symes and Smith, 1987), in
response to Activin depends not on when cells receive an inductive signal, but
on the `age' of the responding tissue
(Cooke and Smith, 1990). The
observation may even relate to the midblastula transition itself
(Newport and Kirschner, 1982a;
Newport and Kirschner, 1982b),
in the sense that gene activation may not occur until transcription factors,
or transcription factor complexes, have gained access to the nucleus.
Preliminary results indicate that a fusion protein consisting of cyan
fluorescent protein fused to three basic nuclear localisation signals (NLS)
also only entered the nucleus after the midblastula transition (see Fig. S1 in
the supplementary material). Together, these results suggest, consistent with
data of Dreyer (Dreyer, 1987),
that the entry of many proteins into the nucleus may be suppressed until MBT
in Xenopus. We are now investigating how general this conclusion
might be, and whether it applies to all transcription factors or indeed to all
nuclear proteins.
BiFC and other Smads
TGFß signalling plays important roles in the development of
Xenopus and of most, if not all, multicellular organisms. If one were
able to follow the immediate response to TGFß signalling in individual
cells and in real time, this would greatly increase our understanding of early
development and also of regeneration and disease. We show here that BiFC
between Smad2 and Smad4 can indeed be used to reveal TGFß signalling,
suggesting that the creation of transgenic lines expressing the two BiFC
constructs might be useful in attempts to follow Smad signalling throughout
development and adult life. This might also assist in attempts to understand
how cells distinguish between different concentrations of TGFß family
members to activate the expression of different genes
(Bourillot et al., 2002;
Green et al., 1992). In
principle, the technique should also be applicable to other members of the
Smad family, although our efforts to study the other R-Smads that respond to
Activin and the Xnrs in the Xenopus embryo have not yet met with
success. These proteins are Smad3 (Howell
et al., 2001), and a shorter isoform of Smad2 that lacks exon 3
(Smad2exon3) (Faure et al.,
2000). In both cases, BiFC complexes with Smad4 are present in the
nuclei of Xenopus animal pole blastomeres even in the absence of
ligand. This behaviour may be related to the ability of the two proteins to
bind DNA directly and to the fact that their nuclear localisation sequences,
unlike that of Smad2, are not masked in any way
(Kurisaki et al., 2001). We
also note that a GFP-tagged version of Smad3 is constitutively nuclear
(Nicolas et al., 2004). In
future work we plan to ask whether mutated versions of Smad3 and
Smad2exon3 might provide more effective reagents, and whether one can
apply BiFC to Smad1, 5 or 8, which respond to members of the BMP family.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/23/4209/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: ASM Scientific Ltd, Building 405, Babraham Research
Campus, Babraham CB2 4AT, UK
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Armes, N. A. and Smith, J. C. (1997). The ALK-2 and ALK-4 activin receptors transduce distinct mesoderm-inducing signals during early Xenopus development but do not co-operate to establish thresholds. Development 124,3797 -3804.[Abstract]
Batut, J., Howell, M. and Hill, C. S. (2007). Kinesin-mediated transport of Smad2 is required for signaling in response to TGF-beta ligands. Dev. Cell 12,261 -274.[CrossRef][Medline]
Bengtsson, L. (2007). What MAN1 does to the Smads. TGFbeta/BMP signaling and the nuclear envelope. FEBS J. 274,1374 -1382.[CrossRef][Medline]
Bourillot, P. Y., Garrett, N. and Gurdon, J. B.
(2002). A changing morphogen gradient is interpreted by
continuous transduction flow. Development
129,2167
-2180.
Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A.,
Baird, G. S., Zacharias, D. A. and Tsien, R. Y. (2002). A
monomeric red fluorescent protein. Proc. Natl. Acad. Sci.
USA 99,7877
-7882.
Cho, K. W., Blumberg, B., Steinbeisser, H. and De Robertis, E. M. (1991). Molecular nature of Spemann's organizer: the role of the Xenopus homeobox gene goosecoid. Cell 67,1111 -1120.[CrossRef][Medline]
Cooke, J. and Smith, J. C. (1990). Measurement of developmental time by cells of early embryos. Cell 60,891 -894.[CrossRef][Medline]
Cooke, J., Smith, J. C., Smith, E. J. and Yaqoob, M.
(1987). The organization of mesodermal pattern in Xenopus
laevis: experiments using a Xenopus mesoderm-inducing factor.
Development 101,893
-908.
Dong, C., Li, Z., Alvarez, R., Jr, Feng, X. H. and Goldschmidt-Clermont, P. J. (2000). Microtubule binding to Smads may regulate TGF beta activity. Mol. Cell 5, 27-34.[CrossRef][Medline]
Dreyer, C. (1987). Differential accumulation of
oocyte nuclear proteins by embryonic nuclei of Xenopus.
Development 101,829
-846.
Faure, S., Lee, M. A., Keller, T., ten Dijke, P. and Whitman, M. (2000). Endogenous patterns of TGFbeta superfamily signaling during early Xenopus development. Development 127,2917 -2931.[Abstract]
Green, J. B., New, H. V. and Smith, J. C. (1992). Responses of embryonic Xenopus cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm. Cell 71,731 -739.[CrossRef][Medline]
Grimm, O. H. and Gurdon, J. B. (2002). Nuclear exclusion of Smad2 is a mechanism leading to loss of competence. Nat. Cell Biol. 4,519 -522.[CrossRef][Medline]
Gruenbaum, Y., Margalit, A., Goldman, R. D., Shumaker, D. K. and Wilson, K. L. (2005). The nuclear lamina comes of age. Nat. Rev. Mol. Cell Biol. 6, 21-31.[CrossRef][Medline]
Hata, A., Lo, R. S., Wotton, D., Lagna, G. and Massague, J. (1997). Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4. Nature 388, 82-87.[CrossRef][Medline]
Heasman, J. (2006). Patterning the early
Xenopus embryo. Development
133,1205
-1217.
Howell, M., Mohun, T. J. and Hill, C. S. (2001). Xenopus Smad3 is specifically expressed in the chordoneural hinge, notochord and in the endocardium of the developing heart. Mech. Dev. 104,147 -150.[CrossRef][Medline]
Hu, C. D. and Kerppola, T. K. (2003). Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat. Biotechnol. 21,539 -545.[CrossRef][Medline]
Hu, C. D., Chinenov, Y. and Kerppola, T. K. (2002). Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol. Cell 9,789 -798.[CrossRef][Medline]
Inman, G. J. and Hill, C. S. (2002).
Stoichiometry of active smad-transcription factor complexes on DNA.
J. Biol. Chem. 277,51008
-51016.
Jayaraman, L. and Massague, J. (2000). Distinct
oligomeric states of SMAD proteins in the transforming growth factor-beta
pathway. J. Biol. Chem.
275,40710
-40717.
Kerppola, T. K. (2006). Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat. Protoc. 1,1278 -1286.[Medline]
Kurisaki, A., Kose, S., Yoneda, Y., Heldin, C. H. and Moustakas,
A. (2001). Transforming growth factor-beta induces nuclear
import of Smad3 in an importin-beta1 and Ran-dependent manner. Mol.
Biol. Cell 12,1079
-1091.
Lee, M. A., Heasman, J. and Whitman, M. (2001).
Timing of endogenous activin-like signals and regional specification of the
embryo. Development 128,2939
-2952.
Nagai, T., Sawano, A., Park, E. S. and Miyawaki, A.
(2001). Circularly permuted green fluorescent proteins engineered
to sense Ca2+. Proc. Natl. Acad. Sci. USA
98,3197
-3202.
Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K. and Miyawaki, A. (2002). A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20,87 -90.[CrossRef][Medline]
Newport, J. and Kirschner, M. (1982a). A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage. Cell 30,675 -686.[CrossRef][Medline]
Newport, J. and Kirschner, M. (1982b). A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription. Cell 30,687 -696.[CrossRef][Medline]
Nicolas, F. J., De Bosscher, K., Schmierer, B. and Hill, C.
S. (2004). Analysis of Smad nucleocytoplasmic shuttling in
living cells. J. Cell Sci.
117,4113
-4125.
Nieuwkoop, P. D. and Faber, J. (1994). Normal Table of Xenopus laevis (Daudin). New York: Garland.
Piepenburg, O., Grimmer, D., Williams, P. H. and Smith, J.
C. (2004). Activin redux: specification of mesodermal pattern
in Xenopus by graded concentrations of endogenous activin B.
Development 131,4977
-4986.
Qin, B., Lam, S. S. and Lin, K. (1999). Crystal structure of a transcriptionally active Smad4 fragment. Structure 7,1493 -1503.[Medline]
Rekas, A., Alattia, J. R., Nagai, T., Miyawaki, A. and Ikura,
M. (2002). Crystal structure of venus, a yellow fluorescent
protein with improved maturation and reduced environmental sensitivity.
J. Biol. Chem. 277,50573
-50578.
Saka, Y. and Smith, J. C. (2004). A Xenopus tribbles orthologue is required for the progression of mitosis and for development of the nervous system. Dev. Biol. 273,210 -225.[CrossRef][Medline]
Sawano, A. and Miyawaki, A. (2000). Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Res. 28,E78 .[CrossRef][Medline]
Schohl, A. and Fagotto, F. (2002).
Beta-catenin, MAPK and Smad signaling during early Xenopus
development. Development
129, 37-52.
Shen, M. M. (2007). Nodal signaling:
developmental roles and regulation. Development
134,1023
-1034.
Shyu, Y. J., Liu, H., Deng, X. and Hu, C. D. (2006). Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. Biotechniques 40, 61-66.[Medline]
Smith, J. C., Symes, K., Hynes, R. O. and DeSimone, D. (1990). Mesoderm induction and the control of gastrulation in Xenopus laevis: the roles of fibronectin and integrins. Development 108,229 -238.[Abstract]
Smith, J. C., Price, B. M., Green, J. B., Weigel, D. and Herrmann, B. G. (1991). Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. Cell 67,79 -87.[CrossRef][Medline]
Symes, K. and Smith, J. C. (1987). Gastrulation movements provide an early marker of mesoderm induction in Xenopus. Development 101,339 -349.[Abstract]
Tada, M., O'Reilly, M. A. and Smith, J. C. (1997). Analysis of competence and of Brachyury autoinduction by use of hormone-inducible Xbra. Development 124,2225 -2234.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  R. Williams Yasushi Saka: Stirring a melting pot of math and morphogens J. Cell Biol., October 20, 2008; 181(5): 716 - 717. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Plaza, F. Prince, Y. Adachi, C. Punzo, D. L. Cribbs, and W. J. Gehring Cross-regulatory protein-protein interactions between Hox and Pax transcription factors PNAS, September 9, 2008; 105(36): 13439 - 13444. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
