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First published online 16 April 2008
doi: 10.1242/dev.019323
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Max-Planck Institute of Immunobiology, Stübeweg 51, D-79108 Freiburg, Germany.
Author for correspondence (e-mail:
oelgeschlaeg{at}immunbio.mpg.de)
Accepted 19 March 2008
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: TGF-β, Mucin, O-linked glycosylation, BMP, Nodal
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Shi and Massague,
2003). TGF-β family members bind to heteromeric complexes
consisting of type I and type II transmembrane serine/threonine kinase
receptors. Upon ligand binding, the type II receptor subunit activates the
type I receptor, which subsequently phosphorylates receptor-associated
mediators of the Smad family (R-Smads). The phosphorylated R-Smad proteins
translocate into the nucleus and activate, in concert with additional
transcriptional co-activators, the transcription of specific target genes
(Attisano and Wrana, 2002;
Feng and Derynck, 2005). The
TGF-β superfamily can be subdivided into two main branches. The BMP
signalling pathway activates Smad1, Smad5 and Smad8, whereas Activin/Nodal
signalling activates Smad2 and Smad3
(Miyazawa et al., 2002).
During early vertebrate embryogenesis, the formation of mesoderm requires the
activity of Nodal-related proteins, whereas BMP activity is required for the
establishment of dorsoventral (or back-to-belly) polarity
(De Robertis and Kuroda, 2004;
Schier and Talbot, 2005;
Stern, 2005).
The glycosylation of cell surface proteins is of central importance for the
regulation of signal transduction during embryonic development. Heparan
sulphate proteoglycans (HSPGs) are essential co-factors for the Wnt, Hedgehog,
Fibroblast growth factor (FGF) and TGF-β signalling pathways in
Drosophila as well as mice
(Perrimon and Bernfield,
2000). Cell surface proteoglycans, including membrane-associated
endoglin and betaglycan, act as additional co-receptors (TGF-β type III
receptors) that bind TGF-β proteins and facilitate the binding of the
ligand to the heteromeric TGF-β receptor complex. In addition, secreted
proteoglycans, like Decorin and Biglycan, bind and sequester TGF-β in the
extracellular matrix (Gumienny and
Padgett, 2002). Modification of epidermal growth factor-like (EGF)
repeats by O-fucosyltransferases and
β1,3-N-acetylglucosaminyltransferases of the Fringe family regulates
Notch as well as Nodal signalling. The O-linked glycosylation of Notch and of
Notch ligands modulates the activation of Notch signalling after binding to
its ligands Delta and Serrate/Jagged
(Haltiwanger and Lowe, 2004).
Mutation of an O-fucosylation site in the Nodal co-receptor Cripto interferes
with the activation of Nodal signalling
(Schiffer et al., 2001;
Yan et al., 2002). More
recently, a β1,4-galactosyltransferase was shown to modulate dorsoventral
patterning and BMP signalling in the early zebrafish embryo
(Machingo et al., 2006).
The mucin-type of O-linked glycosylation, characterised by
-N-acetylgalactosamine (GalNAc) attached to the hydroxyl group of
serine or threonine side chains, is the most abundant form of O-linked
glycosylation in higher eukaryotes (Hang
and Bertozzi, 2005). Over 150 mucin-type glycoproteins have been
annotated in mammals, but a consensus recognition sequence for the
O-glycosyltransferases has not been described
(Julenius et al., 2005). The
initial addition of the GalNAc moiety is catalyzed by members of the
UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNT) family.
Subsequently, downstream glycosyltransferases generate complex O-linked
glycans that can modulate a variety of biological processes
(Haltiwanger and Lowe, 2004;
Hang and Bertozzi, 2005).
Interestingly, changes in the expression levels of GalNT family members and in
the structures of these O-linked glycans have been associated with a number of
human diseases, including immunodeficiencies and cancer
(Tsuboi and Fukuda, 2001;
Hollingsworth and Swanson;
2004; Brockhausen,
2006).
In mammals, 15 members of the GalNT family have been identified that
display different substrate specificities and tissue-specific expression
patterns (Ten Hagen et al.,
2003; Young et al.,
2003; Cheng et al.,
2004). However, a role for these proteins in embryonic development
has not been described. Here, we report the identification of
N-acetylgalactosaminyltransferase-like 1 (Galntl-1) as a novel negative
regulator of TGF-β signalling. Xenopus Galntl-1 (xGalntl-1) is
specifically expressed in neural and dorsal mesodermal tissues and is required
for the proper formation of the neural crest, spinal cord and anterior
notochord. Xenopus and mammalian Galntl-1 can inhibit BMP as well as
Nodal signalling in the early Xenopus embryo and in human HEK 293T
cells. Our biochemical data suggest that Galntl-1 interferes with the
formation of heteromeric TGF-β receptor complexes. This study identifies
a N-acetylgalactosaminyltransferase as a novel and important regulator of
TGF-β signalling in the early Xenopus embryo and might provide a
new paradigm for the regulation of TGF-β signalling in vertebrates.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)]. The
open reading frames as well as epitope-tagged versions were cloned by PCR into
pCS2+. Human ALK3 (BMPR1A - Human Gene Nomenclature Database), mouse Alk6
(Bmpr1b - Mouse Genome Informatics) and human BMPR-II (BMPR2) expression
vectors were kindly provided by Dr L. Attisano (University of Toronto, Canada)
and the coding sequences cloned into the EcoRI and XbaI
sites of pCS2+. For secreted Galntl-1, the nucleotides encoding amino acids
93-563 of xGalntl-1 were introduced in-frame into a pCS2 expression vector
containing the Chordin N-terminus, followed by a FLAG-tag sequence
(Oelgeschläger et al.,
2003a). For mRNA synthesis, the constructs were cut with
NotI and transcribed with SP6 RNA polymerase using the mMessage
mMachine Kit (Ambion, Austin, TX).
Embryos and explants
In vitro fertilisation, embryo and explant culture, microinjection of
synthetic mRNA, in situ hybridisation and RT-PCR analysis were performed as
described (Yamamoto et al.,
2007; Sive et al.,
2000). The in situ probes for msx1, slug and
xbra have been described elsewhere
(Yamamoto et al., 2007;
Tribulo et al., 2003). For
vibratome sections, embryos were embedded in gelatine-albumin
(Gove et al., 1997) and 30
µm sections mounted in Glycergel (DAKO, Denmark). For western blot analysis
of proteins expressed in ectodermal explants, the animal caps were isolated at
stage 9, cultured in 1x Steinberg solution until stage 11 and
transferred into lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM
EDTA, 1% Triton-100, 1 mM DTT, 10% glycerol, supplemented with protease
inhibitor cocktail (Roche)]. The analysis of xGalntl-1 secretion using
dissociated animal cap cells was performed as described
(Oelgeschläger et al.,
2003b).
RT-PCR and morpholinos
RT-PCR analysis was performed as previously described
(Oelgeschläger et al.,
2003a; Yamamoto et al.,
2007; Agius et al.,
2000). Additional primers (forward, reverse) were:
xGalntl-1, 5'-AGCATCCAGCAAGTCTCCGAGC-3' and
5'-GTGATGATGACACTGGTTGAGG-3'; xGalnt-1,
5'-CAAGGTGTTGTGGATGGC-3' and
5'-TTCTCATTAGCGTAGACCAACC-3'; xGalnt-6,
5'-TCACAATGAGGCTTGGTC-3' and
5'-CAACAGCGGTGTAATCTTCTGC-3'; xGalnt-7,
5'-AGGACAGAGTCACGGATAGAGC-3' and
5'-TCGCATCCTTGTAATCTGGTCC-3'; dlx-5,
5'-TCTCTACTGCCACGAACTGAGC-3' and
5'-TCTGGCAATGGTTGGAAGGTCC-3'; and ncad,
5'-TTGTTGTATGGATGAAGCGTCG-3' and
5'-CGAACACTAACAAGGAATCG-3'. The xGalntl-1 morpholino sequences
were 5'-AAGCCTTCAGCTCTTTCCCATTCTC-3' and
5'-CTGATCCTTCTCATGCTGCCGGTAG-3'. In vitro translation of synthetic
mRNA was performed as described (Yamamoto
et al., 2007). In all in vivo experiments, 2 nl of a 1:1 mixture
of both morpholinos was used (1 ng/nl each).
Protein analysis
For western blot analysis of HA-tagged Alk-3, Alk-4, Alk-6 and ActR-IIB
proteins, transiently transfected HEK 293T cells were lysed in lysis buffer
and proteins detected using HRP-coupled anti-HA antibodies (Sigma).
Epitope-tagged proteins were immunoprecipitated in lysis buffer with a
monoclonal anti-FLAG antibody (Sigma), monoclonal anti-FLAG M2 antibody
(Sigma) or a polyclonal anti-Myc antibody (Abcam, Cambridge, UK) and proteins
visualised with HRP-coupled anti-HA, polyclonal anti-FLAG (Sigma) or
monoclonal anti-Myc antibody (Sigma). For phospho-Smad blots, the cells were
lysed in Phosphosafe (Novagen, San Diego, CA) and the transiently expressed
FLAG-tagged Smad proteins immunoprecipitated in lysis buffer. The antibodies
specific for phospho-Smad1, Smad1, phospho-Smad2 and Smad2 (Cell Signaling
Technology, Danvers, MA) were used according to manufacturer's instructions.
For the treatment of HEK 293T with recombinant Activin or BMP4 protein
(R&D Systems, Minneapolis, MN), transiently transfected cells were
incubated overnight in serum-free medium, incubated in serum-free medium
containing 2 ng/ml Activin or 40 ng/ml BMP4 protein for 2 hours and lysed in
Phosphosafe. For immunohistochemical stainings, Cos-7 cells were transfected
with Superfect (Qiagen, Hilden, Germany) on culture slides (BD Falcon), fixed
with 4% paraformaldehyde and proteins stained using a monoclonal anti-HA
antibody or polyclonal anti-FLAG (Sigma) and Alexa Fluor 568-conjugated
anti-mouse or Alexa Fluor 488-conjugated anti-rabbit antibody (Invitrogen).
The detection of mucin-type glycosylation in cell membranes and the Golgi
compartment using fluorescence-coupled Helix pomatia agglutinin (HPA
Alexa Fluor 488, Invitrogen) has been described elsewhere
(Virtanen, 1990).
GenBank accession numbers
The accession numbers for the nucleotide sequences are: xGalntl-1,
BM192636; xGalNT-1, BC060419; xGalNT-4, BC071009; xGalNT-6, BC110706;
xGalNT-7, BC070527; xGalNT-11, BC080006; human GALNTL1, BC036812; GalNT-1/13,
X85018/NM_020474; GalNT-2, BC041120; GalNT-4, BC036390; GalNT-6, BC114505;
GalNT-7, BC047468; GalNT-11, NM_022087; GalNT-14, NM_024572; mouse Galntl1,
CD347785; zGalNT-14, NM_001044995; dmGalNT-2, GA16973.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Wandall et al., 2007). The
N-terminal transmembrane and the central catalytic domain are highly conserved
between Xenopus and human Galntl-1 homologues (>80%), whereas the
C-terminal Ricin domains are more divergent, with only 40% amino acid
identity. To test for N-acetylgalactosaminyltransferase activity, we used
fluorescence-coupled agglutinin from Helix pomatia (HPA) that
specifically binds to GalNAc--Thr/Ser
(Lehtonen et al., 1989;
Virtanen, 1990;
Hang et al., 2003). As
expected, we were able to detect enhanced mucin-type O-linked glycosylation in
the Golgi compartment and at the cell surface of xGalntl-1-expressing cells
(Fig. 1B).
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xGalntl-1 inhibits mesoderm formation and Activin/Nodal signalling
Marginal microinjection of xGalntl-1 mRNA resulted in severe
gastrulation defects (Fig. 2B).
Co-injection of β-galactosidase mRNA (lacZ) revealed a
cell-autonomous inhibition of mesoderm formation at early gastrula stages,
indicated by the reduced expression of the pan-mesodermal marker xbra
(Fig. 2C). In marginal zone
explants, xGalntl-1 strongly reduced the expression of dorsal
(-actin) and ventral (-globin, gata1, xvent1)
mesodermal marker genes and induced the expression of anterior neural marker
genes in ventral marginal zone explants
(Fig. 2D). The formation of
dorsal and ventral mesoderm in the Xenopus embryo at gastrula stages
is mediated by a Nodal activity gradient in the underlying vegetal tissue
(Agius et al., 2000;
Martello et al., 2007). In
addition, members of the FGF family can induce mesodermal cell fate and
directly activate transcription of xbra
(Latinkic et al., 1997;
Wardle and Smith, 2006). The
induction of mesodermal (xbra, chordin) and endodermal
(sox17β) marker genes in ectodermal explants microinjected with
mRNA encoding Activin or Nodal was inhibited by co-injection of
xGalntl-1 mRNA. By contrast, the induction of xbra by
microinjection of eFGF mRNA was unaffected by xGalntl-1
(Fig. 2E and data not shown).
We conclude from these data that xGalntl-1 interfered specifically with the
activity of the Activin/Nodal pathway. The inhibition of Activin signalling by
xGalntl-1 appeared to be direct, as the stimulation of Smad2 phosphorylation
after treatment of transiently transfected HEK 293T cells with recombinant
Activin protein for 2 hours was inhibited in xGalntl-1-expressing cells
(Fig. 2F).
|
;
Heasman, 2002) that inhibited
the in vitro translation and in vivo activity of xGalntl-1 from mRNA
containing the morpholino target sequences in the 5'-UTR
(Fig. 2G and data not shown).
To minimise non-specific effects of the morpholinos, we injected a mixture of
both morpholinos (half the concentration of each) in all subsequent
experiments. To test whether endogenous xGalntl-1 activity modulates
Activin/Nodal signalling in vivo, we analysed the effect of the xGalntl-1
morpholinos on mesoderm induction by Activin mRNA in animal cap
explants. Co-injection of the xGalntl-1 morpholinos stimulated the induction
of mesoderm by Activin. This effect was rescued by co-injection of
xGalntl-1 mRNA lacking the morpholino target sequence, at
concentrations that did not affect the development of the whole embryo or the
differentiation of embryonic explants (Fig.
2H and data not shown). We conclude from these data that xGalntl-1
inhibits the activity of the Activin/Nodal pathway in vivo.
xGalntl-1 inhibits BMP signalling
The neuralisation of ventral marginal zone explants by xGalntl-1
(Fig. 2D) implied that
xGalntl-1 might also inhibit BMP activity. Microinjection of mRNA encoding
xGalntl-1 into animal blastomeres led to a dorsalised phenotype, with enlarged
head and reduced tail and trunk structures
(Fig. 3B). In embryonic
ectodermal explants, xGalntl-1 induced the expression of otx2 and
xag. In contrast to the bona fide BMP antagonist Chordin, xGalntl-1
did not induce the expression of anterior neural marker genes
(Fig. 3E), arguing for a mild
reduction of BMP activity by xGalntl-1
(Wilson and Hemmati-Brivanlou,
1995). Microinjection of mRNA encoding human or mouse Galntl1
generated similar dorsalised phenotypes in whole embryos, induced the
expression of otx2 and xag in animal cap explants and
interfered with mesoderm formation at gastrula stages
(Fig. 3B,G and data not shown).
Similar to other glycosyltransferases
(El-Battari et al., 2003),
xGalntl-1 seemed to be cleaved and secreted from transiently transfected HEK
293T cells and microinjected ectodermal explants. We detected a significant
amount of xGalntl-1 protein in the supernatant of dissociated animal cap
explants that migrated at a slightly lower molecular weight in SDS gels
(Fig. 3F). Replacing the
N-terminal transmembrane domain of xGalntl-1 with the Chordin leader peptide
did not significantly increase the level of secreted xGalntl-1 protein, but
instead abolished xGalntl-1 activity in the embryo
(Fig. 3D,F). Thus, the
N-terminal signal peptide that retains xGalntl-1 in the Golgi compartment is
required for Galntl-1 activity, whereas the differences in the C-terminal
Ricin domain of Xenopus and mammalian Galntl-1 proteins had no
obvious effect in these assays.
The activation of FGF, Activin/Nodal and Wnt signalling also induces the
expression of neural and anterior marker genes such as otx2 and
xag. These signalling pathways can interfere with BMP activity,
stimulating the expression of BMP antagonists, repressing BMP expression, or
by interference with the nuclear translocation of phospho-Smad proteins
(Kretzschmar et al., 1997;
Baker et al., 1999;
Sasai et al., 1994;
Oelgeschläger et al.,
2003a; Kuroda et al.,
2005). Microinjection of BMP7/OP-1 mRNA
prevented the induction of otx2 and xag by xGalntl-1 in
animal cap assays. By contrast, the inhibition of FGF, Activin/Nodal or Wnt
signalling by microinjection of a dominant-negative FGF receptor (dnFGFR-4),
dominant-negative Ras (not shown), dominant-negative Activin/Nodal type I
receptor (tAlk-4) and dominant-negative TCF-3 had no effect
(Fig. 3H see Fig. S2 in the
supplementary material). Furthermore, the expression of xGalntl-1 decreased
phospho-Smad1 levels in embryonic explants at gastrula stages and in
transiently transfected HEK 293T cells treated with recombinant BMP4 protein
(Fig. 3I,J). Interestingly,
xGalntl-1 did not inhibit phospho-Smad1 induction to the same degree in cells
expressing a constitutively active BMP type I receptor
(Fig. 3K). We conclude from
these data that xGalntl-1 interfered with BMP signalling upstream of, or on
the level of, BMP receptor complexes.
|
;
Mizutani et al., 2006).
However, the overall patterning of the spinal cord was unaffected (data not
shown). In more-severely affected embryos, the anterior notochord was
hypoblastic (Fig. 4B) and the
shape of the anterior somites disturbed, probably reflecting reduced notochord
signalling (data not shown). At gastrula stages, we did not observe changes in
the expression of the pan-mesoderm marker xbra, indicating that the
effect of the xGaltnl-1 morpholino on endogenous Activin/Nodal activity was
not strong enough to overcome the intrinsic regulatory network, including
TGF-β inhibitors such as Ectodermin
(Dupont et al., 2005). The
earliest phenotype we observed was a thinning of the neural crest territory at
neurula stages and reduced expression of the neural crest marker genes
msx1 and slug (Fig.
4C). Co-injection of lacZ mRNA revealed a strong
reduction of msx1 expression in the injected area and, importantly,
this effect was rescued by co-injection of xGalntl-1 mRNA lacking the
morpholino target sequence (Fig.
4C). At tailbud stages, the anterior expression of the neural
crest marker slug was reduced and the formation of the branchial
arches disturbed (Fig. 4B and
data not shown). In dorsal marginal zone explants, the xGalntl-1 morpholino
generated stronger phenotypes with significant reduction of anterior
(six3, otx2) and neural crest (snail) tissue, whereas the
expression levels of ventral ectodermal (xvent2) and posterior
mesodermal (xbra) marker genes were unchanged
(Fig. 4D).
BMP signalling plays an important role in the formation of neural and
neural crest tissue. In particular, the expression of msx1 is induced
by a sharp threshold concentration of BMP and is expanded in Xenopus
and zebrafish embryos with reduced BMP activity
(Tribulo et al., 2003). Thus,
the morpholino knock-down of xGalntl-1 generated specific phenotypes in the
tissues that express endogenous xGalntl-1, which, in support of our
gain-of-function data, suggests a regulation of TGF-β signalling by
xGalntl-1.
xGalntl-1 inhibits ActR-IIB activity
To test whether the inhibition of BMP activity by xGalntl-1 in ectodermal
explants was due to inhibition of type I or type II BMP receptor proteins, we
co-expressed xGalntl-1 with Alk-3/BMPR-IA, Alk-6/BMPR-IB, BMPR-II and ActR-IIB
in animal cap explants. As shown in Fig.
5A and Fig. S3 in the supplementary material, only ActR-IIB
inhibited the induction of xag and otx2 by xGalntl-1
completely. ActR-IIB overexpression induces the formation of posterior
mesoderm in animal cap explants (New et
al., 1997). Microinjection of 400 pg ActR-IIB mRNA
induced the expression of posterior mesodermal marker genes and this activity
was strongly inhibited by xGalntl-1 (Fig.
5B). By contrast, similar to the effects observed for Activin, the
xGalntl-1 morpholinos stimulated mesoderm induction by low amounts of
ActR-IIB mRNA that did not induce the expression of mesodermal marker
genes alone (Fig. 5C). This
effect was apparently due to reduced ActR-IIB activity, as xbra
induction by xActR-IIB was blocked by xGalntl-1 already at early gastrula
stages and, importantly, the induction of Smad2 phosphorylation by ActR-IIB
was inhibited by xGalntl-1 in HEK 293T cells
(Fig. 5D). By contrast, the
stimulation of phospho-Smad1 levels by BMPR-II was hardly affected
(Fig. 5D).
|
|
-galactosaminide (benzyl-GalNAc). This small,
chemically synthesized sugar analogue competes for the processing of core
GalNAc residues of mucin-type O-linked glycans
(Kuan et al., 1989;
Prescher and Bertozzi, 2006).
We treated transiently transfected HEK 293T cells with 2 mM benzyl-GalNAc to
test whether the inhibitory activity of xGalntl-1 was dependent on its
glycosyltransferase activity. The benzyl-GalNAc-treated cells had
significantly higher phospho-Smad2 levels in the presence of xGalntl-1
(Fig. 5E). Next, we tested
whether the reduced expression of msx1 in cells lacking endogenous
xGalntl-1 protein at early neurula stages could be due to enhanced ActR-IIB
activity. Microinjection of ActR-IIB mRNA interfered with
msx1 expression, comparable to the effects observed with the
xGalntl-1 morpholinos, and co-injection of xGalntl-1 mRNA was able to
restore msx1 expression in ActR-IIB-overexpressing, as well as in
xGalntl-1-depleted, cells (Fig.
5F). Taken together, these data suggest that mucin-type O-linked
glycosylation interferes with ActR-IIB activity in the early Xenopus
embryo as well as in human HEK 293T cells.
|
). Immunostaining of transiently transfected Cos-7 cells did
not reveal changes in the membrane localisation of ActR-IIB, Alk-3, Alk-6 or
Alk-4 protein that could explain the strong inhibitory effect of xGalntl-1
co-expression on ActR-IIB activity (Fig.
6A and data not shown). In addition, western blot analysis of
ActR-IIB, Alk-3, Alk-4 and Alk-6 proteins expressed in animal cap explants or
HEK 293T cells in the presence or absence of xGalntl-1, did not reveal
significant changes in steady-state protein levels
(Fig. 6B). However, as shown in
Fig. 6B,C, co-expression of
xGalntl-1 in embryonic as well as 293T cells resulted in a migration shift of
the ActR-IIB and Alk-3 proteins. Interestingly, the xGalntl-1-dependent shift
of the ActR-IIB protein was not observed in HEK 293T cells treated with 2 mM
benzyl-GalNAc (Fig. 6C). Finally, we analysed the binding of ActR-IIB to the type I receptor Alk-4, which specifically mediates Activin/Nodal-related signals, and to the BMP-specific type I receptors Alk-3 and Alk-6. Expression constructs for HA-tagged type I receptors (Alk-3, Alk-4 and Alk-6), Myc-tagged ActR-IIB and FLAG-tagged xGalntl-1 were transfected into HEK 293T cells, cell lysates immunoprecipitated with an anti-Myc antibody and co-immunoprecipitated type I receptors visualised using anti-HA antibodies. In these experiments, the binding of ActR-IIB to all three type I receptors was strongly reduced in the presence of xGalntl-1 (Fig. 6D). Similar effects were observed using a Myc-tagged ActR-IIA (data not shown). By contrast, the binding of BMPR-II to Alk-3 was unaffected by xGalntl-1 co-expression (Fig. 6E). Thus, xGalntl-1 specifically interferes with the formation of heteromeric TGF-β receptor complexes containing ActR-IIA and ActR-IIB. The sGalntl-1 protein, which lacks the N-terminal transmembrane domain and did not display any activity in early Xenopus embryos (Fig. 3), also did not interfere with the binding of ActR-IIB to type I receptor proteins (Fig. 6F). In addition, other proteins of the N-acetylgalactosaminyltransferase family that are expressed in the early Xenopus embryo (Fig. 1), including xGalnt-6, had no effect on the binding of ActR-IIB to Alk-4 und did not interfere with mesoderm formation during gastrulation (Fig. 6F and data not shown). In summary, xGalntl-1 does not affect the stability or intracellular trafficking of TGF-β receptor proteins, but specifically interferes with the binding of ActR-IIA and ActR-IIB to type I TGF-β receptor proteins that can mediate BMP as well as Nodal signalling.
|
DISCUSSION |
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|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A common level of control takes place in the extracellular
space, where secreted antagonists prevent TGF-β ligands from binding to
their cognate receptors (Miyazono,
2000; Balemans and Van Hul,
2002). In particular, recent studies have unravelled a complex
system of interacting proteins that regulate the interaction of BMP ligands
with their cognate receptors (De Robertis,
2006). In addition, regulation of TGF-β signals occurs at the
level of the cell membrane, the cytoplasm and the nucleus
(Lutz and Knaus, 2002;
Shi and Massague, 2003) This
study demonstrates an important role for the N-acetylgalactosaminyltransferase
Galntl-1 in the regulation of TGF-β signalling. In the early
Xenopus embryo, Galntl-1 interfered with Nodal/Activin-dependent
mesoderm formation, inhibited BMP signalling and stimulated neural
differentiation (Figs 2 and
3). In addition, Galntl-1
effectively interfered with the phosphorylation of BMP as well as of
Activin/Nodal-specific R-Smads in the Xenopus embryo and in human HEK
293T cells, whereas specific xGaltnl-1 morpholinos stimulated Activin and
ActR-IIB activity in embryonic explants (Figs
2 and
5). Thus, endogenous xGalntl-1
negatively regulates TGF-β signalling in the early Xenopus
embryo. In HEK 293T cells, Xenopus Galntl-1 interfered with the
stimulation of R-Smad phosphorylation by Activin and BMP signals,
demonstrating that Xenopus Galntl-1 can also act on human components
of these signalling pathways. In addition, frog and mammalian Galntl-1
displayed similar activities on TGF-β signalling in the early frog
embryo, HEK 293T cells and in our biochemical assays, despite the significant
differences in the C-terminal Ricin domain
(Fig. 3 and data not shown).
Thus, the regulation of TGF-β signalling by Galntl-1 seems to be an
evolutionarily conserved mechanism that could also be active during mammalian
embryogenesis and adult tissue homeostasis.
Requirements for xGalntl-1 in the early Xenopus embryo
Although mucin-type O-linked glycans are very abundant, little is known
about the function of this type of O-glycosylation in embryonic development.
Targeted deletion of single N-acetylgalactosaminyltransferases has not
revealed obvious functional deficits, arguing that some functional redundancy
might exist (Haltiwanger and Lowe,
2004). We detected the expression of several
N-acetylgalactosaminyltransferases throughout early Xenopus
embryogenesis (Fig. 1).
Nevertheless, our loss-of-function studies revealed a specific requirement of
xGalntl-1 for the formation of the neural crest and the neural tube. The
formation of these tissues is well known to be dependent on a tight regulation
of TGF-β signalling (De Robertis and
Kuroda, 2004; Barembaum and
Bronner-Fraser, 2005; Stern,
2005). The msx1 and msx2 genes are required for
the formation of neural crest cells and stimulate the expression of additional
early neural crest marker genes, including snail, slug and
foxd3 (Tribulo et al.,
2003; Khadka et al.,
2006). The msx1 gene is a direct target of the BMP signal
transduction pathway (Takahashi et al.,
1997; Alvarez-Martinez et al.,
2002), but the transcriptional activation of msx1 is
dose-dependent and requires intermediate levels of BMP signalling activity.
Therefore, the territory expressing msx-1 is expanded in
Xenopus and zebrafish embryos with reduced BMP signalling activity,
and locally applied Noggin protein can induce ectopic msx1 expression
in neighbouring tissues (Nguyen et al.,
2000; Tribulo et al.,
2003). The requirement of endogenous xGalntl-1 for the
determination of the narrow msx1-positive territory implicates an
essential role for xGalntl-1 in the formation of the BMP activity gradient at
neurula stages.
The dorsoventral patterning of the neural tube is dependent on the
establishment of a BMP signalling gradient
(Placzek and Briscoe, 2005;
Mizutani et al., 2006). In
addition, it has been proposed that distinct types of BMP receptors regulate
the switch between proliferation and differentiation of neural precursor cells
in the neural tube (Chizhikov and Millen,
2005). Xenopus Galntl-1 is specifically expressed in the
mantle (differentiating) territory of the spinal cord and is excluded from the
central area containing proliferating neuronal precursors
(Fig. 1E). Thus, the modulation
of BMP receptor complex formation by xGalntl-1 might participate in the
regulation of neural differentiation in the spinal cord. The effects of the
xGalntl-1 morpholinos on the formation of the spinal cord were rather mild,
but the lack of anterior neural crest and the reduction of neural tissue
suggest an important role of xGalntl-1 in the regulation of proliferation and
differentiation of these tissues. Interestingly, the morpholino knock-down of
ActR-IIA and ActR-IIB in zebrafish revealed a requirement for both receptor
subtypes in the formation of cranial neural crest and neural tissue
(Albertson et al., 2005). In
addition, overexpression of xActR-IIB in the early Xenopus embryo
inhibited msx1 expression at neurula stage
(Fig. 5F), similar to the
reduction observed with the xGalntl-1 morpholino. Thus, at least some of the
loss-of-function phenotypes observed for xGalntl-1 could be mediated by
modulation of ActR-IIB activity.
Regulation of heteromeric TGF-β receptor complexes by xGalntl-1
The BMP and Activin/Nodal branches of the TGF-β superfamily signal
through distinct type I receptors, but share type II receptors, including
ActR-IIB (Attisano et al.,
1992). The TGF-β superfamily member BMP3 antagonises BMP and
Activin-like signals, generating phenotypes comparable to those we observed
with xGalntl-1 (Hino et al.,
2003; Gamer et al.,
2005). However, the molecular mechanisms underlying the inhibitory
activities of BMP3 are not completely understood. BMP3 binds to ActR-IIB and
inhibits ActR-IIB activity, without interfering with ligand binding or
receptor complex formation (Gamer et al.,
2005). Similar to the effect of BMP3, xGalntl-1 did not inhibit
BMP signalling completely. The remaining BMP activity might be mediated by
BMPR-II. The binding of BMPR-II to Alk-3 was unaffected and BMPR-II activity
was not inhibited by xGalntl-1 (Fig.
5D and Fig. 6E).
Interestingly, a secreted form of Galntl-1 (sGalntl1) and an additional member
of the family of N-acetylgalactosaminyltransferases (xGalnt-6) expressed
during early Xenopus embryogenesis did not interfere with the
formation of heteromeric receptor complexes in vitro and did not inhibit
TGF-β signalling in vivo (Fig.
3 and Fig. 6F).
Thus, the biological activity of Galntl-1 is specific and correlated well with
our biochemical results.
Galntl-1 did not affect the steady-state protein levels or cellular
localisation of type I or type II receptor proteins in Xenopus
embryonic extracts or transfected cell lines
(Fig. 6A,B). However, xGalntl-1
did induce a migration shift of Alk-3 and ActR-IIB proteins under denaturing
gel conditions. A specific inhibitor of mucin-type O-linked glycosylation,
benzyl-GalNAc, prevented the shift of the ActR-IIB protein as well as the
inhibition of ActR-IIB activity by Galntl-1
(Fig. 5E,
Fig. 6C). This might suggest
that Galntl-1 inhibits TGF-β receptor subunits, in particular ActR-IIB,
by a direct modification. A consensus recognition sequence for the
O-glycosyltransferases is not known, but potential glycosylation sites in
ActR-IIA, ActR-IIB and Alk-3 can be predicted using NetOGlyc 3.1
(Julenius et al., 2005).
However, mutations in the potential glycosylation sites of Alk-3 and ActR-IIB
did not interfere with inhibition by xGalntl-1 (data not shown). It is
possible that additional glycosylation sites exist or that additional proteins
involved in the formation of ActR-II/B receptor complexes are modified by
xGalntl-1. We were unable to co-immunoprecipitate xGalntl-1 with Alk-3, Alk-4,
Alk-6 or ActR-IIB and did not observe Galntl-1 protein in the cell membrane
(Fig. 6A and data not shown).
Thus, it is unlikely that Galntl-1 interferes with the formation of
heteromeric complexes only through a direct interaction with TGF-β
receptor subunits. However, we cannot exclude the possibility that Galntl-1
might interact with additional unknown factors in the Golgi compartment that
are required for the efficient formation of ActR-II/B-containing receptor
complexes.
In summary, we have shown that the activity of Activin and BMP signalling is regulated by a N-acetylgalactosaminyltransferase with a highly specific expression pattern, adding an additional component into the complex regulatory network modulating TGFβ signalling. Importantly, this novel regulatory mechanism is evolutionarily conserved between different vertebrate species. In the future, it will be important to understand whether different members of the N-acetylgalactosaminyltransferase family target different components of TGFβ signalling, providing a new paradigm for regulation in this important pathway.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/10/1813/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
These authors contributed equally to this work
Present address: Division of Developmental Biology, Cincinnati Children's
Hospital Medical Centre, 3333 Burnet Avenue, Cincinnati, OH 45229-3039,
USA
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REFERENCES |
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N-TCF-3 (400 pg).
(I) Western blot for Smad1 or phospho-Smad1 using animal cap lysates
from uninjected embryos or embryos microinjected with 200 pg Smad1-FLAG mRNA
alone or together with 200 pg xGalntl-1 mRNA. (J) Western blot
analysis for Smad1 and phospho-Smad1 from transiently transfected Hek293T
cells treated for 2 hours with 40 ng/ml recombinant BMP4 protein. (K)
Western blot for phospho-Smad1 or Smad1 using lysates from HEK 293T cells
transiently transfected with the indicated expression plasmids. In the RT-PCR
experiments 
