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RESEARCH ARTICLE 1813
Development 135, 1813-1822 (2008) doi:10.1242/dev.019323
Regulation of TGF-β signalling by
N-acetylgalactosaminyltransferase-like 1
Patrick Herr*,†, Ganna Korniychuk†, Yukiyo Yamamoto‡, Kristina Grubisic and Michael Oelgeschläger§
The TGF-β superfamily of secreted signalling molecules plays a pivotal role in the regulation of early embryogenesis, organogenesis
and adult tissue homeostasis. Here we report the identification of Xenopus N-acetylgalactosaminyltransferase-like 1 (xGalntl-1) as a
novel important regulator of TGF-β signalling. N-acetylgalactosaminyltransferases mediate the first step of mucin-type
glycosylation, adding N-acetylgalactose to serine or threonine side chains. xGalntl-1 is expressed in the anterior mesoderm and
neural crest territory at neurula stage, and in the anterior neural crest, notochord and the mediolateral spinal cord at tailbud stage.
Inhibition of endogenous xGalntl-1 protein synthesis, using specific morpholino oligomers, interfered with the formation of
anterior neural crest, anterior notochord and the spinal cord. Xenopus and mammalian Galntl-1 inhibited Activin as well as BMP
signalling in the early Xenopus embryo and in human HEK 293T cells. Gain- and loss-of-function experiments showed that xGalntl-1
interferes with the activity of the common TGF-β type II receptor ActR-IIB in vivo. In addition, our biochemical data demonstrated
that xGalntl-1 specifically interferes with the binding of ActR-IIB to Activin- and BMP-specific type I receptors. This inhibitory activity
of xGalntl-1 was dependent on mucin-type glycosylation, as it was sensitive to the chemical inhibitor benzyl-GalNAc. These studies
reveal an important role of a N-acetylgalactosaminyltransferase in the regulation of TGF-β signalling. This novel regulatory
mechanism is evolutionarily conserved and, thus, might provide a new paradigm for the regulation of TGF-β signalling in
vertebrates.
INTRODUCTION
The TGF-β superfamily of secreted growth factors regulates a
plethora of biological processes in a highly dose-dependent manner
that demands tight regulation of this signalling pathway during
embryogenesis and adult tissue homeostasis (Lutz and Knaus, 2002;
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
Max-Planck Institute of Immunobiology, Stübeweg 51, D-79108 Freiburg, Germany.
*Present address: Institute of Molecular Biology, University of Zurich,
Winterthurerstrasse 190, 8057 Zurich, Switzerland
†
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
§
Author for correspondence (e-mail: [email protected])
Accepted 19 March 2008
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 β1,3N-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 α-Nacetylgalactosamine (GalNAc) attached to the hydroxyl group of
serine or threonine side chains, is the most abundant form of Olinked 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 Oglycosyltransferases 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
DEVELOPMENT
KEY WORDS: TGF-β, Mucin, O-linked glycosylation, BMP, Nodal
1814 RESEARCH ARTICLE
MATERIALS AND METHODS
Constructs
Full-length cDNA encoding Xenopus, human and mouse Galntl-1 and
Xenopus Galnt-6 were obtained from RZPD [IMAGE Consortium (Lennon
et al., 1996)]. 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 1⫻ 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⬘-TTGTTGTATGGATGAAG-
CGTCG-3⬘ and 5⬘-CGAACACTAACAAGGAATCG-3⬘. The xGalntl-1
morpholino sequences were 5⬘-AAGCCTTCAGCTCTTTCCCATTCTC3⬘ 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 ActRIIB 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 568conjugated 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.
RESULTS
Xenopus Galntl-1 is specifically expressed in
neural and dorsal mesoderm tissues
In a screen for genes that are negatively regulated by BMP signals
(our unpublished results), we isolated a Xenopus homologue
(xGalntl-1) of human and mouse N-acetylgalactosaminyltransferase
1. The xGalntl-1 protein shares over 72% amino acid identity with
mouse and human Galntl1, 56% identity with zebrafish Galnt14 and
47% with Drosophila Galnt2 (Fig. 1A and see Fig. S1 in the
supplementary material). Members of the GalNAc-transferase
family are characterised by an N-terminal transmembrane domain
that retains the enzyme in the Golgi compartment, a central catalytic
domain and a C-terminal Ricin domain that facilitates binding to
partially glycosylated substrates (Fritz et al., 2006; Wandall et al.,
2007). The N-terminal transmembrane and the central catalytic
domain are highly conserved between Xenopus and human Galntl1 homologues (>80%), whereas the C-terminal Ricin domains are
more divergent, with only 40% amino acid identity. To test for Nacetylgalactosaminyltransferase activity, we used fluorescencecoupled agglutinin from Helix pomatia (HPA) that specifically binds
to GalNAc-α-Thr/Ser (Lehtonen et al., 1989; Virtanen, 1990; Hang
DEVELOPMENT
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 tissuespecific 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 (Galntl1) 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.
Development 135 (10)
Regulation of TGF-β by Galntl-1
RESEARCH ARTICLE 1815
et al., 2003). As expected, we were able to detect enhanced mucintype O-linked glycosylation in the Golgi compartment and at the cell
surface of xGalntl-1-expressing cells (Fig. 1B).
We identified Xenopus homologues of additional Nacetylgalactosaminyltransferases in the EST databases that share
significant homology with xGalntl-1. Maternal and zygotic
expression of xGalntl-1 and three additional Xenopus Nacetylgalactosaminyltransferases were detectable throughout early
development. However, only xGalntl-1 expression was restricted to
dorsal marginal zone explants at later developmental stages (Fig.
1C). At neurula stages, the xGalntl-1 transcript levels increased and
could be detected in the anterior mesoderm and the neural crest
territory (Fig. 1D). At tailbud stages, xGalntl-1 was specifically
expressed in the forebrain, mediolateral spinal cord, notochord and
in the neural crest cells that are migrating into the head region,
branchial arches and the heart anlage (Fig. 1E). The expression of
xGalntl-1 in the spinal cord was restricted to the mantle
(differentiating) territory of the spinal cord and was excluded from
the central area containing proliferating neuronal precursors. In
addition, the expression of xGalntl-1 in the spinal cord decreased
along the anterior-posterior body axis, whereas the expression in the
notochord was highest in posterior regions.
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 (α-actin) and ventral (α-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
DEVELOPMENT
Fig. 1. Sequence and expression of xGalntl-1. (A) Comparison of Xenopus Galntl-1, human GALNTL1 (hGalntl-1), zebrafish Galnt14 (zGalnt-14)
and Drosophila Galnt2 (dGalnt-2) protein sequences. The transmembrane domain (TM, blue), the catalytic domain (red), the Ricin domain (green)
and amino acid identities/similarities for the different protein domains are indicated. (B) Detection of mucin-type glycosylation at the cell surface
and in the Golgi compartment in xGalntl-1-transfected Cos-7 cells using Helix pomatia agglutinin (HPA) conjugated to Alexa Fluor 488. (C) RT-PCR
analysis of xGalntl-1, xGalnt-1, xGalnt-6 and xGalnt-7 expression, using RNA from whole embryos (left) or dorsal and ventral marginal zone
explants, isolated at gastrula stages and analysed at stage 20 (right). The blood marker α-globin, neural marker β-tubulin, dorsal mesodermal
marker chordin and the nieuwkoop centre-specific gene siamois were used as references for the indicated developmental stages. Dorsal (ncam,
myoD) and ventral (sizzled, msx1) marker genes were used as controls for the marginal zone explants. (D) Whole-mount in situ hybridisation
analysis of xGalntl-1 expression at stage 13. Numbered lines indicate the positions of vibratome sections that reveal xGalntl-1 expression in the
anterior mesoderm and in the deep layer of the lateral neural plate. (E) In situ analysis of xGalntl-1 expression at stage 27. The paraffin sections
below show specific expression of xGalntl-1 in the anterior brain (1), neural crest (2), mediolateral spinal cord (2-4) and notochord (3,4).
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
Fig. 2. Inhibition of mesoderm formation and Activin signalling
by xGalntl-1. (A) Stage 40 uninjected control Xenopus embryo and
(B) sibling embryos microinjected radially with 400 pg xGalntl-1 mRNA
at 2- to 4-cell stages. (C) In situ hybridisation for the pan-mesodermal
marker xbra in gastrula stage embryo microinjected either radially with
xGalntl-1 mRNA alone or into single blastomeres together with lacZ
mRNA at the 4-cell stage. (D) RT-PCR analysis of dorsal (DMZ) and
ventral marginal zone (VMZ) explants at stage 20. The expression of
α-globin (blood), gata1 and xvent1 (ventral mesoderm), msx1 (neural
crest, epidermis) was reduced in VMZ from embryos microinjected with
200 pg xGalntl-1 mRNA, whereas the expression levels of xag (cement
gland), otx2, rx2a (forebrain) and ncam (neural) were increased. The
expression of α-actin (muscle) was reduced in DMZ and slightly
increased in VMZ. (E) RT-PCR analysis for chordin (dorsal mesoderm),
sox17β (endoderm) and xbra (mesoderm) expression using stage 20
animal cap explants from embryos microinjected with mRNA encoding
eFGF (100 pg) or Activin (2 pg), in the presence or absence of 200 pg
xGalntl-1 mRNA. (F) Western blot analysis of Smad and phospho-Smad
proteins from HEK 293T cells that were transiently transfected with
Smad2-FLAG and xGalntl-1 expression vectors and cultured for 2 hours
in the absence or presence of 2ng/ml recombinant Activin protein.
(G) In vitro translation of xGalntl-1 mRNA containing (+5⬘-UTR) or
lacking (–5⬘-UTR) morpholino target sequences. Two different
morpholinos (Galntl-1-MO1 and Galntl-1-MO2, 2.5 μM) as well as a
mixture of both (1.25 μM each) specifically interfered with translation
of RNA containing the morpholino target sequences. (H) RT-PCR
analysis of animal cap explants at stage 20 from embryos microinjected
with 0.5 pg Activin mRNA, xGalntl-1 morpholinos (1 ng each) and 20
pg xGalntl-1 mRNA. The morpholino stimulated the induction of myoD
(muscle) and xbra expression by Activin. In all RT-PCR experiments, odc
served as a loading control.
Development 135 (10)
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).
To address the in vivo function of xGalntl-1, we designed two
specific morpholinos (Summerton, 1999; 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 xGalntl1 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
DEVELOPMENT
1816 RESEARCH ARTICLE
Regulation of TGF-β by Galntl-1
RESEARCH ARTICLE 1817
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.
xGalntl-1 is required for the differentiation of
neural and mesodermal tissue
Embryos microinjected with the specific xGalntl-1 morpholinos
developed with a smaller head and bent trunk. Paraffin sections
revealed that the brain and the spinal cord were reduced in size (Fig.
4A). The patterning of the neural tube is regulated by a dorsoventral
BMP activity gradient (Placzek and Briscoe, 2005; 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 xGalntl1 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.
DEVELOPMENT
Fig. 3. Inhibition of BMP signalling by Galntl-1. (A-D) Stage
36 embryos that were injected animally at the 8-cell stage with a
total of 2 ng of mRNA encoding xGalntl-1, human GALNTL1
(hGalntl-1) or secreted Galntl-1 (sGalntl-1), with the N-terminal
transmembrane domain replaced by the Chordin leader peptide.
(E) RT-PCR analysis of animal cap explants from embryos injected
with 2 ng xGalntl-1 or 40 pg chordin mRNA at stage 20 for
neural (pax6, ncam, otx2), anterior (xag), muscle (myf5) and
epidermal (cytokeratin) marker genes. (F) Western blot of the
supernatant (SN) and cell pellet of dissociated ectodermal cells
expressing the xGalntl-1 or sGalntl-1 with a C-terminal HA-TAG.
(G) RT-PCR analysis of animal cap explants from embryos
microinjected with mRNA encoding human, mouse (m) or
secreted forms of Galntl-1. (H) RT-PCR analysis of animal cap
explants from embryos microinjected with mRNA encoding
xGalntl-1 (1 ng) alone or in combination with mRNA encoding
BMP7/OP-1 (400 pg), dnFGFR-4 (400 pg), dnAlk-4 (400 pg) or
ΔN-TCF-3 (400 pg). (I) Western blot for Smad1 or phosphoSmad1 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 phosphoSmad1 or Smad1 using lysates from HEK 293T cells transiently
transfected with the indicated expression plasmids. In the RTPCR experiments α-actin or myf-5 served as controls for
mesoderm contamination; odc as a loading control.
Development 135 (10)
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).
A commonly used inhibitor of mucin-type glycosylation is
benzyl-N-acetyl-α-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.
Fig. 4. Xenopus Galntl-1 loss-of-function phenotype. (A) Stage 40
uninjected control embryo and embryo injected radially with the
xGalntl-1-specific morpholinos at the 2- to 4-cell stage. Numbered lines
indicate the positions of the paraffin sections shown on the right that
reveal a reduction of neural tissue in the morpholino-injected embryos.
(B) Uninjected control embryo and embryos microinjected with xGalntl1 morpholinos stained with the notochord-specific antibody MZ15 at
stage 40 (left) and analysed by in situ hybridisation for slug expression
at stage 30. The morpholino-injected embryos display defects in the
anterior notochord and reduced anterior expression of slug. (C) Wholemount in situ hybridisation for slug of stage 15 embryos after radial
microinjection of the xGalntl-1 morpholinos at the 2- to 4-cell stage,
and stage 13 embryo stained for msx1 after single injections of the
xGalntl-1 morpholinos into a dorsal-animal blastomere at the 8-cell
stage together with lacZ mRNA. (D) RT-PCR analysis of stage 20 dorsal
marginal zone explants from uninjected embryos or embryos
microinjected with xGalntl-1 morpholinos alone or together with
xGalntl-1 mRNA (20 pg). The expression of otx2 and six3 (forebrain) as
well as of snail (neural crest) was inhibited by the xGalntl-1
morpholinos, whereas the expression of dlx5 (epidermis), xvent2
(ventral mesoderm) and xbra (mesoderm) was unaffected.
Fig. 5. Inhibition of ActR-IIB by Galntl-1. (A) RT-PCR analysis of animal
cap explants analysed at stage 20 from embryos microinjected with 400
pg xGalntl-1 mRNA alone or together with 200 pg mRNA encoding
human ALK3, mouse Alk6, human BMPR-II or Xenopus ActR-IIB. (B) RTPCR analysis of stage 20 animal caps microinjected with 400 pg ActR-IIB
mRNA either alone or together with 1 ng xGalntl-1 mRNA. The
mesodermal marker genes xbra and wnt8 indicated ActR-IIB activity,
whereas otx2 and xag indicated xGalntl-1 activity, and xvent2 served as a
marker for ventral mesoderm and odc as a loading control. (C) RT-PCR
analysis of stage 20 animal caps microinjected with 40 pg ActR-IIB mRNA
alone, together with xGalntl-1 morpholinos, and xGalntl-1 morpholinos
and 100 pg xGalntl-1 mRNA. (D) Western blot analysis of Smad and
phospho-Smad levels from HEK 293T cells transiently transfected with
Smad1-FLAG, BMPR-II and xGalntl-1 (left) or Smad2-FLAG, ActR-IIB and
xGalntl-1 (right). (E) Western blot analysis of phospho-Smad2 and Smad2
levels from HEK 293T cells transiently transfected with Smad2-FLAG,
ActR-IIB and xGalntl-1 and incubated overnight in the presence or
absence of 2 mM benzyl-GalNAc. (F) Whole-mount in situ hybridisation
for msx1 using stage 13 embryos microinjected with xGalntl-1
morpholinos, 100 pg xActR-IIB mRNA and 100 pg xGalntl-1 mRNA into
dorsal-animal blastomeres at the 4- to 8-cell stage.
DEVELOPMENT
1818 RESEARCH ARTICLE
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-1depleted, 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.
Galntl-1 interferes with the formation of
heteromeric TGF-β receptor complexes
It has been reported that mucin-type O-linked glycosylation can
affect protein stability, intracellular protein trafficking and proteinprotein interactions (Hang and Bertozzi, 2005). 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 coexpression on ActR-IIB activity (Fig. 6A and data not shown). In
addition, western blot analysis of ActR-IIB, Alk-3, Alk-4 and Alk6 proteins expressed in animal cap explants or HEK 293T cells in
RESEARCH ARTICLE 1819
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 ActRIIB 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, Alk4 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 coimmunoprecipitated 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 ActRIIA (data not shown). By contrast, the binding of BMPR-II to Alk-
Fig. 6. Galntl-1 inhibits the binding of ActR-IIB to type I receptors. (A) Immunohistochemical staining of transiently transfected Cos-7 cells for
ActR-IIB containing a C-terminal HA-TAG and xGalntl-1 containing a C-terminal FLAG-TAG. (B) Western blot analyses of stage 11 Xenopus animal
cap cells expressing HA-tagged ActR-IIB, Alk-3, Alk-4 and Alk-6 and xGalntl-1. (C) Western blot analyses of transiently transfected HEK 293T cells
expressing HA-tagged xActR-IIB and xGalntl-1 and incubated overnight in the presence or absence of 2 mM benzyl-GalNAc. (D) Coimmunoprecipitation of HA-tagged Alk-3, Alk-4 and Alk-6 with MYC-tagged ActR-IIB in the presence or absence of FLAG-tagged xGalntl-1 from
lysates of transiently transfected HEK 293T cells. (E) Co-immunprecipitation of Alk-3 with Myc-tagged ActR-IIB or FLAG-tagged BMPR-II in the
presence or absence of untagged xGalntl-1. The binding of BMPR-II to Alk-3 does not seem to be inhibited by xGAlntl-1. (F) Co-immunprecipitation
of Alk-4-HA with ActR-IIB-Myc in the presence of xGalntl-1, sGalntl-1 or xGalnt-6. Only xGalntl-1 interfered with the formation of the heteromeric
complex. In all experiments, 10% of the lysates used for co-immunprecipitation was subjected to direct immunprecipitation and the quantity of
protein analysed by western blotting for the different epitopes.
DEVELOPMENT
Regulation of TGF-β by Galntl-1
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 Nacetylgalactosaminyltransferase 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 TGFβ receptor proteins that can mediate BMP as well as Nodal
signalling.
DISCUSSION
Negative regulation of TGF-β signalling by
Galntl-1
The TGF-β signal transduction pathway is regulated on various
levels of the cascade (Lutz and Knaus, 2002). 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 RSmad 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 Galntl1 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 Nacetylgalactosaminyltransferases 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
Development 135 (10)
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 msx1positive 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 ActRIIB 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 ActRIIB 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
DEVELOPMENT
1820 RESEARCH ARTICLE
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 TGFβ 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 ActRII/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 Nacetylgalactosaminyltransferase family target different components
of TGFβ signalling, providing a new paradigm for regulation in this
important pathway.
We thank Drs O. Wessely and F. Rentzsch for comments on the manuscript, Dr
Randy Cassada for critical reading and Drs L. Attisano, E. M. De Robertis and
R. Kemler for reagents. M.O. thanks Dr De Robertis for his invaluable guidance
and support. This work was supported by the Max-Planck Society.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/10/1813/DC1
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