The use of the zebrafish embryo as a reporter organism revealed that Cg-BMPR1, Cg-TGFbsfR2, Cg-ALR I, an activin Type I receptor or their dominant negative acting truncated forms, when ov
Trang 1repertoire of BMP receptor signal transducing proteins in the lophotrochozoan Crassostrea gigas suggests a shared ancestral BMP/activin pathway
Amaury Herpin1,2, Christophe Lelong2, Thomas Becker1, Frederic Rosa3, Pascal Favrel2
and Charles Cunningham1
1 Sars International Centre for Marine Molecular Biology, High Technology Centre, Bergen, Norway
2 Laboratoire de Biologie et Biotechnologies Marines, IBFA, Universite´ de Caen Basse-Normandie, IFREMER UMR 100, Physiologie et Ecophysiologie des mollusques marins, Caen, France
3 U 368 INSERM, Ecole Normale Supe´rieure, Paris, France
The genes governing mesoderm specification have been
extensively studied in vertebrates, arthropods and
nem-atodes The latter two phyla belong to the ecdysozoan
clade but little is understood of these molecules in the
other major protostomal clade, the lophotrochozoa
An increasing amount of comparative data from ecdysozoans as well as from vertebrates suggests that many of the proteins involved in mesodermal
Keywords
Crassostrea gigas; zebrafish; BMP;
TGF-beta; early embyogenesis
Correspondence
A Herpin, University of Wuerzburg,
Physiological Chemistry I, Am Hubland,
97074 Wuerzburg, Germany
Fax: +49 931888 4150
Tel: +49 931888 4165
E-mail: amaury.herpin@biozentrum.
uni-wuerzburg.de
(Received 15 April 2005, accepted 12 May
2005)
doi:10.1111/j.1742-4658.2005.04761.x
The transforming growth factor b (TGF-b) superfamily includes bone mor-phogenetic proteins, activins and TGF-b sensu stricto (s.s) These ligands, which transduce their signal through a heteromeric complex of type I and type II receptors, have been shown to play a key role in numerous biologi-cal processes including early embryonic development in both deuterostomes and ecdyzozoans Lophochotrozoans, the third major group of bilaterian animals, have remained in the background of the molecular survey of metazoan development We report the cloning and functional study of the central part of the BMP pathway machinery in the bivalve mollusc Cras-sostrea gigas(Cg-BMPR1 type I receptor and Cg-TGFbsfR2 type II recep-tor), showing an unusual functional mode of signal transduction for this superfamily The use of the zebrafish embryo as a reporter organism revealed that Cg-BMPR1, Cg-TGFbsfR2, Cg-ALR I, an activin Type I receptor or their dominant negative acting truncated forms, when over-expressed during gastrulation, resulted in a range of phenotypes displaying severe disturbance of anterioposterior patterning, due to strong modula-tions of ventrolateral mesoderm patterning The results suggest that Cg-BMPR1, and to a certain degree Cg-TGFbsfR2 proteins, function in
C gigas in a similar way to their zebrafish orthologues Finally, based on phylogenetic analyses, we propose an evolutionary model within the com-plete TGF-b superfamily Thus, evidence provided by this study argues for
a possible conserved endomesoderm⁄ ectomesoderm inductive mechanism in spiralians through an ancestral BMP⁄ activin pathway in which the singu-lar, promiscuous and probably unique Cg-TGFbsfR2 would be the shared type II receptor interface for both BMP and activin ligands
Abbreviations
BMP, bone morphogenetic protein; BMPR2, type II BMP receptors; TGF-b, transforming growth factor b.
Trang 2patterning are highly conserved with respect to both
structure and function, regardless of diversity and
evo-lution of body plans [1–5]
The transforming growth factor b (TGF-b)
super-family, which includes bone morphogenetic proteins
(BMPs), activin and activin-like proteins such as nodal
and their receptors, has been implicated in multiple
processes during animal development Members of the
TGF-b superfamily transduce signals through
hetero-meric complexes of ligand specific type I and II
ser-ine⁄ threonine kinase receptors [6] Type II receptors
are capable of binding ligand dimers alone, while type
I receptors can only bind ligands in cooperation with
type II receptors Ligand binding induces the
forma-tion of a heterotetracomplex in which the two type II
receptors unidirectionally transphosphorylate a dimer
of type I receptors Activated type I receptors in turn
catalyse the phosphorylation of receptor substrates,
the Smads Smad family members were originally
iden-tified through genetic screens in flies (mad Drosophila
mutants) and worms (sma Caenorhabditis mutants)
These move to the nucleus to associate with
transcrip-tional coactivators and regulate the transcription of
target genes [7] While this pathway is conserved for
most TGF-b superfamily ligands, including BMPs and
activin, nodal binds the activin specific type I receptor
and the cripto coreceptor to stimulate downstream
responses [8,9] In the absence of cripto, the type I
act-ivin receptor can mediate signal transduction
stimula-ted by activin but not nodal Mutations in the gene
encoding the mouse type IB activin receptor, ActRIB,
as well as the ActRIIA⁄ ActRIIB double mutants,
dis-play gastrulation defective phenotypes resembling
those of mouse nodal mutants [10–12]
In Drosophila, decapentaplegic (DPP), screw and a
third BMP ligand Gbb appear to share a common set
of receptors that include the type II receptor punt and
the type I receptors thick veins and saxophone
(reviewed in [13]) The activin type I receptor baboon
also signals in conjunction with punt, though the
acti-vin pathway appears to have little influence on
pattern-ing [14] While punt appears most closely related to the
vertebrate type II activin receptors, another receptor
(wishful thinking) has been identified that is
homolog-ous to the vertebrate type II BMP receptors (BMPR2)
Vertebrate BMPR2 receptors are the only ones that
bind BMP ligands exclusively, and in Drosophila
phe-notypes arising from mutations in the gene encoding
wishful thinking, wit, suggest a role for this protein in
synapse regulation and⁄ or maintenance [15,16]
As part of an ongoing project to understand the role
of the TGF-b superfamily ligands, their receptors and
signal transduction pathways in the lophotrochozoan
bivalve mollusc Crassotrea gigas, we report the cloning and functional study of the central part of the BMP pathway (the Cg-BMPR1 type I receptor and Cg-TGFbsfR2 type II receptor) This shows probably the most ancestral and unusual functional mode of sig-nal transduction for this superfamily, with a duplicate extracellular ligand binding domain TGFbsfR2 type II homologous receptor displaying a unique extracellular structure Because technical limitations relative to our model make direct functional studies difficult, we have tested whether Cg-BMPR1 and Cg-TGFbsfR2 mole-cules can function in the context of a vertebrate TGF-b superfamily signalling pathway by overexpressing them during zebrafish early embryogenesis The molecular nature of dorsoventral and anteroposterior patterning
in molluscs is discussed, in the context of Cg-BMPR1 and Cg-TGFbsfR2 expression patterns during C gigas early development
One piece of evidence from this study suggests that the molecular mechanisms controlling mesodermal pat-terning across all bilateria may be conserved through a complete, original and functional BMP⁄ activin path-way in lophotrochozoans, for which a singular and promiscuous type II receptor would be the shared interface for both BMP and activin ligands
Results
Type I and II TGFb superfamily receptor ortho-logues from C gigas
Four full length cDNA clones were obtained that encode orthologues of three type I and one type II TGFb superfamily receptor(s) from the oyster C gigas Clones encoding a type 1 activin-like receptor (Cg-ALR1: accession number AJ309316) as well as a TGFb sensu stricto type I-like receptor (Cg-TGFbR1: accession number CAD66433) have been described previously [17,18] These clones will not be discussed
in detail here, apart from within the phylogenetic and functional (Cg-ALR1) analyses of the receptor family The characteristics of the two remaining cDNA clones and the proteins their sequences infer are discussed below
A full length 1907 base pair cDNA clone containing
an open reading frame encoding 534 amino acids was isolated from a C gigas mantle edge library The pre-dicted protein contained a number of features charac-teristic of BMP type 1 receptors [19] The protein, named Cg-BMPR1, comprises a leader peptide, an extracellular domain containing 10 cysteines whose positions are conserved in comparison to those of vertebrate BMP type I receptors, and a CCX(5)CN
Trang 3cysteine knot preceding the transmembrane region
(Fig 1A) A glycine-serine domain (GS box) did not
follow the canonical SGSGSGLP consensus sequence
but rather was encoded by a SSGCGSGPP motif The
remaining intracellular catalytic domains are highly
conserved Membership of Cg-BMPR1 to the BMP
type 1 receptor subfamily is clearly established by the
sequence of the L45 loop kinase domain The
Cg-BMPR1 L45 loop sequence differs from the
canon-ical sequence (ASDIKGT⁄ NGSW) by only a single
residue (underlined) This motif plays an important
role across phyla in determining the specificity of type
I receptors for Smad proteins [20] The gene and
inferred protein sequence of Cg-BMPR1 has been
lodged in the GenBank database with the accession
number AJ577293
The second full length cDNA clone encoded a
pro-tein with an expected length of 1174 amino acids The
inferred protein sequence bore most resemblance to
TGF-b superfamily type 2 receptors and was thus
named Cg-TGFbsfR2 Interestingly, the extracellular
domain of Cg-TGFbsfR2 is structurally divergent from
all other type II receptors that have been described
previously Uniquely, it contains two extracellular
domains that we have named C1 and C2 C1 defines
the domain closest to the amino terminal end and C2
defines the domain closest to the cell membrane A
comparison of the inferred amino acid sequence of the
C1 and C2 domains reveals that only the approximate
spacing of the 10 cysteine residues is conserved
(Fig 1B) Both domains contain a typical BMP⁄ activin
type II receptor CCCX(4)CN cysteine knot at their
N-terminal ends (Fig 1B) Phylogenetic analyses
com-paring the extracellular domains of BMP⁄ activin type
II receptors, C gigas C1 and C2 regions, as well as
sequences from sponge receptors for which such
extra-cellular dupli⁄ triplications are observed, showed that
Cg-TGFbsfR2 C1 and C2 domains were not clustering
together but were branching at the root defining BMP
and activin type II receptor clades (Fig 2A) This
characteristic was also shared with the duplicated
domains of the sponge Ephydatia fluviatilis ALK-6
type II receptor (Fig 2A) The C1 and C2 domains
were not directly adjacent but were joined by a linker
sequence The intracellular kinase domain conforms to
the canonical sequence of serine⁄ threonine protein
kin-ase domains seen for these receptors, and exhibits a
singularly long C-terminal extension similar to many
BMP type 2 receptors [21]
A more general phylogenetic tree was generated
using a conserved kinase cytoplasmic protein sequence
of all four C gigas receptors together with selected
protostome and deuterostome TGF-b superfamily
receptor orthologues (Fig 2B) BMPR1 and Cg-TGFbsfR2 cluster reliably with BMP type I and II receptors, respectively, and are closely associated with the Drosophila orthologues thick vein and wishful think-ing, respectively Cg-ALR1 and Cg-TGFbR1 were most closely related to activin and TGF-b type I recep-tors, respectively
The intron–exon organization of the genes encoding Cg-BMPR1 and Cg-TGFbsfR2 is shown in Fig 2C The serine⁄ threonine kinase domain in both proteins is encoded by two exons equivalent to kinase subdomains
X and XI [22] In addition, the GS box and L45 loop
of Cg-BMPR1, as well as the C-terminal extension
of Cg-TGFbsfR2, are encoded by unique exons The C1 and C2 domains of the extracellular region of Cg-TGFbsfR2 are each encoded by one or two exons Interestingly these two domains are separated by a short linker encoded by its own exon (Figs 1B and 2B) Both genes show high levels of phase conservation
in comparison to other oyster TGFb superfamily re-ceptors as well as orthologous rere-ceptors from other species (data not shown)
Expression patterns of Cg-BMPR1 and Cg-TGFbsfR2 in adult tissues, during early embryogenesis and larval development The early origin and high degree of conservation of TGF-b signalling protein orthologues during animal evolution from radiata to highly evolved bilateria sug-gest that they are involved in key biological processes common to most metazoans [23] To gain insight into
a possible physiological role of Cg-BMPR1 and Cg-TGFbsfR2, temporal gene expression patterns in early larval developmental stages and adult tissues were investigated using real time quantitative PCR (Fig 3) mRNAs from adult tissues (haemocytes, man-tle edge, adductor muscle, digestive tract, gills, heart and labial palp) including female gonads (oocytes), and from various stages of embryonic and larval devel-opment (blastula, gastrula, trochophore larvae, D lar-vae, 7 and 14 days post fertilization larlar-vae, pediveliger larvae and metamorphosing larvae) were used as sam-ples Although Cg-BMPR1 and Cg-TGFbsfR2 tran-scripts were ubiquitously expressed at reasonable levels
in all adult tissues, interestingly Cg-BMPR1 transcripts were always expressed about 10-fold more than Cg-TGFbsfR2 basal levels (respectively around 0.1 and 0.01 copies per 1 copy of GAPDH; Fig 3B) Remark-ably, this propensity is even more pronounced (up to
100 fold) when considering embryonic and larval development (Fig 3A) Then, Cg-BMPR1 was around 10-fold more expressed during early development
Trang 4B
Fig 1 Deduced amino acid sequence of
Cg-BMPR1 and Cg-TGFbsfR2 (A) The
implied amino acid sequence of Cg-BMPR1
contains a leader peptide shown in italics.
Cysteine residues characteristic of the TGFb
superfamily type I receptors are in bold and
underlined, and the cysteine knot is boxed.
Also boxed are the transmembrane domain,
the ATP binding site, the L45 loop and the
serine ⁄ threonine kinase domain (B) The
implied amino acid sequence of
Cg-TGFbsfR2 contains a leader peptide
shown in italics Two extracellular domains
were present in Cg-TGFbsfR2 The first (C1)
contained 10 cysteine residues whose
spa-cing was characteristic of TGFb superfamily
type II receptors These are in bold and
underlined, and the cysteine knot is boxed.
The second extracellular domain (C2) also
contained 10 cysteines and these are also
shown in bold and the cysteine knot is
boxed The C1 and C2 domains appeared to
be joined by a ‘linker’ sequence Also boxed
are the transmembrane domain and the
serine ⁄ threonine kinase domain.
Trang 5Daf-1 C elegans
Crassostrea gigas C2 domain
ALK-6 E fluviatilis C2 domain
Crassostrea gigas C1 domain
Wit D melanogaster
Punt D melanogaster ActR-2b H sapiens ActR-2b D rerio ActR-2b C auratus BMPR-2 X laevis BMPR-2 H sapiens Daf-4 C elegans ALK-6 E fluviatilis C1 domain
ALK-4 E fluviatilis C1 domain ALK-4 E fluviatilis C2 domain
Activin
BMP
100
100 100 92
100 94
79
74
88 77
69 76
64
Daf-4 C elegans
ActR-2b D rerio ALK-6 E fluviatilis
ALK-1 E fluviatilis C32D5.2(actr) C elegans
Crassostrea gigas
Cg-BMPR-1 Crassostrea gigas
Wit D melanogaster
ALK-4 E fluviatilis
Cg-ALR1 Crassostrea gigas
Saxophone D melanogaster ALK-8 D rerio Acvrl-1 D rerio ALK-1 H sapiens Hr-BMPR H roretzi
Cg-TβR-1Crassostrea gigas
Baboon (AtR-I) D melanogaster
TβR1 H sapiens
TARAM D rerio STKR1 T rubripes Thick vein D melanogaster
Punt D melanogaster
TGFR B pahangi STPK A caninum
ALK-2 E fluviatilis
ActR-2b C auratus
TβR-II H sapiens
TβR-II G gallus
TβR-IIa X laevis
BMPR-2 X laevis BMPR-2 H sapiens ActR-2b H sapiens
Daf-1 C elegans
BMP-RIa H sapiens BMP-RIa D rerio BMP-RIb D rerio
Raf D melanogaster (out group) B-raf H sapiens (out group)
Activin
TGF-Activin
BMP
BMP
TGF-100 99
100
100 100
100
100
100
98 100
96
88
87 83
91 86 69
86
86 62
87 95 77
82 71
79
95 78
66 87
87
58 62
78
97
A
B
Fig 2.
Trang 6(1.2 Cg-BMPR1 copies for 1 of GAPDH in oocytes)
when compared to adult tissues Cg-BMPR1
expres-sion steadily dropped during early and larval
develop-ment After D larvae stage and up to metamorphosis,
expression levels returned to the basal adult state
(0.1–0.15 copies relative to GAPDH) Although
Cg-TGFbsfR2 transcripts were only detected at moderate
levels, two peaks of expression were observed, the first
during gastrulation and the second just before
meta-morphosis In all cases, Cg-TGFbsrR2 average
expres-sion level is around 10-fold lower than Cg-BMPR1
when referring to adult tissues
Cg-BMPR1 transduces a ventralizing signal
during zebrafish mesoderm induction
To determine whether Cg-BMPR1 and Cg-TGFbsfR2
are able to function in a manner similar to their
ortho-logues, we employed the zebrafish embryo as a ‘repor-ter organism’ Specifically, we wished to analyse how expression of these two molecules was able to perturb the TGFb superfamily ligand–receptor signalling path-way during zebrafish early development Examples of the range and severity of the phenotypes recorded in the following experiments are shown in Fig 4
Injection of 5–200 pg per embryo of full length Cg-BMPR1 transcript produced a range of ventralized embryos (Figs 4A and 5A) Whole mount in situ hybridization showed that tbx6 expression was expan-ded towards the dorsal part of the embryo while gsc expression was slightly reduced and not seen ectopi-cally (Fig 6B1–4) When mRNA encoding a truncated version of Cg-BMPR1 (DN-Cg-BMPR1) was injected,
a range of dorsalized embryos was observed at increas-ing concentrations (Figs 4B and 5B) The tbx6 and gsc expression patterns were congruent with these
observa-Cg-ALR1
(6Kb, 6 introns)
0 1 1 1 1 1 1
0 1
Cg-TGFβR1
(10,5Kb, 9 introns)
Cg-BMPR1
Cg-TGFβsfR2
(>15Kb, 9 introns)
0 1
0
Linker
GS Box
I
Extracellular Domain TM L45 loop
Domain
GS
1
TGF β type 1 receptor
prototype
C
Fig 2 (A) Phylogenetic relationship of the extracellular domain of TGFb superfamily type II receptors Sequences used for the alignment of extracellular parts were truncated to strictly embed the 10 conserved cysteines upstream of the characteristic activin ⁄ BMP cysteine knot CCCX(4)C Split duplicated extracellular domains are reported as C1 and C2 domains from the N-terminal part of the protein This tree was generated by using CLUSTAL X From this alignment a distance-based phylogenetic tree was constructed using the minimum evolution method
of the PAUP package The percentage recovery of the branch in 1000 bootstrap replications is indicated ActR2b Carassius auratus (ABB58749)Daf-1 Caenohabditis elegans (P20792), Daf-4 Caenohabditis elegans (P50488), Cg-TGFbsfR2 C gigas (CAD20574), ActR-2b Danio rerio (NP_571285), Punt Drosophila melanogaster (AAC41566), Wishful thinking D melanogaster (AAF60175), ALK-4 Ephydatia fluvatilis (AB026827), ALK-6 E fluvatilis (AB026829), ActR-2b Homo sapiens (NP_001097), BMPR-2 H sapiens (NP_001195), BMPR-2 Xenopus laevis (AAB39883) (B) Phylogenetic tree showing the relationship of Cg-ALR1, Cg-BMPR1, Cg-TGFbR1 and Cg-TGFbsfR2 to other TGFb superfamily ligand receptors This tree was generated by using the alignment in CLUSTAL X From this alignment a distance-based phylogenetic tree was constructed using the minimum evolution method of the PAUP package The percentage recovery of the branch in 1000 bootstrap repli-cations is indicated STPK Ancylostoma caninum (AAL06642), TGFR Brugia pahangi (ACC47801), C32D5.2(Actr) Caenohabditis ele-gans (NP_495271), Daf-1 Caenohabditis eleele-gans (P20792), Daf-4 Caenohabditis eleele-gans (P50488), ActR2b Carassius auratus (ABB58749), Cg-ALR1 Crassostrea gigas (AJ309316), Cg-TbR1 C gigas (AJ544074), Cg-BMPR1 C gigas (CAE11917), Cg-TGFbsf2 C gigas (CAD20574), ActR-2b Danio rerio (NP_571285), Acvrl-1 Danio rerio (AAM53074), ALK-8 Danio rerio (NP_571420), RIa Danio rerio (BAA32748), BMP-RIb Danio rerio (BAA76408), TARAM Danio rerio (NP_571065), Baboon (Atr-I) Drosophila melanogaster (A55921), Punt D melanogaster (AAC41566), Saxophone D melanogaster (I45712), Thick vein D melanogaster (XP_079689), Wishful thinking D melanogaster (AAF60175), ALK-1 Ephydatia fluvatilis (BAA82601), ALK-2 E fluvatilis (BAA82602), ALK-4 E fluvatilis (AB026827), ALK-6 E fluvatilis (AB026829), TbR-II Gallus gallus (I50429), HrBMPR Halocynthia roretzi (BAB87725), ActR-2b Homo sapiens (NP_001097), ALK-1 H sapiens (CAA80255), BMP-RIa H sapiens (NP_004320), BMPR-2 H sapiens (NP_001195), TbR1 H sapiens (P36897), TbR-II H sapiens (P37173), STKR1 Takifugu rubripes (AAC34382), BMPR-2 X laevis (AAB39883), TbR-IIa X laevis (AAG40577) Outgroups: Raf D melanogaster (X07181), B-raf H sap-iens (M95712) (C) Exon structure and domain organization of TGFb superfamily type I and II receptor genes The intron phase (0, 1 or 2) is indicated above each intron–exon boundary Boxes I, X and XI are representative of kinase subdomains [22] Extracellular and transmem-brane (TM) domains are also shown The L45 loop and the GS box are specific to type I receptors The type II receptor contains two extra-cellular domains (C1 and C2) joined by a linker sequence C1 and the linker are encoded by single exons, C2 by two exons.
Trang 7tions: tbx6 was considerably repressed while gsc was
up-regulated and ectopically expressed throughout the
embryo (Fig 6C1–4)
Expression of Cg-ALR1 results in both posterior
and anterior defects in zebrafish embryos
Expression of Cg-ALR1 resulted in a dose-dependent
range of anterior defects, including in some embryos a
lack of otic vesicles (Fig 7A,B) These defects were
always combined with mild posterior defects In
addi-tion, a significant fraction of the Cg-ALR1 injected
embryos (between 5 and 10% depending on the
mRNA concentration) displayed a bifida chordata
phenotype in combination with severe anterior defects
(Fig 7A,Bc) Cg-ALR1 expression resulted in an
expression of gsc in ventral regions of the embryo
(Fig 6D3,D4) The expression domain of tbx6 was
restricted to the ventral regions and fragmented at the
gastrula stage (Fig 6D1,D2)
When mRNA encoding a truncated version of
Cg-ALRI (DN-Cg-ALR1) was injected at a range of
2–400 pg per embryo, posterior structure defects were observed in a dose-dependent manner (Fig 7C,D) The tbx6 expression pattern was restricted to the ventral side (Fig 6E1,E2) while gsc expression in the dorsal mesoderm was almost completely abolished (Fig 6E3,E4)
Cg-TGFbsfR2 transduces a dorsalizing signal during zebrafish mesoderm induction When injected at concentrations of between 10 and
200 pg per embryo, Cg-TGFbsfR2 induced dorsaliza-tion in a concentradorsaliza-tion-dependent manner (Figs 4B and 8A) Expression of tbx6 was dramatically repres-sed, even if in some cases its expression at the mar-gin of the blastoderm was expanded (Fig 9A1,A2) Expression of gsc was clearly expanded in all cases (Fig 9A3,A4) mRNA encoding a truncated Cg-TGFbsfR2 (DN-Cg-Cg-TGFbsfR2) was generated by inserting a stop codon at the C-terminal side of the transmembrane domain This protein included both extracellular C1 and C2 domains as well as the
Cg-BMPR1
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
s t y O
a l u t s l B
a l u r s G e r o o r T
.
r a D f p
r e il e V
L f p 1
L r e il e V r e il e i d P
L
a t e M m
h r o o
L g i s
0 0.001 0.002 0.003 0.004 0.005 0.006 0.007
s t y O
a l u t s l B
a l u r s G e r o o r T
.
r a D
L r e il e V f
L r e il e V r e il e i d P
L
a t e M m h r o o
L g i s
0
0.05
0.1
0.15
0.2
0.25
Cg-BMPR1
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04
A
B
Fig 3 Differential Cg-BMPRI and Cg-TGFbsfR2 expression patterns during early development (A) and in adult tissues (B) measured by real
time quantitative RT-PCR Each value is the mean ± SE of three animals (tissues) or the mean of a pool of embryos or larva (L) from one spawn ME, mantle edge; DG, digestive gland; LP, labial palps; PAM, posterior adductor muscle; G, gills; He, heart; H, haemocytes The rel-ative level of receptor expression was calculated for one copy of the GAPDH housekeeping gene by using the following formula: N ¼
1 · 2(Ct GAPDH – Ct target).
Trang 8transmembrane region Ectopic expression of this
protein led to phenotypes comparable to those
obtained after injection of truncated Cg-BMPR1
(Figs 8B and 4B) At concentrations of up to 100 pg
mRNA per embryo, the range of dorsalization
observed (Figs 4B and 8B) was comparable with that
obtained after Cg-TGFbsfR2 injection D4 and D5
phenotypes were observed solely with concentrations
of 200 pg mRNA per embryo (Figs 4B and 8B) As
was observed with full length Cg-TGFbsfR2 mRNA,
when the truncated protein was expressed tbx6 was
repressed even if in some cases its expression at
the margin of the blastoderm was expanded
(Fig 9B1,B2) In all cases gsc expression was clearly
expanded (Fig 9B3,B4)
The C1 and C2 domains of TGFbsfR2 have different ligand binding properties
Although the C1 binding domain of Cg-TGFbsfR2 clustered with its BMP type II Drosophila wit homo-logous receptor (Fig 2A), phylogenetic analyses suggested that both C1 and C2 were unspecified (Fig 2A) To clarify the binding properties of each of these domains we generated synthetic mRNA encoding only the C1 or C2 domains Expression of only the C1 domain resulted in the dorsalization of embryos in a concentration-dependent manner (Fig 8C) Similarly, tbx6 and gsc expression (Fig 9C1–4) were repressed and expanded, respectively, through dorso–ventral ter-ritories Although the majority of embryos expressing the C2 domain exhibited weakly dorsalized phenotypes (Figs 8D and 4C), a sizeable minority were weakly ventralized and showed the bifida chordata phenotype
in a manner similar to those obtained after Cg-Tolloid,
a C gigas Tolloid-like orthologue; (A Herpin et al., unpublished results), injections but with additional anterior defects It is not clear from the tbx6 and gsc expression patterns observed in these embryos whether they are dorsalized or ventralized (Fig 9D1–4)
Ventralized embryos
24 Hpf
A
Dorsalized embryos
24 Hpf
B
Fig 4 The range of zebrafish phenotypes observed on
overexpres-sion of TGF-b superfamily receptors (A) Examples showing the
ven-tralized phenotypes obtained after overexpression of Cg-BMPR1.
These phenotypes ranged from the least (Vt1) to the most severe
(Vt3) (B) Examples showing the dorsalized phenotypes obtained
after overexpression of intact and truncated Cg-TGFbsfR2 and its C1
and C2 domains and truncated Cg-BMPR1 These phenotypes
ran-ged from the least (D1) to the most severe (D5).
0 25 50 75 100
2 10 50 100 200
D4 D5 D3 D2 D1 Normal
DN-Cg-BMPR1
n=195 n=209 n=167 n=114 n=182
pg/embryo
Proportion of embryos (%)
Phenotypes
Vt3 Vt2 Vt1 Normal
5 50 100 150 200
0 25 50 75 100
Cg-BMPR1
n=77 n=96 n=80 n=73 n=101
pg/embryo
Proportion of embryos (%)
Phenotypes
A
B
Fig 5 Histograms showing the phenotype distribution after over-expression of (A) Cg-BMPR1 and (B) truncated Cg-BMPR1 (DN-Cg-BMPR1) The proportion of embryos showing an individual phenotype is indicated by colour The number of embryos injected for each concentration of mRNA is indicated above each bar of the histograms.
Trang 9We have described the cloning and functional analyses
of three TGFb superfamily type I and one type II
receptor orthologues These are the first molecules of
this kind to be identified in lophotrochozoans Below
we discuss some of the questions that arise from our
experiments and their analysis
Did the evolution of TGF-b superfamily receptors
occur episodically or gradually?
Phylogenetic analysis of TGFb superfamily receptors
shows them to be clearly divided into two major
clus-ters, containing either type I or type II receptors Each
cluster is further divided into individual clades contain-ing TGF-b sensu strico (s.s.), activin or BMP receptors This observation is congruent, structurally, with the subfamilies already defined by the ligands and suggests
a concerted evolution between ligands and receptors [23] According to the phylogenetic tree shown in Fig 2B, type (I or II) and subtype (TGF-b s.s., activin
or BMP) duplications that gave rise to all known types and subtypes, would predate the divergence between parazoans–eumetazoans and protostomes–deutero-stomes for types and most subtypes, respectively In addition, although the observation of a second extra-cellular domain in the Cg-TGFbsfR2 receptor is unique among protostome and deuterostome type II receptors, multiple extracellular domains are observed
80% epiboly
tbx6
B-80% epiboly
goosecoid
E2 E1
A-D2
Fig 6 In situ hybridization of zebrafish embryos using the ventro-lateral mesoderm marker tbx6 and the dorsal mesoderm marker goosecoid
at 80% epiboly Two examples are shown for each group and each marker Changes in the localization of the tbx6 expression pattern is highlighted DN (dominant negative) indicates that truncated receptor was overexpressed in these experiments.
Trang 10among sponge (parazoan) molecules [24] At this time,
the basic receptor repertoire may have already
consis-ted of BMP and activin type I and II receptors [24]
The type II TGFb s.s receptor has only been identified
in deuterostomes It may therefore have been lost
during protostome evolution or alternatively have been
acquired during the gene explosion that occurred prior
to the emergence of the chordates Finally, detailed
phylogenetic analyses of duplicated extracellular
domains (Fig 2A) observed in sponges and C gigas
receptors showed them to be more closely related to
other extracellular domains than to their duplicated
counterpart, suggesting a very early duplication event,
probably before the one that gave rise to BMP and
activin subtypes
The gene organization of TGF-b superfamily type I
and II receptors suggests evolution by exon
shuffling
Type I and II TGF-b superfamily receptor gene
organ-ization across protostomes and deuterostomes reveals
that domain distribution among exons is conserved
Usually, intron boundaries do not interrupt functional domains Indeed, for both type I and II receptors, both the phase and position of the exon boundaries of the core kinase domain are highly conserved suggesting that these genes were derived from a common ances-tral kinase gene that encoded sections X and XI of the receptor serine⁄ threonine kinase domain [22] Another striking point is the phase conservation of the intron– exon boundaries that lie either side of the exon enco-ding the extracellular domain This lends support to the theory that the ligand specificity of these receptors may have been achieved by exon shuffling
Cg-BMPR1 and Cg-TGFbsfR2 transcripts are maternally supplied and make the oyster early embryo susceptible to respond to a BMP/activin signal
In many animal species members of the TGF-b super-family of growth factors play a crucial role in specific developmental events [25,26] During early embryo-genesis both Cg-BMPR1 and, to a certain degree, Cg-TFGbsfR2 show an apparent accumulation in
C
A1
A2
A3
A4
P1
P2
P3
Bc A
DN-Cg-ALR1
0 25 50 75 100
dead A4
A2 A1 Normal
10 50 100 200 400pg/embryo
n=177 n=250 n=207 n=197 n=218
D
Phenotypes
Proportion of embryos (%)
0 25 50 75 100
P3 P2 P1 Normal Bc
Cg-ALR1
n=99 n=123 n=108 n=99 n=118
pg/embryo
B
Proportion of embryos (%)
Phenotypes
Fig 7 Distribution of phenotypes after
over-expression of Cg-ALR1 and its truncated
form DN-Cg-ALR1 (A) A dose-dependent
range of anterior defects (P1-3) combined
with mild posterior defects was observed
after overexpression of Cg-ALR1 A
signifi-cant fraction of the embryos also displayed
a bifida chordata (Bc) phenotype in
combina-tion with severe anterior defects (B)
Histo-gram showing the proportion of embryos
displaying each phenotype after injection
with Cg-ALR1 The number of embryos
injected for each experiment is also
indica-ted (C) A dose-dependent range of range of
posterior defects (A1-4) was observed after
overexpression of truncated Cg-ALR1
(DN-Cg-ALR1) (D) Histogram showing the
proportion of embryos displaying each
phenotype after injection with DN-Cg-ALR1.
The number of embryos injected for each
experiment is also indicated.