Structural and functional evidence for a singular repertoire 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 Sars International Centre for Marine Molecular Biology, High Technology Centre, Bergen, Norway ´ Laboratoire de Biologie et Biotechnologies Marines, IBFA, Universite de Caen Basse-Normandie, IFREMER UMR 100, Physiologie et Ecophysiologie des mollusques marins, Caen, France ´ U 368 INSERM, Ecole Normale Superieure, Paris, France 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 morphogenetic 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 biological 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 Crassostrea gigas (Cg-BMPR1 type I receptor and Cg-TGFbsfR2 type II receptor), 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 overexpressed during gastrulation, resulted in a range of phenotypes displaying severe disturbance of anterioposterior patterning, due to strong modulations 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 complete 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 singular, promiscuous and probably unique Cg-TGFbsfR2 would be the shared type II receptor interface for both BMP and activin ligands The genes governing mesoderm specification have been extensively studied in vertebrates, arthropods and nematodes 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 Abbreviations BMP, bone morphogenetic protein; BMPR2, type II BMP receptors; TGF-b, transforming growth factor b 3424 FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS A Herpin et al patterning are highly conserved with respect to both structure and function, regardless of diversity and evolution of body plans [1–5] The transforming growth factor b (TGF-b) superfamily, 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 heteromeric complexes of ligand specific type I and II serine ⁄ 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 formation 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 identified through genetic screens in flies (mad Drosophila mutants) and worms (sma Caenorhabditis mutants) These move to the nucleus to associate with transcriptional 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 activin receptor can mediate signal transduction stimulated 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, display 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 activin pathway appears to have little influence on patterning [14] While punt appears most closely related to the vertebrate type II activin receptors, another receptor (wishful thinking) has been identified that is homologous to the vertebrate type II BMP receptors (BMPR2) Vertebrate BMPR2 receptors are the only ones that bind BMP ligands exclusively, and in Drosophila phenotypes 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 FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS BMP/activin pathway in Crassostrea gigas 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 signal 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 molecules 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 patterning across all bilateria may be conserved through a complete, original and functional BMP ⁄ activin pathway 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 orthologues 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 activin-like receptor (CgALR1: 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 predicted protein contained a number of features characteristic of BMP type 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 3425 BMP/activin pathway in Crassostrea gigas cysteine 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 receptor subfamily is clearly established by the sequence of the L45 loop kinase domain The Cg-BMPR1 L45 loop sequence differs from the canonical 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 protein with an expected length of 1174 amino acids The inferred protein sequence bore most resemblance to TGF-b superfamily type 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 comparing 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 extracellular 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 kinase domains seen for these receptors, and exhibits a singularly long C-terminal extension similar to many BMP type 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 3426 A Herpin et al receptor orthologues (Fig 2B) Cg-BMPR1 and CgTGFbsfR2 cluster reliably with BMP type I and II receptors, respectively, and are closely associated with the Drosophila orthologues thick vein and wishful thinking, respectively Cg-ALR1 and Cg-TGFbR1 were most closely related to activin and TGF-b type I receptors, 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 receptors as well as orthologous receptors 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 suggest 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, mantle edge, adductor muscle, digestive tract, gills, heart and labial palp) including female gonads (oocytes), and from various stages of embryonic and larval development (blastula, gastrula, trochophore larvae, D larvae, and 14 days post fertilization larvae, pediveliger larvae and metamorphosing larvae) were used as samples Although Cg-BMPR1 and Cg-TGFbsfR2 transcripts were ubiquitously expressed at reasonable levels in all adult tissues, interestingly Cg-BMPR1 transcripts were always expressed about 10-fold more than CgTGFbsfR2 basal levels (respectively around 0.1 and 0.01 copies per copy of GAPDH; Fig 3B) Remarkably, 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 FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS A Herpin et al BMP/activin pathway in Crassostrea gigas A B Fig 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 CgTGFbsfR2 contains a leader peptide shown in italics Two extracellular domains were present in Cg-TGFbsfR2 The first (C1) contained 10 cysteine residues whose spacing 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 FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS 3427 BMP/activin pathway in Crassostrea gigas A Herpin et al A Crassostrea gigas C2 domain ALK-6 E fluviatilis C2 domain 77 Crassostrea gigas C1 domain 76 88 Wit D melanogaster ActR-2b H sapiens 92 Activin 100 64 ActR-2b D rerio 100 94 ActR-2b C auratus T ype II r ecept or s Punt D melanogaster 69 BMPR-2 X laevis BMP 100 BMPR-2 H sapiens Daf-4 C elegans 79 Ty pe I re c eptors ALK-6 E fluviatilis C1 domain Daf-1 C elegans 74 ALK-4 E fluviatilis C1 domain 100 ALK-4 E fluviatilis C2 domain B ALK-4 E fluviatilis Cg-ALR1 Crassostrea gigas Saxophone D melanogaster 96 62 99 88 Activin ALK-8 D rerio Acvrl-1 D rerio 100 Hr-BMPR H roretzi Cg-TβR-1 Crassostrea gigas Baboon (AtR-I) D melanogaster 86 69 TβR1 H sapiens 91 86 TGF- TARAM D rerio 100 100 Type I receptors ALK-1 H sapiens 86 STKR1 T rubripes Cg-BMPR-1 Crassostrea gigas 87 Thick vein D melanogaster 83 BMP-RIb D rerio 87 BMP BMP-RIa D rerio 100 100 BMP-RIa H sapiens ALK-6 E fluviatilis Daf-1 C elegans 62 Daf-4 C elegans Crassostrea gigas 58 77 Wit D melanogaster Punt D melanogaster 78 ActR-2b H sapiens 87 ActR-2b D rerio 95 100 82 87 BMPR-2 X laevis 79 100 Activin ActR-2b C auratus BMPR-2 H sapiens BMP TβR-IIa X laevis TβR-II G gallus 97 100 TGF- T ype II re cep tors 71 TβR-II H sapiens TGFR B pahangi STPK A caninum 78 95 66 C32D5.2(actr) C elegans ALK-1 E fluviatilis 98 ALK-2 E fluviatilis Raf D melanogaster (out group) B-raf H sapiens (out group) Fig 3428 FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS A Herpin et al BMP/activin pathway in Crassostrea gigas C Cg-ALR1 (6Kb, introns) Cg-TGFβR1 (10,5Kb, introns) Cg-BMPR1 (10Kb, 10 introns) 1 1 1 2 1 1 GS GS Box TGFβ type receptor prototype Extracellular Domain TM Domain I X L45 loop XI Cg-TGFβsfR2 (>15Kb, introns) C1 C2 1 TM Domain 1 Linker Fig (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 replications is indicated STPK Ancylostoma caninum (AAL06642), TGFR Brugia pahangi (ACC47801), C32D5.2(Actr) Caenohabditis elegans (NP_495271), Daf-1 Caenohabditis elegans (P20792), Daf-4 Caenohabditis elegans (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), BMP-RIa Danio rerio (BAA32748), BMPRIb 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 sapiens (M95712) (C) Exon structure and domain organization of TGFb superfamily type I and II receptor genes The intron phase (0, or 2) is indicated above each intron–exon boundary Boxes I, X and XI are representative of kinase subdomains [22] Extracellular and transmembrane (TM) domains are also shown The L45 loop and the GS box are specific to type I receptors The type II receptor contains two extracellular domains (C1 and C2) joined by a linker sequence C1 and the linker are encoded by single exons, C2 by two exons (1.2 Cg-BMPR1 copies for of GAPDH in oocytes) when compared to adult tissues Cg-BMPR1 expression steadily dropped during early and larval development 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 CgTGFbsfR2 transcripts were only detected at moderate levels, two peaks of expression were observed, the first during gastrulation and the second just before metamorphosis In all cases, Cg-TGFbsrR2 average expression 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 orthoFEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS logues, we employed the zebrafish embryo as a ‘reporter organism’ Specifically, we wished to analyse how expression of these two molecules was able to perturb the TGFb superfamily ligand–receptor signalling pathway during zebrafish early development Examples of the range and severity of the phenotypes recorded in the following experiments are shown in Fig 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 expanded towards the dorsal part of the embryo while gsc expression was slightly reduced and not seen ectopically (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 increasing concentrations (Figs 4B and 5B) The tbx6 and gsc expression patterns were congruent with these observa3429 BMP/activin pathway in Crassostrea gigas A A Herpin et al 0.4 0.2 yt es B la st ul a G as Tr tr ul oc a op ho re L D La 7d rv pf ae Ve lig 14 er dp L fV el ig er Pe L di M ve et lig am er or L ph os in g L B 0.004 0.003 0.002 0.001 Cg-BMPR1 Cg-TGFβsfR2 0.04 number of copies of Cg-TGFβsfR2 per copy of GAPDH 0.25 number of copies of Cg-BMPR1 per copy of GAPDH 0.005 st ul a G as Tr tr ul oc a op ho re L D La 7d rv pf ae Ve li g 14 er dp L fV el ig Pe er di L M ve et li g am er or L ph os in g L 0.8 0.6 0.006 yt es 1.0 0.007 B la 1.2 O oc 1.4 number of copies of Cg-TGFβsfR2 per copy of GAPDH Cg-TGFβsfR2 O oc number of copies of Cg-BMPR1 per copy of GAPDH Cg-BMPR1 0.2 0.15 0.1 0.05 0.035 0.03 0.025 0.02 0.015 0.01 0.005 H ME PAM DG G He LP H ME PAM DG G He LP Fig 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 relative level of receptor expression was calculated for one copy of the GAPDH housekeeping gene by using the following formula: N ¼ · 2(Ct GAPDH – Ct target) tions: 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 addition, a significant fraction of the Cg-ALR1 injected embryos (between 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 3430 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 dorsalization in a concentration-dependent manner (Figs 4B and 8A) Expression of tbx6 was dramatically repressed, even if in some cases its expression at the margin of the blastoderm was expanded (Fig 9A1,A2) Expression of gsc was clearly expanded in all cases (Fig 9A3,A4) mRNA encoding a truncated CgTGFbsfR2 (DN-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 FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS A Herpin et al BMP/activin pathway in Crassostrea gigas Dorsalized embryos 24 Hpf Ventralized embryos 24 Hpf A A n=77 100 n=96 n=80 n=73 n=101 Phenotypes B Proportion 75 Vt3 of embryos 50 (%) Vt2 Vt1 25 Normal 50 100 150 200 pg/embryo Cg-BMPR1 B n=195 n=209 n=167 n=114 n=182 100 Phenotypes D5 D4 D3 D2 D1 Normal Proportion 75 of embryos 50 (%) 25 10 50 100 200 pg/embryo DN-Cg-BMPR1 Fig Histograms showing the phenotype distribution after overexpression of (A) Cg-BMPR1 and (B) truncated Cg-BMPR1 (DNCg-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 Fig The range of zebrafish phenotypes observed on overexpression of TGF-b superfamily receptors (A) Examples showing the ventralized 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 ranged from the least (D1) to the most severe (D5) transmembrane 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) FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS 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 homologous 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 territories 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) 3431 BMP/activin pathway in Crassostrea gigas A Herpin et al 80% epiboly goosecoid 80% epiboly tbx6 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4 D1 D2 D3 D4 E1 E2 E3 E4 DN(Cg-ALR1) Cg-ALR1 DN(Cg-BMP-R1) Cg-BMP-R1 Control A1 Fig 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 Discussion We 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 clusters, containing either type I or type II receptors Each 3432 cluster is further divided into individual clades containing 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–deuterostomes for types and most subtypes, respectively In addition, although the observation of a second extracellular domain in the Cg-TGFbsfR2 receptor is unique among protostome and deuterostome type II receptors, multiple extracellular domains are observed FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS A Herpin et al BMP/activin pathway in Crassostrea gigas A C P1 Fig Distribution of phenotypes after overexpression 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 significant fraction of the embryos also displayed a bifida chordata (Bc) phenotype in combination with severe anterior defects (B) Histogram showing the proportion of embryos displaying each phenotype after injection with Cg-ALR1 The number of embryos injected for each experiment is also indicated (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 P2 n=99 n=123 n=99 Proportion 75 of embryos 50 (%) A4 n=118 Phenotypes Bc P3 P2 P1 25 Normal D 100 n=177 n=250 n=207 n=197 n=218 Phenotypes dead Proportion 75 of embryos 50 (%) A4 A3 A2 A1 25 Normal 0 10 50 100 200 Cg-ALR1 among sponge (parazoan) molecules [24] At this time, the basic receptor repertoire may have already consisted 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 organization across protostomes and deuterostomes reveals that domain distribution among exons is conserved FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS n=108 A3 Bc 100 A2 P3 B A1 400 pg/embryo 10 50 100 200 400 pg/embryo DN-Cg-ALR1 Usually, intron boundaries 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 ancestral 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 encoding 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 superfamily of growth factors play a crucial role in specific developmental events [25,26] During early embryogenesis both Cg-BMPR1 and, to a certain degree, Cg-TFGbsfR2 show an apparent accumulation in 3433 BMP/activin pathway in Crassostrea gigas A n=176 n=97 n=123 n=110 A Herpin et al n=155 100 Phenotypes 75 D5 D4 D3 D2 D1 Normal Proportion of embryos 50 (%) 25 10 50 75 100 200 pg/embryo Cg-TGFβsfR2 B n=183 n=362 n=300 n=225 n=364 100 75 D5 D4 D3 D2 D1 Normal Proportion of embryos 50 (%) 25 10 50 75 100 200 pg/embryo DN-Cg-TGFβsfR2 C 100 n=145 n=182 n=147 n=165 n=117 Phenotypes D5 D4 D3 D2 D1 Normal 75 Proportion of embryos 50 (%) 25 10 50 100 300 pg/embryo 200 DN-Cg-TGFβsfR2C1 D 100 n=152 n=216 n=170 n=216 n=148 Phenotypes Bc D5 D4 D3 D2 D1 Normal 75 Proportion of embryos 50 (%) 25 10 50 100 200 300 pg/embryo DN-Cg-TGFβsfR2C2 Fig Histograms showing the proportion of embryos displaying each phenotype after injection with (A) Cg-TGFbsfR2 mRNA, (B) DN-Cg-TGFbsfR2 mRNA, (C) DN-Cg-TGFbsf2RC1 domain mRNA, and (D) DN-Cg-TGFbsfR2 C2 domain mRNA The number of embryos injected for each experiment is also indicated unfertilized oocytes (up to 10-fold more than the average level for Cg-BMPR1) This demonstrates that newly fertilized eggs could have the aptitude to respond to a BMP-like signal before zygotic transcription initiation After mid-blastula transition, while Cg-BMPR1 3434 transcripts are steadily decreasing until metamorphosis, two peaks of Cg-TFGbsfR2 mRNA occur at the time of the two major organomorphogenetic events arising during lophotrochozoan larval development, namely early morphogenesis (gastrula) and metamorphosis [27] Such expression patterns, especially during early embryogenic development, are consistent with that of several serine ⁄ threonine kinase receptors [28–30] implying a way in which Cg-BMPR1 and Cg-TFGbsfR2 may be involved as morphogens for early development in C gigas If we consider the whole repertoire of TGF-b pathway components including ligands and receptors so far identified in C gigas (reviewed in [23]), all of the receptors (activin type I receptor [17], TGF-b type I sensu stricto receptor [18] and now BMP type I receptor) are without exception expressed in a synexpression group throughout early development On the other hand mGDF [1], a C gigas BMP2 ligand orthologue, like Cg-TFGbsfR2 is specifically highly expressed at a later stage, mainly prior to or during metamorphosis (Fig 3A) Taken together, these complementary expression fluctuations between type I and II receptors and ligands probably reflect a relatively lax system for which receptor ⁄ receptor, as well as ligand ⁄ receptor, interactions would be flexible across TFG-b subfamilies [14,31,32] Hence, ligands/receptors versatility and type I/type II receptors variegation, leading in some cases to cross talk between TGF-b subfamily pathways, are probably answers to compensate for the relatively low number of primary interacting components observed in protostome species when compared to their deuterostome counterparts [23] Cg-BMPR1 partially ventralizes zebrafish embryos during gastrulation by transducing a BMP-like ventralizing signal in the mesoderm Expression of Cg-BMPR1 resulted in moderate ventralization of zebrafish embryos with the loss of anterior structures together with slightly enlarged posterior somites This phenotype is similar to those induced by Cg-Tolloid ⁄ tolloid ⁄ xolloid overexpression in fish and frog (A Herpin et al., unpublished results; [33,34]) This phenotype also resembles that of the zebrafish chordin loss of function mutant [35] and suggests that the mesoderm may be partially ventralized This hypothesis was supported by analysis of tbx6 and gsc expression by the gastrula Expression of tbx6, which specifies ventral-type mesoderm [36], was expanded towards the dorsal region, showing that Cg-BMPR1 induced ventral mesoderm in zebrafish In contrast, the gsc expression pattern showed the dorsal mesoderm to be more diffuse and clearly redistributed towards FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS A Herpin et al BMP/activin pathway in Crassostrea gigas 80% epiboly goosecoid 80% epiboly tbx6 A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4 D1 D2 D3 D4 DN (Cg-TGFβsf-R2-C2) DN (Cg-TGFβsf-R2-C1) DN (Cg-TGFβsf-R2) Cg-TGFβsf-R2 Control Fig 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 ventral regions Unsurprisingly, when Cg-BMPR1 was overexpressed, a similar outcome to that observed with previously characterized oyster Cg-Tolloid was seen, although the phenotypes were not usually so severe (A Herpin et al., unpublished results) This probably reflects the fact that, even in the presence of an abnormally high concentration of type I BMP receptor, enhanced signalling is limited by the endogenous ligand concentration Expression of truncated CgBMPR1 predictably produced a range of dose-dependent dorsalized phenotypes, as the dominant negative BMP type I receptor probably sequestered BMP ligands The phenotypes associated with the abrogation of BMP pathway signalling were comparable to those obtained in BMP2-deficient zebrafish [37] Moreover, FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS the pattern of tbx6 and gsc expression indicated that the observed dorsalization was due to inappropriate expansion of the dorsal mesoderm with a concomitant reduction in the ventral mesoderm These observations suggest that Cg-BMPR1 is capable of functioning in a similar way to its zebrafish orthologues by promoting ventral mesoderm induction and repressing dorsal mesoderm formation during gastrulation Cg-ALRI interferes with endogenous BMP/activin pathways during zebrafish gastrulation Expression of full length Cg-ALRI in zebrafish resulted in embryos with disruption of the anteroposterior 3435 BMP/activin pathway in Crassostrea gigas axis In contrast to overexpression of either endogenous TbRI [38] or mouse ActRIA and ActRIB [39] in zebrafish, Cg-ALRI expression did not induce a duplication of anterior structures but rather a bifida chordata phenotype where the posterior part of the notochord is divided around the yolk This phenotype is probably due to a delay in blastopore closure during gastrulation Cg-ALRI expression induced gsc expression ectopically towards ventral regions while repressing tbx6 expression Expression of truncated Cg-ALRI generally resulted in dorsalized phenotypes combined with some anterior defects and analysis of the expression of the mesodermal markers showed repression of both the dorsal and ventrolateral mesoderm at gastrula stage These observations are in agreement with those of Thisse and colleagues following overexpression of the activin specific competitive inhibitor, antivin [40,41] Expression of Cg-TGFbsfR2 in zebrafish embryos While the structure and function of oyster type I BMP and activin receptors appears to be conserved in comparison to those of other invertebrates and vertebrates, the structure of the oyster type II receptor was more surprising Cg-TGFbsfR2 possess two extracellular domains joined by a linker sequence, an observation so far unique to type II TGFb superfamily receptors from protostomes or deuterostomes, although it is a characteristic shared with sponges for which, among seven receptors cloned in Ephydatia fluviatilis, five possess a single ‘conventional’ domain, one a duplicated and another one a triplicated extracellular domain [24] Unfortunately, our experiments not clarify whether this receptor is active or not in its full length form Expression of both the wild type and dominant negative forms of Cg-TGFbsfR2 give rise to the same phenotype and modified tbx6 ⁄ gsc marker expression patterns We believe that this is probably due to the fact that the oyster type II receptor is incompatible with the zebrafish TGF-b superfamily type I receptors The observation that expression of the truncated oyster type II receptor is able to disrupt mesoderm formation certainly suggests that this receptor is capable of ligand binding More surprising is the apparent differential ligand affinity of the C1 and C2 extracellular domains Expression of C1 led to the apparent sequestration of BMP-like ligands, resulting in the production of dorsalized phenotypes Injection of relatively high concentrations of mRNA encoding the C2 domain, however, resulted in embryos with a combination of features of both dorsalized and ventralized phenotypes This is suggestive of an oyster type II 3436 A Herpin et al receptor able to interact with both BMPs and activins via its double extracellular ligand binding domains Such promiscuous behaviour has already been described for the Drosophila sax [42] and Caenohabditis elegans Daf-1 proteins [43], but these two receptors are conventional single extracellular receptors It is of course feasible that TGFbsfR2 acts similarly to the TGF-b silencing pseudoreceptor bambi [44]; however, the highly conserved cytoplasmic domains suggests that its encoding gene is still under evolutionary pressure to maintain its integrity This hitherto single C gigas type II receptor may represent an undefined evolutionary stage in TGF-b ⁄ activin ⁄ BMP pathway signal transduction involving a pleiotropic and promiscuous receptor Evidence for the conservation of the mechanism of mesoderm induction in molluscs and across bilaterians Although Lartillot and colleagues have observed for a number of developmental genes involved in anteroposterior patterning that their spatial and temporal expression pattern is highly conserved across bilaterians [5,45], the molecular mechanisms in lophotrochozoans remain unclear Unequal cleaving spiralian adult mesoderm is derived from two split territories The endomesoderm arises from the fourth quartet micromere of the D quadrant, the mesentoblast [46]; subsequently, the ectomesoderm is derived from the second and ⁄ or third quartets of micromeres (reviewed in [47]) For the first time in a lophotrochozoan we have shown that two key members of the BMP pathway Cg-BMPR1 and the recently characterized Cg-Tolloid (A Herpin et al unpublished results) are present and expressed as a synexpression group throughout early development as well as in adult tissues of C gigas Moreover, using the zebrafish embryo as a reporter system we have demonstrated that these two pathway components, as well as the activin-like type I receptor Cg-ALRI, are able to regulate mesoderm induction during gastrulation and early development stages, despite the considerable evolutionary distance between spiralians and chordates The results of these functional studies when combined with our expression analyses and phylogenetic data, would suggest a conserved mechanism for both ectomesoderm and mesendoderm induction in spiralians in comparison with ecdysozoans and protostomes Further support for the conservation of such an inductive mechanism has been provided by Lambert and Nagy [48] who have shown asymmetric inheritance of mRNA encoding a DPP orthologue during embryonic cleavage by Ilyanassa obsolete, a gastroFEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS A Herpin et al pod mollusc These events produce a complex pattern of mRNA localization in which the DPP transcripts, after being distributed diffusely in the cytoplasm, become localized in all four of the macromere cells at the eight-cell stage These transcripts then segregate into the second and third quartet of micromeres but decay in the second quartet cells, resulting in a specific localization in the third quartet cells as well as in macromere 3D Interestingly, the third quartet cells of spiralians, as well as macromere 3D, have been shown through cell lineage studies to give rise to the endomesoderm and ectomesoderm [47] Together with the evidence provided by this study, this argues for a possible conserved endomesoderm ⁄ ectomesoderm inductive mechanism in spiralians through an ancestral BMP ⁄ activin pathway, in which the singular and promiscuous Cg-TGFbsfR2 could be the shared type II receptor interface for both BMP and activin ligands This hypothesis is supported by the fact that different methods, such as RT-PCR with degenerate primers directed against the highly conserved intracellular kinase domain of these receptors, and low stringency screening of cDNA, genomic and BAC libraries (10 times coverage) using different probes, failed to isolate any new type I or II receptors Cg-TGFbsfR2 would represent the only (TGF-b) type II receptor encoded within the C gigas genome Experimental procedures Cloning and sequencing of TGFb superfamily receptor cDNAs We previously reported the cloning of two TGF-b superfamily receptors, a type I activin-like receptor (Cg-ALR1) [17] as well as another type I TGF-b s.s receptor (Cg-TGFbRI) from C gigas [18] cDNAs encoding novel type I receptors and one type II receptor were cloned from C gigas in an identical manner Gene-specific primers were then used to isolate a full length cDNA from an oyster mantle edge library The resulting clones were designated Cg-BMPR1 and Cg-TGFbsfR2 Phylogenetic analysis Sequences encoding TGFb superfamily receptor proteins were chosen to represent a range of protostomes and deuterostomes in phylogenetic analyses Preference was given to sequences for which functional data were available The sequences were aligned using clustal x version 1.81 and by manual inspection Alignments used for phylogenetic relationships of the cytoplasmic domain were carried out, making use of the whole receptor intracellular part amputa- FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS BMP/activin pathway in Crassostrea gigas ted of its highly variable C-terminal domain after the terminal conserved arginine of the cytoplasmic serine ⁄ threonine kinase domain Sequences for phylogenetic relationships of the extracellular part were truncated to strictly take into consideration the 10 conserved cysteines upstream of the characteristic cysteine knot CCCX(4)C From these alignments, distance-based phylogenetic trees were constructed using the minimum evolution method of the paup package version 4.0b4a One thousand bootstrap trials were run using the neighbor-joining algorithm for each node Isolation and characterization of Cg-BMPR1 and Cg-TGFbsfR2 TGFb genes A C gigas genomic library was constructed in kDASH II (Stratagene, La Jolla, CA, USA) A total of 1.8 · 106 independent clones were recovered After amplification to 4.5 · 1010 pfmL)1, a total of 2.5 · 105 recombinant phages were screened at high stringency with digoxigenin-11dUTP labelled probes encoding full length TGF-b superfamily receptor cDNAs Unique clones containing each of the two novel receptor genes were sequenced and the intron–exon structures of the genes determined by comparing the genomic sequence with the cDNA sequence Quantification of Cg-BMPR1 and Cg-TGFbsfR2 receptor transcripts Because technical limitations relative to oyster embryos and larvae make whole-mount in situ hybridizations less reliable than RT-PCR [17], quantitative real time RT-PCR was the best alternative The larval developmental stage pattern and tissue distribution of Cg-BMPR1 and Cg-TGFbsfR2 mRNA was investigated by quantitative RT-PCR using an iCycler (Bio-Rad, Hercules, CA, USA) Total RNA from various developmental stages and adult tissues was isolated using Tri-Reagent (Sigma-Aldrich, Munich, Germany) according to the manufacturer’s instructions After treatment for 20 at 37 °C with U of DNase I (Sigma) to prevent genomic DNA contamination, lg of total RNA was reversed transcribed using lg of random hexanucleotide primers (Promega, Madison, WI, USA), 0.5 mm dNTPs and 200 U M-MLV reverse transcriptase (Promega) at 37 °C for h in the appropriate buffer The reaction was stopped by incubation at 70 °C for 10 iQTM SYBR Green supermix PCR kit (Bio-Rad) was used for real time monitoring of amplification (5 ng of template cDNA, 40 cycles: 95 °C for 15 s, 60 °C for 15 s) with 250 nm of the following primers: QsBMPR1, 5¢-AGCTTGCCCCCAACCTC-3¢; QaBMPR1, 5¢-ATGGTCTCTGCGGGTTGA-3¢; QsTGFbsfR2, 5¢-GCC AGATCCCAAATTAGTGC-3¢; QaTGFbsfR2, 5¢-TGAA ACCACAGCCTCAGTTG-3¢, where ‘s’ and ‘a’ indicate sense and antisense primers, respectively Accurate amplification of the target amplicon was checked by performing melt- 3437 BMP/activin pathway in Crassostrea gigas ing curve analysis Using QsGAPDH (5¢-TTCTCTT GCCCCTCTTGC-3¢) and QaGAPDH (5¢-CGCCCAATCC TTGTTGCTT-3¢) primers, a parallel amplification of oyster GAPDH transcript (EMBL CG548886) was carried out to normalize the expression data of the receptor transcripts The relative level of receptor expression was calculated for copy of the GAPDH housekeeping gene by using the formula N ¼ · 2(Ct GAPDH – Ct target) Zebrafish maintenance and preparation of eggs TAB zebrafish were reared and maintained on a light ⁄ dark cycle essentially as described by Westerfield [49] All experiments and fish maintenance comply with current Norwegian law Synchronized eggs were obtained by mixing previously separated female and male fish at a ratio of : Eggs were then collected as described by Culp et al [50] Self-digested Pronase E (Sigma) at 0.5 mgỈmL)1 was used to remove the egg chorion The reaction was stopped by removing the enzyme and transferring the eggs into Holtfreter’s solution (85 mm NaCl, mm KCl, mm CaCl2 and 3.5 mm NaHCO3) and rinsing Microinjections Initially, full length cDNAs encoding TGFb superfamily receptors were subcloned into the plasmid pRN3 [51] Receptor cDNAs for use as dominant-negative controls were obtained by inserting a stop codon either immediately up- or downstream of the sequence encoding the transmembrane domain Capped synthetic mRNAs encoding both full length and truncated proteins were transcribed in vitro using the Stratagene mCAP RNA capping kit and linearized plasmids as templates The capped mRNAs were dissolved in RNAse-free 0.2 m KCl containing 0.5% (w ⁄ v) phenol red to monitor injection mRNA or plasmid DNA was injected into the cytoplasm of a single-cell embryo After injection, embryos were transferred into 24-well tissue culture plates containing Holtfreter’s solution (5–7 embryos per well), incubated at 28 °C for up to 24 h and then examined by microscopy for phenotype analysis Unfertilized eggs were removed after h Alternatively, embryos were fixed for in situ hybridization at 80% epiboly Uninjected and control embryos injected with 0.2 m KCl were analysed under the same conditions Whole-mount in situ hybridization Antisense probes were transcribed in vitro and labelled using digoxigenin-labelled UTP (Roche, Indianapolis, IN, USA) The anteroaxial goosecoid mesodermal marker [52], and the ventrolateral tbx6 mesodermal marker [36] were used as probes Whole-mount staining of embryos was performed as described by Rissi et al [53] 3438 A Herpin et al Acknowledgements This work was performed with the aid of grants from the Norwegian Research Council, Basse-Normandie Regional Council, France, and the French-Norwegian Foundation for Scientific, Technical and Industrial Research We would like to acknowledge the help provided by the Zebrafish facility technical staff at the Sars Centre We would also like to thank Prof Patrick ´ ´ Lemaire (Laboratoire de Genetique et Physiologie du ´ ´ Developpement, IBDM, CNRS ⁄ INSERM ⁄ Universite ´ diterranee ⁄ AP de Marseille) for provision of ´ de la Me the pRN3 plasmid We acknowledge J Laisney and A H Hansen for their technical assistance We also thank Prof Manfred Schartl (Physiological Chemistry I, University of Wuerzburg, Germany) for his comments 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Journal 272 (2005) 3424–3440 ª 2005 FEBS ... FEBS BMP/ activin pathway in Crassostrea gigas 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. .. transmembrane domain and the serine ⁄ threonine kinase domain FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS 3427 BMP/ activin pathway in Crassostrea gigas A Herpin et al A Crassostrea gigas C2 domain ALK-6... intracellular part amputa- FEBS Journal 272 (2005) 3424–3440 ª 2005 FEBS BMP/ activin pathway in Crassostrea gigas ted of its highly variable C-terminal domain after the terminal conserved arginine of the