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Embryonic expression and evolutionary analysis of the amphioxus Dickkopf and Kremen family genes
JOURNAL OF
GENETICS AND GENOMICS J. Genet. Genomics 37 (2010) 637−645
www.jgenetgenomics.org
Embryonic expression and evolutionary analysis of the amphioxus Dickkopf and Kremen family genes Yujun Zhang a, b, Bingyu Mao a, * a
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China b Graduate University of Chinese Academy of Sciences, Beijing 100049, China Received for publication 8 July 2010; revised 3 August 2010; accepted 17 August 2010
Abstract The secreted Wnt signaling inhibitor Dickkopf1 (Dkk1) plays key role in vertebrate head induction. Its receptor Kremen synergizes with Dkk1 in Wnt inhibition. Here we have carried out expression and functional studies of the Dkk and Kremen genes in amphioxus (Branchiostoma belcheri). During embryonic and larval development, BbDkk1/2/4 is expressed in the posterior mesoendoderm, anterior somatic mesoderm and the pharyngeal regions. Its expression becomes restricted to the pharyngeal region on the left side at larval stages. In 45 h larvae, BbDkk1/2/4 is expressed specifically in the cerebral vesicle. BbDkk3 was only detected at larval stages in the mid-intestine region. Seven Kremen related genes were identified in the genome of the Florida amphioxus (Branchiostoma floridae), clustered in 4 scaffolds, and are designated Kremen1-4 and Kremen-like 1-3, respectively. In B. belcheri, Kremen1 is strongly expressed in the mesoendoderm during early development and Kremen3 is expressed asymmetrically in spots in the larval pharyngeal region. In luciferase reporter assays, BbDkk1/2/4 can strongly inhibit Wnt signaling, while BbDkk3, BbKremen1 and BbKremen3 can not. No co-operative effect was observed between amphioxus Dkk1/2/4 and Kremens, suggesting that the interaction between Dkk and Kremen likely originated later during evolution. Keywords: amphioxus; Dickkopf; Kremen; evolution; expression pattern
Introduction Cannonical Wnt signaling pathway is well conserved during evolution and plays essential roles in the formation and patterning of the body axes in different animals (Nascone and Mercola, 1997; Wikramanayake et al., 1998; Yamaguchi, 2001; Croce and McClay, 2006). It also plays roles in germ layer formations in various animals (Rocheleau et al., 1997; Thorpe et al., 1997; Imai et al., 2000; Wikramanayake et al., 2003). The Dickkopf (Dkk) * Corresponding author. Tel: +86-871-519 8989; Fax: +86-871-519 3137. E-mail address: [email protected] DOI: 10.1016/S1673-8527(09)60082-5
family of secreted inhibitors of canonical Wnt signaling function in the patterning of the body plan from hydra to vertebrates (Glinka et al., 1998; Krupnik et al., 1999; Guder et al., 2006; Caneparo et al., 2007). In human, there are 4 Dkks: Dkk1, 2 and 4 can inhibit Wnts, while Dkk3 can not (Krupnik et al., 1999; Mao and Niehrs, 2003; Niehrs, 2006). The developmental role of Dkk3 in vertebrates is still poorly understood. Although widely expressed during embryonic development (Monaghan et al., 1999), Dkk3-deficient mice develop normally, are fertile and have only a mild phenotype (Barrantes Idel et al., 2006). Dkk1, 2 and 4 work through binding the Wnt receptors LRP5/6 and sequestering the Wnt ligands (Mao et
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al., 2001; Semenov et al., 2001). In vertebrates, Kremen1 and 2, two novel coreceptors for Dkk1 and Dkk2, have been shown to enhance the Wnt inhibitory role of Dkks (Mao et al., 2002; Mao and Niehrs, 2003; Nakamura and Matsumoto, 2008). Kremens are single-transmembrane proteins and have a unique extracellular structure containing a Kringle, a WSC and a CUB domain (Nakamura et al., 2001). However, Kremen-like genes have not been reported in invertebrates. How its unique structure evolved during evolution is an interesting question. In a previous study, Yu et al. (2007) studied the expression pattern of Dkk1/2/4 and Dkk3 in the Florida amphioxus (Branchiostoma floridae) and suggested that the anterior patterning mechanism through Wnt inhibition by Dkk is conserved in amphioxus. However, functional proof has been lacking. Here, we have cloned the Dickkopf genes in amphioxus Branchiostoma belcheri and studied their developmental expression patterns. We also studied the Kremen related genes in the genome of the Florida amphioxus and 7 genes were identified. The expression patterns of two Kremen genes were studied in B. belcheri embryos and larvae.
Materials and methods Embryos Adult amphioxus (B. belcheri) (Zhang et al., 2006; Zhong et al., 2009) were collected during the breeding season from the South China Sea near Beihai (Guangxi Province, China) and maintained in the laboratory. Naturally fertilized eggs were collected and cultured at room temperature. The developing embryos and larvae at desired stages were fixed in fresh 4% paraformaldehyde at room temperature for 30 min or at 4°C overnight, dehydrated in graded methanol and stored in 70% methanol at −20°C.
Cloning of amphioxus Dkk and Kremen genes The full open reading frames of BbDkk1/2/4 and 3 were cloned by RT-PCR using primers designed according to the BfDkk1/2/4 sequence (Yu et al., 2007) and the Dkk3 sequence (GenBank accession No. AY608670) of B. japonicum (Zhang et al., 2006; Zhong et al., 2009). The primers used are:
BbDkk1/2/4: 5′-CCGTAACTTGTCCACACCGTAGA-3′ and 5′-GTGCTTTGATGCGTCTTGTTGGG-3′; BbDkk3: 5′-CTCACTACTCACATCCCTGTCTCTA-3′ and 5′-ACCAGTGAGACATACATCGAAGAGT-3′. The BbDkk1/2/4 and BbDkk3 sequences have been submitted to GenBank under accession Nos. HM590023 and HM590024. The genome of amphioxus (B. floridae, http://genome. jgi-psf.org/Brafl1) was BLASTed using mouse Kremen proteins and 10 hits were found. GenomeScan (http://genes. mit.edu/genomescan.html) was used for predicting the Kremen genes aided by EST database searches and manual correction (Supplemental document). Phylogenetic tree was constructed using MAGA4 (Tamura et al., 2007) using Neigbour-Joining method. Full length sequences of BbKremen1 and 2 and partial sequences of BbKremen4 and BbKremen-like 1 and 3 were cloned using RT-PCR. The primers used are: BbKremen1: 5′-ATGGGGCCAGGAGAGTTCGGAG-3′ and 5′-TCACAATTCTCCCAGCACAGCA-3′; BbKremen2: 5′-ATGGCATCTACCCACGTTTGGATGT-3′ and 5′-CTATTCGCTACTGTAACCATGAGCG-3′; BbKremen4: 5′-CTACAGAGGAAACCAGTCGGAGC-3′ and 5′-ACCCGCCACACCGTCCCATCTTC-3′; BbKremen-like1: 5′-CCTACCGATCACCGACTGTGCT-3′ and 5′-GTACTCTACAATCGTCAAACTATCG-3′; BbKremen-like3: 5′-TGACCCAGACAAGGTTATCAA GTTC-3′ and 5′-CTCGTAGTGCGCATGCTCGTTCTGT-3′. The PCR products were cloned into pBS-T vector (Tiangen, China) and sequenced. The BbKremens sequences have been submitted to GenBank under accession No. HM590025–HM590029. For functional analysis, the full length open reading frames of BbDkk1/2/4, BbDkk3, BbKremen1 and BbKremen2 were cloned into the pCS2 + vector.
In situ hybridization and luciferase reporter assays In situ hybridization in amphioxus embryos and larvae was carried out as described (Zhang and Mao, 2009). Luciferase reporter assays in HEK 293T cells were done in 96 well plates at least in triplicates as described (Mao et al., 2001). Plasmid concentrations used (ng/well): superTOPFLASH, 80; pTK-Renilla, 8; mWnt1, 64; mfrizzled8, 8; hLRP6, 2, unless indicated otherwise. Luciferase activity was normalized against Renilla activity.
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Results Cloning and expression of amphioxus Dkk genes Two Dkk genes were cloned using RT-PCR from B. belcheri, one homologous to vertebrate Dkk1/2/4 and the other one homologous to vertebrate Dkk3. The predicted amphioxus Dkk1/2/4 protein contains two typical cysteine-rich domains (CRD), as its vertebrate ho mologs. In a phylogenetic tree analysis, BbDKK1/2/4 is clustered together with the Dkk-1, -2 and -4 genes while BbDkk3 is clustered in the Dkk3 branch (data not shown; Yu et al., 2007). The amphioxus Dkk3, however, contains only the second CRD domain, unlike the Dkk3 proteins in any other species including hydra and sea urchin (Croce et al., 2006). The amphioxus Dkk3 might have undergone lineage specific diversifycation and lost the first CRD domain. Interestingly, the Dkk1/2/4 in hydra and sea urchin also contain only the CRD2 domain (Croce et al., 2006). To gain insight into their developmental roles, in situ hybridization was carried out using amphioxus embryos and larvae. Similar as in B. floridae (Yu et al., 2007), BbDkk1/2/4 was detected in the mesendoderm around the blastopore at the gastrula stage (Fig. 1, A and A′). At early neurula stage (2−4 somites), the expression of Dkk1/2/4 is confined to the posterior mesoendoderm (Fig.1, B and B′). At 6-somite stage, its expression remains strong in the posterior blastopore region and also turns on in the anterior mesendoderm, forming two stripes anterior to the first and second somites, respectively (Fig. 1, C and C′). In 14 h larvae, its expression in the posterior mesoderm turns into two stripes on both sides. The expression stripes in the anterior mesoderm become stronger. Interestingly, its expression in the anterior region starts to become asymmetric, its expression on right side becomes slightly weaker than on the left side (Fig. 1, D and D′). Later on, its expression becomes undetectable on the right side in the anterior region and only weakly at the posterior region, while remains strong on the left side in both anterior and posterior mesoderm (Fig. 1, E and E′). In 18 h larvae, its expression is no longer detected in the posterior mesoderm, and remains only in two patches in the anterior pharyngeal region on the left side (Fig. 1, F and F′). In 45 h larvae, BbDkk1/2/4 expresses specifically in the cerebral vesicle (Fig. 1G).
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BbDkk3, however, is not expressed in the embryonic developmental stages (data not shown). From 24 h larval stage, it becomes strongly expressed in the mid-intestine region (Fig. 1, H and I). In B. floridae, Dkk3 is strongly expressed in the anterior ectoderm at the onset of gastrulation and also weakly in the mesendoderm by mid-gastrula stage (Yu et al., 2007). The different expression pattern of Dkk3 in the two species likely reflects different roles of Dkk3 in the two species. In fact, the protein sequence identity between BbDkk3 and BfDkk3 is only 51% (data not shown), suggesting strong lineage diversification of amphioxus Dkk3.
The amphioxus Kremen genes We extracted the Kremen related sequences from the genome of the Florida amphioxus (B. floridae). Surprisingly, 10 Kremen like sequences were found in the amphioxus genome, located in four scaffolds (Fig. 2A). According to the predicted domain structures, amphioxus Kremen proteins can be divided into 2 groups. One group contains all three types of domains (Kringle, WSC and CUB, Fig. 2B). The 4 genes form a cluster locating on scaffold 36, and are designated Kremen1, 2, 3 and 4 sequentially (Fig. 2A). Interestingly, 3 of the 4 Kremen proteins in this group contain two Kringle domains instead of one. The predicted BfKremen proteins show less than 50% sequence identity between each other and the 4 genes should represent independent ones. In phylogenetic analysis, these proteins show similar homology to vertebrate Kremen1 and Kremen2 (Fig. 2C). The second group of predicted Kremen related proteins are lack of the Kringle domain (Fig. 2B) and designated Kremen-like (Krl) proteins. The 3 genes on scaffold 149 show close homology to the one on scaffold 152 and the two genes on scaffold 89 respectively, both in sequence and gene structure (Supplemental Table1). Since the B. floridae genome database contains sequences of two haplotypes (Li et al., 2007), we suggest that the 6 genes might represent different alleles of three independent genes. The six genes are named Krl1a, Krl1b, Krl2a, Krl2b, Krl3a and Krl3b, respectively (Fig. 2, A and B). Structurally, the amphioxus Kremen-like proteins are more similar to the Kremen-like proteins in sea urchin (Strongylocentrotus purpuratus), which also contain only the WSC and CUB domains (Fig. 2, B and C; Croce et al., 2006).
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Fig. 1. Developmental expression of Dkk1/2/4 (A–G) and Dkk3 (H, I) in B. belcheri embryos and larvae. A, A′: mid-gastrula stage. A: lateral view, animal pole to the left. A′: vegetal view. B, B′: neurula stage. C, C′: late neurula stage. D, D′: 14 h larvae. E, E′: 16 h larvae. F, F′: 18 h larvae. G: 45 h larva. H: 24 h larva. I: 45 h larva. B−G, lateral views, anterior to the left; B′–F′: dorsal views, anterior to the left. The arrowheads indicate the expression domains. Scale bar = 50 μm.
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Fig. 2. Structures and phylogenetic analysis of amphioxus Kremens. A: the distribution of the predicted Kremen related genes on the scaffolds of B. floridae genome. B: domain structures of the predicted amphioxus Kremen and Kremen-like proteins. C: phylogenetic analysis of the Kremen related proteins in sea urchin, amphioxus, tunicate, Xenopus, mouse and human. Bf, Branchiostoma belcheri; Ci, Ciona intestinalis; Homo, Homo sapiens; Mus, Mus musculus; Sp, Strongylocentrotus purpuratus; Xl, Xenopus laevis. The Kremen proteins used are: Mus Kremen1, NP_115772 (GenBank accession No.); Homo Kremen1, CAQ07690; Xl Kremen1, NP_001082145; Mus Kremen2, NP_082692; Homo Kremen2, BAC00872; Xl Kremen2, NP_001082352; Ci Kremen, XP_002125526; Sp Kremen-like 1 and 2, Glean IDs 25250 and 25249 (http://goblet.molgen.mpg.de/cgi-bin/seaurchin-genombase.cgi).
Developmental expression of amphioxus Kremens Primers were designed according to predicted Bf-Kremen sequences to amplify the Kremen sequences in B. belcheri. Using cDNA from 10-somite stage amphioxus embryos, 5 fragments of BbKremen cDNAs were cloned: Krm1, Krm2, Krm4, Krl1 and Krl3. In situ hybridization was performed in amphioxus embryos and larvae at different developmental stages using different probes. Krm1 is expressed widely in the mesendoderm from early neurula stage on. At later stages, it is expressed in the axial somatic mesoderm, the notochord and dorsal endoderm (Fig. 3, A–D). Krm2, however, shows restricted expression domains in the amphioxus larvae. At early larval stage (18 h), Krm2 is expressed in 3 spots in the pharyngeal region asymmetrically, with two spots on the right side and one on the left side (Fig. 3, E and F). Similar expression pattern remains in 24 h larvae (Fig. 3, G and H). The differential expression of Krm1 and 2 suggests clear functional diversification of the
two genes. For Krm4, Krl1 and Krl3, no clear expression pattern was detected at the stages examined (data not shown).
Amphioxus Kremens do not synergize with Dkk1/2/4 in Wnt inhibition We cloned the full-length open reading frame of BbKrm1 and BbKrm2 for further functional studies, since their encoded protein structures are similar to the vertebrate ones. We first tested whether amphioxus Dkk1/2/4, Dkk3, Krm1 or Krm2 could inhibit Wnt signaling in 293T cells and in Xenopus embryos. As expected, BbDkk1/2/4 strongly inhibits Wnt signaling in luciferase reporter assay while BbDkk3 can not (Fig. 4A). When ectopically expressed in Xenopus embryos, BbDkk1/2/4 induces big-head phenotype (data not shown), indicating strong Wnt signaling inhibition. In vertebrates, Kremen strongly enhances the activity of Dkk1/2/4 in Wnt inhibition. We
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tested whether this is the case with amphioxus Dkk1/2/4 and Kremen. Amphioxus Dkk1/2/4 is able to cooperate with Xenopus Kremen1 to inhibit Wnt signaling but not
amphioxus Krm1 or Krm2 (Fig. 4B). Similarly, Xenopus Dkk1 also fails to cooperate with the two amphioxus Kremens in this assay (Fig. 4B).
Fig. 3. Developmental expression of Kremen1 (A–D) and Kremen2 (E–H) in B. belcheri embryos and larvae. A: early neurula stage. B: 14 h larva. C: 16 h larva. D: 20 h larva. E: 18 h larva. F: dorsal view of E. G: 24 h larva. H: dorsal view of the head region of the larva in G. A–E, G, lateral views, anterior to the left. The arrowheads indicate the expression domains.
Fig. 4. Luciferase assays showing the activities of amphioxus Dkk1/2/4 and Kremens in Wnt inhibition. A: BbDkk1/2/4 but not Dkk3 can inhibit Wnt signaling in 293T cells. W/F/L, the cells were transfected with combined mWnt1, mFz8 and hLRP6 to stimulate the Wnt signaling. B: amphioxus Kremens fail to synergize with amphioxus or Xenopus Dkks to inhibit Wnt signaling.
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Discussion In amphioxus, the Wnt genes are widely expressed in various tissues during embryonic development, including the blastopore (Wnt-8, Wnt-11, Wnt-1, Wnt-4) and mesoendoderm (Wnt-4, Wnt-7) at gastrula stage (Holland et al., 2000; Schubert et al., 2000, 2001; Holland, 2002; Yu et al., 2007). The high level distribution of nuclear β-catenin around the blsatopore at gastrula stage also suggested the activation of Wnt signaling in this area (Holland et al., 2005). The expression of BbDkk1/2/4 in the mesendoderm at gastrula stage suggests that it might be involved in germ layer formation at this stage. The local expression of BbDkk1/2/4 in the posterior mesoendoderm at early neurula stage (Fig. 1B) might help to create a Wnt-free region required for proper cell differentiation. In vertebrates, canonical Wnt signaling plays crucial role in the anterior-posterior patterning of the embryo. Its inhibition in the anterior region by secreted Wnt inhibitors (including Dkk1 and sFRPs) is essential for the development of the anterior structures, including the forebrain, heart and foregut. In Xenopus, Dkk1 is expressed in the prechodal plate mesoderm at neurula stage (Glinka et al., 1998), which lies under the developing forebrain. In the mouse, it is expressed in the anterior mesendoderm at gastrula stage (Glinka et al., 1998). In both species, blocking the function of Dkk1 by specific antibody or genetic knockout leads to loss of the head structure. In amphioxus, however, Dkk1/2/4 is not expressed in the anterior midline mesoenderm at neurula stages (Fig. 1, B and C). Whether it is involved in the anterior patterning in amphioxus awaits further functional studies. Although Dkk3 is expressed in the anterior mesoendoderm in Florida amphioxus (Yu et al., 2007), we did not detect such expression patterns in B. Belcheri. Anyway, Dkk3 cannot inhibit Wnt signaling and is less likely to be involved in anterior-posterior patterning. Amphioxus Dkk1/2/4 is expressed in the posterior forming somites at neurula and early larval stages, which is also the case in Xenopus, suggesting conserved roles for Dkk1 in somite formation. At larval stage, BbDkk1/2/4 and Kremen2 are asymmetrically expressed in the pharyngeal region. Many genes show left-right asymmetric expression pattern in amphioxus, including members of the Hedgehog, Pax, Nkx and Notch gene families (Boorman and Shimeld, 2002). Our data suggest that the regulation of Wnt signaling might be involved in the control of the left-right asymmetry in amphioxus.
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The evolution of the Kremen genes is an interesting issue. The vertebrate Kremen proteins have typical modular structures and contain 3 extracellular domains, the Kringle, WSC and CUB domains. The predicted sea urchin Kremen-like proteins contain only the WSC and CUB domains. In amphioxus, the Kremen related genes has clearly undergone lineage specific expansion and contain both kinds of structures. Amphioxus Kremen-like proteins lack the Kringle domain, which is closer to sea urchin Kremen, while the 4 Kremen proteins contain all the 3 type of domains (Kringle, WSC and CUB). In amphioxus, a fourth kind of domain (the CCP domain) also present in all the Kremen related proteins, which is not seen in any other species. Interestingly, each protein domain (including the Kringle, WSC, CUB, and CCP domains) is encoded by one or two independent exons (Supplemental Table 1). This situation is also true in Ciona and human (Supplemental Table 2), suggesting that this family of genes might have evolved through the exon shuffling mechanism (Patthy, 1999). A possible model for the evolution of this family of genes is proposed in Fig. 5. The prototype of the Kremen protein might be similar to the WSC proteins in yeast, which are single transmembrane proteins containing an extracellular WSC domain and are required for the maintenance of cell wall integrity (Verna et al., 1997). In the deuterostome (including sea urchin), the CUB domain encoding exon shuffled into the gene to create the Kremen-like genes. During the origin of chordates, the Kringle domain encoding exon was further added to the Kremen-like genes to create the typical Kremen structures in tunicates and vertebrate. The amphioxus Kremen genes seem to have taken an independent evolution pathway. The CCP domain was first added to create the Kremen-like proteins in amphioxus, and a second exon-shuffling event created the Kremen genes in amphioxus today. This scenario supports the view that tunicates are evolutionarily closer than amphioxus to vertebrates (Delsuc et al., 2006). The developmental roles of amphioxus Kremens await further studies. In Xenopus, Kremen is involved in neural crest development (Hassler et al., 2007). In mouse, double knockout of Kremen1 and 2 leads to limb defects and high bone density (Ellwanger et al., 2008). Although genetic interaction between Dkk1 and Kremen has been confirmed in mouse (Ellwanger et al., 2008), Kremens are not universally required for Dkk1 function. In amphioxus, Dkk1/2/4 and Kremens show different embryonic expression patterns and lack cooperation in Wnt inhibition assay.
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Fig. 5. A model for the evolution of the Kremen related proteins in eukaryotes.
The interaction between Dkk1 and Kremen is more likely an accidental event during vertebrate origin. It will be of interest to test whether Dkk1 and Kremen interact in tunicates, which have a typical vertebrate type Kremen.
Acknowledgements This work was supported by the grant from the Innovation Project of Chinese Academy of Sciences (No. KSCX2-YW-R-090). We thank Prof. Junyuan Chen (Nanjing University) for amphioxus materials.
Supplemental data Supplemental document and Tables 1 and 2 associated with this article can be found in the online version at www.jgenetgenomics.org.
References Barrantes Idel, B., Montero-Pedrazuela, A., Guadano-Ferraz, A., Obregon, M.J., Martinez de Mena, R., Gailus-Durner, V., Fuchs, H., Franz, T.J., Kalaydjiev, S., Klempt, M., Holter, S., Rathkolb, B., Reinhard, C., Morreale de Escobar, G., Bernal, J., Busch, D.H., Wurst, W., Wolf, E., Schulz, H., Shtrom, S., Greiner, E., Hrabe de Angelis, M., Westphal, H., and Niehrs, C. (2006). Generation and characterization of dickkopf3 mutant mice. Mol. Cell. Biol. 26:
2317−2326. Boorman, C.J., and Shimeld, S.M. (2002). The evolution of left-right asymmetry in chordates. Bioessays 24: 1004−1011. Caneparo, L., Huang, Y.L., Staudt, N., Tada, M., Ahrendt, R., Kazanskaya, O., Niehrs, C., and Houart, C. (2007). Dickkopf-1 regulates gastrulation movements by coordinated modulation of Wnt/beta catenin and Wnt/PCP activities, through interaction with the Dally-like homolog Knypek. Genes Dev. 21: 465−480. Croce, J.C., and McClay, D.R. (2006). The canonical Wnt pathway in embryonic axis polarity. Semin. Cell Dev. Biol. 17: 168−174. Croce, J.C., Wu, S.Y., Byrum, C., Xu, R., Duloquin, L., Wikramanayake, A.H., Gache, C., and McClay, D.R. (2006). A genome-wide survey of the evolutionarily conserved Wnt pathways in the sea urchin Strongylocentrotus purpuratus. Dev. Biol. 300: 121−131. Delsuc, F., Brinkmann, H., Chourrout, D., and Philippe, H. (2006). Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 439: 965−968. Ellwanger, K., Saito, H., Clement-Lacroix, P., Maltry, N., Niedermeyer, J., Lee, W.K., Baron, R., Rawadi, G., Westphal, H., and Niehrs, C. (2008). Targeted disruption of the Wnt regulator Kremen induces limb defects and high bone density. Mol. Cell. Biol. 28: 4875−4882. Glinka, A., Wu, W., Delius, H., Monaghan, A.P., Blumenstock, C., and Niehrs, C. (1998). Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391: 357−362. Guder, C., Pinho, S., Nacak, T.G., Schmidt, H.A., Hobmayer, B., Niehrs, C., and Holstein, T.W. (2006). An ancient Wnt-Dickkopf antagonism in Hydra. Development 133: 901−911. Hassler, C., Cruciat, C.M., Huang, Y.L., Kuriyama, S., Mayor, R., and Niehrs, C. (2007). Kremen is required for neural crest induction in Xenopus and promotes LRP6-mediated Wnt signaling. Development 134: 4255−4263. Holland, L.Z. (2002). Heads or tails? Amphioxus and the evolution of
Yujun Zhang et al. / Journal of Genetics and Genomics 37 (2010) 637−645
anterior-posterior patterning in deuterostomes. Dev. Biol. 241: 209−228. Holland, L.Z., Holland, N.N., and Schubert, M. (2000). Developmental expression of AmphiWnt1, an amphioxus gene in the Wnt1/wingless subfamily. Dev. Genes Evol. 210: 522−524. Holland, L.Z., Panfilio, K.A., Chastain, R., Schubert, M., and Holland, N.D. (2005). Nuclear beta-catenin promotes non-neural ectoderm and posterior cell fates in amphioxus embryos. Dev. Dyn. 233: 1430−1443. Imai, K., Takada, N., Satoh, N., and Satou, Y. (2000). β-catenin mediates the specification of endoderm cells in ascidian embryos. Development 127: 3009−3020. Krupnik, V.E., Sharp, J.D., Jiang, C., Robison, K., Chickering, T.W., Amaravadi, L., Brown, D.E., Guyot, D., Mays, G., Leiby, K., Chang, B., Duong, T., Goodearl, A.D., Gearing, D.P., Sokol, S.Y., and McCarthy, S.A. (1999). Functional and structural diversity of the human Dickkopf gene family. Gene 238: 301−313. Li, G., Zhang, Q.J., Ji, Z.L., and Wang, Y.Q. (2007). Origin and evolution of vertebrate ABCA genes: a story from amphioxus. Gene 405: 88−95. Mao, B., and Niehrs, C. (2003). Kremen2 modulates Dickkopf2 activity during Wnt/LRP6 signaling. Gene 302: 179−183. Mao, B., Wu, W., Li, Y., Hoppe, D., Stannek, P., Glinka, A., and Niehrs, C. (2001). LDL-receptor-related protein 6 is a receptor for Dickkopf proteins. Nature 411: 321−325. Mao, B., Wu, W., Davidson, G., Marhold, J., Li, M., Mechler, B.M., Delius, H., Hoppe, D., Stannek, P., Walter, C., Glinka, A., and Niehrs, C. (2002). Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature 417: 664−667. Monaghan, A.P., Kioschis, P., Wu, W., Zuniga, A., Bock, D., Poustka, A., Delius, H., and Niehrs, C. (1999). Dickkopf genes are co-ordinately expressed in mesodermal lineages. Mech. Dev. 87: 45−56. Nakamura, T., and Matsumoto, K. (2008). The functions and possible significance of Kremen as the gatekeeper of Wnt signalling in development and pathology. J. Cell. Mol. Med. 12: 391−408. Nakamura, T., Aoki, S., Kitajima, K., Takahashi, T., and Matsumoto, K. (2001). Molecular cloning and characterization of Kremen, a novel kringle-containing transmembrane protein. Biochim. Biophys. Acta 1518: 63−72. Nascone, N., and Mercola, M. (1997). Organizer induction determines left-right asymmetry in Xenopus. Dev. Biol. 189: 68−78. Niehrs, C. (2006). Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene 25: 7469−7481. Patthy, L. (1999). Genome evolution and the evolution of exon-shuffling—a review. Gene 238: 103−114. Rocheleau, C.E., Downs, W.D., Lin, R., Wittmann, C., Bei, Y., Cha,
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Y.H., Ali, M., Priess, J.R., and Mello, C.C. (1997). Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos. Cell 90: 707−716. Schubert, M., Holland, L.Z., Stokes, M.D., and Holland, N.D. (2001). Three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) associated with the tail bud: the evolution of somitogenesis in chordates. Dev. Biol. 240: 262−273. Schubert, M., Holland, L.Z., Panopoulou, G.D., Lehrach, H., and Holland, N.D. (2000). Characterization of amphioxus AmphiWnt8: insights into the evolution of patterning of the embryonic dorsoventral axis. Evol. Dev. 2: 85−92. Semenov, M.V., Tamai, K., Brott, B.K., Kuhl, M., Sokol, S., and He, X. (2001). Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Curr. Biol. 11: 951−961. Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596−1599. Thorpe, C.J., Schlesinger, A., Carter, J.C., and Bowerman, B. (1997). Wnt signaling polarizes an early C. elegans blastomere to distinguish endoderm from mesoderm. Cell 90: 695−705. Verna, J., Lodder, A., Lee, K., Vagts, A., and Ballester, R. (1997). A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 94: 13804−13809. Wikramanayake, A.H., Huang, L., and Klein, W.H. (1998). beta-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo. Proc. Natl. Acad. Sci. USA 95: 9343−9348. Wikramanayake, A.H., Hong, M., Lee, P.N., Pang, K., Byrum, C.A., Bince, J.M., Xu, R., and Martindale, M.Q. (2003). An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation. Nature 426: 446−450. Yamaguchi, T.P. (2001). Heads or tails: Wnts and anterior-posterior patterning. Curr. Biol. 11: R713−R724. Yu, J.K., Satou, Y., Holland, N.D., Shin, I.T., Kohara, Y., Satoh, N., Bronner-Fraser, M., and Holland, L.Z. (2007). Axial patterning in cephalochordates and the evolution of the organizer. Nature 445: 613−617. Zhang, Q.J., Zhong, J., Fang, S.H., and Wang, Y.Q. (2006). Branchiostoma japonicum and B. belcheri are distinct lancelets (cephalochordata) in Xiamen waters in China. Zool. Sci. 23: 573−579. Zhang, Y., and Mao, B. (2009). Developmental expression of an amphioxus (Branchiostoma belcheri) gene encoding a GATA transcription factor. Zoological Research 30: 137−143. Zhong, J., Zhang, Q.J., Xu, Q.S., Schubert, M., Laudet, V., and Wang, Y.Q. (2009). Complete mitochondrial genomes defining two distinct lancelet species in the West Pacific Ocean. Mar. Biol. Res. 5: 278−285.
GENETICS AND GENOMICS J. Genet. Genomics 37 (2010) 637−645
www.jgenetgenomics.org
Embryonic expression and evolutionary analysis of the amphioxus Dickkopf and Kremen family genes Yujun Zhang a, b, Bingyu Mao a, * a
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China b Graduate University of Chinese Academy of Sciences, Beijing 100049, China Received for publication 8 July 2010; revised 3 August 2010; accepted 17 August 2010
Abstract The secreted Wnt signaling inhibitor Dickkopf1 (Dkk1) plays key role in vertebrate head induction. Its receptor Kremen synergizes with Dkk1 in Wnt inhibition. Here we have carried out expression and functional studies of the Dkk and Kremen genes in amphioxus (Branchiostoma belcheri). During embryonic and larval development, BbDkk1/2/4 is expressed in the posterior mesoendoderm, anterior somatic mesoderm and the pharyngeal regions. Its expression becomes restricted to the pharyngeal region on the left side at larval stages. In 45 h larvae, BbDkk1/2/4 is expressed specifically in the cerebral vesicle. BbDkk3 was only detected at larval stages in the mid-intestine region. Seven Kremen related genes were identified in the genome of the Florida amphioxus (Branchiostoma floridae), clustered in 4 scaffolds, and are designated Kremen1-4 and Kremen-like 1-3, respectively. In B. belcheri, Kremen1 is strongly expressed in the mesoendoderm during early development and Kremen3 is expressed asymmetrically in spots in the larval pharyngeal region. In luciferase reporter assays, BbDkk1/2/4 can strongly inhibit Wnt signaling, while BbDkk3, BbKremen1 and BbKremen3 can not. No co-operative effect was observed between amphioxus Dkk1/2/4 and Kremens, suggesting that the interaction between Dkk and Kremen likely originated later during evolution. Keywords: amphioxus; Dickkopf; Kremen; evolution; expression pattern
Introduction Cannonical Wnt signaling pathway is well conserved during evolution and plays essential roles in the formation and patterning of the body axes in different animals (Nascone and Mercola, 1997; Wikramanayake et al., 1998; Yamaguchi, 2001; Croce and McClay, 2006). It also plays roles in germ layer formations in various animals (Rocheleau et al., 1997; Thorpe et al., 1997; Imai et al., 2000; Wikramanayake et al., 2003). The Dickkopf (Dkk) * Corresponding author. Tel: +86-871-519 8989; Fax: +86-871-519 3137. E-mail address: [email protected] DOI: 10.1016/S1673-8527(09)60082-5
family of secreted inhibitors of canonical Wnt signaling function in the patterning of the body plan from hydra to vertebrates (Glinka et al., 1998; Krupnik et al., 1999; Guder et al., 2006; Caneparo et al., 2007). In human, there are 4 Dkks: Dkk1, 2 and 4 can inhibit Wnts, while Dkk3 can not (Krupnik et al., 1999; Mao and Niehrs, 2003; Niehrs, 2006). The developmental role of Dkk3 in vertebrates is still poorly understood. Although widely expressed during embryonic development (Monaghan et al., 1999), Dkk3-deficient mice develop normally, are fertile and have only a mild phenotype (Barrantes Idel et al., 2006). Dkk1, 2 and 4 work through binding the Wnt receptors LRP5/6 and sequestering the Wnt ligands (Mao et
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al., 2001; Semenov et al., 2001). In vertebrates, Kremen1 and 2, two novel coreceptors for Dkk1 and Dkk2, have been shown to enhance the Wnt inhibitory role of Dkks (Mao et al., 2002; Mao and Niehrs, 2003; Nakamura and Matsumoto, 2008). Kremens are single-transmembrane proteins and have a unique extracellular structure containing a Kringle, a WSC and a CUB domain (Nakamura et al., 2001). However, Kremen-like genes have not been reported in invertebrates. How its unique structure evolved during evolution is an interesting question. In a previous study, Yu et al. (2007) studied the expression pattern of Dkk1/2/4 and Dkk3 in the Florida amphioxus (Branchiostoma floridae) and suggested that the anterior patterning mechanism through Wnt inhibition by Dkk is conserved in amphioxus. However, functional proof has been lacking. Here, we have cloned the Dickkopf genes in amphioxus Branchiostoma belcheri and studied their developmental expression patterns. We also studied the Kremen related genes in the genome of the Florida amphioxus and 7 genes were identified. The expression patterns of two Kremen genes were studied in B. belcheri embryos and larvae.
Materials and methods Embryos Adult amphioxus (B. belcheri) (Zhang et al., 2006; Zhong et al., 2009) were collected during the breeding season from the South China Sea near Beihai (Guangxi Province, China) and maintained in the laboratory. Naturally fertilized eggs were collected and cultured at room temperature. The developing embryos and larvae at desired stages were fixed in fresh 4% paraformaldehyde at room temperature for 30 min or at 4°C overnight, dehydrated in graded methanol and stored in 70% methanol at −20°C.
Cloning of amphioxus Dkk and Kremen genes The full open reading frames of BbDkk1/2/4 and 3 were cloned by RT-PCR using primers designed according to the BfDkk1/2/4 sequence (Yu et al., 2007) and the Dkk3 sequence (GenBank accession No. AY608670) of B. japonicum (Zhang et al., 2006; Zhong et al., 2009). The primers used are:
BbDkk1/2/4: 5′-CCGTAACTTGTCCACACCGTAGA-3′ and 5′-GTGCTTTGATGCGTCTTGTTGGG-3′; BbDkk3: 5′-CTCACTACTCACATCCCTGTCTCTA-3′ and 5′-ACCAGTGAGACATACATCGAAGAGT-3′. The BbDkk1/2/4 and BbDkk3 sequences have been submitted to GenBank under accession Nos. HM590023 and HM590024. The genome of amphioxus (B. floridae, http://genome. jgi-psf.org/Brafl1) was BLASTed using mouse Kremen proteins and 10 hits were found. GenomeScan (http://genes. mit.edu/genomescan.html) was used for predicting the Kremen genes aided by EST database searches and manual correction (Supplemental document). Phylogenetic tree was constructed using MAGA4 (Tamura et al., 2007) using Neigbour-Joining method. Full length sequences of BbKremen1 and 2 and partial sequences of BbKremen4 and BbKremen-like 1 and 3 were cloned using RT-PCR. The primers used are: BbKremen1: 5′-ATGGGGCCAGGAGAGTTCGGAG-3′ and 5′-TCACAATTCTCCCAGCACAGCA-3′; BbKremen2: 5′-ATGGCATCTACCCACGTTTGGATGT-3′ and 5′-CTATTCGCTACTGTAACCATGAGCG-3′; BbKremen4: 5′-CTACAGAGGAAACCAGTCGGAGC-3′ and 5′-ACCCGCCACACCGTCCCATCTTC-3′; BbKremen-like1: 5′-CCTACCGATCACCGACTGTGCT-3′ and 5′-GTACTCTACAATCGTCAAACTATCG-3′; BbKremen-like3: 5′-TGACCCAGACAAGGTTATCAA GTTC-3′ and 5′-CTCGTAGTGCGCATGCTCGTTCTGT-3′. The PCR products were cloned into pBS-T vector (Tiangen, China) and sequenced. The BbKremens sequences have been submitted to GenBank under accession No. HM590025–HM590029. For functional analysis, the full length open reading frames of BbDkk1/2/4, BbDkk3, BbKremen1 and BbKremen2 were cloned into the pCS2 + vector.
In situ hybridization and luciferase reporter assays In situ hybridization in amphioxus embryos and larvae was carried out as described (Zhang and Mao, 2009). Luciferase reporter assays in HEK 293T cells were done in 96 well plates at least in triplicates as described (Mao et al., 2001). Plasmid concentrations used (ng/well): superTOPFLASH, 80; pTK-Renilla, 8; mWnt1, 64; mfrizzled8, 8; hLRP6, 2, unless indicated otherwise. Luciferase activity was normalized against Renilla activity.
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Results Cloning and expression of amphioxus Dkk genes Two Dkk genes were cloned using RT-PCR from B. belcheri, one homologous to vertebrate Dkk1/2/4 and the other one homologous to vertebrate Dkk3. The predicted amphioxus Dkk1/2/4 protein contains two typical cysteine-rich domains (CRD), as its vertebrate ho mologs. In a phylogenetic tree analysis, BbDKK1/2/4 is clustered together with the Dkk-1, -2 and -4 genes while BbDkk3 is clustered in the Dkk3 branch (data not shown; Yu et al., 2007). The amphioxus Dkk3, however, contains only the second CRD domain, unlike the Dkk3 proteins in any other species including hydra and sea urchin (Croce et al., 2006). The amphioxus Dkk3 might have undergone lineage specific diversifycation and lost the first CRD domain. Interestingly, the Dkk1/2/4 in hydra and sea urchin also contain only the CRD2 domain (Croce et al., 2006). To gain insight into their developmental roles, in situ hybridization was carried out using amphioxus embryos and larvae. Similar as in B. floridae (Yu et al., 2007), BbDkk1/2/4 was detected in the mesendoderm around the blastopore at the gastrula stage (Fig. 1, A and A′). At early neurula stage (2−4 somites), the expression of Dkk1/2/4 is confined to the posterior mesoendoderm (Fig.1, B and B′). At 6-somite stage, its expression remains strong in the posterior blastopore region and also turns on in the anterior mesendoderm, forming two stripes anterior to the first and second somites, respectively (Fig. 1, C and C′). In 14 h larvae, its expression in the posterior mesoderm turns into two stripes on both sides. The expression stripes in the anterior mesoderm become stronger. Interestingly, its expression in the anterior region starts to become asymmetric, its expression on right side becomes slightly weaker than on the left side (Fig. 1, D and D′). Later on, its expression becomes undetectable on the right side in the anterior region and only weakly at the posterior region, while remains strong on the left side in both anterior and posterior mesoderm (Fig. 1, E and E′). In 18 h larvae, its expression is no longer detected in the posterior mesoderm, and remains only in two patches in the anterior pharyngeal region on the left side (Fig. 1, F and F′). In 45 h larvae, BbDkk1/2/4 expresses specifically in the cerebral vesicle (Fig. 1G).
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BbDkk3, however, is not expressed in the embryonic developmental stages (data not shown). From 24 h larval stage, it becomes strongly expressed in the mid-intestine region (Fig. 1, H and I). In B. floridae, Dkk3 is strongly expressed in the anterior ectoderm at the onset of gastrulation and also weakly in the mesendoderm by mid-gastrula stage (Yu et al., 2007). The different expression pattern of Dkk3 in the two species likely reflects different roles of Dkk3 in the two species. In fact, the protein sequence identity between BbDkk3 and BfDkk3 is only 51% (data not shown), suggesting strong lineage diversification of amphioxus Dkk3.
The amphioxus Kremen genes We extracted the Kremen related sequences from the genome of the Florida amphioxus (B. floridae). Surprisingly, 10 Kremen like sequences were found in the amphioxus genome, located in four scaffolds (Fig. 2A). According to the predicted domain structures, amphioxus Kremen proteins can be divided into 2 groups. One group contains all three types of domains (Kringle, WSC and CUB, Fig. 2B). The 4 genes form a cluster locating on scaffold 36, and are designated Kremen1, 2, 3 and 4 sequentially (Fig. 2A). Interestingly, 3 of the 4 Kremen proteins in this group contain two Kringle domains instead of one. The predicted BfKremen proteins show less than 50% sequence identity between each other and the 4 genes should represent independent ones. In phylogenetic analysis, these proteins show similar homology to vertebrate Kremen1 and Kremen2 (Fig. 2C). The second group of predicted Kremen related proteins are lack of the Kringle domain (Fig. 2B) and designated Kremen-like (Krl) proteins. The 3 genes on scaffold 149 show close homology to the one on scaffold 152 and the two genes on scaffold 89 respectively, both in sequence and gene structure (Supplemental Table1). Since the B. floridae genome database contains sequences of two haplotypes (Li et al., 2007), we suggest that the 6 genes might represent different alleles of three independent genes. The six genes are named Krl1a, Krl1b, Krl2a, Krl2b, Krl3a and Krl3b, respectively (Fig. 2, A and B). Structurally, the amphioxus Kremen-like proteins are more similar to the Kremen-like proteins in sea urchin (Strongylocentrotus purpuratus), which also contain only the WSC and CUB domains (Fig. 2, B and C; Croce et al., 2006).
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Fig. 1. Developmental expression of Dkk1/2/4 (A–G) and Dkk3 (H, I) in B. belcheri embryos and larvae. A, A′: mid-gastrula stage. A: lateral view, animal pole to the left. A′: vegetal view. B, B′: neurula stage. C, C′: late neurula stage. D, D′: 14 h larvae. E, E′: 16 h larvae. F, F′: 18 h larvae. G: 45 h larva. H: 24 h larva. I: 45 h larva. B−G, lateral views, anterior to the left; B′–F′: dorsal views, anterior to the left. The arrowheads indicate the expression domains. Scale bar = 50 μm.
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Fig. 2. Structures and phylogenetic analysis of amphioxus Kremens. A: the distribution of the predicted Kremen related genes on the scaffolds of B. floridae genome. B: domain structures of the predicted amphioxus Kremen and Kremen-like proteins. C: phylogenetic analysis of the Kremen related proteins in sea urchin, amphioxus, tunicate, Xenopus, mouse and human. Bf, Branchiostoma belcheri; Ci, Ciona intestinalis; Homo, Homo sapiens; Mus, Mus musculus; Sp, Strongylocentrotus purpuratus; Xl, Xenopus laevis. The Kremen proteins used are: Mus Kremen1, NP_115772 (GenBank accession No.); Homo Kremen1, CAQ07690; Xl Kremen1, NP_001082145; Mus Kremen2, NP_082692; Homo Kremen2, BAC00872; Xl Kremen2, NP_001082352; Ci Kremen, XP_002125526; Sp Kremen-like 1 and 2, Glean IDs 25250 and 25249 (http://goblet.molgen.mpg.de/cgi-bin/seaurchin-genombase.cgi).
Developmental expression of amphioxus Kremens Primers were designed according to predicted Bf-Kremen sequences to amplify the Kremen sequences in B. belcheri. Using cDNA from 10-somite stage amphioxus embryos, 5 fragments of BbKremen cDNAs were cloned: Krm1, Krm2, Krm4, Krl1 and Krl3. In situ hybridization was performed in amphioxus embryos and larvae at different developmental stages using different probes. Krm1 is expressed widely in the mesendoderm from early neurula stage on. At later stages, it is expressed in the axial somatic mesoderm, the notochord and dorsal endoderm (Fig. 3, A–D). Krm2, however, shows restricted expression domains in the amphioxus larvae. At early larval stage (18 h), Krm2 is expressed in 3 spots in the pharyngeal region asymmetrically, with two spots on the right side and one on the left side (Fig. 3, E and F). Similar expression pattern remains in 24 h larvae (Fig. 3, G and H). The differential expression of Krm1 and 2 suggests clear functional diversification of the
two genes. For Krm4, Krl1 and Krl3, no clear expression pattern was detected at the stages examined (data not shown).
Amphioxus Kremens do not synergize with Dkk1/2/4 in Wnt inhibition We cloned the full-length open reading frame of BbKrm1 and BbKrm2 for further functional studies, since their encoded protein structures are similar to the vertebrate ones. We first tested whether amphioxus Dkk1/2/4, Dkk3, Krm1 or Krm2 could inhibit Wnt signaling in 293T cells and in Xenopus embryos. As expected, BbDkk1/2/4 strongly inhibits Wnt signaling in luciferase reporter assay while BbDkk3 can not (Fig. 4A). When ectopically expressed in Xenopus embryos, BbDkk1/2/4 induces big-head phenotype (data not shown), indicating strong Wnt signaling inhibition. In vertebrates, Kremen strongly enhances the activity of Dkk1/2/4 in Wnt inhibition. We
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tested whether this is the case with amphioxus Dkk1/2/4 and Kremen. Amphioxus Dkk1/2/4 is able to cooperate with Xenopus Kremen1 to inhibit Wnt signaling but not
amphioxus Krm1 or Krm2 (Fig. 4B). Similarly, Xenopus Dkk1 also fails to cooperate with the two amphioxus Kremens in this assay (Fig. 4B).
Fig. 3. Developmental expression of Kremen1 (A–D) and Kremen2 (E–H) in B. belcheri embryos and larvae. A: early neurula stage. B: 14 h larva. C: 16 h larva. D: 20 h larva. E: 18 h larva. F: dorsal view of E. G: 24 h larva. H: dorsal view of the head region of the larva in G. A–E, G, lateral views, anterior to the left. The arrowheads indicate the expression domains.
Fig. 4. Luciferase assays showing the activities of amphioxus Dkk1/2/4 and Kremens in Wnt inhibition. A: BbDkk1/2/4 but not Dkk3 can inhibit Wnt signaling in 293T cells. W/F/L, the cells were transfected with combined mWnt1, mFz8 and hLRP6 to stimulate the Wnt signaling. B: amphioxus Kremens fail to synergize with amphioxus or Xenopus Dkks to inhibit Wnt signaling.
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Discussion In amphioxus, the Wnt genes are widely expressed in various tissues during embryonic development, including the blastopore (Wnt-8, Wnt-11, Wnt-1, Wnt-4) and mesoendoderm (Wnt-4, Wnt-7) at gastrula stage (Holland et al., 2000; Schubert et al., 2000, 2001; Holland, 2002; Yu et al., 2007). The high level distribution of nuclear β-catenin around the blsatopore at gastrula stage also suggested the activation of Wnt signaling in this area (Holland et al., 2005). The expression of BbDkk1/2/4 in the mesendoderm at gastrula stage suggests that it might be involved in germ layer formation at this stage. The local expression of BbDkk1/2/4 in the posterior mesoendoderm at early neurula stage (Fig. 1B) might help to create a Wnt-free region required for proper cell differentiation. In vertebrates, canonical Wnt signaling plays crucial role in the anterior-posterior patterning of the embryo. Its inhibition in the anterior region by secreted Wnt inhibitors (including Dkk1 and sFRPs) is essential for the development of the anterior structures, including the forebrain, heart and foregut. In Xenopus, Dkk1 is expressed in the prechodal plate mesoderm at neurula stage (Glinka et al., 1998), which lies under the developing forebrain. In the mouse, it is expressed in the anterior mesendoderm at gastrula stage (Glinka et al., 1998). In both species, blocking the function of Dkk1 by specific antibody or genetic knockout leads to loss of the head structure. In amphioxus, however, Dkk1/2/4 is not expressed in the anterior midline mesoenderm at neurula stages (Fig. 1, B and C). Whether it is involved in the anterior patterning in amphioxus awaits further functional studies. Although Dkk3 is expressed in the anterior mesoendoderm in Florida amphioxus (Yu et al., 2007), we did not detect such expression patterns in B. Belcheri. Anyway, Dkk3 cannot inhibit Wnt signaling and is less likely to be involved in anterior-posterior patterning. Amphioxus Dkk1/2/4 is expressed in the posterior forming somites at neurula and early larval stages, which is also the case in Xenopus, suggesting conserved roles for Dkk1 in somite formation. At larval stage, BbDkk1/2/4 and Kremen2 are asymmetrically expressed in the pharyngeal region. Many genes show left-right asymmetric expression pattern in amphioxus, including members of the Hedgehog, Pax, Nkx and Notch gene families (Boorman and Shimeld, 2002). Our data suggest that the regulation of Wnt signaling might be involved in the control of the left-right asymmetry in amphioxus.
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The evolution of the Kremen genes is an interesting issue. The vertebrate Kremen proteins have typical modular structures and contain 3 extracellular domains, the Kringle, WSC and CUB domains. The predicted sea urchin Kremen-like proteins contain only the WSC and CUB domains. In amphioxus, the Kremen related genes has clearly undergone lineage specific expansion and contain both kinds of structures. Amphioxus Kremen-like proteins lack the Kringle domain, which is closer to sea urchin Kremen, while the 4 Kremen proteins contain all the 3 type of domains (Kringle, WSC and CUB). In amphioxus, a fourth kind of domain (the CCP domain) also present in all the Kremen related proteins, which is not seen in any other species. Interestingly, each protein domain (including the Kringle, WSC, CUB, and CCP domains) is encoded by one or two independent exons (Supplemental Table 1). This situation is also true in Ciona and human (Supplemental Table 2), suggesting that this family of genes might have evolved through the exon shuffling mechanism (Patthy, 1999). A possible model for the evolution of this family of genes is proposed in Fig. 5. The prototype of the Kremen protein might be similar to the WSC proteins in yeast, which are single transmembrane proteins containing an extracellular WSC domain and are required for the maintenance of cell wall integrity (Verna et al., 1997). In the deuterostome (including sea urchin), the CUB domain encoding exon shuffled into the gene to create the Kremen-like genes. During the origin of chordates, the Kringle domain encoding exon was further added to the Kremen-like genes to create the typical Kremen structures in tunicates and vertebrate. The amphioxus Kremen genes seem to have taken an independent evolution pathway. The CCP domain was first added to create the Kremen-like proteins in amphioxus, and a second exon-shuffling event created the Kremen genes in amphioxus today. This scenario supports the view that tunicates are evolutionarily closer than amphioxus to vertebrates (Delsuc et al., 2006). The developmental roles of amphioxus Kremens await further studies. In Xenopus, Kremen is involved in neural crest development (Hassler et al., 2007). In mouse, double knockout of Kremen1 and 2 leads to limb defects and high bone density (Ellwanger et al., 2008). Although genetic interaction between Dkk1 and Kremen has been confirmed in mouse (Ellwanger et al., 2008), Kremens are not universally required for Dkk1 function. In amphioxus, Dkk1/2/4 and Kremens show different embryonic expression patterns and lack cooperation in Wnt inhibition assay.
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Fig. 5. A model for the evolution of the Kremen related proteins in eukaryotes.
The interaction between Dkk1 and Kremen is more likely an accidental event during vertebrate origin. It will be of interest to test whether Dkk1 and Kremen interact in tunicates, which have a typical vertebrate type Kremen.
Acknowledgements This work was supported by the grant from the Innovation Project of Chinese Academy of Sciences (No. KSCX2-YW-R-090). We thank Prof. Junyuan Chen (Nanjing University) for amphioxus materials.
Supplemental data Supplemental document and Tables 1 and 2 associated with this article can be found in the online version at www.jgenetgenomics.org.
References Barrantes Idel, B., Montero-Pedrazuela, A., Guadano-Ferraz, A., Obregon, M.J., Martinez de Mena, R., Gailus-Durner, V., Fuchs, H., Franz, T.J., Kalaydjiev, S., Klempt, M., Holter, S., Rathkolb, B., Reinhard, C., Morreale de Escobar, G., Bernal, J., Busch, D.H., Wurst, W., Wolf, E., Schulz, H., Shtrom, S., Greiner, E., Hrabe de Angelis, M., Westphal, H., and Niehrs, C. (2006). Generation and characterization of dickkopf3 mutant mice. Mol. Cell. Biol. 26:
2317−2326. Boorman, C.J., and Shimeld, S.M. (2002). The evolution of left-right asymmetry in chordates. Bioessays 24: 1004−1011. Caneparo, L., Huang, Y.L., Staudt, N., Tada, M., Ahrendt, R., Kazanskaya, O., Niehrs, C., and Houart, C. (2007). Dickkopf-1 regulates gastrulation movements by coordinated modulation of Wnt/beta catenin and Wnt/PCP activities, through interaction with the Dally-like homolog Knypek. Genes Dev. 21: 465−480. Croce, J.C., and McClay, D.R. (2006). The canonical Wnt pathway in embryonic axis polarity. Semin. Cell Dev. Biol. 17: 168−174. Croce, J.C., Wu, S.Y., Byrum, C., Xu, R., Duloquin, L., Wikramanayake, A.H., Gache, C., and McClay, D.R. (2006). A genome-wide survey of the evolutionarily conserved Wnt pathways in the sea urchin Strongylocentrotus purpuratus. Dev. Biol. 300: 121−131. Delsuc, F., Brinkmann, H., Chourrout, D., and Philippe, H. (2006). Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 439: 965−968. Ellwanger, K., Saito, H., Clement-Lacroix, P., Maltry, N., Niedermeyer, J., Lee, W.K., Baron, R., Rawadi, G., Westphal, H., and Niehrs, C. (2008). Targeted disruption of the Wnt regulator Kremen induces limb defects and high bone density. Mol. Cell. Biol. 28: 4875−4882. Glinka, A., Wu, W., Delius, H., Monaghan, A.P., Blumenstock, C., and Niehrs, C. (1998). Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391: 357−362. Guder, C., Pinho, S., Nacak, T.G., Schmidt, H.A., Hobmayer, B., Niehrs, C., and Holstein, T.W. (2006). An ancient Wnt-Dickkopf antagonism in Hydra. Development 133: 901−911. Hassler, C., Cruciat, C.M., Huang, Y.L., Kuriyama, S., Mayor, R., and Niehrs, C. (2007). Kremen is required for neural crest induction in Xenopus and promotes LRP6-mediated Wnt signaling. Development 134: 4255−4263. Holland, L.Z. (2002). Heads or tails? Amphioxus and the evolution of
Yujun Zhang et al. / Journal of Genetics and Genomics 37 (2010) 637−645
anterior-posterior patterning in deuterostomes. Dev. Biol. 241: 209−228. Holland, L.Z., Holland, N.N., and Schubert, M. (2000). Developmental expression of AmphiWnt1, an amphioxus gene in the Wnt1/wingless subfamily. Dev. Genes Evol. 210: 522−524. Holland, L.Z., Panfilio, K.A., Chastain, R., Schubert, M., and Holland, N.D. (2005). Nuclear beta-catenin promotes non-neural ectoderm and posterior cell fates in amphioxus embryos. Dev. Dyn. 233: 1430−1443. Imai, K., Takada, N., Satoh, N., and Satou, Y. (2000). β-catenin mediates the specification of endoderm cells in ascidian embryos. Development 127: 3009−3020. Krupnik, V.E., Sharp, J.D., Jiang, C., Robison, K., Chickering, T.W., Amaravadi, L., Brown, D.E., Guyot, D., Mays, G., Leiby, K., Chang, B., Duong, T., Goodearl, A.D., Gearing, D.P., Sokol, S.Y., and McCarthy, S.A. (1999). Functional and structural diversity of the human Dickkopf gene family. Gene 238: 301−313. Li, G., Zhang, Q.J., Ji, Z.L., and Wang, Y.Q. (2007). Origin and evolution of vertebrate ABCA genes: a story from amphioxus. Gene 405: 88−95. Mao, B., and Niehrs, C. (2003). Kremen2 modulates Dickkopf2 activity during Wnt/LRP6 signaling. Gene 302: 179−183. Mao, B., Wu, W., Li, Y., Hoppe, D., Stannek, P., Glinka, A., and Niehrs, C. (2001). LDL-receptor-related protein 6 is a receptor for Dickkopf proteins. Nature 411: 321−325. Mao, B., Wu, W., Davidson, G., Marhold, J., Li, M., Mechler, B.M., Delius, H., Hoppe, D., Stannek, P., Walter, C., Glinka, A., and Niehrs, C. (2002). Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature 417: 664−667. Monaghan, A.P., Kioschis, P., Wu, W., Zuniga, A., Bock, D., Poustka, A., Delius, H., and Niehrs, C. (1999). Dickkopf genes are co-ordinately expressed in mesodermal lineages. Mech. Dev. 87: 45−56. Nakamura, T., and Matsumoto, K. (2008). The functions and possible significance of Kremen as the gatekeeper of Wnt signalling in development and pathology. J. Cell. Mol. Med. 12: 391−408. Nakamura, T., Aoki, S., Kitajima, K., Takahashi, T., and Matsumoto, K. (2001). Molecular cloning and characterization of Kremen, a novel kringle-containing transmembrane protein. Biochim. Biophys. Acta 1518: 63−72. Nascone, N., and Mercola, M. (1997). Organizer induction determines left-right asymmetry in Xenopus. Dev. Biol. 189: 68−78. Niehrs, C. (2006). Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene 25: 7469−7481. Patthy, L. (1999). Genome evolution and the evolution of exon-shuffling—a review. Gene 238: 103−114. Rocheleau, C.E., Downs, W.D., Lin, R., Wittmann, C., Bei, Y., Cha,
645
Y.H., Ali, M., Priess, J.R., and Mello, C.C. (1997). Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos. Cell 90: 707−716. Schubert, M., Holland, L.Z., Stokes, M.D., and Holland, N.D. (2001). Three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) associated with the tail bud: the evolution of somitogenesis in chordates. Dev. Biol. 240: 262−273. Schubert, M., Holland, L.Z., Panopoulou, G.D., Lehrach, H., and Holland, N.D. (2000). Characterization of amphioxus AmphiWnt8: insights into the evolution of patterning of the embryonic dorsoventral axis. Evol. Dev. 2: 85−92. Semenov, M.V., Tamai, K., Brott, B.K., Kuhl, M., Sokol, S., and He, X. (2001). Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Curr. Biol. 11: 951−961. Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596−1599. Thorpe, C.J., Schlesinger, A., Carter, J.C., and Bowerman, B. (1997). Wnt signaling polarizes an early C. elegans blastomere to distinguish endoderm from mesoderm. Cell 90: 695−705. Verna, J., Lodder, A., Lee, K., Vagts, A., and Ballester, R. (1997). A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 94: 13804−13809. Wikramanayake, A.H., Huang, L., and Klein, W.H. (1998). beta-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo. Proc. Natl. Acad. Sci. USA 95: 9343−9348. Wikramanayake, A.H., Hong, M., Lee, P.N., Pang, K., Byrum, C.A., Bince, J.M., Xu, R., and Martindale, M.Q. (2003). An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation. Nature 426: 446−450. Yamaguchi, T.P. (2001). Heads or tails: Wnts and anterior-posterior patterning. Curr. Biol. 11: R713−R724. Yu, J.K., Satou, Y., Holland, N.D., Shin, I.T., Kohara, Y., Satoh, N., Bronner-Fraser, M., and Holland, L.Z. (2007). Axial patterning in cephalochordates and the evolution of the organizer. Nature 445: 613−617. Zhang, Q.J., Zhong, J., Fang, S.H., and Wang, Y.Q. (2006). Branchiostoma japonicum and B. belcheri are distinct lancelets (cephalochordata) in Xiamen waters in China. Zool. Sci. 23: 573−579. Zhang, Y., and Mao, B. (2009). Developmental expression of an amphioxus (Branchiostoma belcheri) gene encoding a GATA transcription factor. Zoological Research 30: 137−143. Zhong, J., Zhang, Q.J., Xu, Q.S., Schubert, M., Laudet, V., and Wang, Y.Q. (2009). Complete mitochondrial genomes defining two distinct lancelet species in the West Pacific Ocean. Mar. Biol. Res. 5: 278−285.