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Dev Genes Evol (2003) 212:563–570 DOI 10.1007/s00427-002-0272-x ORIGINAL ARTICLE Corinna B. Scholz · Ulrich Technau The ancestral role of Brachyury: expression of NemBra1 in the basal cnidarian Nematostella vectensis (Anthozoa) Received: 27 June 2002 / Accepted: 3 September 2002 / Published online: 20 November 2002 Springer-Verlag 2002 Abstract The T-Box transcription factor Brachyury plays important roles in the development of all bilateral animals examined so far. In order to understand the ancestral function of Brachyury we cloned NemBra1, a Brachyury homolog from the anthozoan sea anemone Nematostella vectensis. Anthozoa are considered the basal group among the Cnidaria. First NemBra1 expression could be detected at the blastula/gastrula transition and gene activity persists until adulthood of the animals. In situ hybridization shows that NemBra1 expression in gastrulae and early planula larvae is restricted to a circle around the blastopore. When the larvae begin to metamorphose into primary polyps, the expression zone extends into the developing mesenteries. In adult polyps Brachyury expression persists in the mesenteries, but is excluded from the septal filament and the differentiated retractor muscles, which also develop from the mesenteries. We conclude that the ancestral function of Brachyury was in specifying the blastopore and its endodermal derivatives. Keywords Mesoderm · Cnidaria · Brachyury · Blastopore · Nematostella vectensis Introduction The evolutionary transition between the diploblastic and the triploblastic animals is marked by the emergence of the mesoderm. This major step in evolution is correlated with an enormous diversification of animal body plans. It is therefore conceivable that genes involved in the evolution of the mesoderm were also important players in the evolution of animal body plans. In order to Edited by D. Tautz C.B. Scholz · U. Technau ()) Molecular Cell Biology, Institute of Zoology, Darmstadt University of Technology, Schnittspahnstrasse 10, 64287 Darmstadt, Germany e-mail: [email protected] Tel.: +49-6151-166244 Fax: +49-6151-166077 understand the diplo-triploblastic transition on a molecular level, we started cloning genes involved in mesoderm formation in Bilateria from diploblastic animals. In vertebrates one of the key transcription factors involved in regulation of mesoderm differentiation is the T-box gene Brachyury (Hermann et al. 1990; Beddington et al. 1992; Kispert et al. 1995; O’Reilly et al. 1995; reviewed in Smith 1997, 1999). Brachyury is first expressed pan-mesodermally in the marginal zone and becomes restricted to the axial mesoderm, i.e. the notochord, upon gastrulation (Wilkinson et al. 1990). Vertebrate Brachyury has a conserved dual role in differentiation of the posterior mesoderm and axis elongation which seems to be a consequence of its role in cell motility (Wilkinson et al. 1990; Smith et al. 1991; Conlon and Smith 1999; Beddington et al. 1992). While notochord expression is conserved among all chordates (Yasuo and Satoh 1998; Bassham and Postlethwait 2000) comparative expression analysis of Brachyury homologs in basal deuterostomes (echinoderms and hemichordates) suggest that the ancestral expression in basal deuterostomes was in fore- and hindgut formation (Peterson et al. 1999a, b; Shoguchi et al. 1999; Tagawa et al. 1998; Croce et al. 2001). After metamorphosis Brachyury expression appears also in meso- and metacoel, the more posterior parts of the mesoderm (Peterson et al. 1999b). Thus, Brachyury expression in mesodermal tissues and in the developing gut appears to be ancestral within the deuterostomes. In the protostome Drosophila and other insects the Brachyury homolog T-related gene (Trg) is expressed in the hindgut of developing larvae (Kispert et al. 1994). Recent studies showed that Trg is also required for the specification of the visceral caudal mesoderm (Kusch and Reuter 1999). The ancestral function of Brachyury in the Bilateria became evident by the recent expression analysis in a member of the second major protostome clade, the Lophotrochozoa. In the polychaete annelid Platynereis dumerilii the Brachyury homolog Pd-bra is expressed around the slit-like blastopore, which develops into foreand hindgut, similar to the gut formation in basal 564 deuterostomes, indicating a homology of these structures (Arendt et al. 2001). The marine gastropod Patella vulgata also shows Brachyury expression at the posterior edge of the mouth-forming blastopore and later along the AP axis (Lartillot et al. 2002). Likewise, the arrow worm Paraspadella gotoi expresses Brachyury at the blastopore, similar to embryos of both Deuterostomia and Protostomia (Takada et al. 2002). In conclusion, the common ancestor of all bilaterians most likely first expressed Brachyury in the blastopore which developed into foreand hindgut (Arendt et al. 2001; Technau 2001). As a step to resolve the evolutionary origin of this gene and its basic function before its implication in mesoderm formation, a Brachyury homolog (HyBra1) was isolated from the diploblastic cnidarian Hydra (Technau and Bode 1999). HyBra1 is expressed in the endoderm of the polyp’s head (i.e. the hypostome) and acts very early in head formation. However, the hydrozoan Hydra is considered a rather derived organism among the Cnidaria and a connection between the hypostome of the adult hydra polyp and the blastopore of Bilateria is not obvious. Also, embryogenesis in Hydra does not offer any clue in this respect. Hydra embryos gastrulate by multipolar immigration, then form a cuticle and go into a diapause of variable length. The young polyps hatch directly from the cuticle stage, thereby skipping the planula stage, which is the typical larva of cnidarians (Martin et al. 1997). A more suitable organism to study gene expression in early embryos and for tracing back ancestral gene functions is the sea anemone Nematostella vectensis (Hand and Uhlinger 1992; Fritzenwanker and Technau 2002). Nematostella belongs to the Anthozoa, which are considered more basal than the hydrozoan Hydra (Bridge et al. 1992, 1995; Collins 2002). The Nematostella embryo develops via a coeloblastula and an invagination gastrula into a planula larva. During metamorphosis into young polyps they develop the hypostome with mouth and tentacles at their former posterior end, which is the site of gastrulation (Hand and Uhlinger 1992). Here we present the isolation of the Nematostella Brachyury homolog, NemBra1, and show the first in situ hybridizations in Nematostella. The analysis supports the view of an ancestral function of Brachyury in designating the blastoporal region and the tissue derived from the blastopore. Materials and methods Animal culture Adult polyps of N. vectensis were kept in Nematostella medium (NM; 1/3 artificial seawater; Tropic Marin, Dreieich) at 18C and fed four times per week with nauplius larvae of Artemia salina. Spawning was induced as described (Fritzenwanker and Technau 2002). The medium was changed once a week. Cloning of NemBra1 and a b-actin fragment The NemBra1 fragment was amplified from embryonic first strand cDNA by PCR with degenerate primers (Bra-1: 5' AYGGNMGNMGNATGTTYCC 3') and Bra-3: 5' RAANSCYTTNGCRAANGG 3'). PCR conditions were: 3 min at 94C (1 cycle); 1 min at 94C, 1 min at 42C, 1 min at 72C (35 cycles); 10 min at 72C. A fragment of the expected size was obtained, cloned into TA cloning vector TOPOII (Invitrogen) and sequenced. Full length NemBra1 (GenBank accession AF540387) was obtained by 3' and 5' RACE (Frohman 1995; Gene Racer Kit, Invitrogen). The resulting RACE sequences were used to design specific primers and to amplify the full-length clone from first strand cDNA. The actin fragment (GenBank accession AF327845) was obtained by nested PCR with degenerate primers (Act5': 5' GGNGTNATGGTNGGNATG 3'; Act3': 5' DATCCACATYTGYTGRAA 3'; nested ActDeg5.2: 5' AARATGACNCARATHATGTT 3'; nested ActSpez3: 5' GCGGTCGGCGATGCCTGG 3') from first strand cDNA. PCR was carried out as follows: 3 min at 94C (1 cycle); 1 min at 94C, 1 min at 44C, 1 min at 72C (35 cycles); 5 min at 72C. The amplified fragment was cloned into TA cloning vector pGEM-T (Promega) and sequenced. RT-PCR expression analysis RT-PCR expression analysis was performed on cDNA of different embryonic stages using specific primers against NemBra1 and actin [NemBra1: NemBra2/5 (5' ATAAACAAAACTCTGGGGGAC 3') and NemBra2/31 (5' TTTAGACTCGTGATCTCTTCG 3'); actin: ActSpez5 (5' GCTAACACTGTCCTGTCT 3') and ActSpez3'A (5' TGGAAGGTGGACAGGGAA 3')]. The PCR conditions for NemBra1 were: 3 min at 94C (1 cycle); 1 min at 94C, 1 min at 46C, 1 min at 72C (35 cycles); 5 min at 72C. In situ hybridization The in situ hybridization protocol developed for Nematostella was based on a protocol for Hydra (Grens et al. 1995), however, with several important modifications. The jelly surrounding the embryos was dissolved by treatment with 2% cysteine in NM pH 7.6 for 15– 20 min on a rotary shaker. Isolated embryos were fixed in 1.25% glutaraldehyde/4% paraformaldehyde (in NM) overnight at 4C. The samples were stored in methanol at –20C. Primary polyps were allowed to relax in NM prior to fixation. The animals were anesthetized by carefully adding an equal volume of 7.13% MgCl2 and incubating the animals for 8–10 min at room temperature. Then the animals were cauterized with 2% HCl in NM for 5 min at 4C, followed by several washes in NM, and were fixed with 4% PFA/ 1.25% glutaraldehyde. Fixed animals were rehydrated by a series of washes in methanol/PBS/0.02% Triton X-100 (PBT; 100%, 75%, 50%, 25%, twice at 0% methanol/PBT) for 5 min each step and were treated with proteinase K (20 g/ml) for 18 min, followed by 0.4% glycine/PBT, two washes in 0.1 M triethanolamine and two washes in 0.25% (v/v) acetic anhydride/0.1 M triethanolamine. The specimens were washed twice in PBT and refixed in 4% paraformaldehyde for 20 min. After several PBT washes the animals were transferred into hybridization solution (50% formamide, 4 SSC, 5 Denhardt’s, 0.1% CHAPS, 200 g/ml yeast RNA, 100 g/ml heparin and 0.1% Tween 20) incubating for 10 min in PBT/hybridization solution (1:1) and 10 min in hybridization solution at room temperature (RT). Prehybridization was done at 55C for at least 2 h. Hybridization was carried out at 55C for 36– 60 h with a final probe concentration of 0.25 ng/l. Free probe was removed by nine changes of 50% formamide/4 SSC/0.1% Tween at 55C for 1 h each. In the last washing step samples were cooled down to RT. After two washes in PBT and one washing step in MAB (100 mM maleic acid/150 mM NaCl; pH 7.5) the samples were blocked in blocking solution (Roche) at RT for at least 2 h. Animals were incubated overnight at 4C in anti DIG-APconjugated antibody (Roche; 1:2,000 in blocking solution). After 565 several washes with MAB over 24 h the specimens were transferred into NTMT (100 mM NaCl; 100 mM Tris-HCl, pH 9.5; 50 mM MgCl2; 0.1% Tween 20; 1 mM levamisol). Detection was carried out with NBT/BCIP (Roche) in a 1:50 dilution in NTMT at 37C. The color reaction was stopped in ethanol. Vibratome sections After in situ hybridization adult polyps were mounted in 14% BSA/ 0.44% gelatine in PBS/2.5% glutaraldehyde. Fifteen minutes after fixation, blocks were cut in the vibratome in slices of 50–100 m thickness. Results Isolation of the Brachyury homolog NemBra1 and an actin homolog Nemactin from N. vectensis Using PCR and RACE we cloned a homolog of the T-box gene Brachyury from N. vectensis. As a positive control in expression analysis by RT-PCR and in situ hybridization experiments, we also cloned a 585-bp fragment of an actin by PCR. The Nematostella actin fragment shows 97% amino acid identity to Xenopus cytosolic b-actin. The cDNA sequence of the Brachyury homolog contains 1,763 bp. Conceptual translation of the single open reading frame predicts a protein of 480 amino acids. An alignment with T-domains of other Brachyury proteins shows that the T-domain of Nematostella Brachyury is 82% identical to that of Xenopus Brachyury and shares all conserved motifs characteristic for Brachyury homologs (Fig. 1A). By comparison, the carboxyterminal activation domain shows only 21% amino acid identity to the Xenopus protein. A phylogenetic analysis clearly shows that Nematostella Brachyury belongs to the Brachyury subfamily of T-box proteins (Fig. 1B). Within the Brachyury proteins, Nematostella Brachyury clusters with other cnidarian Brachyury homologs, for instance the Hydra ortholog HyBra1 (Fig. 1B). However, it is not closely related to a recently identified second Brachyury gene from Hydra, HyBra2 (Technau, unpublished data). We therefore termed the Nematostella Brachyury gene NemBra1. Spatial and temporal expression analysis of NemBra1 during early embryogenesis Embryogenesis of N. vectensis begins with two to four rounds of radial cleavage. Cleavage then becomes somewhat irregular until a coeloblastula has formed about 8–10 h after fertilization. Subsequently, gastrulation occurs by epiboly (or invagination) at one pole, forming the blastopore. By about 24 h of development the postgastrula has differentiated cilia on its ectodermal layer and starts moving around as a planula larva with the former blastopore at its posterior end. At about 7 days of development the planula starts to metamorphose into a primary polyp. During this process, tissue from the Fig. 1A, B Sequence analysis of NemBra1. A Amino acid sequence alignment (ClustalW) with Brachyury T-domains of different organisms. B Phylogenetic analysis of T-domains using the Maximum likelihood program PUZZLE (Strimmer and von Haeseler 1997). JTT was used as model of substitution, the parameter alpha of the gamma distribution was 0.68 indicating a strong rate of heterogeneity. One thousand replica calculations were performed. Numbers are percent of statistical support (Ce Caenorhabditis elegans, M mouse, As Halocynthia roretzi, Dm Drosophila melanogaster, X Xenopus, Am Amphioxus, Zf zebrafish, Pd Platynereis dumerilii, Pf Ptychodera flava, Hp Hemicentrotus pulcherrimus, Nem Nematostella vectensis, Hy Hydra vulgaris, Trg T-related gene (Drosophila), Pod Podocoryne carnea, He Hydractinia echinata) 566 Fig. 2 RT-PCR expression analysis with NemBra1 and Nemactin. PCR primers for NemBra1 are flanking an intron, ruling out contamination of cDNA with genomic DNA when the RT-PCR produced one band in the expected size blastopore has retracted inside the body cavity and develops into the mouth. The first four tentacles then develop from the outer margins of the former blastopore. At the same time, two mesenteries (septae) form from the endodermal cell layer starting at two opposing parts of the blastopore, which now is more oval or slit-like. The primary polyps are able to feed and rapidly grow to larger sizes, developing additional mesenteries and tentacles, always between existing ones. During growth of the juvenile polyps retractor muscles develop on one side of the endodermal mesenteries (Hand and Uhlinger 1992). In adults, cells of the mesenteries also contribute to the differentiation of gametes. We first analyzed the temporal expression profile of NemBra1 and Nematostella cytosolic b-actin during embryogenesis by RT-PCR on pooled embryos of defined stages. While Nemactin is expressed at constant levels throughout embryogenesis including unfertilized eggs, NemBra1 is not expressed maternally and gene expression is first detectable at the blastula/gastrula transition and persists until adulthood (Fig. 2). In larger juvenile polyps that were cut in the middle of the body column, NemBra1 transcripts can be equally detected in both halves, suggesting that expression is not restricted to one side of the animal. In order to analyze the spatial expression pattern of NemBra1 we established an in situ hybridization protocol for embryos and primary polyps of N. vectensis. In situ hybridization shows that actin is expressed evenly in embryos of all stages and primary polyps (Fig. 3A–D). By comparison, no expression of NemBra1 could be detected in early cleavage embryos and morula stages (Fig. 4A–C), consistent with the RT-PCR data. A clear signal of NemBra1 expression could be detected in late gastrula and early planula larvae. The expression zone is restricted to a circle around the blastopore of the developing larvae (Fig. 4E, F). During metamorphosis into primary polyps the NemBra1 expression is found in the endodermal part of the blastopore, where it concentrates at two opposing ends, the anlagen for the first two mesenteries (Fig. 5A). In primary polyps, NemBra1 is expressed around the polyp’s mouth and in the growing mesenteries, including the progenitors of muscle tissue (Fig. 5B, C). Vibratome cross sections of adult polyps revealed that NemBra1 Fig. 3A–E Expression of the Nematostella b-actin during embryogenesis. A Unfertilized egg, B blastula, C planula, D metamorphosing planula, E primary polyp. Stronger signals are due to denser tissue. Bars correspond to 100 m expression persists in the mesenteries, however, the expression is usually restricted to one side of the twolayered mesenteries only. Expression is excluded from the retractor muscle cells that form on one side of the mesenteries and it is also excluded from the distal tip, the septal filament (Fig. 5D–F). Discussion Homologs of the T-box gene Brachyury have been isolated from a wide range of metazoan phyla (reviewed in Technau 2001). We recently cloned the first Brachyury homolog from the cnidarian Hydra (Technau and Bode 1999). The diploblastic Cnidaria evolved at least 600 million years ago and are thought to be descendents of one of the most basal metazoans. However, no Brachyury gene has been reported so far from the Anthozoa, which are considered the more basal class of Cnidaria compared to the Hydrozoa such as Hydra, Podocoryne or Hydractinia (Bridge et al. 1992, 1995; Collins 2002). 567 Fig. 4A–F Spatial expression analysis of NemBra1 by in situ hybridization. A Eight-cell-stage embryo, B early cleavage, C blastula, D gastrula, E planula, view on the blastopore, F planula, lateral view. Bars correspond to 100 m No T-box genes have been reported so far from protists and none are found in the yeast genome. Hence, it is feasible that the T-box gene family arose with the evolution of animal multicellularity. The anthozoan N. vectensis is to date the most basal metazoan from which the T-box gene Brachyury has been isolated and might therefore give clues about the ancestral role of this gene in metazoan evolution. NemBra1 is more than 80% identical in the T-domain on the amino acid level to orthologous proteins of vertebrates. This strikingly high degree of sequence conservation indicates that a strong selection pressure must exist to maintain the precise structure of the Tdomain, which is the DNA-binding domain. All amino acids that have been shown to be implicated in dimerization and DNA-binding of the T-domain of Xenopus Brachyury (Mller and Herrmann 1997) are conserved in NemBra1, suggesting that the NemBra1 T-domain also functions similarly on a molecular level, i.e. that it binds to similar binding sites on the DNA. The comparison of basal deuterostome and protostome primary larvae suggests that in Urbilateria Brachyury specified the fore- and hindgut as well as parts of the posterior mesoderm (reviewed in Technau 2001 and references therein). This gut developed as part of the blastopore (Arendt et al. 2001). In Hydra expression is confined to the hypostome, the tissue surrounding the mouth (Technau and Bode 1999). According to the gastraea theory of Haeckel (1896), Hydra’s mouth corresponds to the blastopore of other animals. Unfortunately, Hydra embryos gastrulate through multipolar ingression of single cells (Martin et al. 1997), and hence do not have a well-defined blastopore. It is therefore not clear, how the mouth of the adult Hydra polyp relates to the blastopore of Bilateria. By contrast, the anthozoan Nematostella has a welldefined blastopore (Hand and Uhlinger 1992), which marks the posterior end of the planula larva and later develops into the mouth of the polyp. NemBra1 is expressed continuously around the blastopore. From these data it seems obvious that the hypostome of Hydra is a derived structure of the blastopore. We therefore propose that the blastoporal expression is ancestral for all diploblasts and triploblasts. No information on Brachyury is currently available from the sponges, so it is therefore still unclear whether the blastoporal expression is also ancestral for metazoans as a whole. However, given the strong conservation of the cnidarian T-box, we also expect the presence of Brachyury in sponges. Interestingly, Nematostella Brachyury has a second expression domain in the mesenteries. Mesenteries are endodermal lappets that start developing from two 568 Fig. 5A–F NemBra1 expression in metamorphosing larvae and polyps. A Metamorphosing planula, B primary polyp, C sense control with primary polyp, D schematic cross-section through an adult sea anemone (modified after Barnes 1963), E schematic drawing of vibratome cross-section (F) through the gastric region of a juvenile polyp showing NemBra1 expression in one of the mesenteries. The arrow (B) indicates the polyp’s mouth opening, the arrowheads (B) point to the first two mesenteries (ec ectoderm, en endoderm, m mesogloea, sf septal filament, rmf retractor muscle filament). Bars correspond to 100 m opposing poles of the blastopore in the metamorphosing planula larva. In these mesenteries the endodermal retractor muscle cells of Nematostella develop. How does the blastoporal expression relate to the muscle differentiation and to mesoderm specification? The analysis of all larval stages in Nematostella reveals a contiguous expression of Brachyury in the blastopore and the developing mesenteries. It is therefore conceivable that during bilaterian evolution the mesoderm originated from a subpopulation of cells in the blastoporal region, specified by Brachyury. In all Bilateria, muscles are a major derivative of the mesoderm and in vertebrates specification of the mesoderm by Brachyury is required for later expression of myogenic factors (for review see Smith 1997, 1999). In fact, ectopic expression of Brachyury in animal caps of Xenopus embryos can induce the formation of muscle tissue in a dose-dependent manner (O’Reilly et al. 1995). A specification of muscle tissue by Brachyury might therefore already exist in the diploblasts. Interestingly, in the marine hydrozoan Podocoryne carnea, Brachyury, the bHLH transcription factor twist, the zinc finger gene snail and the myogenic factor mef2 are all expressed in presumptive smooth and striated muscle cells in the manubrium and in the bell (Spring et al. 2000, 2002). A role of “mesodermal” and myogenic genes of Bilateria in the differentiation of muscle tissue in diploblasts therefore seems plausible. Yet, in adult polyps of Nematostella Brachyury expression in the mesenteries is excluded from the differentiated muscle tissue but present in the adjacent tissue. Brachyury might therefore be required for the competence of the tissue to differentiate into muscle tissue, but in later stages of muscle differentiation. Alternatively, Brachyury might function on a different level. Instead of acting as a determinant of a specific type of tissue it might have a role in morphogenetic movements involving changes in cell adhesion and cell motility. In the sea urchin Lytechinus variegatus, Brachyury is expressed in a dynamic manner around the blastopore, i.e. cells of the invaginating endoderm turn on Brachyury transiently as they are moving through the blastopore, but turn it off as soon as they are involuted (Gross and McClay 2001). Consistently, blocking Brachyury function results in a perturbation of gastrulation movements in these organisms, but not in the expression of endodermal and mesodermal marker genes (Gross and McClay 2001). Also, in Xenopus and zebrafish, Brachyury seems to be involved in convergence and extension of the notochord via one of its target genes, Wnt11 (Heisenberg et al. 2000; Tada and Smith 2000). Hence, Brachyury expression in Nematostella could also reflect a role in morphogenetic movements during gastrulation. Studies to test these ideas are currently underway. 569 The expression pattern of NemBra1 might also have implications for the evolution of bilaterian body axes. A number of genes involved in anterior-posterior (AP) and dorso-ventral (DV) axis formation in Bilateria have been cloned from Cnidaria and some of them are expressed in a localized manner (Schummer et al. 1992; Shenk et al. 1993; Broun et al. 1999; Technau and Bode 1999; Hobmayer et al. 2000; Yanze et al. 2001; Hayward et al. 2002), suggesting that genes of the AP and DV axis are present in these basal metazoans and may be involved in the formation of the single apparent body axis in Cnidaria. Whether this apparent body axis corresponds to the DV or AP axis of Bilateria is unclear and a matter of debate (Technau 2001; Meinhardt 2002). One possibility is that a symmetry break occurred between the Cnidaria and the Bilateria. An intermediate stage of such a symmetry break would be a biradial pattern. In Nematostella the transition from a radially symmetrical to a biradial pattern happens during metamorphosis, when the circular blastoporal Brachyury expression dissolves into two opposing expression domains, which mark the position of the first pair of mesenteries. However, the recent identification and expression analysis of the dpp homolog from the anthozoan coral Acropora millepora suggests that the symmetry break might already have occurred at the level of the Anthozoa. In late gastrulae of Acropora, Am-dpp is expressed asymmetrically at one margin of the blastopore (Hayward et al. 2002). Anatomists have claimed for decades that adult anthozoan polyps display features of bilaterality: the retractor muscles in the mesenteries are formed in an asymmetrical pattern and the slit-like mouth has a differentiated siphonoglyph on one side (Barnes 1963). These morphological characters may be set up during embryogenesis by genes involved in the formation of bilaterian DV and AP axis. 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