Dev Genes Evol (2002) 212:551–561 DOI 10.1007/s00427-002-0274-8
ORIGINAL ARTICLE
Nicolas Lartillot · Martine Le Gouar · Andr Adoutte
Expression patterns of fork head and goosecoid homologues in the mollusc Patella vulgata supports the ancestry of the anterior mesendoderm across Bilateria Received: 5 August 2002 / Accepted: 16 September 2002 / Published online: 18 October 2002 Springer-Verlag 2002
Abstract We have characterised orthologues of the genes fork head and goosecoid in the gastropod Patella vulgata. In this species, the anterior-posterior (AP) axis is determined just before gastrulation, and leads to the specification of two mesodermal components on each side of the presumptive endoderm, one anterior (ectomesoderm), and one posterior (endomesoderm). Both fork head and goosecoid are expressed from the time the AP axis is specified, up to the end of gastrulation. fork head mRNA is detected in the whole endoderm, as well as in the anterior mesoderm, whereas goosecoid is only expressed anteriorly, in the three germ layers. The two genes are thus coexpressed in the anterior mesoderm, suggesting the latter’s homology with vertebrate prechordal mesoderm. In addition, since prechordal plate is known to belong to an anterior, so called “head organiser”, and since its inductive role is dependent on the function of the vertebrate fork head and goosecoid orthologues, we further suggest that the anterior mesoderm may also have a role in anterior inductive patterning in Spiralia. Finally, we propose that a mode of axial development involving two organisers, one anterior and one posterior, is ancestral to the Bilateria, and that both organisers evolved from the single head organiser of a putative hydra-like ancestor. Keywords fork head · goosecoid · Mollusc · Gastrulation · Ectomesoderm
Introduction Molecular analysis of metazoan development has proven useful for comparing body plans on the large evolutionary scale, and has led some authors to the conclusion that the common ancestor of all bilaterally symmetrical animals Edited by D. Tautz N. Lartillot ()) · M. Le Gouar · A. Adoutte Centre de Gntique Molculaire, C.N.R.S batiment 26, 1 avenue de la Terrasse, 91198 Gif Sur Yvette, France e-mail:
[email protected]
(Urbilateria) might have been much more complex than traditionally claimed (De Robertis 1997; Pennisi and Roush 1997). Yet, beyond the comparison of the adult body plans, one would also like to understand the evolution of the processes themselves underlying early development. In this respect, the main objective of this paper is to perform a comparative analysis of the molecules involved in the patterning of the early gastrula and in axial development across bilaterians. According to current phylogenetic knowledge (Adoutte et al. 2000), comparing vertebrates with either ecdysozoans or lophotrochozoans, or both, should yield informative insights into the development of Urbilateria. Until now, most comparative analyses have relied on ecdysozoans and deuterostomes, however, pointing to the necessity of acquiring new data in lophotrochozoan species. Classically, in Bilateria, three germ layers are recognised: ectoderm, endoderm, mesoderm. The latter is generally defined as the tissue situated between the basement membrane of ectoderm and endoderm, and differentiates into a variety of organs such as muscles, metanephridia, blood vessels, and mesenchymes. From a comparative point of view, however, mesoderm has always been a puzzling aspect of bilaterian evolution, mainly because of the diversity of ways through which mesoderm originates during early development across, and even within, phyla. This has led many authors to argue that mesoderm would probably not be homologous across Bilateria. To make the situation worse, in many species, mesodermal derivatives do not even stem from a single embryonic anlage. This is conspicuous in spiral cleaving animals (Spiralia), like annelids and molluscs, where it has long been recognised that mesoderm has at least a double origin (Lillie 1895). In spiralians, the major mesodermal contribution comes from a vegetal blastomere, 4d, which gives rise to the mesodermal germ bands. But in addition, some mesodermal cells originate from more animal cells, from the second and third quartets of micromeres. Since other micromeres of the second and third quartets are classically thought to give rise to ectoderm in Spiralia, this second mesodermal contribu-
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tion is called ectomesoderm (Verdonk and van den Biggelaar 1983; Wierzejski 1905). In vertebrates, mesoderm is initially specified as a single field, which in Xenopus corresponds to the marginal zone. Since this field also contributes to endoderm, we might call it mesendoderm. During gastrulation, the mesendoderm involutes through the blastopore lips, and differentiates inside the embryo. Here, it becomes subdivided into two components. Anteriorly is the prechordal plate, that lies beneath the anterior part of the central nervous system (Adelmann 1922; Seifert et al. 1993), and finally contributes to very few adult structures. Posteriorly, the rest of the mesendoderm will form the endoderm, the somites, the notochord, and the lateral plate mesoderm of the trunk and the tail. Interestingly, this anterior-posterior (AP) subdivision of vertebrate mesoderm and endoderm has a now wellunderstood developmental basis: essentially, the AP axis is patterned by two organisers, one for the head, and one for the trunk and the tail. The head organising signal partly comes from the prechordal plate (Kiecker and Niehrs 2001), and is involved in the induction and the dorso-ventral patterning of the anterior nervous system, whereas dorsal-axial mesendoderm stemming from the upper lip of the blastopore, or the node in amniotes, patterns the more caudal part of the axis. These two organisers share common molecular markers, like HNF3b, a homologue of the Drosophila transcription factor fork head, which is expressed in the whole endoderm, in axial mesoderm along the entire AP axis, and is required for the inductive properties of both anterior and trunk-tail mesendoderm (Nag et al. 1993; Ruiz i Altaba and Jessel 1992; Monaghan et al. 1993). In addition, during gastrulation, the whole mesendoderm displays a transient ERK MAP kinase activity (Christen and Slack 1999), inducing a transient expression of the T-box transcription factor Brachyury (Amaya et al. 1993). Subsequently, however, Brachyury expression is quickly downregulated in the prechordal mesoderm, by goosecoid-mediated transcriptional repression (Artinger et al. 1997; Latinkic and Smith 1999), while its expression is maintained along the axial mesoderm posterior to the prechordal region and in the tailbud. We have previously shown that, in Patella, a Brachyury homologue is transiently expressed in the 3D blastomere, under the direct transcriptional control of ERK MAP kinase (Lartillot et al. 2002), a situation very similar to that of vertebrate mesoderm induction. Since 3D is the organiser of bilateral symmetry of the early gastrula, and the posterior mesendoderm mother cell, these results suggest that the posterior organiser might be an ancient feature of Bilateria. Here we extend our observations by characterising, in Patella, a fork head and a goosecoid orthologue. We have determined their expression patterns during gastrulation, and resolved part of their lineage of expression. In particular, we show that both fork head and goosecoid are expressed in the ectomesoderm. On the basis of the combined results obtained with Brachyury, MAP kinase, fork head and
goosecoid, we suggest a one to one homology between spiralian ecto- and endomesoderm, with vertebrate prechordal plate and trunk-tail mesoderm, respectively. Finally, we propose a model for the evolution of AP axis development since the last common ancestor of Bilateria (Urbilateria).
Materials and methods Patella in vitro fertilisation and embryo rearing Adult animals were obtained from the Station Biologique de Roscoff, France, and kept in artificial sea water. Embryos were obtained as described previously (van den Biggelaar 1977). Cloning and sequencing Nested PCR was used to amplify fragments of Patella fork head and goosecoid genes. PCR were performed on a mass-zapped cDNA library (of 16-h-stage trochophores) kindly provided by A. E. van Loon (van Loon et al. 1991). The following degenerate primers were used; for fork head: TNATNACNATGGCNATNCA and TARCANCCRTTYTCRAACATRTTNCC for the first round, and GARATNTAYCARTTYATNATGGA and AARTARCANCCRTTYTCRAACAT for the second round; for goosecoid: GGGACNATNTTYACNGANGARCA and GGGCKNCKRTTYTTRAACCANAC for the first round, and GGGTTYACNGANGARCARCTNGA and GGGTTYTTRAACCANACYTCNAC for the second round of amplification. The PCR fragments were cloned into the pCRII vector (Invitrogen). Twelve clones were sequenced in the case of fork head. Two different classes of sequences were obtained, one corresponding to a divergent, non fork head, winged helix gene, which was not considered further, and one very similar to Drosophila fork head. In the case of goosecoid, nine clones were sequenced, six displaying the same sequence, and three not corresponding to homeobox sequences. The 3' and 5' ends of the corresponding genes were amplified from the same zapped cDNA library, using vector- and gene-specific (non degenerate) primers (sequences are available upon request). The sequences of both cDNA clones are available on the EMBL nucleotide sequence database (sequence numbers AJ507423 and AJ507424). Alignment and phylogenetic analysis Sequences of fork head and goosecoid homologues were gathered using the NCBI PubMed database (http://www.ncbi.nlm.nih.gov). All sequences were aligned with clustalX (Thompson et al. 1997), and only the conserved domains were kept for subsequent analysis. Phylogeny reconstruction was performed by Maximum Parsimony, using the software PAUP (Swofford 2002). In situ hybridisation and Hoechst staining RNA in situ hybridisations were performed as described previously (Lartillot et al. 2002), and embryos were dehydrated and mounted in benzyl alcohol-benzyl benzoate. In some cases, following the in situ hybridisation, the embryos were stained with Hoechst (5 g/ml in TBS-T; 10 min incubation) to highlight the nuclei of their cells, and mounted in 80% glycerol. A detailed protocol is available upon request.
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Fig. 1A, B Phylogenetic analysis of the fork head and goosecoid families, as inferred by maximum parsimony. In both cases, several equiparsimonious trees were obtained, of which the strict consensus was built. A Tree based on the amino acid sequences of the winged helix domain of several fork head orthologues. In addition to the
Fox A class (fork head class), several members of the classes B, C and D (Kaestner et al. 2000) were used to root the tree. B Tree reconstructed using an alignment of several goosecoid and other paired type homeodomains. Non goosecoid paired type sequences have been used to root the tree
Results
symmetry of the whole embryo (Fig. 2B; van den Biggelaar 1977). 3D also gives birth to 4d, the endomesoderm mother cell (Fig. 2C, red), which is situated at the posterior edge of the set of endodermal blastomeres (vegetal plate; Fig. 2C, yellow). Meanwhile, ectomesoderm is specified on the anterior side of the vegetal plate, stemming from the 3a, 3b and 2b2 blastomeres (Fig. 2C, orange; Dictus and Damen 1997). Subsequently, we will adopt a terminology based on the differential positioning of the two mesodermal components along the AP axis, and thus call ecto- and endomesoderm, anterior and posterior mesoderm, respectively (for justifications, see Discussion). Finally, Patella gastrulation is protostomous (Verdonk and van den Biggelaar 1983). The cells of the vegetal plate are recovered by epiboly of the rim ectoderm encircling them. This rim is equivalent to a blastopore, which gives rise to the mouth. During gastrulation, the ectoderm of the posterior edge of the blastopore, deriving from 2d, contributes to the ventral structures of the trunk, whereas ectodermal cells of the anterior and lateral edges participate in the morphogenesis of the mouth region. Meanwhile, mesodermal cells of both anlagen freely mix, and contribute to a variety of larval and adult muscles (Damen, in press).
fork head and goosecoid homologues in P. vulgata Using degenerate primers, we have amplified and cloned, from a 16-h cDNA library, fragments of putative Patella fork head and goosecoid homologues. Primers were designed to allow the amplification of the most conserved regions of the two genes, corresponding to the winged helix domain for fork head, and the homeodomain in the case of goosecoid. Next, vector-anchored race PCR was performed, allowing full length cDNAs of both genes to be obtained (1,513 bp and 1,398 bp, respectively). The coding sequences were aligned with other metazoan winged helix and homeobox proteins. Only regions displaying an unequivocal alignment, roughly corresponding to the DNA binding domains, were kept for further analysis. A maximum parsimony reconstruction shows that the two genes thus identified can be confidently considered molluscan fork head and goosecoid orthologues (Fig. 1). Both sense and antisense DIG-labelled RNA probes were synthesised for each clone, and were used to determine the expression patterns of the two genes during early development and larval stages, by in situ hybridisation. Expression profiles were interpreted with the help of the exhaustive fate map determined by Dictus and Damen (1997). In Patella, AP axis specification occurs at the initially fourfold symmetrical 32-cell stage (Fig. 2A), and consists of the specification of one of the four quadrants of the embryo as the posterior (D) quadrant. Just before gastrulation, the macromere of the D quadrant (3D) acts as an organiser that sets up the bilateral
Expression pattern of fork head during embryogenesis Patella fork head transcripts can be detected as soon as the mid-32-cell stage (3 h post first cleavage, h.p.f.c.), very weakly, in the vegetal region of the blastula, in the anterior and lateral macromeres, 3A, 3B and 3C (Fig. 3A, D). Expression is sharply upregulated during the follow-
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Fig. 2A–C Overview of Patella early development before gastrulation. Embryos are approximately 200 m in size, and are depicted as seen from the vegetal side, D (posterior) quadrant at the bottom. A 32-cell stage, displaying fourfold symmetry along the animal vegetal axis. At this stage, the four quadrants are still equivalent. B 60-cell stage (3.5–4 hours post first cleavage). 3D induction has
taken place, and 3D looks smaller, since most of its mass is internalised. Cleavages are now bilateral, and the AP axis is now visible. C 64-cell stage; the germ layers are almost segregated. Red posterior mesoderm (endomesoderm), orange anterior mesoderm (ectomesoderm), yellow endoderm. Note that 2b2 has been coloured in orange here, although it might contribute to the three germ layers
ing hour, so that 30 min after 64-cell stage (4.30 h.p.f.c.), fork head is strongly expressed in these three cells’ derivatives, 4A, 4a, 4B, 4b, 4C and 4c (Fig. 3B, E). Together with 4D, all these cells make up the complete endodermal anlage but, notably, fork head is not expressed in 4D. Anterior to the presumptive endoderm, two other cells, 3a2 and 3b2, display an even stronger expression (Fig. 3B, E). As depicted in Fig. 2, these cells represent the major part of the presumptive anterior mesoderm (see Fig. 2C). At 6 h.p.f.c., (past 88-cell stage), fork head is still present in the endoderm, except for 4D, as well as in the mesodermal 3a and 3b daughter cells, 3a1, 3a2, 3b1 and 3b2 (Fig. 3C,F, small arrowheads). fork head is also expressed in 2a22, 2b22 and 2c22 (Fig. 3C, arrows). These three blastomeres contribute to the ectoderm around the mouth. In addition, 2b22 also yields mesodermal derivatives and contribute to the foregut roof. Finally, a faint expression is seen posteriorly, in one isolated blastomere situated markedly to the right of the embryo (Fig. 3C, large arrowhead). This blastomere might be derived from 2d22, which has been proposed previously as playing the role of a stem cell for the ectodermal midline, and which also expresses Brachyury (Lartillot et al. 2002). At 8 h.p.f.c., the expression pattern can be described as a broad and weak endodermal expression, combined with a very strong expression in two pairs of cells situated anteriorly in a bilateral fashion (Fig. 3G, arrowheads). These four cells stem from 3a2 and 3b2, which express fork head as early as the 64-cell stage. The mesodermal expression of fork head allows one to follow the gastrulation movements taking place on the anterior edge of the blastopore (Fig. 3H–J). In a first step, the fork head expression domain spreads along the lateral sides of the blastopore, although it is difficult to tell the
relative contributions of the divisions of cells already expressing fork head at 8 h.p.f.c., and of the spread of expression to neighbouring cells. This group of cells then progressively takes the shape of a horseshoe (Fig. 3H), that invaginates only at the very end of gastrulation (around 12–14 h.p.f.c., Fig. 3I), so as to form a collar of mesoderm around the larval mouth. Finally, fork head expression spreads to the whole anterior mesodermal field, and is still visible in the 16-h (Fig. 3J), and 24-h (Fig. 3K, L) trochophore larva. During all these stages, fork head remains expressed in the endoderm, albeit weakly. In conclusion, fork head is expressed in endoderm and anterior mesoderm, throughout gastrulation and in the young trochophore larva. This fork head mesodermal expression is reminiscent of the transient expression of HNF-3b, the vertebrate fork head homologue, in the prechordal mesoderm of vertebrates (Filosa et al. 1997). Since the prechordal plate displays a combined expression of HNF-3b and goosecoid, we searched for a goosecoid homologue in Patella, and determined its expression pattern during gastrulation.
Fig. 3A–L Expression of fork head during early development, as determined by in situ hybridisation. Unless mentioned explicitly, all views are vegetal, D quadrant to the bottom. Embryos and trochophore larvae are approximately 200 m in size. A, D 3hours post first cleavage (h.p.f.c.), mid-32-cell stage. Faint expression is detected in 3A, 3B and 3C. B 4.00 h.p.f.c. (64-cell stage) in situ hybridisation was combined with Hoechst staining, to highlight the nuclei of cells expressing fork head: 4A, 4a, 4B, 4b, 4C, 4c and 3a2 and 3b2. E 4.30 h.p.f.c. (30 min after 64-cell stage). fork head is expressed in endodermal derivatives, except for 4D. Expression is also detected in a few cells anterior to endoderm, which are ectomesodermal. C, F 6 h.p.f.c. embryo, showing fork
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head expression in endodermal blastomeres, in 2a22, 2b22 and 2c22 (arrows) and in 3a2 and 3b2 derivatives (small arrowheads). Posterior, and to the right of the embryo (to the left on the figure, large arrowhead), a faint expression is detected in 2d212 (midline stem cell). G 8 h.p.f.c. embryo, displaying faint expression in the presumptive endoderm, and strong expression in two pairs of two cells (small arrowheads), situated on the anterior edge of the
vegetal plate. H, I fork head is transcribed at high levels in ectomesoderm during late gastrulation. H 8 h.p.f.c. I 12 h.p.f.c. J fork head expression in a 15-h larva, ventral view. K, L fork head expression in a 24-h-old larva. Ventral (K), and side (L) view (a.p. apical pole, end. endoderm, pt. prototroche, s.g. shell gland, st. stomodeum, v.p. ventral plate)
556 Fig. 4A–H Expression profile of goosecoid during gastrulation. Embryos and trochophore larvae are approximately 200 m in size. A 63-cell stage (shortly before 4 h.p.f.c.), vegetal view. goosecoid is expressed in 2m2 derivatives. Faint expression is also detected in 3a2 and 3b2. B 4.30 h.p.f.c., vegetal view. goosecoid expression has been down regulated in 2m2 derivatives, except for 2a22, 2b22 and 2c22 (arrows). Transcripts are still detected in 3a2 and 3b2 (small arrowheads), as well as in two cells on the opposite (posterior) edge of the vegetal plate (large arrowheads). C 6 h.p.f.c., vegetal view. goosecoid transcripts are present in two pairs of cells derived from 3a2 and 3b2, anterior to the vegetal plate (small arrowheads), and in a pair of cells posterior to the vegetal plate (large arrowheads). D, E 8 h.p.f.c. embryo. D Anterior view; E dorsal posterior view. goosecoid is expressed in the same cells as at 6 h.p.f.c: the anterior pairs (D small arrowheads), and the posterior pair (E large arrowheads). In addition, it is expressed in a dorsal row of cells, and a faint expression is also detected in two dorsal lateral cells, just below the prototroche. F–H 16-h-old trochophore. Ventral (F), dorsal (G), and three quarter left (H) views, showing goosecoid expression in the ectoderm around the stomodeum, and along the mantle edge (a.p. apical pole, m.e. mantle edge, pt. prototroche, s.g. shell gland, v.p. vegetal plate)
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goosecoid is expressed in ectomesoderm during gastrulation goosecoid is expressed in part or all of the 2a2, 2b2, 2c2, and 2d2 derivatives from the 32- to the 64-cell stage (Fig. 4A). At the 64-cell stage, goosecoid seems to remain expressed in 2a22, 2b22, and 2c22 (Fig. 4B, arrows), as well as in 3a2 and 3b2 (Fig. 4B, small arrowheads). Goosecoid, as for fork head, is therefore expressed in the anterior mesoderm (3a, 3b and 2b), but not in the posterior one (4d). By 6 h.p.f.c., goosecoid can be detected in the derivatives of 3a and 3b, situated on the anterior edge of the vegetal plate (Fig. 3C, small arrowheads), that are also expressing fork head at that stage (Fig. 3G), as well as in two small cells on the posterior edge of the blastopore (Fig. 4C, large arrowheads), which stem from 3c22 and 3d22 and will contribute to the basal part of the mouth. As gastrulation proceeds, the expression of goosecoid is maintained in 3a- and 3b-derived anterior mesoderm (Fig. 4D, small arrowheads), as well as in the other pair of cells identified at 6 h.p.f.c. (Fig. 4E, large arrowheads). By 8 h.p.f.c., an additional domain of expression appears dorsally, as a row of ectodermal cells initially transversal (Fig. 4E), but which progressively bends posteriorly, and extends laterally, forming the edge of the mantle. In the young 15-h trochophore, the mesodermal staining has disappeared. goosecoid expression is seen in the ectoderm around the mouth (Fig. 4F), and in the cells of the mantle edge (Fig. 4G, H). Of note are the cells contributing to the ectoderm around the stomodeum which probably stem from cells that have expressed goosecoid earlier during gastrulation: 2b2 on the upper side, 2a2 and 2c2 on the right and on the left, and 2d2 for the lower part. The two lateral aspects of this ectodermal expression are very similar to what is seen in the closely related Platynereis trochophore larva, and are thus likely to contribute to the stomodeal nervous system, as in Platynereis (Arendt et al. 2001). In summary, Goosecoid, the homologue of goosecoid in Patella is co-expressed with fork head in anterior mesoderm, early on during gastrulation but, in contrast to fork head, this goosecoid mesodermal expression is not maintained in the early larval stages, where goosecoid is detected only in cells committed to the ectodermal cells around the mouth and along the forming mantle edge.
Discussion Ecto- and endomesoderm and the AP subdivision of the spiralian third germ layer We have cloned fork head and goosecoid orthologues in P. vulgata, and have characterised their expression profiles. Both genes are expressed before and during gastrulation, in the anterior but not the posterior mesoderm. The anterior mesodermal component is classically called ectomesoderm, a name which comes from the fact that the equivalent cells from the other quadrants (i.e. 3c,
3d, 2a, 2c and 2d) are supposedly of ectodermal fate (Wierzejski 1905). In contrast, since 4a, 4b and 4c belong to endoderm, the 4d-derived mesoderm is thus called endomesoderm. This terminology, originally due to Lillie (1895), was chosen in a context where a very strong interpretative value was given to cell lineages, all the more so as invertebrate development was supposed to be completely mosaic (Wilson 1893). We now know that spiralian early development is much more regulative than previously thought, however (van den Biggelaar 1977), suggesting that such lineage considerations might not be essential, after all. In our view, at least in the frame of the comparative perspective which we would like to develop, an alternative terminology referring to the position with respect to the blastopore [i.e. anterior (3a/3b/2b2) and posterior (4d) mesoderm] might turn out to be much more significant. Indeed, this differential positioning along the AP axis is still visible later during development, as can be seen in the larva where the anterior mesoderm forms the collar of mesenchyme around the mouth (Fig. 3K), while the 4d-derived germ bands run along the lateral sides, up to the anus (Dictus and Damen 1997). In addition, it is very stable across Spiralia: in molluscs, whereas the endomesoderm is almost always derived from 4d and gives birth to the mesodermal bands (Verdonk and van den Biggelaar 1983), ectomesoderm generally stems from 2a2 and 2c2 in lamellibranches and scaphopods, or from 3a and 3b cells in gastropods (Verdonk and van den Biggelaar 1983); all these blastomeres are situated on the anterior and lateral border of the endodermal plate. At a broader evolutionary scale, recent lineage studies have been performed in the polyclade Hoploplana (Boyer et al. 1998), and in the nemertean Cerebratulus (Henry and Martindale 1998). In both species, mesoderm originates from 4d, but also from 2b in Hoploplana, and from 3a and 3b in Cerebratulus, which in both cases corresponds to subsets of Patella’s mesodermal lineage. Altogether, mesodermal anlagen in Spiralia are always in the marginal zone, wedged in between endoderm and ectoderm, and easily conform to a subdivision into a posterior 4d-derived component and an anterior one of a more variable lineage. Ancestry of mesoderm AP subdivision More importantly, our nomenclature, based on AP positioning, is also meaningful at a larger evolutionary scale. Indeed, the combined expression of goosecoid and fork head in anterior mesoderm in Patella, and of HNF3b and goosecoid in the prechordal plate of vertebrates, suggests that the early specification of an anterior mesodermal component is ancestral to the bilaterians. A similar argument can be made about the posterior mesoderm: we have shown previously that Brachyury was expressed in 3D right after its induction, and under the direct transcriptional control of the ERK MAP kinase, resulting in a transient expression of Brachyury in 4d, the posterior mesoderm stem cell (Lartillot et al. 2002).
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Likewise, ERK is activated in involuting mesoderm in vertebrates, and transiently activates the expression of Brachyury, suggesting molecular conservation in the specification of posterior mesoderm as well. Note that in vertebrates, ERK activation and Brachyury expression occurs in the whole mesodermal anlage, including the prechordal mesoderm (Christen and Slack 1999), whereas it takes place only in posterior mesoderm in Patella. However, in vertebrates, the subsequent repression of Brachyury by goosecoid (Artinger et al. 1997; Latinkic and Smith 1999), leading to complementary expression patterns on the axial mesoderm, suggests that this difference may not be essential. Hence, the results presented here and previously suggest a one to one homology of spiralian anterior and posterior mesoderm, with vertebrate prechordal plate and trunk-tail mesoderm, respectively. Are there other molecular evidences in favour of this hypothesis? A twist orthologue has been identified in Patella (Nederbragt et al. 2002) and the expression pattern reported indicates that this gene is also expressed in anterior mesoderm, a fact reminiscent of the anterior expression of twist in vertebrates, more specifically in cephalic mesoderm (Wolf et al. 1991). In addition, an orthologue of the vertebrate genes Mox1 and Mox2 has been characterised in the gastropod Haliotis, where it is likely to be expressed in the 4d-derived mesoderm (Hinman and Degnan 2002). Mox genes are expressed during early somitogenesis (Candia et al. 1992), and might be implicated in the differentiation of trunk mesoderm (Candia and Wright 1995), further supporting the homology of trunk-tail mesoderm between Spiralia and vertebrates. Finally, in the tunicate Molgula, two mesodermal components can be recognised as well, one on the anterior and one on the posterior edge of the endodermal anlage. And, as in Patella, the Molgula fork head orthologue is expressed in the anterior mesoderm but not in the posterior one, and is required for its involution during gastrulation (Olsen and Jeffery 1997). Altogether, these data suggest that the specification of two mesodermal components along the AP axis, even before gastrulation has started, is a very ancient feature, already present in the common ancestor of all bilaterian phyla.
foregut and in the perioral ectoderm in the polychaete annelid Platynereis dumerilii (Arendt et al. 2001). Hence, in every case, the expression of the two genes seem to overlap extensively, defining a broad anterior domain across the three germ layers. Interestingly, experimental evidence in vertebrates suggests that substantial inductive interactions take place in this anterior region. In particular, prechordal plate and foregut endoderm are responsible for anterior neural induction, dorso-ventral patterning of the anterior neural ectoderm, and also have anteriorising capabilities (Kiecker and Niehrs 2001). In Xenopus and in the mouse, prechordal mesendoderm ablation results in anterior deletions, suggesting, among many other experimental data, the existence of a vertebrate head organiser distinct from the organiser of the trunk and the tail (Spemann organiser proper). Foregut and anterior visceral mesoderm have also been implicated in brain patterning in Drosophila (Page 2002), raising the possibility that the head organiser might be a very ancient feature of Bilateria. This is corroborated by genetic data: HNF-3b is required in mouse prechordal mesendoderm for head morphogenesis (Weinstein et al. 1994), and likewise, fork head expression in foregut is necessary for proper brain patterning in Drosophila (Page 2002). goosecoid might also be involved in these inductive patterning events, at least in mouse, where embryos having a HNF-3b+/– goosecoid –/– genotype show defects in anterior nervous system patterning similar to those seen in a HNF-3b–/– genetic context (Filosa et al. 1997). In the light of all these observations, our findings relating to the anterior expression of fork head and goosecoid in Spiralia are consistent with the hypothesis that an anterior inductive centre would also be present in Spiralia. So far, inductive properties of the anterior mesoderm and endoderm have not been characterised in Patella, nor in any other trochozoan, although they cannot be excluded. Interestingly, 2b2, one of the cells co-expressing fork head and goosecoid early during gastrulation, gives rise to a thin endodermal stripe making up the roof of the foregut, up to the apical pole of the larva (Dictus and Damen 1997), where the brain later develops. Head and trunk-tail organisers: a dipole orchestrating axial development
Conservation of anterior inductive patterning across Bilateria Neither in vertebrates nor in Patella, is fork head and goosecoid expression restricted to mesoderm, however. In vertebrates, during gastrulation, both genes are transiently expressed in the foregut endoderm, the prechordal plate, and the anterior floorplate (Filosa et al. 1997). In Patella, at the 64-cell stage, goosecoid and fork head are coexpressed in 3a and 3b (mesoderm), 2a2 and 2c2 (ectoderm) and 2b2, which participate in the anterior (pretrochal) ectoderm, anterior mesoderm and foregut roof. A goosecoid homologue is also expressed in the
We have advocated previously that the trunk-tail or posterior organiser would be ancestral to the Bilateria as well, as was suggested by the expression pattern of Brachyury in Bilateria (Lartillot et al. 2002). Hence, the entire axial development of Bilateria would be orchestrated by two organisers, both inherited from the last common ancestor of Bilateria. Interestingly, in vertebrates these two organisers share common molecular properties. Thus, both head and trunk-tail axial mesoderm express HNF-3b (Ang et al. 1993; Monaghan et al. 1993; Ruiz i Altaba and Jessell 1992), as well as the hedgehog homologue sonic hedgehog (Krauss et al. 1993). As
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noted before, HNF-3b has an essential function in prechordal mesendoderm, but it is also required for trunk-tail organiser maintenance and AP axis elongation (Ang and Rossant 1994; Weinstein et al. 1994), while sonic hedgehog mediates part of the inductive role of the axial tissues, in particular, the dorso-ventral patterning of the neural tube (Chiang et al. 1996). A hedgehog homologue has been recently identified in Patella. It is expressed along the entire ventral midline, including the foregut and the terminal structures (Nederbragt et al. 2002). Likewise, fork head is expressed at both poles, and in a series of patches along the ventral midline of Drosophila (Weigel et al. 1989), and its posterior expression is necessary for the specification of the caudal visceral mesoderm (Kusch and Reuter 1999). Altogether, these data support the hypothesis of an ancestral role of fork head and hedgehog in both anterior and axialposterior inductive patterning. Accordingly, one has to interpret the absence of axial expression of hedgehog in Drosophila, and of fork head in Patella, as secondary losses (although in Patella, a very faint and transient posterior expression of fork head is seen, by 6 h after first cleavage; see Fig. 3C). Conversely, Brachyury and goosecoid repress each other in vertebrates, leading to the segregation of goosecoid in the anterior organiser, and of Brachyury in the posterior one, and similarly the complementary expression patterns of these two genes in Patella suggest that this epistatic antinomic relationship between the two genes might be inherited from the last common ancestor. Altogether, both shared and antinomic molecular features seem to be involved in the twofold organising system inherited from the last common ancestor of Bilateria. The shared elements are particularly interesting, since they could betray a common origin for the two inductive structures. The AP axis might have originated from a split of the organiser of the radial ancestor, by amphistomy The emergence of such a dipolar system, responsible for the entire axial development, must have been a fundamental aspect in bilaterian origins. Is it possible to find equivalent systems in non bilaterian animals? Hydra have a radial symmetry, and accordingly, they have only one organiser, the hypostome, which is the single opening of their gastric cavity (Broun and Bode 2002; Browne 1909). In certain cnidarian species, like Nematostella, the hypostome corresponds to the blastoporal ring. Interestingly, in Hydra, the homologues of Brachyury (Technau and Bode 1999), fork head (Martinez et al. 1997) and goosecoid (Broun et al. 1999) are all expressed in the hypostome, suggesting that the bilaterian head and trunktail organiser could have indeed originated by a split of the cnidarian, ring-like organising centre. During this process, some genes, like fork head and maybe also hedgehog, would have been co-opted in both substructures, while others, like goosecoid and Brachyury, would have segregated. Topologically speaking, such a split can
Fig. 5A–D Diagram illustrating how, according to our scenario, mesoderm induction, AP axis specification, and amphistomous gastrulation would have occurred at some point in the lineage leading to the last common ancestor of all Bilateria. A A homogeneous mesodermal field is induced radially in the marginal zone, by a signal emitted by the neighbouring endoderm (arrows). B A process of symmetry breaking leads to the specification of an AP axis, first in the vegetal region, thereby subdividing mesoderm into two components, one anterior (orange), and one posterior (red). C Gastrulation is amphistomous, and thus enforces the separation of the two mesodermal fields into two rings, around the mouth and the anus. D Posterior growth leads to the basic bilaterian ground plan. Here, the posterior mesoderm is depicted as segmented, although the ancestry of segmentation is not a necessary aspect of the present scenario
be done easily by fusion of the lateral lips of the blastopore ring. This mode of blastopore closure, called amphistomy, has been proposed several times previously as an evolutionary scenario allowing the derivation of typical bilaterian bodyplan features, such as a digestive tube with two openings, an extended AP axis and a paired ventral nerve cord, from a blind-ended and radial Gastraea (Arendt and Nubler-Jung 1997; Jgersten 1955; Naef 1927; Sedgwick 1884). The idea that amphistomy would be ancestral to the Bilateria is further supported by the expression patterns of Brachyury homologues in metazoans (Lartillot et al. 2002; Technau 2001). In the present context, amphistomy also yields a natural explanation for the evolution of bilaterian axial development, which can be summarised according to the following sequence (Fig. 5): the centre of developmental activity is the vegetal pole of the blastula, which is the centre of the endodermal plate. First, the endoderm induces mesoderm in the marginal zone (Fig. 5A), and orchestrates a radial gastrulation. Second, a symmetry breaking event subdivides the blastopore ring into an anterior and a posterior component (Fig. 5B), whereby an AP axis is specified. Third, amphistomous blastopore closure subdivides the mesoderm into its anterior and posterior components (Fig. 5C), and alongside, creates the dipolar organising structure. Finally, the anterior loop gives birth to the mouth and organises head morphogenesis, while the posterior loop becomes a growth zone, laying down trunk and tail and organising progressive axial development (Fig. 5D).
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This scenario not only accounts for the origins of the dipolar organiser, but also gives an explanation for the apparent composite nature of mesoderm. Indeed, the double origin of mesoderm in Spiralia had been one of the most confounding facts faced by classical embryologists, as they were trying to synthesise their observations into an evolutionarily consistent germ layer theory (Conklin 1897; Wilson 1892), and finally, most embryologists turned to the idea that mesoderm would not be a natural germ layer but rather a collection of developmentally and evolutionarily heterogeneous tissues (Nielsen 2001). Here, in contrast, the scenario that we propose predicts that mesoderm is in fact a bona fide germ layer, although in some cases this might have been obscured by subsequent evolution during which the step by step process proposed above would have evolved into a direct, one-step, induction of a two-component mesoderm, as seen in extant spiralians. Finally, this scenario gives us a clue as to the anatomical origin of the prechordal plate. According to our hypothesis, the prechordal mesoderm is homologous to the anterior mesoderm. By its position along the anterior edge of the blastopore, in Patella but also in Urbilateria, the anterior mesoderm has an obvious topological connection with the mouth suggesting that, in addition to its inductive role, it might have contributed to the mesodermal mouth tissues. This is supported by the role of the Caenorhabditis fork head orthologue, pha-4, which functions as a selector gene for the pharynx (Azzaria et al. 1996; Horner et al. 1998; Mango et al. 1994). In the nematode, the pharynx develops as a completely integrated developmental unit, that generates its own mesodermal structures (mainly the pharyngeal muscles; Horner et al. 1998). The nematode is thus an extreme case where the anterior mesoderm appears to have a well-defined anatomical role in mouth development. Conversely, and concomitantly with the inversion along the dorsal-ventral axis, vertebrates have lost their ancestral mouth (Arendt and Nubler-Jung 1997). Yet, mesoderm and endoderm of this ancestral mouth are still present in vertebrate embryos forming the prechordal plate, whose role in inductive patterning has remained the only reason for its conservation across vertebrates, in spite of the loss of its anatomical function. Altogether, these two cases, exemplified by the nematode and the vertebrates, yield us the best evidence suggesting that Urbilateria’s anterior mesoderm was fulfilling a dual function: anatomical, and developmental. Acknowledgements We wish to thank Jo van den Biggelaar, Lex Nederbragt, Hans Goedemans and Andre van Loon for making available the cDNA libraries, and for all the work that has been done with their help. We are grateful to Benjamin Prudhomme, Michel Vervoort, Renaud de Rosa, Jean-Franois Julien and Guillaume Balavoine, as well as two anonymous referees, for their useful comments on the manuscript. Work in the Centre de Gntique Molculaire is financially supported by the Centre National de la Recherche Scientifique and the Universit Paris-VI. This work was also supported by the Genome Project (CNRS), the Fondation de la Recherche Mdicale, and the Institut Franais de la Biodiversit.
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