Deuterostomic Development in the Protostome Priapulus Caudatus

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provided by Elsevier - Publisher Connector Current Biology 22, 2161–2166, November 20, 2012 ª2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.09.037 Report Deuterostomic Development in the Protostome Priapulus caudatus

Jose´ M. Martıń-Durań,1 Ralf Janssen,2 Sofia Wennberg,2 blastopore in the last common bilaterian ancestor (Figure 1A). Graham E. Budd,2 and Andreas Hejnol1,* The relationship between the blastopore, the mouth, and the 1Sars International Centre for Marine Molecular Biology, anus in the bilaterian ancestor plays a pivotal role in hypoth- University of Bergen, Bergen 5008, Norway eses about the evolution of bilateral animals from an ancestor 2Department of Earth Sciences, Palaeobiology, Uppsala with a single gut opening (as seen in extant cnidarians) [2, 3, University, Villava¨ gen 16, Uppsala 75236, Sweden 10, 11]. Therefore, reconstructing the ancestral fate of the blastopore in the Protostomia is a crucial step in understanding the early evolution and diversification of the Bilateria. Summary Priapulid worms are a group of marine animals that belong to the Ecdysozoa, which forms, together with the Spiralia The fate of the blastopore during development in the bilater- (Lophotrochozoa), the Protostomia. Most recent phylogenies ian ancestor is currently not well understood. In deutero- place priapulids in the third main group of ecdysozoans (the stomes, the blastopore forms the anus, but its fate in Scalidophora; Figure S1), separate from the other two well- protostome groups is variable [1]. This variability, combined studied lineages that include nematodes and arthropods, with an absence of information from key taxa, hampers the respectively [6]. The adult priapulid is a sausage-shaped, reconstruction of the ancestral developmental mode of the mud-dwelling, annulated worm of large size (0.5–20 cm), with Protostomia and the Bilateria. The blastopore fate of an anterior proboscis (or introvert) and a terminal mouth the bilaterian ancestor plays a crucial role in understanding (Figures 1B and 1C). Their fossil record indicates that the pria- the transition from radial to bilateral symmetric organisms pulid body plan was among the most abundant and wide- [2, 3]. Priapulids have a conservative morphology, an abun- spread in the Early Cambrian [4, 12, 13]. Priapulids mostly dant Cambrian fossil record, and a phylogenetic position reproduce by external fertilization, and the embryos undergo that make them a key group in understanding protostome holoblastic radial cleavage [14], considered to be the ancestral evolution [4, 5]. Here, we characterize gastrulation and the cleavage mode in ecdysozoans [15]. Correspondingly, nuclear embryonic expression of genes involved in bilaterian fore- and mitochondrial genes tend to show high evolutionary gut and hindgut patterning in Priapulus caudatus.We conservation in extant priapulids [5, 16]. In this way, priapulids show that the blastopore gives rise to the anus at the vegetal appear to be a conservatively evolving lineage and, as such, pole and that the hindgut markers brachyury and caudal are a key group to consider for reconstructing the ancestral condi- expressed in the blastopore and anus, whereas the foregut tion of the Ecdysozoa. markers foxA and goosecoid are expressed in the mouth in Gastrulation in the priapulid Priapulus caudatus starts at the the animal hemisphere. Thereby, gastrulation in the conser- 64-cell stage with the invagination of the vegetal-most cells vatively evolving protostome P. caudatus follows strictly into a small blastocoel and the epibolic movement of the a deuterostomic pattern. These results are more compatible animal blastomeres toward the vegetal pole [14]. Three days with a deuterostomic rather than protostomic (blastopore after fertilization, the invaginated endomesodermal cells forms the mouth) or amphistomic (mouth and anus are define a small archenteron that opens to the exterior through formed simultaneously) mode of development in the last a blastopore in the vegetal pole (Figures 2A and 2A0), which common bilaterian ancestor. narrows by the end of gastrulation (Figures 2B and 2B0). Five days after fertilization, the introvert-trunk boundary (Figures Results and Discussion 1B and 1C) is established. Vegetal ectodermal cells below the equator of the embryo invaginate and form an indentation The division of bilaterally symmetric animals (Bilateria) into the (Figure 2E) that divides the embryo into a larger animal and Protostomia and the Deuterostomia [6] was traditionally based a smaller vegetal hemisphere, which correspond to the intro- on the fate of the blastopore [7], which is the orifice of invagi- vert and trunk of the larva, respectively (Figure 2C). Along nation of the endomesodermal cells [8]. In deuterostomes, the the upper ectodermal layer of the equatorial indentation, we blastopore becomes the anus, whereas in protostomes the detect actin-positive cell outgrowths in seven cells (Figure 2E) fate of the blastopore is highly variable (Figure 1A and see that will form the presumptive epidermal buccal tooth-like also Figure S1 available online). In some protostome lineages structures of the larval introvert (‘‘scalids’’; Figure 1C). The the blastopore forms the mouth, in others it becomes the animal-vegetal axis of the egg thus corresponds to the ante- anus, whereas in others it elongates and, by midclosure, rior-posterior axis of the larva and adult. We have named this simultaneously forms the mouth and the anus (‘‘amphistomy’’) stage ‘‘introvertula’’. At this point, on the ventral (see below) [1, 2, 9]. The common shared fate of the blastopore in the Deu- side of the introvert (animal hemisphere), a group of ecto- terostomia indicates that deuterostomy is the ancestral state dermal cells ingresses and forms the mouth of the embryo to this group. However, the diversity of blastopore fates (Figure 2C). The developing anus is identified by a strong observed in the Protostomia makes it difficult to identify the actin-positive bundle at the vegetal pole (Figure S2). After ancestral mode of development in this group. As a conse- 6 days of development, the introvert-trunk boundary is more quence, not much is known regarding the fate of the pronounced (Figure 2D). Actin filaments in the introvert and in the trunk indicate the developing musculature of the embryo (Figures 2D and 2F). The mouth is now at an anterior terminal *Correspondence: [email protected] position (Figure 2F), and the gut lumen runs from the mouth Current Biology Vol 22 No 22 2162

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Figure 1. Fate of the Blastopore in the Bilateria and the Adult and Larva of Priapulus caudatus (A) Diversity of blastoporal fates in the Bilateria. Whereas in the Deuterostomia the blastopore forms the anus, the Protostomia exhibits a diversityof blastoporal fates. This diversity hampers the establishment of an ancestral fate of the blastopore to the Protostomia and, thus, to the last common bilaterian ancestor. Nevertheless, deciphering the ancestral mode of development to the Bilateria is crucial to our understanding of the transition from animals with a blind gut, like modern cnidarians, to bilateral symmetrical animals with a through gut. (B and C) General appearance of an adult (B) (picture courtesy of www.kahikai.org) and a hatching larva (C) of the priapulid Priapulus caudatus. The annulated body (cuticle rings pointed to by the white arrow in B) is divided into an anterior introvert and a posterior trunk separated by a ring-like constriction of the cuticle (the introvert-trunk boundary, white arrowheads in B and C). The introvert can retract inside the trunk, which bears a caudal appendage of unknown function at its posterior end in the adult. The mouth (asterisk) is located at the anterior end of the introvert and is surrounded by complex cuticu- larized toothlike structures called scalids (C). The hatching priapulid gradually matures into the adult form through a series of molts. ca, caudal appendage; in, introvert; tr, trunk; sc, scalids. Scale bars in (B) represent 1 cm and in (C) 50 mm. to the anus, which is formed by four ectodermal cells at the The foregut markers foxA and goosecoid (gsc) are ex- vegetal/posterior pole of the trunk (Figures 2D0, 2F, and S2). pressed during gastrulation in the animal hemisphere at the The direct formation of the anus from the blastopore site of the future mouth, before the ingression of ectodermal (deuterostomy) is supported by the embryonic expression of cells becomes visible. Orthologs of foxA are expressed in the the hindgut/posterior markers brachyury (bra) and caudal pharynx of cnidarians and in the foregut, the endodermal (cdx)(Figure 3). Orthologs of bra are expressed in the blasto- midgut, and the hindgut of deuterostomes and protostomes pore and gut opening of cnidarians; in the blastopore, foregut, (Table S1). During gastrulation, the priapulid ortholog of foxA and hindgut of ambulacrarians, spiralians, and chaetognaths; is expressed in a cluster of ectodermal cells on the ventral and in the blastopore and hindgut of arthropods (Table S1). side of the embryo (Figures 3I–3V) and in a subset of endome- In P. caudatus, bra is expressed in the blastopore and in sodermal cells. At the introvertula stage, foxA is expressed in a subset of endomesodermal cells during gastrulation (Figures the oral ectoderm and in the gut (Figures 3K and 3L). gsc is 3A and 3B). In the introvertula stage, bra is only detected in expressed in the pharynx of cnidarians and in the foregut of the anus (Figures 3C and 3D), which mirrors the lack of oral a broad range of deuterostomes and protostomes (Table brachyury expression in arthropods and suggests that this is S1). In P. caudatus, gsc is detected in a ventral cluster of ecto- ancestral to the whole Ecdysozoa. The gene cdx is primarily dermal cells (Figures 3M–3Z) and in an anterior ectodermal expressed in the hindgut of the deuterostomes and proto- domain that includes the mouth of the introvertula (Figures stomes (Table S1). The ortholog of cdx in P. caudatus is 3O and 3P). Orthodenticle (otx) has a conserved function in widely expressed in the vegetal pole of the gastrula (Figures specifying anterior body regions and neural structures in 3E and 3F), and in the trunk (posterior) of the introvertula bilaterians (Table S1), and it is expressed in the presumptive (Figures 3G and 3H). developing ventral nervous system of P. caudatus [17] Deuterostomic Development in Priapulus caudatus 2163

Figure 2. Development, Gastrulation, and Early Organogenesis in Priapulus caudatus z projections of confocal stacks of embryos at 3, 4, 5, and 6 days of development stained with phallacidin (green) and propidium iodide (magenta). Gastru- lation (A and A0) occurs at the vegetal pole and involves the formation of a small archenteron and a conspicuous blastopore (asterisk). After the invagination of the endomesoderm (B and B0), the archenteron collapses (white arrowhead in B), and the blastopore (asterisk) narrows. A high number of mitoses (white arrows) in the ectoderm is observed, in particular in the animal hemisphere. At day 5 (C and C0), an inward ectodermal infolding appears at the vegetal pole (white arrowheads in C and D), which marks the introvert-trunk boundary (magniﬁed in E from the white rectangle in C). At 7 points of this infolding, an ectodermal cell of the introvert region forms an actin-rich outgrowth (E) that will become an oral scalid. Note that the introvert shows a higher number of cells and smaller cells than the trunk. The mouth is formed on the ventral side of the introvert independently from the site of gastrulation. (D, D0, and F) After 6 days of development, the mouth shifts to an anterior terminal position, and an actin-positive bundle that corresponds to the gut lumen (central phallacidin spot in F) runs from the mouth to the anus. The former vegetal pole, where the blastopore opened, becomes the anus (D0). Schematic drawings of priapulid embryos at days 3, 4, 5, and 6 of development (A00–D00), respectively, placed in the general context of priapulid embryonic development. The black arrowheads indicate the position of the introvert-trunk boundary and its movement during introvert retraction. Drawings are oriented with the animal/anterior pole to the top and the ventral side to the left. Drawings are not to scale. In (A–E), animal/anterior to the top and vegetal/posterior to the bottom. In (F), the introvert projection is to the top and the trunk to the bottom; in (C–D0), ventral is to the left. an, anus; ac, archenteron; bp, blastopore; dg, digestive tract; ec, ectoderm; em, endomesoderm; mc, muscle; mo, mouth; sc, scalid. Scale bars in (A–D) and (F) represent 50 mm and in (A0–D0) and (E) represent 10 mm.

(Figures 3Q–3T). The colocalization of foxA and gsc with either the closure of the blastopore [24] or amphistomy otx (Figures 3U–3AB) indicates that the mouth is formed [25, 26]. In accordance with their extreme ecological and ventrally, a position that is considered to be ancestral in bilat- morphological diversity, divergent modes of cleavage and erians [2, 3, 10, 11]. gastrulation are observed among terrestrial hexapods, myria- There is little consensus about the primary mode of gastrula- pods, and chelicerates [18]. On the other hand, the marine tion of the Ecdysozoa, due to the diversity and divergence pycnogonids and some crustaceans retain the putative ances- observed in the main ecdysozoan lineages, namely the Nema- tral holoblastic cleavage. Unlike in most arthropods and toida (Nematoda + Nematomorpha) and the Panarthropoda ecdysozoans, the blastopore of pycnogonids forms in the (Tardigrada + Onychophora + Arthropoda) [18, 19](Figure S1). future dorsal side of the embryo and forms the mouth Similar to P. caudatus, nematomorphs display holoblastic (protostomy) or closes [15, 27]. Most crustaceans exhibit cleavage and deuterostomy, with the blastopore forming in deuterostomy with the blastopore forming in a ventral-poste- the ventral-posterior region of the embryo [20]. Although nema- rior position [18, 28, 29], which may be considered ancestral todes show holoblastic cleavage, gastrulation and gut forma- to the Panarthropoda given the divergence of tardigrades, tion can occur either by amphistomy (e.g., Tobrilus stefanskii) onychophorans, and most arthropods and the similarity to [19] or by the ventral-posterior ingression of the founder endo- the development of the Nematoida and Priapulida. dermal cell(s) and subsequent closure of the blastopore (e.g., Enoplus brevis and C. elegans)[19, 21]. Therefore, the nemato- Conclusions morph and nematode conditions suggest that in the last We show deuterostomic development in P. caudatus, common ancestor of the Nematoida, gastrulation occurred in a member of the third major lineage of the Ecdysozoa, the the ventral-posterior region and the blastopore likely formed Scalidophora. The expression of bra, cdx, foxA, gsc, and otx posterior/anal structures (deuterostomy). during early development in P. caudatus is similar to that of Tardigrades show holoblastic cleavage followed by gastru- the nematodes and arthropods where it has been described lation and blastopore closure [22, 23]. Onychophorans exhibit (Table S1). These ﬁndings indicate an ancestral ecdysozoan mostly superﬁcial cleavage [18], and gastrulation involves expression pattern (Figure 4) and, together with the particular Current Biology Vol 22 No 22 2164

Figure 3. Expression of Hindgut and Foregut Markers during Priapulus caudatus Gastrulation and Mouth Formation Whole-mount in situ hybridization in priapulid embryos at the gastrula and introvertula stage. During gastrulation, the hindgut markers bra (A and B) and cdx (E and F) are expressed at the vegetal pole and in the blastopore (magnified in the insets of B and F). At the introvertula stage, bra is expressed in the anus (C and D) and cdx in the trunk posterior ectoderm (G and H). cdx also shows expression on a small cluster of cells of the introvert, probably associated with the nervous system (black arrow in G). The foregut markers foxA and gsc are expressed in an equatorial ectodermal domain of the gastrula (I and J; M and N) (black arrows in I and M). At the introvertula stage, foxA is expressed in the terminal oral ectoderm of the introvert (inset in L) and in the digestive tract (K and L), and gsc is expressed in the mouth (inset in P) and in a lateral ectodermal domain of the introvert (O and P). otx is detected on one lateral of the priapulid gastrula (black arrow in Q) in a U-shaped pattern (Q and R) and in the anterior nervous structures of the introvertula (S and T): one lateral of the introvert (slashed line in S–AB), the mouth (inset in T), and a ring around the introvert-trunk boundary (black arrows in S, W, and AA). The simultaneous detection of foxA and gsc with otx shows overlap- ping expression patterns and, thus, indicates that the specification of the mouth occurs in the ventral side of the embryo. In all panels, the asterisk indicates the vegetal/posterior region. In (C), (G), (K), (O), (S), (W), and (AA), the black arrowheads mark the introvert-trunk boundary, and anterior is to the top and ventral to the left. In (I), (M), (Q), (U), and (Y), ventral is to the left. In (W), the white arrowhead points to the foxA expression in the digestive tract. The schematic drawings of the gastrula and introvertula stage depict the reported expression domains. Draw- ings are not to scale. The slight elongation in the animal-vegetal axis of the embryos at the gastrula stage is an artifact of fixation.

conservatively evolving features of priapulids, strongly the mouth, and the anus during embryogenesis led to blasto- suggest that the anus is the ancestral fate of the blastopore pore closure, and, inversely, the simultaneous activation of in this major clade of protostomes. the mouth and anal gene regulatory networks in different Deuterostomy in the ecdysozoan ancestor has major evolu- regions of the blastopore, together with the extension of the tionary implications. Given that chaetognaths [30] (an enig- embryo along the anterior-posterior axis, originated amphis- matic group of protostomes not belonging to the Ecdysozoa tomy. This sequence of evolutionary events differs from tradi- nor the Spiralia [31, 32]) and deuterostomes also exhibit tional hypotheses about the origin of the Bilateria [2, 10, 11], deuterostomic development, it is likely that deuterostomy but agrees with the hypothesis that explains the transition was the ancestral developmental program in bilaterians (Fig- from radial to bilateral animals through the shift of the blasto- ure 4). Furthermore, distinct and independent gene regulatory poral gene regulatory network, from the animal to the vegetal networks to specify the blastopore, the anus, and the mouth pole, that allowed the independent development of the meta- were present in the last common bilaterian ancestor, as it is zoan mouth from the blastopore in bilaterians [3]. The present observed in extant priapulids, chaetognaths, acoels [33], and findings thereby challenge our classic ideas about the origin deuterostomes (Table S1). and diversification of bilaterian animals and highlight the This scenario can easily explain the variability of fates of the importance of studying the great diversity of animal forms in blastopore observed in the Protostomia by the spatial and/or our pursuit of a better understanding of animal evolution. temporal coupling or decoupling of the blastoporal, oral, and anal gene regulatory networks as new egg structures, modes of cleavage, and reproductive strategies evolved. Accordingly, Experimental Procedures protostomy is the secondary coupling of the genes specifying Adult gravid specimens of P. caudatus Lamarck 1816 were collected from the blastopore and the mouth, which appeared independently Gullmarsfjorden (Fiskeba¨ ckskil, Sweden) in November and spawned as in several lineages of ecdysozoans and the Spiralia. The described previously [14]. Embryos were permeabilized with 0.05% thiogly- complete dissociation of the genes specifying the blastopore, colate and 0.01% pronase in filtered deep-sea water (FDSW) for 45 min at Deuterostomic Development in Priapulus caudatus 2165

Bilateria Figure 4. Evolution of the Bilaterian Through Gut Related to the Expression of bra, cdx, gsc, and Protostomia foxA and the Deuterostomic Development of * P. caudatus The expression of bra, cdx, gsc, and foxA indi- * cates the homology between the mouth and the anus of P. caudatus and other bilaterians, as well as the homology of the oral opening of cnidarians and the mouth of bilaterians. The * * comparison of the embryogenesis and expres- Cnidaria Deuterostomia Ecdysozoa Spiralia sion data in P. caudatus with the other ecdyso- (Nematostella vectensis) (Ptychodera flava) (Priapulus caudatus) (Platynereis dumerilii) zoan lineages support considering deuterostomy as the ancestral mode of development in the - Evolution of protostomy Ecdysozoa and, therefore, suggest that the last in certain groups common ancestor to the Deuterostomia and Pro- tostomia exhibited deuterostomic development. This scenario differs from traditional hypotheses that considered either protostomy or amphis- bra tomy as the ancestral mode of development, cdx but agrees with recent molecular results and - Separation of mouth development foxA a hypothesis that indicates the separation of the from the blastopore (deuterostomy) gsc development of the mouth from the site of gastru- - Evolution of the through gut lation as a key step in the evolution of the Bilate- ria. Embryos are oriented with the animal pole (also anterior, in bilaterians) to the top and the ventral side (in bilaterians) to the left. The asterisk indicates the fate of the blastopore in each embryo. Only gene expression related to the through gut is indicated in this diagram (see Table S1 for references). Drawings are not to scale.

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