Evolutionary implications of morphogenesis and ...

Developmental Biology 352 (2011) 164–176

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Developmental Biology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / d e v e l o p m e n t a l b i o l o g y

Evolution of Developmental Control Mechanisms

Evolutionary implications of morphogenesis and molecular patterning of the blind gut in the planarian Schmidtea polychroa José María Martín-Durán ⁎, Rafael Romero Universitat de Barcelona, Departament de Genètica, Avda. Diagonal 645, E-08028 Barcelona, Spain

a r t i c l e

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Article history: Received for publication 22 September 2010 Revised 20 January 2011 Accepted 25 January 2011 Available online 3 February 2011 Keywords: Planarian Evolution Development Foregut Midgut Hindgut Digestive system

a b s t r a c t The formation of a through-gut was a key innovation in the evolution of metazoans. There is still controversy regarding the origin of the anus and how it may have been either gained or lost during evolution in different bilaterian taxa. Thus, the study of groups with a blind gut is of great importance for understanding the evolution of this organ system. Here, we describe the morphogenesis and molecular patterning of the blind gut in the sexual triclad Schmidtea polychroa. We identify and analyze the expression of goosecoid, commonly associated with the foregut, and the GATA, ParaHox and T-box genes, members of which commonly are associated with gut regionalization. We show that GATA456a is expressed in the blind gut of triclads, while GATA456b is localized in dorsal parenchymal cells. Goosecoid is expressed in the central nervous system, and the unique ParaHox gene identified, Xlox, is detected in association with the nervous system. We have not isolated any brachyury gene in the T-box complement of S. polychroa, which consists of one tbx1/10, three tbx2/3 and one tbx20. Furthermore, the absence of genes like brachyury and caudal is also present in other groups of Platyhelminthes. This study suggests that GATA456, in combination with foxA, is a gut-specific patterning mechanism conserved in the triclad S. polychroa, while the conserved gut-associated expression of foregut, midgut and hindgut markers is absent. Based on these data and the deviations in spiral cleavage found in more basal flatworms, we propose that the lack of an anus is an innovation of Platyhelminthes. This may be associated with loss of gut gene expression or even gene loss. © 2011 Elsevier Inc. All rights reserved.

Introduction The development of a through-gut was one of the key innovations in the evolution of metazoans, as it allowed the directional movement of ingested nutrients along the body, thereby enabling the diversification and adaptation of regions of the digestive tract to deal with food at different stages of digestion. Despite its importance, the evolutionary origin of the through-gut and sequence of events that led to its appearance are still not well understood. Recently, the study of this question in acoels, bilateral worms with a single gut opening that are placed at the base of all remaining bilateria (Ruiz-Trillo et al., 1999; Hejnol et al., 2009), suggested that the through-gut could have evolved multiple times during animal diversification (Hejnol and Martindale, 2008). According to the latter study, the mouth of acoels, protostomes, deuterostomes, with the possible exception of chordates, and therefore, of the last common ancestor of the Bilateria, was homologous. The origin of the anus is more complex. The expression of conserved hindgut genes in the posterior region of the acoel worm could mean either that this is a remnant of a posterior anal opening

⁎ Corresponding author. Fax: + 34 934 034 420. E-mail address: [email protected] (J.M. Martín-Durán). 0012-1606/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2011.01.032

present in the last common ancestor of all bilaterians, or that the co-option of these genes in the male gonopore predated the formation of a posterior ectodermal–endodermal connection. Thus, it is evident that further study of those bilateral taxa that possess a blind gut is necessary to better understand the origin and subsequent diversification of this ancient organ system. Platyhelminthes, together with the Acoelomorpha (acoels and nemertodermatids), Gnathostomulida, Micrognathozoa and Xenoturbellida, form the bilateral phyla whose plesiomorphic trait is the lack of an anus (Hyman, 1951; Kristensen, 2002). Whether these taxa simply retained the ancestral blind gut organization (and thus did not evolve from ancestors with a through-gut) or they represent secondary losses of a conserved through-gut is a question that remains to be answered. Among free-living Platyhelminthes, triclads (commonly referred to as planarians) are the most commonly studied group, given their extensive regenerative capabilities and the increasing availability of molecular data (Robb and Sánchez Alvarado, 2008) and methodological resources (Newmark and Sánchez Alvarado, 2002; Saló et al., 2009). Study of their embryogenesis has, however, been hindered by difficulties in the handling of, and experimentation with, embryos, and only in recent years has a thorough description of their embryogenesis been accomplished in the species Schmidtea polychroa (Schmidt, 1861) (Cardona et al., 2005; Cardona et al., 2006; MartínDurán et al., 2010). Triclads have ectolecithal embryos, characterized by

J.M. Martín-Durán, R. Romero / Developmental Biology 352 (2011) 164–176

the presence of embryonic nutrients in a separate cell lineage, the yolk cells, packaged together with the oocytes in the same egg capsule. Despite being placed within the Lophotrochozoans (Halanych et al., 1995; Aguinaldo et al., 1997) triclads have lost the ancestral spiral cleavage. Their embryonic development has been divided into eight stages (Cardona et al., 2005). Following cleavage, some blastomeres differentiate into transient structures (stages 1 and 2), which are required to ingest the maternally supplied nutrients (stage 3). The remaining undifferentiated blastomeres proliferate (stage 4) and give rise to the definitive axial pattern and organ primordia (stages 5 and 6). Finally, the definitive organ systems and body proportions are established (stages 7 and 8). Despite its divergence, this pattern of embryonic development still shows conservation with the ancestral spiralian pattern at the level of the molecular mechanisms controlling germ layer segregation and axial patterning (Martín-Durán et al., 2010). Triclads are therefore an attractive system among free-living Platyhelminthes in which to address fundamental questions in metazoan development and evolution. As expected in such a primary organ system, a significant level of conservation among the molecular mechanisms that specify the cells and tissues of the digestive system is observed across taxa (Roberts, 2000; Stainier, 2005; Hinman and Davidson, 2007). Conserved expression of blastoporal and foregut markers, such as brachyury, foxA, orthodenticle (otx) and goosecoid (gsc), is detected, for instance, in ambulacrarians (hemichordates and echinoderms), protostomes, acoels, cnidarians and ctenophores (Peterson et al., 1999; Arendt et al., 2001; Davidson et al., 2002; Scholz and Technau, 2003; Martindale et al., 2004; Boyle and Seaver, 2008; Hejnol and Martindale, 2008; Yamada et al., 2009; Boyle and Seaver, 2010). Similarly, a conserved expression of the GATA456 subfamily of transcription factors (Patient and McGhee, 2002; Technau and Scholz, 2003) and ParaHox genes (Holland, 2001; Fröbius and Seaver, 2006; Kulakova et al., 2008; Cole et al., 2009; Samadi and Steiner, 2010) during the specification, differentiation and regionalization of the gut into foregut, midgut and hindgut in most metazoans has been demonstrated. In triclads, the function of these conserved gut-related genes during embryogenesis and regeneration is still poorly understood. Only foxA (Koinuma et al., 2000; Martín-Durán et al., 2010) and otx (Umesono et al., 1999) have been studied to date, and only the former was shown to be expressed in the pharynx during embryonic development and regeneration. In this work, we use the sexual species S. polychroa to shed light on the evolution of the digestive tract of bilaterians through the study of the formation and molecular patterning of the blind gut during embryonic development. We thoroughly describe the morphological events leading to the formation of a mature digestive system in triclads, and complement these findings with the expression patterns of triclad gut genes (orthologs of porcupine and ßcatenin) at key developmental stages. In addition, we identify and characterize the expression domains during embryogenesis of those genes involved in bilaterian endoderm specification and regionalization, namely, GATA456 transcription factors, goosecoid, the ParaHox complement and the T-box complement. Finally, we discuss our data in relation to previous knowledge on these genes in different metazoan taxa and put forward a possible scenario to explain the evolution of a blind gut within the Platyhelminthes.

Material and methods Animal culture A collection of S. polychroa from Sant Celoni (Barcelona, Spain) was maintained in the lab as described previously (Martín-Durán et al., 2008). Egg capsules were regularly collected and kept in Petri dishes

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at 20 °C in culture water (distilled water:tap water, 1:1, v:v) until the desired developmental stage was reached. Histology Egg capsules were fixed in 4% paraformaldehyde, 1% glutaraldehyde in PBS (phosphate buffer saline) for 4 h at 4 °C and washed overnight in PBS. Hatchlings and juveniles were fixed in Steinmann's fixative (concentrated nitric acid:saturated solution of mercuric chloride in 5% sodium chloride:distilled water, 1:1:1, v:v:v) for a few minutes and washed several times in distilled water. All specimens for histological studies were embedded in paraffin and sectioned as described elsewhere (Cardona et al., 2005), counterstained with Mallory's solution (Sluys, 1989) and mounted in DPX mounting medium (44581, Sigma, St Louis, MO). Identification and cloning of S. polychroa genes S. polychroa is a closely related species to the triclad model S. mediterranea. Partial fragments of the genes Spol-ßcatenin-2 and Spolporcupine-1 (GenBank accession numbers HQ230963 and HQ230962, respectively) were cloned using interspecific primers designed against the respective S. mediterranea sequences. Spol-GATA123a, Spol-GATA123b, Spol-GATA456a, Spol-GATA456b, Spol-goosecoid and Spol-xlox (GenBank accession numbers HQ230964, HQ230965, HQ230966, HQ230967, HQ230968 and HQ230969, respectively) were cloned with primers designed against the predicted S. mediterranea genes. These predictions were established via BLAST searches (http://ncbi.nlm.nih.gov) and genomic annotations available in SmedGD (http://smedgd.neuro.utah. edu). The T-box-containing genes Spol-tbx1/10, Spol-tbx2/3a, Spol-tbx2/3b and Spol-tbx2/3c (GenBank accession numbers HQ230973, HQ230970, HQ230971 and HQ230972) were cloned using the degenerate forward primer GRRMFP (5′-GGNMGNMGNATGTTYCC-3′) and the degenerate reverse primer PFAKGFR (5′-CKRAANCCYTTNGCRAANGG-3′). The gene Spol-tbx20 (GenBank accession number HQ230974) was cloned with the degenerate primers NKPTDW (5′-AAYAARCCNACNGAYTGG-3′) and PETIFIA (5′-GCDATRAADATNGTYTCNGG-3′). Elongation of the cloned fragments and identification of full-length transcripts were achieved by classic RACE-PCR as described in Frohman (1994). Identification of S. mansoni and M. lignano genes S. mansoni ParaHox genes and T-box genes were identified via BLAST searches of the genome of this organism (http://www.sanger. ac.uk/cgi-bin/blast/submitblast/s_mansoni) using the scaffolds version 3.1 as the database. Predictions were compared with the T-box predictions available in the NCBI database, and the latter were used for the phylogenetic analysis. M. lignano ParaHox genes and T-box genes were identified via BLAST searches of its genome (http://www. macgenome.org/blast/index.html) using the ML100625 assembly, and results were supplemented with searches of the M. lignano EST database (http://flatworm.uibk.ac.at/macest/blast.php). Phylogenetic analysis Amino acid alignments were carried out using MAFFT (Katoh et al., 2002) and corrected by hand for obvious alignment errors. Nexus files of any of these are available upon request. In each case, ProtTest (Abascal et al., 2005) was used to select the best-fit model of protein evolution and MrBayes v3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003) was used to conduct the Bayesian phylogenetic analysis. The models used for each analysis were: JTT with GATA factors, JTT + I + G with goosecoid, xlox and T-box genes, RtREV with flatworm ParaHox genes and WAG with flatworm T-box genes. The results are a consensus of two converged runs of 2,000,000 (goosecoid), 2,300,000 (T-box proteins), 3,500,000 (xlox), 4,500,000

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(flatworm ParaHox genes), 6,000,000 (GATA transcription factors) or 13,000,000 (flatworm T-box genes) generations sampled every 1000 generations. Trees were visualized in FigTree and edited in Adobe Illustrator. Taxon names for gene sequences are abbreviated as follows: Aa Aedes aegypti, Ag Anopheles gambiae, Am Apis mellifera, An Aspergillus niger, As Archaster typicus, Bf Branchiostoma floridae, Cht Chaetopterus sp., Ci Ciona intestinalis, Cl Convolutriloba longifissura, Ct Capitella teleta, Dj Dugesia japonica, Dl Diplosoma listerianum, Dm Drosophila melanogaster, Dr Danio rerio, Dt Discocelis tigrina, Es Euprymna scolopes, Gv Gibbula varia, He Hydroides elegans, Hr Halocynthia roretzi, Hs Homo sapiens, Lg Lottia gigantea, Ml Macrostomum lignano, Mm Mus musculus, Nc Neurospora crassa, Nv Nematostella vectensis, Od Oikopleura dioica, Pc Podocoryna carnea, Pd Platynereis dumerilii, Pf Ptychodera flava, Ps Phascolion strombi, Pv Patella vulgata, Sk Saccoglossus kowalevskii, Smar Strigamia maritima, Sp S. polychroa, Spur Strongylocentrotus purpuratus, Sr Symsagittifera roscoffensis, Tc Tribolium castaneum, Tl Themiste lageniformis, Um Ustilago maydis, and Xl Xenopus laevis. Gene expression studies Single colorimetric or fluorescent whole-mount in situ hybridizations in triclad embryos were carried out as previously described (Martín-Durán et al., 2010). Whole-mount in situ hybridizations in hatchlings and juveniles were performed as described in Umesono et al. (1997), using an in situ robot (InsituPro VSi, Intavis), and developed with an alkaline phosphatase–NBT/BCIP colorimetric assay. For all experiments, a sense riboprobe was used as the negative control (supplementary file 1, Fig. S1). Juveniles with a specific expression pattern were embedded in a gelatin/albumin mixture and solidified with glutaraldehyde. The specimens were subsequently sectioned at 30 μm thickness using a vibratome (Vibratome 1000 plus). The sections were then mounted in 70% glycerol. Anti-αtubulin AA4.3 mouse monoclonal antibody from the Developmental Studies Hybridoma Bank was used at 1:50 dilution to detect the cephalic ganglia, nerve cords and pharynx. In situ hybridization on paraffin sections of embryos at stage 7 were performed as described in Martín-Durán et al. (2010). Imaging Whole-mount embryos, hatchlings and juveniles were observed under a Leica MZ16F stereomicroscope. Images from representative specimens of each experiment were captured with a ProgRes®C3 camera from Jenoptik. Histological sections were observed under a Zeiss Axiophot microscope equipped with a Leica DFC300FX camera. Fluorescent preparations were observed under a Leica TCS-SPE confocal microscope. Images were analyzed using Helicon Focus, Adobe Photoshop, ImageJ and Adobe Illustrator. Results Gut morphogenesis during S. polychroa embryonic development The digestive system of an adult S. polychroa consists of a blind intestine (divided into three branches, one anterior and two posterior, with many lateral diverticula) connected through a short esophagus to a plicate pharynx. The pharynx projects posteriorly and is usually located in the mid part of the body. It rests inside of a cylindrical pharyngeal cavity with a single opening, the mouth. During embryonic development, two different digestive systems are formed (Hallez, 1887; Cardona et al., 2005; Cardona et al., 2006): the transient embryonic pharynx of the yolk-feeding embryo, and the definitive blind gut and pharynx of the hatchling. The transition between these two systems takes place after about 6 days of development, when the primordium of the definitive pharynx starts

to form ventrally to the degenerating embryonic pharynx (Fig. 1AI–AVI). One day later, the embryonic pharynx disappears completely and the pharynx anlage appears as a rounded organ with a lumen running throughout and a rudiment of the pharynx cavity, not yet connected to the exterior (Fig. 1BI–BVI). By the tenth day of development, the pharynx primordium becomes a cylindrical fold of complex histological structure and the pharynx cavity connects to the outer ventral surface of the embryo, thus opening the mouth of the embryo. Upon hatching (~day 15), the pharynx is fully developed, showing different muscle and nervous layers and a well-developed ciliated epithelium (Fig. 1C). In 10-day-old embryos, the parenchyma and the gut cells are restricted to the outermost area of the embryo (Fig. 1D). At this point, groups of cells project towards the inside of the yolk mass, forming the primordia of the lateral intestine diverticula (Fig. 1DI). After fifteen days of development, when the embryo hatches, the yolk cells still form a compact mass, but are now delimited by a thin epithelium of cells (Fig. 1EI). The diverticula are more defined and the posterior gut branches start to become apparent (Fig. 1E). The embryo thus hatches with an immature digestive system, but with the blind intestine filled with nutrients. By 3 days after hatching, the typical shape of the blind intestine of a triclad is evident (Fig. 1F) and the yolk content has been absorbed by the immature gut cells, which have grown in size and present many digestive vesicles inside (Fig. 1FI). The pharynx elongates and the esophagus becomes discernible. Six days after hatching, the gut cells are still full of yolk vesicles (Fig. 1GI), but the gut lumen is clear of yolk (Fig. 1G). After 9 days, the juvenile has completely digested the maternally-supplied yolk content (Fig. 1H) and has a fully developed digestive system with all the distinct parts observed in the adult (Fig. 1I). This process of maturation observed by means of histological sections is also partially visible in living hatchlings of a few days (supplementary file 2, Fig. S2). Fig. 1J summarizes the main events during the development of the definitive gut in S. polychroa. Expression of the triclad gut genes porcupine-1 and ß-catenin-2 To overcome the difficulties encountered in the histological studies with regards to defining how the gut cells arise and organize the digestive system during embryonic development, we decided to study the expression of triclad gut genes in embryos of different stages. The genes Smed-porcupine-1 and Smed-ßcatenin-2 have been reported to be expressed diffusely in the blind intestine of adult specimens of the triclad species Schmidtea mediterranea (Gurley et al., 2008; Iglesias et al., 2008). We have partially cloned both genes in the sexual species S. polychroa, showing that they share high similarity with their orthologous genes in S. mediterranea (supplementary file 3, Table S1). By whole-mount in situ hybridization, Spol-porcupine-1 is detected in clumps of cells in stage 4 embryos (Fig. 2A). At stage 5, when the embryo adopts its definitive axial identities (Martín-Durán et al., 2010), its expression is mostly localized in the lateral margins of the embryo and ventrally to the degenerating embryonic pharynx in the cells that correspond to the early stages of the definitive pharynx primordium (Fig. 2B). This expression is maintained during stage 6 and stage 7, when Spol-porcupine-1 is detected in the cells surrounding the inner mass of yolk and in the parenchymal projections that will define the future gut diverticula (Fig. 2C). Similar expression patterns are observed for Spol-ßcatenin-2. At stage 5, it is detected in a ring of cells located more towards the future ventral side of the embryo, but still engulfing the yolk (Fig. 2E). In contrast to Spol-porcupine-1, Spol-ßcatenin-2 is not detected in the definitive pharynx anlage at this stage. At stage 7, Spol-ßcatenin-2 is detected in those cells nearer to the ingested yolk, the definitive pharynx and the growing gut diverticula (Fig. 2F). All in all, morphology and the expression of triclad gut genes provide a framework in which to address how the evolutionarily conserved genes determining and patterning the endoderm in other metazoans are expressed during the development of the digestive system of triclads.


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Fig. 1. Gut formation during the embryonic development of Schmidtea polychroa. Paraffin sections of embryos (A–B, D), hatchlings (C, E–H) and an adult (I) stained with Mallory's solution. (A–C, E–H) Horizontal sections. (D, I) Sagittal sections. (AI–AVI) In 6-day-old embryos, the definitive pharynx primordium appears as a clump of cells developing ventrally to the degenerating embryonic pharynx. (BI–BVI) One day later, the pharynx anlage develops a primitive pharynx cavity and the lumen of the plicate pharynx. At this point, the embryonic pharynx has been completely reabsorbed. (C) Appearance of the plicate pharynx in a newborn. The asterisk indicates the distal opening. (D, DI) Ten-day-old embryo and detail of the gut and parenchymatic cells. At this stage, the embryo consists of a large mass of yolk and a thin peripheral layer of embryonic cells in which the different organs are developing. Note the projections towards the yolk mass that will form the lateral gut diverticula (black arrowheads in DI). (E–H, EI–HI) Sections of hatchlings at day 0, day 3, day 6 and day 9, respectively, and details of their digestive system. As the days pass, the hatchling absorbs the yolk content (see food vesicles in FI–HI , indicated by a black arrow) and the original thin gut surrounding the yolk (black arrowheads in EI) transforms into a mature columnar epithelium. (I, II) Transverse section of an adult, showing the different parts of the digestive system, part of the copulatory organ and detail of the adult gut (II, food vesicles denoted by a black arrow). (J) Schematic summary of developmental events that take place in embryogenesis and after hatching during the development of the digestive system of triclads. In blue, the primary gut cells and the gastrodermis (based on this study). In green, the definitive pharynx anlage and the plicate pharynx. Drawings of different stages are not to scale. co, copulatory apparatus; dpp, definitive pharynx primordium; e, eye; es, esophagus; ep, embryonic pharynx; gd, gut diverticula; p, pharynx; pc, pharynx cavity; pl, pharynx lumen; y, yolk. Scale bars, AI–BI 100 μm, C 50 μm, D–E 500 μm, F–I 1 mm. In A and B, superscripts indicate the relative position of the section from the dorsal to the ventral side of the embryo.

Characterization of the GATA complement and expression of GATA456 genes Owing to their role in triggering endoderm and mesoderm development, we decided to identify and characterize the expression of the GATA456 complement during the embryonic development of

S. polychroa. A previous study proposed the existence of four different GATA genes in triclads, three GATA456 and a single GATA123, based on in silico predictions over the genome of S. mediterranea (Gillis et al., 2008). We have identified and partially cloned four GATA genes in S. polychroa, which correspond to the four genes already predicted in S. mediterranea. We have assigned their orthology to either of the two

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Fig. 2. Embryonic expression of the triclad gut genes Spol-porcupine-1 and Spol-ßcatenin-2. (A–C, E–F) Whole-mount in situ hybridization of Spol-porcupine-1 and Spol-ßcatenin-2 in embryos at stage 4 (S4), stage 5 (S5) and stage 7 (S7). (A) Spol-porcupine-1 is first expressed in groups of cells in the germ band (black arrowheads). (B) In stage 5 embryos, it is mostly detected in the lateral margins of the embryo (white arrowheads) and in the definitive pharynx primordium ventrally to the degenerating embryonic pharynx (dashed circle). (C) At stage 7, Spol-porcupine-1 is detected in the cells closest to the yolk and in the growing gut diverticula (white arrowheads). Staining in the pharynx is most likely artefactual, since it occurs with the sense riboprobe at this stage (see supplementary file 1, Fig. S1A). (E) At stage 5, Spol-ßcatenin-2 is detected in a ventro-lateral ring-like pattern (dashed black circle). (F) At stage 7, it is expressed in the pharynx and in those cells enclosing the yolk and forming the gut diverticula (white arrowheads). (D–G) Diagrams representing the changes in the expression of each gene during development. Drawings of different stages are not to scale. In C and F, the anterior is to the left. e, eye; ep, embryonic pharynx; dp, definitive pharynx.

classes of GATA factors by phylogenetic analysis using the alignment of the zinc finger domains, a strategy also followed in Gillis et al. (2008). As seen in Fig. 3A, the GATA complement of S. polychroa consists of two GATA123 genes (Spol-GATA123a and Spol-GATA123b) and two GATA456 genes (Spol-GATA456a and Spol-GATA456b). SpolGATA123b corresponds to the in silico predicted Smed-GATA456Nc. An amino-acid alignment of the full-length sequences of SpolGATA456a and Spol-GATA456b with other genes of the class GATA456 shows the presence of common features and motifs among these proteins (supplementary file 4, Fig. S3). As observed in other Spiralians, such as the annelids Capitella teleta or Chaetopterus, or the sipunculan worm T. lageniformis (Boyle and Seaver, 2008; Boyle and Seaver, 2010), triclad GATA456 genes present short-lengthresidue spans between their two class IV zinc finger domains (supplementary file 4, Fig. S3A). Additionally, divergent GATA456specific motifs upstream from the dual-zinc finger domains (Gillis et al., 2007) are aligned in the S. polychroa GATA456 genes (supplementary file 4, Figs. S3B–D). Given the high divergence exhibited by triclad GATA456 genes, we have applied a 20% identity cut-off between the triclad sequence and the consensus sequence of the motif, as in Gillis et al. (2007), to test for their presence or absence. Using this threshold, Spol-GATA456a has retained a divergent N2 motif (28.57% of identity) and has lost the N1, N3 and N4 motifs (Fig. 3B). In contrast, Spol-GATA456b has lost the N1 and N2 motif, but has retained divergent N3 and N4 motifs (22.22% and 25% identity, respectively) (Fig. 3B). Analysis of the expression of the GATA456 genes during the embryonic development of S. polychroa reveals that Spol-GATA456a is first detected in stage 3–4 embryos (Fig. 3C), in isolated cells located in the germ band (thin layer of syncytial yolk delimited externally by the embryonic epidermis and internally by the ingested yolk). It is not expressed in the embryonic pharynx, and the positive cells show no common pattern among different embryos. At stage 5, the number of positive cells increases, especially in the lateral margin of the embryo (Fig. 3D). However, it is not expressed in the degenerating pharynx or the definitive pharynx anlage. By stage 7, Spol-GATA456a is detected in isolated cells all around the yolk mass and in the growing gut diverticula (Figs. 3E–F).

Spol-GATA456b is also first detected in stage 3–4 embryos, but in fewer isolated cells of the germ band (Fig. 3G). At stage 5, the number of positive cells is still low and most of them are located in the future dorsal side of the definitive embryo, particularly around the degenerating embryonic pharynx (Fig. 3H). During anterior–posterior elongation and dorsoventral flattening of stage 6 embryos, SpolGATA456b is detected in many dorsal parenchymal cells and few ventral cells (Fig. 3H), a pattern also observed in stage 7 embryos (Figs. 3J–K). Complementary expression of Gata456 genes and porcupine-1 in embryos and hatchlings To further characterize the expression of the triclad GATA456 genes in the development of the digestive system, we decided to study the complementary localization of these two genes and that of the triclad gut gene Spol-porcupine-1, in embryos (Fig. 4) and hatchlings (Fig. 5). Figs. 4A–DI shows the colocalization by means of double fluorescent whole-mount in situ hybridization of Spol-GATA456a and Spolporcupine-1 in those cells closer to the ingested mass of yolk. As also observed in the single in situ hybridizations (Figs. 2C and 3E), Spol-porcupine-1 presents a broader localization (Fig. 4A), while SpolGATA456a is specifically expressed in isolated cells placed in close contact with the yolk (Fig. 4B). In contrast, Spol-GATA456b and Spolporcupine-1 do not show overlapping patterns (Fig. 4H). The cells expressing Spol-GATA456b (Fig. 4E) do not colocalize with the expression of Spol-porcupine-1 (Fig. 4F). Similarly, Spol-GATA456a and Spol-GATA456b do not colocalize either (Figs. 4I–L), as expected. The expression of Spol-porcupine-1 and Spol-GATA456a in newborns and in 10-day-old hatchlings also confirms the coexpression of both genes in the gastrodermis (Fig. 5). In newborns, the gut staining is more diffuse: the three gut branches are visible (especially a large anterior one), but the lateral gut diverticula are not completely defined (Figs. 5A and D). However, 7 to 10 days after hatching, the expression of these two genes corresponds to the expression expected in a mature gut (Figs. 5B and E). Vibratome cross sections confirm the expression of Spol-porcupine-1 and SpolGATA456a in the gut branches (Figs. 5C and F). Similarly, the


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Fig. 3. Complement of GATA factors and embryonic expression of Spol-GATA456a and Spol-GATA456b. (A) Phylogenetic analysis of the orthology of GATA factors. The support values of the branches are posterior probabilities of Bayesian likelihood (only those ≥0.99 are shown and indicated as an asterisk; see supplementary Fig. S4 for all the support values of the branches). S. polychroa GATA sequences are highlighted in red. (B) Sequence characterization of each S. polychroa GATA456 gene. Intron positions are represented as inverted triangles (in red those that are evolutionarily conserved). The Platynereis dumerilii GATA456 factor (Pd GATA456, on the left) exemplifies the putative complete GATA456 gene, with the four GATA456-specific motifs and a compact class IV dual-zinc finger domain. By contrast, S. polychroa GATA456 factors present a split dual-zinc finger domain (light blue boxes) and only certain subsets of specific GATA456 motifs. (C–E, G–J) Whole-mount in situ hybridization of Spol-GATA456a and Spol-GATA456b in embryos at stage 4 (S4), stage 5 (S5), stage 6 (S6) and stage 7 (S7). (EI, JI) Higher magnifications of the whole-mount in situ patterns. (F–K) In situ hybridization on paraffin sections of stage 7 embryos of Spol-GATA456a and Spol-GATA456b, respectively. (C) At stage 3–4, Spol-GATA456a is expressed in discrete cells (white arrowheads) in the germ band. (D) At stage 5, it is still detected in isolated cells (white arrowheads), particularly in the lateral margin of the embryo (white arrowheads). (E–F) At stage 7, Spol-GATA456a is expressed in isolated cells close to the yolk (white arrowheads) and in the forming gut diverticula (black arrow in EI, black arrowheads in F). (G) Spol-GATA456b is detected in a few cells in the germ band at stage 3–4 (white arrowheads), but not in the embryonic pharynx. (G) At stage 5, it is localized in some cells of the future dorsal side of the embryo, especially around the degenerating embryonic pharynx (white arrowhead). (I–K) Finally, at stage 6 and 7, it is still detected in scattered dorsal cells (white arrowheads in I and J, black arrowheads in K). (L–M) Diagrams representing the changes in the expression of each gene during development. Drawings of different stages are not to scale. In E, I and H, the anterior is to the left. d, dorsal side; dp, definitive pharynx; e, eye, ep, embryonic pharynx; gb, germ band; iy, ingested yolk; ob, outlying blastomeres; v, ventral side.

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Fig. 4. Complementary expression of Spol-GATA456a, Spol-GATA456b and Spol-porcupine-1 in embryos of S. polychroa. (A–L) z-projections of confocal stacks from whole-mount double fluorescent in situ hybridizations on embryos at stage 7. (A–D) Spol-porcupine-1 and Spol-GATA456a exhibit a similar distribution in the cells surrounding the ingested yolk (white arrowheads denote cells expressing both genes). (E–H) On the contrary, dorsal cells positive to Spol-GATA456b (white arrowhead) do not co-express Spol-porcupine-1. (I–L) Similarly, cells expressing Spol-GATA456a (white arrows) do not co-express Spol-GATA456b (white arrowheads). In all panels, the dashed white lines delimit the boundaries of the embryo and the forming gut diverticula. Scale bars, A–H 100 μm, I–L 250 μm.

expression studies in hatchlings demonstrate the localization of SpolGATA456b in isolated cells mostly on the dorsal side of the animal (Figs. 5G and H), located in close contact with the mature gut branches (Fig. 5I). Expression of goosecoid and the ParaHox gene xlox in embryogenesis Having demonstrated the relation between the GATA456 genes and the blind gut of triclads, we studied the expression of genes involved in the regionalization of the bilaterian gut. We identified the full-length sequence of the foregut-marker goosecoid in the triclad species S. polychroa, and assigned its orthology by means of phylogenetic analysis (Fig. 6A). Since previous studies were unable to identify any ParaHox gene in different species of triclad flatworms (Saló et al., 2001), we predicted the putative ParaHox complement of triclads by in silico searches of the genome of S. mediterranea, the sister species of S. polychroa. We were able to identify only one out of the three ParaHox classes (gsx, xlox and caudal) in the genome. Based on this prediction we cloned the full-length sequence of this ParaHox gene in S. polychroa and assessed its orthology to the xlox subgroup through a phylogenetic analysis (Fig. 6B). We then characterize the expression domains of Spol-goosecoid and Spol-xlox during embryonic development. Spol-goosecoid is not detected at stage 5, when the definitive digestive system starts to be formed (Fig. 6C). No expression is detected in the developing definitive pharynx anlage. At stage 7, when the immature digestive system of the triclad is already defined, Spol-goosecoid is localized in

the brain, in a few isolated cells (Figs. 6D–E). Spol-xlox is first detected in stage 5 embryos, as a small population of cells in the germ band (Fig. 6G). However, as soon as the embryo elongates and flattens (Fig. 6H), Spol-xlox is expressed in two symmetrical rows of cells running anterior–posteriorly, in close association with the ventral nerve cords (Fig. 6I–II). Characterization of the complement of T-box genes in S. polychroa To further characterize the molecular patterning of the digestive system of triclad flatworms, we wanted to study the expression of the T-box-containing gene brachyury. By means of degenerate primers against the T-box domain we were able to clone five different T-boxcontaining genes in S. polychroa. This result is supported by the in silico prediction of the same set of genes in the genome of S. mediterranea. Phylogenetic analysis shows that the complement of T-box genes in S. polychroa comprises three different Tbx2/3 genes (Spol-tbx2/3a, Spol-tbx2/3b and Spol-tbx2/3c), one Tbx1/10 (Spol-tbx1/ 10) and one Tbx20 (Spol-tbx20) (Fig. 7A). Thus we have not identified any representative of the brachyury subfamily. Nevertheless, we decided to study the expression patterns of these five T-box genes and their possible relationship with the development and molecular patterning of the triclad digestive system. As observed in Figs. 7B–F, they exhibit a wide variety of expression domains. Spol-tbx2/3a shows a similar pattern to that of triclad gut genes (Spol-porcupine-1 and Spol-ßcatenin-2), being detected, additionally, in the most proximal region of the definitive pharynx lumen (Fig. 7B). We confirm its gut-


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Fig. 5. Expression of Spol-GATA456a, Spol-GATA456b and Spol-porcupine-1 during the maturation of the blind gut. (A–B, D–E, G–H) Whole-mount in situ hybridization on newborns and 10-day-old hatchlings. (C, F, I) Vibratome cross sections from the whole-mount in situ hybridization on 10-day-old hatchlings. (A–C) Spol-porcupine-1 is detected in the maturing blind gut of the hatchlings, along with Spol-GATA456a (D–F). Note that after 10 days, the diffuse signal from the gut in the newborns becomes defined into the three gut branches and lateral diverticula. (G–I) In contrast, Spol-GATA456b is detected in dorsal cells (black arrowheads) distributed in a gut-like fashion (HI) and in close contact with the gut branches (I). In A–B, D–E, G–H, the anterior is to the left. The asterisk marks the pharynx and the dashed black line in B, E, H the position of the sections shown. In C, F and I the dashed black lines circumscribe the gut branches. In all sections, dorsal is up and ventral is down. e, eye; gb, gut branches. Scale bars, A–B, D–E, G–H 1 mm, C, F, I 50 μm.

related expression in juveniles of S. polychroa (supplementary file 6, Fig. S5A). In contrast, Spol-tbx2/3b is detected in parenchymal cells all around the embryo (Fig. 7C) and Spol-tbx2/3c is localized in the brain and in the esophagus in juveniles (Fig. 7D and supplementary file 6, Fig. S5B). Spol-tbx1/10 is expressed in parenchymal cells on the dorsal side of the embryo (Fig. 7E), and finally, Spol-tbx20 is detected all around the lateral margin of the embryo and in two ventral rows of cells running from anterior to posterior (Fig. 7F).

Loss of ParaHox and brachyury genes in other platyhelminth lineages To determine whether the absence of the brachyury and ParaHox genes is specific to triclads or, on the contrary, is also evident in other groups of Platyhelminthes, we searched for these genes in the genome of the basal macrostomid flatworm M. lignano and the divergent digenean parasite Schistosoma mansoni. Although these genomes are not yet fully assembled, we were able to identify different short sequences with homology to either the T-box family or the ParaHox class (supplementary file 7, Table S2, and supplementary file 8, Fig. S6A). In the case of S. mansoni, we have identified only one ParaHox gene, belonging to the gsx subfamily (supplementary file 9, Fig. S7A). Therefore, digeneans share the absence of caudal with triclads, though the specific loss of gsx in triclads and xlox in digeneans seems to be specific to each group. In contrast, we were able to predict the full set of ParaHox genes in the basal flatworm M. lignano (supplementary files 7, 8 and 9, Table S2, Fig. S6A, and Fig. S7A). None of the predictions found in S. mansoni corresponds to a T-box gene of the brachyury subfamily (supplementary files 7, 8 and 9, Table S2, Fig. S6B, and Fig. S7B). Similarly, we did not find any gene with a similarity to brachyury in the genome of M. lignano (supplementary files 7, 8 and 9, Table S2, Fig. S6B, and Fig. S7B), suggesting that the loss of this key developmental gene was basal to Platyhelminthes or at least to Rhabditophora.

Discussion Schmidtea polychroa gut development Among triclads, the gut system has a uniform structure and is composed of a blind gastrodermis forked into three branches and a complex pharynx. Our study shows that the definitive adult digestive system is formed de novo from stage 5 onwards during embryogenesis. At this stage, which also corresponds to the point at which the definitive axial properties of the embryo are established (MartínDurán et al., 2010), the primordial organization of the future gut arises (Fig. 1J). In particular, the formation of the definitive pharynx shows great similarity with the regeneration of the pharynx in adult triclads (Bueno et al., 1997; Kobayashi et al., 1999). In both cases, the pharynx muscle and epithelial cells arise from the primordium itself, delimiting first the pharynx cavity and pharynx lumen and, later on, opening the cavity to the exterior through the mouth (Figs. 1A–B). The timing of development is similar to the timing of pharynx regeneration, and the maturation of the definitive pharynx also follows a proximo–distal direction (Figs. 1E–I). All these findings support the idea that the mechanisms acting during the development of the definitive triclad embryo are those that are later redeployed during adult regeneration and homeostasis (Martín-Durán et al., 2010). The specific development of the gut cells has been much less well studied. It has been proposed that the gastrodermis is formed by large, yolk-rich cells placed in the periphery of the ingested yolk mass (Cardona et al., 2005). Our histological observations (Figs. 1A–I) indicate that the gastrodermis is formed within the thin germ band, in small cells with no distinctive morphology that are placed in close contact with the yolk. Their definitive morphology as columnar apicobasally polarized cells is acquired after hatching when they absorb the yolk. This post-embryonic maturation of the digestive system offers a new perspective on the ecological significance of ectolecithic development in triclads. Most of the maternally-supplied nutrients

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Fig. 6. Complement of ParaHox genes and embryonic expression of Spol-goosecoid and Spol-xlox. Phylogenetic analysis of the orthology of Spol-goosecoid (A) and Spol-xlox (B), both highlighted in red. The support values of the branches are posterior probabilities of Bayesian likelihood (only those above 0.5 are shown and those ≥0.99 are indicated by an asterisk). In A the Otx family was used as the outgroup. In B the nodes marked with a α, ß and χ have posterior probabilities of 0.55, 0.97 and 0.94, respectively. (C–D, G–I) Wholemount in situ hybridization of Spol-goosecoid and Spol-xlox in embryos at stage 5 (S5) and stage 7 (S7). (E) In situ hybridization of Spol-goosecoid on paraffin sections of stage 7 embryos. (C) Spol-goosecoid is not detected at stage 5 and only at later stages (D-DI) is it expressed in the cephalic ganglia (white arrowheads), in few isolated cells (E, black arrowhead). (G) At stage 5, Spol-xlox is detected in a few cells in the germ band of the embryo (black arrowheads). (H) At stage 7, as the embryo elongates and flattens, expression is detected in isolated cells running from the anterior to the posterior end in two parallel rows (white arrowheads), overlapping with the ventral nerve cords (I–II). (F–J) Diagrams representing the changes in the expression of each gene during development. Drawings of different stages are not to scale. In D and H, the anterior is to the left. In I, the dashed rectangle indicates the amplified area in II. dp, definitive pharynx; e, eye; ep, embryonic pharynx; vnc, ventral nerve cords.

are not used during embryonic development, but are gathered inside the embryo and absorbed after hatching, during the first days of life of the juvenile (Fig. 1J). Thus, a primary advantage of ectolecithal development may be that it provides a dependable energy source to young hatchlings, promoting their survival. Molecular regionalization of the gut The subfamily of GATA456 factors is related to endoderm development in a wide variety of bilaterians, from nematodes and arthropods (Maduro and Rothman, 2002; Murakami et al., 2005) to deuterostomes (Stainier, 2002). In spiralians, the expression of GATA factors has been reported in the polychaete annelids Capitella teleta, Chaetopterus sp. and P. dumerilii, and in the sipunculan T. lageniformis (Boyle and Seaver, 2008; Boyle and Seaver, 2010; Gillis et al., 2008). In most of these

organisms GATA456 factors are expressed in the endoderm, particularly in the midgut, and in a mesodermal sublineage. Our work shows that the GATA complement of S. polychroa consists of four genes, two GATA123 and two GATA456 (Fig. 3A). Spol-GATA456a is clearly associated with the blind gut, but not with the definitive pharynx (Figs. 3C–F, Figs. 4A–D, Figs. 5A–F). Accordingly, Spol-GATA456a and Spol-foxA exhibit complementary expression in the digestive system of triclads, since the first is restricted to the blind gut and the latter is present in the pharynx (Martín-Durán et al., 2010). This mechanism of gut regionalization seems to be conserved in spiralians, suggesting the presence of a common gut-specific gene regulatory network in these organisms (Boyle and Seaver, 2010). The interpretation of the Spol-GATA456b expression pattern in triclad embryos is less clear. It is mostly detected in dorsal cells that are tightly associated with the gut but with no co-expression of endodermal genes


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Fig. 7. Complement of T-box genes and embryonic expression of Spol-tbx2/3a, Spol-tbx2/3b, Spol-tbx2/3c, Spol-tbx1/10 and Spol-tbx20. (A) Phylogenetic analysis of the orthology of T-box factors. The support values of the branches are posterior probabilities of Bayesian likelihood (only those above 0.5 are shown and those ≥ 0.99 are indicated by an asterisk). S. polychroa Tbx sequences are highlighted in red and each Tbx subfamily is framed in a different color. (B–F) Whole-mount in situ hybridization of the different Tbx genes in embryos at stage 7. (B) Spol-tbx2/3a is expressed in a gut-like fashion, in those cells surrounding the inner yolk, the forming gut diverticula (white arrowheads) and the inner lumen of the definitive pharynx. (C) Spol-tbx2/3b is expressed in parenchymatic cells all along the triclad body, both dorsally and ventrally. (D) In contrast, Spol-tbx2/3c is specifically detected in the brain of the embryo (white arrowheads). (E) Spol-tbx1/10 is localized in discrete dorsal cells. (F) Finally, Spol-tbx20 is detected in the body margin and in two parallel rows of cells running from anterior to posterior, probably the ventral nerve cords (dashed lines). (BI–FI) Diagrams representing the expression of each gene in stage 7 embryos. Drawings are not to scale. In B–F the anterior is to the left. dp, definitive pharynx; e, eye.

(Figs. 3G–K, Figs. 4E–L, Figs. 5G–I). A similar expression pattern was reported for the conserved foregut marker NK2.1 in the triclad Girardia tigrina (Garcia-Fernàndez et al., 1993). These cells could either be gutassociated mesoderm (parenchymal cells), or a specific type of gut cells. Two cell types have been described in the digestive system of triclads: the most abundant phagocytic cells, with a large clavate shape, and the smaller granular cells, usually thought to be of a glandular nature (Hyman, 1951). With the current data, neither of these two possibilities can be completely dismissed. Our study demonstrates that several developmental genes associated with different regions of the through-gut in other bilaterians do not exhibit such a gut-associated expression in the triclad S. polychroa (Figs. 6 and 7). In spiralians, goosecoid has been studied in the

polychaete annelid P. dumerilii and in the mollusk P. vulgata (Arendt et al., 2001; Lartillot et al., 2002). In both organisms it is expressed early in the larval foregut, in the ectoderm around the mouth, where it contributes to the development of the stomodeal nervous system. Additionally, in P. vulgata goosecoid is expressed in the mantel edge (Lartillot et al., 2002). In S. polychroa, Spol-goosecoid is expressed in the central nervous system (Figs. 6D–E). The late detection of goosecoid in the brain in S. polychroa is consistent with a conserved neural expression of this gene. However, we have not observed an early expression in the definitive pharynx, suggesting that in triclads goosecoid is not associated with the early specification of the foregut region. In spiralians, the ParaHox genes have been studied in the mollusks P. vulgata and G. varia (Samadi and Steiner, 2010; Le Gouar et al.,

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2003), the polychaete annelids P. dumerilii, Capitella teleta and Nereis virens (de Rosa et al., 2005; Fröbius and Seaver, 2006; Kulakova et al., 2008; Hui et al., 2009), the oligochaete Tubifex tubifex (Matsuo et al., 2005) and the leeches Helobdella triserialis and Hirudo medicinalis (Wysocka-Diller et al., 1995; Wedeen and Shankland, 1997). In all species in which Xlox has been studied, it is detected in the midgut region of the digestive system. Additionally, in the polychaetes P. dumerilii and N. virens and in the mollusk G. varia, Xlox is expressed in ventral nervous structures. Neural expression is also found in chordates (Brooke et al., 1998), and probably represents an ancestral expression of this gene (Hui et al., 2009). We show that the ParaHox complement in the triclad species S. polychroa consists of a single gene, Xlox (Fig. 6B). While the basal flatworm M. lignano exhibits the whole set of ParaHox genes, the absence of particular subfamilies is also present in another Neoophora Platyhelminth (Fig. 8B, supplementary file 9, Fig. S7A). Surprisingly, Spol-xlox is not expressed in the digestive system during gut morphogenesis, but instead is detected in association with the ventral nerve cords (Figs. 6H–I). In Platyhelminthes, Xlox was also identified in one species of polyclad flatworm, D. tigrina (Saló et al., 2001), but no expression has been reported. It will be interesting to study its localization during embryonic development in order to address whether or not the expression of Xlox in the gut was already lost in more basal Platyhelminthes. Finally, most studies in spiralians have focused on the role of brachyury during embryonic development and little attention has been paid to the other Tbx genes. The only data available are the expression of the gene T-brain and one Tbx2/3 in the polychaete annelid H. elegans (Arenas-Mena, 2008; Arenas-Mena, 2010). In this organism, the transcription factor Tbx2/3 is expressed in the dorsal side of the embryo. We show that the T-box complement in the triclad species S. polychroa consists of three Tbx2/3 genes, one Tbx1/10 and one Tbx20 (Fig. 7A). This implies a dramatic reduction in the number of T-box subfamilies within this lineage (Brachyury, T-brain, Tbx4/5, Tbx15/18/22 and Tbx6/16), a characteristic that is also present in other groups of flatworms (Fig. 8B, supplementary file 9, Fig. S7B). In S. polychroa, T-box genes are expressed in various organs during embryonic development (Figs. 7B–F). Only Spol-tbx2/3a shows clear gut-associated expression, in the pharynx and in parenchymal cells close to the gut branches (Fig. 7B and supplementary file, 6, Fig. S5). In C. elegans, a role for tbx2 (the only clear member of the Tbx2/3/4/5

subfamily) during the development of the anterior pharyngeal muscle has been reported (Smith and Mango, 2007). This may suggest a conserved expression of tbx2 genes during the development of gutrelated mesoderm in protostomes. Further investigation of Tbx genes in other taxa will help to decipher the evolution and function of these genes among bilaterians. Altogether, our data indicate that only a subset of the conserved bilaterian gut developmental mechanisms is present in the freshwater triclad S. polychroa. The expression of foxA suggests that the pharynx of triclads could be considered homologous to either the foregut or the anterior midgut of other bilaterians with a through-gut. However, important foregut markers like brachyury or gsx have been lost, and other foregut-related genes including NK2.1 and otx (GarciaFernàndez et al., 1993; Umesono et al., 1999) are not expressed in the pharynx (Fig. 8A). The determination of the endoderm through a GATA456-dependent mechanism seems to be conserved in triclads, but not the specification of a main midgut region, since Xlox is not expressed in the digestive system. Finally, other hindgut-associated genes do not show gut-associated expression either, whether due to the physical loss of the gene in the genome (the case of brachyury or caudal) or the absence of the expression domain in the developing embryo (loss of the hindgut domain of foxA or orthopedia) (Umesono et al., 1997). Accordingly, we propose that the antero-posterior gutregionalization present in those bilaterians with a through-gut is not present in triclads. In our model, the digestive system is only patterned in two main regions, namely, the pharynx and the blind gut. Evolution of the digestive system in Platyhelminthes Great morphological variability in the digestive system is observed between groups of Platyhelminthes and, in general, increasing complexity and diversity is observed (Fig. 8B). Basal groups like catenulids or macrostomids have a simple digestive system, with an anterior mouth and a simple non-protrusible pharynx that connects with a non-branched blind gut. However, in more derived groups, such as polyclads, triclads or parasitic flatworms, the mouth can open anywhere along the antero-posterior axis and the pharynx is a complex organ of plicate or bulbous type. Additionally, the blind gut branches to increase the digestive surface. In these taxa, a few species have developed an anus, which usually appears as multiple lateral

Fig. 8. Evolution of the molecular patterning of the digestive system in Platyhelminthes. (A) Comparative expression of genes involved in gut regionalization between a prototypic bilaterian with a through-gut and a triclad. (B) Evolutionary changes in the morphology of the digestive system within the Platyhelminthes and evolution of the ParaHox and T-box complements. Only the basal taxon Catenulida and those groups for which molecular data are available are presented. Red crosses indicate gene absence and question marks uncertainty in the molecular data. References in A: a Martín-Durán et al. (2010), b this study, c,d Umesono et al. (1997, 1999), e Garcia-Fernàndez et al. (1993).


connections of the gut diverticula with the epidermis (as in the polyclad genus Cycloporus, Thysanozoon or Yungia, or the triclad species Incapora weyrachi) or as a single posterior pore (dorsal, as in the polyclad species Leptoteredra maculata, or ventral, as in the Haplopharyngida) (Hyman, 1951). The presence of an anal pore, which soon closes, during the embryonic development of parasitic monogenean Polystoma integerrimum is even more puzzling (Halkin, 1901). Nevertheless, the general common feature to the different digestive systems of Platyhelminthes is the lack of an anus. The absence of a second gut opening and the lack of the evolutionarily conserved antero-posterior gut patterning mechanisms in triclads can be explained in two different ways. Flatworms could have retained a hypothetical ancestral spiralian state in which only the GATA456/FoxA patterning mechanism was present, but a throughgut had not yet evolved. The anus in other spiralians would therefore be the result of different gains, a scenario similar to that proposed by Hejnol and Martindale (2008). The most parsimonious explanation, however, is to assume that the ancestral spiralian had a through-gut and it was lost in the lineage leading to Platyhelminthes. It has been proposed that the copulatory bursa and female gonoduct arose from the hindgut in the ancestor of flatworms, with concomitant loss of the anus (Remane, 1951; reviewed by Reisinger, 1961), adding an alternative view to theories on the evolution of the through-gut. Recent phylogenies support a close relationship between Platyhelminthes and rotifers and gastrotrichs, both of which possess a through-gut (Hejnol, 2010). Embryological data also support the hypothesis of the loss of an anus. Catenulids and polyclads are the only groups of flatworms that have retained the ancestral spiral cleavage. In most spiralians, the descendants of the blastomeres 3A–C (or in particular the macromeres 4A–4C) and the blastomere 4D give rise to the cellular components of the gut (Nielsen, 2004, 2005). However, during the development of the polyclad Hoploplana inquilina, these cells degenerate without giving rise to any larval structure (Surface, 1907). We propose that the lack of an anus in Platyhelminthes is a secondary loss that was accompanied by changes in spiralian development and the loss of conserved antero-posterior gut patterning mechanisms. However, further investigation in other groups of Platyhelminthes, particularly catenulids and polyclads, is required to better understand the developmental events that led to the formation of a blind gut within this lineage. Conclusions This study investigates the morphological and molecular development of the digestive system in the triclad flatworm S. polychroa. The expression of GATA456 genes during its embryonic development suggests that these genes have retained their evolutionarily conserved role in endoderm specification and development in this lineage. Moreover, the complementary expression of Spol-GATA456a and Spol-foxA in the blind gut and the pharynx, respectively, supports the hypothesis that both transcription factors are a key part of an ancient gut-specific gene regulatory network conserved in spiralians. Our data indicate, however, that the digestive system of triclads has lost the gut-associated expression of most of the foregut, midgut and hindgut markers, suggesting that the triclad gut system is not axially patterned as a through-gut, but instead only a pharyngeal region and a blind gut are molecularly distinguishable. In addition, important developmental genes associated with specific gut regions, such as caudal or brachyury, have been lost in triclads and even in more basal groups of flatworms. These data together with the phylogenetic position of flatworms within spiralians, and the deviations observed in basal groups in the specification of the gut, suggest that the lack of an anus and the formation of a blind gut are secondary innovations of Platyhelminthes. Supplementary materials related to this article can be found online at doi:10.1016/j.ydbio.2011.01.032.

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Acknowledgments We thank T. López-Hernández, M. Vila-Farré and F. Monjo for support and comments on this manuscript. Special thanks to A. Hejnol, B. Egger, I. Somorjai and the two anonymous reviewers for comments that helped to improve the manuscript. The monoclonal antibody AA4.3 developed by Charles Walsh was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. JMM-D is an FPU fellow from the MICINN (Ministerio de Ciencia e Innovación), Spain. This work was supported by MEC BFU-2007-63209, Spain, to R.R. References Abascal, F., Zardoya, R., Posada, D., 2005. ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21, 2104–2105. Aguinaldo, A.M., Turbeville, J.M., Linford, L.S., Rivera, M.C., Garey, J.R., Raff, R.A., Lake, J.A., 1997. Evidence for a clade of nematodes, arthropods and other moulting animals. Nature 387, 489–493. 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