patterning - Core

Developmental Biology 354 (2011) 173–190

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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

Ventralization of an indirect developing hemichordate by NiCl2 suggests a conserved mechanism of dorso-ventral (D/V) patterning in Ambulacraria (hemichordates and echinoderms) E. Röttinger, M.Q. Martindale ⁎ Kewalo Marine Laboratory, PBRC, University of Hawaii, Honolulu, HI, USA

a r t i c l e

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Article history: Received for publication 28 September 2010 Revised 7 March 2011 Accepted 28 March 2011 Available online 3 April 2011 Keywords: Hemichordate Enteropneust Nickel chloride (NiCl2) Dorso-ventral patterning Indirect development

a b s t r a c t One of the earliest steps in embryonic development is the establishment of the future body axes. Morphological and molecular data place the Ambulacraria (echinoderms and hemichordates) within the Deuterostomia and as the sister taxon to chordates. Extensive work over the last decades in echinoid (sea urchins) echinoderms has led to the characterization of gene regulatory networks underlying germ layer specification and axis formation during embryogenesis. However, with the exception of recent studies from a direct developing hemichordate (Saccoglossus kowalevskii), very little is known about the molecular mechanism underlying early hemichordate development. Unlike echinoids, indirect developing hemichordates retain the larval body axes and major larval tissues after metamorphosis into the adult worm. In order to gain insight into dorso-ventral (D/V) patterning, we used nickel chloride (NiCl2), a potent ventralizing agent on echinoderm embryos, on the indirect developing enteropneust hemichordate, Ptychodera flava. Our present study shows that NiCl2 disrupts the D/V axis and induces formation of a circumferential mouth when treated before the onset of gastrulation. Molecular analysis, using newly isolated tissue-specific markers, shows that the ventral ectoderm is expanded at expense of dorsal ectoderm in treated embryos, but has little effect on germ layer or anterior–posterior markers. The resulting ventralized phenotype, the effective dose, and the NiCl2 sensitive response period of Ptychodera flava, is very similar to the effects of nickel on embryonic development described in larval echinoderms. These strong similarities allow one to speculate that a NiCl2 sensitive pathway involved in dorso-ventral patterning may be shared between echinoderms, hemichordates and a putative ambulacrarian ancestor. Furthermore, nickel treatments ventralize the direct developing hemichordate, S. kowalevskii indicating that a common pathway patterns both larval and adult body plans of the ambulacrarian ancestor and provides insight in to the origin of the chordate body plan. © 2011 Elsevier Inc. All rights reserved.

Introduction One of the earliest steps in the development of an animal's body plan is the specification of the future body axes. Although the mechanisms and timing of axis specification can vary at the species level (Prodon et al., 2004), in most metazoans, the primary egg axis, the so-called animal–vegetal (A/V) axis is established during oogenesis. This axis plays an important role in the subsequent establishment of the embryonic anterior–posterior (A/P), dorso-ventral (D/V, also called the oral–aboral (O/A) axis in larval echinoderms) and the consequent left–right (L/R) axes. Axial reorganizations may have played crucial roles in major evolutionary events, like the transition from radial to bilaterally symmetric animals (Finnerty et al., 2004; Matus et al., 2006) or the proposal that a D/V inversion (shift of the invertebrate mouth to the

⁎ Corresponding author. Fax: +1 808 599 4817. E-mail address: [email protected] (M.Q. Martindale). 0012-1606/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2011.03.030

opposite side of the embryo) associated with the origin of chordates (Arendt and Nubler-Jung, 1994; Benito-Gutierrez and Arendt, 2009; De Robertis and Sasai, 1996; Gerhart, 2000; Lacalli, 1995). Understanding the mechanisms that drive axial patterning during both larval and adult life history stages and how they have been altered is therefore crucial for understanding possible evolutionary scenarios involved in body plan reorganization. In this study we describe the alteration of axial patterning in an indirect developing hemichordate, a representative of an early branching deuterostome with potential chordate-like features. Chordates, a group of animals that consists of vertebrates, urochordates (ascidians), and cephalochordates (Amphioxus) are the most intensely studied group of multicellular organisms. However, the evolutionary origin of this group of animals and their unique morphological traits are largely unresolved and remain one of the more controversial questions in metazoan evolutionary biology. The chordates belong to a larger group of metazoan animals called deuterostomes. The deuterostomes consist of two major clades, the chordates and their sister group called the Ambulacraria (Swalla and

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Smith, 2008), traditionally formed by two taxa, echinoderms (e.g. sea urchins, sea stars, sea cucumbers) and hemichordates (e.g. acorn worms). A potential but highly controversial third taxon, Xenoturbella, has also been proposed to belong to this group, however they currently do not bear a single morphological synapomorphy with any other deuterostome (Bourlat et al., 2003, 2006; Dunn et al., 2008; Hejnol et al., 2009; Perseke et al., 2007; Philippe et al., 2007) (Fig. 1A). Although the morphology and life history of echinoderms and hemichordates is well studied, the variation between these two groups does not provide a clear view of the origin of chordate features. For example, extant adult echinoderms provide little insight into the problem because they are highly derived pentaradial forms that are difficult to homologize morphologically with any of the defining characteristics of chordates. Although there is a large body of work regarding the development of echinoid larvae from decades of molecular work (Angerer and Angerer, 2000; Croce et al., 2006; Davidson et al., 2002a,b; Duboc et al., 2004; Ettensohn and Sweet, 2000; McClay, 2000; Röttinger et al., 2008; Smith and Davidson, 2008; Sodergren et al., 2006) including the sequencing of the purple sea urchin genome (Sodergren et al., 2006), the relationship between embryonic patterning and adult body plan formation in echinoderms relative to chordate origins remains problematic due to their radical metamorphosis of a bilaterally symmetrical larva to the pentaradial adult. Adult enteropneust hemichordates (acorn worms) are better candidates for understanding chordate origins in that, like chordates, both their larval and adult body plan are bilaterally symmetric and the adult shares some adult chordate specific traits, such as a filter feeding pharynx with gills slits and a transient post anal tail (only in harrimaniids). However adult hemichordates also lack other defining chordate features such as a clearly homologous dorsal hollow nerve cord

or notochord that runs the length of the body. Recently, the direct developing hemichordate Saccoglossus kowalevskii (Harrimana) has been developed as a key species for studying the origin of the deuterostome body plan (Cameron et al., 2000; Lowe et al., 2006, 2003; Rychel et al., 2006). Hemichordates contain about 100 living species, divided into three classes. Although the exact taxonomic relationship of the three hemichordate classes is currently under debate (Fig. 1A) it appears that the indirect developing ptychoderidae enteropneust worms (e.g. P. flava) are representatives of the basal branch of hemichordates and may retain ancestral deuterostome life history characteristics (Cameron et al., 2000; Cannon et al., 2009; Swalla and Smith, 2008). In order to identify features crucial for the understanding of ancestral characters present in basal deuterostomes, a detailed understanding of the fate maps and early development of echinoderms, hemichordates and chordates is required. Fate mapping studies on indirect developing echinoderms (e.g. (Cameron et al., 1987)), and direct and indirect developing hemichordates (Colwin, 1953; Henry et al., 2001) revealed strong similarities between hemichordates and indirect developing echinoids (Fig. 1B). In fact, hemichordates and indirect developing echinoderms both generate their mesoderm at the vegetal pole, which differs from that in chordates, where the vegetal most region gives rise to endoderm (Kumano and Smith, 2002). Comparisons of the adult bilateral hemichordate body plan to pentaradial echinoderms are difficult, but one feature that could unite both groups is the bilaterally symmetrical “dipleurula” pelagic larval stage. An important life history characteristic of the ptychoderid hemichordates is that they undergo indirect development with the formation of a larval form that metamorphoses into a benthic adult worm after an extended pelagic phase (Hadfield, 1975; Nielsen and Hay-Schmidt, 2007; Tagawa et al.,

Fig. 1. (A) Phylogenetic position of the Hemichordata. Echinodermata and Hemichordata comprise the Ambulacraria, a group of non-chordate deuterostomes. Among Hemichordata (green box), the ptychoderidae enteropneust worms (red box) are representatives of the basal branch of hemichordates (Cannon et al., 2009). The dashed line connecting Xenoturbella indicates that their position within deuterostomes remains controversial (Hejnol et al., 2009). (B) Diagram representing the similarities in fate maps and larval morphology between echinoderms and hemichordates. Saccoglossus kowalevskii is a direct developing hemichordate, Ptychodera flava, an indirect developing hemichordate and Paracentrotus lividus an indirect developing echinoderm. All represented stages are oriented animal up, vegetal down, ventral (oral) to the left and dorsal to the right. Red and yellow shading represents the presumptive mesoderm and endoderm, respectively. (Henry et al., 2001; Logan and McClay, 1997; Lowe et al., 2003). (C) Schematic representation of P. flava metamorphosis. The adult form retains the same symmetry properties and most of the same tissues as the larval form.


1998a). The phylogenetic position, together with the similarities of morphology and cell fate maps of echinoderm and ptychoderid larvae (Fig. 1B, (Henry et al., 2001; Nielsen and Hay-Schmidt, 2007; Swalla, 2006) suggest that a biphasic life history was an ancestral trait for the Ambulacraria. However, not enough molecular data are currently known to provide a compelling argument for the homology of any larval (or adult) structures or the mechanisms of cell fate specification between echinoderms and hemichordates. An interesting fact to bear in mind is that, unlike indirect developing sea urchins (e.g. Paracentrotus lividus, Strongylocentrotus purpuratus) that undergo a radical metamorphosis with the adult arising from a rudiment of “set aside” cells on the left side of the larvae, the “dipleurula-like” larva of indirect developing hemichordates like P. flava undergoes a less dramatic morphological change at metamorphosis (Fig. 1C). The adult acorn worm retains the same symmetry (bilateral) and axial properties (A/P and D/V axes) as its larva and virtually all tissues (mouth, anus, gut, coeloms, etc.) are preserved through metamorphosis, except for some fields of ciliation and components of the larval nervous system (Nielsen and HaySchmidt, 2007). Thus, the molecular basis for larval cell fate specification and axial patterning in indirect developing hemichordates has a direct impact on adult body plan formation. Although the morphology and life cycle of hemichordates was initially described in the late 1880s (Bateson, 1884, 1886a,b; Morgan, 1891, 1894), molecular studies, investigating patterning of the enteropneust body plans started only a decade ago focusing on the larvae of the indirect developing hemichordate species Ptychodera flava (Harada et al., 2000, 2002; Nomaksteinsky et al., 2009; Okai et al., 2000; Peterson et al., 1999a; Taguchi et al., 2000, 2002). More recently, compelling work in the direct developing species, S. kowalevskii, has provided new insight into patterning mechanisms of the A/P (Lowe et al., 2003) and D/V (Lowe et al., 2006) axes of juvenile/adult enteropneusts. For example, bmp2/4 is expressed in the dorsal midline throughout all stages of development, while its antagonist chordin, is expressed ventrally. While functional analysis of Bmp2/4 in S. kowalevskii suggests no role in adult neural patterning, this study showed that this pathway plays a central and critical role in D/V patterning (Lowe et al., 2006). In contrast, nothing is known about the mechanism of D/V axis patterning in Ptychodera flava. In this indirect developing species, Pf-bmp2/4 has been reported to be expressed exclusively in the hydropore well after the establishment of the D/V axis, suggesting that Pf-bmp2/4 in this species is not at all involved in dorsoventral patterning (Harada et al., 2002). Thus, little reliable information is known about the molecular mechanism of D/V axis formation in Ptychodera flava. One way to disrupt D/V patterning in echinoderms is to treat zygotes with nickel chloride (NiCl2), resulting in radialization of the larvae (Duboc et al., 2004; Hardin et al., 1992; Lallier, 1956; Minsuk and Raff, 2005; Rulon, 1953; Timourian and Watchmaker, 1972). In indirect developing echinoderms, NiCl2 causes the expansion of ventral (oral) ectoderm at the expense of dorsal (aboral) tissues (Hardin et al., 1992). In direct developing echinoids, it promotes the formation of the left side vestibular ectoderm at the expense of right side tissue, therefore sinistralizing the embryo (Minsuk and Raff, 2005). Those results suggest that although the molecular mechanism by which NiCl2 acts is not yet elucidated, a nickel-sensitive pathway required for D/V larval patterning appears to have been co-opted in direct developing echinoderms to regulate the adult L/R axis (Minsuk and Raff, 2005). Although NiCl2 has been extensively used for toxicology studies for decades in various phyla (Calabrese et al., 1977; Clary, 1975; Hunter and Vergnano, 1953; Lu et al., 1979; Storeng and Jonsen, 1980), such a profound effect of NiCl2 on body plan patterning has never been described outside the echinoids. In the present study, we addressed the question of how and when the dorso-ventral axis is specified in an indirect developing hemichordate, Ptychodera flava. We treated embryos after fertilization with NiCl2 and assayed for effects during axis specification and patterning at the

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morphological and molecular levels. Our results show that NiCl2 affects ectodermal, mesodermal, and endodermal patterning and induces the formation of radialized embryos, similar to what has been described in echinoderms (Lallier, 1956; Minsuk and Raff, 2005; Rulon, 1953; Timourian and Watchmaker, 1972). Furthermore, molecular analysis using newly isolated tissue-specific markers shows that formation of dorsal ectoderm is abolished, ventral ectoderm is expanded, while the A/P axis and the endo- and mesoderm (EM) remain largely unaffected. Materials and methods Animals, embryos and treatments Adult P. flava were collected from shallow sand flats located either at Paiko lagoon on the leeward shore, or in Kaneohe Bay on the windward coast of Oahu, Hawaii. These were cultured in coarse sand in bowls submerged in the lab sea tables supplied with fresh running seawater. Spawning was induced following the methods of Tagawa et al. (1998b), which is triggered by elevated water temperatures. The eggs were filtered several times in 0.22 μm-filtered seawater (FSW) in order to dilute the mucus and remove particles. Mature eggs were then fertilized by addition of motile sperm. Embryos were cultured at room temperature (RT, 22–25 °C) in glass bowls. Treatment with NiCl2 was carried out by exposing embryos to 50 μM of NiCl2 (Sigma, #N-5756) in FSW at various time intervals. Experiments have been repeated at least three times with a minimum of n = 45. If not indicated otherwise, representative phenotypes present in at least 80% of the treated embryos are shown. Cloning of P. flava hnf3, brachyury, foxQ2, pea, sfrp1-like, fgfr1-like, chordin, bmp2/4, dlx, smad6 and frizzled 5/8 The Pf-foxA/hnf3, Pf-brachyury, Pf-bmp2/4 and Pf-dlx sequences have been described previously (Harada et al., 2002; Tagawa et al., 1998a; Taguchi et al., 2000) and were used to sub-clone into pGemT® (Promega Corp.) from mixed stage cDNA. All other sequences used in this study were isolated in the course of a transcriptome analysis (454/GS FLX Sequencing performed by SeqWright, Inc.) on RNA extracted from a mix of nine developmental stages (4, 16, 20, 24, 36, 40, 44, 52 and 68 hpf). We obtained 535,353 reads, with an average length of 314 bp, which assembled to 297,351 unique reads. The initial analysis was carried out using Blast2Go (www.blast2go.org) and the results of the batch-BLAST manually searched for signaling molecules and transcriptions factors of interest. The assembled reads were also uploaded to a local server and used for individual gene specific BLAST searches. To obtain sequences containing the full-length open reading frames (ORF) of Pf-foxQ2, Pf-fz5/8 and Pf-sfrp1-like we performed 5′ and 3′ RACE PCR on mixed stage RACE cDNA using the SMART™ RACE cDNA Amplification Kit (Clontech Inc.). Sequences corresponding to the full length ORF (or the longest possible ORF for foxQ2) were used to design primers in order to sub-clone as described above. The accession numbers for the P. flava cDNA sequences described here and used for the phylogenetic analysis have been submitted to Genbank. Pf-pea: HQ291270, Pf-ets1/2: HQ291271, Pf-sfrp1-like: HQ291269, Pffrizzled1/2/7: HQ291267, Pf-frizzled5/8: HQ291268, Pf-foxQ2: HQ291266, Pf-fgfr1l: HQ904073, Pf-chordin: HQ904075, Pf-smad6: HQ904074. Phylogenetic analysis The P. flava gene sequences described in this paper were translated and protein domains identified using SMART (Letunic et al., 2006; Schultz et al., 1998) (http://smart.embl-heidelberg.de/). They were aligned with orthologous sequences (obtained by database searches using BlastP (http://www.ncbi.nlm.nih.gov/BLAST/) in MacVector using the ClustalW

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Fig. 2. (A–J) Embryonic development of Ptychodera flava. DIC images of characteristic developmental stages: (A) 2 cell (note polar body on animal pole of the cleaved cell), (B) blastula, (C) early gastrula, (D) late gastrula, (E) hydropore formation, late gastrula, (F) mouth formation, late gastrula (pre-hatching), (G) newly hatched tonaria larvae, (H) late Krohn stage larva, (I) Agassiz stage larva, and (J) Juvenile. The axial orientation is the same as in Fig. 1. (hpf) hours post fertilization, (mpf) month post fertilization, (hpm) hours post metamorphosis, (dpm) days post metamorphosis. (✫) Protocoel/Mesoderm, (■) Hydropore, (*) Mouth.

tool and corrected by hand. A Bayesian phylogenetic analysis was performed using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) using the “mixed” amino acid model with two runs of 2,000,000 generations, sampled every 100 generations with 40 chains. A summary consensus tree was produced in MrBayes from the last 10,000 trees of each run (20,000 total trees), representing 1,000,000 stationary generations. Posterior probabilities were calculated from this consensus. Abbreviations and accession number for the sequences used in the analysis are shown in Supplementary Table 1.

in situ Hybridization Whole-mount in situ hybridizations were performed following a protocol adapted from Pang and Martindale (2008). Inserts were PCRamplified using SP6 and T7 primers and probes where synthesized with the T7 (Pf-sfrp1-like, Pf-foxQ2) or SP6 (Pf-pea, Pf-frizzled5/8, Pf-brachyury and Pf-foxA/hnf3) RNA polymerase. A control Pf-pea sense probe was used to verify the specificity of the hybridization. cDNA sequences, ranging from 1000 to 2500 base pairs, were used to transcribe digoxigenin-labeled antisense RNA probes (Megascript Kit, Ambion, Inc.). Embryos were fixed for 1 h in 4% paraformaldehyde (in DEPC treated milliq filtered water) with 0.1 M MOPS and 0.5 M NaCl. They were rinsed five times in phosphate-buffered saline (PBS) and stored in methanol at −20 °C. Prior to hybridization, embryos were gradually rehydrated into PTw (PBS plus 0.1% Tween-20). Pre-hatching stages were then digested with 10 μg/ml Proteinase K (Invitrogen, #AM2546) for 10 min at room temperature, washed twice with 2 mg/ml glycine, washed twice with 1% (w/v) triethanolamine, and treated with 0.3% acetic anhydride. Following two washes in PTw, they were refixed in 4% paraformaldehyde for 1 h at room temperature. After undergoing five PTw washes, they were washed twice in hybridization buffer (50% formamide, 5× SSC pH 4.5, 50 μg/ml heparin, 0.1% Tween-20, 1% SDS, 100 μg/ml salmon sperm DNA). Following an overnight pre-hybridization, they were hybridized with digoxigenin-labeled RNA probes (1 ng/ml) at 62 °C for 24–48 h. They were gradually washed out of hybridization buffer through a series of washes as follows: 20 min each of 75%:25% (hybridization buffer:2× SSC pH 7.0), 50%:50%, 25%:75%, twice for 20 min in 100% 2× SSC pH 7.0, and three times for 10 min in 0.05× SSC pH 7.0. Hybridized embryos were then brought back into PTw. After a 1-h incubation in

blocking buffer (Roche, Inc.), they were incubated overnight at 4 °C with an alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche, Inc). Following ten 10-min washes with PTw and five 10-min washes with PBS, embryos were washed once with AP Buffer 1 (100 mM Tris pH 9.5, 100 mM NaCl, 0.5% Tween-20) and then twice with AP Buffer 2 (AP Buffer 1 plus 50 mM MgCl2). They were then stained with the substrates NBT and BCIP to achieve a purple precipitate. When staining reached optimal levels, they were washed five times with PTw and then transferred to 70% glycerol for mounting. All images were taken on a Zeiss Axioskop with an Axiocam camera triggered by Axiovision software. All expression patterns (wild-type and NiCl2 treated) described here have been submitted to Kahi Kai, the comparative marine invertebrate gene expression database hosted at http://www.kahikai.org/index.php? content=genes.

Results Ptychodera flava development Cleavage of P. flava is holoblastic and radial (Hadfield, 1975; Tagawa et al., 1998b) and a hollow blastula forms 12 h post fertilization (hpf) (Fig. 2B). The following stages represent characteristic features of embryonic development of P. flava, but slight variation can be observed in different embryonic batches and due to temperature of culturing. The thickening of the vegetal plate, the precursor of the endomesoderm (Henry et al., 2001), indicates the beginning of gastrulation when the archenteron of the embryo forms (Fig. 2C). Toward the end of gastrulation the mesoderm forms by enterocoely as the protocoel pinches off from the anterior end of the embryonic gut (Fig. 2D). The single mesodermal protocoel (anterior coelom) elongates asymmetrically until it reaches the dorsal ectoderm where it fuses to form the hydropore (Fig. 2E). Following hydropore formation, the endodermal archenteron bends toward the ventral ectoderm where it fuses with the presumptive stomodeum (Fig. 2F), before giving rise to the mouth and hatching (Fig. 2G). After hatching from the chorion, the tonaria larvae posses a tri-partite gut composed of a pharynx, stomach, and intestine (fore-, mid-, hindgut), the triangular protocoel with dorsal hydropore, and the anterior apical plate (Fig. 2G). The larvae feed in the water


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Fig. 3. Spatial expression of Pf-brachyury, Pf-foxA/hnf3, Pf-bmp2/4 and Pf-dlx in Ptychodera flava. Spatial distribution of Pf-bra (A, E, I, M, Q), Pf-foxA/hnf3 (B, F, J, N, R), bmp2/4 (C, G, K, O, S), and Pf-dlx (D, H, L, P, T) transcripts during normal development analyzed by in situ hybridization. (A–D) Egg, (E–H) Blastula, (I–L) Gastrula, (M–P) Pre-hatching, (Q–T) tonaria larva. Expression of Pf-foxA/hnf3 in the stomach/intestine boundary is indicated by a black arrow in (R) and expression of Pf-dlx in individual cells at the ventral-dorsal boundary is indicated with an arrow in T (for a surface view see Fig. 7Zf). (*) indicates stomodeal expression of Pf-foxA/hnf3 (N). All embryos are oriented animal to the top, vegetal to the bottom, ventral to the left and dorsal to the right. Additional images and views of those expression patterns can be viewed at: http://www.kahikai.org/index.php?content=genes.

column for several months (Fig. 2H) prior to metamorphosis (Fig. 2I, J) (Hadfield, 1975; Nielsen and Hay-Schmidt, 2007; Tagawa et al., 1998b). Identification of tissue and region specific markers in Ptychodera flava In order to identify tissue and region specific markers (ventral, dorsal, endodermal and mesodermal), we performed a transcriptome analysis of RNA extracted from a mix of nine developmental stages using the 454 GS FLX Titanium sequencing method, followed by an in situ hybridization screen. We identified sequences encoding predicted

hemichordate orthologs of Forkhead, Ets and Smad transcription factors, members of the FGF and Frizzled receptor families and the secreted glycoprotein Chordin. In order to obtain additional information required for full-length sequences of the Forkhead transcription factor and one of the Frizzled receptors, we performed 5′ RACE-PCR. Analysis of the protein structure confirmed their putative orthologies by the presence of a characteristic Forkhead domain in the Forkhead-like sequence (Supplementary Fig. 1A), one Pea and one Ets domain in the Ets-like prediction (Supplementary Fig. 2A) and a signal peptide, a cysteine rich domain (CRD) followed by seven transmembrane (TM) domains and

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the KTXXXW motif essential for the activation of the Wnt/β-catenin pathway (Huang and Klein, 2004; Umbhauer et al., 2000) in a first frizzled-like sequence (Supplementary Fig. 3A). A fourth sequence, another Frizzled-like prediction, contains a signal peptide and a CRD domain but lacks the TM domains and the KTXXXW motif (Supplementary Fig. 3A). To make sure that the absence of TM domains and the KTXXXW motif is not caused by a sequencing or assembly error of the 454 sequencing method, we performed a 3′ RACE-PCR and confirmed the predicted stop codon. The predicted protein structure for a FGF receptor prediction revealed the presence of a signal peptide, three immunoglobulin (IG) domains and one transmembrane domain but lacking a fibronectin domain and a intracellular catalytic domain (Supplemental Fig. 4A) essential for signal transduction of the FGF receptor family (Kostich et al., 2002; Manning et al., 2002). Nevertheless, this structure is characteristic of an untypical Fgf Receptor, called Fgf Receptor like 1 (or Nou-darake in the planarian) described as an antagonist of the Fgf pathway (Bertrand et al., 2009; Steinberg et al., 2009; Wiedemann and Trueb, 2000). The Chordin-like sequence contains four CR domains and three chordin domains (Supplemental Fig. 5A) while the last sequence, a Smad6 prediction, contains one MH1 as well as one MH2 domain (Supplemental Fig. 6A). Blast analysis indicates that the amino acid sequences of the Forkhead, Ets or Smad transcription factors, the two frizzled receptors, the secreted glycoprotein as well as the FGF receptor like gene are most closely related to FoxQ2, Pea (Etv4), Smad6, Frizzled1-like, Frizzled5/8, Chordin and Fgfr-like1 respectively. Phylogenetic analysis confirmed the protein orthologies for FoxQ2 (Supplementary Fig. 1B), Pea (Supplementary Fig. 2B), Frizzled 5/8 (Fz5/8) (Supplementary Fig. 3B), Fgfr1-like (Supplementary Fig. 4B), Chordin (Supplementary Fig. 5B) and Smad6 (Supplementary Fig. 6B), however, this analysis was un-conclusive for Frizzled1-like. It appears that this gene groups best with Secreted frizzled related proteins (Sfrp) (Supplementary Fig. 3B), a family of genes described as Wnt pathway regulators and characterized by their similarities to Frizzled receptors. As in Frizzleds, the Sfrps contain a signal peptide and a CRD but lack the TM domains and instead terminate with a netrin-like domain. However, the netrinlike domain appears to be absent in the P. flava sequence (Supplementary Fig. 3A), so we named this gene sfrp1-like with additional analysis required to better understand its exact orthology and role during hemichordate development. Re-examination of previously described molecular markers Identification of Ptychodera flava orthologs of Pf-brachyury (Pf-bra) and Pf-foxA/hnf3 have been reported previously (Tagawa et al., 1998a; Taguchi et al., 2000). We re-examined these expression patterns and confirmed expression of Pf-bra in the blastoporal region (Fig. 3I, M, Q), the posterior edge of the stomodeum (Fig. 3M) and in the oral-most portion of the pharynx (Fig. 3Q). Similarly, Pf-foxA/hnf3 expression is first detected in the vegetal plate (presumptive endomesoderm) of the blastula stage (Fig. 3F) and in all invaginating cells after the onset of gastrulation (Fig. 3J). After the formation of the protocoel, when the archenteron bends toward the ventral (oral) ectoderm, Pf-foxA/hnf3 transcripts are detected strongly in the anterior region of the endoderm, while the signal appears to diminish at the base of the archenteron (Fig. 3N). After hatching, the tonaria larva expresses Pf-foxA/hnf3 in two distinct regions, the entire pharynx and the boundary between the stomach and intestine (Fig. 3R). Double in situ hybridization using probes against both genes have led Taguchi and colleagues to the

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conclusion that Pf-foxA/hnf3 expression is restricted to the embryonic endoderm (Taguchi et al., 2000). However, we observed that at the end of gastrulation, Pf-foxA/hnf3 transcripts are also detected in the ventral ectodermal region that will form the mouth and in the pharynx after its fusion with the apical region of the archenteron (Fig. 3N). Thus, in addition to their endodermal and blastoporal expression, both Pfbrachyury and Pf-foxA/hnf3 are expressed in the ventral ectoderm before hatching, and in the mouth/pharynx after hatching. We also re-examined expression of two previously reported dorsal markers, Pf-bmp2/4 and Pf-dlx (Harada et al., 2001, 2002). We confirmed expression of Pf-dlx in a presumptive ectodermal territory at the blastula stage (Fig. 3H, Supplementary Fig. 8E, F), the dorsal endo- and ectoderm during gastrulation (Fig. 3L) and a progressive confinement of its expression into individual cells at the boundary between the ventral and dorsal ectoderm by the end of gastrulation through hatching (Fig. 3P, T and Fig. 7Zc). Satoh and colleagues failed to detect Pf-bmp2/4 expression during embryogenesis and found it localized in the hydroporal region of the tonaria larva (Fig. 3O, S), proposing that Pf-Bmp2/4 may not be involved in axis formation in P. flava (Harada et al., 2001). In contrast to these findings, we detect faint expression in the egg (Fig. 3C) and blastula stages (Fig. 3G) and observe a clear asymmetric endo- and ectodermal expression of Pf-bmp2/4 starting at the gastrula stage (Fig. 3K) that becomes restricted to the dorsal part of the protocoel and later the hydropore after hatching (Fig. 3O, S). These observations are important as they suggest a putative role of the Bmp pathway in patterning the D/V axis in P. flava. Novel molecular markers for dorsal and ventral ectoderm, apical plate, endoderm and mesoderm Pf-chordin and Pf-fgfr1-like are expressed in the ventral ectoderm The earliest stage in which Pf-chordin expression can be detected is at the early blastula stage (Fig. 4H). During gastrulation, the intensity of the asymmetric signal increases and remains restricted to the ventral ectoderm (the opposite side of Pf-bmp2/4 expression) until the end of the gastrula stage (Fig. 4O, V). Pf-chordin transcripts are no longer detected after hatching (Fig. 4Zc). Pf-fgfr1-like expression is observed after the onset of gastrulation in the stomodeum (ventral ectoderm) as well as the apical region of the endoderm that will fuse with the stomodeum to form the mouth and pharynx of the larva (Fig. 4P, W). The tonaria larva expresses Pf-fgfr1-like in the entire pharynx after hatching (Fig. 4Zd), reminiscent of Pf-foxA/hnf3 expression. Pf-smad6 and Pf-pea are expressed transiently in dorsal territories Pf-smad6 transcripts are first detected at the gastrula stage, localized asymmetrically within the dorsal endo- and ectoderm (Fig. 4Q). After the formation of the hydropore, Pf-smad6 expression is observed dorsally in all three germ layers; the dorsal ectoderm, the dorsal part of the protocoel (mesoderm) and the dorsal region of the endoderm (Fig. 4X). No expression is detected after hatching (Fig. 4Ze). Localized Pf-pea transcripts are not detected before the gastrula stage or after larval hatching, however Pf-pea expression appears transiently in four highly localized ectodermal regions toward the end of gastrulation (Fig. 4R). At the late gastrula stage Pf-pea transcripts are detected in the ventral ectoderm close to the blastopore, in the lateral ectoderm, in an animal region between the stomodeum and the apical plate and a broad domain in the dorsal ectoderm (Fig. 4R, http://www.kahikai.org/index. php?content=gene_view&geneid=219). Expression in all domains

Fig. 4. Expression of Pf-chordin, Pf-fgfr1-like, Pf-smad6, Pf-pea, Pf-foxQ2, Pf-sfrp-like and Pf-frizzled5/8 (Pf-fz5/8) in Ptychodera flava. Spatial distribution of Pf-chordin (A, H, O, V, Zc), Pf-fgfr1like (B, I, P, W, Zd), Pf-smad6 (C, J, Q, X, Ze), Pf-pea (D, K, R, Y, Zf), Pf-foxQ2 (E, L, S, Z, Zg), Pf-sfrp1-like (F, M, T, Za, Zh) and Pf-fz5/8 (G, N, U, Zb, Zi) transcripts during normal development analyzed by in situ hybridization. (A–G) Egg, (H–N) Blastula, (O–U) Gastrula, (V–Zb) Pre-hatching, (Zc–Zi) tonaria larva. Expression of three distinct ectodermal expression domains for pea are indicated by black arrowheads in (R) and the inter apical-stomodeum domain expression of pea by a single black arrowhead in (Y). Dotted ellipsoid circles in (T) indicate the regions where Pf-sfrp1-like transcripts are absent and the dotted outline in (X) indicate the dorsal expression of Pf-smad6 in the endo-, meso- and ectoderm. Ectodermal, mesodermal and endodermal expression domains of Pf-fz5/8 are indicated by white arrows in (Zb and Zi). (*). All embryos are oriented animal to the top, vegetal to the bottom, ventral to the left and dorsal to the right. Additional images and views of those expression patterns can be viewed at: http://www.kahikai.org/index.php?content=genes.

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except the inter apical-stomodeum ectoderm is strongly reduced at the pre-hatching stage (Fig. 4Y) and no expression is detected after hatching (Fig. 4Zf). Based on the ventral expression of Pf-chordin and Pf-fgfr1-like as well as the dorsal expression of Pf-pea, Pf-smad6, Pf-bmp2/4 and Pf-dlx detected by late gastrula stages (Figs. 3O, P and 4V, W, X, Y) we propose that earlier asymmetric expression of these genes are also localized to the same ventral or dorsal sides of the embryo respectively. Nonetheless, double in situ hybridization experiments using probes for both ventral and dorsal markers simultaneously are required to confirm these observations. Pf-foxQ2 expression in the apical plate The transcription factor Pf-foxQ2 is locally expressed in approximately half of the embryo at the early blastula stage (Fig. 4L). Double in situ hybridization with probes for Pf-foxQ2 and Pf-foxA/ hnf3, which is expressed in the vegetal plate at that stage (Fig. 3F), shows that the Pf-foxQ2 expression domain corresponds to the animal hemisphere (Supplementary Fig. 8A, C). After the onset of gastrulation, Pf-foxQ2 expression becomes progressively restricted to the apical region of the larvae (Fig. 4S) and remains in the apical plate after hatching (Fig. 4Z,Zg). Pf-sfrp1-like is expressed transiently in the endoderm Transient and localized expression of Pf-sfrp1-like is only detected at the end of gastrulation in a broad presumptive endodermal domain that excludes the apex and the base of the archenteron (Fig. 4T). After the separation of the mesoderm from the endoderm by the formation of the protocoel, Pf-sfrp1-like transcripts are restricted to the presumptive fore-, and mid-gut region and excluded from mesodermal derivatives (Fig. 4Za). Endodermal expression of Pf-sfrp1-like can be weakly observed in newly hatched larvae, but is undetectable in tonaria stage larvae (Fig. 4Zh). Pf-fz5/8 transcripts are detected in forming mesoderm Pf-fz5/8 transcripts are first detected maternally in the egg (Fig. 4G), however localized zygotic expression appears in a restricted region at the blastula stage (Fig. 4N). Double in situ hybridization with probes for Pf-fz5/8 and Pf-foxA/hnf3, which is expressed in the vegetal plate at that stage (Fig. 3F), shows that, similar to Pf-foxQ2, expression of Pf-fz5/8 is restricted to the animal hemisphere (Supplementary Fig. 8B, D). At the end of gastrulation, transcripts are localized to the apical region of the ectoderm as well as the forming archenteron, in a region that will give rise to the mesoderm (protocoel) (Fig. 4U). After the segregation of endoderm from mesoderm, Pf-fz5/8 transcripts are observed in both the mesodermal protocoel and adjacent apical endoderm (Fig. 4Zb). Expression in all three domains remains detectable in the tonaria larvae shortly after hatching (Fig. 4Zi) but disappears in the endo- and mesoderm at later stages (data not shown). Taken together, we have identified and re-analyzed molecular markers for the ventral ectoderm (Pf-chordin, Pf-fgfr1-like, Pf-hnf3/ foxA, Pf-bra), dorsal ectoderm (Pf-smad6, Pf-pea, Pf-bmp2/4, Pf-dlx), apical plate (Pf-foxQ2 and Pf-fz5/8), blastopore (Pf-bra and Pf-pea), endoderm (Pf-sfrp1-like and Pf-hnf3/foxA) and mesoderm (Pf-fz5/8). Detection of transcripts in the eggs by in situ hybridization suggests the presence of maternal expression of Pf-foxQ2, Pf-fz5/8 and possibly Pf-bmp2/4 and Pf-dlx, although more sensitive experiments (e.g. qPCR) are required to confirm these observations. NiCl2 radializes Ptychodera flava embryos In order to determine whether the D/V axis in indirect developing hemichordates is determined in the same way as it is in indirect developing echinoids, we treated P. flava embryos with NiCl2. In sea urchins, treatments with NiCl2 cause the disruption of the D/V axis

and a radialization of the larvae. P. flava embryos were treated continuously after fertilization with different concentrations of NiCl2 (10–300 μM) and examined morphologically through the tonaria stage. The large majority (N95%) of the treated animals at the lowest concentration resembled control larvae, while embryos treated with 60 μM or higher concentrations of NiCl2 resulted in high mortality. In contrast, treatments at 30 μM induced severe phenotypes in the majority or embryos (N70%) without any significant mortality. Therefore we re-analyzed the effects of NiCl2 in P. flava for morphological defects at different developmental stages using a slightly elevated concentration of 50 μM to improve uniformity of phenotype (Fig. 5G–O). No morphological effects of treatment are visible compared to control embryos before the segregation of mesoderm and endoderm (Fig. 5G, H). In normal embryos, the first D/V symmetry-breaking event is the formation of the hydropore on the dorsal side of the embryo. In treated embryos, the mesoderm forms by enterocoely at the expected developmental stage but fails to elongate and fuse with the dorsal ectoderm to form the hydropore (Fig. 5I). Instead, the endoderm merges with the ectoderm in an extended domain to form a circumferential mouth (Fig. 5K, L). Hatching occurs but is delayed approximately 24 h (±8 h) (Fig. 5L) and swimming movements appear reduced compared to control larvae (data not shown), suggesting NiCl2 treatment affects cilia formation or activity. After hatching, the larvae consist of two thick ectodermal halves, one animal (containing the mesodermal protocoel), and one vegetal (containing the presumptive archenteron), separated by a mouth (Fig. 5M–O). In approximately 20% of the treated larvae, the circumferential mouth leads to the separation of the animal and the vegetal domains (Fig. 5O). The absence of the hydropore and the formation of a radialized mouth suggest that NiCl2 in P. flava interferes with D/V patterning and ventralizes all of the ectoderm. NiCl2 sensitive period ranges from cleavage to late blastula In echinoderms, the NiCl2 sensitive period begins at the blastula stage and ends at the early/mid gastrula stage (summarized in Minsuk and Raff, 2005). In order to define the NiCl2 sensitive period in P. flava, we treated groups of embryos for various periods of time starting at different times after fertilization. Embryos were scored for the formation of the hydropore, radialization of the mouth and for time of hatching at 96 hpf (Fig. 6). As described for treatments starting at the 2-cell stage (Fig. 5G–O), NiCl2 added before 12 hpf blocked the formation of the hydropore, induced a circumferential mouth and delayed hatching in more than 90% of cases (Fig. 6C). Depending on the batch of embryos used, we observed slightly extended periods of sensitivity inducing phenotypes with a comparable penetrance, up to 15 hpf (light green box). However, when NiCl2 was added shortly after the onset of gastrulation (24 hpf), hatching was delayed in all, but formation of the hydropore was inhibited in only approximately 30% of the embryos (Fig. 6D), and an expanded mouth observed in only 15% of the treated embryos (data not shown, light grey box). Finally, treatments starting at the end of gastrulation (36 hpf) did not interfere with either hydropore or mouth formation and had only slight delays in hatching (Fig. 6E). These observations suggest that the prime period of sensitivity to NiCl2 in axial patterning ranges from cleavage to the blastula stage (green box in Fig. 6A) that tapers off by the beginning of gastrulation (grey box in Fig. 6A). NiCl2 treatment targets D/V patterning The main morphological defects caused by NiCl2 are in structures formed along the D/V axis. To determine if NiCl2 interferes with the A–P axis or with the specification of the endoderm or mesoderm, we compared the expression of gene markers (described in Figs. 3 and 4) in control and NiCl2 treated embryos before and after hatching (Fig. 7).

E. Röttinger, M.Q. Martindale / Developmental Biology 354 (2011) 173–190 Fig. 5. NiCl2 treatment radializes embryos and expands ventral (oral) ectoderm. (A–F) Control embryos. (G–O) Embryos treated with 50 μM NiCl2. (A, G) early blastula, (B, H) early gastrula, (C, D, I, K) late gastrula, (E, L) newly hatched tonaria, (F, M–O) tonaria. All images are lateral views, except L, M, and O, which are (vv) ventral views. (*) Mouth, (■) Hydropore.

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182 E. Röttinger, M.Q. Martindale / Developmental Biology 354 (2011) 173–190

Fig. 6. NiCl2 sensitivity period of P. flava. (A) Embryos were incubated at the indicated times with 50 μM of NiCl2 and scored for phenotypes at the tonaria stage (96 hpf). Horizontal blue or grey bars represent treatment period according to the developmental time scale at the top of the figure, and the resulting phenotype in each case is indicated by the color of the bars: blue, radialized phenotype; grey, no phenotype. The letters next to the end of the bars indicate the image and phenotype the various treatments periods are corresponding to. The NiCl2 sensitive period for P. flava (6–12 hpf), is indicated by the green box. The dashed line and the light green box indicate the variability observed in different batches of embryos, while the light grey box indicates the sensitivity period with a reduced penetrance (approx. 30%). (2c) 2 cell stage, (eB) early blastula, (eG) early gastrula, (lG) late gastrula, (vlG) very late gastrula. (B) Control embryos. Representative phenotypes observed after NiCl2 treatments starting (C) before 14 hpf, (D) at 24 hpf and (E) at 36 hpf. (■) Hydropore, (*) Mouth.


Analysis of Pf-fz5/8 (Fig. 7G, H), Pf-sfrp1-like (Fig. 7I, J) and Pf-foxQ2 (Fig. 7K, L) expression in NiCl2 treated embryos revealed that their endodermal, mesodermal or apical plate expression is not affected. In untreated embryos, Pf-bra and Pf-foxA/hnf3 (in addition to their endodermal blastoporal expression) are co-expressed together with Pf-fgfr1-like in the stomodeum (Fig. 7M–R). Pf-chordin expression is restricted to a wider territory than just the stomodeum but is still confined to the ventral surface (Fig. 7S). As expected ventral expression of those genes is radialized in treated embryos and define a clear circumferential boundary between the animal and vegetal parts of the embryo/larvae (Fig. 7U–Za), with Pf-chordin maintaining its broader expression domain (Fig. 7Za). In contrast, dorsal expression of Pf-bmp2/4, Pf-dlx, Pf-pea and Pf-smad6 (Fig. 7Zc–Zi) is completely abolished while the inter-apical-stomodeum expression and the ventral blastoporal expression of Pf-pea is extended (Fig. 7Zo). Taken together, these observations demonstrate that NiCl2 treatments appear to only affect gene expression along the D/V axis, while the A/P axis and formation of the endo- and mesodermal germ layers remain largely unaffected. Interestingly, dorsal mesodermal as well as dorsal endodermal expression of Pf-smad6 is also abolished, suggesting that the NiCl2 affects D/V patterning in all three germ layers. Discussion NiCl2 ventralizes P. flava We have shown that in this indirect developing hemichordate NiCl2 treatments induce the formation of radialized embryos similar to observations made in echinoderms (Hardin et al., 1992; Minsuk and Raff, 2005). We also report an additional ventral ectodermal expression of Pf-foxA/hnf3 as well as asymmetric embryonic expression of Pf-bmp2/4 (Fig. 3) that were not reported in previous studies (Taguchi et al., 2000), and present new molecular markers, Pf-chordin, Pf-fgfr1-like, Pf-smad6, Pf-pea, Pf-foxQ2, Pf-sfrp-like and Pf-fz5/8 that are primarily expressed in the ventral ectoderm, dorsal ectoderm, apical plate, endoderm and mesoderm respectively (Fig. 4). Furthermore, we show that NiCl2 treatment before the onset of gastrulation inhibits the formation of the dorsal hydropore and induces the formation of a circumferential mouth, indicating that NiCl2 causes a ventralization of the larval and possibly adult body plan (Figs. 5, 6). Finally, using molecular markers, we show that NiCl2 transforms lateral and dorsal ectoderm cells into ventral fate (Fig. 7), and that appears to affect all three germ layers. This study reveals precise temporal and spatial activation of gene expression during embryogenesis. Orthologs of Hnf3 (FoxA), Brachyury, Fz5/8, FoxQ2, Sfrp, Smad6 and Ets related factors have been identified in the hemichordate S. kowalevskii in the course of an EST analysis (Freeman et al., 2008) but no spatial expression analysis for these genes has been reported. In contrast, bmp2/4, chordin and dlx expression have been recently published for S. kowalevskii (Lowe et al., 2006). Echinoderm orthologs for all genes mentioned above, except sfrp1-like, have also been identified and expression patterns reported (Angerer et al., 2000; Bertrand et al., 2009; Bradham et al., 2009; Croce et al., 2001, 2006; Duboc et al., 2004; Gross and McClay, 2001; Harada et al., 1995, 1996; Howard-Ashby et al., 2006; Lapraz et al., 2006, 2009; Oliveri et al., 2006; Peterson et al., 1999b; Rast et al., 2002; Rizzo et al., 2006; Saudemont et al., 2010; Shoguchi et al., 1999; Tu et al., 2006). In the following section we will review and compare the reported expression patterns in other ambulacrarians to our findings. Strong similarities of expression of bra, foxA/hnf3, foxQ2, chordin, dlx, smad6, and fz5/8 exist between larval hemichordates and echinoderms Initial analyses of bra expression in indirect developing Ambulacraria suggested that stomodeal expression of that gene may be

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shared between hemichordates (P. flava) and starfish (Shoguchi et al., 1999; Tagawa et al., 1998a), but not with sea urchin embryos (Harada et al., 1995; Peterson et al., 1999b). Earlier reports of hnf3/foxA expression in a sea urchin (Harada et al., 1996) and in P. flava (Taguchi et al., 2000) did not describe any stomodeal expression in either species, however, more recent re-examinations of bra and hnf3/foxA expression in various echinoderms (Croce et al., 2001; Gross and McClay, 2001; Oliveri et al., 2006; Rast et al., 2002) and in P. flava (this study, Fig. 3) show that in the vast majority of analyzed Ambulacraria, the two genes are indeed expressed in a stomodeal domain prior to the formation of the mouth. The only remaining exception is the lack of stomodeal bra (HpTa) expression in the echinoderm Hemicentrotus pulcherrimus (Harada et al., 1996). A detailed re-examination of this expression pattern in this species might reveal if the stomodeal expression has been previously overseen or been lost in this species, and support the hypothesis that bra and hnf3/foxA are expressed in the ventral ectoderm of all ambulacrarians. The strong similarity of gene expression across Ambulacraria is also observed for five other genes. Sea urchin foxQ2 expression is nearly identical to the P. flava expression described here. After an initial phase of foxQ2 expression in the animal hemisphere during cleavage/early blastula stages, transcripts are restricted to the animal pole domain where they remain detectable during late larval stages in both taxa (Yaguchi et al., 2008, this study Fig. 4). Interestingly, echinoderm FoxQ2 function seems to be required for spatial coordination of the A/V and D/V axes in echinoderms, although foxQ2 expression is not altered by NiCl2 treatments in P. flava (Fig. 7K, L). Functional experiments are required to determine if there is a conserved role in axis formation in hemichordates for this gene. Pf-chordin encodes a putative Bmp antagonist from P. flava and can be detected asymmetrically as early as the blastula stage. During gastrulation, chordin is expressed in a broad domain within the ventral ectoderm (Fig. 4). Orthologs in S. kowalevskii (Lowe et al., 2006) and echinoderms (Bradham et al., 2009; Lapraz et al., 2009) are both also expressed exclusively in the ventral ectoderm starting at the blastula/early gastrula stages. The significance of chordin expression with respect to bmp2/4 expression within ambulacrarians will be discussed below. Another, ventrally expressed gene identified in this study is Pffgfr1-like. Its localized expression within the stomodeum and the apical endoderm starts during gastrulation and persists through hatching (Fig. 4). FgfrR1-like is a protein closely related to fibroblast growth factor receptors (FgfrR) that has been not only been identified in a wide variety of metazoans (Bertrand et al., 2009; Wiedemann and Trueb, 2000) but shown to block FGF signaling in both planarian (Cebria et al., 2002) and vertebrates (Steinberg et al., 2009). Curiously, expression patterns for this gene have been reported from only a limited number of taxa. For echinoderms, no whole mount expression pattern is available, but RT-PCR on tissue extracted from adult sea urchins suggests that fgfr1-like is expressed at high levels in the mouth, esophagus and intestine (Zhuang et al., 2009). One cannot make assumptions about embryonic expression based on adult echinoderm expression data, therefore, a thorough embryonic expression analysis is required in echinoderms to verify if the ventral (oral) expression of fgfr1-like is shared in both taxa. In the direct developing hemichordate S. kowalevskii, dlx (distal-less, a homeobox transcription factor) is expressed in the dorsal ectoderm and in scattered cells in the anterior ectoderm of the prosome (Lowe et al., 2003), a structure absent in P. flava embryos. In P. flava, Pf-dlx is locally expressed in the presumptive dorsal ectoderm starting at the blastula stage (Fig. 3H) and in individual cells of the dorso-ventral boundary of the tonaria larvae (Harada et al., 2001, Fig. 3). In the sea urchin, dlx is expressed in the presumptive dorsal ectoderm starting at the mesenchyme blastula stage (Howard-Ashby et al., 2006). Interestingly, dlx is a downstream target of Bmp2/4 in the dorsal midline of S. kowalevskii (Lowe et al., 2006) and the dorsal ectoderm of the sea urchin

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P. lividus (Saudemont et al., 2010) suggesting that the function of this transcriptional factor may be conserved as well. We have shown that Pf-smad6 is transiently expressed in the dorsal ectoderm at the beginning of gastrulation as well as in the dorsal, endo-, meso- and ectoderm prior to hatching (Fig. 4). Expression of this gene has recently been reported in the context of a detailed gene regulatory network (GRN) analysis of ectoderm specification in a sea urchin. The authors have shown that, similar to its hemichordate ortholog, smad6 is expressed in the presumptive dorsal ectoderm starting at the mesenchyme blastula stage. So far no dorsal endo- or mesodermal expression has been observed. Interestingly, this study shows that similar to vertebrates, smad6 is a direct downstream target of Bmp2/4 and knock-down and overexpression experiments show that Smad6 acts in a negative feedback loop to control Bmp activity (Saudemont et al., 2010). During echinoderm (P. lividus) embryogenesis, fz5/8 is expressed in the animal hemisphere and the presumptive secondary mesenchyme prior to gastrulation and in the animal pole domain and the definitive secondary mesenchyme that forms the tip of the archenteron at the end of gastrulation (Croce et al., 2006). In P. flava, fz5/ 8 expression is also initiated in the animal hemisphere during cleavage stages and is restricted to the animal pole in larval stages (Fig. 4). However, mesodermal expression at the tip of the forming archenteron is initiated only at the end of gastrulation suggesting a different temporal control in fz5/8 expression and perhaps mesodermal specification between these two taxa.

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ventral or the dorsal ectoderm. Interestingly, functional studies carried out in both S. kowalevskii and sea urchins have shown that regardless of the position of bmp2/4 expression, the target of Bmp2/4 signaling in both systems is dorsal ectoderm (Angerer et al., 2000; Duboc et al., 2004; Lowe et al., 2006). dlx is an indirect downstream target of Bmp2/4 in S. kowalevskii and P. lividus and expressed in dorsal ectoderm. In P. flava, expression of Pf-dlx is also confined to the dorsal ectoderm, suggesting that the target of Bmp signaling will be on the dorsal side of the embryo in this indirect developing hemichordate. Supporting this hypothesis is that chordin, an antagonist of Bmp signaling, is expressed in the ventral ectoderm in all ambulacrarians studied to date (Bradham et al., 2009; Duboc et al., 2004; Lowe et al., 2006; this study). The strong similarities of the majority of these expression patterns suggest a shared character of echinoderms and hemichordates in specifying and patterning the body plan. However, the heterochrony observed for Pf-fz5/8 expression and the fact that Pf-pea, as well as Pfbmp2/4, are expressed in completely opposite ectodermal territories compared to their sea urchin orthologs also suggests important changes in the transcriptional regulation of these genes in the two ambulacrarian taxa. Comparative studies of other indirect developing hemichordates or echinoderms (asteroids, holothurids, crinoids, etc.) and analysis of the underlying GRN are required to obtain a better picture about conservation of transcriptional regulation in Ambulacraria.

NiCl2 radializes Ambulacraria Differences in expression of pea and bmp2/4 between hemichordates and echinoderms Even more drastic changes in gene expression is observed for pea. The echinoderm pea gene is expressed at the tip of the invaginating archenteron (presumptive secondary mesenchyme), around the apical pole ectodermal domain and in the ventral/lateral ectoderm (Rizzo et al., 2006; Rottinger et al., 2008). Interestingly, Pf-pea is transiently expressed only in the ectoderm. Pf-pea transcripts are excluded from the apical plate and are detected predominantly in the dorsal ectoderm (Fig. 4R, Y) exactly the opposite of its echinoderm counterpart. This observation suggests a divergent temporal and spatial transcriptional control of the echinoderm and hemichordate pea genes. However, we cannot be certain that there are no additional pea orthologs until genome sequencing is complete. A previous study reported that Pf-bmp2/4 is first expressed late in dorsal larval structures of P. flava, suggesting that Bmp2/4 signaling may not play a role in embryonic axial patterning (Harada et al., 2002). We show that Pf-bmp2/4 is clearly asymmetrically expressed during gastrulation, well before any morphological manifestations of the D/V axis are visible (Fig. 3), suggesting that Bmp2/4 could indeed play a causal role in axial patterning in P. flava. The site of bmp2/4 expression relative to its role in D/V patterning in ambulacrarians is an interesting example of the evolution of axial patterning mechanisms. bmp2/4 of the sea cucumber is expressed in dorsal structures (Harada et al., 2002), as is bmp2/4 in the direct developing hemichordate S. kowalevskii (Lowe et al., 2006). However, bmp2/4 expression in sea urchin larva is expressed in the ventral ectoderm of the embryo, not on the dorsal side (Angerer et al., 2000; Duboc et al., 2004). These observations make it uncertain whether the ancestral location of bmp2/4 expression in Ambulacraria was on the

Several studies have shown that in echinoderms NiCl2 interferes with axial patterning by radializing the embryo by promoting ventralization in indirect- or sinistralization in direct developing echinoderms (Emlet, 1995; Hardin et al., 1992; Minsuk and Raff, 2005). Fig. 8 is a schematic representation of the phenotypes observed in Ambulacraria after NiCl2 treatments, underlying the strong similarities caused by this reagent. In both indirect developing species, lateral and dorsal ectoderm is transformed into a ventral fate leading to ventralized larvae. In P. flava, smad6 expression in dorsal mesoderm and endoderm is also affected by NiCl2 treatment, suggesting that similar mechanisms are patterning multiple germ layers in this species. In agreement with this hypothesis, a dorsoventral gradient of Notch in the sea urchin embryo is abolished in the vegetal plate and the forming archenteron following treatment with nickel chloride (Sherwood and McClay, 1997). Although radialized by NiCl2 treatments, the only direct developing echinoderm that has been studied, the echinoid H. erythrogramma, exhibits a sinistralizing phenotype characterized by the extension of the left-sided vestibular ectoderm in expense of the right-sided one. Direct developing echinoids possess only a transient larval D/V axis indicated by a remnant ciliary band, a vestigial larval skeleton, and a transient expression of gsc on the ventral side (Emlet, 1995; Wilson et al., 2005). Minsuk and Raff have proposed that the sinistralizing effect of NiCl2 may be a sign of co-option of the ectodermal patterning mechanism from D/V to L/R that has occurred during evolution leading to direct development (Minsuk and Raff, 2005). In S. kowalevskii (direct developing hemichordate) the D/V axis is well defined and an initial study focusing on the role of Bmp2/4 in D/V axis establishment has been recently published (Lowe et al., 2006). Preliminary data kindly provided by Sebastien Darras & Christopher Lowe on S. kowalevskii embryos show that NiS04 treatments cause the

Fig. 7. Effects of NiCl2 on specification of ectoderm, mesoderm and endoderm along the D/V axis. Embryos were treated with 50 μM NiCl2 starting at the 2-cell stage and whole mount in situ hybridization was then performed at the late gastrula stage or on tonaria larvae. The effects of NiCl2 on the gene expression program of the ectoderm, mesoderm of endoderm were analyzed by in situ hybridization with the indicated probes. (A, B, G, H) Pf-frizzled5/8 (Pf-fz5/8), (C, D, I, J) Pf-sfrp1-like, (E, F, K, L) Pf-foxQ2, (M, N, U, V) Pf-brachyury (Pf-bra), (O, P, W, X) PffoxA/hnf3, (Q, R, Y, Z) Pf-fgfr1-like, (S, T, Za, Zb) Pf-chordin, (Zc, Zd, Zk, Zl) Pf-bmp2/4, (Ze, Zf, Zm, Zn) Pf-dlx, (Zg, Zh, Zo, Zp) Pf-pea and (Zi, Zj, Zq, Zr) Pf-smad6. (A–F, M–T, Zc–Zj) Control embryos, (G-L,U-Zb,Zk–Zr) NiCl2 treated embryos, (A, C, E, G, I, K, M, O, Q, S, U, W, Y, Za, Zc, Ze, Zg, Zi, Zk, Zm, Zo, Zq) late gastrula and (B, D, F, H, J, L, N, P, R, T, V, X, Z, Zb, Zd, Zf, Zh, Zj, Zl, Zn, Zp, Zr) tonaria larvae. The three arrows in (Zf) indicate the expression of dlx in isolated cells within the dorso-ventral boundary and the dotted outline in (Zi) indicates the dorsal expression of Pf-smad6 in the endo-, meso- and ectoderm. (■) Hydropore, (*) Mouth.

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Fig. 8. Diagram representing phenotypes observed after NiCl2 treatment in various ambulacarians. (A, E) (Pf, Ptychodera flava, an indirect developing hemichordate, (B, F) (Pl, Paracentrotus lividus an indirect developing echinoderm, (C, G) (Sk, Saccoglossus kowalevskii) a direct developing hemichordate and (C, F) (He, Heliocidaris erythrogramma, a direct developing echinoderm. For each species a late gastrula and a fully formed larvae are shown (control and NiCl2 treated). All represented embryos are lateral views oriented animal up, vegetal down and ventral (oral, blue domain) to the left and dorsal (aboral) to the right, except the late larval stage of (He) where the green domain indicates the vestibular ectoderm on the left side of the animal. The notion of dorsal or ventral (or left and right for He) looses its significance in NiCl2 treated, in which the D/V patterning has been disrupted. Red and yellow represent the presumptive mesoderm and endoderm, respectively. Blue represents the ventral ectoderm and green, the vestibular ectoderm (Heliocidaris only).

extension of ventral structures to lateral and dorsal regions clearly ventralizing the juveniles (Supplementary Fig. 7). Although these treatments also slightly affect A/P patterning in the developing embryos, these results reinforce the idea of the existence of a nickelsensitive pathway that patterns the D/V axis in larval and adult life history stages in both echinoderms and hemichordates. Cell lineage experiments, where single cells of the developing P. flava embryo are labeled with DiI at the 2- to 16-cell stage, indicate that in indirect developing enteropneusts the D/V axis does not appear to be closely linked to the early cleavage program but is likely set up by cell interactions following the 16-cell stage (Henry et al.,

2001). In fact, random D/V labels are observed when a single cell is labeled before the 16-cell stage and embryonic fragments can regulate to form normal larvae as long as they contain cells derived from the vegetal hemisphere (Henry et al., 2001). In agreement with these observations, our analysis of the sensitivity period for NiCl2 shows that treatments starting before the onset of gastrulation are able to interfere with D/V patterning showing that the dorso-ventral axis is still labile before the late blastula stage. The observations from both studies indicate that the D/V axis is specified between 6 hpf (cleavage) and 12 hpf (late blastula). In Table 1 we have summarized the main effects of nickel treatments on the Ambulacraria. In all

Table 1 Comparison of phenotypes observed after NiCl2 treatment in various ambulacarians. The values in the bottom row represent the concentration of nickel chloride needed for full radialization. Literature cited: (Agca et al., 2009; Di Bernardo et al., 1999; Duboc et al., 2004; Hardin et al., 1992; Lallier, 1956; Minsuk and Raff, 2005; Poustka et al., 2004). NiCl2 phenotype

Indirect developing Hemichordate (P. flava)

Indirect developing Echinoderm (P. lividus, L. variegatus, S. purpuratus, H. tuberculata)

Direct developing Echinoderm (H. erythrogramma)

Radially symmetrical ectoderm NiCl2 sensitive period Dosage for full radialization (μM)

yes (ventralized) cleavage to late blastula 50

yes (ventralized) blastula to early gastrula 50–5000

yes (sinistralized) blastula to mid gastrula 2–10


currently studied species, NiCl2 causes radially symmetrical ectoderm (ventral or left side) and has a similar sensitivity period (slightly earlier for P. flava). Dosage for full radialization in P. flava is approximately 5 times higher than in H. erythrgramma (direct developer) but lower or equivalent to indirect developing echinoderms. Taken together, these observations suggest that NiCl2 targets the same molecular pathway(s) in echinoderms and indirect developing hemichordates involved with D/V (or L/R) patterning. Mechanism of NiCl2 action Nickel is a ubiquitous component in the biosphere and the vast majority of nickel related studies concern the understanding of its toxicity, physiology and role in cancers (Calabrese et al., 1977; Clary, 1975; Doll, 1958; Doll et al., 1970; Hunter and Vergnano, 1953; Lu et al., 1979; Salnikow and Costa, 2000; Storeng and Jonsen, 1980; Takumi et al., 2010; Zamponi et al., 1996). These studies present a basic understanding of how nickel could affect living cells. Compounds of nickel (and other metals) have been associated with carcinogenesis for quite a while (reviewed in Sunderman, 1977). In addition to genetic damage via both oxidative and nonoxidative mechanisms, metals can also cause significant changes in DNA methylation and histone modifications, leading to epigenetic silencing or reactivation of gene expression. Carcinogenic effects of nickel are likely to result from its ability to interfere with DNA repair processes (reviewed in Salnikow and Zhitkovich, 2008). In addition to the role of nickel at the DNA level, metals can also directly interfere with transcriptional regulation by modifying the DNA binding capacity of Zn-finger transcription factors (Hartwig, 2001; Nagaoka et al., 1993). The Ca2+ system Nickel has been shown to interfere with T-type Ca2+ channels and cause an elevation of intracellular Ca2+ (Mlinar and Enyeart, 1993; Satoh, 1995; Zamponi et al., 1996). Inhibition of T-type channels in Xenopus animal caps using nickel at concentrations ranging from 10 to 100 μM (similar to this study) induces the formation of a cement gland, a mucus-secreting organ located at the anterior end of Xenopus embryo (Huang and Ding, 1999). Interestingly, formation of the cement gland can be positively or negatively influenced by dorsalizing, ventralizing, neuralizing or posteriorizing factors (Bradley et al., 1996). It has been suggested that gradients of intracellular Ca2+ help establish the dorso-ventral axis of various developing embryos (Jaffe, 1999; Palma et al., 2001; Webb and Miller, 2006). In fact, a Ca2+ signaling system mediates the ventralizing signal in patterning early Xenopus embryos and inhibition of this ventral signal leads to dorsal differentiation. Disruption of the Ca2+ gradient results in a perturbed D/V axis and converts ventral to dorsal fates (Kuhl et al., 2000; Kume et al., 2000). It would be interesting to know if nickel treatment, through its Ca2+ channel inhibitory effect, interferes with axial patterning in Xenopus that would result in an induction of the cement gland in amphibian embryos. Analyzing this question in Xenopus could also provide insight in to whether all deuterostomes share a nickel sensitive pathway that patterns the body axis or if our observations are Ambulacraria-specific. The hypoxia response pathway At least part of nickel's cancer inducing effect is mediated by stabilization of hypoxia inducible factor (HIF-1a) and activating its downstream signaling. Under normal oxygen conditions, prolyl hydroxylases target HIF-1a for degradation and block the pathway. Hypoxia in turn, blocks the activity of hydroxylases leading to the activation of HIF-1a and downstream transcription (Salnikow et al., 2000a). Nickel compounds have been shown to mimic hypoxia. Therefore ectopically activating the hypoxia-signaling pathway can lead to the above described diseases (Salnikow et al., 2003b). A largescale approach to identify nickel inducible genes in cell culture

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showed that hypoxic stress can induce expression of two sets of downstream targets, HIF dependent and HIF independent genes (Salnikow et al., 2003a). In addition, independently of HIF activation, hypoxia (or nickel) can induce a redox state in live cells (Salnikow et al., 2000b; Sun and Oberley, 1996). A growing number of proteins such as bZip, Pax, Ets, homeodomain or bHLH transcription factors are known to be redox-regulated (Sun and Oberley, 1996). The relationship, if any, of the HIF independent regulation downstream of NiCl2 induction and the redox pathway has not been shown, but these observations show that nickel, through its hypoxic effect, can activate at least two sets of downstream targets. Several studies have shown the existence of a respiratory gradient and the crucial role of redox signaling in dorso-ventral patterning in echinoderms (Child, 1941; Coffman and Davidson, 2001; Coffman and Denegre, 2007; Czihak and Meyer, 1964; Pease, 1941, 1942). Recently two HIF orthologs have been identified in a sea urchin (Sodergren et al., 2006), but have not been further characterized. As nickel also affects the dorso-ventral axis of indirect developing echinoderms and hemichordates, the redox mechanism could be a plausible explanation for the mode of action of NiCl2 during early development. A redox gradient has not yet been reported for hemichordates, however, the similarity of the NiCl2 effect suggest that similar mechanisms may be involved in patterning the dorso-ventral axis in those two taxa. The Nodal-signaling pathway Nodal factors, members of the TGFß family of signaling molecules, play crucial roles in chordate development. They have been implicated in various developmental processes such as endo- and mesoderm formation and axial patterning along the A/P, D/V, and L/R axis of the developing embryo (Hamada et al., 2002; Morokuma et al., 2002; Stainier, 2002; Whitman, 2001; Whitman and Mercola, 2001; Yu et al., 2002). In echinoderms, this pathway plays an essential role during embryogenesis in the establishment of the D/V (oral–aboral) and L/R axis (Duboc et al., 2004, 2005; Flowers et al., 2004; Smith et al., 2008). Interestingly, Nodal overexpression mimics the effects of NiCl2 causing ventralized or sinistralized embryos in indirect or direct developing echinoderms respectively and nodal expression is expanded in nickel treated P. lividus embryos (Duboc et al., 2004; Smith et al., 2008). In order to improve the understanding of the relationship between NiCl2 and the Nodal pathway in echinoderms, Venuti and colleagues analyzed the effects of NiCl2 in embryos where the Nodal pathway has been previously disrupted. Although the phenotype from this double treatment is different than control, NiCl2, or Nodal disrupted embryos, they lack any sign of ventral (oral) ectoderm specification, suggesting that NiCl2 requires proper Nodal signaling to ventralize the embryos (Agca et al., 2009). Lepage and colleagues proposed that NiCl2 acts “early and upstream of nodal expression” based on the observation that nodal expression was radialized in NiCl2 treated P. lividus embryos approximately 1–2 h after the onset of its endogenous expression (Duboc et al., 2004). NiCl2 is not sufficient to induce nodal expression based on the absence of nodal expression (analyzed by qPCR) 1 h before and 2 h after the onset of its endogenous expression in S. purpuratus (Agca et al., 2009). These two studies show a clear discrepancy of the data in that nodal expression remains insensitive to NiCl2 treatments in S. purpuratus (Agca et al., 2009) but is radialized in P. lividus (Duboc et al., 2004) 2 h after initial expression. Re-examination of the effects of NiCl on nodal expression before, during or shortly after endogenous initiation in the two species using both in situ hybridization and qPCR analysis would help to gain a better understanding in the real effects of NiCl2 on nodal initiation, and to shed light on the question if there may be a species specific response to nickel. One of the earliest observable asymmetries along the sea urchin D/V (O/A) axis is a redox gradient established by a higher activity (or density) of mitochondria (Coffman and Davidson, 2001). A possible mechanism that could explain how mitochondrial activity affects nodal expression is

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via the production of reactive oxygen species (ROS), such as H2O2 (Coffman et al., 2009). These ROS are known to activate the stress activated protein p38 required for nodal expression in a third sea urchin species L. variegatus (Bradham and McClay, 2006). Coffman and colleagues recently analyzed the role of the redox signal on nodal expression in S. purpuratus. Quenching of mitochondrial H2O2 caused a transient but significant reduction of nodal expression, suggesting that initial nodal expression is partially redox-dependant (Coffman et al., 2009). This observation would be in disagreement with the idea that nickel acts through the redox gradient on nodal expression and D/V patterning, if NiCl2 has no effect on initiating nodal expression (Agca et al., 2009). Interestingly, the augmentation of mitochondrial H202 has no observable effect on the initiation of nodal expression (Coffman et al., 2009) similar to the steady level of initial nodal expression after nickel treatments (Agca et al., 2009). Thus, one cannot rule out the possibility that NiCl2 acts on nodal expression via the cellular redox state (initiation and/or maintenance) resulting in the perturbation of the D/V axis in echinoderms. A causal link between NiCl2 treatments and this asymmetric redox gradient has never been experimentally shown in the sea urchin embryo. Therefore additional experiments are required to gain more insight into the mechanism of action of NiCl2 and its effect on patterning the larval D/V axis in Ambulacraria. Interestingly, cisregulatory analysis of nodal in echinoderms, has identified binding sites for putative downstream targets of redox signaling involved in transcriptional regulation (Nam et al., 2007; Range et al., 2007) that would support the idea that nickel acts through the redox pathway and nodal expression on axial patterning. Nodal factors have yet to be identified and characterized in hemichordates and nothing is known about their function in this taxon. This functional study presents the first attempt to gain insight into the axial patterning mechanism of an indirect developing hemichordate. We have shown that NiCl2 induces a similar phenotype to the one described in its sister taxon, the echinoderms. The exact molecular mechanism by which this metal acts remains unknown, but comparison of our hemichordate results to existing echinoderm data suggest that a conserved NiCl2 sensitive pathway, involved in D/V patterning of the embryo, is shared across Ambulacraria. The phylogenetic position, together with the morphological and fate mapping similarities of indirect developing echinoids and P. flava led to the hypothesis that a biphasic life history was ancestral for Ambulacraria. Henry and colleagues also proposed that “… some key developmental events/mechanisms…” may be shared by the ancestor of echinoderms and hemichordates (Henry et al., 2001). The existence of a NiCl2 sensitive pathway involved in D/V patterning in both taxa, as well as the strong similarities observed in the expression patterns of key developmental genes with those from echinoderms strongly support the common ancestry of these two groups. Nevertheless we also found and confirmed a profound difference in the ectodermal expression of pea and bmp2/4 (e.g., ventral in echinoids versus dorsal in P. flava) that suggests differences in the transcriptional control of certain genes that may play a role in body plan diversification. Identification and characterization of the role of the Nodal pathway in both direct and indirect developing hemichordates as well as the investigation of the gene regulatory network underlying ectoderm patterning in P. flava will hopefully provide new insights into the fundamental mechanisms of deuterostome evolution. Supplementary materials related to this article can be found online at doi:10.1016/j.ydbio.2011.03.030. Acknowledgments We thank Billie Swalla, and Jean-François Brunet for their help in the initial phase of the project, Kevin Pang for careful reading of the manuscript, Aldine Amiel and the members of the Martindale lab for fruitful discussions. We also thank Richard Chock, Aldine Amiel, Guy

Lhomond, Timothy DuBuc, Mattias Ormestad, Lionel Guidi and Pascal Gerdsmeier for their help collecting adult animals. We particularly thank Sebastien Darras (IBDML, Marseille) and Chris Lowe (Stanford Univ.) for sharing unpublished data and for kindly providing Supplementary Fig. 7. This project was supported by the European Molecular Biology Organization (EMBO), the Hawaii Community Foundation (HCF) and the National Science Foundation (NSF), (to ER and MQM). References Agca, C., Klein, W.H., Venuti, J.M., 2009. Respecification of ectoderm and altered Nodal expression in sea urchin embryos after cobalt and nickel treatment. Mech. Dev. 126, 430–442. Angerer, L.M., Angerer, R.C., 2000. Animal–vegetal axis patterning mechanisms in the early sea urchin embryo. Dev. Biol. 218, 1–12. Angerer, L.M., Oleksyn, D.W., Logan, C.Y., McClay, D.R., Dale, L., Angerer, R.C., 2000. A BMP pathway regulates cell fate allocation along the sea urchin animal–vegetal embryonic axis. 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A Discrete Model of Drosophila Eggshell Patterning Reveals ... - Core

Patterning of silicon surfaces with noncontact atomic force ... - Core

Patterning in Recent Radiocarbon Dates from Southern Africa ... - Core

Micro-patterning of Polymer-based Optical Oxygen Sensors ... - CORE

Conservation of ParaHox genes' function in patterning of the ... - Core

orabona - GO patterning - arXiv

Patterning the insect eye www.researchgate.net › publication › fulltext › Patterning

Patterning the spine - eLife

Computational approaches to developmental patterning

Double Patterning Combined with Shrink

Modeling the Developmental Patterning of

Patterning of the mammalian cochlea

Retinoic acid and hindbrain patterning

Direct Laser Interference Patterning - MDPI

3D patterning of ZnO nanostructures

Nanoscale patterning with block copolymers

Patterning in EnglishWritten Medical Leaflets

Dynamics of evaporative colloidal patterning

nano-patterning on ... - Springer Link