Developmental Biology 410 (2016) 108–118
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Developmental Biology journal homepage: www.elsevier.com/locate/developmentalbiology
Evolution of Developmental Control Mechanisms
Regulatory circuit rewiring and functional divergence of the duplicate admp genes in dorsoventral axial patterning Yi-Cheng Chang a,b, Chih-Yu Pai a, Yi-Chih Chen a, Hsiu-Chi Ting a, Pedro Martinez c,d, Maximilian J. Telford e, Jr-Kai Yu a, Yi-Hsien Su a,b,n a
Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan c Department de Genètica, Universitat de Barcelona, Barcelona, Spain d Institució de Recerca I Estudis Avançats (ICREA), Barcelona, Spain e Department of Genetics, Evolution and Environment, University College London, London, UK b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 December 2015 Accepted 18 December 2015 Available online 21 December 2015
The spatially opposed expression of Antidorsalizing morphogenetic protein (Admp) and BMP signals controls dorsoventral (DV) polarity across Bilateria and hence represents an ancient regulatory circuit. Here, we show that in addition to the conserved admp1 that constitutes the ancient circuit, a second admp gene (admp2) is present in Ambulacraria (Echinodermata þHemichordata) and two marine worms belonging to Xenoturbellida and Acoelomorpha. The phylogenetic distribution implies that the two admp genes were duplicated in the Bilaterian common ancestor and admp2 was subsequently lost in chordates and protostomes. We show that the ambulacrarian admp1 and admp2 are under opposite transcriptional control by BMP signals and knockdown of Admps in sea urchins impaired their DV polarity. Over-expression of either Admps reinforced BMP signaling but resulted in different phenotypes in the sea urchin embryo. Our study provides an excellent example of signaling circuit rewiring and protein functional changes after gene duplications. & 2015 Elsevier Inc. All rights reserved.
Keywords: BMP Admp Sea urchin Acorn worm Ambulacraria
1. Introduction The DV polarity of bilaterians is established by a BMP gradient generated through interactions between BMP ligands, including BMP and Admp, and their antagonists, such as chordin and noggin (De Robertis, 2008). The BMP/Admp regulatory circuit, in which admp expression is repressed by BMP signaling and the Admp protein generated on the low BMP side further promotes BMP signaling, has been suggested to be a conserved feature used by bilaterians to establish or regenerate their DV axes (De Robertis, 2008; Gavino and Reddien, 2011; Reversade and De Robertis, 2005; Srivastava et al., 2014). The Echinodermata and Hemichordata form a clade called Ambulacraria (Cannon et al., 2014), and together with Chordata constitute the three major phyla in Deuterostomes. In echinoderms such as sea urchins, although their adults are pentaradially symmetric, they nevertheless develop initially as a bilaterally symmetric gastrula, in which the DV axis is patterned by BMP n Corresponding author at: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan. E-mail address:
[email protected] (Y.-H. Su).
http://dx.doi.org/10.1016/j.ydbio.2015.12.015 0012-1606/& 2015 Elsevier Inc. All rights reserved.
signals as in other bilaterians (Lapraz et al., 2009; McClay, 2011). The sea urchin bmp2/4 gene, one of the major contributors of the BMP gradient, is transcriptionally activated on the ventral side of the blastula by Nodal signals (Ben-Tabou de-Leon et al., 2013; Duboc et al., 2004; Saudemont et al., 2010; Su, 2009). Intriguingly, both bmp2/4 and chordin genes are under the control of Nodal signaling and thus are co-expressed in the ventral ectoderm of sea urchin embryos (Bradham et al., 2009; Lapraz et al., 2009; Su et al., 2009). A BMP activity gradient nevertheless forms that is high on the dorsal side of the embryo, opposite to the co-expression domain of bmp2/4 and chordin (Chen et al., 2011; Lapraz et al., 2009). In the hemichordate acorn worm embryos, bmp2/4 is expressed on the dorsal side, opposite to the expression domain of chordin on the ventral side, and together they also generate a BMP gradient that is high on the dorsal side and control the DV polarity of the embryos (Lowe et al., 2006; Rottinger et al., 2015; Rottinger and Martindale, 2011). The sea urchin genome contains two admp genes, admp1 and admp2 (Lapraz et al., 2006). The sea urchin admp2 has been shown to be expressed in the dorsal ectoderm with BMP signals required for its expression (Saudemont et al., 2010). Similarly, an admp2 gene identified from a hemichordate acorn worm is expressed in the dorsal ectoderm, where high BMP activity is observed (Rottinger
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et al., 2015). The expression of the ambulacrarian admp genes in the domain with high BMP activity is completely opposite to what have been observed in other bilaterians that have been studied, in which admp genes are always expressed on the low BMP side due to the repression of admp transcription by BMP signals. These results brought doubts on whether the conserved BMP/Admp regulatory circuit is altered in Ambulacraria. In this study, we aim to investigate the evolutionary origin and transcriptional regulation of the two admp genes, with a further analysis of the functions of their gene products during sea urchin
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embryogenesis. We survey the presence of admp genes in various animal genomes/transcriptomes and reveal their possible evolutionary history. We discover that the two admp genes, in both sea urchin and acorn worm embryos, are under opposite transcriptional control by BMP signals. Both admp genes are required for establishing DV polarity in the sea urchin embryo, although the two Admp proteins have distinguishable functions when overexpressed. We also discuss the transcriptional rewiring of the admp genes and the functional divergence of their gene products after gene duplications.
Fig. 1. Phylogenetic tree of Admp proteins and other TGFβ family members. Protein sequences were aligned and trimmed, and the tree was constructed using the LG model under a Maximum likelihood analysis. Each TGFβ subfamily forms a distinct group (colored boxes). The Admp1 sequences of bilaterians form a group (purple box) while the Admp2 sequences of ambulacrarians and xenacoelomorphs form another distinct group (pink box). Cnidarian Admp-like proteins (green box) form a group, which is the sister to all bilaterian Admp sequences. No admp genes were found in the ctenophore genome, although members of the TGFβ family were identified (red arrow). The species names and accession numbers corresponding to sequence names in the tree are given in Table S1. The level of bootstrap support is indicated. Scale bar shows substitutions per position.
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2. Materials and methods 2.1. Phylogenetic analyses The sequences were gathered from the National Centre for Biotechnology Information databases, various genomes, EST/transcriptome databases, or obtained by PCR cloning. The accession numbers or gene IDs of the sequences used in our analyses are listed in Table S1. The protein sequences were aligned using ClustalW in Mega6 (Tamura et al., 2013), and the ambiguously aligned regions were detected and removed with Gblocks (Castresana, 2000) to generate the tree in Fig. 1. The maximum likelihood analysis was performed using the RAxML Black Box program with the LG model provided by CIPRES Science Gateway (http://www.phylo.org/) with 1000 bootstrap replicates. Bootstrap values higher than 50% are shown. 2.2. Molecular cloning and QPCR analysis To amplify the sea urchin and acorn worm admp1 and admp2 from embryonic cDNA, PCR primers were designed based on the sea urchin genome database (Cameron et al., 2009) and the acorn worm transcriptome database (Chen et al., 2014), respectively. The amphioxus (Branchiostoma floridae) admp cDNA clone was identified from the EST database (Yu et al., 2008). The coding sequences of admp genes were cloned into the pCS2 þ vector and in vitro transcribed using an mMessage Machine kit (Ambion, Austin, TX, USA). QPCR analysis was performed as previously described (Chen et al., 2011) and the primer sequences are listed in Table S2. 2.3. Embryo preparation and manipulation Sea urchin (Strongylocentrotus purpuratus), acorn worms (Ptychodera flava), and zebrafish adults were obtained, spawned, and fertilized as described (Chen et al., 2011; Ikuta et al., 2013; Tsai et al., 2014; Lin et al., 2016). The sea urchin and zebrafish embryos were microinjected following published procedures (Ben-Tabou de-Leon et al., 2013; Tsai et al., 2014). The sequences of MOs used to inject sea urchin embryos are listed in Table S3. To perturb BMP signaling, recombinant BMP4 protein (R&D Systems) or DMH1 (Sigma) were added to the embryo cultures after fertilization, and the same volumes of solvents, 0.1% BSA or DMSO, respectively, were added as controls. The in situ hybridization and immunohistochemistry were performed, and the embryos were imaged as described (Chen et al., 2011; Ikuta et al., 2013). All images were generated on a Zeiss Axio Imager A1 or a Leica TCS-SP5 APBS inverted confocal system.
3. Results 3.1. Ambulacrarians have two admp genes Two admp genes, admp1 and admp2, have been annotated in the sea urchin genome (Lapraz et al., 2006). We searched the genomes and transcriptomes of a wide range of animals for genes and transcripts encoding Admp-like proteins and performed a phylogenetic analysis of these proteins together with other TGFβ family members (Fig. 1 and Table S1). Admp orthologs could not be identified in the ctenophore, sponge, and Trichoplax genomes, suggesting that admp genes have arisen in the common ancestor of Cnidaria and Bilateria: the cnidarian Admp-related proteins are placed at the base of all bilaterian Admps. We have been able to find a single admp gene in most bilaterian and cnidarian genomes that we have analyzed; the exceptions are the sea urchins, hemichordates, the xenoturbellid Xenoturbella
bocki, the acoel Symsagittifera roscoffensis, and the vertebrates Xenopus and chick, all of which contain two admp genes. The ambulacrarian Admp1s group with other bilaterian Admps, and the orthology is corroborated by the conserved linkage with the pinhead gene, as it occurs in various bilaterian genomes in which a pinhead gene is found (Fig. S1). This linkage has been shown to be responsible for controlling the mutually exclusive expression of admp and pinhead in an ascidian embryo (Imai et al., 2012). In addition to the Admp1 group, the Admp2s of the sea urchins, hemichordates, Xenoturbella, and the acoel form a distinct group with a high support value (Fig. 1). The early branching position of the single cnidarian Admp-related protein prior to the separation of the bilaterian Admp1 and Admp2 genes suggests that the genes encoding Admp1 and Admp2 arose from a gene duplication event after the divergence of Cnidaria and Bilateria. Because the ambulacrarian Admp2s group independently from all the Admp1s, we propose that the duplication occurred in the common ancestor of Bilateria and admp2 genes were subsequently lost in the protostome and chordate lineages. Xenopus and chick genomes also contain two admp genes (Kumano et al., 2006). The phylogenetic analysis revealed that the vertebrate Admp2 proteins are not closely related to the ambulacrarian Admp2s, but are within the Admp1 group along with their paralogs, supporting their origin in a specific duplication event that took place in the vertebrate lineage (Fig. S2). 3.2. Expression of the two admp genes on the opposite sides along the DV axis To investigate the roles of the admp1 and admp2 genes in Ambulacraria, we first analyzed their expression patterns during embryogenesis of the sea urchin Strongylocentrotus purpuratus and the acorn worm Ptychodera flava. Quantitative RT-PCR (QPCR) analysis of the sea urchin admp1 revealed that the transcript was first detected at 26 h post fertilization (hpf) and the expression level continued to increase (Fig. S3a). Consistent with the QPCR data, the admp1 transcript was not observed at the mesenchyme blastula stage (24 hpf) (Fig. 2a); at the early gastrula stage the transcript was detected in a few cells of the ventral ectoderm (Fig. 2b–b′). As gastrulation proceeds, the admp1 expression domain expands along the animal-vegetal axis with the expression being stronger in the ventral midline of the ectoderm and weaker in the ventral side of the archenteron (Fig. 2c–e′). Double fluorescent in situ hybridization (FISH) analysis confirmed that the sea urchin admp1 is co-expressed with brachyury (bra) (Li et al., 2013), the transcript of which marks the stomodeal domain on the ventral side of the mid gastrula (Fig. 2f). In the indirectly developing hemichordate acorn worm P. flava, admp1 is expressed on the ventral side of the embryo (Fig. 2g–k), similar to the situation seen in the sea urchin. Double FISH confirmed that the Ptychodera admp1 expression domain is more towards the animal pole, when compared to the chordin expression domain in the central ventral ectoderm (Rottinger and Martindale, 2011) (Fig. 2l). The expression of admp1 in the ventral ectoderm resembles that seen in the late gastrula of the direct developing acorn worm, Saccoglossus kowalevskii (Lowe et al., 2006). During sea urchin embryogenesis, the high BMP activity on the dorsal side specifies dorsal epidermal cell fate, and the presence of the BMP antagonist Chordin maintains BMP activity at a low level on the ventral side (Angerer et al., 2000; Chen et al., 2011; Lapraz et al., 2009). High BMP activity is also observed on the dorsal side of the P. flava embryos (Rottinger et al., 2015). The expression of admp1 on the side of ambulacrarian embryos with low BMP activity is similar to what is seen in chordates; in the invertebrate chordate amphioxus (Branchiostoma floridae), the admp gene is expressed along the dorsal midline of the ectoderm and mesendoderm, where
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Fig. 2. Expression of sea urchin and acorn worm admp genes during embryogenesis. In situ hybridizations were performed at various developmental stages as indicated on top (EG, early gastrula; MG, mid gastrula; LG, late gastrula). (a–e”) Sea urchin admp1 transcript is detected initially in the ventral ectodermal cells (solid arrowheads) during gastrulation and later in the ventral endodermal cells (open arrowheads). (f) Double FISH of sea urchin admp1 and bra was performed at the mid gastrula stage. (g–j) Expression of the acorn worm admp1 gene (solid arrowheads) begins at the early blastula stage and the expression stays on the ventral side throughout gastrulation. (k) Admp1 expression in three patches of the ventral ectoderm is evident at the tornaria larval stage. (l) Double FISH of the acorn worm admp1 and chordin was performed at the early gastrula stage. (m–q′) The sea urchin admp2 is expressed in the dorsal vegetal ectodermal cells (arrows) at the mesenchyme blastula stage and later in the anal plate of the aboral ectoderm at the LG and larval stages. (r) Double FISH of the sea urchin admp2 and irxA were performed at the mesenchyme blastula stage. (s–w) The acorn worm admp2 transcript (arrows) was detected in the dorsal ectoderm from the blastula to the larval stages. (x) Double FISH of the acorn worm admp2 and dlx was performed at the blastula stage. All embryos/larvae were viewed from the lateral side, except b′-e′ and f from the oral/ventral side, m′–q′ from the vegetal pole, and r from the aboral/dorsal side. The asterisks denote the mouth opening at the larval stages.
BMP activity is low (Yu et al., 2007). The expression of ambulacrarian admp1 genes on the ventral side of the embryos, where BMP activity is low, suggests that the BMP/Admp regulatory circuit is conserved in this lineage. However, we found that the admp2 genes
of both sea urchin and acorn worm are expressed not ventrally (as is admp1), but on the dorsal side, from the blastula to the larval stages, where BMP signaling is highest (Fig. 2m–x; Fig. S3b). Double FISH analysis confirmed that the sea urchin admp2 is expressed within
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the vegetal part of the irxA expression domain in the dorsal ectoderm (Chen et al., 2011) (Fig. 2r), while the acorn worm admp2 is expressed in a broader region of the dorsal ectoderm, similar to the dlx expression domain (Harada et al., 2001) (Fig. 2x). Equivalent expression patterns of admp2 genes on the dorsal side of the embryos have also been described in the European sea urchin Paracentrotus lividus (Saudemont et al., 2010) and in embryos from a Hawaiian population of P. flava (Rottinger et al., 2015). The ambulacrarian admp1 and admp2 genes are therefore expressed on opposite sides along the DV axis of the developing embryos. 3.3. Opposite transcriptional control of the two admp genes by BMP and Nodal signals We next investigated whether these admp genes are regulated by BMP signals. As expected for the conserved BMP/Admp regulatory circuit, the expression of the admp1 gene in both sea urchin and hemichordate embryos was expanded when the BMP
activity was down-regulated, either by injecting a bmp2/4 genespecific antisense morpholino (MO) in the sea urchin embryo or by treating the acorn worm embryos with DMH1, a BMP signaling inhibitor (Hao et al., 2010) (Fig. 3a–b′). Conversely, elevating BMP activity by treating the embryos with exogenous recombinant BMP proteins abolished the expression of admp1 genes (Fig. 3c–d′). In striking contrast, the expression of the novel admp2 genes in both ambulacrarians is regulated by BMP signaling in completely the opposite way compared to the conserved BMP/Admp regulatory circuit. Ambulacrarian admp2 expression was abolished when the BMP activity was down-regulated and increased if the BMP activity was elevated (Fig. 3e–h′). These results show that the ambulacrarian admp1 and admp2 genes are under opposite transcriptional control by BMP signals; BMP signaling represses admp1 expression but activates that of admp2. It is important to mention that we found that short treatments of recombinant BMP proteins failed to perturb expression of both admp1 and admp2, but were able to induce the expression of its direct target gene tbx2/3 (Fig.
Fig. 3. Opposite transcriptional control of admp1 and admp2 genes by BMP signals. The in situ hybridization of admp genes were performed in sea urchin embryos injected with the bmp2/4 MO (50 μM), in acorn worm embryos treated with DMH1 (2 μM), and in embryos treated with the recombinant mouse (mBMP4, 250 ng/ml) or zebrafish (zBMP4, 100 ng/ml) BMP4 protein. Almost 100% of the embryos showed the phenotype displayed. The embryos were observed from the oral/ventral (ov), lateral (lv; ventral side to the left and dorsal side to the right), or vegetal (vv) side, as indicated at the bottom left corner of each panel.
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S4a–f′), suggesting that both admp1 and admp2 are indirectly regulated by BMP signals. Because Nodal signaling is a major player in DV patterning in sea urchin embryos, we further investigated whether the two admp genes are regulated by Nodal signaling. In embryos injected with nodal MO, admp1 expression was abolished while the admp2 expression domain was expanded to the whole vegetal ectoderm (Fig. S4g–n). These results suggest that, similar to the opposite effects of BMP signals on the expression of admp genes, Nodal signals induce the expression of the admp1 and repress admp2 expression in the ventral ectoderm. Because bmp2/4 is downstream of Nodal signals and required for admp2 expression (Fig. 3e–e′), the fact that the expression domain of admp2 is expanded in the nodal morphants suggests that an additional factor is able to activate admp2 expression in the vegetal ectoderm.
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3.4. Admp and BMP molecules act synergistically to generate BMP activity gradient along the DV axis To investigate the roles of Admp1 and Admp2 during sea urchin embryogenesis, we conducted knockdown experiments by injecting MOs to block RNA splicing or translation. Several spliceblocking (sMO) and translation-blocking (tMO) MOs were designed against admp1 and admp2, and the MO efficacy was validated by RT-PCR and the test of GFP fusion constructs, respectively (Fig. S5a–f). In embryos injected with MOs against admp1 or admp2, the elongation of the embryos along the DV axis was slightly affected and the two skeletal rods failed to converge at the apex of the embryo, although the DV polarity remained recognizable with the mouth opening located on the ventral side (Fig. 4a–c′). Embryos injected with a control MO, with a random
Fig. 4. Admp and BMP molecules act synergistically to generate BMP activity gradient and regulate DV patterning. (a–c′) Sea urchin embryos were injected with admp1 sMO (100 μM) or admp2 tMO (300 μM), and the phenotypes were observed at the pluteus stage. More than 90% of the embryos showed the phenotype displayed. The embryos were viewed from the lateral (lv) or vegetal (vv) sides with the ventral side to the left. (d–i) The in situ hybridizations of admp1 were performed in embryos injected with MOs against admp1, admp2, and (or) bmp5-8 (100 μM) as indicated. The gastrula embryos were observed from the ventral side, and the animal pole was on top. (j) The number of cell rows containing the admp1 transcript was quantified. The sample sizes are indicated inside each column. The injected MOs are listed at the bottom of the panel. The error bars are the standard deviation, and the p value is o 0.001 (T test, ***).
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sequence at the same concentration, extended normally along the DV axis (Fig. S5g). Because two BMP ligands, BMP2/4 and BMP5-8, reportedly play a significant role in enhancing dorsal regulatory gene transcription (Ben-Tabou de-Leon et al., 2013), our results support the proposition that the two sea urchin Admp proteins play, at least, partially redundant roles in patterning the DV axis. We confirmed the synergistic effects of BMP2/4 and BMP5-8 by analyzing the expansion of the admp1 expression domain as an indicator of the BMP activity level after blocking the translation of bmp2/4 and bmp5-8 by MO injections (Fig. S5h). Similar synergistic effects were also observed when MOs against admp1 or admp2 were injected together with bmp5-8 MO (Fig. 4d–h). The maximum expansion of the admp1 expression domain was observed when embryos were co-injected with all three MOs (Fig. 4i–j). Taken together, these results suggest that the dorsal and ventral BMP signals act synergistically to pattern the DV axis in the sea urchin
embryo, similarly to what has been observed in the Xenopus embryos (Reversade and De Robertis, 2005). 3.5. The two Admps have evolved different functions recognized by the sea urchin embryo but not by zebrafish embryos Although the endogenous Admp1 and Admp2 play similar roles in patterning the DV axis during sea urchin embryogenesis, it is intriguing to examine whether they have evolved different functions by introducing them ectopically into the embryos. The overexpression of either admp1 or admp2 in the sea urchin embryo expanded BMP activity, as evidenced by phospho-Smad1/5/8 (pSmad) antibody staining, although the effects differed slightly between these two genes. Similar to previously published results (Chen et al., 2011; Lapraz et al., 2009), pSmad signal was detected on the dorsal half of the blastula embryos (Fig. 5a–a′). In admp1over-expressing embryos, pSmad was detected in an equatorial
Fig. 5. Functional analyses of Admp1 and Admp2 in the sea urchin and zebrafish embryos. Upon injection of mRNAs (300 ng/μl) encoding sea urchin Admp1 or Admp2, sea urchin mesenchyme blastula embryos were stained with an anti-pSmad antibody (a–c′) or hybridized with probes against the ventral marker chordin (d–f), the dorsal marker dlx (g–i), or the pigment cell precursor marker gcm (j–l). The white arrowhead in c indicates the animal pole. Pigment cells (black arrowheads) were restricted to the dorsal side of the normal pluteus larva (m), but were more broadly distributed in the admp1 mRNA-injected (n) and absent in the admp2 mRNA-injected embryos (o). The sea urchin embryos were viewed from the lateral side (lv) with the ventral side to the left or from the animal pole (av). Almost 100% of the injected embryos showed the phenotype displayed. (p-z6) Zebrafish embryos were injected with 200 pg of mRNAs encoding sea urchin Admp1, Admp2, or amphioxus Admp (Bf-Admp). The in situ hybridizations of the dorsal organizer markers gsc and chordin, and the ventral marker eve were performed at 70% epiboly, and the embryos were viewed from the dorsal (p– v) or ventral (w–y) sides. The length of the gsc expression domain is indicated by bars. The white arrow indicates the expression of chordin in the axial mesoderm. Zebrafish phenotypes were observed at 24 hpf (z-z6). The arrowheads and arrows indicate the reduced tail and absence of tail, respectively. The numbers in the bottom left-hand corners indicate the ratios of the displayed phenotypes.
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belt of the ectodermal cells (Fig. 4b–b′). Over-expression of admp2 expanded the ectodermal pSmad signal further towards the animal pole and the pSmad signal was also detected in the ingressed mesenchyme cells (Fig. 4c–c′; Fig. S6a). We also noticed that, although pSmad signal was expanded when the embryos over-expressing admp1 or admp2, the pSmad signal was generally weaker than in the controls. It is possible that over-expression of admps affects the endogenous BMP signal transduction and that these Admp molecules are not as efficient as the BMP ligands in transducing signals via Smad. When a higher concentration of admp1 mRNA was injected, pSmad signal was even weaker (Fig. S6a). Admp2 was more effective than Admp1 at expanding pSmad signal to the animal pole of the embryo and its effects on strengthening BMP signals were dose-dependent. Genes that are normally expressed in the ventral ectoderm, such as chordin, gsc, and bmp2/ 4, were down-regulated in injected embryos, while the expression of the dorsal ectodermal genes dlx and tbx2/3 was expanded (Fig. 5d–i; Fig. S6b), coinciding with the requirement of BMP signaling for their transcriptional activation (Ben-Tabou de-Leon et al., 2013; Saudemont et al., 2010). Clear differences between admp1 and admp2 mRNA-injected embryos were also observed in the expression of gcm (Fig. 4j–l), a gene that controls the specification of pigment cell precursors located in the dorsal half of the vegetal plate (Ransick and Davidson, 2012). The expression of gcm expands to the ventral side in the admp1-over-expressing embryos but disappears when admp2 is over-expressed. Other distinct roles of Admp1 and Admp2 include their effects on the expression of the dorsal ectodermal marker hox7 and the ciliary band marker hnf6 (Fig. S6b). Embryos injected with admp1 or admp2 mRNAs were observed to lose their DV polarity, as demonstrated by the failure of the gut to bend towards the ventral side (Fig. 5m–o). Pigment cells were broadly distributed in the admp1-over-expressing embryos but absent when admp2 was over-expressed. These phenotypic changes upon admp mRNA injections showed a dose-dependency (Fig. S7). To examine whether the number of pigment cells was increased in embryos over-expressing admp1, we performed in situ hybridization with a polyketide synthase (pks) probe; pks is a pigment cell-specific marker (Calestani et al., 2003). The results showed that the number of pigment cells remained unaltered in the admp1-over-expressed embryos, compared to the control embryos injected with the same amount of gfp mRNA (Fig. S8a–b), suggesting that only the distribution of pigment cells but not their number was affected. To extend the comparison, sea urchin embryos injected with mRNA encoding the amphioxus Admp were observed and they displayed phenotypes similar to that seen in sea urchin admp1 mRNA-injected embryos (Figs. S6b and S7). This functional similarity is consistent with their phylogenetic affinities (Fig. 1) and suggests that the sea urchin Admp1 retains the function seen in other bilaterian Admp proteins and that, by contrast, Admp2 has acquired a different role. In admp2-over-expressing embryos, the non-skeletogenic mesoderm-derived pigment and blastocoelar cells, as well as the expression of their respective specification genes gcm and ese, completely disappear (Materna et al., 2013) (Fig. 5l and Fig. S8c). In order to examine whether the different functions of the sea urchin Admp1 and Admp2 can be recognized in a vertebrate system in which admp2 gene is absent, we over-expressed either of these paralogs in zebrafish embryos. Compared to control zebrafish embryos, all embryos injected with mRNA encoding sea urchin Admp1, Admp2, or amphioxus Admp have smaller organizers, as revealed by the expression of the organizer marker gsc, and are shorter due to the partial loss of dorsal structures (Fig. 5p– s). Similarly, the extension of chordin expression in the axial mesoderm was not observed in the admp1- or admp2-over-expressing embryos (Fig. 5t–v), while the expression of the ventral marker eve
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was not affected (Fig. 5w–y). The phenotypes of embryos overexpressing different Admp proteins are indistinguishable (Fig. 5z– z6), and resemble those of zebrafish embryos over-expressing its own admp mRNA (Lele et al., 2001; Willot et al., 2002). Control embryos injected with the same amount of gfp mRNA developed normally (Fig. S8d). Therefore, although Admp1 and Admp2 have different effects when over-expressed in the sea urchin embryo, our results show that these Admp family members are functionally interchangeable in zebrafish embryos, suggesting that the corresponding signaling receiving system for Admp2 has not been evolved or has been lost in the chordate lineage. 3.6. Both prodomains and TGFβ domains of the Admp proteins are important for their functional divergence Admp proteins, similar to other TGFβ family members, contain a conserved C-terminal TGFβ domain and a less well-conserved N-terminal prodomain (Harrison et al., 2011). To investigate if either domain is likely to be responsible for the difference in the function of Admp1 and Admp2 in sea urchin embryos, we created chimeric constructs by swapping the two functional domains of both proteins (Fig. 6). Embryos injected with the chimeric construct containing the Admp1 prodomain and the Admp2 TGFβ domain showed a phenotype similar to that of the admp1-over-expressing embryos, with a disrupted DV axis and a broad distribution of pigment cells (Fig. 6a–b′ and e–f'). Conversely, when injecting, at high concentration, the chimeric construct consisting of the Admp2 prodomain and the Admp1 TGFβ domain, half of the injected embryos lost their pigments cells, a phenotype similar to that of the admp2over-expressing embryos (Fig. 6c–d′ and g–j′). These results suggest that the functional divergence of the two sea urchin Admps is contributed mainly by their prodomains. We next examined the expression of DV markers in the embryos injected with the chimeric mRNAs (Fig. S9). The results showed that the distinct effects on marker gene expression observed in admp1 and admp2-overexpressing embryos (Fig. 5j–l; Fig. S6b) were absent in the embryos expressing chimeras. Therefore, we concluded that both the prodomains and TGFβ domains are important for the functional divergence of Admp1 and Admp2.
4. Discussion In this study, we have shown that the conserved BMP/Admp1 regulatory circuit in which BMP signals repress admp1 expression and Admp1 contributes to the BMP activity is present in ambulacrarian embryos. In addition, ambulacrarian embryos also use a second BMP/Admp2 regulatory circuit, in which BMP signals activate admp2 transcription, and Admp2 further boosts BMP activity. The opposite transcriptional control of the two admp genes by BMP signals shown here confirmed the results seen in another species of sea urchins (Lapraz et al., 2015). Our results strongly suggest that both regulatory circuits were present in the common ancestor of the Ambulacraria. Based on our phylogenetic analysis, we propose that a single admp gene was duplicated into admp1 and admp2 in the bilaterian ancestor (Fig. 7). Due to the still uncertain phylogenetic positions of Xenoturbellida and Acoelomorpha (together are called Xenacoelomorpha) as an early branching group of Bilateria or a sister group of Ambulacraria (Dunn et al., 2014), admp2 genes may have been retained independently or present in the common ancestor of Ambulacraria and Xenacoelomorpha, respectively, and then lost in the protostome and chordate lineages. Our study further provides a nice example for understanding the evolution of transcriptional control and the changes of protein
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Fig. 6. Phenotypes of sea urchin embryos injected with mRNAs encoding Admp1 (a–b′), Admp2 (c–d′), hybrid of the prodomain from Admp1 and the TGFβ domain from Admp2 (Admp1P2T) (e–f′), or hybrid of the prodomain from Admp2 and the TGFβ domain from Admp1 (Admp2P1T) (g–j′). The domain structures of Admp proteins translated from the injected mRNAs are illustrated. Pigment cells are indicated with arrowheads. Live pluteus larvae (72 hpf) were observed from the lateral or vegetal side, and the ventral side is to the left. The black box represents the two phenotypes observed in Admp2P1T-over-expressed embryos. The ratios of the displayed phenotypes are indicated at the bottom left corner.
functions after duplication of genes encoding signaling ligands. In Cnidaria, the sister group of Bilateria, BMP signaling is also important for establishing their secondary axis during gastrulation (Saina et al., 2009). Prior to gastrulation, the admp-related gene of the cnidarian Nematostella vectensis is expressed in a central domain located at the animal pole, bmp2/4 is expressed in the central ring that surrounds this central domain, and chordin is expressed in the external ring that lies adjacent to the internal ring (Rottinger et al., 2012). It is unclear whether the expression of the Nematostella admp-related gene is regulated by BMP signals. It is also unknown whether the two admp genes of Xenoturbella and acoels are under similar opposite regulation by BMP signals. Nevertheless, our results suggest that, after the ancient admp gene duplicated into admp1 and admp2 in the bilaterian ancestor, the two ambulacrarian paralogs were transcriptionally regulated by
the BMP signals in opposite ways (Fig. 7). Changes in transcriptional control after gene duplications are also found in bmp2 and bmp4 genes, after they duplicated from their ancestral bmp2/4 gene. During zebrafish gastrulation, the expression of bmp2b and bmp4 is restricted to the future ventral side (Langdon and Mullins, 2011; Ramel and Hill, 2013). Interestingly, in the Xenopus embryos, bmp2 is expressed on the dorsal side, opposite to the expression domain of its paralog bmp4 on the ventral side (De Robertis and Colozza, 2013; Inomata et al., 2008). Nevertheless, in both animals, the regulatory circuits involving BMPs and their antagonists robustly generate a ventral to dorsal BMP activity gradient. In most bilaterians, expression of bmp and chordin are on opposite sides of embryos. However, in both sea urchin blastulae (Lapraz et al., 2009; Su, 2009) and the Nematostella gastrula (Matus et al., 2006), bmp2/4 and chordin are co-
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Fig. 7. Evolutionary scenarios of the admp genes. The common ancestor at each node is indicated with colored circles. The dashed lines indicate two possible phylogenetic positions of Xenoturbella and acoels. The ovals near the admp loci denote possible enhancers; the purple ovals are negatively and the pink ovals are positively regulated by BMP signals, respectively. The dashed admp2 loci indicate the lost of admp2 genes in the protostomes and chordates.
expressed on the same side of the embryos, and their BMP activity gradients are still perfectly formed on the opposite side of the chordin expression domain (Chen et al., 2011; Lapraz et al., 2009; Leclere and Rentzsch, 2014). These examples show that transcriptional regulation of bmp genes were modified during evolution without affecting the generation of BMP activity gradients. These data also support the predictions from a mathematical model in which the expression of BMP antagonists (such as chordin), and not the bmp themselves (such as bmp4), is needed to be spatially restricted to generate a signaling gradient (Genikhovich et al., 2015). The duplication of the admp genes not only gave rise to two opposite regulatory circuits, but the functions of their gene products have also changed. Our study shows that the two sea urchin Admps have different effects on the nonskeletogenic mesodermal cells when they are over-expressed in the sea urchin embryos. It is thus clear that functions of the two Admp paralogs diversified after the duplication and we speculate that one of the two paralogs has kept the ancestral function and the other has evolved new functions (neofunctionalization) (Carroll, 2005; Force et al., 1999). One clear example of the functional changes in signaling ligands after gene duplications is the opposite effect of the two Nematostella paralogous FGFs on apical organ formation (Rentzsch et al., 2008). Neofunctionalization is probably an important and common feature for duplicated genes to be retained in genomes (Assis and Bachtrog, 2013). Taken together, our study on admp genes provides a good example of both the rewiring of signaling circuits and the changes of protein functions resulting from the duplication of genes encoding signaling molecules.
Author Contributions Y.C. Chang designed and performed most of the experiments conducted in sea urchin and zebrafish embryos in consultation with Y.H.S. C.Y.P. generated chimeric constructs, and Y.C. Chen and H.C.T. performed experiments in hemichordate embryos in consultation with Y.H.S. and J.K.Y. P.M, M.J.T, and J.K.Y provided the sequences obtained from database mining. Y.C. Chang and Y.H.S. wrote the manuscript, and P.M., M.J.T, and J.K.Y commented on it.
Acknowledgments We thank Yu-Hsiu Liu in Dr. Sheng-Ping L. Hwang's laboratory for her technical assistance in microinjection of zebrafish embryos and Drs. Jen-Chieh Lin and Bon-chu Chung for providing zebrafish cDNA clones for in situ hybridization. Y.H.S and J.K.Y. are supported by the intramural fund from Institute of Cellular and Organismic Biology, Academia Sinica and grants from Ministry of Science and Technology, Taiwan (103-2311-B-001-030-MY3 and 103-2627-B001-001 to Y.H.S. and 102-2311-B-001-011-MY3 and 104-2923-B001-002-MY3 to J.K.Y.). M.J.T is supported by a Royal Society Wolfson Research Merit Award. P.M. is supported by a Grant of the Spanish “Ministerio de Economia y Competitividad (MINECO) (BFU2012-32806)”.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ydbio.2015.12.015.
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