- Open Access
Genomic insights into Wnt signaling in an early diverging metazoan, the ctenophore Mnemiopsis leidyi
© Pang et al; licensee BioMed Central Ltd. 2010
- Received: 4 June 2010
- Accepted: 4 October 2010
- Published: 4 October 2010
Intercellular signaling pathways are a fundamental component of the integrating cellular behavior required for the evolution of multicellularity. The genomes of three of the four early branching animal phyla (Cnidaria, Placozoa and Porifera) have been surveyed for key components, but not the fourth (Ctenophora). Genomic data from ctenophores could be particularly relevant, as ctenophores have been proposed to be one of the earliest branching metazoan phyla.
A preliminary assembly of the lobate ctenophore Mnemiopsis leidyi genome generated using next-generation sequencing technologies were searched for components of a developmentally important signaling pathway, the Wnt/β-catenin pathway. Molecular phylogenetic analysis shows four distinct Wnt ligands (MlWnt6, MlWnt9, MlWntA and MlWntX), and most, but not all components of the receptor and intracellular signaling pathway were detected. In situ hybridization of the four Wnt ligands showed that they are expressed in discrete regions associated with the aboral pole, tentacle apparati and apical organ.
Ctenophores show a minimal (but not obviously simple) complement of Wnt signaling components. Furthermore, it is difficult to compare the Mnemiopsis Wnt expression patterns with those of other metazoans. mRNA expression of Wnt pathway components appears later in development than expected, and zygotic gene expression does not appear to play a role in early axis specification. Notably absent in the Mnemiopsis genome are most major secreted antagonists, which suggests that complex regulation of this secreted signaling pathway probably evolved later in animal evolution.
- Adenomatous Polyposis Coli
- Apical Organ
- Armadillo Repeat
- Aboral Pole
- Apical Sensory Organ
Fortunately, genomic data (gene content and complexity) and information on overall genomic structure can prove useful in resolving the relationship of these clades to one another. The genomes of the anthozoan cnidarian Nematostella vectenis , the hydrozoan cnidarian Hydra magnipapillata , the placozoan Trichoplax adhaerens  and the sponge Amphimedon queenslandica  have already proven to be invaluable resources in the effort to understand the genomic makeup of the earliest metazoans. Along with data from other sponges , the genomic data from choanoflagellates [17, 18] (the sister group of metazoans) have provided significant insight into the molecular complexity present in the closest extant unicellular ancestor of animals. Nonetheless, the available data from ctenophores (that is, the modest expressed sequence tag (EST) sets from two species, Mnemiopsis leidyi and Pleurobrachia pileus) is far from sufficient to resolve the placement of this enigmatic lineage.
Although ctenophores have proven to be exceptional experimental embryological material, very little is known about the identity of the exact genes and proteins involved in specifying the body axes. To date, work on ctenophores has focused mainly on different families of transcription factors, including Sox , Fox , T-box  and Homeobox [28, 29], yet nothing is known about the cell signaling pathways. Bilaterian model systems have identified a limited number of cell signaling pathways, including the Wnt/β-catenin, TGF-β, Hedgehog, Notch, receptor tyrosine kinase, and Jak/STAT pathways. These pathways generally involve an extracellular (and often diffusible) ligand, transmembrane receptor, intracellular signal transduction/amplification system and, interestingly, a system of antagonists that can be used to further regulate informational content. These systems are used repeatedly in different tissues throughout the life history of organisms , with the basic elements of these systems arising early in animal evolution .
We recently used next-generation technologies to sequence the genome of the lobate ctenophore Mnemiopsis leidyi, in an effort to better understand early animal evolution. In this paper, we look at one particular aspect, the evolution of the canonical Wnt signaling pathway. We found a near-complete Wnt signaling pathway present, including four Wnt ligands. However, part of the 'destruction complex' appears to be incomplete, and many Wnt antagonists are not recognizable in the genome. In situ hybridization studies showed that transcripts for all four Wnt genes are detected relatively late in development in discrete domains of the developing tentacles and apical organ.
Wnt/β-catenin pathway members present in the Mnemiopsis leidyi genome
Mle scaffold or accession number
DIX, PDZ, DEP
Fz, Fri 7TM
Fz, Fri 7TM
PHD zinc finger
NP_073736.2: Porc isoA
EGF(2), LY, EGF, LY(4), EGF
NP_001139628.1: GSK-3 beta
ANK(10), SAM, PARP, C2
ZnF, KIX, BROMO, DUF906, ZnF
AAC51770.1: CREB-binding protein
WD40(6), LDLa(3), transmembrane
Receptors and downstream components of the Wnt signaling pathway
In addition to the four Wnt ligands, the Mnemiopsis genome contains the receptors Fzd and LRP5/6 (Table 1). We were able to clone and identify two Fzd genes (MlFzdA and MlFzdB), both containing the extracellular Frizzled cysteine-rich domain (CRD), which binds Wnt ligands, and the transmembrane domain (Figure 5B). MlFzdA also has a signal peptide and the intracellular KTXXXW motif (KTASNW), which is thought to bind the PDZ domain of Dsh and is therefore required for canonical Wnt signaling . MlFzdB does not appear to have the signal peptide or the KTXXXW motif based on cloned RACE PCR fragments. There was only a single LRP5/6 identified in the genome.
For functional canonical Wnt signaling, key intracellular components of the pathway are required. In Mnemiopsis, there are single genes encoding Dishevelled (MlDsh) and β-catenin (MlBcat). MlDsh contains all three key domains found in Dishevelled proteins of other animals (DIX, PDZ and DEP) (Figure 5B). The full-length MlBcat sequence was cloned from a mixed stage cDNA template. Similar to β-catenin from other metazoans, there is a highly conserved GSK-3 phosphorylation site and a conserved N-terminal motif (Figure 5B). Centrally, there are 12 armadillo repeats that are clearly detectable but widely divergent compared with other metazoan sequences. Surprisingly, based on the homology of predicted protein sequences, MlBcat appears to lack both C-terminal motifs (motifs A and B), which are thought to serve as transactivational domains . When Wnt signaling is inactive, the 'destruction complex', composed of axin, APC and GSK-3, binds cytoplasmic β-catenin and targets it for degradation . Although we found a clear GSK-3 ortholog, in silico searches found only a partial match to APC (low similarity to armadillo repeat domain and lacking all other domains) and did not find any evidence of axin. It is known that GSK-3 can phosphorylate β-catenin without requiring the other members of the complex . We did find that the transcription factor TCF/LEF (MlTcf), the binding partner of stabilized nuclear β-catenin, is required for the activation of downstream target genes. MlTcf contains the β-catenin binding domain at its amino terminus and also contains the the Sox-Tcf high mobility group domain, which binds DNA (Figure 5B).
Although we found Wnt pathway genes from all parts of the pathway, including ligand modification/secretion, receptors and other membrane-associated proteins, and cytoplasmic and nuclear factors (Table 1; see Additional file 2), we failed to identify the important antagonists Dickkopf (DKK), Wnt Inhibitory Factor (WIF) and Cerberus (CER), which are characteristic of bilaterian Wnt signaling. We were able to identify a possible Secreted Frizzled-related gene (MlSfrp) that may be involved in regulating Wnt signaling; it contains the extracellular Frizzled CRD but not the transmembrane domain (Figure 5B). Unlike bilaterian Sfrp, MlSfrp lacks a Netrin-like (NTR) domain.
Wnt/β-catenin expression patterns
Whereas MlWntA and MlWnt9 are primarily expressed in small regions of the tentacle bulb, the other two Wnt genes are associated with the apical sensory organ and its surrounding regions. The apical organ is primarily a gravity-sensing organ , although it possibly also acts as a photoreceptor , and it is highly innervated, as evidenced by ultrastructural analysis . MlWnt6 is expressed in the apical organ floor, primarily in a central region along the tentacular plane (Figure 6C). There is also faint diffuse expression of MlWnt6 within the tentacle bulb. MlWntX is expressed in a region surrounding the floor of the apical organ, except for two areas in the pharyngeal plane, where it is excluded (Figure 6D). There is also expression in cells of the ciliated groove, the ciliated connection pathway between the gravity-sensing cells of the apical organ, and the locomotory comb rows.
The key Wnt modulator MlDsh is expressed maternally in a uniform pattern and throughout development in almost all cells (Figure 7D). By contrast, MlBcat is first detected during mid-gastrulation, and is localized to the region surrounding the blastopore (Figure 7E). However, the blastopore, which corresponds to the animal pole and the future location of the mouth, is already fully formed, and the endodermal macromeres have already been internalized by the time zygotic transcripts are detectable. The cells that express MlBcat remain on the surface, and expression is not seen in endodermal precursors. We are not able to detect any transcripts before this stage by in situ hybridization; however, we cannot rule out that there are low levels of expression or maternally deposited protein present. As development proceeds, MlBcat is expressed almost ubiquitously in both the ectoderm and the endoderm. The only region in which it is not expressed (or is expressed at low levels) is in the cells that form the comb plates. It is possible that this widespread expression is due to the role of β-catenin in cell adhesion. The onset of MlBcat expression occurs earlier than that of all four Wnt genes and, in contrast to the expression of MlBcat, which is initially localized to the oral (animal) pole, all four Wnt genes are localized primarily to the aboral (vegetal) pole of the embryo. Finally, the transcription factor MlTcf is expressed after gastrulation diffusely in the ectoderm and more intensely around the blastopore (Figure 7F). Similar to MlBcat, it also is not expressed in cells that form the comb plates. Late expression of MlTcf is confined to individual cells of the apical organ and parts of the tentacle bulb.
To date, existing studies have offered only a partial view of a limited number of gene families in ctenophores [25–29, 49–51]. Using next-generation sequencing, we were able to investigate complete gene families and signaling pathways in the ctenophore Mnemiopsis leidyi. Although results from full genome analyses of gene families are not yet available, we examine in this paper the comprehensiveness of an important developmental signaling pathway: the Wnt/β-catenin pathway. To ensure that the genomic searches were complete and that false negatives were minimized, we examined the published 15,752 Mnemiopsis ESTs and found that there was a match of approximately 97 to 99% to genomic contigs, depending on stringency conditions (data not shown), suggesting that our genomic sequencing is fairly complete.
Ancestral metazoan gene complement
Wnt/β-catenin pathway evolution
Core components of the Wnt pathway are present in all non-bilaterians (Table 2) , suggesting the existence of functionally active signaling at the base of the animal tree of life. Many genes in the Wnt pathway appear to be animal-specific novelties. However, proteins containing Armadillo repeats (such as those in β-catenin) are found in all eukaryotes; these proteins have cytoskeletal functions in fungi and protists, and are involved in intracellular signaling in plants . Certain pathway components are also present in organisms such as the slime mold Dictyostelium, which contains a β-catenin-like gene called aardvark, a GSK-3 homolog and Frizzled-like receptors [53, 54]. Aardvark has 10 armadillo repeats (compared with 12 in β-catenin), potential N-terminal GSK-3 phosphorylation sites, and no C-terminus motifs. Functional work has shown aardvark to have roles in both adherens junctions and cell signaling (in the form of stalk formation); however this is independent of GSK-3 activity . All other metazoan β-catenin proteins examined to date have the two C-terminus motifs (A and B) that are thought to be transactivational domains, except for Caenorhabtidis elegans, which seems to have lost or modified them . Thus, depending on the phylogenetic positions, ctenophores either lost these two motifs or they evolved after the ctenophore divergence. Additionally, bilaterian β-catenins also have other C-terminal motifs, which appear to be lineage-specific innovations.
The lack of certain Wnt pathway components in Mnemiopsis that are present in other non-bilaterians is an intriguing result. For instance, axin is found in Amphimedon, Trichoplax, Nematostella and bilaterians, but appears to be missing from Mnemiopsis (Table 2). Whether this gene appeared after ctenophores diverged from later metazoan lineages or was lost in the Mnemiopsis lineage is not yet clear. Likewise, there seems to be a paucity of diffusible antagonists in Mnemiopsis, Amphimedon and Trichoplax. Whereas Amphimedon has several Sfrp-like genes [15, 31], Mnemiopsis has only a single Sfrp; however, in both species the netrin domain is lacking. A DKK ortholog has been reported only for the sponge Oscarella carmela , as well as cnidarians and deuterostomes . Trichoplax does not appear to have any of the known antagonists. Whereas DKK appears to be relatively ancient and lost in the protostome lineage, WIF is probably a bilaterian novelty and CER is only found in vertebrates. It is likely that antagonists were relatively recent additions to the pathway, providing an extra mechanism to control the activity. Alternatively, there could be additional novel antagonists in Mnemiopsis or in the other early lineages, whose identities can only be discovered through functional experiments.
Based on the gene content observed in the early-branching phyla, we can begin to deduce the key steps that led to the complexity observed in the bilaterian Wnt signaling pathway. It appears that the core components were present in the metazoan ancestor, including a Wnt ligand, Frizzled receptor, Dsh and β-catenin. Before the cnidarian-bilaterian ancestor developed, a series of duplication and divergent events, especially among the Wnt genes themselves, led to significant expansion of the components in the pathway. This expansion, coupled with the origin of the Wnt antagonists DKK, WIF and CER was probably the catalyst for the acquisition of additional roles of the pathway.
Based on gene content and diversity, our results are incongruent with a sister relationship between cnidarians and ctenophores (that is, the Coelenterata hypothesis). Firstly, in our phylogenies the genes of ctenophores do not group closely with those of cnidarians. Moreover, if cnidarians and ctenophores were sister phyla, a tremendous amount of gene loss (including the loss of multiple Wnt ligands, axin and DKK) would have been required in the Mnemiopsis lineage. These results are consistent with previous analyses of homeobox and nuclear receptor gene families (Ryan et al., submitted; Reitzel et al., submitted) in rejecting the Coelenterata hypothesis. Unfortunately, the comparison of Wnt signaling components between Mnemiopsis, Amphimedon and Trichoplax is not sufficient to identify the relationships between Ctenophora, Porifera and Placozoa. Additionally, it is not known how well Mnemiopsis represents the ancestral ctenophore gene complement, therefore data from other ctenophores would be of great benefit.
In Mnemiopsis, all of the Wnt genes are expressed at the aboral (vegetal) pole in a striking pattern that suggests they are playing some role in patterning the body. However, they are expressed at such a late stage in development that many cell fates have already been specified. The expression patterns of the Wnt genes in the apical organ and tentacle bulb would suggest they might be involved in neural specification. In cnidarians, it has been suggested that Wnt genes expressed in staggered ectodermal and endodermal domains are patterning the oral-aboral axis in a 'Wnt code' [35, 56]. By contrast, most of the Wnt genes in Nematostella are primarily expressed at the oral pole, whereas in Mnemiopsis, they are expressed at the aboral pole. In the sponge Amphimedon, a Wnt gene is expressed at the posterior pole of the swimming larvae . A major similarity is that Wnt genes appear to be expressed at the posterior pole of most animals [57, 58]. Mnemiopsis locomotes primarily with the oral end to the front (as do most ctenophores), as this aids in feeding; however, they are capable of moving in both directions. Cnidarians, such as Nematostella, swim in the direction of the aboral end, as this is the location of their apical tuft. The observed Wnt expression patterns could suggest that the aboral pole of ctenophores corresponds to the posterior pole of bilaterians.
It is difficult to determine whether the Wnt/β-catenin pathway is functioning early in development based on in situ expression patterns alone. Whereas MlFzdA and MlDsh are both expressed maternally and persist through early cleavage, MlBcat and the Wnt genes are not expressed until after gastrulation. Furthermore, whereas the Wnts are detected primarily at the aboral pole, MlBcat is initially expressed at the oral (or animal) pole. Either this pathway is not involved in early axis specification or there may be a maternal β-catenin protein that is functioning before onset of zygotic expression. Protein localization (particularly that of β-catenin) would help to determine whether the pathway is involved in axial patterning; however, we have not generated an antibody to β-catenin and have yet to find one that crossreacts. Because MlBcat appears to lack transactivational domains (at least as determined by sequence comparison), further experiments are necessary to determine whether it can actually function as a transcriptional activator. Attempts to activate Wnt signaling via GSK-3 inhibition (for example, with lithium chloride or alsterpaullone treatments) have not produced any obvious phenotype [Pang K, personal observation]. Functional experiments to knock down gene expression (morpholino antisense oligonucleotides or dominant negative constructs) would provide much-needed insight into whether canonical Wnt signaling is actually active in the developing embryo.
The canonical Wnt signaling pathway evolved at the base of the animal tree of life. We searched through the genome of the ctenophore Mnemiopsis leidyi, and identified most of the components of this well-known developmental signaling pathway. Conspicuously absent from ctenophores is axin, a member of the 'destruction complex', which is present in all other animals. Wnt antagonists also appear to be lacking or scarce in early diverging metazoans, with Sfrp present only in ctenophores, sponges and cnidarians, and DKK present only in sponges and cnidarians, with vertebrates possessing the entire array of Wnt antagonists (Sfrp, DKK, WIF and CER). Wnt genes evolved early in animal evolution, but did not radiate and diversify until the Cnidarian-Bilaterian ancestor. However, it is also not clear if Wnt signaling has direct effects on the regulation of gene expression in ctenophores, as key transactivational domains in a downstream target of the Wnt pathway, β-catenin, appear to be absent, and pharmacological treatments that lead to the stimulation of β-catenin activity in other metazoans produce no visible phenotype with these.
Although most of the canonical Wnt pathway components are present, their mRNA expression patterns would suggest that this pathway is not involved in early axis specification in Mnemiopsis. Both the late expression patterns (after the axes have been specified) and the expression of Wnt and β-catenin at opposite poles of the embryo suggest that this pathway may not required for fate specification. The rapid development of ctenophores could imply that asymmetric segregation of maternally loaded protein, rather than zygotic gene expression, is responsible for precocious cell fate specification in these embryos. Further genomic, expression and functional analyses are necessary to determine what genes and/or determinants are involved in axis specification in this unique early diverging animal lineage. Moreover, once the Mnemiopsis axial patterning system has been deciphered, it will become increasingly important to reach a consensus regarding the branching position of Ctenophora relative to other early-branching metazoans to place this unique developmental program within a phylogenetic context.
Animal collection and gene expression
Mnemiopsis leidyi adults were collected (from Eel Pond or the NOAA Rock Jetty, Woods Hole, MA, USA) during the months of June and July and spawned as previously described . RNA was extracted from embryos at regular intervals from fertilization to 36 hours (TRI Reagent; Molecular Research Center, Cincinnati, OH, USA) . RNA was reverse transcribed to generate cDNA (SMART RACE cDNA Amplification Kit; BD Biosciences, San Jose, CA, USA). This cDNA was used as template to isolate the genes of interest. The following genes were isolated and fully sequenced, and are described in this paper: MlWntA (HM448813), MlWnt6 (HM448814), MlWnt9 (HM448815), MlWntX (HM448816), MlBcat (HM448817), MlDsh (HM448818), MlFzdA (HM448819), MlFzdB (HM448820), MlSfrp (HM448821) and MlTcf (HM448822). Additionally sequences were isolated for MlPygopus (HM448823), MlChibby (HM448824), MlPorc (HM448825) and MlDIXD (HM448826).
For whole-mount in situ hybridization, embryos were fixed at various stages from freshly collected uncleaved embryos (0 HPF) to cydippids (24 to 36 HPF). They were stored in methanol at -20°C until used. Digoxygenin-labeled riboprobes (0.1 ng/ul) (Ambion/Applied Biosystems, Austin, TX, USA) were hybridized for 48 hours at 60°C, and detected using an alkaline phosphatase-conjugated antibody (Roche Applied Science, Indianapolis, IN, USA) and the colorimetric substrate nitro-blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) . After detection, specimens were washed with phosphate-buffered saline and transferred through a glycerol series up to 70% glycerol. They were then mounted, viewed under a compound microscope (Zeiss Axioskop 2, Jena, Germany), and imaged using a digital imaging system (AxioCam HRc with Axiovision software; Zeiss). Color balance and brightness were adjusted using Photoshop software (Adobe Systems Incorporated, San Jose, CA, USA). The only modification to the in situ protocol was a change in acetic anhydride treatment (treated in 0.1 mol/L triethanolamine rather than 1% w/v) (for most recently updated protocols, contact the authors). All in situ images presented here and additional developmental stages and/or views, are available online via the comparative gene expression database, Kahikai http://www.kahikai.com.
Genome sequencing and searches
Mnemiopsis genomic DNA was collected from the self-fertilized spawning of two separate adult animals. One pool of genomic DNA was used to construct a library for 454 sequencing and the other used for Illumina paired-end sequencing. The 454 sequencing resulted in 8.1 million reads (2.7 Gb), which were assembled into contigs using the Phusion assembler . The Illumina run resulted in 2.8 million paired end reads, which combined with the 454 data, was used to generate 5,100 scaffolds (scaffold N50 of 187 kb), resulting in a total coverage of ~50×.
The Mnemiopsis genome was scanned in silico for genes of interest using a reciprocal BLAST approach. Human, frog, Drosophila and Nematostella orthologs were used as queries for TBLASTN searches. Candidate matches were then used in BLASTP searches of the human genome to find the closest hit. If the closest match was not the original ortholog or if the E-value was greater than 0.001, then it was coded as being absent from the genome. A gene model was created by scanning the genomic region using Genscan . This predicted protein sequence was then searched for conserved Pfam domains using SMART . For certain genes of interest, gene-specific primers were designed for RACE PCR (MacVector, Cary, NC, USA). RACE PCR fragments were then conceptually spliced and aligned back to genomic contigs for comparison of exon-intron boundaries, using Sequencher (Gene Codes, Ann Arbor, MI, USA).
The Mnemiopsis predicted amino acid sequences were aligned with the sequences of other organisms. The predicted domains or regions of interest were trimmed and aligned using Muscle, then corrected by hand for alignment errors (see Additional file 3, Additional file 4). Bayesian phylogenetic analyses were performed using MrBayes 3.1.2  using the 'mixed' amino acid model with four independent runs of 5 million generations each, sampled every 100 generations with four chains. A summary consensus tree was produced in MrBayes from the last 49,000 trees of each run (196,000 trees in total), representing 4,900,000 stationary generations. Posterior probabilities were calculated from this consensus. Maximum likelihood analyses were performed using PhyML , using the WAG model with 1000 bootstraps. Alignments and nexus files are available upon request.
This work was supported by an NSF Graduate Research Fellowship to KP, NASA and NSF grants to MQM, and the Intramural Research Program of the National Human Genome Research Institute (National Institutes of Health). We thank Alice Young, Brian Schmidt, Natalie Gurson, Richelle Legaspi and Betsy Novotny, who were involved with the Mnemiopsis genomic sequencing at NISC. This manuscript was greatly improved due to the many discussions and comments from members of the Martindale Lab. Special thanks to William E. Browne and Eric Roettinger for images in Figure 1A, and the Embryology Course at the Marine Biological Laboratory (Woods Hole, MA) for summer research space.
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