David K. Simmons

The genome of the ctenophore Mnemiopsis leidyi and its implications for cell type evolution

Introduction An understanding of ctenophore biology is critical for reconstructing events that occurred early in animal evolution. The phylogenetic relationship of ctenophores (comb jellies) to other animals has been a source of long-standing debate. Until recently, it was thought that Porifera (sponges) was the earliest diverging animal lineage, but recent reports have instead suggested Ctenophora as the earliest diverging animal lineage. Because ctenophores share some of the same complex cell types with bilaterians (such as neural and mesodermal cells), the phylogenetic position of ctenophores affects how we think about the early evolution of these cell types. The phylogenetic position of the ctenophore Mnemiopsis leidyi and its implications regarding the origin of mesodermal cell types. (A) Adult M. leidyi. (B) Summary of the relationships of the five main branches of animals and the outgroup Choanoflagellata

Genomic organization, evolution, and expression of photoprotein and opsin genes in Mnemiopsis leidyi: a new view of ctenophore photocytes

BackgroundCalcium-activated photoproteins are luciferase variants found in photocyte cells of bioluminescent jellyfish (Phylum Cnidaria) and comb jellies (Phylum Ctenophora). The complete genomic sequence from the ctenophore Mnemiopsis leidyi, a representative of the earliest branch of animals that emit light, provided an opportunity to examine the genome of an organism that uses this class of luciferase for bioluminescence and to look for genes involved in light reception. To determine when photoprotein genes first arose, we examined the genomic sequence from other early-branching taxa. We combined our genomic survey with gene trees, developmental expression patterns, and functional protein assays of photoproteins and opsins to provide a comprehensive view of light production and light reception in Mnemiopsis.ResultsThe Mnemiopsis genome has 10 full-length photoprotein genes situated within two genomic clusters with high sequence conservation that are maintained due to strong purifying selection and concerted evolution. Photoprotein-like genes were also identified in the genomes of the non-luminescent sponge Amphimedon queenslandica and the non-luminescent cnidarian Nematostella vectensis, and phylogenomic analysis demonstrated that photoprotein genes arose at the base of all animals. Photoprotein gene expression in Mnemiopsis embryos begins during gastrulation in migrating precursors to photocytes and persists throughout development in the canals where photocytes reside. We identified three putative opsin genes in the Mnemiopsis genome and show that they do not group with well-known bilaterian opsin subfamilies. Interestingly, photoprotein transcripts are co-expressed with two of the putative opsins in developing photocytes. Opsin expression is also seen in the apical sensory organ. We present evidence that one opsin functions as a photopigment in vitro, absorbing light at wavelengths that overlap with peak photoprotein light emission, raising the hypothesis that light production and light reception may be functionally connected in ctenophore photocytes. We also present genomic evidence of a complete ciliary phototransduction cascade in Mnemiopsis.ConclusionsThis study elucidates the genomic organization, evolutionary history, and developmental expression of photoprotein and opsin genes in the ctenophore Mnemiopsis leidyi, introduces a novel dual role for ctenophore photocytes in both bioluminescence and phototransduction, and raises the possibility that light production and light reception are linked in this early-branching non-bilaterian animal.

Lim homeobox genes in the Ctenophore Mnemiopsis leidyi: the evolution of neural cell type specification.

BackgroundNervous systems are thought to be important to the evolutionary success and diversification of metazoans, yet little is known about the origin of simple nervous systems at the base of the animal tree. Recent data suggest that ctenophores, a group of macroscopic pelagic marine invertebrates, are the most ancient group of animals that possess a definitive nervous system consisting of a distributed nerve net and an apical statocyst. This study reports on details of the evolution of the neural cell type specifying transcription factor family of LIM homeobox containing genes (Lhx), which have highly conserved functions in neural specification in bilaterian animals.ResultsUsing next generation sequencing, the first draft of the genome of the ctenophore Mnemiopsis leidyi has been generated. The Lhx genes in all animals are represented by seven subfamilies (Lhx1/5, Lhx3/4, Lmx, Islet, Lhx2/9, Lhx6/8, and LMO) of which four were found to be represented in the ctenophore lineage (Lhx1/5, Lhx3/4, Lmx, and Islet). Interestingly, the ctenophore Lhx gene complement is more similar to the sponge complement (sponges do not possess neurons) than to either the cnidarian-bilaterian or placozoan Lhx complements. Using whole mount in situ hybridization, the Lhx gene expression patterns were examined and found to be expressed around the blastopore and in cells that give rise to the apical organ and putative neural sensory cells.ConclusionThis research gives us a first look at neural cell type specification in the ctenophore M. leidyi. Within M. leidyi, Lhx genes are expressed in overlapping domains within proposed neural cellular and sensory cell territories. These data suggest that Lhx genes likely played a conserved role in the patterning of sensory cells in the ancestor of sponges and ctenophores, and may provide a link to the expression of Lhx orthologs in sponge larval photoreceptive cells. Lhx genes were later co-opted into patterning more diversified complements of neural and non-neural cell types in later evolving animals.

Expression of multiple Sox genes through embryonic development in the ctenophore Mnemiopsis leidyi is spatially restricted to zones of cell proliferation

BackgroundThe Sox genes, a family of transcription factors characterized by the presence of a high mobility group (HMG) box domain, are among the central groups of developmental regulators in the animal kingdom. They are indispensable in progenitor cell fate determination, and various Sox family members are involved in managing the critical balance between stem cells and differentiating cells. There are 20 mammalian Sox genes that are divided into five major groups (B, C, D, E, and F). True Sox genes have been identified in all animal lineages but not outside Metazoa, indicating that this gene family arose at the origin of the animals. Whole-genome sequencing of the lobate ctenophore Mnemiopsis leidyi allowed us to examine the full complement and expression of the Sox gene family in this early-branching animal lineage.ResultsOur phylogenetic analyses of the Sox gene family were generally in agreement with previous studies and placed five of the six Mnemiopsis Sox genes into one of the major Sox groups: SoxB (MleSox1), SoxC (MleSox2), SoxE (MleSox3, MleSox4), and SoxF (MleSox5), with one unclassified gene (MleSox6). We investigated the expression of five out of six Mnemiopsis Sox genes during early development. Expression patterns determined through in situ hybridization generally revealed spatially restricted Sox expression patterns in somatic cells within zones of cell proliferation, as determined by EdU staining. These zones were located in the apical sense organ, upper tentacle bulbs, and developing comb rows in Mnemiopsis, and coincide with similar zones identified in the cydippid ctenophore Pleurobrachia.ConclusionsOur results are consistent with the established role of multiple Sox genes in the maintenance of stem cell pools. Both similarities and differences in juvenile cydippid stage expression patterns between Mnemiopsis Sox genes and their orthologs from Pleurobrachia highlight the importance of using multiple species to characterize the evolution of development within a given phylum. In light of recent phylogenetic evidence that Ctenophora is the earliest-branching animal lineage, our results are consistent with the hypothesis that the ancient primary function of Sox family genes was to regulate the maintenance of stem cells and function in cell fate determination.

Expanded Functional Diversity of Shaker K + Channels in Cnidarians Is Driven by Gene Expansion

The genome of the cnidarian Nematostella vectensis (starlet sea anemone) provides a molecular genetic view into the first nervous systems, which appeared in a late common ancestor of cnidarians and bilaterians. Nematostella has a surprisingly large and diverse set of neuronal signaling genes including paralogs of most neuronal signaling molecules found in higher metazoans. Several ion channel gene families are highly expanded in the sea anemone, including three subfamilies of the Shaker K+ channel gene family: Shaker (Kv1), Shaw (Kv3) and Shal (Kv4). In order to better understand the physiological significance of these voltage-gated K+ channel expansions, we analyzed the function of 18 members of the 20 gene Shaker subfamily in Nematostella. Six of the Nematostella Shaker genes express functional homotetrameric K+ channels in vitro. These include functional orthologs of bilaterian Shakers and channels with an unusually high threshold for voltage activation. We identified 11 Nematostella Shaker genes with a distinct “silent” or “regulatory” phenotype; these encode subunits that function only in heteromeric channels and serve to further diversify Nematostella Shaker channel gating properties. Subunits with the regulatory phenotype have not previously been found in the Shaker subfamily, but have evolved independently in the Shab (Kv2) family in vertebrates and the Shal family in a cnidarian. Phylogenetic analysis indicates that regulatory subunits were present in ancestral cnidarians, but have continued to diversity at a high rate after the split between anthozoans and hydrozoans. Comparison of Shaker family gene complements from diverse metazoan species reveals frequent, large scale duplication has produced highly unique sets of Shaker channels in the major metazoan lineages.

Ether-à-go-go family voltage-gated K + channels evolved in an ancestral metazoan and functionally diversified in a cnidarian- bilaterian ancestor

We examined the evolutionary origins of the ether-à-go-go (EAG) family of voltage-gated K+ channels, which have a strong influence on the excitability of neurons. The bilaterian EAG family comprises three gene subfamilies (Eag, Erg and Elk) distinguished by sequence conservation and functional properties. Searches of genome sequence indicate that EAG channels are metazoan specific, appearing first in ctenophores. However, phylogenetic analysis including two EAG family channels from the ctenophore Mnemiopsis leidyi indicates that the diversification of the Eag, Erg and Elk gene subfamilies occurred in a cnidarian/bilaterian ancestor after divergence from ctenophores. Erg channel function is highly conserved between cnidarians and mammals. Here we show that Eag and Elk channels from the sea anemone Nematostella vectensis (NvEag and NvElk) also share high functional conservation with mammalian channels. NvEag, like bilaterian Eag channels, has rapid kinetics, whereas NvElk activates at extremely hyperpolarized voltages, which is characteristic of Elk channels. Potent inhibition of voltage activation by extracellular protons is conserved between mammalian and Nematostella EAG channels. However, characteristic inhibition of voltage activation by Mg2+ in Eag channels and Ca2+ in Erg channels is reduced in Nematostella because of mutation of a highly conserved aspartate residue in the voltage sensor. This mutation may preserve sub-threshold activation of Nematostella Eag and Erg channels in a high divalent cation environment. mRNA in situ hybridization of EAG channels in Nematostella suggests that they are differentially expressed in distinct cell types. Most notable is the expression of NvEag in cnidocytes, a cnidarian-specific stinging cell thought to be a neuronal subtype.

Major diversification of voltage-gated K+ channels occurred in ancestral parahoxozoans

Significance We examined the origin and evolution of two major families of voltage-gated K+ channels, Shaker and KCNQ, which regulate action potential repolarization, patterning, and threshold. Shaker family channels evolved in a basal metazoan ancestor of ctenophores and parahoxozoans (including cnidarians and bilaterians), but functional diversification of the Shaker family and the emergence of the KCNQ family occurred specifically within the parahoxozoan lineage. Our results suggest that many major innovations in the regulation of cellular excitability by voltage-gated K+ channels are unique to parahoxozoans and that these innovations occurred before the divergence of cnidarians and bilaterians. Ctenophores and sponges separated prior to this burst of innovation and thus either lack major mechanisms for action potential regulation or evolved such mechanisms independently. We examined the origins and functional evolution of the Shaker and KCNQ families of voltage-gated K+ channels to better understand how neuronal excitability evolved. In bilaterians, the Shaker family consists of four functionally distinct gene families (Shaker, Shab, Shal, and Shaw) that share a subunit structure consisting of a voltage-gated K+ channel motif coupled to a cytoplasmic domain that mediates subfamily-exclusive assembly (T1). We traced the origin of this unique Shaker subunit structure to a common ancestor of ctenophores and parahoxozoans (cnidarians, bilaterians, and placozoans). Thus, the Shaker family is metazoan specific but is likely to have evolved in a basal metazoan. Phylogenetic analysis suggested that the Shaker subfamily could predate the divergence of ctenophores and parahoxozoans, but that the Shab, Shal, and Shaw subfamilies are parahoxozoan specific. In support of this, putative ctenophore Shaker subfamily channel subunits coassembled with cnidarian and mouse Shaker subunits, but not with cnidarian Shab, Shal, or Shaw subunits. The KCNQ family, which has a distinct subunit structure, also appears solely within the parahoxozoan lineage. Functional analysis indicated that the characteristic properties of Shaker, Shab, Shal, Shaw, and KCNQ currents evolved before the divergence of cnidarians and bilaterians. These results show that a major diversification of voltage-gated K+ channels occurred in ancestral parahoxozoans and imply that many fundamental mechanisms for the regulation of action potential propagation evolved at this time. Our results further suggest that there are likely to be substantial differences in the regulation of neuronal excitability between ctenophores and parahoxozoans.

Cas9-mediated excision of Nematostella brachyury disrupts endoderm development, pharynx formation and oral-aboral patterning

ABSTRACT The mesoderm is a key novelty in animal evolution, although we understand little of how the mesoderm arose. brachyury, the founding member of the T-box gene family, is a key gene in chordate mesoderm development. However, the brachyury gene was present in the common ancestor of fungi and animals long before mesoderm appeared. To explore ancestral roles of brachyury prior to the evolution of definitive mesoderm, we excised the gene using CRISPR/Cas9 in the diploblastic cnidarian Nematostella vectensis. Nvbrachyury is normally expressed in precursors of the pharynx, which separates endoderm from ectoderm. In knockout embryos, the pharynx does not form, embryos fail to elongate, and endoderm organization, ectodermal cell polarity and patterning along the oral-aboral axis are disrupted. Expression of many genes both inside and outside the Nvbrachyury expression domain is affected, including downregulation of Wnt genes at the oral pole. Our results point to an ancient role for brachyury in morphogenesis, cell polarity and the patterning of both ectodermal and endodermal derivatives along the primary body axis. Summary: Brachyury is a key regulator of mesoderm, but is also found in diploblasts, which lack this tissue layer. Knockout experiments reveal its widespread effects on the endomesodermal gene regulatory network.

Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

Significance We report the characterization of two novel glutamate receptor subunits from recently sequenced ctenophore genomes. The origin of vertebrate NMDA subtype ionotropic glutamate receptors (iGluRs), which play a major role in synaptic plasticity and which require both glutamate and glycine for activation of ion channel gating, is not well understood. Using X-ray crystallography of the ligand binding domains of iGluRs from the comb jelly Mnemiopsis leidyi and the sea gooseberry Pleurobrachia bachei, candidates for the earliest lineage metazoans, we discovered that a large subset of these iGluR subunits form glycine receptors. Similarities to modern-day NMDA receptors suggest NMDA and ctenophore receptors may provide clues to the role of iGluRs in the evolution of neural systems in metazoa. Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.

Antagonistic BMP–cWNT signaling in the cnidarian Nematostella vectensis reveals insight into the evolution of mesoderm

Significance Definitive mesoderm (e.g., muscle cells) evolved in the ancestor of the bilateria but is not present in their sister group, Cnidaria. Forward transcriptomics and gene knockdown in the anthozoan Nematostella vectensis show that both cWnt and BMP 2/4 signaling pathways reciprocally regulate components of the endomesodermal gene regulatory network (GRN) during development to set up distinct regional territories prior to the onset of gastrulation. Furthermore, the conserved “kernel” of the bilaterian heart mesoderm GRN is operational in the endomesoderm of the anthozoan N. vectensis. This GRN fails to have a “lockdown” feedback loop found in bilaterian GRNs that may help explain the highly regenerative potential of adult cnidarian endomesoderm, suggesting that the endoderm and mesoderm arose from the bifunctional endomesoderm. Gastrulation was arguably the key evolutionary innovation that enabled metazoan diversification, leading to the formation of distinct germ layers and specialized tissues. Differential gene expression specifying cell fate is governed by the inputs of intracellular and/or extracellular signals. Beta-catenin/Tcf and the TGF-beta bone morphogenetic protein (BMP) provide critical molecular signaling inputs during germ layer specification in bilaterian metazoans, but there has been no direct experimental evidence for a specific role for BMP signaling during endomesoderm specification in the early branching metazoan Nematostella vectensis (an anthozoan cnidarian). Using forward transcriptomics, we show that beta-catenin/Tcf signaling and BMP2/4 signaling provide differential inputs into the cnidarian endomesodermal gene regulatory network (GRN) at the onset of gastrulation (24 h postfertilization) in N. vectensis. Surprisingly, beta-catenin/Tcf signaling and BMP2/4 signaling regulate a subset of common downstream target genes in the GRN in opposite ways, leading to the spatial and temporal differentiation of fields of cells in the developing embryo. Thus, we show that regulatory interactions between beta-catenin/Tcf signaling and BMP2/4 signaling are required for the specification and determination of different embryonic regions and the patterning of the oral–aboral axis in Nematostella. We also show functionally that the conserved “kernel” of the bilaterian heart mesoderm GRN is operational in N. vectensis, which reinforces the hypothesis that the endoderm and mesoderm in triploblastic bilaterians evolved from the bifunctional endomesoderm (gastrodermis) of a diploblastic ancestor, and that slow rhythmic contractions might have been one of the earliest functions of mesodermal tissue.