The ctenophore genome and the evolutionary origins of neural systems

Abstract

The origins of neural systems remain unresolved. In contrast to other basal metazoans, ctenophores (comb jellies) have both complex nervous and mesoderm-derived muscular systems. These holoplanktonic predators also have sophisticated ciliated locomotion, behaviour and distinct development. Here we present the draft genome of Pleurobrachia bachei, Pacific sea gooseberry, together with ten other ctenophore transcriptomes, and show that they are remarkably distinct from other animal genomes in their content of neurogenic, immune and developmental genes. Our integrative analyses place Ctenophora as the earliest lineage within Metazoa. This hypothesis is supported by comparative analysis of multiple gene families, including the apparent absence of HOX genes, canonical microRNA machinery, and reduced immune complement in ctenophores. Although two distinct nervous systems are well recognized in ctenophores, many bilaterian neuron-specific genes and genes of 'classical' neurotransmitter pathways either are absent or, if present, are not expressed in neurons. Our metabolomic and physiological data are consistent with the hypothesis that ctenophore neural systems, and possibly muscle specification, evolved independently from those in other animals.

Extended Data Figure 1

a–e, Anatomy of the ctenophore, Pleurobrachia bachei A. Agassiz, 1860. Natural coloration of the major organs in live animal are shown. a, Details of the transparent Pleurobrachia body are shown including, b, the pharynx and tentacle sheaths (pockets). Eight rows of comb plates, called ctenes, are made of giant compound cilia that diffract light – creating iridescence. c, Combs rows in Pleurobrachia are constantly beating. The mouth and the aboral organ (AO) are located at the opposite poles of the animal (a, c). The AO controls complex coordinated behaviors of the animal; d, Ciliated furrows connect the AO and the ctenes to mediate behavior. e, Tentacles have numerous contractile tentillae used to capture food with specialized glue cells or colloblasts (See also Fig. 1e of the main text). f–h, Pleurobrachia neural nets and muscles. f, Comb plate muscles (red) were revealed using in situ hybridization for β-tubulin and subepithelial neural net (green) revealed by tyrosinated α-tubulin immunostaining. g, In this image comb cilia (green) were stained using tyrosinated α-tubulin antibodies (green) where as underlying comb plate muscles were visualized by phalloidin (a muscle marker) that did not stain neurons. h, Organization of the subepithelial neural net around the Mouth as revealed by tyrosinated α-tubulin antibodies (whole mount preparation). Scale: 120 μm (f); 100 μm (g); 200 μm (h). See Supplementary Methods SM10 and SM11.

Extended Data Figure 2. DNA methylation and active DNA demethylation in Pleurobrachia bachei

CpG DNA methylation facilitates the elimination of CpG dinucleotides over evolutionary time a, Histogram shows relative occurrences of different dinucleotides in genomes of P. bachei (red bars), Drosophila melanogaster (green bars, no DNA methylation) and Homo sapiens (blue bars). The P. bachei genome contains 2.3 % CpG dinucleotides, which is much lower than the expected random frequency and, therefore, indicative of a genome that undergoes methylation compared to humans. b, DNMT genealogy tree. The enzyme DNA methyltransferase (DNMT), which catalyzes transfer of a methyl group to DNA to form 5- methyl cytosine (5-mC)¹⁴⁷, is present in Pleurobrachia. c, TET family of enzymes catalyzes active DNA demethylation via formation of 5-hydroxymethyl cytosine (5-hmC, the 6^th DNA base). RNA-seq profiling reveals differential expression for DNMT and TET-like genes during development and in adult P. bachei. Both DNMT and TET-like genes are predominantly expressed during cleavage starting from the 1^st division. However, the TET-like gene is also highly expressed in adult combs (asterisk). Y-axis shows a normalized expression level for each transcript. d, ELIZA based colorimetric assays validate the presence of both 5-mC and 5-hmC in the P. bachei genome (the rat brain is used as a positive control; n=6 for Pleurobrachia and n=3 for rat; data shown as mean ± s.e.m, see the Supplementary Methods SM8 and Supplementary Data section SD3 for details).

Extended Data Figure 3

a, Phylogeny of Metazoa based on 586 genes. Topology inferred using RAxML 7.2.7 and maximum likelihood (ML) with the CAT +WAG + F model with all taxa from the Supplementary Table 12S. Bootstrap support values are listed at each node. Color coding: purple –Ctenophora yellow – Porifera, pink – Cnidaria, light blue – Bilateria. b, Removal of fast-evolving taxa Trichoplax and Caenorhabditis improves topological robustness. Topology inferred using RAxML 7.2.7 and maximum likelihood (ML) with the CAT +WAG + F model with all taxa from Supplementary Table 12S except Trichoplax and Caenorhabditis. Bootstrap support values are listed at each node. c, Removal of distant out-groups such as Fungi and Filasterea further improves topological robustness. Topology inferred using RAxML 7.2.7 using maximum likelihood (ML) with the CAT +WAG + F model with all taxa from Supplementary Table 12S except Trichoplax, Caenorhabditis, and non-choanoflagellate outgroups. Bootstrap support values are listed at each node. d, Analysis with improved ctenophore taxon sampling based on 114 genes. Topology inferred using RAxML 7.2.7 using maximum likelihood (ML) with the CAT +WAG + F model with all taxa from Supplementary Table 13S. Bootstrap support values are listed at each node.

Extended Data Figure 4

a, Identification of tentacle-specific transcripts. The left photo shows scanning electron micrograph of a Pleurobrachia tentacle with two branching tentillae densely covered with hundreds of colloblasts or glue cells. Comparative transcriptome (RNA-seq) profiling among major organs allowed us to identify several dozen genes differentially or uniquely expressed in tentacles. The histogram shows illustrative examples of some of these genes with a normalized expression level (Y-axis) for each represented transcript. One of these Pleurobrachia-specific genes we named Tentillin (green arrow). In situ hybridization experiments (n=9) revealed a remarkable cell-specificity expression patterns for Tentillin in all main tentacle branches and tentillae, possible labeling colloblasts or associated secretory cells. b, Identification of comb-specific transcripts. The left photo shows a microscopic image of one comb row from an intact animal. The natural coloration is a reflection of the beautiful iridescence patterns produced from large cilia forming combs. Comparative transcriptome (RNA-seq) profiling among major organs allowed us to identify several hundreds of genes differentially or uniquely expressed in combs. The histogram shows illustrative examples of some of these genes with a normalized expression level (Y-axis) for each represented transcript (see Supplementary Methods S4.2.3.6, S4.2.3.7 and SM10, all sequences used in the analysis can be found in Supplementary Tables 29S, 30S and 32S).

Extended Data Figure 5

a, b, Dicer and Argonaut, are predominatly expressed in structures associated to sensory and integrative functions. These include the aboral organ, polar fields and combs. Note, a relatively weak staining of other cell types in the skin and following ciliated furrows in Dicer and Argonaut preparations (top two images). c, d, Pleurobrachia ELAV is Expressed in Combs and not in neurons. ELAVs are RNA binding proteins and they are considered as pan-neuronal markers (see Supplementary Data SD5.6.1). However, in Pleurobrachia ELAVs’ expression has not been detected in neural tissues or cells with recognizable neuronal like appearances. In situ hybridization for Pleurobrachia ELAV3 (c–d) shows the highest levels of expression in the adult comb plate but not in any of the neural tissues or organs enriched with neurons such as the aboral organ and polar fields. e, WntX is selectively expressed in the aboral organ (AO) and major conductive pathways of Pleurobrachia suggesting its involvements in integrative and neural-like functions (in situ hybridization on a whole-mount preparation). A. One of the highest WntX expressions is found in AO and ciliated furrows whereas the polar fields showed a moderate expression level associated to their central regions. In situ hybridization was performed on whole mounts using DIG labeled probes (see details in the Supplementary Methods, all in situ hybridization were performed at least on 4–5 different animals and these are representative photos for these experiments). Scales: 500 μm (a–d). 800 μm (e).

Extended Data Figure 6

a–c, Absence of Serotonin in ctenophores. Here, we used nanoliter volume sampling, capillary electrophoresis separation, and wavelength-resolved native fluorescence detection as described for ultra-sensitive assay of 5-hydroxytryptamine (serotonin or 5-HT) and related metabolites (a, the top electropherogram and the table with standards used). Limits of detection (LODs) range from the low attomole to the femtomole range, with 5-HT LODs being approximately 20–50 attomoles. b, Using this assay we failed to detect 5-HT in Pleurobrachia (bottom left, n=6) but c, 5-HT was reliable detected in the hemichordate Saccoglossus (bottom right) and molluscs. See details in the Supplementary Methods SM17 and Supplementary Table 22S for quantification.

Extended Data Figure 7

a, The Ionotropic glutamate receptors (iGluRs) are diverse and underwent substantial adaptive radiation within the Ctenophora lineage. Phylogenetic analysis shows Pleurobrachia iGluRs share highest identity to each other forming a distinct branch on the tree topology (Supplementary Data SD5.9). b, Differential expressed of iGluR subtypes in Pleurobrachia bachei (red and green labeling with fluorescent in situ hybridization protocols). Dark blue fluorescence is DAPI nuclear staining. Aboral organ –AO. Scale 100 μm (b1-2), 60 μm (b3), 50 μm (b4), 30 μm (b5), 200 μm (b6). c–f, Glutamate induced action potentials and currents in muscle cells. c, Typical responses of ctenophore muscle cells to glutamate pulses recorded extracellulary (as individual action potentials/contractions from a single muscle cell in response to local application of Glutamate, 1mM), and d, from the same cell in whole-cell current clamp mode with clear action potentials. e, Isolated muscle cell. Scale 25μm. f, Glutamate activated whole-cell currents recorded from the same cell (as in c). Time course of application is depicted by the diagram below the voltage signal. Two responses (inward current) are shown. The holding potential was −70mV (Supplementary Methods SM13-16). g, Representative electropherograms show capillary electrophoresis separation with laser induced fluorescence detection from different organs in Pleurobrachia bachei (n=5) for transmitter candidate identification. The bottom electropherograms are standards (Supplementary Methods SM17 and Supplementary Tables 23–25S for quantification).

Extended Data Figure 8

a, Computational pipeline for prediction of secretory products in Pleurobrachia and the overview of secretory products predicted from the Pleurobrachia gene models (Supplementary Method and Data sections SM4.2.3.7 and SD5.11, respectively). b, Expression of novel secretory molecules in Ctenophores (DIG-labeled in situ hybridization, Supplementary Data SD5.11 and Methods SM 10). Each of predicted secretory prohormone was selected based upon its unique and/or highly differential expression pattern as revealed by RNA-seq profiling. Ctenophorin is uniquely expressed in polarized cells around the mouth of Pleurobrachia and we found its homologs in all ctenophore species we sequenced- here is its name. Tentillin is a Plerobrachia-specific gene, which is uniquely expressed in polarized secretory-like cells in tentillae and tentacles. Jansonin’s expression is primarily restricted to polarized cells located in the aboral organ and polar fields. b4, For comparison, we showed different but also cell-specific expression pattern of BarX Transcription Factor in cells of unknown identify localized in polar fields, comb plates and tentacles. c–d, Majority of predicted secretory products are expressed later in development and in adult organs of Pleurobrachia (RNA-seq). c, Expression patterns of 72 predicted prohormones in P. bachei indicates that 20 of them are present and differentially expressed in development (Supplementary Table 32S for all Pleurobrachia precursor sequences). Surprisingly 5 of these precursor mRNAs were found starting from the 2^nd cleavage stage whereas the rest are predominantly expressed on day 3 of development. This correlates to the first appearance of neurons in Pleurobrachia cydippid larva (see Supplementary Data SD5.11 and Supplementary Method section S4.2.3.6 for the RNA-seq analysis).

Extended Data Figure 9

a, Metazoan Ion Channel Complement. The 112 ion channels identified in the Pleurobrachia genome are classified as voltage gated (v) or other gating such as second messengers. Receptors channels (R) are ligand-gated or ionotropic (iGluR, ChRN, HTR3, GABA and CLR) and indicated in the grey. Metazoan novelties indicate type of ion channels absent in the choanoflagellates, the sister group to all animals. Colored squares show channels: (i) primarily absent in Ctenophores (pink), (ii) secondarily lost in sponges or placozoans (dark yellow), (iii) eumetazoan (Cnidaria+Bilareria) innovations (blue), or (iv) examples of expansion of certain class of channels in some animal lineages (red). All Pleurobrachia sequences used in the analysis can be found in Supplementary Table 31S. b, Ion Channels are Predominantly Expressed in Tentacles, Combs and Aboral Organ. Hierarchical Clustering of 112 identified ion channels in developmental stages and adult tissues of Pleurobrachia. Adult organs involved in food capture and ciliated locomotion and integrative functions show significantly higher diversity and overall higher level of expression levels for most of ion channel types. Mobile tentacles had the highest expression of voltage gated channels, in particular Ca_v and Na_v. The legend shows relative expression levels based on RNA-seq data (see Supplementary Methods S4.2.3.6).

Extended Data Figure 10. Two alternative scenario of neuronal evolution

a, Single origin of the neural system (Monophyly) with possible loss of some neural molecular components in Ctenophores as well as the possible secondarily loss of the entire nervous systems in sponges and placozoans; b, Multiple origins of neurons in animals as introduced and supported by this manuscript (see main text discussion section for details).

Figure 1. Ctenophores and their innovations

a, The sea gooseberry, Pleurobrachia bachei (Fig. 1S) was selected as a target for genome sequencing due to preservation of traits ancestral for this lineage and since in situ hybridization/immunolabeling is possible. b–d: Major ctenophore innovations. b, Nervous system revealed by tyrosinated α-tubulin immunolabeling; c, Scanning electron microscopy (SEM) imaging of nerve net in a tentacle pocket (scale:15μm). d, Locomotory ciliated combs (SEM, scale:100μm). e, Glue-secreting cells – Colloblasts in tentacles (SEM, scale:50μm). f, Relationships among major animal clades with Choanoflagellates sister to all Metazoa.

Figure 2. Phylogenomic reconstruction among major ctenophore lineages

Cydippid (Euplokamis, Pleurobrachia, Dryodora, and Mertensiidae) and lobate (Beroe, Mnemiopsis and Bolinopsis) ctenophores were polyphyletic, suggesting independent loss of both cydippid larval stage and tentacle apparatus as well as independent development of bilateral symmetry in benthic/aberrant ctenophores, Vallicula and Coeloplana (Supplementary_Data SD4).

Figure 3. Gene gain and gene loss in ctenophores

a, Predicted scope of gene loss (blue numbers – e.g.–4,952 in Placozoa) from the common metazoan ancestor. Red and green numbers indicate genes shared between bilaterians and ctenophores (7,771), as well as between ctenophores and other eukaryotic lineages sister to animals, respectively. Text on tree indicates emergence of complex animal traits and gene families. b, Uniquely shared and lineage-specific genes among basal metazoans. Values under species names indicate number of genes (*) without any recognized homologs (e-value is 10⁻⁴) vs the total number of predicted gene models in are relevant species (Supplementary Table_14bS).

Figure 4. Nature of Ctenophore Innovations

a, Main developmental stages in Pleurobrachia from eggs to the cleavage (2–64 cells), gastrulation (1–3 hrs) and formation of cydippid larvae (~24 hours). b, Hierarchical clustering of approximately 400 ctenophore-specific genes differentially expressed among different development stages and adult structures as revealed by RNA-seq experiments. Color index as follows: black indicates highest level of expression, followed by purple, red then down to white indicating no expression. Most of these ctenophore-specific genes are primarily expressed during 4–32 cell stages (asterisks). The red circle indicates a subset of novel genes uniquely expressed in combs, tentacles and the aboral organ. These genes lack recognized homologs in other organisms. c, Diversity and differential expression of RNA editing genes in Pleurobrachia development and adult tissues (RNA-seq). ADAR1 has highest expression level in early cleavage stages while ADAR2-3 and ADAT1-2 are most abundant in the combs. d, Morphological appearance of neurons during 3^rd day of development (the top insert, neuronal cell bodies are stained tyrosinated α-tubulin antibodies, red arrows) correlates with abundant expression of multiple iGluR receptors suggesting that Glutamate plays an important role as an intercellular messenger. Muscles formed well before neuronal differentiation at end of 1^st day of development (the bottom insert, phalloidin staining, yellow arrow); white arrow points to the embryonic mouth with hundreds of cilia inside. In c and d expression levels of RNA editing or iGluRs genes shown as a normalized frequency of sequence reads for a given transcript from all RNA-seq data for each developmental stage (Supplementary Methods).

Figure 5. Emergence of Neural Organization in Pleurobrachia

a, Two neural nets in Pleurobrachia as revealed by tyrosinated α-tubulin immunostaining. Top image shows subepithelial net with concentrations of neuronal elements in the polar fields and ciliated furrows, known as structures involved in sensory and motor functions respectively (blue arrow in right insert indicates location of a neuronal somata with individual neurites marked by red arrows). The bottom image shows neurons of mesogloeal net (arrows are neuronal somatas; arrowheads are neuronal processes). Note, phalloidin (a muscle marker) did not stain these cells. Scale: 120μm (top); 10μm (bottom images). b, L- glutamate (10⁻⁷–10⁻³ M) induced action potentials in muscle cells whereas other transmitter candidates were ineffective even at concentrations up to 5mM. Typical responses of ctenophore muscle cells to local pulses of a transmitter application were recorded both as individual action potentials (whole-cell current-clamp mode) and video contractions from a single muscle cell. The graph shows normalized responses from the same muscle cell indicating L-glutamate is the most potential excitatory molecule compared to D-glutamate or L-/D-aspartate (Supplementary Methods). c, Key molecular innovations underlying neural organization in ctenophores. Bars indicate presence or relative expansions of selected gene families in all basal metazoan lineages from the inferred urmetazoan ancestor. The data suggests that sponges and placozoan never developed neural systems, or, assuming that pre-neuronal organization in the urmetazoan ancestor, sponges and placozoans lost their nervous systems. Either hypothesis point toward extensive parallel evolution of neural systems in ctenophores vs the Bilateria+Cnidaria clade. d, The aboral organ has the greatest diversity and highest expression levels of 12 gap junction proteins suggesting unmatched expansion of electrical signalling in this complex integrative organ - an analog of an elementary brain in ctenophores. Expression of different innexins shown as a summation of normalized frequencies of respective sequencing reads in RNA-seq data obtained from each developmental stage and adult tissues (Supplementary Methods).