. Author manuscript; available in PMC: 2018 Jun 13.

Published in final edited form as:Nature. 2017 Dec 13;553(7686):45–50. doi:10.1038/nature25030

Convergent evolution of bilaterian nerve cords

José M Martín-Durán^1,^#,Kevin Pang^1,^#,Aina Børve¹,Henrike Semmler Lê^1,²,Anlaug Furu¹,Johanna Taylor Cannon³,Ulf Jondelius³,Andreas Hejnol^1,^*

¹Sars Centre for Marine Molecular Biology, University of Bergen. Thørmohlensgate 55. 5006 Bergen. Norway

²Natural History Museum of Denmark, Biosystematics Section. Universitetsparken 15. DK-2100 Copenhagen. Denmark

³Naturhistoriska Riksmuseet, P.O. Box 50007, SE-104 05 Stockholm. Sweden

Correspondence and requests for materials should be addressed to AH (andreas.hejnol@uib.no).

Contributed equally.

Issue date 2018 Jan 4.

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PMCID: PMC5756474 EMSID: EMS74897 PMID:29236686

Abstract

It has been hypothesized that a condensed nervous system with a medial ventral nerve cord is an ancestral character of Bilateria. The presence of similar dorsoventral molecular patterns along the nerve cords of vertebrates, flies, and an annelid has been interpreted as support for this scenario. Whether these similarities are generally found across the diversity of bilaterian neuroanatomies is unclear, and thus the evolutionary history of the nervous system is still contentious. To assess the conservation of the dorsoventral nerve cord patterning, we studied representatives of Xenacoelomorpha, Rotifera, Nemertea, Brachiopoda, and Annelida. None of the studied species show a conserved dorsoventral molecular regionalization of their nerve cords, not even the annelidOwenia fusiformis, whose trunk neuroanatomy parallels that of vertebrates and flies. Our findings restrict the use of molecular patterns to explain nervous system evolution, and suggest that the similarities in dorsoventral patterning and trunk neuroanatomies evolved independently in Bilateria.

The nervous systems of Bilateria, and in particular their trunk neuroanatomies, are morphologically diverse¹ (Fig. 1a). Groups such as arthropods, annelids, and chordates exhibit a medially condensed nerve cord, which is ventral in arthropods and annelids, and dorsal in chordates. In contrast, other lineages have multiple paired longitudinal nerve cords distributed at different dorsoventral (DV) levels. There are even bilaterians with only weakly condensed basiepidermal nerve nets, similar to those in cnidarians (Fig. 1a), which supports that this net-like neural arrangement predates the Cnidaria–Bilateria split²^,³ (Fig. 1a). However, the earliest configuration of the bilaterian central nervous system (CNS) is still vividly debated²^,⁴^–⁷ (Fig. 1a), and thus it is unclear when and how often nerve cords evolved in Bilateria.

(a) A nerve net is ancestral for Cnidaria and Bilateria. The neuroanatomical diversity hampers the reconstruction of the CNS evolution in Bilateria. (b) A central argument for an ancestral medially condensed VNC for Bilateria is the similar deployment of DV-TFs in vertebrates,*Drosophila*, and theP. dumerilii larva. The staggered expression of these genes concurs with specific neuronal populations.

The conserved deployment of signaling molecules and transcription factors (TFs) along the bilaterian anteroposterior and dorsoventral axes grounds most scenarios for the evolution of the CNS²^,⁷^–¹³. In particular, the similar expression of the TFsnkx2.1/nkx2.2,nkx6,pax6,pax3/7, andmsx in the ventral neuroectoderm of the flyDrosophila melanogaster and the annelidPlatynereis dumerilii, and the dorsal neural plate of vertebrates (Fig. 1b) is a core argument to propose an ancestral CNS comprising a medial ventral nerve cord (VNC) in Bilateria²^,⁴^,⁷^,⁸^,¹³^,¹⁴. InP. dumerilii and vertebrates, and to some extent inDrosophila, the staggered expression of these genes correlates with the spatial location of neuronal cell types along their trunks⁸^,¹⁰^,¹³. Serotonergic neurons form in the ventromedialnkx2.2⁺/nkx6⁺ region, cholinergic motoneurons develop in thenkx6⁺/pax6⁺ area, anddbx⁺ interneurons and lateral sensory trunk neurons differentiate in the more dorsolateralpax6⁺/pax3/7⁺ andpax3/7⁺/msx⁺ domains, respectively (Fig. 1b). The dorsoventral arrangement of these TFs and neuronal cell types is absent in hemichordates¹¹^,¹²^,¹⁵, nematodes¹⁶^,¹⁷, and planarians¹⁸ (Supplemental Data Table 1), consistent with the idea that the most recent ancestor of Bilateria had a dorsoventrally patterned, medially condensed VNC that has been repeatedly lost in these and perhaps other groups¹³. However, there is an alternative explanation – that a CNS with a single nerve cord and the similar dorsoventral patterning is the trait that repeatedly evolved, and thus was absent in the most recent common bilaterian ancestor⁵^,⁹^,¹¹^,¹².

Neuroectodermal patterning in Xenacoelomorpha

To explore the conservation of neuroectodermal patterning systems in Bilateria, we first studied Xenacoelomorpha (Extended Data Fig. 1), which is the sister group to all remaining bilaterian lineages¹⁹^,²⁰ (i.e. Nephrozoa). We focused our analyses onXenoturbella bocki, the nemertodermatidsMeara stichopi andNemertoderma westbladi, and the acoelIsodiametra pulchra. As in the acoelHofstenia miamia²¹ and most other bilaterians⁷^,¹¹, these xenacoelomorphs differentially express anteroposterior marker genes along their primary body axis²²^,²³ (Extended Data Fig. 2a, c;Extended Data Fig. 3). The BMP pathway, which has an ancestral DV patterning role²¹^,²⁴ and an anti-neural role inDrosophila and vertebrates¹⁰, is also similarly deployed in all studied xenacoelomorphs²¹, withbmp ligands expressed dorsally and antagonists located more ventrolaterally (Fig. 2a, d;Extended Data Fig. 2d;Extended Data Fig. 4). However, the DV-TFs that we found in our genomic resources (Supplementary Information Table 1) did not show a clear staggered arrangement (Fig. 2b, e). Therefore, Xenacoelomorpha only exhibits the anteroposterior and BMP ectodermal patterning systems, which is reminiscent of the cnidarian condition²⁵.

(a)*bmp* ligands andadmp are expressed dorsally;*chd* is expressed ventroposteriorly. (b)*nkx2.1*,*nkx6*, andmsx are expressed ventrally;*pax6* is expressed broadly;*Hb9* andChAT are in the nerve cords. (c)*M. stichopi* CNS (green arrowheads indicate the anterior commissures; red arrowheads indicate the nerve cords). (d)*bmp* ligands are expressed dorsally;*admp-a* is expressed posteroventrally;*admp-b* is expressed anterolaterally. (e)*nkx2.1* paralogs andnkx2.2 are expressed ventrally;*nkx6* is expressed laterally;*pax6* throughout the body;*msx* in isolated cells;*Hb9* andChAT are in the brain. (f)*I. pulchra* CNS (green arrowheads indicate the brain; red arrowheads indicate the nerve cords). Insets are lateral views. ac, anterior commissure; bnn, basiepidermal nerve net; dnc, dorsal nerve cord; lnc, lateral nerve cord; np, neuropile; pc, posterior commissure; st, statocyst; vlnc, ventrolateral nerve cord; vnc, ventral nerve cord. Scale bars, 100 µm.

Importantly, ectodermal patterning systems are deployed independently of the trunk neuroanatomy in Xenacoelomorpha. Similar to cnidarians, xenacoelomorphs have a uniformly distributed, diffuse basiepidermal nerve net³^,²⁶^–²⁸.Xenoturbella species have only this network²⁷. However, nemertodermatids have additional longitudinal basiepidermal nerve cords²⁶, located dorsally inM. stichopi²⁹ (Fig. 2c), and ventrally inN. westbladi (Extended Data Fig. 2e). The acoelI. pulchra has also four pairs of subepidermal nerve cords distributed along the DV axis²⁸ (Fig. 2f). Genes commonly involved in neurogenesis (Extended Data Fig. 5a, d) and neural transmission (Extended Data Fig. 2b, f;Extended Data Fig. 5b, c, e) are consistently expressed in the sensory structures and neural condensations in these species. However, the DV-TFnkx6 does not co-localize with the motoneuron markerChAT in the trunk ofM. stichopi andI. pulchra, while the relation ofpax6+ cells to this and another motoneuron marker (Hb9) is unclear in both species (Fig. 2b, e). Therefore, the diversity of neuroanatomies of Xenacoelomorpha contrasts with the more conserved deployment of ectodermal anteroposterior and BMP patterning systems. This, together with the observation that disruption of BMP signalling does not affect CNS development (Extended Data Fig. 6) support that the anti-neural role of the BMP pathway evolved after the Xenacoelomorpha-Nephrozoa split. Likewise, the expression of DV-TFs unrelated to the distinct trunk neuroanatomies suggests that the dorsoventral patterning of the nerve cords also evolved after the Xenacoelomorpha–Nephrozoa split.

DV patterning in Brachiopoda

To investigate the conservation of the dorsoventral nerve cord patterning in Nephrozoa, we focused on Spiralia³⁰, one of the three major nephrozoan clades. Although some lineages have a medially condensed VNC (i.e. Annelida), a main pair of VNCs is widespread and probably homologous in Spiralia⁵. We first studied the brachiopodTerebratalia transversa, where we identified staggered expression of DV-TFs in the anterior ventral midline of the larval trunk. At this stage,nkx2.1³¹ andpax6³² are expressed in the apical lobe, albeitpax6 expression projects slightly into the mantle lobe. However, there is a medialnkx2.2⁺/nkx6⁺ domain, a more lateralnkx6⁺/pax6⁺/pax3/7⁺ region, and a broad, dorsolateralmsx⁺ area in the anterior ventral ectoderm of the larval ‘trunk’ (i.e. mantle and pedicle lobes) (Fig. 3a, b;Extended Data Fig. 7a). Additionally, a narrow line of cells below the apical-mantle boundary crossing the ventral midline expressespax3/7 (Fig. 3a, b;Extended Data 7a). These expression domains disappear in the highly modified adult body (Extended Data Fig. 7a–c). The staggered expression of DV-TFs in the ventral anterior ectoderm of the trunk only partially correlates with the larval neuroanatomy, which consists of an anterior condensation and a medial accumulation of serotonergic cells on the ventral side, from where pairs of neurites innervate the chaetae and posterior end (Fig. 3c). The DV-TFs do not co-express with most neuronal markers¹³, which are mostly expressed in the anterior region (Fig. 3a, d;Extended Data Fig. 7a, d). Only twotph⁺ clusters in the medial serotonergic condensation of the larval trunk co-localise with thenkx2.2⁺/nkx6⁺ medial domain. Therefore, the brachiopodT. transversa resembles vertebrates, arthropods, andP. dumerilii in the presence of a ventral serotonergicnkx2.2⁺/nkx6⁺ area⁸^,¹⁰^,¹³^,³³, as well as in thenkx6,pax6,pax3/7 andmsx dorsolateral domains, which are however not apparently connected to any neural trunk structure.

(a)*nkx2.2* andnkx6 are in the trunk midline (arrowheads), posterior tip (arrows), gut, and apical cells (*nkx6*);*pax3/7* is expressed laterally (arrowheads) and in the apical lobe (arrow);*msx* is in the mantle and ventral pedicle. (b) There is annkx2.2⁺/*nkx6*⁺ medioventral region, and a more lateralnkx6⁺/*pax6*⁺/*pax3/7*⁺ anterior trunk domain. (c)*T. transversa* larval CNS (green arrowheads indicate the neuropile ini, and the trunk serotonergic condensation inii; red arrowheads mark the VNCs; pink arrowheads indicate the innervation of the chaetae). (d) Onlytph is expressed in the trunk (arrows and arrowheads indicate expression areas). (e)*nkx2.2* andnkx6 are in the trunk ventral midline (arrowheads), apical lobe (arrows), and gut;*pax3/7* is in the mesoderm (arrows), and in two ventrolateral trunk domains (arrowheads);*msx* is in the trunk, shell epithelium (arrowhead), and mesoderm (arrows). (f)*N. anomala* larval CNS (green arrowhead indicates the neuropile; red arrowheads ini mark the VNCs; red arrowheads inii indicate the innervation of the chaetae). ao, apical organ; bp, blastopore; ch, chaetae; mo, mouth; np, neuropile; vnc, ventral nerve cord. Scale bars, 50 µm.

The staggered ectodermal expression of DV-TFs in the anteroventral trunk ofT. transversa is largely conserved in the brachiopodNovocrania anomala. In this brachiopod,nkx2.1³¹ andpax6³² are expressed in the apical lobe, andnkx2.2 andnkx6 are expressed medially in the trunk (Fig. 3e). As inT. transversa, nkx6 extends more laterally at the anterior trunk, where it co-localizes withpax3/7 in the early larva, andmsx is broadly detected in the trunk (Fig. 3e;Extended Data Fig. 7e). Therefore,N. anomala has also a medial ventralnkx2.2⁺/nkx6⁺ domain, but remarkably, this domain does not co-localise with any serotonergic condensation, which is lacking in the larval CNS of this brachiopod (Fig. 3f). Therefore, the conserved staggered expression of the DV-TFs in the anteroventral larval trunk is not necessarily connected to the CNS, suggesting that this patterning system may rather be patterning only the ectoderm in Brachiopoda.

DV patterning in Nemertea

Similar to brachiopods, some DV-TFs show staggered expression along the trunk ventral side of the nemerteanLineus ruber. In this worm, DV-TFs are first detected in the larval imaginal discs (Extended Data Fig. 8a). In metamorphic and definitive juveniles,nkx2.1 is expressed in the head and proboscis, andpax3/7 is broadly expressed (Fig. 4a;Extended Data Fig. 8a). However,nkx2.2,nkx6, andpax6 are detected in isolated ventrolateral cells, as well as in cephalic domains (nkx2.2,nkx6,pax6) and isolated trunk cells (nkx2.2) (Fig. 4a;Extended Data Fig. 8a). Remarkably,nkx2.2 andnkx6 do not co-localise, butnkx6 andpax6 do (Fig 4b). These staggered domains relate to the disposition of the VNCs ofL. ruber (Fig. 4c). Furthermore,nkx2.2⁺ cells co-express the serotonergic markertph, andnkx6⁺ cells express the motoneuron markerHb9, but notVAchT (Fig. 4a, b). Therefore, the staggered expression of the DV-TFsnkx2.2,nkx6, andpax6 are linked to the ventral trunk CNS and some neuronal cell type markers inL. ruber, which is similar to the situation described in vertebrates andP. dumerilii⁸^,¹⁰^,¹³^,³³.

(a)*nkx2.1* andnkx2.2 are in the head (arrows), proboscis (*nkx2.1*), and trunk cells (*nkx2.2*);*nkx6* andpax6 are in the head (arrows) and VNCs (arrowheads);*pax3/7* is broadly expressed. Neuronal markers are in the brain (arrows) and VNCs (arrowheads). (b) In the VNCs,*nkx2.2*⁺ cells expresstph, but notnkx6;*nkx6*⁺ cells expresspax3/7 andHb9, but notVAchT. (c)*L. ruber* CNS (green arrowheads indicate the brain; red arrowheads mark the VNCs and the dorsal neurite in the upper inset). br, brain; dnc, dorsal nerve cord; mo, mouth; tr, trunk; pb, proboscis; vnc, ventral nerve cord. Scale bar, 100 µm.

DV patterning in Rotifera

To explore the conservation of the dorsoventral patterning in Spiralia, we studied the rotiferEpiphanes senta, a member of the sister lineage to all remaining Spiralia³⁰. Different from the brachiopod larvae and the nemertean juvenile,E. senta juveniles lack a staggered expression of DV-TFs along their trunks. The threenkx2.1 paralogs,nkx2.2, andpax6 are all in distinct brain domains of the juvenile rotifer (Fig. 5a). Only the genenkx6 is detected in two posterior trunk cells (Fig. 5a). As in brachiopods and nemerteans, the trunk CNS comprises two VNCs, and additional paired dorsolateral nerves (Fig. 5b). The trunk expression ofnkx6 probably corresponds to the vesicle ganglia¹, but it is not related to motoneurons, as inferred by the expression ofHb9 andChAT (Extended Data Fig. 9a). Therefore, spiralians with paired VNCs deploy the DV-TFs without a consistent association with their trunk neuroanatomies.

(a)*nkx2.1* paralogs,*nkx2.2*, andpax6 show brain domains (arrowheads);*nkx6* is detected posteriorly (arrowheads). (b)*E. senta* CNS (green arrowhead indicates the brain; red arrowheads indicate the VNCs, and additional neurites). (c)*nkx2.2* shows gut expression (arrowhead);*nkx6* is in the ventral midline (arrowheads);*pax6* is in two lateral larval bands (arrowheads), and juvenile head;*pax3/7* is in two ventrolateral larval clusters and midline (arrowheads), but in two trunk clusters in juveniles (arrowheads);*msx* paralogs are in ventral larval domains and the juvenile VNC (arrowheads). (d)*O. fusiformis* CNS (green arrowheads indicate the apical larval FMRF-amide cell and juvenile brain; red arrowheads indicate the larval anterior axon and juvenile medial VNC). ao, apical organ; br, brain; cb, ciliary band; cg, caudal ganglion; ch, chaetae; ln, lateral neurites; mo, mouth; ms, mastax; np, neuropile; vg, vesicle ganglia; vnc, ventral nerve cord. Scale bars, 50 µm.

DV patterning in Annelida

To investigate the conservation of the dorsoventral patterning in Annelida, the only spiralian lineage with a medially condensed VNC¹^,⁵, we studied the annelidOwenia fusiformis, which belongs to the sister lineage to all remaining annelids³⁴. Remarkably, this annelid deploys the DV-TFs differently fromP. dumerilii¹³^,³⁵. Besides the gut-related expression ofnkx2.1³¹,nkx2.2, andnkx6 in embryos and larvae, the ventral ectodermal midline expressesnkx6,pax3/7, and twomsx paralogs (Fig. 5c;Extended Data Fig. 9b). Additionally,pax6 andpax3/7 show more lateral larval expression domains (Fig. 5c). However, the ventral ectoderm of the juvenile only expressesnkx6 andmsx-b (Fig. 5c;Extended Data Fig. 9c). As in most other annelids¹, the adult CNS includes a VNC inO. fusiformis, which is not yet present in the early larva³⁶ (Fig. 5d). In the juvenile, only the expression ofnkx6 andmsx-b relate to the location of serotonin (Fig. 5d) and motoneuronal markers (Extended Data Fig. 9d). Therefore, the dorsoventral patterning system varies also among annelids with a homologous condensed VNC, and between larval¹³ and adult stages³⁵ (Extended Data Fig. 10a).

Discussion

Our study provides compelling evidence that the genes involved in the dorsoventral patterning of vertebrate,Drosophila, andP. dumerilii nerve cords do not show a similar staggered expression in the nerve cords of xenacoelomorphs and many spiralian lineages (Fig. 6a;Extended Data Fig. 10a, b). Although DV-TFs define ectodermal domains in the larval brachiopod trunks and the nemertean juvenile (Fig. 6a), these do not necessarily correlate with the trunk CNS and the location of neuronal markers (Fig. 6a). Indeed, the cell lineage relationships between the early ectodermal expression domains and specific neuronal cell types⁸^,¹⁰^,¹³ are unclear, even inDrosophila¹⁰^,³³, and still need to be broadly and functionally tested. Our findings demonstrate that the expression of DV-TFs not only differs between species with multiple nerve cords but also between spiralians that share a medially condensed homologous VNC. A similar case is observed among chordates, where the cephalochordate³⁷ and tunicate³⁸ neural plates only partially show the vertebrate molecular arrangement (Extended Data Fig. 10b;Supplementary Information Table 2), which is likely not a secondary loss given the absence of the dorsoventral patterning in Hemichordata¹¹^,¹². Therefore, the expression of DV-TFs evolved independently from the trunk neuroanatomy at least in certain bilaterian lineages, which restricts the use of this patterning system to homologize CNS anatomies⁷^,⁸^,¹⁴ and neuronal cell types²^,⁸.

(a) Gene expression summary. Dotted lines indicate that the expression does not extend along the entire trunk. (b) Proposed scenario for the evolution of neuroectodermal patterning systems in Bilateria. A nerve net, and the anteroposterior (AP) and BMP axial patterning predate the Cnidaria-Bilateria split, and were present in the Bilateria ancestor. The ancestral nephrozoan neuroanatomy remains unclear (question mark). The dorsoventral (DV) patterning system is not tied to the CNS arrangement in Bilateria (as in Chordata and Annelida). In red, lineages analysed in this study. The green dot with red border indicates that there are annelids with and without the DV patterning.

The similarities in the expression of anteroposterior and BMP patterning systems in Cnidaria and Bilateria⁷^,²¹^,²⁵ suggest that these patterning mechanisms predate the Cnidaria–Bilateria split (Fig. 6b). However, these systems are deployed in organisms within these clades with diffuse nerve nets and/or centralized nervous systems, which indicates that their ancient role was probably general body plan regionalization⁹, and not CNS patterning and neurogenesis²^,⁷. This also limits their use to homologize CNS anatomies. However, the evolution of the dorsoventral patterning of the nerve cords is more complicated (Extended Data Fig. 10c). If the similarities in dorsoventral CNS patterning between vertebrates, flies, andP. dumerilii are homologous and thus reflect the ancestral bilaterian (or nephrozoan) state⁷^,⁸^,¹³^,¹⁴, then this patterning system was independently lost/modified a great number of times. The differences between vertebrates andDrosophila in the upstream modulators of DV-TFs and in their functional integration³³^,³⁹ should thus be regarded as a case of developmental system drift⁴⁰ over large phylogenetic distances. Alternatively, and more parsimoniously, these differences may indicate that the commonalities in dorsoventral nerve cord organization between vertebrates, arthropods and some annelids evolved convergently (Fig. 6b;Extended Data Fig. 10c). The similar staggered expression domains of DV-TFs in these three lineages, together with the ones uncovered by our study (Figs. 3,4) may reflect the existence of ancient ectodermal gene regulatory sub-modules¹⁷^,³⁸^,⁴¹^,⁴² that got repeatedly assembled for the patterning of bilaterian nerve cords and neuronal cell type specification. Therefore, advancing our understanding of CNS evolution largely relies on functionally identifying the developmental implications of the anteroposterior and dorsoventral patterning systems in diverse bilaterians, before they can be used to homologize particular morphological structures and cell types⁵^,⁴³.

Methods

Animal collections and sample fixations

Gravid adults were collected from the coasts near Friday Harbor Laboratories, U.S.A. (T. transversa), Espeland Marine Biological Station, Norway (M. stichopi andN. anomala), Fanafjorden, Norway (L. ruber), Station Biologique de Roscoff, France (O. fusiformis), and Gullmarsfjord, Sweden (N. westbladi andX. bocki). Peter Ladurner (University of Innsbruck) kindly provided a stable culture ofI. pulchra, which was maintained as previously described⁴⁴. A stable laboratory culture ofE. senta was maintained in glass bowls with 25 mL Jaworski’s medium in controlled environment of 20 °C and a 14:10 h light-dark cycle. They were fedad libitum with the algaeRhodomonas sp.,Cryptomonas sp. andChlamydomonas reinhardtii. Brachiopod, nemertean and annelid adults were spawned as described elsewhere⁴⁵^–⁴⁸. Acoelomorph eggs were collected year round (I. pulchra) and in September–October (M. stichopi)²⁹. All samples were fixed in 4% paraformaldehyde in culture medium for 1 h at room temperature. After fixation, samples were washed in 0.1% Tween-20 phosphate buffer saline (PTw), dehydrated through a graded series of methanol, and stored at -20 ºC in pure methanol. Samples used for immunohistochemistry were store in PTw at 4 ºC. Before fixation, larval and juvenile stages were relaxed in 7.4% magnesium chloride,E. senta were relaxed in 10% EtOH and 1% bupivacaine. The eggshells ofM. stichopi andI. pulchra eggs were permeabilised with 1% sodium thioglycolate and 0.2 mg/ml protease for 20 min before fixation.

DMH1 treatments

M. stichopi andI. pulchra embryos were collected at the 1–2 cell stage and cultured with regular water changes in cell culture dishes until the desired developmental stage. Control embryos were treated with 0.1% DMSO and experimental embryos were treated with DMH1 (Sigma) up to 10 µM. Seawater containing the DMH1 was changed every day until fixation. Embryos and hatchlings were fixed as described above, and stored in PTw at 4 ºC.

Gene identification and expression analyses

RNAseq data obtained from mixed developmental stages and juveniles/adults was used for gene identification. Gene orthology was based on reciprocal best BLAST hit. For particular gene families, maximum likelihood phylogenetic analyses were conducted with RAxML v8.2.6⁴⁹, after building multiple protein alignments with MAFFT v7⁵⁰ and trimming poorly aligned regions with gblocks v0.91b⁵¹ (Supplementary Fig. 1). Whole-mount colorimetricin situ hybridization on brachiopod embryos,L. ruber,O. fusiformis and juvenileE. senta was performed following an already established protocol³¹^,⁴⁵. Probe concentrations ranged 0.1–1 ng/µl and permeabilisation time was 15 min forM. stichopi and post-metamorphic brachiopod juveniles, 5 min forI. pulchra, and 10 min for the other species. Double fluorescent whole-mountin situ hybridization was performed as described elsewhere³¹.

Immunohistochemistry

Samples were permeabilised in 0.1–0.5% Triton X-100 phosphate buffer saline (PTx), and blocked in 0.1–1% bovine serum albumin (BSA) in PTx. The antibodies anti-tyrosinated tubulin (Sigma), anti-serotonin (Sigma), and anti-FMRFamide (Immunostar) were diluted in 5% normal goat serum (NGS) in PTx at a concentration of 1:500, 1:200, and 1:200, respectively. Samples were incubated with the primary antibody solutions for 24-72 h at 4 ºC. Followed by several washes in 1% BSA in PTx, samples were incubated overnight with Alexa-conjugated secondary antibodies at a 1:250 dilution in 5% NGS in PTx. Before mounting and imaging, samples were washed several times in 1% BSA in PTx. Nuclei and actin filaments were counterstained with DAPI (Molecular Probes) and BODIPY FL Phallacidin (Molecular Probes).

Imaging

Representative embryos from colorimetricin situ hybridization experiments were cleared in 70% glycerol and imaged with a Zeiss Axiocam HRc connected to a Zeiss Axioscope Ax10 using bright field Nomarsky optics. Fluorescently labelled samples were cleared and mounted in benzyl benzoate/benzyl alcohol (2:1) and scanned in a Leica SP5 confocal laser-scanning microscope. Images were analysed with Fiji and Photoshop CS6 (Adobe) and figure plates were assembled with Illustrator CS6 (Adobe). Brightness/contrast and colour balance adjustments were applied to the whole image, not parts.

Data availability

All newly determined sequences have been deposited in GenBank (accession numbersKY809717–KY809754,KY709718–KY709823, andMF988103–MF988108). Multiple protein alignments used for orthology assignment are available upon request.Extended Data Figure 6c has associated source data.

Extended Data

Extended Data Figure 3 — (a,b) Expression of anteroposterior markers in adult specimens ofM. stichopi andI. pulchra. In both species,*sFRP1/5*,*vax*,*six3/6*, andBarH are expressed in anterior territories (black arrowheads). InM. stichopi,Rx is also expressed anteriorly, but broadly along the animal body inI. pulchra. In this acoel,*emx* is detected in the anterior part of the animal (background staining close to the gonads). In the nemertodermatid, the anterior neural markersotx,*otp*,*pax2/5/8*, andfezf are expressed along the entire AP axis, in association with the dorsal nerve cords (black dotted lines inotp). InI. pulchra,*otx*,*pax2/5/8-a*, andpax2/5/8-b are broadly expressed. InM. stichopi, anirx ortholog is detected in the posterior tip, while it is detected in the anterior tip and around the mouth and copulatory apparatus in the acoel (arrowheads). Thegbx ortholog ofM. stichopi is expressed posteriorly, and the trunk-related Hox genes are expressed in two lateral rows (*anterior Hox*) and anteriorly to the mouth and in the posterior tip (*posterior Hox*). In the nemertodermatid and the acoel, Wnt ligand genes are expressed posteriorly (arrowheads). All images are dorsoventral views with anterior to the left. (c) Schematic summary of anteroposterior expression in the nemertodermatidM. stichopi and the acoelI. pulchra. Drawings are not to scale and the extent of the expression domains are only approximate. The expression of posterior Hox inI. pulchra is based on²³.

Extended Data Figure 4 — (a) In the nemertodermatidMeara stichopi, the BMP pathway antagoniststwisted gastrulation (*tsg*), andcrossveinless 2 (*cv2*) are expressed dorsally, while the antagonistBAMBI is broadly detected in the ventral side. The genetolloid (*tld*) is expressed both dorsally and ventrally. The BMP receptorsbmpR-I is expressed dorsolaterally and thebmpR-II is detected more broadly. The genessmad1 andsmad4 are expressed broadly andsmad6 is expressed along the dorsal nerve cords. (b)*bmp2/4* is not expressed in neuronal cells (*elav1*⁺ cells), but in cells located medially to the nerve cords (tubulin positive). The cells expressingbmp2/4 also expresstsg, andcv2 is expressed dorsally along the nerve cords. (c) In the acoelIsodiametra pulchra, the BMP antagonisttld is expressed ventrally, thebmpR-I is detected in the inner body, andbmpR-II is expressed anteriorly and posteriorly around the copulatory organ. The genessmad1 andsmad4 are expressed generally, whilesmad6 is expressed in two bilaterally symmetrical anterior clusters. All main panels are dorsoventral views, and the insets are lateral views.

Extended Data Figure 5 — (a) In the nemertodermatidMeara stichopi, the genes associated with neuronal fate commitmentelav1,*soxB2*,*ash1*,*ash2*,*atonal*, andneuroD are detected along the dorsal nerve cords. (b) Similarly, the neuronal markerssynaptotagmin (*syt*),*tyrosine hydroxylase* (*tyr*),*vesicular monoamine transporter* (*VMAT*),*choline acetyltransferase* (*ChAT*),*vesicular acetylcholine transporter* (*VAchT*), andtryptophan hydroxylase (*tph*) are mostly expressed dorsally, along the dorsal nerve cords. (c) Morphology ofI. pulchra embryos stained against tyrosinated tubulin (Tyr tub) and serotonin (5-HT), and counterstained with phallacidin (actin bundles) and DAPI (nuclei). The first tubulin-positive cells that resemble neurons appear anteriorly (arrowheads) at 24 hours post-fertilization (hpf). By 32 hpf, the anterior and posterior lobes of the brain, as well as some neurite bundles are visible. Similarly, the first serotonergic cells are detected at 24 hpf in the anterior end (arrowheads). (d) InI. pulchra, the pro-neural markerelav1 is broadly expressed,*soxB* is detected in the head region (arrowhead), andash1b in the anterior tip (arrowhead). (e) InI. pulchra, the neuronal markersyt is highly expressed in the anterior neuropile. The markertyr is detected in the statocyst and isolated cells.VMAT is detected in isolated dorsal cell clusters in the juvenile that concentrate along the adult brain.ChAT andVAchT are expressed in the brain in juveniles and adults (gonadal staining in the adult is background). The genetph is expressed in isolated ventral cells of the adult. All panels are dorsoventral views with anterior to the left. Scale bars, 50 µm inc.

Extended Data Figure 6 — (a) Schematic overview of dorsomorphin homologue 1 (DMH1) treatments inM. stichopi and % of hatching embryos for each experimental condition. (b)*M. stichopi* embryos incubated with DMH1 from 3 to 8 weeks and after hatching show more serotonergic commissures that control animals. (c) The differences in the number of commissures are significant in both pre-hatching (asterisk; two-tail t-test; p-value < 0.0001) and post-hatching (asterisk; two-tail t-test; p-value < 0.0014) treated embryos. In contrast, the number of serotonin positive neurite bundles is not significantly increased in any of the treatments. (d) Despite the abnormal development of serotonergic axonal tracts,*slit* androbo genes are expressed similarly. The differences in signal intensity are due to technical variability. (e) Schematic overview of DMH1 treatments inI. pulchra and % of hatching embryos for each experimental condition. (f) Morphological analyses of DMH1 treated embryos. Treatment in early stages affects normal development, while treatments from 4 hours onwards does not significantly compromises embryogenesis. (g) Embryos treated between 0–4 hours post fertilization and fixed at 24 hours of development show expanded expression of the ventral markernkx2.1, reduced expression of the dorsal genebmp2/4, and unaffected expression of the anterior markersFRP1/5. The embryo shows a disorganized morphology, as revealed by actin staining. (h) The expression of the ventral markernkx2.1 is expanded in early treated embryos (0–48 h), but unaffected in embryos treated after 4 hours of development. In (b,d,f–h), the asterisk marks the anterior pole. In (b,d,f), panels are dorsoventral views, and in (g,h) the panels are lateral views.

Extended Data Figure 7 — (a) Gene expression during early gastrulation and elongation, and in late larvae ofT. transversa. The genenkx2.2 is expressed ventroposteriorly (black arrowhead) and in the pedicle lobe of the larva (arrow).nkx6 is detected in two bilateral symmetrical ectodermal posterior clusters (arrowheads) and in the archenteron wall. In the larva,*nkx6* is expressed in the pedicle lobe (arrow) and midgut.pax3/7 is first detected in two ventrolateral domains at the prospective apical-trunk boundary (arrowheads), and in the ventral anterior region of the larva. The genemsx is first expressed dorsally, in the future mantle ectoderm (arrowheads), and in the mantle of the larva. (b) In 2-days old post-metamorphic juveniles, the CNS comprises a main serotonergic anterior commissure (white arrowhead; dorsoventral view) that innervates the developing lophophore. The schematic drawing is not to scale, and the blue line represent the commissure. (c) The genenkx2.1 is expressed in the anterior region (arrowhead), between the lophophores in 2-day old juveniles. The genesnkx2.2 andnkx6 are expressed in the pedicle (arrowheads), andnkx6 is also detected in the gut (arrow). The genepax6 shows no expression,*pax3/7* is detected in the neural commissure (arrowhead), andmsx is expressed in the cells at the edge of the mantle (dotted line). (d) Neuronal markers in late larvae ofT. transversa. The serotonergic markertph is expressed in the anteroventral condensation of the mantle lobe (arrowhead) and in dorsal ectodermal cells of the apical lobe (arrow). No expression is detected forHb9, and the genesdbx,*VAchT* andChAT are all detected in the anterior apical neuroectoderm (arrowheads). (e) Gene expression during early gastrulation and elongation, and in late larvae ofN. anomala. The genenkx2.2 is expressed in the anterior blastoporal lip at the onset of axial elongation, and it is not detected in the late larva.nkx6 is asymmetrically expressed around the blastopore, in the putative anteroventral ectoderm (arrowhead). As the blastopore closes, the expression extends posteriorly and concentrates along the midline of the larva (arrowhead). The genepax3/7 is detected in the posterior mesoderm at the onset of axial elongation (arrow). The genemsx is expressed in the prospective mantle lobe ectoderm (arrowheads) and in the dorsal shell-forming epithelium of the late larva. The asterisks indicate the animal/anterior pole and white dotted lines in (a,d) mark the region of background noise caused by probe trapping in the shell forming ectoderm. Panel orientations are indicated in the first row/column and apply to the rest of the panels in the same column/row. Scale bar, 100 µm inb.

Extended Data Figure 8 — (a) None of the nerve cord patterning genes is expressed during gastrulation inL. ruber. In the intracapsular larva,*nkx2.1* is expressed in the cephalic imaginal discs (arrowheads),*nkx2.2* andnkx6 in an anterior and a posterior domain of the trunk imaginal discs (arrowheads) respectively, andpax6 is detected both in the cephalic and the anterior trunk imaginal discs (arrowheads).pax3/7 is broadly expressed. With metamorphosis,*nkx2.1* is detected in the head and proboscis,*nkx2.2* is detected in the nerve cords and isolated trunk cells (arrowheads),*nkx6* is expressed in the nerve cords (arrowheads),*pax6* is observed in the head and nerve cords (arrowheads), andpax3/7 remains broadly expressed. All gastrulae are vegetal views. For larvae and early juveniles, the left column is a dorsoventral view and the right column is a lateral view (anterior to the left). All late juvenile pictures are lateral views, with anterior to the left. (b) Lateral views (anterior to the left) of neuronal markers in juveniles. They are all expressed in the ventral nerve cords (arrowheads), and not in the dorsal neurite bundle. In all panels, the asterisk indicates the position of the mouth opening. bp, blastopore; mo, mouth; pb, proboscis.

Extended Data Figure 9 — **(a)** Expression of the motoneuron markersHb9 andChAT in juveniles of the rotiferEpiphanes senta. The geneHb9 is detected in neurons of the mastax (arrowheads) and weakly in isolated cells of the brain (arrow). The geneChAT is detected in the brain (arrow), cells of the corona and mastax (arrowheads). (b) Expression of dorsoventral patterning genes in gastrulae and elongating embryos ofOwenia fusiformis.nkx2.2 andnkx6 are expressed in the internalized endomesoderm (arrowheads). The genepax6 is expressed in two lateral rows during elongation (arrowhead) andpax3/7 in two lateral cells (arrowhead). Of the two paralogs,*msx-a* is first detected in a posterior ectodermal domain (arrowhead) and in two additional bilaterally symmetrical posterior cells (arrowheads) during elongation. The genemsx-b is only detected during elongation in a posterior domain (arrowhead). (c) Ventral view of the expression ofnkx2.1 in the juvenile of the annelidO. fusiformis. This gene is detected in the foregut (arrowheads) and hindgut (arrow). (d) Expression of the motoneuron markersHb9 andChAT inO. fusiformis.Hb9 is first detected in lateral domains of the archenteron/gut during embryogenesis and larva, and in isolated cells of the ventral trunk of the juvenile. The geneChAT is detected in three cells of the apical region of the embryo and larva, and in the neuropile and two lateral ventral cords of the juvenile. bp, blastopore; mo, mouth; ms, mastax. The asterisk in (a) marks the position of the mouth.

Extended Data Figure 10 — (a,b) Schematic drawings of trunk neuroanatomy (nerve cords in blue) and expression of patterning genes in spiralian (a) and bilaterian (b) lineages. The overall location of patterning genes expression domains with respect to the DV axis and nerve cords is indicated by light green. In (a), the red dashed squared expression ofpax6 andpax3/7 in brachiopods indicates that these expression domains are only in the anterior region of the mantle lobe, not all along the trunk. Similarly, the red dashed squared expression ofnkx6 in rotifers highlights that this gene is only expressed posteriorly in the trunk. In (b), the red dashed squared expression ofnkx2.1,*nkx2.2* andnkx6 in Cnidaria indicates that these genes are expressed in the pharynx ectoderm. The red dashed squared expression ofnkx6 inM. stichopi shows that this gene is only expressed posteriorly. In the acoelI. pulchra, the red dashed squared expression ofnkx2.2 specifies that this gene is only expressed between mouth and copulatory organ. Red circles imply that a gene is not expressed in the trunk or is missing. Question marks indicate that there is no available data about the expression of that particular gene. See Extended Data Table 1 and main text for references. Schematic drawings are not to scale and only represent approximate relative expression domains. (c) Alternative scenarios for the evolution of the dorsoventral patterning and bilaterian nerve cords. In scenario A, the medially condensed nerve cords of vertebrates, arthropods, and annelids are homologous. Therefore, the dorsoventral patterning was lost multiple times both in lineages with medially condensed nerve cords (e.g. the annelidOwenia fusiformis, cephalochordates, and tunicates) and in lineages with multiple nerve cords and diffuse nerve nets. In scenario B, which is supported by this study and is more parsimonious, the similarities in dorsoventral patterning and trunk neuroanatomies of vertebrates, arthropods, and some annelids evolved convergently. The diversity of nerve cord arrangements in nephrozoan lineages hampers reconstructing the ancestral neuroanatomy for this group (question mark). Animal phylogeny based on¹⁹

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Acknowledgements

We thank the staff at the marine stations, current and former members of the Hejnol lab, and Casey Dunn. The Sars Core Budget, the FP7-PEOPLE-2009-RG, and the ERC Community’s Framework Program Horizon 2020 to AH funded this work. The NSF IRFP Postdoctoral Fellowship funded KP. The Carlsberg Foundation funded HSL. The Swedish Research Council funded UJ and JTC.

Footnotes

Author Contributions

JMMD, KP, HSL, AH designed the study. JMMD, KP, AB, AF, AH, UJ, JTC collected the animals. JMMD, KP, AB, HS, AF, AH performed the experiments. JMMD, KP, AH wrote the manuscript. All authors read and approved the final manuscript.

Author information

All sequences have been deposited in GenBank (accession numbersKY809717–KY809754,KY709718–KY709823,MF988103–MF988108). Reprints and permissions information is available atwww.nature.com/reprints.

The authors declare no competing financial interests.

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Associated Data

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Supplementary Materials

Reporting summary

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Supplemental information guide

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Movatterモバイル変換

PERMALINK

Convergent evolution of bilaterian nerve cords

José M Martín-Durán

Kevin Pang

Aina Børve

Henrike Semmler Lê

Anlaug Furu

Johanna Taylor Cannon

Ulf Jondelius

Andreas Hejnol

Abstract

Figure 1. CNS evolution and dorsoventral patterning.

Neuroectodermal patterning in Xenacoelomorpha

Figure 2. Dorsoventral patterning in Xenacoelomorpha.

DV patterning in Brachiopoda

Figure 3. Dorsoventral patterning in Brachiopoda.

DV patterning in Nemertea

Figure 4. Dorsoventral patterning in Nemertea.

DV patterning in Rotifera

Figure 5. Dorsoventral patterning in Rotifera and Annelida.

DV patterning in Annelida

Discussion

Figure 6. Dorsoventral patterning and CNS evolution.

Methods

Animal collections and sample fixations

DMH1 treatments

Gene identification and expression analyses

Immunohistochemistry

Imaging

Data availability

Extended Data

Extended Data Figure 1. Studied species.

Extended Data Figure 2. Gene expression inXenoturbella bocki andNemertoderma westbladi.

Extended Data Figure 3. Anteroposterior patterning in Xenacoelomorpha.

Extended Data Figure 4. Expression of BMP components in Nemertodermatida and Acoela.

Extended Data Figure 5. Expression of neuronal markers in Nemertodermatida and Acoela.

Extended Data Figure 6. DMH1 treatments inMeara stichopi andIsodiametra pulchra.

Extended Data Figure 7. Gene expression in Brachiopoda.

Extended Data Figure 8. Gene expression in the nemerteanLineus ruber.

Extended Data Figure 9. Molecular patterning and motoneuron markers in Rotifera and Annelida.

Extended Data Figure 10. Dorsoventral patterning and the evolution of bilaterian trunk neuroanatomy.

Supplementary Information

Acknowledgements

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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