Convergent evolution of bilaterian nerve cords.

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. Here we study representatives of Xenacoelomorpha, Rotifera, Nemertea, Brachiopoda, and Annelida to assess the conservation of the dorsoventral nerve cord patterning. None of the studied species show a conserved dorsoventral molecular regionalization of their nerve cords, not even the annelid Owenia 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 localization of Toll and Imd pathway and complement system components and their response to Vibrio infection in the nemertean Lineus ruber.

BACKGROUND: Innate immunity is the first line of defense against pathogens. In animals, the Toll pathway, the Imd pathway, the complement system, and lectins are well-known mechanisms involved in innate immunity. Although these pathways and systems are well understood in vertebrates and arthropods, they are understudied in other invertebrates. RESULTS: To shed light on immunity in the nemertean Lineus ruber, we performed a transcriptomic survey and identified the main components of the Toll pathway (e.g., myD88, dorsal/dif/NFκB-p65), the Imd pathway (e.g., imd, relish/NFκB-p105/100), the complement system (e.g., C3, cfb), and some lectins (FreD-Cs and C-lectins). In situ hybridization showed that TLRß1, TLRß2, and imd are expressed in the nervous system; the complement gene C3-1 is expressed in the gut; and the lectins are expressed in the nervous system, the blood, and the gut. To reveal their potential role in defense mechanisms, we performed immune challenge experiments, in which Lineus ruber specimens were exposed to the gram-negative bacteria Vibrio diazotrophicus. Our results show the upregulation of specific components of the Toll pathway (TLRα3, TLRß1, and TLRß2), the complement system (C3-1), and lectins (c-lectin2 and fred-c5). CONCLUSIONS: Therefore, similarly to what occurs in other invertebrates, our study shows that components of the Toll pathway, the complement system, and lectins are involved in the immune response in the nemertean Lineus ruber. The presence of these pathways and systems in Lineus ruber, but also in other spiralians; in ecdysozoans; and in deuterostomes suggests that these pathways and systems were involved in the immune response in the stem species of Bilateria.

Molecular and morphological analysis of the developing nemertean brain indicates convergent evolution of complex brains in Spiralia.

BACKGROUND: The brain anatomy in the clade Spiralia can vary from simple, commissural brains (e.g., gastrotrichs, rotifers) to rather complex, partitioned structures (e.g., in cephalopods and annelids). How often and in which lineages complex brains evolved still remains unclear. Nemerteans are a clade of worm-like spiralians, which possess a complex central nervous system (CNS) with a prominent brain, and elaborated chemosensory and neuroglandular cerebral organs, which have been previously suggested as homologs to the annelid mushroom bodies. To understand the developmental and evolutionary origins of the complex brain in nemerteans and spiralians in general, we investigated details of the neuroanatomy and gene expression in the brain and cerebral organs of the juveniles of nemertean Lineus ruber. RESULTS: In the juveniles, the CNS is already composed of all major elements present in the adults, including the brain, paired longitudinal lateral nerve cords, and an unpaired dorsal nerve cord, which suggests that further neural development is mostly related with increase in the size but not in complexity. The ultrastructure of the juvenile cerebral organ revealed that it is composed of several distinct cell types present also in the adults. The 12 transcription factors commonly used as brain cell type markers in bilaterians show region-specific expression in the nemertean brain and divide the entire organ into several molecularly distinct areas, partially overlapping with the morphological compartments. Additionally, several of the mushroom body-specific genes are expressed in the developing cerebral organs. CONCLUSIONS: The dissimilar expression of molecular brain markers between L. ruber and the annelid Platynereis dumerilii indicates that the complex brains present in those two species evolved convergently by independent expansions of non-homologous regions of a simpler brain present in their last common ancestor. Although the same genes are expressed in mushroom bodies and cerebral organs, their spatial expression within organs shows apparent differences between annelids and nemerteans, indicating convergent recruitment of the same genes into patterning of non-homologous organs or hint toward a more complicated evolutionary process, in which conserved and novel cell types contribute to the non-homologous structures.

Clustered brachiopod Hox genes are not expressed collinearly and are associated with lophotrochozoan novelties.

Temporal collinearity is often considered the main force preserving Hox gene clusters in animal genomes. Studies that combine genomic and gene expression data are scarce, however, particularly in invertebrates like the Lophotrochozoa. As a result, the temporal collinearity hypothesis is currently built on poorly supported foundations. Here we characterize the complement, cluster, and expression of Hox genes in two brachiopod species, Terebratalia transversa and Novocrania anomalaT. transversa has a split cluster with 10 genes (lab, pb, Hox3, Dfd, Scr, Lox5, Antp, Lox4, Post2, and Post1), whereas N. anomala has 9 genes (apparently missing Post1). Our in situ hybridization, real-time quantitative PCR, and stage-specific transcriptomic analyses show that brachiopod Hox genes are neither strictly temporally nor spatially collinear; only pb (in T. transversa), Hox3 (in both brachiopods), and Dfd (in both brachiopods) show staggered mesodermal expression. Thus, our findings support the idea that temporal collinearity might contribute to keeping Hox genes clustered. Remarkably, expression of the Hox genes in both brachiopod species demonstrates cooption of Hox genes in the chaetae and shell fields, two major lophotrochozoan morphological novelties. The shared and specific expression of Hox genes, together with Arx, Zic, and Notch pathway components in chaetae and shell fields in brachiopods, mollusks, and annelids provide molecular evidence supporting the conservation of the molecular basis for these lophotrochozoan hallmarks.

Development and juvenile anatomy of the nemertodermatid Meara stichopi (Bock) Westblad 1949 (Acoelomorpha).

INTRODUCTION: Nemertodermatida is the sister group of the Acoela, which together form the Acoelomorpha, a taxon that comprises bilaterally symmetric, small aquatic worms. While there are several descriptions of the embryology of acoel species, descriptions of nemertodermatid development are scarce. To be able to reconstruct the ground pattern of the Acoelomorpha it is crucial to gain more information about the development of several nemertodermatid species. Here we describe the development of the nemertodermatid Meara stichopi using light and fluorescent microscopic methods. RESULTS: We have collected Meara stichopi during several seasons and reconstruct the complex annual reproductive cycle dependent on the sea cucumber Parastichopus tremulus. Using common fluorescent markers for musculature (BODIPY FL-phallacidin) and neurons (antibodies against FMRFamide, serotonin, tyrosinated-tubulin) and live imaging techniques, we followed embryogenesis which takes approximately 9-10 weeks. The cleavage pattern is stereotypic up to the 16-cell stage. Ring- and longitudinal musculature start to develop during week 6, followed by the formation of the basiepidermal nervous system. The juvenile is hatching without mouth opening and has a basiepidermal nerve net with two dorsal neurite bundles and an anterior condensation. CONCLUSIONS: The development of Meara stichopi differs from the development of Acoela in that it is less stereotypic and does not follow the typical acoel duet cleavage program. During late development Meara stichopi does not show a temporal anterior to posterior gradient during muscle and nervous system formation.

Peripheral and central employment of acid-sensing ion channels during early bilaterian evolution.

Nervous systems are endowed with rapid chemosensation and intercellular signaling by ligand-gated ion channels (LGICs). While a complex, bilaterally symmetrical nervous system is a major innovation of bilaterian animals, the employment of specific LGICs during early bilaterian evolution is poorly understood. We therefore questioned bilaterian animals' employment of acid-sensing ion channels (ASICs), LGICs that mediate fast excitatory responses to decreases in extracellular pH in vertebrate neurons. Our phylogenetic analysis identified an earlier emergence of ASICs from the overarching DEG/ENaC (degenerin/epithelial sodium channel) superfamily than previously thought and suggests that ASICs were a bilaterian innovation. Our broad examination of ASIC gene expression and biophysical function in each major bilaterian lineage of Xenacoelomorpha, Protostomia, and Deuterostomia suggests that the earliest bilaterian ASICs were probably expressed in the periphery, before being incorporated into the brain as it emerged independently in certain deuterostomes and xenacoelomorphs. The loss of certain peripheral cells from Ecdysozoa after they separated from other protostomes likely explains their loss of ASICs, and thus the absence of ASICs from model organisms Drosophila and Caenorhabditis elegans. Thus, our use of diverse bilaterians in the investigation of LGIC expression and function offers a unique hypothesis on the employment of LGICs in early bilaterian evolution.

Most animals on Earth, from worms to chimpanzees, belong to a group known as the bilaterians. Despite their rich variety of shapes and lifestyles, all these creatures share similarities ­ in particular, a complex nervous system where neurons can quickly relay electric signals. This is made possible by a class of proteins, known as ligand-gated ion channels, which are studded through the membrane of cells. There, they help neurons efficiently communicate with each other by converting external chemical information into internal electrical signals. Yet despite their importance, how and when these proteins have evolved remains poorly understood. Marti-Solans et al. decided to explore this question by focusing on acid-sensing ion channels, a family which often forms the linchpin of bilaterian neural networks. They examined when these proteins first evolved (that is, in which putative ancestral animals) and where in the body. To do so, they combed through genetic data from all major bilaterian lineages as well as from non-biletarian groups; this included previously unexplored datasets that give insight into the type of cells in which a particular gene is active. The analyses revealed that the channels are specific to bilaterians, but that they appeared earlier than previously thought, being present in the very first members of this group. However, at this stage, the proteins were mainly located in cells at the periphery of the body rather than in those from emerging neural circuits. This suggests that the channels were co-opted by nerve cells later on, when the nervous systems became more complex. The proteins being initially located in cells at the outer edge of the body could also explain why they are absent in bilaterian creatures such as fruit flies and nematode worms; these animals all belong to a lineage where growth takes place by shedding their external layers. Acid-sensing ion channels are an important group of potential drug targets, often being implicated in pain and diseases of the nervous system. The work of Marti-Solans et al. offers an insight into the diversity of roles these proteins can play in the body, demonstrating once again how evolution can repurpose the same biophysical functions to serve a range of needs inside an organism.

The evolution of the metazoan Toll receptor family and its expression during protostome development.

BACKGROUND: Toll-like receptors (TLRs) play a crucial role in immunity and development. They contain leucine-rich repeat domains, one transmembrane domain, and one Toll/IL-1 receptor domain. TLRs have been classified into V-type/scc and P-type/mcc TLRs, based on differences in the leucine-rich repeat domain region. Although TLRs are widespread in animals, detailed phylogenetic studies of this gene family are lacking. Here we aim to uncover TLR evolution by conducting a survey and a phylogenetic analysis in species across Bilateria. To discriminate between their role in development and immunity we furthermore analyzed stage-specific transcriptomes of the ecdysozoans Priapulus caudatus and Hypsibius exemplaris, and the spiralians Crassostrea gigas and Terebratalia transversa. RESULTS: We detected a low number of TLRs in ecdysozoan species, and multiple independent radiations within the Spiralia. V-type/scc and P-type/mcc type-receptors are present in cnidarians, protostomes and deuterostomes, and therefore they emerged early in TLR evolution, followed by a loss in xenacoelomorphs. Our phylogenetic analysis shows that TLRs cluster into three major clades: clade α is present in cnidarians, ecdysozoans, and spiralians; clade ß in deuterostomes, ecdysozoans, and spiralians; and clade γ is only found in spiralians. Our stage-specific transcriptome and in situ hybridization analyses show that TLRs are expressed during development in all species analyzed, which indicates a broad role of TLRs during animal development. CONCLUSIONS: Our findings suggest that a clade α TLR gene (TLR-Ca) and a clade ß/γ TLR gene (TLR-Cß/γ) were already present in the cnidarian-bilaterian common ancestor. However, although TLR-Ca was conserved in cnidarians, TLR-Cß/γ was lost during the early evolution of these taxa. Moreover, TLR-Cß/γ duplicated to generate TLR-Cß and TLR-Cγ in the lineage to the last common protostome-deuterostome ancestor. TLR-Ca, TLR-Cß and TLR-Cγ further expanded generating the three major TLR clades. While all three clades radiated in several spiralian lineages, specific TLRs clades have been presumably lost in other lineages. Furthermore, the expression of the majority of these genes during protostome ontogeny suggests a likely role in development.

Mesodermal gene expression in the acoel Isodiametra pulchra indicates a low number of mesodermal cell types and the endomesodermal origin of the gonads.

Acoelomorphs are bilaterally symmetric small marine worms that lack a coelom and possess a digestive system with a single opening. Two alternative phylogenetic positions of this group within the animal tree are currently debated. In one view, Acoelomorpha is the sister group to all remaining Bilateria and as such, is a morphologically simple stepping stone in bilaterian evolution. In the other, the group is a lineage within the Deuterostomia, and therefore, has derived a simple morphology from a more complex ancestor. Acoels and the closely related Nemertodermatida and Xenoturbellida, which together form the Acoelomorpha, possess a very limited number of cell types. To further investigate the diversity and origin of mesodermal cell types we describe the expression pattern of 12 orthologs of bilaterian mesodermal markers including Six1/2, Twist, FoxC, GATA4/5/6, in the acoel Isodiametra pulchra. All the genes are expressed in stem cells (neoblasts), gonads, and at least subsets of the acoel musculature. Most are expressed in endomesodermal compartments of I. pulchra developing embryos similar to what has been described in cnidarians. Our molecular evidence indicates a very limited number of mesodermal cell types and suggests an endomesodermal origin of the gonads and the stem cell system. We discuss our results in light of the two prevailing phylogenetic positions of Acoelomorpha.