The brittle star genome illuminates the genetic basis of animal appendage regeneration.

Species within nearly all extant animal lineages are capable of regenerating body parts. However, it remains unclear whether the gene expression programme controlling regeneration is evolutionarily conserved. Brittle stars are a species-rich class of echinoderms with outstanding regenerative abilities, but investigations into the genetic bases of regeneration in this group have been hindered by the limited genomic resources. Here we report a chromosome-scale genome assembly for the brittle star Amphiura filiformis. We show that the brittle star genome is the most rearranged among echinoderms sequenced so far, featuring a reorganized Hox cluster reminiscent of the rearrangements observed in sea urchins. In addition, we performed an extensive profiling of gene expression during brittle star adult arm regeneration and identified sequential waves of gene expression governing wound healing, proliferation and differentiation. We conducted comparative transcriptomic analyses with other invertebrate and vertebrate models for appendage regeneration and uncovered hundreds of genes with conserved expression dynamics, particularly during the proliferative phase of regeneration. Our findings emphasize the crucial importance of echinoderms to detect long-range expression conservation between vertebrates and classical invertebrate regeneration model systems.

Genomic signatures of exceptional longevity and negligible aging in the long-lived red sea urchin.

The red sea urchin (Mesocentrotus franciscanus) is one of the Earth's longest-living animals, reported to live more than 100 years with indeterminate growth, life-long reproduction, and no increase in mortality rate with age. To understand the genetic underpinnings of longevity and negligible aging, we constructed a chromosome-level assembly of the red sea urchin genome and compared it to that of short-lived sea urchin species. Genome-wide syntenic alignments identified chromosome rearrangements that distinguish short- and long-lived species. Expanded gene families in long-lived species play a role in innate immunity, sensory nervous system, and genome stability. An integrated network of genes under positive selection in the red sea urchin was involved in genomic regulation, mRNA fidelity, protein homeostasis, and mitochondrial function. Our results implicated known longevity genes in sea urchin longevity but also revealed distinct molecular signatures that may promote long-term maintenance of tissue homeostasis, disease resistance, and negligible aging.

Immunological Diversity Is a Cornerstone of Organismal Defense and Allorecognition across Metazoa.

The ongoing arms race between hosts and microbes has fueled the evolution of novel strategies for diversifying the molecules involved in immune responses. Characterization of immune systems from an ever-broadening phylogenetic range of organisms reveals that there are many mechanisms by which this diversity can be generated and maintained. Diversification strategies operate at the level of populations, genomes, genes, and even individual transcripts. Lineage-specific innovations have been cataloged within the immune systems of both invertebrates and vertebrates. Furthermore, somatic diversification of immune receptor genes has now been described in jawless vertebrates and some invertebrate species. In addition to pathogen detection, immunological diversity plays important roles in several distinct allorecognition systems. In this Brief Review, we highlight some of the evolutionary innovations employed by a variety of metazoan species to generate the molecular diversity required to detect a vast array of molecules in the context of both immune response and self/nonself-recognition.

Bioinformatics Approaches for Analyzing Multigene Families Encoding Immune Receptors.

Genome sequences are quickly becoming available from a variety of organisms, providing researchers with an abundance of previously inaccessible information and an important source of insight into immune mechanisms. There are a variety of methods to accurately characterize genes from new genome sequences, but immune receptors pose special challenges for these techniques. Immune receptors, particularly those that directly recognize pathogens, often diverge rapidly among species and are commonly found in large, complex multigene families. Because of these characteristics, immune receptors tend to be overlooked or misannotated in large-scale genomic surveys. We describe here a strategy to characterize homologs of immune receptors and to identify putative receptors from newly assembled genome or transcriptome sequences. The description of these protocols is aimed at a typical immunologist and does not rely on substantial a priori knowledge of bioinformatics. The approach is based on using low-stringency sequence searches to identify divergent homologs. For receptors with multiple domains, the intersection of low-stringency searches can be used to identify divergent receptor sequences with high confidence. For multigene families, these predictions can be refined using sequence conservation among gene family paralogs. Assembled genome sequences serve as a critical foundation for subsequent functional characterization and remove long-standing barriers in understanding the evolution of immune recognition systems.

Extracellular Vesicle Signatures and Post-Translational Protein Deimination in Purple Sea Urchin (Strongylocentrotus purpuratus) Coelomic Fluid-Novel Insights into Echinodermata Biology.

The purple sea urchin (Strongylocentrotus purpuratus) is a marine invertebrate of the class Echinoidea that serves as an important research model for developmental biology, cell biology, and immunology, as well as for understanding regenerative responses and ageing. Peptidylarginine deiminases (PADs) are calcium-dependent enzymes that mediate post-translational protein deimination/citrullination. These alterations affect protein function and may also play roles in protein moonlighting. Extracellular vesicles (EVs) are membrane-bound vesicles that are released from cells as a means of cellular communication. Their cargo includes a range of protein and RNA molecules. EVs can be isolated from many body fluids and are therefore used as biomarkers in physiological and pathological responses. This study assessed EVs present in the coelomic fluid of the purple sea urchin (Strongylocentrotus purpuratus), and identified both total protein cargo as well as the deiminated protein cargo. Deiminated proteins in coelomic fluid EVs were compared with the total deiminated proteins identified in coelomic fluid to assess putative differences in deiminated protein targets. Functional protein network analysis for deiminated proteins revealed pathways for immune, metabolic, and gene regulatory functions within both total coelomic fluid and EVs. Key KEGG and GO pathways for total EV protein cargo furthermore showed some overlap with deimination-enriched pathways. The findings presented in this study add to current understanding of how post-translational deimination may shape immunity across the phylogeny tree, including possibly via PAD activity from microbiota symbionts. Furthermore, this study provides a platform for research on EVs as biomarkers in sea urchin models.

A nomenclature for echinoderm genes.

Echinoderm embryos and larvae are prominent experimental model systems for studying developmental mechanisms. High-quality, assembled, annotated genome sequences are now available for several echinoderm species, including representatives from most classes. The increased availability of these data necessitates the development of a nomenclature that assigns universally interpretable gene symbols to echinoderm genes to facilitate cross-species comparisons of gene functions, both within echinoderms and across other phyla. This paper describes the implementation of an improved set of echinoderm gene nomenclature guidelines that both communicates meaningful orthology information in protein-coding gene symbols and names and establishes continuity with nomenclatures developed for major vertebrate model organisms, including humans. Differences between the echinoderm gene nomenclature guidelines and vertebrate guidelines are examined and explained. This nomenclature incorporates novel solutions to allow for several types of orthologous relationships, including the single echinoderm genes with multiple vertebrate co-orthologs that result from whole-genome-duplication events. The current version of the Echinoderm Gene Nomenclature Guidelines can be found at https://www.echinobase.org/gene/static/geneNomenclature.jsp Database URL https://www.echinobase.org/.

Sensing the world and its dangers: An evolutionary perspective in neuroimmunology.

Detecting danger is key to the survival and success of all species. Animal nervous and immune systems cooperate to optimize danger detection. Preceding studies have highlighted the benefits of bringing neurons into the defense game, including regulation of immune responses, wound healing, pathogen control, and survival. Here, we summarize the body of knowledge in neuroimmune communication and assert that neuronal participation in the immune response is deeply beneficial in each step of combating infection, from inception to resolution. Despite the documented tight association between the immune and nervous systems in mammals or invertebrate model organisms, interdependence of these two systems is largely unexplored across metazoans. This review brings a phylogenetic perspective of the nervous and immune systems in the context of danger detection and advocates for the use of non-model organisms to diversify the field of neuroimmunology. We identify key taxa that are ripe for investigation due to the emergence of key evolutionary innovations in their immune and nervous systems. This novel perspective will help define the primordial principles that govern neuroimmune communication across taxa.

Mitochondrial copper and phosphate transporter specificity was defined early in the evolution of eukaryotes.

The mitochondrial carrier family protein SLC25A3 transports both copper and phosphate in mammals, yet in Saccharomyces cerevisiae the transport of these substrates is partitioned across two paralogs: PIC2 and MIR1. To understand the ancestral state of copper and phosphate transport in mitochondria, we explored the evolutionary relationships of PIC2 and MIR1 orthologs across the eukaryotic tree of life. Phylogenetic analyses revealed that PIC2-like and MIR1-like orthologs are present in all major eukaryotic supergroups, indicating an ancient gene duplication created these paralogs. To link this phylogenetic signal to protein function, we used structural modeling and site-directed mutagenesis to identify residues involved in copper and phosphate transport. Based on these analyses, we generated an L175A variant of mouse SLC25A3 that retains the ability to transport copper but not phosphate. This work highlights the utility of using an evolutionary framework to uncover amino acids involved in substrate recognition by mitochondrial carrier family proteins.

Zinc protection of fertilized eggs is an ancient feature of sexual reproduction in animals.

One of the earliest and most prevalent barriers to successful reproduction is polyspermy, or fertilization of an egg by multiple sperm. To prevent these supernumerary fertilizations, eggs have evolved multiple mechanisms. It has recently been proposed that zinc released by mammalian eggs at fertilization may block additional sperm from entering. Here, we demonstrate that eggs from amphibia and teleost fish also release zinc. Using Xenopus laevis as a model, we document that zinc reversibly blocks fertilization. Finally, we demonstrate that extracellular zinc similarly disrupts early embryonic development in eggs from diverse phyla, including Cnidaria, Echinodermata, and Chordata. Our study reveals that a fundamental strategy protecting human eggs from fertilization by multiple sperm may have evolved more than 650 million years ago.

Immune activity at the gut epithelium in the larval sea urchin.

The embryo of the purple sea urchin has been a fruitful model for the study of developmental gene regulatory networks. For similar reasons, the feeding sea urchin larva provides a gene regulatory model to investigate immune interactions at the gut epithelium. Here we describe what is known of the gut structure and immune cells of the sea urchin larva, and the cellular and gene expression response of the larva to gut-associated immune challenge. As a focused example of how the sea urchin larva can be compared with vertebrate systems, we discuss the expression and function of the IL-17 signalling system in the course of the larval immune response.

The Axial Organ and the Pharynx Are Sites of Hematopoiesis in the Sea Urchin.

Background: The location of coelomocyte proliferation in adult sea urchins is unknown and speculations since the early 1800s have been based on microanatomy and tracer uptake studies. In adult sea urchins (Strongylocentrotus purpuratus) with down-regulated immune systems, coelomocyte numbers increase in response to immune challenge, and whether some or all of these cells are newly proliferated is not known. The gene regulatory network that encodes transcription factors that control hematopoiesis in embryonic and larval sea urchins has not been investigated in adults. Hence, to identify the hematopoietic tissue in adult sea urchins, cell proliferation, expression of phagocyte specific genes, and expression of genes encoding transcription factors that function in the conserved regulatory network that controls hematopoiesis in embryonic and larval sea urchins were investigated for several tissues. Results: Cell proliferation was induced in adult sea urchins either by immune challenge through injection of heat-killed Vibrio diazotrophicus or by cell depletion through aspiration of coelomic fluid. In response to either of these stimuli, newly proliferated coelomocytes constitute only about 10% of the cells in the coelomic fluid. In tissues, newly proliferated cells and cells that express SpTransformer proteins (formerly Sp185/333) that are markers for phagocytes are present in the axial organ, gonad, pharynx, esophagus, and gut with no differences among tissues. The expression level of genes encoding transcription factors that regulate hematopoiesis show that both the axial organ and the pharynx have elevated expression compared to coelomocytes, esophagus, gut, and gonad. Similarly, an RNAseq dataset shows similar results for the axial organ and pharynx, but also suggests that the axial organ may be a site for removal and recycling of cells in the coelomic cavity. Conclusions: Results presented here are consistent with previous speculations that the axial organ may be a site of coelomocyte proliferation and that it may also be a center for cellular removal and recycling. A second site, the pharynx, may also have hematopoietic activity, a tissue that has been assumed to function only as part of the intestinal tract.

Techniques for analyzing gene expression using BAC-based reporter constructs.

To characterize the complex regulatory control of gene expression using fluorescent protein reporters, it is often necessary to analyze large genomic regions. Bacteria artificial chromosome (BAC) vectors, which are able to support DNA fragments of up to 300kb, provide stable platforms for experimental manipulation. Using phage-based systems of homologous recombination, BACs can be efficiently engineered for a variety of aims. These include expressing fluorescent proteins to delineate gene expression boundaries using high-resolution, in vivo microscopy, tracing cell lineages using stable fluorescent proteins, perturbing endogenous protein function by expressing dominant negative forms, interfering with development by mis-expressing transcription factors, and identifying regulatory regions through deletion analysis. Here, we present a series of protocols for identifying BAC clones that contain genes of interest, modifying BACs for use as reporter constructs, and preparing BAC DNA for microinjection into fertilized eggs. Although the protocols here are tailored for use in echinoderm embryonic and larval stages, these methods are easily adaptable for use in other transgenic systems. As fluorescent protein technology continues to expand, so do the potential applications for recombinant BACs.

Analysis of immune response in the sea urchin larva.

Sea urchin larvae deploy a complex immune system in the context of relatively simple morphology. Several types of phagocytic or granular immune cells respond rapidly to microbes and microbial components within the body cavity. Many of these cells also respond to microbial disturbances in the gut lumen. In the course of immune response, hundreds of genes are up- and downregulated, many of which have homologs involved in immunity in other species. Thus, the larval sea urchin provides an experimentally advantageous model for investigating the response to immune challenge at the level of cell behavior and gene regulatory networks. Importantly, the morphological simplicity and optical clarity of these larvae allow studies to be carried out within the intact animal. Here, we outline techniques to probe and visualize the immune system of the feeding sea urchin larva, particularly for quantifying gene expression and cell migration as the animal responds to both pathogens and symbionts. Techniques addressed in this chapter include (1) exposure of larvae to microbes and microbial products in sea water and by blastocoelar microinjection, (2) time-lapse imaging of immune response, (3) isolation of culturable bacteria associated with feeding larvae, (4) quantification of larval associations with isolated bacterial strains and (5) preparation of secreted products from isolated bacteria for testing in larval culture.

AID/APOBEC-like cytidine deaminases are ancient innate immune mediators in invertebrates.

In the course of both innate and adaptive immunity, cytidine deaminases within the activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) family modulate immune responses by mutating specific nucleic acid sequences of hosts and pathogens. The evolutionary emergence of these mediators, however, seems to coincide precisely with the emergence of adaptive immunity in vertebrates. Here, we show a family of genes in species within two divergent invertebrate phyla-the echinoderm Strongylocentrotus purpuratus and the brachiopod Lingula anatina-that encode proteins with similarities in amino acid sequence and enzymatic activities to the vertebrate AID/APOBECs. The expression of these invertebrate factors is enriched in tissues undergoing constant, direct interactions with microbes and can be induced upon pathogen challenge. Our findings suggest that AID/APOBEC proteins, and their function in immunity, emerged far earlier than previously thought. Thus, cytidine deamination is probably an ancient innate immune mechanism that predates the protostome/deuterostome divergence.

Bacterial artificial chromosomes as recombinant reporter constructs to investigate gene expression and regulation in echinoderms.

Genome sequences contain all the necessary information-both coding and regulatory sequences-to construct an organism. The developmental process translates this genomic information into a three-dimensional form. One interpretation of this translation process can be described using gene regulatory network (GRN) models, which are maps of interactions among regulatory gene products in time and space. As high throughput investigations reveal increasing complexity within these GRNs, it becomes apparent that efficient methods are required to test the necessity and sufficiency of regulatory interactions. One of the most complete GRNs for early development has been described in the purple sea urchin, Strongylocentrotus purpuratus. This work has been facilitated by two resources: a well-annotated genome sequence and transgenes generated in bacterial artificial chromosome (BAC) constructs. BAC libraries played a central role in assembling the S. purpuratus genome sequence and continue to serve as platforms for generating reporter constructs for use in expression and regulatory analyses. Optically transparent echinoderm larvae are highly amenable to transgenic approaches and are therefore particularly well suited for experiments that rely on BAC-based reporter transgenes. Here, we discuss the experimental utility of BAC constructs in the context of understanding developmental processes in echinoderm embryos and larvae.

An Organismal Model for Gene Regulatory Networks in the Gut-Associated Immune Response.

The gut epithelium is an ancient site of complex communication between the animal immune system and the microbial world. While elements of self-non-self receptors and effector mechanisms differ greatly among animal phyla, some aspects of recognition, regulation, and response are broadly conserved. A gene regulatory network (GRN) approach provides a means to investigate the nature of this conservation and divergence even as more peripheral functional details remain incompletely understood. The sea urchin embryo is an unparalleled experimental model for detangling the GRNs that govern embryonic development. By applying this theoretical framework to the free swimming, feeding larval stage of the purple sea urchin, it is possible to delineate the conserved regulatory circuitry that regulates the gut-associated immune response. This model provides a morphologically simple system in which to efficiently unravel regulatory connections that are phylogenetically relevant to immunity in vertebrates. Here, we review the organism-wide cellular and transcriptional immune response of the sea urchin larva. A large set of transcription factors and signal systems, including epithelial expression of interleukin 17 (IL17), are important mediators in the activation of the early gut-associated response. Many of these have homologs that are active in vertebrate immunity, while others are ancient in animals but absent in vertebrates or specific to echinoderms. This larval model provides a means to experimentally characterize immune function encoded in the sea urchin genome and the regulatory interconnections that control immune response and resolution across the tissues of the organism.

Perturbation of gut bacteria induces a coordinated cellular immune response in the purple sea urchin larva.

This corrects the article DOI: 10.1038/icb.2016.51.

Corrigendum: Whole genome analysis of a schistosomiasis-transmitting freshwater snail.

This corrects the article DOI: 10.1038/ncomms15451.

Whole genome analysis of a schistosomiasis-transmitting freshwater snail.

Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis.