Katherine M. Buckley

Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution.

Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ∼500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms.

Distinctive expression patterns of 185/333 genes in the purple sea urchin, Strongylocentrotus purpuratus : an unexpectedly diverse family of transcripts in response to LPS, β-1,3-glucan, and dsRNA

BackgroundA diverse set of transcripts called 185/333 is strongly expressed in sea urchins responding to immune challenge. Optimal alignments of full-length 185/333 cDNAs requires the insertion of large gaps that define 25 blocks of sequence called elements. The presence or absence of individual elements also defines a specific element pattern for each message. Individual sea urchins were challenged with pathogen associated molecular patterns (PAMPs) (lipopolysaccharide, β-1,3-glucan, or double stranded RNA), and changes in the 185/333 message repertoire were followed over time.ResultsEach animal expressed a diverse set of 185/333 messages prior to challenge and a 0.96 kb message was the predominant size after challenge. Sequence analysis of the cloned messages indicated that the major element pattern expressed in immunoquiescent sea urchins was either C1 or E2.1. In contrast, most animals responding to lipopolysaccharide, β-1,3-glucan or injury, predominantly expressed messages of the E2 pattern. In addition to the major patterns, extensive element pattern diversity was observed among the different animals before and after challenge. Nucleotide sequence diversity of the transcripts increased in response to β-1,3-glucan, double stranded RNA and injury, whereas diversity decreased in response to LPS.ConclusionThese results illustrate that sea urchins appear to be able to differentiate among different PAMPs by inducing the transcription of different sets of 185/333 genes. Furthermore, animals may share a suite of 185/333 genes that are expressed in response to common pathogens, while also maintaining a large number of unique genes within the population.

Highly diversified innate receptor systems and new forms of animal immunity

Detailed understanding of animal immunity derives almost entirely from investigations of vertebrates, with a smaller, but significant, contribution from studies in fruit flies. This limited phylogenetic scope has artificially polarized the larger view of animal immunity toward the complex adaptive immune systems of vertebrates on the one hand and systems driven by relatively small, stable families of innate receptors of insects on the other. In the past few years analyses of a series of invertebrate deuterostome genome sequences, including those from echinoderms and cephalochordates, sharply modify this view. These findings have far-reaching implications for characterizing the potential range of animal immunity and for inferring the evolutionary pathway that led to vertebrate immune systems.

Extraordinary diversity among members of the large gene family, 185/333, from the purple sea urchin, Strongylocentrotus purpuratus

BackgroundRecent analysis of immune-related genes within the sea urchin genome revealed a number of large gene families with vertebrate homologues, such as the Toll-like and NOD/NALP-like receptor families and C-type lectins in addition to a rudimentary complement system. Therefore, the immune response of the purple sea urchin appears to be more complex than previously believed. Another component of the sea urchin immune response is an unusual family of mRNAs, known as 185/333, which is strongly upregulated in response to pathogen challenge. The work presented here indicates that this family of transcripts is derived from an unexpectedly diverse gene family.ResultsThe 185/333 genes are small (< 2 kb) with only two exons. Their extraordinary diversity was exemplified by 121 unique sequences identified from 171 cloned genes. Sequences from the second exons were aligned optimally by introducing large gaps, which defined blocks of sequence known as elements. Genes were defined by the presence or absence of elements. Phylogenetic analysis defined five intron types which, when combined with the exon element patterns resulted in 31 gene patterns, 14 of which were not described previously. Sequence diversity was present in all elements, and was higher in the intron than the exons. Repeats within the sequence facilitated multiple alignments, of which two were analyzed in detail. Although the two alignments differed in length, number of elements, and number of patterns, both were about equally accurate at describing the 185/333 sequences. The genes were closely linked and flanked by short repeats. The repeats within and between the genes may promote their diversification through gene conversion, recombination, and meiotic mispairing.ConclusionThe diversity of the 185/333 gene family represents an intriguing addition to what is known about the S. purpuratus immune response, and provides further evidence that invertebrate immune systems are neither simple nor static.

Sp185/333: A novel family of genes and proteins involved in the purple sea urchin immune response

The Sp185/333 system of genes, messages and proteins are expressed in the coelomocytes of the purple sea urchin, Strongylocentrotus purpuratus, and is an extraordinary example of diversification of a putative innate immune response system in an invertebrate. Reviewed here, is the current understanding of this complex system as illustrated by sequence comparisons of the genes, messages and deduced proteins with descriptions of diversity, including preliminary results on genomic organization and descriptions of 185/333 in other echinoids. Sp185/333 gene expression in adults and embryos occurs in response to immune challenge and includes changes in the frequencies of Sp185/333-positive coelomocytes in the adults. The diversity of the Sp185/333 protein repertoire in coelomocytes is far greater than the sequence diversity encoded in the genes, which may be the result of rapid gene recombination, RNA editing and/or low-fidelity transcription, plus post-translational modifications. This review concludes with preliminary results and speculations on protein function.

Diversity of animal immune receptors and the origins of recognition complexity in the deuterostomes.

Invertebrate animals are characterized by extraordinary diversity in terms of body plan, life history and life span. The past impression that invertebrate immune responses are controlled by relatively simple innate systems is increasingly contradicted by genomic analyses that reveal significant evolutionary novelty and complexity. One accessible measure of this complexity is the multiplicity of genes encoding homologs of pattern recognition receptors. These multigene families vary significantly in size, and their sequence character suggests that they vary in function. At the same time, certain aspects of downstream signaling appear to be conserved. Here, we analyze five major classes of immune recognition receptors from newly available animal genome sequences. These include the Toll-like receptors (TLR), Nod-like receptors (NLR), SRCR domain scavenger receptors, peptidoglycan recognition proteins (PGRP), and Gram negative binding proteins (GNBP). We discuss innate immune complexity in the invertebrate deuterostomes, which was first recognized in sea urchins, within the wider context of emerging genomic information across animal phyla.

Sequence variations in 185/333 messages from the purple sea urchin suggest posttranscriptional modifications to increase immune diversity.

The 185/333 gene family is highly expressed in two subsets of immune cells in the purple sea urchin in response to immune challenges. The genes encode a surprisingly diverse set of transcripts, which is a function of the variable presence or absence of blocks of shared sequences, known as elements that generate element patterns. Diversity is also the result of a significant level of point mutations. Together, variable element patterns and single nucleotide polymorphisms result in many unique transcripts. The 185/333 genes only have two exons, with the variable element patterns encoded entirely within the second exon. The diversity of the gene family may be the result of frequent recombination among the 185/333 genes that generates a mosaic distribution of element sequences among the genes. A comparative analysis of the sequences for the genes and messages from individual sea urchins indicates that these two sequence sets have largely different nucleotide sequences and appear to use different element patterns. Furthermore, the nucleotide substitution patterns between genes and messages reveal a strong bias toward transitions, particularly cytidine to uridine conversions. These data are consistent with cytidine deaminase activity and may represent a novel form of immunological diversification in an invertebrate immune response system.

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

The purple sea urchin (Strongylocentrotus purpuratus) genome sequence contains a complex repertoire of genes encoding innate immune recognition proteins and homologs of important vertebrate immune regulatory factors. To characterize how this immune system is deployed within an experimentally tractable, intact animal, we investigate the immune capability of the larval stage. Sea urchin embryos and larvae are morphologically simple and transparent, providing an organism‐wide model to view immune response at cellular resolution. Here we present evidence for immune function in five mesenchymal cell types based on morphology, behavior and gene expression. Two cell types are phagocytic; the others interact at sites of microbial detection or injury. We characterize immune‐associated gene markers for three cell types, including a perforin‐like molecule, a scavenger receptor, a complement‐like thioester‐containing protein and the echinoderm‐specific immune response factor 185/333. We elicit larval immune responses by (1) bacterial injection into the blastocoel and (2) seawater exposure to the marine bacterium Vibrio diazotrophicus to perturb immune state in the gut. Exposure at the epithelium induces a strong response in which pigment cells (one type of immune cell) migrate from the ectoderm to interact with the gut epithelium. Bacteria that accumulate in the gut later invade the blastocoel, where they are cleared by phagocytic and granular immune cells. The complexity of this coordinated, dynamic inflammatory program within the simple larval morphology provides a system in which to characterize processes that direct both aspects of the echinoderm‐specific immune response as well as those that are shared with other deuterostomes, including vertebrates.

IL17 factors are early regulators in the gut epithelium during inflammatory response to Vibrio in the sea urchin larva

IL17 cytokines are central mediators of mammalian immunity. In vertebrates, these factors derive from diverse cellular sources. Sea urchins share a molecular heritage with chordates that includes the IL17 system. Here, we characterize the role of epithelial expression of IL17 in the larval gut-associated immune response. The purple sea urchin genome encodes 10 IL17 subfamilies (35 genes) and 2 IL17 receptors. Most of these subfamilies are conserved throughout echinoderms. Two IL17 subfamilies are sequentially strongly upregulated and attenuated in the gut epithelium in response to bacterial disturbance. IL17R1 signal perturbation results in reduced expression of several response genes including an IL17 subtype, indicating a potential feedback. A third IL17 subfamily is activated in adult immune cells indicating that expression in immune cells and epithelia is divided among families. The larva provides a tractable model to investigate the regulation and consequences of gut epithelial IL17 expression across the organism. DOI: http://dx.doi.org/10.7554/eLife.23481.001

Characterizing immune receptors from new genome sequences.

Genome sequences are quickly being generated from a variety of organisms and provide 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 computational strategy to characterize homologs of immune receptors and also to identify putative novel receptors from newly assembled genome sequences. The description of these protocols is aimed at a typical immunologist, and a substantial knowledge of bioinformatics is not expected. 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. This strategy has recently been useful in identifying novel expansions in immune receptors in a number of animal genomes and will likely continue to revolutionize our view of animal immunity as new genomes emerge.