Hagfish genome elucidates vertebrate whole-genome duplication events and their evolutionary consequences.

Polyploidy or whole-genome duplication (WGD) is a major event that drastically reshapes genome architecture and is often assumed to be causally associated with organismal innovations and radiations. The 2R hypothesis suggests that two WGD events (1R and 2R) occurred during early vertebrate evolution. However, the timing of the 2R event relative to the divergence of gnathostomes (jawed vertebrates) and cyclostomes (jawless hagfishes and lampreys) is unresolved and whether these WGD events underlie vertebrate phenotypic diversification remains elusive. Here we present the genome of the inshore hagfish, Eptatretus burgeri. Through comparative analysis with lamprey and gnathostome genomes, we reconstruct the early events in cyclostome genome evolution, leveraging insights into the ancestral vertebrate genome. Genome-wide synteny and phylogenetic analyses support a scenario in which 1R occurred in the vertebrate stem-lineage during the early Cambrian, and 2R occurred in the gnathostome stem-lineage, maximally in the late Cambrian-earliest Ordovician, after its divergence from cyclostomes. We find that the genome of stem-cyclostomes experienced an additional independent genome triplication. Functional genomic and morphospace analyses demonstrate that WGD events generally contribute to developmental evolution with similar changes in the regulatory genome of both vertebrate groups. However, appreciable morphological diversification occurred only in the gnathostome but not in the cyclostome lineage, calling into question the general expectation that WGDs lead to leaps of bodyplan complexity.

Evolution of two distinct variable lymphocyte receptors in lampreys: VLRD and VLRE.

Jawless vertebrates possess an alternative adaptive immune system in which antigens are recognized by variable lymphocyte receptors (VLRs) generated by combinatorial assembly of leucine-rich repeat (LRR) cassettes. Three types of receptors, VLRA, VLRB, and VLRC, have been previously identified. VLRA- and VLRC-expressing cells are T cell-like, whereas VLRB-expressing cells are B cell-like. Here, we report two types of VLRs in lampreys, VLRD and VLRE, phylogenetically related to VLRA and VLRC. The germline VLRD and VLRE genes are flanked by 39 LRR cassettes used in the assembly of mature VLRD and VLRE, with cassettes from chromosomes containing the VLRA and VLRC genes also contributing to VLRD and VLRE assemblies. VLRD and VLRE transcription is highest in the triple-negative (VLRA-/VLRB-/VLRC-) population of lymphocytes, albeit also detectable in VLRA+ and VLRC+ populations. Tissue distribution studies suggest that lamprey VLRD+ and VLRE+ lymphocytes comprise T-like sublineages of cells.

Evolution of variable lymphocyte receptor B antibody loci in jawless vertebrates.

Three types of variable lymphocyte receptor (VLR) genes, VLRA, VLRB, and VLRC, encode antigen recognition receptors in the extant jawless vertebrates, lampreys and hagfish. The somatically diversified repertoires of these VLRs are generated by serial stepwise copying of leucine-rich repeat (LRR) sequences into an incomplete germline VLR gene. Lymphocytes that express VLRA or VLRC are T cell-like, while VLRB-expressing cells are B cell-like. Here, we analyze the composition of the VLRB locus in different jawless vertebrates to elucidate its configuration and evolutionary modification. The incomplete germline VLRB genes of two hagfish species contain short noncoding intervening sequences, whereas germline VLRB genes in six representative lamprey species have much longer intervening sequences that exhibit notable genomic variation. Genomic clusters of potential LRR cassette donors, fragments of which are copied to complete VLRB gene assembly, are identified in Japanese lamprey and sea lamprey. In the sea lamprey, 428 LRR cassettes are located in five clusters spread over a total of 1.7 Mbp of chromosomal DNA. Preferential usage of the different donor cassettes for VLRB assemblage is characterized in our analysis, which reveals evolutionary modifications of the lamprey VLRB genes, elucidates the organization of the complex VLRB locus, and provides a comprehensive catalog of donor VLRB cassettes in sea lamprey and Japanese lamprey.

Post-translational protein deimination signatures in sea lamprey (Petromyzon marinus) plasma and plasma-extracellular vesicles.

Lampreys are a jawless vertebrate species belonging to an ancient vertebrate lineage that diverged from a common ancestor with humans ~500 million years ago. The sea lamprey (Petromyzon marinus) has a filter feeding ammocoete larval stage that metamorphoses into a parasitic adult, feeding both on teleost and elasmobranch fish. Lampreys are a valuable comparative model species for vertebrate immunity and physiology due to their unique phylogenetic position, unusual adaptive immune system, and physiological adaptions such as tolerance to salinity changes and urea. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family which catalyses post-translational deimination/citrullination in target proteins, enabling proteins to gain new functions (moonlighting). The identification of deiminated protein targets in species across phylogeny may provide novel insights into post-translational regulation of physiological and pathophysiological processes. Extracellular vesicles (EVs) are membrane vesicles released from cells that carry cargos of small molecules and proteins for cellular communication, involved in both normal and pathological processes. The current study identified deimination signatures in proteins of both total plasma and plasma-EVs in sea lamprey and furthermore reports the first characterisation of plasma-EVs in lamprey. EVs were poly-dispersed in the size range of 40-500 nm, similar to what is observed in other taxa, positive for CD63 and Flotillin-1. Plasma-EV morphology was confirmed by transmission electron microscopy. Assessment of deimination/citrullination signatures in lamprey plasma and plasma-EVs, revealed 72 deimination target proteins involved in immunity, metabolism and gene regulation in whole plasma, and 37 target proteins in EVs, whereof 24 were shared targets. Furthermore, the presence of deiminated histone H3, indicative of gene-regulatory mechanisms and also a marker of neutrophil extracellular trap formation (NETosis), was confirmed in lamprey plasma. Functional protein network analysis revealed some differences in KEGG and GO pathways of deiminated proteins in whole plasma compared with plasma-EVs. For example, while common STRING network clusters in plasma and plasma-EVs included Peptide chain elongation, Viral mRNA translation, Glycolysis and gluconeogenesis, STRING network clusters specific for EVs only included: Cellular response to heat stress, Muscle protein and striated muscle thin filament, Nucleosome, Protein processing in endoplasmic reticulum, Nucleosome and histone deacetylase complex. STRING network clusters specific for plasma were: Adipokinetic hormone receptor activity, Fibrinogen alpha/beta chain family, peptidase S1A, Glutathione synthesis and recycling-arginine, Fructose 1,6-bisphosphate metabolic process, Carbon metabolism and lactate dehydrogenase activity, Post-translational protein phosphorylation, Regulation of insulin-like growth factor transport and clotting cascade. Overall, for the EV citrullinome, five STRING network clusters, 10 KEGG pathways, 15 molecular GO pathways and 29 Reactome pathways were identified, compared with nine STRING network clusters, six KEGG pathways, two Molecular GO pathways and one Reactome pathway specific for whole plasma; while further pathways were shared. The reported findings indicate that major pathways relevant for immunity and metabolism are targets of deimination in lamprey plasma and plasma-EVs, with some differences, and may help elucidating roles for the conserved PAD enzyme family in regulation of immune and metabolic function throughout phylogeny.

Ancient BCMA-like Genes Herald B Cell Regulation in Lampreys.

The TNF superfamily ligands BAFF and APRIL interact with three receptors, BAFFR, BCMA, and TACI, to play discrete and crucial roles in regulating B cell selection and homeostasis in mammals. The interactions between these ligands and receptors are both specific and redundant: BAFFR binds BAFF, whereas BCMA and TACI bind to either BAFF or APRIL. In a previous phylogenetic inquiry, we identified and characterized a BAFF-like gene in lampreys, which, with hagfish, are the only extant jawless vertebrates, both of which have B-like and T-like lymphocytes. To gain insight into lymphocyte regulation in jawless vertebrates, in this study we identified two BCMA-like genes in lampreys, BCMAL1 and BCMAL2, which were found to be preferentially expressed by B-like lymphocytes. In vitro analyses indicated that the lamprey BAFF-like protein can bind to a BCMA-like receptor Ig fusion protein and to both BCMAL1- and BCMAL2-transfected cells. Discriminating regulatory roles for the two BCMA-like molecules are suggested by their differential expression before and after activation of the B-like lymphocytes in lampreys. Our composite results imply that BAFF-based mechanisms for B cell regulation evolved before the divergence of jawed and jawless vertebrates.

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.

Mitigating Anticipated Effects of Systematic Errors Supports Sister-Group Relationship between Xenacoelomorpha and Ambulacraria.

Xenoturbella and the acoelomorph worms (Xenacoelomorpha) are simple marine animals with controversial affinities. They have been placed as the sister group of all other bilaterian animals (Nephrozoa hypothesis), implying their simplicity is an ancient characteristic [1, 2]; alternatively, they have been linked to the complex Ambulacraria (echinoderms and hemichordates) in a clade called the Xenambulacraria [3-5], suggesting their simplicity evolved by reduction from a complex ancestor. The difficulty resolving this problem implies the phylogenetic signal supporting the correct solution is weak and affected by inadequate modeling, creating a misleading non-phylogenetic signal. The idea that the Nephrozoa hypothesis might be an artifact is prompted by the faster molecular evolutionary rate observed within the Acoelomorpha. Unequal rates of evolution are known to result in the systematic artifact of long branch attraction, which would be predicted to result in an attraction between long-branch acoelomorphs and the outgroup, pulling them toward the root [6]. Other biases inadequately accommodated by the models used can also have strong effects, exacerbated in the context of short internal branches and long terminal branches [7]. We have assembled a large and informative dataset to address this problem. Analyses designed to reduce or to emphasize misleading signals show the Nephrozoa hypothesis is supported under conditions expected to exacerbate errors, and the Xenambulacraria hypothesis is preferred in conditions designed to reduce errors. Our reanalyses of two other recently published datasets [1, 2] produce the same result. We conclude that the Xenacoelomorpha are simplified relatives of the Ambulacraria.

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.

Bacterial Exposure Mediates Developmental Plasticity and Resistance to Lethal Vibrio lentus Infection in Purple Sea Urchin (Strongylocentrotus purpuratus) Larvae.

Exposure to and colonization by bacteria during development have wide-ranging beneficial effects on animal biology but can also inhibit growth or cause disease. The immune system is the prime mediator of these microbial interactions and is itself shaped by them. Studies using diverse animal taxa have begun to elucidate the mechanisms underlying the acquisition and transmission of bacterial symbionts and their interactions with developing immune systems. Moreover, the contexts of these associations are often confounded by stark differences between "wild type" microbiota and the bacterial communities associated with animals raised in conventional or germ-free laboratories. In this study, we investigate the spatio-temporal kinetics of bacterial colonization and associated effects on growth and immune function in larvae of the purple sea urchin (Strongylocentrotus purpuratus) as a model for host-microbe interactions and immune system development. We also compare the host-associated microbiota of developing embryos and larvae raised in natural seawater or exposed to adult-associated bacteria in the laboratory. Bacteria associated with zygotes, embryos, and early larvae are detectable with 16S amplicon sequencing, but 16S-FISH indicates that the vast majority of larval bacterial load is acquired after feeding begins and is localized to the gut lumen. The bacterial communities of laboratory-cultured embryos are significantly less diverse than the natural microbiota but recapitulate its major components (Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes), suggesting that biologically relevant host-microbe interactions can be studied in the laboratory. We also demonstrate that bacterial exposure in early development induces changes in morphology and in the immune system. In the absence of bacteria, larvae grow larger at the 4-arm stage. Additionally, bacteria-exposed larvae are significantly more resistant to lethal infection with the larva-associated pathogen Vibrio lentus suggesting that early exposure to high levels of microbes, as would be expected in natural conditions, affects the immune state in later larvae. These results expand our knowledge of microbial influences on early sea urchin development and establish a model in which to study the interactions between the developing larval immune system and the acquisition of larval microbiota.

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.

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.

A conserved alternative form of the purple sea urchin HEB/E2-2/E2A transcription factor mediates a switch in E-protein regulatory state in differentiating immune cells.

E-proteins are basic helix-loop-helix (bHLH) transcription factors with essential roles in animal development. In mammals, these are encoded by three loci: E2-2 (ITF-2/ME2/SEF2/TCF4), E2A (TCF3), and HEB (ME1/REB/TCF12). The HEB and E2-2 paralogs are expressed as alternative (Alt) isoforms with distinct N-terminal sequences encoded by unique exons under separate regulatory control. Expression of these alternative transcripts is restricted relative to the longer (Can) forms, suggesting distinct regulatory roles, although the functions of the Alt proteins remain poorly understood. Here, we characterize the single sea urchin E-protein ortholog (SpE-protein). The organization of the SpE-protein gene closely resembles that of the extended HEB/E2-2 vertebrate loci, including a transcript that initiates at a homologous alternative transcription start site (SpE-Alt). The existence of an Alt form in the sea urchin indicates that this feature predates the emergence of the vertebrates. We present additional evidence indicating that this transcript was present in the common bilaterian ancestor. In contrast to the widely expressed canonical form (SpE-Can), SpE-Alt expression is tightly restricted. SpE-Alt is expressed in two phases: first in aboral non-skeletogenic mesenchyme (NSM) cells and then in oral NSM cells preceding their differentiation and ingression into the blastocoel. Derivatives of these cells mediate immune response in the larval stage. Inhibition of SpE-Alt activity interferes with these events. Notably, although the two isoforms are initially co-expressed, as these cells differentiate, SpE-Can is excluded from the SpE-Alt(+) cell population. This mutually exclusive expression is dependent on SpE-Alt function, which reveals a previously undescribed negative regulatory linkage between the two E-protein forms. Collectively, these findings reorient our understanding of the evolution of this transcription factor family and highlight fundamental properties of E-protein biology.

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.

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.

The ctenophore genome and the evolutionary origins of neural systems.

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

An ancient role for Gata-1/2/3 and Scl transcription factor homologs in the development of immunocytes.

Although vertebrate hematopoiesis is the focus of intense study, immunocyte development is well-characterized in only a few invertebrate groups. The sea urchin embryo provides a morphologically simple model for immune cell development in an organism that is phylogenetically allied to vertebrates. Larval immunocytes, including pigment cells and several blastocoelar cell subtypes, emerge from a population of non-skeletal mesodermal (NSM) precursors that is specified at the blastula stage. This ring of cells is first partitioned into oral and aboral fields with distinct blastocoelar and pigment cell gene regulatory programs. The oral field is subsequently specified into several distinct immune and non-immune cell types during gastrulation. Here we characterize the oral NSM expression and downstream function of two homologs of key vertebrate hematopoietic transcription factors: SpGatac, an ortholog of vertebrate Gata-1/2/3 and SpScl, an ortholog of Scl/Tal-2/Lyl-1. Perturbation of SpGatac affects blastocoelar cell migration at gastrulation and later expression of immune effector genes, whereas interference with SpScl function disrupts segregation of pigment and blastocoelar cell precursors. Homologs of several transcription regulators that interact with Gata-1/2/3 and Scl factors in vertebrate hematopoiesis are also co-expressed in the oral NSM, including SpE-protein, the sea urchin homolog of vertebrate E2A/HEB/E2-2 and SpLmo2, an ortholog of a dedicated cofactor of the Scl-GATA transcription complex. Regulatory analysis of SpGatac indicates that oral NSM identity is directly suppressed in presumptive pigment cells by the transcription factor SpGcm. These findings provide part of a comparative basis to understand the evolutionary origins and regulatory biology of deuterostome immune cell differentiation in the context of a tractable gene regulatory network model.