View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Developmental Biology 300 (2006) 321–334 www.elsevier.com/locate/ydbio

The genomic underpinnings of in Strongylocentrotus purpuratus

Anthony J. Robertson a, Jenifer Croce b, Seth Carbonneau a, Ekaterina Voronina c, Esther Miranda b, ⁎ David R. McClay b, James A. Coffman a,

a Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672, USA b Duke University, Developmental, Molecular, and Cellular Biology Group, Durham, NC 27708, USA c Brown University, Department of Molecular Biology, Cellular Biology, and Biochemistry, Providence, RI 02912, USA Received for publication 11 May 2006; revised 21 August 2006; accepted 22 August 2006 Available online 30 August 2006

Abstract

Programmed cell death through apoptosis is a pan-metazoan character involving intermolecular signaling networks that have undergone substantial lineage-specific evolution. A survey of apoptosis-related proteins encoded in the sea urchin genome provides insight into this evolution while revealing some interesting novelties, which we highlight here. First, in addition to a typical CARD-carrying Apaf-1 homologue, sea urchins have at least two novel Apaf-1-like proteins that are each linked to a , suggesting that have evolved unique apoptotic signaling pathways. Second, sea urchins have an unusually large number of . While the set of effector caspases (caspases-3/7 and - 6) in sea urchins is similar to that found in other basal deuterostomes, signal-responsive initiator caspase subfamilies (caspases-8/10 and 9, which are respectively linked to DED and CARD adaptor domains) have undergone -specific expansions. In addition, there are two groups of divergent caspases, one distantly related to the interleukin converting enzyme (ICE)-like subfamily, and a large clan that does not cluster with any of the vertebrate caspases. Third, the complexity of proteins containing an anti-apoptotic BIR domain and of Bcl-2 family members approaches that of , and is greater than that found in protostome model systems such as Drosophila or Caenorhabditis elegans. Finally, the presence of Death receptor homologues, previously known only in vertebrates, in both Strongylocentrotus purpuratus and Nematostella vectensis suggests that this family of apoptotic signaling proteins evolved early in and was subsequently lost in the and arthropod lineage(s). Our results suggest that cell survival is contingent upon a diverse array of signals in sea urchins, more comparable in complexity to vertebrates than to arthropods or , but also with unique features that may relate to specific requirements imposed by the biphasic life cycle and/or immunological idiosyncrasies of this organism. © 2006 Elsevier Inc. All rights reserved.

Keywords: Apoptosis; Evolution; Apaf-1; Caspase; BIR domain; IAP; Bcl-2; TNFR; Genome; Sea Urchin

Introduction notion, the genomes of all animals thus far examined encode the basic apoptotic toolkit (Koonin and Aravind, 2002). This Apoptosis, a type of eukaryotic cell suicide defined by a toolkit generally contains the following proteins or protein characteristic set of morphological and biochemical features, domains: the caspases, a family of cysteine aspartyl proteases plays critical roles in both development and homeostasis (Kerr at the core of the apoptotic apparatus that execute many of the et al., 1972; Hengartner, 2000; Twomey and McCarthy, 2005). activities that bring about disintegration of the cell; a family of Its emergence in our primordial multicellular ancestors was 6-alpha helical “adaptor” domains [CARD (caspase recruit- likely a pre-requisite for metazoan evolution, providing a ment domain), death, and DED ()] means for the elimination of pathogenic cells and for the contained within apoptosis-associated signaling proteins as genomic control of cell survival in response to environmental well as some of the caspases, which transmit cell death signals or ontogenetically specified signals. Consistent with this to the caspases via homotypic or heterotypic protein–protein interactions; Apaf-1, a CARD-containing protein that initiates ⁎ Corresponding author. caspase-mediated apoptosis in response to cytochrome c E-mail address: [email protected] (J.A. Coffman). released from mitochondria; the IAP (inhibitor of apoptosis)

0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.08.053 322 A.J. Robertson et al. / Developmental Biology 300 (2006) 321–334 proteins which antagonize the caspases; and the Bcl-2 family rise to the proto-mitochondrial endosymbiont (Koonin and of pro- and anti-apoptotic proteins that regulate the release of Aravind, 2002). In response to stress or specific signals, apoptotic signals from the mitochondria (reviewed in Hen- mitochondria undergo a permeability transition and release gartner, 2000; Wang, 2001; Koonin and Aravind, 2002). On cytochrome c into the cytosol (reviewed in Armstrong, 2006). top of these core mechanisms, numerous pro- and anti- Cytochrome c thenassembleswithApaf-1toformthe apoptotic systems have evolved that add additional layers of apoptosome, which oligimerizes with the CARDs of caspase-9 regulation, involving for example networks of signal transdu- family members, thereby activating caspase-9 and the caspase cing kinases and transcription factors that modulate the cascade (Liu et al., 1996; Li et al., 1997). Mitochondria also activity of the apoptotic apparatus. release other pro-apoptotic proteins, including AIF (apoptosis Signals leading to apoptosis can originate either from inside inducing factor) and Endo-G (Susin et al., 1999; Li et al., 2001; or outside of a cell. In vertebrates at least (Wang, 2001; Wang et al., 2002), which contribute to caspase-independent Kornbluth and White, 2005; Armstrong, 2006), mitochondria lie apoptosis in response to intrinsic stress signals; and SMAC/ at the core of the cell-intrinsic signaling pathways and also DIABLO, which counters the anti-apoptotic IAP proteins (Du et participate in some of the extrinsic signaling pathways (Fig. 1), al., 2000; Verhagen et al., 2000; Fig. 1). Intrinsic signals leading and comparative genomic analyses suggest that much of the to apoptosis include genotoxic and oxidative stress. Extrinsic apoptotic apparatus originated in the bacterial lineage that gave signals in the form of secreted ligands are generally received by

Fig. 1. Simplified schematic illustrating representative examples of intrinsic and extrinsic apoptotic signaling pathways. The intrinsic pathway (left) can be activated by various intracellular signals such as DNA damage. These signals down-regulate pro-survival family proteins such Bcl-2 and Bcl-xl, allowing pro-apoptotic molecules such as Bak and Bax to induce mitochondrial permeability transition leading to the release of cytochrome c and other pro-apoptotic molecules including Smac/DIABLO, which counters the anti-apoptotic activity of IAPs (see text). Cytochrome c then binds Apaf-1 to form the apoptosome leading to the activation of caspase-9, which in turn activates the effector caspase cascade leading to cell death. The extrinsic pathways (right) are activated by the binding of secreted ligands members of the TNF family to their cognate receptors members of the TNFR family. After activation receptor homotrimers or homomers recruit cytoplasmic adaptor molecules that trigger cell death via different pathways. Downstream of the classic cell death receptors (e.g., FAS, DR4/5, TNFR1), adaptor molecules directly bind and activate the cleavage of the pro-caspases-8 and -10, which in turn activate the effector caspases that execute cell death. In contrast, p75NTR adaptor molecules activate the Jnk pathway, which then triggers cell death using the intrinsic pathway (Barker, 2004). However, in some cells the classic death receptors can also lead to cell death via the intrinsic pathway by activating the pro-apoptotic protein Bid. Proteins for which no direct sea urchin orthologues were found are shaded gray. A.J. Robertson et al. / Developmental Biology 300 (2006) 321–334 323 death domain-containing transmembrane receptors in the tumor identify proteins containing the following domains: Peptidase_C14 (PF00656); factor receptor (TNFR) superfamily (Bridgham et al., BIR (PF00653); Bcl-2 (PF00452); TNFR_c6 (PF00020); NACHT (PF05729); NB-ARC (PF00931); Death (PF00531); DED (PF01335); and CARD 2003), which engage death domain-containing adaptor proteins (PF00619). Most caspase genes were identified by similarity to the PFAM that in turn activate DED-containing members of the caspases-8/ Peptidase_C14 (caspase) domain, although a few were initially found by 10 family (Tibbetts et al., 2003), resulting in direct activation of similarity to either CARD or DED and subsequently found to contain partial the caspase cascade and/or induction of the mitochondrial caspase domain sequences. TNF and TNFR proteins were obtained by BLASTP permeability transition (Fig. 1). or TBLASTN against the S. purpuratus genome using as queries full or partial sequences of known vertebrate or ascidian proteins. ESTs and/or tiling array data Both intrinsically and extrinsically activated apoptosis occurs (Samanta et al., 2006) validating exon assignments and indicating gene normally in the ontogeny of many organisms. Well-known expression were obtained from the Baylor Genboree website (http://www. examples include the elimination of supernumerary cells during genboree.org/java-bin/PurpleUrchin/index.jsp). embryogenesis in nematodes (Ellis and Horvitz, 1986); pruning of neurons during nervous system development wherein survival Collection of apoptosis-related genes from other genomes of neurons is often contingent upon formation of functional synapses (which provide pro-survival signals; reviewed in Vertebrate (Homo sapiens or Mus musculus), urochordate (Ciona intestina- lis), mollusk (Lymnaea stagnalis), arthropod (D. melanogaster), and nematode Davies, 2003); removal of webbing between the digits in (C. elegans) gene sequences used in multiple sequence alignments and vertebrate limb development; and loss of the tail during anuran phylogenetic tree construction were obtained from Genbank (NCBI). Apopto- metamorphosis (Zuzarte-Luis and Hurle, 2002). Extrinsically sis-related genes from the sea anemone N. vectensis were obtained by using the activated apoptosis also plays crucial roles in the vertebrate PFAM search engine on the Stellabase website (www.stellabase.org; Sullivan et immune system, wherein lymphocyte survival is contingent on al., 2006). both a lack of self-recognition and permissive signaling via Multiple sequence alignments and tree construction interleukins (withdrawal of which leads to apoptosis), while apoptosis of activated lymphocytes in response to specific ligand Multiple sequence alignments were carried out using Clustal W or Clustal X binding by TNFR family members is used to turn off the immune programs. Phylogenetic analyses were performed using MEGA version 3.1 response (reviewed in Gupta et al., 2005). (Kumar et al., 2004) or PAUP version 4.0 (Swofford, 1998). For the Clustal W It has been noted that the complexity of the apoptotic alignments in MEGA 3.1, the PAM protein weight matrix was used for caspase domains; GONNET was used for Apaf-1 proteins and Bcl-2 domains, and apparatus in vertebrates is substantially greater than it is in BLOSUM for BIR proteins. The trimmed caspase domain and Bcl-2 domain invertebrate model organisms such as Drosophila melanogaster sequences used in the alignments are listed in Supplemental data file 1. Apaf-1, and Caenorhabditis elegans, leading to the speculation that caspase, BIR domain and Bcl-2 family trees were constructed in MEGA 3.1 there has been a vertebrate-specific expansion and evolutionary using the Neighbor-Joining method and 1000 bootstrap replicates. TNF, TNFR diversification of apoptosis-related genes (Aravind et al., 2001). and TRAF protein alignments were generated by Clustal X and saved as nexus files, and trees were then obtained with the PAUP 4.0 program using the The sequenced genome of the sea urchin Strongylocentrotus neighbor-joining method and 5000 bootstrap replicates. The trees displayed in purpuratus allows us to explore this hypothesis and ask what the figures represent the bootstrap consensus, with the percentage of bootstrap parts of the vertebrate apoptotic toolkit are also present in replicates obtained indicated at each node. invertebrate deuterostomes. Furthermore, the availability of the recently sequenced genome of the sea anemone Nematostella Results and discussion vectensis (Sullivan et al., 2006), a cnidarian that provides an outgroup of the bilateria, allows us to ask which parts of the Evolution of the bilaterian apoptotic toolkit apoptotic toolkit evolved prior to the emergence of bilaterians. We find that the complexity of the genomic toolkit for apoptosis To broadly survey the sea urchin apoptotic toolkit and in sea urchins is comparable to, but qualitatively different from, compare it to that of other animals, searches of PFAM domains that of vertebrates, and greater than that of sea anemones, which among the predicted sea urchin proteins as well as reports in the is in turn greater than that of arthropods or nematodes. literature were used to quantify the complexity of selected Moreover, homologues of death domain-linked TNFR family apoptosis-associated domains encoded in various metazoan members, previously thought to be confined to vertebrates genomes (Table 1). The domains surveyed included the major (Bridgham et al., 2003), are present in both sea urchins and sea apoptosis-associated families described in the Introduction anemones, indicating that the absence of this family in (Apaf-1,caspase,BIR,Bcl-2,deathdomain,DED,and nematodes and arthropods is actually due to gene loss. CARD), as well as the NACHT (NAIP, CIIA, HET-E and TP1) NTPase domain, a protein–protein interaction domain found in Materials and methods many vertebrate proteins that transduce signals involved in innate immunity as well as apoptosis (Koonin and Aravind, Annotation of apoptosis-related genes in S. purpuratus 2000). The recently sequenced genome of the cnidarian N. vectensis (Sullivan et al., 2006) was included in this analysis to The genome of S. purpuratus was sequenced and annotated as described discriminate between gain and loss of genes in different (The Sea Urchin Genome Sequencing Consortium, submitted for publication). bilaterian clades. This survey generated two major findings: Computationally predicted models of apoptosis-related genes were collected by searching the GLEAN3 gene set at Baylor, either by BLASTP or TBLASTN first, the complexity of apoptosis-related domains in sea urchins using homologous sequences from known apoptosis-related genes, is comparable to that of vertebrates and considerably higher than or by using the PFAM search engine set up on the Baylor annotation site to that of all the other invertebrate organisms surveyed; and second, 324 A.J. Robertson et al. / Developmental Biology 300 (2006) 321–334

Table 1 Apaf-1 homologues Complexity of selected apoptosis-related genes in various genomes Domain/ Echinoderms Vertebrates Arthropods Nematodes Cnidarians As described in the Introduction, vertebrate Apaf-1 con- a b b b c Family (Sp) (Hs) (Dm) (Ce) (Nv) tributes to activation of apoptosis by nucleating formation of the Apaf-1 4 1 1 1 0 “apoptosome” in combination with cytochrome c released from Caspase 31 14 7 4 5 mitochondria, leading to oligomerization and hence activation BIR 7 8 4 2 4 of caspase-9 (Fig. 1; Li et al., 1997; Zou et al., 1997). The Apaf- Bcl-2 d 10 11 2 1 7 Death 47 (101) 30 9 6 6 1 family in animals is defined by the NB-ARC domain, a DED 4 (5) 7 1 0 5 nucleotide binding P-loop motif also found in various plant CARD 5 (10) 20 1 0 8 resistance (R) genes that promote cell death in response to e NACHT 129 (145) 18 1 1 2 pathogens (van der Biezen and Jones, 1998). Apaf-1 is a The number of annotated genes. Some of the domains identified by the represented by a single orthologue each in humans, fruit flies, PFAM search were not annotated due to problems such as fragmentary sequence and nematodes (and no obvious homologues in sea anemones); or lack of additional diagnostic domains in the models; for families where this in addition to the NB-ARC domain, each of these homologues was the case the total number of PFAM domains identified in the genome (annotated+unannotated) are indicated in parentheses. contains a CARD, whereas human and fly Apaf-1 also have a b Numbers obtained from Koonin and Aravind (2002). For Apaf-1, caspases, set of WD40 repeats required for cytochrome c binding BIR-domain proteins, and Bcl-2 domain proteins the numbers were verified by (reviewed in Kornbluth and White, 2005). Interestingly, there manual searches using the PFAM search engine at StellaBase (www.stellabase. are gene models for four Apaf-1-like proteins in S. purpuratus org) to validate the comparison. (two of which are nearly identical in sequences that probably c Obtained from Stellabase. The number of caspases was revised from that listed at StellaBase after manual inspection of hits. represent haplotypes). Of these, one (SPU_025776) carries a d Proteins with Bcl-2 PFAM domains; does not include “BH3-only” Bcl-2 CARD and seven WD40 repeats, with a domain architecture family members. similar to that of human Apaf-1. The others (SPU_023776 and e Note that PFAM searches with a relaxed expectation value indicate that the SPU_005441/003281) lack the CARD, and instead have a death number of NACHT domains is likely to be even higher than that given here (see domain associated with the NB-ARC domain and one to four Hibino et al., 2006). WD40 repeats (Fig. 2). A multiple sequence alignment produced a neighbor-joining tree that suggests these latter the complexity of these domains in arthropods and nematodes is proteins represent either an ancient Apaf-1 architecture which similar to, and in some cases lower than, that of cnidarians. The arose in deuterostomes and was lost in and/or findings suggest that during bilaterian evolution, a trend has vertebrates, or alternatively, a divergent echinoderm apomorphy been for the basic metazoan toolkit for apoptosis to be expanded (Fig. 2). It is tempting to speculate that the death domain present with diversification in the deuterostome lineages leading to in the novel sea urchin Apaf-1-like proteins generates a greater echinoderms and chordates, and pruned in the protostome diversity of apoptotic pathways, perhaps involving interactions lineages leading to arthropods and nematodes. between the Apaf-1-like proteins and death domain-containing Of particular note is the relative plethora of caspase, death, immune system proteins (Hibino et al., 2006)orDED- and NACHT domains encoded in the sea urchin genome (see containing caspase-8-like proteins. also Hibino et al., 2006). Whereas the protostome models Drosophila and C. elegans each have a single NACHT Caspases domain protein that is involved in telomerase function, in vertebrates this family has undergone an expansion associated The caspase gene family has two major branches: the with a role in immune response and apoptosis (Koonin and apoptotic “CED-3”-like subfamily, and the pro-inflammatory Aravind, 2002). From the data summarized in Table 1, an even “ICE-like” subfamily, hitherto known only in vertebrates (Wang more extreme expansion appears to have occurred in and Gu, 2001). The apoptotic caspases can be categorized as echinoderms, or possibly early in the deuterostome lineage being either upstream initiators or downstream effectors of followed by gene loss in vertebrates. Similarly, caspase and apoptosis, depending on where they function in the apoptotic death domain proteins have significantly increased in numbers cascade (reviewed in Riedl and Shi, 2004). The initiator in both vertebrates and echinoderms. A significant proportion caspases contain regulatory domains that when engaged by of the sea urchin NACHT and death domains are likely signals activate the catalytic caspase domain, which in turn involved in innate immunity (Hibino et al., 2006), and may or proteolytically activates the caspase domain of an effector may not be involved in apoptosis (although there are deep caspase. The activated effector caspase then cleaves numerous evolutionary and functional linkages between the regulatory cellular substrates, including other effector caspases in an networks that control programmed cell death and defense amplification cascade that leads to dismantling of the cell against pathogens). These findings are consistent with the (reviewed in Hengartner, 2000). Initiator caspases fall into two proposition that apoptosis is linked to a greater complexity of classes, based both on domain architecture and similarity signals in echinoderms and vertebrates than it is in nematodes between the sequences of the caspase domain. The caspase-9 and arthropods, a scenario that is underscored by the more family is linked to an N-terminal CARD, which associates with detailed analysis of specific gene families described in the the CARD of Apaf-1 to transduce the mitochondrial signal for following. apoptosis. The caspases-8/10 family is linked to a tandem A.J. Robertson et al. / Developmental Biology 300 (2006) 321–334 325

Fig. 2. Apaf-1 family tree and protein architecture. A Neighbor-Joining tree calculated from multiple sequence alignment of sea urchin Apaf-1-like proteins with human (Apaf-1), Drosophila (DARK) and C. elegans (CED-4) homologues. Protein architectures are indicated schematically. The sea urchin Apaf-1-like proteins with novel architectures (containing a death domain) are highlighted in gray.

N-terminal pair of DEDs, which associate with the death domain Of the caspase-9-like genes, at least one (SPU_000882) is of an adaptor molecule that is in turn activated in response to linked with a CARD, whereas linkage with CARDs could not be extracellular signal transduction. The known effector caspases ascertained for the others in the current sequence assembly. A include members of the caspases-3/7/6 family, which lack similar situation obtains for the caspase-8-like genes: two were adaptor domains and are activated proteolytically. The ICE-like found that are linked to 2 DEDs (SPU_020623 and vertebrate caspases are also linked to a CARD, but their caspase SPU_024371; the latter is not included in the tree, as it lacked domain is structurally divergent from that of the pro-apoptotic a complete caspase domain), whereas the linkage to DEDs for caspases, and functionally they are largely dedicated to the others remains uncertain. The multiplicity of caspase-8-like, and mounting an immune response (Wang and caspase-9-like, ICE-like, and caspase-N genes suggests that Gu, 2001). these families underwent echinoderm-specific expansions, A search of the GLEAN3 gene predictions from the S. probably as tandem arrays, as suggested in several cases by purpuratus genome found 34 matches to the caspase domain, the presence of multiple genes on a single scaffold. An 31 of which were annotated as distinct genes. All but three of analogous expansion of caspase genes was previously reported the models encode proteins containing a conserved cysteine for the urochordates C. intestinalis and C. savignyi (Weill et al., within the catalytic center sequence motif QACRG, or a variant 2005). An alignment of the sea urchin caspase protein sequences thereof (QACQG, QSCRG, QCCRG, and DACWG, Supple- with those of C. intestinalis shows that the multiple caspases on mental Fig. S1). The three models lacking the catalytic cysteine each branch of the tree tend to cluster within each (i.e., (SPU_002921, SPU_002923, and SPU_005875) may represent are paralogous), indicating that the caspase gene family non-proteolytic caspase-like regulatory proteins analogous to expanded independently in these two deuterostome lineages the mammalian protein CLARP (caspase-like apoptosis-regu- (Supplemental Fig. S2). Echinoderms and urochordates are both latory protein; Inohara et al., 1997), or may have novel animals that undergo indirect development via a planktonic functions unrelated to apoptosis. A tree derived from a multiple larva, and it is possible that the expansions of caspase genes in sequence alignment of the sea urchin caspase domains with these clades are adaptations that facilitate the developmental those of human caspases revealed that they fall into five distinct remodeling that occurs during adult metamorphosis (Weill et al., subfamilies (Fig. 3): four caspase-9-like genes; two genes in the 2005). caspases-3/7/6 subfamily (one caspases-3/7-like and the other The presence of a single caspases-3/7-like gene is caspase-6-like); five caspase-8-like genes (note that four of consistent with previous reports indicating that this family these have the catalytic center sequence variant QACQG, as is diversified into distinct caspase-3 and caspase-7 genes within characteristic of their human counterparts caspase-8 and 10; see the vertebrate lineage (Bayascas et al., 2002) whereas the Supplemental Fig. S1); five ICE-like genes; and fourteen genes presence of a clear caspase-6-like gene indicates that the split that cluster together but not closely with any of the known between caspase-6 and caspases-3/7 occurred prior to the vertebrate caspases, which we designate as caspase-N (for divergence of the echinoderm and lineages. Finally, “Novel”). The novel gene that lacks a catalytic cysteine the sea urchin ICE-like set of caspases cluster with themselves (SPU_005875) contains six tandem leucine-rich repeats and do not have any individual orthologues among the (LRRs) encoded in a single N-terminal exon (with the caspase chordate ICE-like caspases (human caspases-1, -4, -5—see domain encoded in the second of two exons). The model is Fig. 3; C. intestinalis CSP4a-c—see Supplemental Fig. S2), validated by an EST (NCBI CX684859) that encompasses both suggesting that this subfamily of caspases may have arisen exons. LRRs are a protein–protein interaction domain often early within deuterostomes and then independently diversified found in extracellular or transmembrane proteins involved in within chordates and echinoderms [and indeed, the ICE-like immune response and defense against environmental pathogens genes underwent independent radiations within different (Pancer and Cooper, 2006). vertebrate lineages (Wang and Gu, 2001)]. While none of 326 A.J. Robertson et al. / Developmental Biology 300 (2006) 321–334 the sea urchin ICE-like caspases is linked to a CARD, at least and another (SPU_002921) has a region with low sequence one (SPU_011872), and possibly a second (SPU_021141) similarity to the DED. The latter (and possibly its sister contains sequence exhibiting similarity to the death domain, SPU_002923 as well) is/are also linked to immunoglobulin

Fig. 3. Family tree of the sea urchin caspases, and their relationship to the vertebrate (human) caspases, derived by the Neighbor-Joining method using a multiple sequence alignment of caspase domains. Nearly identical topologies were obtained using Minimum Evolution and Maximum Parsimony methods. For simplicity, only the last five digits of the sea urchin gene model number are given (i.e., the prefix “SPU_0” has been omitted); the human caspase is designated as Hs followed by the caspase number. Evidence for expression of each sea urchin gene from array tiling data (Samanta et al., 2006) is indicated next to the gene number by an asterisk (*), and from EST data by the symbol ^. Branches contained within a dashed box may represent variants of a single gene (i.e., haplotypes). Adaptor domains (DED and CARD) linked to the caspase domains are indicated by colored ovals. A.J. Robertson et al. / Developmental Biology 300 (2006) 321–334 327

(Ig) I-set and Ig V-set domains, protein–protein interaction (Fig. 4A). A tree based on the BIR domain alignment serves as a domains often associated with immune functions, intercellular guide to graphically represent the set of S. purpuratus BIR recognition, or cell adhesion (Smith and Xue, 1997). As protein architectures relative to sequence similarity between the noted above, SPU_002921 and SPU_002923 lack a catalytic BIR domains in the full proteins (Fig. 4B). Sequence similarity cysteine (having the sequence QGNRE instead of QACRG), and among the genes is largely confined to the sequence of the BIR thus are not likely to have apoptotic functions. domains. The alignment and tree (Figs. 4A, B) indicate that the Most of the CED-3-like caspases display low but detectable type I BIR domains fall into the three subtypes alluded to above, levels of expression in the embryo, as indicated both by tiling with the most downstream (or in one case, only) BIR domain of array signals (Samanta et al., 2006) and by a small number of each protein representing a subfamily most closely related to the EST sequences (evidence for expression from ESTs and/or more primitive type II BIR. The first BIR domain of all of the tiling array data is noted next to each model depicted in Fig. multi-BIR proteins falls into a second subfamily, whereas the 3). While as yet little is known of their functionality in middle BIR of the two 3-BIR proteins is the third subfamily apoptosis, there is abundant experimental evidence for (Fig. 4B). apoptosis in sea urchins, both in the late larva during normal There are two predicted S. purpuratus type II BIR domain metamorphosis (Roccheri et al., 1997, 2002) and in the proteins, SPU_008878 and SPU_001262. SPU_008878 is a embryo in response to stress or specific molecular perturba- small protein with a single survivin/Birc5-like BIR domain of tions (Roccheri et al., 1997; Voronina and Wessel, 2001; 147 amino acids (Fig. 4D). Evidence for its expression in the Lesser et al., 2003; Vega and Epel, 2004; Dickey-Sims et al., embryo is provided by full-length ESTs. The gene sequence 2005). Moreover, there is immunological evidence for encoding the single BIR domain in this gene is split by two activated caspase-3 in apoptotic cells in sea urchin embryos introns, the positions of which are conserved in human and sea (Dickey-Sims et al., 2005). About half of the novel caspases anemone survivin homologues (Fig. 4C). SPU_001262 predicts also display low levels of expression in the embryo and/or a 5224 amino acid protein with a single BIR domain similar to larva (indicated in Fig. 3), whereas the ICE-like caspases do that found in human Bruce/Birc6 (see tree in Fig. 4D). The gene not. It may be that the latter are functionally dedicated to the model is supported by two separate ESTs with coverage of the N- adult phase of the life cycle, possibly as part of the immune terminal region of the predicted gene. SPU_001262 also has a system (Hibino et al., 2006), although it is not currently predicted C-terminal UBCc (ubiquitin-conjugating enzyme E2, known whether or not they are expressed in the adult immune catalytic domain), which together with conserved intron cells. positions (Fig. 4C) further support its orthology with human Bruce/Birc6. Anti-apoptotic BIR domain/IAP proteins The remaining S. purpuratus BIR domain gene predictions are IAP homologues. SPU_014350 and SPU_017593 are The BIR (Baculovirus IAP Repeat) domain is a 70–80 amino highly similar sequences that may be haplotypes. There is full acid sequence found in a number of proteins that function to coverage of a consensus in the EST dataset for SPU_014350/ directly inhibit caspases or otherwise suppress apoptosis, 017593. As shown in Fig. 4 both models encode a protein providing additional layers of regulation (reviewed in Hengart- with three BIR domains, with a c-terminal zf-C3HC4 ring ner, 2000; Wang, 2001). The BIR domain, defined structurally finger domain present in SPU_017593. SPU_020648, by three conserved cysteines and one conserved histidine that SPU_014056 and SPU_014047 are three highly similar BIR coordinate a zinc ion, is found in , yeasts, and metazoans, domain gene sequences with a C-terminal ring finger domain, and exists in two types (Uren et al., 1998; Miller, 1999). Proteins and the latter two are present on the same scaffold and in the with a type I BIR domain are commonly referred to as IAPs. same orientation, indicating that they are probably tandem IAPs also typically contain a C3HC4 ring finger domain, and are duplications. A tree derived from multiple sequence alignment found in arthropods, vertebrates, and baculoviruses, but not in C. of S. purpuratus BIR domain proteins to those of other elegans. They are also not found in Saccaromyces cerevisiae, metazoans and yeast (Fig. 4D) suggests that S. purpuratus suggesting that they may be a metazoan invention. The type I IAPs compose a sea urchin-specific subfamily most similar to BIR domains of IAPs are often repeated, and fall into three H. sapiens Birc 2 and Birc 3. Unlike the situation in different subtypes based on sequence homology (Verhagen et al., , S. purpuratus apparently does not contain BIR 2001). Type II BIR domains are several amino acids larger than domain proteins associated with a CARD or NACHT domain. type I, and are found in the proteins survivin (Birc5) and Bruce In sum, the common ancestor of bilaterians appears to have (Birc6). Survivin is conserved from yeast to humans, which had both a primordial survivin-like type II BIR and a Birc6/ together with its conserved functions in mitosis, meiosis, and the Bruce orthologue containing a type II BIR associated with a cell cycle suggests that it represents the primitive BIR domain UBC, which has been lost in C. elegans. The bilaterian ancestor protein (reviewed in Miller, 1999; Verhagen et al., 2001; Yoo, also had a type I BIR/IAP associated with a ring domain (as 2005). found in N. vectensis SB_16819), which was also lost in the There are seven S. purpuratus GLEAN3 gene models that lineage leading C. elegans, whereas in other bilaterians it encode at least one BIR domain. An alignment of the translated underwent independent duplications/expansions, producing a BIR domain sequences present in these models reveals the complexity of architectures in sea urchins that is intermediate invariant residues in the C-terminal region of each BIR domain between that of H. sapiens and D. melanogaster. Given that 328 A.J. Robertson et al. / Developmental Biology 300 (2006) 321–334 A.J. Robertson et al. / Developmental Biology 300 (2006) 321–334 329

IAPs typically inhibit apoptosis by direct interaction with transmembrane domain. The three mammalian anti-apoptotic caspases, an interesting area for future research is the structural Bcl-2 paralogues (Bcl-2, Bcl-xL, and Bcl-2L2) are represented and functional co-evolution of the caspase and BIR domain by a single sea urchin homologue (SPU_024469; Fig. 5). The protein families in sea urchins. latter protein lacks a well-defined BH3 motif, but carries a sequence with similarity to the BH4 domain, further establish- Bcl-2 proteins ing its identity as an anti-apoptotic Bcl-2 homologue. SPU_024469 also lacks a transmembrane domain, but the The Bcl-2 family of proteins has both pro-apoptotic and anti- model contains only on exon and may be incomplete. Two apoptotic members that regulate apoptosis by controlling genes (SPU_006124 and SPU_021416) are most closely related permeabilization of the outer mitochondrial membrane (and to mammalian Bok, which also has the highest similarity to the hence release of cytochrome c and other apoptosis-inducing two fly Bcl-2 family members (Debc1 and Buffy). Two highly mitochondrial proteins; Gross et al., 1999; Daniel et al., 2003; similar sea urchin gene predictions containing an identical Bcl-2 Armstrong, 2006). Strictly speaking, the “Bcl-2” domain domain (SPU_001916 and SPU_016028; probably the same (PFAM PF00452) or “Bcl” domain (SMART00337) is gene) are orthologous to vertebrate Mil-1/Bcl2L13, whereas the composed of Bcl homology (BH) motifs BH1 and BH2. final two (SPU_010786 and SPU_017154) are not closely Many Bcl-2 domain proteins contain a BH3 motif as well, related to any of the other bilaterian representatives (Fig. 5). No which is required for dimerization and the killing function of clear homologues of the mammalian BH3-only proteins Bad, pro-apoptotic Bcl-2 family members, and a transmembrane Bid, and Bim were identified by BLAST searches. domain (Daniel et al., 2003). In vertebrates the Bcl-2 domain is There are seven Bcl-2 domains contained in gene predictions found in the pro-apoptotic Bcl-2 family proteins Bak and Bax, from the sea anemone (N. vectensis) genome, two of which are which promote mitochondrial permeabilization, and in the anti- identical and probably the same gene. Two of the anemone genes apoptotic proteins Bcl-2 and Bcl-xL, which inhibit apoptosis by are most closely related to the pro-apoptotic vertebrate proteins heterodimerizing with the pro-apoptotic proteins (reviewed in Bak and Bax, one is most similar to vertebrate Bok, and three Daniel et al., 2003; Kornbluth and White, 2005). The latter cluster with themselves and the sea urchin gene SPU_010786 proteins also contain an N-terminal BH4 domain that is (Fig. 5). Interestingly, none of them cluster with the anti- necessary for their anti-apoptotic activity (Daniel et al., 2003). apoptotic BH4-containing proteins, suggesting that the latter A number of “BH3-only” proteins add further layers of may be a deuterostome invention. The Bcl-2 family tree (Fig. 5) regulation; for example, the BH3-only proteins Bid and Bim suggests that there were at least four Bcl-2 domain genes in the activate the function of Bad and Bax (Hengartner, 2000; Wang, common bilaterian ancestor, and that some of these expanded in 2001; Armstrong, 2006). The human genome encodes at least deuterostomes and then diversified further independently in eleven proteins with a Bcl-2 domain, whereas Drosophila has chordates and echinoderms. In contrast, there was a severe two (pro-apoptotic Debc1/Drob-1/dBorg-1/dBok, and anti- reduction of Bcl-2 domain proteins in flies and worms, likely apoptotic Buffy/dBorg-2) and C. elegans only one (the anti- down to a single gene that subsequently duplicated once in apoptotic Ced-9) (Koonin and Aravind, 2002; Kornbluth and arthropods, with subsequent functional divergence of the two White, 2005). Although the C. elegans protein Egl-1 as well as duplicates (as suggested by the relatively high similarity the large set of vertebrate BH3-only proteins are often referred between the two fly genes). to as Bcl-2 family members, they share little sequence similarity outside of the short (9–16 amino acid) BH3 motif, and they Cell-extrinsic apoptotic and anti-apoptotic signaling were not systematically considered in our analysis. Ten proteins with the Bcl-2 domain were found in the gene As discussed in the Introduction, apoptosis can be induced by predictions from the S. purpuratus genome, representing a Bcl- extracellular signals (Fig. 1). These signals are typically carried 2 domain gene complexity similar to that of mammals. Of these, by ligands belonging to the tumor necrosis factor (TNF) family, only four are clearly orthologous to human counterparts, as which bind to their cognate transmembrane receptors, members shown by a neighbor-joining tree obtained from a CLUSTAL W of the tumor necrosis factor receptor (TNFR) family. TNF and multiple sequence alignment (Fig. 5). The pro-apoptotic Bcl-2 TNFR molecules can activate several distinct pathways that lead domain proteins Bax and Bak each have a single sea urchin to cell death. Among the best known are pathways involving the orthologue (SPU_010641 and SPU_014028, respectively), each death domain-containing death receptors Fas, DR4 and DR5, of which like their vertebrate counterparts carries a BH3 motif at and TNFR1 (for reviews see Bhardwaj and Aggarwal, 2003; the N-terminus of the Bcl-2 domain, as well as a C-terminal Khosravi-Far and Esposti, 2004; Jin and El-Deiry, 2005). These

Fig. 4. BIR domain protein phylogeny and domain architecture. (A) Multiple sequence alignment of the BIR domains from the seven predicted sea urchin BIR domain proteins. Numbers refer to the last five digits of the model number. (B) Neighbor-Joining tree calculated from the sequence alignment shown in panel A, and a schematic of the domain architecture of each predicted protein shown on the right (with orthologous BIR domains aligned); numbers as in panel A. BIR domains are represented by green ovals; a UBC-ligase domain by a purple rectangle; and ring fingers by a yellow pentagon. (C) Exon structure of the BIR domain encoding regions of type II BIR genes from H. sapiens, S. purpuratus, and N. vectensis. Amino acid sequences from different exons are shaded different colors. Note that the survivin orthologues have a slightly different exon architecture than the birc6/bruce orthologues. Sea urchin genes are listed as “Sp” followed by last five digits of the SPU model number. (D) The BIR domain protein family tree, produced by Neighbor-Joining from a Clustal W sequence alignment. The domain architecture of each protein is shown on the right. Domains are indicated as in panel B; additionally they include a CARD domain (orange parallelogram) and NACHT domain (blue oval). 330 A.J. Robertson et al. / Developmental Biology 300 (2006) 321–334

Fig. 5. Bcl-2 family tree, produced by Neighbor-Joining of a multiple sequence alignment of Bcl-2 domain (BH1+BH2 motif) sequences from S. purpuratus, H. sapiens, D. melanogaster, and N. vectensis. Unambiguous orthologues are shaded. Additional domains [BH3, BH4 and transmembrane (TM)] found linked to each Bcl-2 domain are indicated as colored boxes. receptors function in a similar way. After activation by the N. vectensis, as well as from the sequenced genomes of other binding of their cognate ligands, respectively FasL, Trail and model organisms. Seven potential TNFRs and four TNF ligands TNF-α, they combine together to form homotrimers that recruit were found in the sea urchin genome, two TNFRs and two via their cytoplasmic death domain-specific adaptor molecules, TNFs in C. intestinalis, and two TNFRs and one TNF in Ne- which allow them to interact with and activate DED-containing matostella (Fig. 6A and Supplemental Table S1). Although initiator caspases (i.e., caspases-8/10 family members; Fig. 1). In most of the TNFR sequences were partial, encoding either a addition to these “classic” death receptors, there are other TNFR death domain or cysteine-rich domains characteristic of the family members that contain a cytoplasmic death domain and TNFRs, we were able to classify each by reverse BLAST and can induce apoptosis, although they act through distinct phylogenetic analysis. No orthologues of either the death pathways. This latter group includes p75NTR/NGFR (p75 receptors Fas, DR4, DR5 or TNFR1, or of the ligands FasL, neurotrophin receptor or NGFR, nerve growth factor receptor) Trail or TNF-α were identified in the sea urchin, the ascidian or and Edar (Ectodysplasin-A receptor; Casaccia-Bonnefil et al., sea anemone genomes, suggesting that these classic death 1999; Bhardwaj and Aggarwal, 2003; Barker, 2004). receptors are vertebrate innovations. However, an orthologue A TBLASTN search was performed to identify TNF ligands of Edar, and two homologues of p75NTR/NGFR (one linked and TNF receptors in S. purpuratus genome. For comparison, to a death domain) were identified in the sea urchin we also collected predicted TNF and TNFR sequences from the (Edar, SPU_020955; p75NTR/NGFR, SPU_025699 and genomes of the urochordate C. intestinalis and the sea anemone SPU_026216) (Fig. 6B). Two homologues of p75NTR/NGFR A.J. Robertson et al. / Developmental Biology 300 (2006) 321–334 331 were found in the sea anemone, only one of which contains a LtBR, SPU_010180) as were their ligands (HEVM-L, death domain (shown in the alignment in Fig. 6B). Thus, death SPU_030072; TL1A, SPU_009528). domain-containing TNFR proteins evolved early during the Another set of players important for the transduction of evolution of metazoans, before the cnidarian/bilaterian diver- extrinsic apoptotic signals are the adaptor molecules. These gence, and were lost in the protostome lineages represented by molecules represent the link between the transmembrane flies and worms. A p75 ligand has been characterized in the TNFRs and the cytoplasmic initiator caspases with DEDs mollusk L. stagnalis (Fainzilber et al., 1996), which in our (i.e., caspases-8/10). Based on their domain organization, analysis showed most similarity to a TNF ligand called CiTNF these adapters can be separated in two groups: the adaptors isolated in the C. intestinalis genome (Fig. 6C). Orthologues of that contain a death domain, such as FADD, TRADD, several TNFRs lacking death domains, but which are implicated RAIDD (or CRADD) and Edaradd, and the adaptor without in either induction or inhibition of apoptosis (Bhardwaj and a death domain represented by the TRAF family (Fesik, Aggarwal, 2003; Hehlgans and Pfeffer, 2005), were also found 2000; Inoue et al., 2000). The death domain adaptors in the sea urchin genome (Supplemental Table S1) (Troy, interact directly with the death domain of the classic death SPU_020740; HVEM, SPU_024584; XEDAR, SPU_018915; receptors, whereas the TRAF proteins are more broadly

Fig. 6. Homologues of genes involved in extrinsic apoptotic signaling. (A) Distribution of TNF, TNFR and adaptor molecules throughout the animal kingdom. (B) Neighbor-Joining tree of TNFR family. The TNFR (cysteine-rich) and death domain architecture that typifies each subfamily is shown schematically. (C) Neighbor- Joining tree of the TNF family. Each TNFR or TNF subfamily is delimited by a colored rectangle. Accession numbers of all sequences used for these trees are indicated in Supplemental Tables S1 and S2. Hs, Homo sapiens; Mm, Mus musculus;Fr,Fugu rubripes; Gg, Gallus gallus; Ci, Ciona intestinalis; Sp, Strongylocentrotus purpuratus; Dm, Drosophila melanogaster; Ls, Lymnaea stagnalis;Nv,Nemanostella vectensis. 332 A.J. Robertson et al. / Developmental Biology 300 (2006) 321–334 used, interacting with death domain adaptors as well as further diversified in vertebrates (Fig. 6). In contrast, the directly with p75/NTR. cytoplasmic signaling machinery has been more conserved than In vertebrates, five death domain adaptors and six TRAF the extracellular signal transduction machinery, with nearly half proteins have been reported (Fig. 6A and Supplemental Table of the vertebrate adaptor molecules having orthologues in S1). Four of these are also present in flies (Fig. 6A) (Hu and cnidarians, echinoderms, arthropods and nematodes. Yang, 2000; Zapata et al., 2000; Horng and Medzhitov, 2001; Shen et al., 2001). Sequences encoding death domain adaptors Apoptosis-associated proteins with key roles in multiple and TRAF proteins are found in both sea urchins and sea cellular processes anemones. Orthologues of FADD (SPU_010777), RAIDD (SPU_023153) and MyD88 (SPU_007342) and of four of the The control of apoptosis is tightly linked to other six known TRAF proteins were identified (SPU_012840, fundamental cellular processes such as cell cycle control, 026495, 008332, and 028898). However, no orthologues of DNA metabolism, and cellular/immune defense, allowing for TRADD or Edaradd were found. Some adaptors have been elimination of mutant, genomically unstable, or infected cells. described as specifically interacting with particular TNFRs, for The linkage between these processes is typically mediated by example TRADD or Edaradd are only recruited by TNFR1 and well-known networks of kinases [e.g., mitogen-activated EDAR receptors respectively (Hsu et al., 1995; Headon et al., protein kinases (MAPKs), protein kinase A, B, and C family 2001). On the other hand, it has been reported that FADD and members, and checkpoints such as ATM, ATR, chk1 and chk2] RAIDD proteins can function as general adaptors that transduce and transcription factors [e.g., NF-κB and p53], most of which signals from multiple TNFRs, including TNFR1 and p75NTR have identifiable sea urchin homologues. These are not (Nichols et al., 1998; Shearwin-Whyatt et al., 2000). Thus, discussed here, as they are described in other papers in this whereas vertebrates have many adaptors, some of which may be issue (Bradham et al., 2006; Fernandez-Guerra et al., 2006; specific for a cognate receptor, echinoderms appear to have a Hibino et al., 2006). smaller number of more generalized adaptors. The N. vectensis genome encodes a similar set of adaptors, with orthologues of Conclusions every sea urchin adaptor except RAIDD. In sum, our analysis reveals a complexity of TNF/TNFR From this initial survey of the sea urchin genome it is genes in sea urchins that is intermediate between that of apparent that many of the protein families constituting the core ecdysozoan protostomes (flies and worms) and vertebrates. apoptotic toolkit have expanded and diversified in echino- Although the sea urchin apparently lacks orthologues of the derms, to an extent that mirrors their independent diversifica- classic Death receptors and their adaptor molecules, its genome tion in chordates. Furthermore, TNFR proteins with a death encodes homologues of p75NTR/NGFR and EDAR, indicating domain – previously thought to be a chordate invention – are that death domain-TNFRs are not specific to the vertebrates as found encoded in the genomes of both sea urchin and sea previously proposed (Bridgham et al., 2003). Further, the anemone, and hence were present prior to the emergence of existence of p75NTR homologues in sea anemone, as well as in bilaterians. These findings are consistent with the proposition echinoderms and urochordates, indicates that this family is the that early in animal evolution apoptosis became contingent most ancestral. Whereas the TNF/TNFR family tree was pruned upon a diverse array of environmental and/or developmental in the protostome lineage(s) leading to arthropods and signaling pathways, which continued to increase in complexity nematodes, it was expanded in the basal deuterostomes, and along different trajectories in the deuterostome lineages

Fig. 7. Summary of evolutionary insights provided by this study. The appearance or loss of different gene families, domains or domain architectures is indicated by shapes mapped onto a schematic of a phylogenetic tree, with appearances in color and losses in gray. A.J. Robertson et al. / Developmental Biology 300 (2006) 321–334 333 represented by sea urchins and mammals, while becoming Bhardwaj, A., Aggarwal, B.B., 2003. Receptor-mediated choreography of life – simplified somewhat in the specialized protostome lineages and death. J. Clin. Immunol. 23, 317 332. Bridgham, J.T., Wilder, J.A., Hollocher, H., Johnson, A.L., 2003. All in the represented by fruit flies and nematodes (Fig. 7). This scenario family: evolutionary and functional relationships among death receptors. is underscored by the fact that in addition to a typical CARD- Cell Death Differ. 10, 19–25. carrying Apaf-1 homologue, the sea urchin genome encodes Bradham, C.A., Foltz, K.R., Beane, W.S., Arnone, M.I., Rizzo, F., Coffman, J.A., two novel Apaf-1-like proteins that are each linked to a death Mushegian, A., Goel, M., Morales, J., Geneviere, A.-M., Lapraz, F., domain, suggesting the presence of unique apoptotic signaling Robertson, A.J., Kelkar, H., Loza-Coll, M., Townley, I.K., Raisch, M., Roux, M.M., Lapage, T., Gache, C., McClay, D.R., Manning, G., 2006. The sea pathways; by the fact that the caspase and Bcl-2 domain urchin kinome: a first look. Dev. Biol. 300, 180–193. families have undergone independent expansions in echino- Casaccia-Bonnefil, P., Gu, C., Khursigara, G., Chao, M.V., 1999. p75 derms and chordates, while the Bcl-2 family has been pruned neurotrophin receptor as a modulator of survival and death decisions. in ecdysozoans; and by the presence of death domain- Microsc. Res. Tech. 45, 217–224. containing TNF receptors in deuterostomes and cnidarians, Daniel, P.T., Schulze-Osthoff, K., Belka, C., Guner, D., 2003. Guardians of cell death: the Bcl-2 family proteins. Essays Biochem. 39, 73–88. and their absence in flies and worms. It is possible that the Davies, A.M., 2003. Regulation of neuronal survival and death by extracellular relatively complex apoptotic toolkit found in S. purpuratus,in signals during development. EMBO J. 22, 2537–2545. particular the expanded set of caspases, is an adaptation that Dickey-Sims, C., Robertson, A.J., Rupp, D.E., McCarthy, J.J., Coffman, J.A., facilitates extensive apoptosis that occurs during metamor- 2005. Runx-dependent expression of PKC is critical for cell survival in the phosis. Another intriguing possibility is that the sea urchin- sea urchin embryo. BMC Biol. 3, 18. Du, C., Fang, M., Li, Y., Li, L., Wang, X., 2000. Smac, a mitochondrial protein specific diversification of apoptosis-associated genes is related that promotes cytochrome c-dependent caspase activation by eliminating to the remarkable complexity of its immune system, which is IAP inhibition. Cell 102, 33–42. only now beginning to be revealed (Hibino et al., 2006). Ellis, H.M., Horvitz, H.R., 1986. Genetic control of programmed cell death in Finally, many of the genes described here may have enigmatic the nematode C. elegans. Cell 44, 817–829. functions related to unique aspects of sea urchin biology that Fainzilber, M., Smit, A.B., Syed, N.I., Wildering, W.C., Herman, van der Schors, R.C., Jimenez, C., Li, K.W., van Minnen, J., Bulloch, A.G., Ibanez, C.F., have yet to be discovered. These possibilities will provide Geraerts, W.P., 1996. CRNF, a molluscan neurotrophic factor that interacts interesting avenues for future research. with the p75 neurotrophin receptor. Science 274, 1540–1543. Fernandez-Guerra, A., Aze, A., Morales, J., Mulner-Lorillon, O., Cosson, B., Acknowledgments Cormier, P., Bradham, C.A., Adams, N., Robertson, A.J., Marzluff, W.F., Coffman, J.A., Geneviere, A.M., 2006. The genomic repertoire for cell cycle control and DNA metabolism in S. purpuratus. Dev. Biol. 300, We thank Dr. Chunying Du (Stowers Institute for Medical 238–251. Research), and Dr. Jonathan Rast and Cynthia Messier Fesik, S.W., 2000. Insights into programmed cell death through structural (Sunnybrook and Women's College Health Sciences Centre, biology. Cell 103, 273–282. University of Toronto) for reviewing the manuscript and Gross, A., McDonnell, J.M., Korsmeyer, S.J., 1999. BCL-2 family members and – providing helpful comments that led to its improvement prior the mitochondria in apoptosis. Genes Dev. 13, 1899 1911. Gupta, S., Su, H., Bi, R., Agrawal, S., Gollapudi, S., 2005. Life and death of to submission. Additional improvements were made based on the lymphocytes: a role in immunosenescence. Immun. Ageing 2, 12. perspicacious critiques of two anonymous reviewers. Apoptosis Headon, D.J., Emmal, S.A., Ferguson, B.M., Tucker, A.S., Justice, M.J., is a large and very active field of research, and many important Sharpe, P.T., Zonana, J., Overbeek, P.A., 2001. Gene defect in ectodermal papers were not cited here due to space limitations or lack of dysplasia implicates a death domain adapter in development. Nature 414, – scholarship on our part; we apologize to those authors. This work 913 916. Hehlgans, T., Pfeffer, K., 2005. The intriguing biology of the tumour necrosis was supported by NIH grants GM070840 (J.A.C.), GM61464 factor/tumour necrosis factor receptor superfamily: players, rules and the (D.R.M.) and HD14483 (D.R.M.). games. Immunology 115, 1–20. Hengartner, M.O., 2000. The biochemistry of apoptosis. Nature 407, Appendix A. Supplementary data 770–776. Hibino, T.S., Loza-Coll, M.A., Messier, C., Majeske, A., Cohen, A., Terwilliger, D., Buckley, K., Brockton, V., Nair, S., Berney, K., Fugmann, S.D., Supplementary data associated with this article can be found, Anderson, M.K., Pancer, Z., Cameron, R.A., Smith, L.C., Rast, J.P., 2006. in the online version, at doi:10.1016/j.ydbio.2006.08.053. The immune gene repertoire encoded in the purple sea urchin genome. Dev. Biol. 300, 349–365. Horng, T., Medzhitov, R., 2001. Drosophila MyD88 is an adapter in the Toll References signaling pathway. Proc. Natl. Acad. Sci. U. S. A. 98, 12654–12658. Hsu, H., Xiong, J., Goeddel, D.V., 1995. The TNF receptor 1-associated Aravind, L., Dixit, V.M., Koonin, E.V., 2001. Apoptotic molecular machinery: protein TRADD signals cell death and NF-kappa B activation. Cell 81, vastly increased complexity in vertebrates revealed by genome comparisons. 495–504. Science 291, 1279–1284. Hu, S., Yang, X., 2000. dFADD, a novel death domain-containing adapter protein Armstrong, J.S., 2006. Mitochondrial membrane permeabilization: the sine qua for the Drosophila caspase DREDD. J. Biol. Chem. 275, 30761–30764. non for cell death. BioEssays 28, 253–260. Inohara, N., Koseki, T., Hu, Y., Chen, S., Nunez, G., 1997. CLARP, a death Barker, P.A., 2004. p75NTR is positively promiscuous: novel partners and new effector domain-containing protein interacts with caspase-8 and regulates insights. Neuron 42, 529–533. apoptosis. Proc. Natl. Acad. Sci. U. S. A. 94, 10717–10722. Bayascas, J.R., Yuste, V.J., Benito, E., Garcia-Fernandez, J., Comella, J.X., Inoue, J., Ishida, T., Tsukamoto, N., Kobayashi, N., Naito, A., Azuma, S., 2002. Isolation of AmphiCASP-3/7, an ancestral caspase from amphioxus Yamamoto, T., 2000. Tumor necrosis factor receptor-associated factor (Branchiostoma floridae). Evolutionary considerations for vertebrate (TRAF) family: adapter proteins that mediate cytokine signaling. Exp. Cell caspases. Cell Death Differ. 9, 1078–1089. Res. 254, 14–24. 334 A.J. Robertson et al. / Developmental Biology 300 (2006) 321–334

Jin, Z., El-Deiry, W.S., 2005. Overview of cell death signaling pathways. Finnerty, J.R., 2006. StellaBase: the Nematostella vectensis Genomics Biol. Ther. 4, 139–163. Database. Nucleic Acids Res. 34, D495–D499. Kerr, J.F., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo, I., Snow, B.E., Brothers, phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer G.M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., Larochette, 26, 239–257. N., Goodlett, D.R., Aebersold, R., Siderovski, D.P., Penninger, J.M., Khosravi-Far, R., Esposti, M.D., 2004. Death receptor signals to mitochondria. Kroemer, G., 1999. Molecular characterization of mitochondrial Cancer Biol. Ther. 3, 1051–1057. apoptosis-inducing factor. Nature 397, 441–446. Koonin, E.V., Aravind, L., 2000. The NACHT family—A new group of Swofford, D.L., 1998. PAUP* Phylogenetic Analysis Using Parsimony, Version predicted NTPases implicated in apoptosis and MHC transcription 4 work. Sunderland, MA: Sinauer. activation. Trends Biochem. Sci. 25, 223–224. The Sea Urchin Genome Sequencing Consortium, submitted for publication. Koonin, E.V., Aravind, L., 2002. Origin and evolution of eukaryotic apoptosis: The genome of the sea urchin Strongylocentrotus purpuratus. Science. the bacterial connection. Cell Death Differ. 9, 394–404. Tibbetts, M.D., Zheng, L., Lenardo, M.J., 2003. The death effector domain Kornbluth, S., White, K., 2005. Apoptosis in Drosophila: neither fish nor fowl protein family: regulators of cellular homeostasis. Nat. Immunol. 4, (nor man, nor worm). J. Cell Sci. 118, 1779–1787. 404–409. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for Twomey, C., McCarthy, J.V., 2005. Pathways of apoptosis and importance in molecular evolutionary genetics analysis and sequence alignment. Brief. development. J. Cell. Mol. Med. 9, 345–359. Bioinform. 5, 150–163. Uren, A.G., Coulson, E.J., Vaux, D.L., 1998. Conservation of baculovirus Lesser, M.P., Kruse, V.A., Barry, T.M., 2003. Exposure to ultraviolet radiation inhibitor of apoptosis repeat proteins (BIRPs) in viruses, nematodes, causes apoptosis in developing sea urchin embryos. J. Exp. Biol. 206, vertebrates and yeasts. Trends Biochem. Sci. 23, 159–162. 4097–4103. van der Biezen, E.A., Jones, J.D., 1998. The NB-ARC domain: a novel Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S., signalling motif shared by plant resistance gene products and regulators of Wang, X., 1997. Cytochrome c and dATP-dependent formation of Apaf-1/ cell death in animals. Curr. Biol. 8, R226–R227. caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489. Vega, R.L., Epel, D., 2004. Stress-induced apoptosis in sea urchin embryogen- Li, L.Y., Luo, X., Wang, X., 2001. Endonuclease G is an apoptotic DNase when esis. Mar. Environ. Res. 58, 799–802. released from mitochondria. Nature 412, 95–99. Verhagen, A.M., Coulson, E.J., Vaux, D.L., 2001. Inhibitor of apoptosis Liu, X., Kim, C.N., Yang, J., Jemmerson, R., Wang, X., 1996. Induction of proteins and their relatives: IAPs and other BIRPs. Genome Biol. 2 apoptotic program in cell-free extracts: requirement for dATP and (REVIEWS3009). cytochrome c. Cell 86, 147–157. Verhagen, A.M., Ekert, P.G., Pakusch, M., Silke, J., Connolly, L.M., Reid, G.E., Miller, L.K., 1999. An exegesis of IAPs: salvation and surprises from BIR Moritz, R.L., Simpson, R.J., Vaux, D.L., 2000. Identification of DIABLO, a motifs. Trends Cell Biol. 9, 323–328. mammalian protein that promotes apoptosis by binding to and antagonizing Nichols, A., Martinou, I., Maundrell, K., Martinou, J.C., 1998. The p75 IAP proteins. Cell 102, 43–53. neurotrophin receptor: effects on neuron survival in vitro and interaction Voronina, E., Wessel, G.M., 2001. Apoptosis in sea urchin oocytes, eggs, and with death domain-containing adaptor proteins. Apoptosis 3, 289–294. early embryos. Mol. Reprod. Dev. 60, 553–561. Pancer, Z., Cooper, M.D., 2006. The evolution of adaptive immunity. Annu. Wang, X., 2001. The expanding role of mitochondria in apoptosis. Genes Dev. Rev. Immunol. 24, 497–518. 15, 2922–2933. Riedl, S.J., Shi, Y., 2004. Molecular mechanisms of caspase regulation during Wang, Y., Gu, X., 2001. Functional divergence in the caspase gene family and apoptosis. Nat. Rev., Mol. Cell Biol. 5, 897–907. altered functional constraints: statistical analysis and prediction. Genetics Roccheri, M.C., Barbata, G., Cardinale, F., Tipa, C., Bosco, L., Oliva, O.A., 158, 1311–1320. Cascino, D., Giudice, G., 1997. Apoptosis in sea urchin embryos. Biochem. Wang, X., Yang, C., Chai, J., Shi, Y., Xue, D., 2002. Mechanisms of AIF- Biophys. Res. Commun. 240, 359–366. mediated apoptotic DNA degradation in Caenorhabditis elegans. Science Roccheri, M.C., Tipa, C., Bonaventura, R., Matranga, V., 2002. Physiological 298, 1587–1592. and induced apoptosis in sea urchin larvae undergoing metamorphosis. Int. Weill, M., Philips, A., Chourrout, D., Fort, P., 2005. The caspase family in J. Dev. Biol. 46, 801–806. urochordates: distinct evolutionary fates in ascidians and larvaceans. Biol. Samanta, M.P., Tongprasit, W., Istrail, S., Cameron, R.A., Tu, Q., Davidson, Cell 97, 857–866. E.H., and Stolc, V., 2006. A high-resolution transcriptome map of the sea Yoo, S.J., 2005. BIR containing proteins (BIRPs): more than just cell death urchin embryo. (Submitted for publication). inhibitors. Integr. Biosci. 9, 181–190. Shearwin-Whyatt, L.M., Harvey, N.L., Kumar, S., 2000. Subcellular localiza- Zapata, J.M., Matsuzawa, S., Godzik, A., Leo, E., Wasserman, S.A., Reed, J.C., tion and CARD-dependent oligomerization of the death adaptor RAIDD. 2000. The Drosophila tumor necrosis factor receptor-associated factor-1 Cell Death Differ. 7, 155–165. (DTRAF1) interacts with Pelle and regulates NFkappaB activity. J. Biol. Shen, B., Liu, H., Skolnik, E.Y., Manley, J.L., 2001. Physical and functional Chem. 275, 12102–12107. interactions between Drosophila TRAF2 and Pelle kinase contribute to Zou, H., Henzel, W.J., Liu, X., Lutschg, A., Wang, X., 1997. Apaf-1, a human dorsal activation. Proc. Natl. Acad. Sci. U. S. A. 98, 8596–8601. protein homologous to C. elegans CED-4, participates in cytochrome c- Smith, D.K., Xue, H., 1997. Sequence profiles of immunoglobulin and dependent activation of caspase-3. Cell 90, 405–413. immunoglobulin-like domains. J. Mol. Biol. 274, 530–545. Zuzarte-Luis, V., Hurle, J.M., 2002. Programmed cell death in the developing Sullivan, J.C., Ryan, J.F., Watson, J.A., Webb, J., Mullikin, J.C., Rokhsar, D., limb. Int. J. Dev. Biol. 46, 871–876.