The echinoderm larval skeleton as a possible model system for experimental evolutionary biology
out of 7
Post on 04-Feb-2017
REVIEWThe Echinoderm Larval Skeleton as a Possible ModelSystem for Experimental Evolutionary BiologyHiroyuki Koga,* Yoshiaki Morino, and Hiroshi WadaGraduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, JapanReceived 28 November 2013; Revised 12 February 2014; Accepted 14 February 2014Summary: The evolution of various body plans resultsfrom the acquisition of novel structures as well as theloss of existing structures. Some novel structuresnecessitate multiple evolutionary steps, requiringorganisms to overcome the intermediate steps, whichmight be less adaptive or neutral. To examine thisissue, echinoderms might provide an ideal experimen-tal system. A larval skeleton is acquired in some echi-noderm lineages, such as sea urchins, probably via theco-option of the skeletogenic machinery that wasalready established to produce the adult skeleton. Theacquisition of a larval skeleton was found to requiremultiple steps and so provides a model experimentalsystem for reproducing intermediate evolutionarystages. The fact that echinoderm embryology has beenstudied with various natural populations also presentsan advantage. genesis 52:186192. VC 2014 Wiley Period-icals, Inc.Key words: echinoderm; larval skeleton; evolution of anovel structure; co-optionSince Aristotles description of the incredible variationof animals in his History of Animals (Peck, 1965, 1970,1991), more than 2000 years had passed before humansbegan to understand the diversity of life using the con-cept of evolution. What keeps people from acceptingthe concept of evolution? Perhaps one obstacle is theexistence of large gaps between animal body plans.This issue is now being overcome by recent progress inresearch on the evolution of development. Based onconserved molecular genetic tools for building animalbodies, much commonality exists among genetic mech-anisms for body construction (Carroll et al., 2010;Denes et al., 2007; Sasai and De Robertis, 1997). Withthe growing understanding of animal developmentmodes as well as increasing paleontological evidence,we are now filling the gaps and reconstructing the com-mon ancestors of multicellular animals.To fill the gaps completely, we must address the issueof the origin of novel structures. Co-option, the rede-ployment of an existing gene or organ to a new devel-opmental context, is a key concept that enables simpleexplanations for the evolution of novelty (True and Car-roll, 2002). The Dlx genes have been deployed repeat-edly for the evolution of body wall outgrowths(Panganiban et al., 1997). The co-option of a singlewingless gene was suggested to be sufficient for a novelwing color pattern in Drosophila species (Werneret al., 2010). As Darwin pointed out, however, under-standing how the evolution of novel organs of extremeperfection and complication is achieved is difficult,referring to the vertebrate eye as one of the difficultieswith his theory (Darwin, 1859). If the acquisition of anovel structure requires multiple evolutionary steps,how do creatures overcome the intermediate steps,which are apparently less adaptive (or neutral)?This article discusses how the echinoderm pluteuslarva is a good system for addressing the issue of theevolution of novel structures. Echinoderms have twotypes of larva: pluteus and auricularia types. The for-mer, which are seen in sea urchins and brittle stars, pos-sess well-developed skeletons that help to extend thelarval arms. The latter type is almost devoid of a larvalskeleton, and so the larval arms are not supported by askeleton. The latter type is seen in starfish and seacucumbers, and 10 years ago, a species of sea lily was* Correspondence to: Hiroyuki Koga, Graduate School of Life and Envi-ronmental Sciences, University of Tsukuba, Tennodai, Tsukuba 3058572, Japan. E-mail: email@example.comPublished online 18 February 2014 inWiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/dvg.22758VC 2014 Wiley Periodicals, Inc. genesis 52:186192 (2014)reported to have auricularia-type larva (Nakano et al.,2003). Because the sister group of echinoderms (hemi-chordates) and the basal group of echinoderms (the sealily) have auricularia-type larvae, the pluteus type isregarded as a derived state (Fig. 1). One key event inthe evolutionary transition from auricularia type to plu-teus type is the acquisition of a larval skeleton.CO-OPTION OF THE GENETIC MACHINERY FORSKELETOGENESISThe larval spicule does not develop, or is possibly degen-erative, in auricularia-type larvae. No larval spicule isobserved in starfish or hemichordates. Although a smallspicule(s) exists in the posterior part of the sea cucumberlarvae, it might represent a secondarily derived state froma pluteus form, as noted below. Conversely, all echino-derm species possess a calcitic endoskeleton called thestereom in adults. Among echinoderm characteristics,such as the pentaradial body plan and water vascular sys-tem, the endoskeleton of adults is the oldest charactershared by extinct species. The basal group of echino-derms (stylophorans) is classified as echinoderms becauseof their stereom, although they lack the pentaradial bodyplan and water vascular system (Clausen and Smith, 2005;Smith, 2005). In sea urchins, the larval skeletons are usu-ally derived from primary mesenchyme cells (PMCs),while Yajima (2007) revealed that PMCs do not contributeadult skeletal elements, indicating that larval and adultskeletons are derived from distinct cell populations.Nevertheless, structural and chemical similarities existbetween the larval and adult skeletons of sea urchins (Ber-man et al., 1993; Killian and Wilt, 1989; Killian et al.,2010; Kitajima et al., 1996; Livingston et al., 2006; Mannet al., 2008a, 2008b, 2010; Richardson et al., 1989). Gaoand Davidson (2008) showed that several transcriptionfactors are expressed in common during larval and adultskeletogenesis in sea urchins. These findings suggestedthat acquisition of the larval skeleton is a product of co-option of the adult skeletogenic machinery into larvalcells (Ettensohn, 2009; Gao and Davidson, 2008; Sharmaand Ettensohn, 2010). Here, to document the similarity ofskeletogenesis between adults and pluteus larvae, wedescribe adult skeletogenesis in starfish in more detail.SKELETOGENESIS DURING METAMORPHOSISIN STARFISHThe starfish Patiria (Asterina) pectinifera undergoestypical indirect development, undergoing two larvalstages: bipinnaria and brachiolaria. Adult spicules beginto form before metamorphosis. At the onset of metamor-phosis, larvae already possess 11 large spicules and manysmall spicules in the adult rudiment (Hamanaka et al.,2011; Hyman, 1955). When the adult rudiment isobserved from the future aboral side, the large spiculesalign in a concentric pattern: five on the outside, anotherfive more internally, and one in the center (Fig. 2e;Hamanaka et al., 2011). In juveniles, the outer spiculescompose the tips of the star arms, whereas the inner andcenter spicules develop into the aboral ossicles, includ-ing the hydropore (Hyman, 1955). Here, we brieflydescribe how this pattern of spicules becomes estab-lished. The first sign of spiculogenesis is observed in 1-week-old bipinnaria larvae (Fig. 2a). Mineralization isobserved beside the left somatocoel as tiny deposits ofcalcite. As the larva develops, more spicules arise andgrow into a mesh-like arrangement along the left sideand then the right side of the stomach, resulting in tworows of spicules (Fig. 2b,c). The fact that spicules formon the left first might reflect the developmental progressof the somatocoel; the left somatocoel expands towardFIG. 1. Phylogenetic framework of the echinoderm larvae. Sea urchins and brittle stars have pluteus larvae with developed skeletons,whereas starfish and sea cucumbers have auricularia larvae. The basal echinoderm (the sea lily) and the sister group of echinoderms (acornworms) have auricularia larvae, which are regarded as the ancestral type.EVOLUTION OF ECHINODERM LARVAL SKELETON 187the right side to surround the stomach. Note that the col-linear expression of sea urchin Hox genes was observedalong the somatocoel from the left-hand side (Arenas-Mena et al., 2000). Clumps of round mesenchymal cellsare observed around the growing spicules (Fig. 2f; Hama-naka et al., 2011), which is reminiscent of the larval skel-etogenic mesenchymal cells in the sea urchin. Similarclumps of mesenchymal cells were observed surround-ing the spicules in juvenile sea urchins and adult seacucumbers (MacBride, 1903; Smith et al., 2008; Wood-land, 1906). Finally, spicules from the left row becomethe five outer ones and spicules from the right rowbecome the inner ones and center one (Fig. 2d,e).From a molecular perspective, some homologs of seaurchin skeletogenic genes have been reported to markthese skeletogenic cells in adult juvenile starfish, includ-ing Ets1, Alx1, Hex (Gao and Davidson, 2008), andvegfr (Fig. 3ce; Morino et al., 2012). Perhaps undercontrol of these genes, the effector genes of skeletogen-esis, such as the carbonic anhydrase gene ApCA1, showspecific expression in skeletogenic cells (Fig. 3a,b; Mor-ino et al., 2012).COMPARISON OF GENE REGULATORYNETWORKS BETWEEN SEA URCHINSKELETOGENESIS AND STARFISH MESODERMThe above comparison of the gene regulatory machin-ery supports the idea that acquisition of the pluteuslarval skeleton was achieved via co-option of thegenetic machinery for adult skeletogenesis. Therefore,to search for the key molecular events in co-option, wefocused on the genes that are involved in both adultand pluteus larval skeletogenesis of the sea urchin, butnot in the mesoderm differentiation of starfish larvae.Several research groups, including ours, have searchedfor the key molecules. Unexpectedly, however, most ofthe transcription factors involved in sea urchin larvalskeletogenesis are also expressed in the mesoderm cellsof starfish larvae, which do not develop a skeleton (Hin-man and Davidson, 2007; Hinman et al., 2009; Kogaet al., 2010; McCauley et al., 2010; Shoguchi et al.,2000). Vascular endothelial growth factor (VEGF) sig-naling is the only potential candidate responsible forthe co-option so far (Morino et al., 2012).MOLECULAR ASPECTS OF CO-OPTION OF THESKELETOGENIC MACHINERYIn sea urchin larvae, the VEGF receptor is expressed inskeletogenic mesenchymal cells; the ligand is expressedmainly in epidermal cells adjacent to the skeletogenicmesenchymal cells and later in the tips of the larvalarms toward which the skeletal rods elongate. Inhibi-tion of VEGF signaling by either VEGF ligand or recep-tor led to a loss of skeleton (Duloquin et al., 2007). Adetailed study by Adomako-Ankomah and EttensohnFIG. 2. Spiculogenesis in the starfish Patiria (Asterina) pectinifera. (ad) Dorsal confocal images of starfish larva that were raised in artifi-cial seawater containing calcein: (a) tiny spicules (arrowheads) stained by calcein were observed along the left somatocoel of 1-week-oldlarvae; (b) the spicules grew in a branching manner, and new spicules arose along the right side of the stomach in late bipinnaria; (c) in earlybrachiolaria, a two-row pattern of major spicules was observed. The arrow indicates spicules located on the oral side; (d) in late brachio-laria, 11 major spicules have formed (eight are seen in the figure: the left three are outer spicules (arrowhead); the right five are inner spi-cules (arrow) and centric spicule (double arrow)); (e) view from adult aboral side of a late brachiolaria (lipid membranes were stained inmagenta). Arrowheads indicate five outer spicules. (f) Round mesenchymal cells crowded around developed spicules in early brachiolarialarva. The scale bars represent 50 mm.188 KOGA ET AL.(2013) provided evidence that VEGF signaling isrequired not only for initiating skeletogenesis but alsofor elongation of the skeletal rods toward animal poles.Morino et al. (2012) examined the expression of ligandand receptors in starfish and found that both wereexpressed during adult skeletogenesis in starfish, butnot in larval development (Fig. 3e,f). VEGF expressionwas also observed in sea urchin adult skeletogenesis(Gao and Davidson, 2008). Therefore, VEGF is likelyone of the key factors responsible for the acquisition ofa larval skeleton.Notably, both the VEGF ligand and receptor areexpressed during larval skeletogenesis in brittle stars(Morino et al., 2012). Because brittle stars are not phy-logenetically close to sea urchins within echinoderms,as sea urchins are perhaps more closely related to seacucumbers (Fig. 1; Janies, 2001; Littlewood et al., 1997;Paul and Smith, 1984; Wada and Satoh, 1994), the larvalskeleton is thought to have been acquired independ-ently in sea urchins and brittle stars (Smith, 1984). Con-sequently, the co-option of VEGF signaling must haveoccurred independently in brittle stars and sea urchins.Alternatively, the common ancestor of brittle stars andsea urchins acquired a larval skeleton, which wasreduced secondarily in sea cucumbers. This alternativehypothesis is equally parsimonious with the hypothesisof independent acquisition. The hypothesis of second-ary loss in sea cucumbers is favored if the activation ofVEGF signaling requires activation of receptors in skele-togenic cells as well as a ligand in adjacent ectodermcells. This idea is more consistent with the discoverythat Alx1 expression in larval mesenchyme cells isshared between sea urchins and sea cucumbers, butnot starfish (McCauley et al., 2012).FROM ESSENTIAL TO SUFFICIENTWe argue that the genetic regulatory network is similarbetween the skeletogenic mesoderm of sea urchins andthe nonskeletogenic larval mesoderm of starfish. Thissimilarity encourages us to perform trials to reproducethe evolutionary process by inducing development of alarval skeleton in starfish larvae. Using ascidians, Abituaet al. (2012) recently reported a notable study in whichthey induced a motile neural crest-like cell by ectopi-cally expressing a single transcription factor, twist, inmelanocytes. Freitas et al. (2012) succeeded in induc-ing an autopod-like structure in the zebrafish fin via theforced expression of Hoxd13. Similarly, we can inducethe ectopic expression of genetic material lacking forskeletogenesis in the starfish embryo. In this case, theVEGF ligand and receptor is an immediate candidate forthis strategy. A series of comparative developmentalstudies provided a list of essential conditions for acquisi-tion of the novel structure, but this synthetic experi-mental evolution (Erwin and Davidson, 2009)FIG. 3. Gene expression correlated with spicules. (a) Gene expression pattern of ApCA1 from the aboral side of brachiolaria. The arrow-heads indicate the staining along left side of stomach, whereas the arrows indicate the staining along right side of stomach. The doublearrowhead indicates staining at oral side. The two-row pattern of spicules was detected consistently. Asterisks indicate nonspecific signalsby dusts. (bd) Gene expression pattern from the left lateral side of brachiolaria: (b) ApCA1 was expressed in clumps of mesenchymal cellslocated between the coelom and epidermis in a pentaradial pattern (arrowheads). (c) Apets1/2 was also expressed in mesenchymal cellswithin the adult rudiment. (d) Apalx1 was expressed in mesenchymes of the adult rudiment with a slightly more restricted manner. (e)Apvegfr was expressed in mesenchymal cells located between the coelom and epidermis. (f) Apvegf was expressed in the epidermis sur-rounding the stomach. The scale bars represent 100 mm.EVOLUTION OF ECHINODERM LARVAL SKELETON 189approach tells us that sufficient steps exist for the novelstructure.A MODEL FOR MULTISTEP EVOLUTIONDetermining how many elementary steps are sufficientfor a certain form of morphological evolution would beinsightful. However, the final goal is not to list therequired steps for morphological evolution, but toresolve Darwins dilemma: how to overcome an inter-mediate stage that is considered to be less adaptive.When thinking about the larval activation of VEGF sig-naling in sea urchin larvae, one may reasonably assumethat an intermediate stage occurred in which eitheronly the ligand was expressed in the epidermis or onlythe receptor was expressed in mesenchyme cells, sothat VEGF signaling was not activated. Two possibleexplanations can be considered. First, the expression ofeither the ligand or receptor is almost neutral or at leastnot seriously deleterious and can consequently beretained for some duration to await activation of thecounterpart. Most echinoderm species have large popu-lations, which should harbor genetic variation, asreported in the sea urchin (Garfield et al., 2012, 2013;Pespeni et al., 2012, 2013a,b). In addition, the activa-tion of the counterpart did not have to occur in thesame individuals, as the activation of VEGF signalingcan be achieved via sexual reproduction (Fig. 4a).Second, if either the ligand or receptor is adaptive, itcan spread in a population more rapidly (Fig. 4b). Thisidea is testable in echinoderm species in which mRNAis easily injected, so an intermediate stage can bemimicked relatively easily. Recent advances in next-generation sequencing will enable us to detect frac-tional gene expression, even if no morphological phe-notype is observed.ECHINODERMS AS A MODEL FOR EVOLUTIONFinally, we discuss the advantageous features of echino-derms for studying evolutionary development, such astheir utility in studying experimental embryology, vari-ous morphologies, and availability of natural popula-tions. In terms of embryology, echinoderms, especiallysea urchins and starfish, are among the best-studiedmarine invertebrates. Therefore, we benefit fromdetailed information and elaborate techniques of exper-imental embryology. On this basis, studies have beenconducted using various classes of echinoderm(Dupont et al., 2009; Hara et al., 2006; Hirokawa et al.,2008; Koga et al., 2010; McCauley et al., 2012; Morinoet al., 2012). Furthermore, echinoderm embryology hasbeen investigated using individuals from natural popula-tions, while studies of other model animals have mostlyinvolved laboratory strains. This allows us to combinepopulation genetics and embryology readily (Garfieldet al., 2013; Pespeni et al., 2013a). As we discussedabove, the variation in gene expression within a popula-tion might be an important condition for achieving mul-tistep evolution. To test this idea, echinoderms offer anexcellent system.In Ontogeny and Phylogeny, Gould (1977) wrote,There may be nothing new under the sun, but permu-tation of the old within complex systems can doFIG. 4. Evolutionary scheme for the activation of VEGF signaling. To activate VEGF signaling, the simultaneous expression of ligand andreceptor genes is required. (a) Assuming that ectopic expression of the ligand or receptor alone is neutral, it can be retained as a variant ina population by chance. Sexual reproduction might create a combination of VEGF- and VEGFr-expression variants, so that individuals inwhich VEGF signaling is activated can occur. (b) If the ectopic expression of either the ligand or receptor is adaptive, it can spread among apopulation by selection. After fixation of the ligand or receptor, VEGF signaling can be activated by a change in the other.190 KOGA ET AL.wonders. . . This is the chief joy of our science. Howdoes nature allow permutations? Perhaps the key issueis how creatures overcome transitory, intermediatestages. To study this issue, we believe that echinodermsprovide a model experimental system.LITERATURE CITEDAbitua PB, Wagner E, Navarrete IA, Levine M. 2012.Identification of a rudimentary neural crest in anon-vertebrate chordate. Nature 492:104107.Adomako-Ankomah A, Ettensohn CA. 2013. Growthfactor-mediated mesodermal cell guidance and skel-etogenesis during sea urchin gastrulation. Develop-ment 140:42144225.Arenas-Mena CS, Cameron AR, Davidson EH. 2000. Spa-tial expression of Hox cluster genes in the ontogenyof a sea urchin. Development 127:46314643.Berman A, Hanson J, Leiserowitz L, Koetzle TF, WeinerS, Addadi L. 1993. Biological control of crystal tex-ture: A widespread strategy for adapting crystalproperties to function. Science 259:776779.Carroll SB, Grenier JK, Weatherbee SD. 2010. FromDNA to diversity: Molecular genetics and the evolu-tion of animal design. Malden: Blackwell.Clausen S, Smith AB. 2005. Palaeoanatomy and biologi-cal affinities of a Cambrian deuterostome (Stylo-phora). Nature 438:351354.Darwin C. 1859. On the origin of species by means ofnatural selection: Or the preservation of favoredraces in the struggle for life. London: John Murray.Denes AS, Jekely G, Steinmetz PR, Raible F, Snyman H,Prudhomme B, Ferrier DE, Balavoine G, Arendt D.2007. Molecular architecture of annelid nerve cordsupports common origin of nervous system central-ization in bilateria. Cell 129:277288.Duloquin L, Lhomond G, Gache C. 2007. LocalizedVEGF signaling from ectoderm to mesenchyme cellscontrols morphogenesis of the sea urchin embryoskeleton. Development 134:22932302.Dupont S, Thorndyke W, Thorndyke MC, Burke RD.2009. Neural development of the brittlestarAmphiura filiformis. Dev Genes Evol 219:159166.Erwin DH, Davidson EH. 2009. The evolution of hier-archical gene regulatory networks. Nat Rev Genet10:141148.Ettensohn CA. 2009. Lessons from a gene regulatorynetwork: Echinoderm skeletogenesis providesinsights into evolution, plasticity and morphogene-sis. Development 136:1121.Freitas R, Gomez-Marn C, Wilson Jonathan M, CasaresF, Gomez-Skarmeta Jose L. 2012. Hoxd13 contribu-tion to the evolution of vertebrate appendages. DevCell 23:12191229.Gao F, Davidson EH. 2008. Transfer of a large gene regu-latory apparatus to a new developmental address inechinoid evolution. Proc Natl Acad Sci USA 105:60916096.Garfield D, Haygood R, Nielsen WJ, Wray GA. 2012.Population genetics of cis-regulatory sequences thatoperate during embryonic development in the seaurchin Strongylocentrotus purpuratus. Evol Dev14:152167.Garfield DA, Runcie DE, Babbitt CC, Haygood R,Nielsen WJ, Wray GA. 2013. The impact of geneexpression variation on the robustness and evolv-ability of a developmental gene regulatory network.PLoS Biol 11:e1001696.Gould SJ. 1977. Ontogeny and phylogeny. Cambridge:Belknap Press of Harvard University Press.Hamanaka G, Hosaka E, Kuraishi R, Hosoya N,Matsumoto M, Kaneko H. 2011. Uneven distributionpattern and increasing numbers of mesenchymecells during development in the starfish, Asterinapectinifera. Dev Growth Differ 53:440449.Hara Y, Yamaguchi M, Akasaka K, Nakano H, Nonaka M,Amemiya S. 2006. Expression patterns of Hox genesin larvae of the sea lily Metacrinus rotundus. DevGenes Evol 216:797809.Hinman VF, Davidson EH. 2007. Evolutionary plasticityof developmental gene regulatory network architec-ture. Proc Natl Acad Sci USA 104:1940419409.Hinman VF, Yankura KA, McCauley BS. 2009. Evolutionof gene regulatory network architectures: Examplesof subcircuit conservation and plasticity betweenclasses of echinoderms. Biochim Biophys Acta1789:326332.Hirokawa T, Komatsu M, Nakajima Y. 2008. Develop-ment of the nervous system in the brittle starAmphipholis kochii. Dev Genes Evol 218:1521.Hyman LH. 1955. The invertebrates: Echinodermata:The coelomate bilateria, Vol. IV. New York:McGraw-Hill Book C.Janies D. 2001. Phylogenetic relationships of extantechinoderm classes. Can J Zool 79:12321250.Killian CE, Croker L, Wilt FH. 2010. SpSM30 gene familyexpression patterns in embryonic and adult biomin-eralized tissues of the sea urchin, Strongylocentro-tus purpuratus. Gene Expr Patterns 10:135139.Killian CE, Wilt FH. 1989. The accumulation and transla-tion of a spicule matrix protein mRNA during seaurchin embryo development. Dev Biol 133:148156.Kitajima T, Tomita M, Killian CE, Akasaka K, Wilt FH.1996. Expression of spicule matrix protein geneSM30 in embryonic and adult mineralized tissues ofsea urchin Hemicentrotus pulcherrimus. DevGrowth Differ 38:687695.Koga H, Matsubara M, Fujitani H, Miyamoto N, KomatsuM, Kiyomoto M, Akasaka K, Wada H. 2010. Func-tional evolution of Ets in echinoderms with focusEVOLUTION OF ECHINODERM LARVAL SKELETON 191on the evolution of echinoderm larval skeletons.Dev Genes Evol 220:107115.Littlewood DTJ, Smith AB, Clough KA, Emson RH.1997. The interrelationships of the echinodermclasses: Morphological andmolecular evidence. BiolJ Linn Soc 61:409438.Livingston BT, Killian CE, Wilt F, Cameron A, LandrumMJ, Ermolaeva O, Sapojnikov V, Maglott DR,Buchanan AM, Ettensohn CA. 2006. A genome-wideanalysis of biomineralization-related proteins in thesea urchin Strongylocentrotus purpuratus. DevBiol 300:335348.MacBride E W. 1903. The development of Echinus escu-lentus, together with some points in the develop-ment of E. miliaris and E. acutus. Philos Trans RSoc Lond B Biol Sci 195:285327.Mann K, Poustka AJ, Mann M. 2008a. In-depth, high-accuracy proteomics of sea urchin tooth organicmatrix. Proteome Sci 6:33..Mann K, Poustka AJ, Mann M. 2008b. The sea urchin(Strongylocentrotus purpuratus) test and spineproteomes. Proteome Sci 6:22.Mann K, Wilt FH, Poustka AJ. 2010. Proteomic analysisof sea urchin (Strongylocentrotus purpuratus) spi-cule matrix. Proteome Sci 8:33.McCauley BS, Weideman EP, Hinman VF. 2010. A con-served gene regulatory network subcircuit drivesdifferent developmental fates in the vegetal pole ofhighly divergent echinoderm embryos. Dev Biol340:200208.McCauley BS, Wright EP, Exner C, Kitazawa C, HinmanVF. 2012. Development of an embryonic skeleto-genic mesenchyme lineage in a sea cucumberreveals the trajectory of change for the evolution ofnovel structures in echinoderms. EvoDevo 3:111.Morino Y, Koga H, Tachibana K, Shoguchi E, KiyomotoM, Wada H. 2012. Heterochronic activation of VEGFsignaling and the evolution of the skeleton in echi-noderm pluteus larvae. Evol Dev 14:428436.Nakano H, Hibino T, Oji T, Hara Y, Amemiya S. 2003.Larval stages of a living sea lily (stalked crinoid echi-noderm). Nature 421:158160.Panganiban G, Irvine SM, Lowe C, Roehl H, Corley LS,Sherbon B, Grenier JK, Fallon JF, Kimble J, WalkerM, Wray GA, Swalla BJ, Martindale MQ, Carroll SB.1997. The origin and evolution of animal appen-dages. Proc Natl Acad Sci USA 94:51625166.Paul CRC, Smith AB. 1984. The early radiation and phy-logeny of echinoderms. Biol Rev 59:443481.Peck AL. 1965. Aristotle history of animals books IIII.Cambridge: Harvard University Press.Peck AL. 1970. Aristotle history of animals books IVVI.Cambridge: Harvard University Press.Peck AL. 1991. Aristotle history of animals books VIIX.Cambridge: Harvard University Press.Pespeni MH, Barney BT, Palumbi SR. 2013a. Differencesin the regulation of growth and biomineralizationgenes revealed through long-term common-gardenacclimation and experimental genomics in the pur-ple sea urchin. Evolution 67:19011914.Pespeni MH, Garfield DA, Manier MK, Palumbi SR.2012. Genome-wide polymorphisms show unex-pected targets of natural selection. Proc Biol Sci279:14121420.Pespeni MH, Sanford E, Gaylord B, Hill TM, Hosfelt JD,Jaris HK, LaVigne M, Lenz EA, Russell AD, YoungMK, Palumbi SR. 2013b. Evolutionary change duringexperimental ocean acidification. Proc Natl AcadSci USA 110:69376942.Richardson W, Kitajima T, Wilt F, Benson S. 1989.Expression of an embryonic spicule matrix gene incalcified tissues of adult sea urchins. Dev Biol 132:266269.Sasai Y, De Robertis EM. 1997. Ectodermal patterning invertebrate embryos. Dev Biol 182:520.Sharma T, Ettensohn CA. 2010. Activation of the skele-togenic gene regulatory network in the early seaurchin embryo. Development 137:11491157.Shoguchi E, Satoh N, Maruyama YK. 2000. A starfishhomolog of mouse T-brain-1 is expressed in thearchenteron of Asterina pectinifera embryos: Possi-ble involvement of two T-box genes in starfish gas-trulation. Dev Growth Differ 42:6168.Smith AB. 1984. Classification of the echinodermata.Palaeontology 27:431459.Smith AB. 2005. The pre-radial history of echinoderms.Geol J 40:255280.Smith MM, Cruz Smith L, Cameron RA, Urry LA. 2008.The larval stages of the sea urchin, Strongylocentro-tus purpuratus. J Morphol 269:713733.True JR, Carroll SB. 2002. Gene co-option in physiologi-cal and morphological evolution. Annu Rev CellDev Biol 18:5380.Wada H, Satoh N. 1994. Phylogenetic relationshipsamong extant classes of echinoderms, as inferredfrom sequences of 18s rdna, coincide with relation-ships deduced from the fossil record. J Mol Evol 38:4149.Werner T, Koshikawa S, Williams TM, Carroll SB. 2010.Generation of a novel wing colour pattern by theWingless morphogen. Nature 464:11431148.Woodland W. 1906. The Scleroblastic Development ofthe Spicules in Cucumariidae. Q J Microsc Sci 49:533559.Yajima M. 2007. A switch in the cellular basis of skeleto-genesis in late-stage sea urchin larvae. Dev Biol 307:272281.192 KOGA ET AL.