The echinoderm larval skeleton as a possible model system for experimental evolutionary biology

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    The Echinoderm Larval Skeleton as a Possible ModelSystem for Experimental Evolutionary Biology

    Hiroyuki Koga,* Yoshiaki Morino, and Hiroshi Wada

    Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, Japan

    Received 28 November 2013; Revised 12 February 2014; Accepted 14 February 2014

    Summary: 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-option

    Since 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 305

    8572, Japan. E-mail: hiro1224koga@gmail.comPublished online 18 February 2014 in

    Wiley Online Library (

    DOI: 10.1002/dvg.22758

    VC 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.


    The 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 transcription

    factors 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.


    The 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 toward

    FIG. 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.


  • the 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).


    The above comparison of the gene regulatory machin-ery supports the idea that acquisition of the pluteus

    larval 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).


    In 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 Ettensohn

    FIG. 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 of

    VEGF 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).


    We 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...


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