Overview The rise of the starlet sea anemone Nematostella ...inbios/Faculty/Layden/PDF/...Overview The rise of the starlet sea anemone Nematostella vectensis as a model system to investigate

Overview

The rise of the starlet sea anemoneNematostella vectensis as a modelsystem to investigate developmentand regenerationMichael J. Layden,1* Fabian Rentzsch2 and Eric Röttinger3

Reverse genetics and next-generation sequencing unlocked a new era in biology.It is now possible to identify an animal(s) with the unique biology most relevantto a particular question and rapidly generate tools to functionally dissect thatbiology. This review highlights the rise of one such novel model system, the star-let sea anemone Nematostella vectensis. Nematostella is a cnidarian (corals, jellyfish,hydras, sea anemones, etc.) animal that was originally targeted by EvoDevoresearchers looking to identify a cnidarian animal to which the development ofbilaterians (insects, worms, echinoderms, vertebrates, mollusks, etc.) could becompared. Studies in Nematostella have accomplished this goal and informed ourunderstanding of the evolution of key bilaterian features. However, Nematostellais now going beyond its intended utility with potential as a model to betterunderstand other areas such as regenerative biology, EcoDevo, or stressresponse. This review intends to highlight key EvoDevo insights from Nematos-tella that guide our understanding about the evolution of axial patterningmechanisms, mesoderm, and nervous systems in bilaterians, as well as to discussbriefly the potential of Nematostella as a model to better understand the relation-ship between development and regeneration. Lastly, the sum of research to datein Nematostella has generated a variety of tools that aided the rise of Nematostellato a viable model system. We provide a catalogue of current resources and tech-niques available to facilitate investigators interested in incorporating Nematostellainto their research. © 2016 Wiley Periodicals, Inc.

How to cite this article:WIREs Dev Biol 2016. doi: 10.1002/wdev.222

INTRODUCTION

Many EvoDevo researchers wished to under-stand how evolutionary radiation gave rise to

the impressive array of biodiversity present in agroup of animals called the bilaterians. To answerthis question, biologists sought to infer the bilaterianmolecular and morphological ‘starting point’ by iden-tifying ancestral traits likely present in the ancestorof all bilaterians (the urbilaterian) through compari-sons to non-bilaterian species. Cnidarian animals(hydras, jellyfish, corals, siphonophores, and sea ane-mones) are widely accepted to be the sister taxon tobilaterians1–3 (Figure 1(b)). The sister relationshipmakes cnidarians the most closely related non-bilaterians and the obvious group to target for devel-oping a model animal. The Cnidaria have two mainclades, the Medusazoa (hydras, jellyfish, and sipho-nophores) and the Anthozoa (e.g., corals, sea

*Correspondence to: [email protected] of Biological Sciences, Lehigh University, Bethlehem,PA, USA2Sars Centre for Marine Molecular Biology, University of Bergen,Bergen, Norway3Institute for Research on Cancer and Aging (IRCAN), CNRSUMR 7284, INSERM U1081, Université de Nice-Sophia-Antipolis,Nice, France

Conflict of interest: The authors have declared no conflicts of inter-est for this article.

© 2016 Wiley Per iodica ls , Inc.

anemones) (Figure 1(b)). Hydra had long been usedas an animal model to better understand regenerationand principles of patterning; however, they producesmall numbers of embryos in culture, and thoseembryos are poorly accessible during most of theirdevelopment.4,5 In 1992, Hand and Uhlingerreported a protocol to maintain the complete lifecycle of Nematostella in culture, which allowed forrobust investigation of cnidarian development.4 AsNematostella, a number of other cnidarian speciessuch as Clytia and Hydractinia have been developedas models.5,6 However, Nematostella is the mostdeveloped and commonly used anthozoan cnidarianmodel. Together investigations in multiple cnidariansystems will complement and strengthen our under-standing of cnidarian biology, and thus provideinsight into the ancestral tool kit that gave rise to thecomplex bilaterian body plans. Here we focus onNematostella and provide brief summaries of the cur-rent state of research on three well-studied aspectsof Nematostella development in regards to bilaterians(axial patterning, emergence of mesoderm, andnervous system evolution). We also provide refer-ences to the tools available for researchers interestedin introducing Nematostella into their research pro-gram, and briefly comment on additional areas,such as regeneration, where Nematostella offers a

novel model system to better understand animalbiology.

OVERVIEW OF NEMATOSTELLAVECTENSIS BIOLOGY

Morphology and Life Cycleof NematostellaNematostella vectensis lives in estuarine environ-ments like salt marshes or brackish water pools, andis thus normally exposed to significant fluctuations intemperature and salinity. The physiological tolerancerequired for life in an unsteady environment maycontribute to the relative ease with which Nematos-tella can be cultured. The life cycle of Nematostellatakes approximately 12 weeks in culture.4 They aredioecious, and fertilization occurs in the externalenvironment after spawning. Zygotes initiallyundergo a series of reductive cleavages to form ablastula. Gastrulation occurs via invagination andinitiates at the animal pole (Figure 2(a)). The gastrulastage is followed by the planula stage, which lastsfrom approximately 24 h post fertilization (hpf ) to96 hpf (temperature dependent, unpublished obser-vations). Planulae are free swimming and during thisstage the developing anemone undergoes a slight

BILATERIA

VERTEBRATA

HEMICHORDATA

ECHINODERMATA

LOPHOTROCHOZOA

ECDYSOZOA

HYDROZOA

SCYPHOZOA

CUBOZOA

SCLERACTINIA

ACTINARIA

CNIDARIA

ME

DU

SO

ZO

AA

NT

HO

ZO

A

(a) (b)

FIGURE 1 | Nematostella vectensis is an anthozoan cnidarian sea anemone. (a) Image of adult Nematostella polyp. Image taken by EricRöttinger. (b) Phylogeny showing the sister relationship of cnidarians to bilaterians and Nematostella’s position within the cnidarians.

Overview wires.wiley.com/devbio

© 2016 Wiley Per iodicals , Inc.

elongation and formation of pharynx and mesenter-ies occurs.7 The planula undergoes a subtle type ofmetamorphosis, which is first apparent by the arrestof swimming movement as well as the formationof tentacle buds, and results in formation of thefour tentacle juvenile polyp. At this point growthand maturation of the polyp occurs in a nutrientdependent manner until they reach sexual maturity.Development from egg to juvenile polyp occurs overroughly 6 days.4,7–10

The morphology of Nematostella is superfi-cially simple. They are diploblastic animals, meaningthat they consist of two germ layers, a bifunctionalinternal endoderm also referred to as the entoderm,endomesoderm, or gastrodermis as well as an outerectoderm (Figure 2(a)). They possess a sac-like gutwith a single oral opening that corresponds to thesite of gastrulation7,8 (Figure 2(b)). The adult mouthis surrounded by as many as 16 tentacles11 (Figure 1(a)). They have a noticeable pharynx and eight radi-ally repeated mesentery structures that run along thelong oral–aboral axis12 (Figure 2(b)). The mesenteriesare comprised of gonads, cnidocytes (stinging cells),and myoepithelial cells that allow the animal toquickly contract along the long axis.12–14 The twoearliest forming mesenteries are termed the primary(previously termed directive) mesenteries; they aresituated ca 160� from one another.15 There are twomain axes to the anthozoan body plan, the oral–aboral axis, which is the primary axis and runs fromthe oral opening to the opposite aboral side, and thedirective axis. The directive axis is oriented orthogo-nal to the oral–aboral axis. Although, cnidarians

have been described as radially symmetric around thelong oral–aboral axis of the animal, is has long beenknown that this is not the case for anthozoans. Thearrangement of retractor muscles on the mesenteriesand the presence of a ciliated groove (the siphono-glyph) on one side of the pharynx establish a clear,although subtle bilateral symmetry (summarized inRef 16). The exact relationship between oral–aboraland directive axes and the anterior–posterior anddorsal-ventral axes are still unclear, but numerousinvestigations have shed a great deal of light on thisissue, and they are discussed below.

The ectoderm and endoderm have differen-tiated epithelial cells and specialized cells (neurons,gland cells, myoepithelial cells, etc.) intermixed witheach other. This is in contrast to most bilaterians,which organize specialized cell types into organs witha dedicated function. However, it should be notedthat distinct regions of the animal are enriched forparticular types of differentiated cells. For example,tentacles, which serve to capture and move prey tothe mouth, are highly enriched for offensive cnido-cyte stinging cells, which envenomate prey.17 In addi-tion, regional patterning is observed for neuralmarkers.18–20 The ectoderm serves as a protectivebarrier between the animal and environment. Differ-entiated cell types found in the ectoderm include sen-sory neurons, cnidocytes stinging cells, gland cells,and interneurons with as of yet unknown function.The endoderm contains absorptive cells that serve indigestion and nutrient uptake. The non-mesenteryendoderm contains neurons, myoepithelial cells (mus-culature), and gland cells.12,14,18 Between the

(a) (b)Egg Cleavage Blastula Early gastrula Mid gastrula Late gastrula Early planula

Planula

Tent.

Or

Ph

Mes

ORL

NSS SS

RAB

Late planula Tentacle bud Early juvenile Juvenile/adult

FIGURE 2 | Development and morphology of Nematostella. (a) Schematic of Nematostella developmental stages. Modified from Ormestadet al. Ref 90. Orange represents presumptive endoderm in blastula and the endoderm in all subsequent stages. The outer ectoderm is white, andthe aboral ectodermal domain is in light blue. (b) DIC image taken by Aldine Amiel of juvenile polyp. The mouth (oral opening) is indicated by(Or), the tentacles by (tent), the pharynx by (Ph), and the mesenteries by (mes). Oral is up in all images.

WIREs Developmental Biology Overview of starlet sea anemone Nematostella vectensis


endoderm and ectoderm lies a mostly acellular meso-glea. The mesoglea contains extracellular matrix aswell as some neurites extending from both the endo-dermal and ectodermal neurons.14,21 The mesogleaalso contains amoebocyte cells that appear to bemigratory, but as of yet their function is unknown.14

Nematostella Genome andExperimental ToolsIn 2007, the genome of Nematostella was released.Based on early comparisons between protostome anddeuterostome genomes, it was believed that the cni-darian genome would be relatively simple (e.g., lack-ing many genes or gene families present inbilaterians). Surprisingly, the Nematostella genome isincredibly complex. Nearly all of the gene familiespresent in bilaterians were found in Nematostella. Ofnote, is a nearly complete catalog of the Wnt ligandspresent in deuterostome bilaterians.22,23 In addition,there was considerable syntenic conservation betweenstretches of cnidarian and vertebrate genomes andhigh conservation of intron-exon boundaries withvertebrate genomes.23 All told at the genomic level,vertebrates and Nematostella are more similar toeach other than either group is to ecdysozoan modelanimals (Drosophila and nematodes).24 Thus, thefirst insight about evolution of bilaterian complexitywas that complex molecular architecture predatedthe emergence of bilaterians and that there is no sim-ple correlation between organismal and genomiccomplexity. The quest for such a correlation is stillattracting attention and has driven the adaptation ofnew experimental tools for Nematostella: micro-RNAs and other smallRNAs have been isolated sys-tematically.25,26 ChIP-seq has been used to identifyepigenetic marks and predict regulatory elements,27

and improved transcriptome resources can provideinsight into the prevalence of alternative splicing andlong non-coding RNAs.28–30

Because of the sequencing of the Nematostellagenome, tool development has occurred on a rela-tively rapid scale providing new investigators anarray of mechanisms for incorporating Nematostellainto their research. Table 1 highlights all the toolsand resources available for Nematostella and pro-vides references and hyperlinks to resource pages forprotocols and genomic tools. First, the sequencedannotated genome offers the ability to rapidly iden-tify gene homologs.23 Although a second generationgenome is still in the works, transcriptome studiesprovide refined gene models.28,29 Tools for visualiz-ing gene expression and protein localization usingtransgenics, mRNA in situ hybridization, injection of

mRNA fusion constructs, and antibody staining havebeen developed.12,31,38,42 The ability to knockdowngene function using morpholinos or knockout genefunction using CRISPR/Cas9 or TALEN-Fokapproaches is now available.35–38 Overexpression viamRNA injection allows for misexpression in earlyembryos.19,30,38,39 Recently, a heat-shock responsivepromoter was identified paving the way for morecomplex inducible constructs and the ability to testgain of function phenotypes at later stages of devel-opment.35 Technology improving conditional generegulation would strengthen Nematostella’s utility asa model system. Protocols for ChIP-seq and RNA-seq have also been developed.27–29 To date Nematos-tella has proven amenable for the adaption of meth-ods deployed in other animal systems to disrupt genefunction and analyze phenotypes in the sea anemone,and with advances like CRISPR/Cas9 technology itseems reasonable that tool development in Nematos-tella will continue to be relatively straight-forward.

OVERVIEW OF AXIAL PATTERNING

Formation and Patterning of theOral–Aboral AxisHow the oral–aboral primary body axis of cnidar-ians relates to the anterior–posterior axis of bilater-ians is a longstanding question. Cnidarian polypslack a brain-like concentration of the nervous systemat one pole of the axis (see below) and a through gutin which the mouth would be located anterior to theanus, which are key morphological features that cor-relate with the orientation of the anterior–posterioraxis in most bilaterians. The lack of these features isusually considered to represent an evolutionarilyearly body plan, and accordingly studies on extantcnidarian polyps can provide insights into the groundstate from which traits like cephalization and athrough gut evolved. Despite the dual function asmouth and anus, the single body opening of cnidar-ian polyps is by convention called oral opening andthe opposite end of the primary body axis is calledthe aboral pole or physa (Figure 2). Labeling experi-ments in Nematostella demonstrate that the blasto-pore and thus the body opening derive from theanimal pole of the egg, i.e., the site where the polarbodies are extruded and where the first cleavage initi-ates.7,8 This had previously been observed inhydrozoans,43 which suggests that the relationbetween oocyte polarity and the oral–aboral axis isconserved in different cnidarians clades. Gastrulationfrom the animal hemisphere is found in cnidariansand ctenophores, a second non-bilataterian clade,



but it is strikingly different from bilaterians, whichgastrulate from the vegetal hemisphere. Despite thisdifference a general similarity exists in the molecularsystems that control the development of axial polar-ity and patterning in cnidarian and bilaterianlineages.

Hox Genes and Axial Patterningin CnidariansInitially, Hox genes were used to understand the rela-tion of the oral–aboral axis and the anterior–posterior axis, but no consensus emerged from thesestudies. Cnidarians possess anterior hox and parahoxgenes, but the presence of central and posterior hoxgenes is controversial. Hox genes that do not belongto the anterior group have either been classified asposterior, as central or posterior, or as without affin-ity to anterior, central, or posterior groups.44–48 InNematostella, one of the five anterior hox genes isexpressed in the pharynx (which is derived from theoral domain) at the planula stage,44 whereas threeother anterior hox genes are expressed in a unilateraldomain in the endoderm.44,45 Of the two potentialposterior hox genes, one is expressed at the aboralpole and the other one in a unilateral endodermaldomain of the same oral–aboral extension as the

three anterior hox genes.44 Interestingly, the domainsof the endodermally expressed genes have clearly dif-ferent extensions along the secondary body axis (seealso below).45 These expression patterns suggest thathox genes are involved in axial patterning in Nema-tostella; however, they also show that they are not areliable tool to clarify the relation of the cnidarianand bilaterian primary body axes. This is furtherillustrated by the observation that orthologs of theaborally expressed putative posterior hox gene areexpressed at the opposite pole in the hydrozoansClytia hemispherica and Podocoryne carnea.47,49 Also,it should be noted that further functional characteriza-tion of cnidarian hox genes coupled with improvedsampling of cnidarian genomes for putative hoxhomologs will likely prove insightful in regards tounderstanding how hox genes became so prominent inpatterning the bilaterian anterior–posterior axis.

Wnt Signaling and the Formation ofEmbryonic PolarityIn most bilaterians, intracellular components of theWnt/β-catenin signaling pathway are key players forthe establishment of axial polarity and consequently,the role of this pathway has been studied extensivelyin Nematostella. In the unfertilized egg, NvDsh

TABLE 1 | List of Techniques and Tools Available for Culturing and Experimental Manipulation of Nematostella

Culture and Care of Nematostella

Culture and spawning Hand and Uhlinger4; Fritzenwanker and Technau9; Genikhovich et al.,31

Inducing and stagingregeneration

Amiel et al.,32; Dubuc et al.,33; Bossert et al.,34

Spatiotemporal gene expression

mRNA in situ Wolenski et al.,13; Genikhovich et al.,31

Immunolocalization Wolenski et al.,13

Transgenic reporters Renfer et al.,12; Ikmi et al.,35

Gene function

Morpholino Magie et al.,36; Rentzsch et al.,37; Layden et al.,38

mRNA misexpression Wikramanayake et al.,39; Layden et al.,38

CRISPR/Cas9, TALEN/Fok1 Ikmi et al.,35

Inducible promoters Ikmi et al.,35

Genome and ‘omics’ level analysis

Annotated genome (Putnam et al.,23) http://genome.jgi-psf.org/Nemve1/Nemve1.home.html, http://cnidarians.bu.edu/stellabase/index.cgi, http://metazoa.ensembl.org/Nematostella_vectensis/Info/Index

Transcriptomes Tulin et al.,29; Helm et al.,28 http://figshare.com/articles/Nematostella_vectensis_transcriptome_and_gene_models_v2_0/807696

ChIP-Seq protocol Schwaiger et al.,27

RNA-seq protocol Helm et al.,28; Tulin et al.,29

Microarray approaches Röttinger et al.,40; Fritz et al.,11; Sinigaglia et al.,41



http://genome.jgi-psf.org/Nemve1/Nemve1.home.html

http://cnidarians.bu.edu/stellabase/index.cgi

http://cnidarians.bu.edu/stellabase/index.cgi

http://metazoa.ensembl.org/Nematostella_vectensis/Info/Index

http://figshare.com/articles/Nematostella_vectensis_transcriptome_and_gene_models_v2_0/807696

http://figshare.com/articles/Nematostella_vectensis_transcriptome_and_gene_models_v2_0/807696

protein localizes predominantly to the cell cortexadjacent to the pronucleus and after fertilization itassociates with the zygotic nucleus (Figure 3(a)).NvDsh remains enriched at the first cleavage furrowand on one side (presumably the animal domain) ofthe blastula.8 Consistent with an involvement of theβ-catenin branch of Wnt signaling (canonical Wnt),an antibody against Xenopus β-catenin and injectionof mRNA encoding a Nvβ-catenin-GFP fusion con-struct revealed preferential stabilization and nucleari-zation on one side of the embryo from the 32-cellstage on and later in the invaginating part of the gas-trula.39 Interference with the function of Nvdsh,Nvtcf, and Nvβ-catenin led to conflicting resultsabout their requirement for the development of axialpolarity. Depending on the experimental approach,either complete absence of morphological polarityand failure to gastrulate; or only a defect in the speci-fication of endoderm and pharynx wasobserved.8,39,40,50 Interestingly, a component of thenon-canonical Wnt/PCP signaling pathway, NvStra-bismus (NvStbm), is also enriched at the animal pole

from zygote to gastrula stage (Figure 3(a)). Knock-down of Nvstbm by morpholino injection preventedgastrulation, but not the localized expression ofendodermal markers,50 indicating that this branch ofWnt signaling is specifically required for the morpho-genetic movements at gastrulation.

Wnt signaling appears to be important in thedevelopment of early asymmetry, but despite the ani-mal pole localization of NvDsh and Nvβcat it is stillnot clear how the polarity of the oocyte is initiallydetermined. Future work focused on identification ofthe mechanisms that stabilize NvDsh at the animalpole will likely yield critical cues as to the mostupstream cues that regulate oral–aboral patterning inNematostella.

Wnt Signaling and the Patterning of theOral–Aboral AxisWhile the role of Wnt/β-catenin signaling in the ini-tial formation of the oral–aboral axis remainsunclear, several studies support a role for this

FIGURE 3 | Gene expression during axial patterning. Localization of transcripts (in italic) and proteins involved in the patterning of the oral–aboral (a) and directive (b) axis, respectively. The developmental stages are indicated above each cartoon. Note that not all details of theexpression patterns are captured. In (a), the expression domain of wnt4 is broader than that of wntA and wnt1. frizzled5/8 is at planula stagestrongly expressed at the aboral pole (green) and weakly in a broader aboral domain (pink). hoxF/anthox1 is at planula stage expressed in thesmall aboral pole domain (green) and in individual cells within the broader aboral domain (pink). It is not clear whether the expression domainsdepicted in blue, yellow, and pink are directly abutting each other. In the zygote, Disheveled protein is localized at the cortex and around thenucleus. Disheveled and Strabismus protein remain preferentially localized to the animal/blastoporal region at least until gastrula stage. In (b),BMP5-8 is at gastrula stage co-expressed in the ectoderm with bmp2/4, chordin, and rgm (i.e., the domain in light green). pSmad1/5/8 stainingforms a gradient with highest levels on the side opposite to the bmp expressing side and low levels on the bmp expressing side. See main text forreferences.



pathway in the subsequent patterning along this axis.Starting from mid-blastula stage on, the expressionof individual Wnt genes demarcates distinct, stag-gered domains within the oral half of theembryo22,40,51 (Figure 3(a)). Both in the ecto-andendoderm, these expression domains show little over-lap and could in principle determine separate terri-tories along this part of the oral–aboral axis. Overactivation of Wnt/β-catenin signaling by GSK3 inhi-bitors has shown that different levels of β-catenin canindeed elicit shifts in the expression of regional mar-kers, indicating that high levels of Wnt/β-catenin sig-naling promote oral pole identity.20,40,52

Consistently, knockdown of Nvtcf leads to an expan-sion of the aboral marker gene Nvfgfa1 and to theloss of the oral ectoderm markers Nvwnt2 andNvchordin.40 Surprisingly, there are no reports onthe function of individual Wnt ligands in Nematos-tella, but it will be important to analyze these mole-cules in an endogenous context to understandpotential differences in their signaling mechanisms.These studies together with data from other cnidar-ian species53–55 clearly suggest that Wnt signalinghas ancient functions in the determination of the siteof gastrulation and the patterning of the oral–aboral axis.

The Development of the AboralDomain—Clues to the Evolution of BrainFormation?Brain-like nervous system centralization occurs inmost bilaterian clades close to the anterior end of thebody,56 but a readily comparable neural structure isnot present in cnidarian polyps. Whether a molecularprogram that resembles anterior patterning is presentin cnidarians, and thus whether they have a bodyregion from which cephalization might have evolved,has been an important question in studies on themolecular control of cnidarian development.57

Identification of unambiguous markers to com-pare the anterior end of the bilaterian body axisto the oral or aboral pole of cnidarians is compli-cated; however, the transcription factors six3,foxQ2, and the Wnt regulator SFRP1 are among thebest candidates currently available. In Nematostella,these genes are expressed at the aboral pole20,41

(Figure 3(a)), and Nvsix3/6 has been shown to be akey regulator of the development of the aboral terri-tory. Knockdown of Nvsix3/6 leads to a progressiverespecification of the aboral area to a more oral iden-tity, as indicated by the expanded expression ofNvwnt2 toward the aboral pole and the failure of theaboral ectodermal cells to acquire their typical

columnar shape. While the available functional andexpression data suggest that the aboral pole ofNematostella develops under the control ofanterior patterning genes, there are also noticeabledifferences at a more detailed level, for examplein the exact relation of the expression domains,which likely reflect differences in the regulatory inter-actions between the conserved aboral/anteriorgenes.58–62

Taken together, the development of the oraland aboral domains in Nematostella is regulated byconserved patterning genes and signaling pathwayswith clear similarities of oral to bilaterian posterior,and aboral to bilaterian anterior development.Despite this overall conservation many questionsremain: How is Disheveled protein localized to theanimal pole and how does this compare to bilater-ians? What is the function of the different Wnt genesalong the axis and what are their signaling mechan-isms? Are other bilaterian anterior genes integratedin the aboral patterning system in Nematostella?Addressing these questions will allow a better under-standing of the evolution of basic features of animalbody plans and they will provide new insights intothe logic and the diversity of patterning systems.

BMP Signaling and the SecondaryBody AxisTraditionally, cnidarians have been described as radi-ally symmetric animals, although bilaterally arrangedinternal structures have been observed in anthozoansalready more than a century ago.16,63,64 After the dis-covery of BMP signaling as a unifying mechanism forthe patterning of the bilaterian dorso-ventral axisand the re-positioning of the anthozoans as the sistergroup to the other cnidarian clades, it became con-ceivable that bilaterality was already present in thelast common ancestor of cnidarians and bilaterians.Studies in Nematostella and in the coral Acroporamillepora indeed showed that BMP pathway genesare expressed asymmetrically along the secondaryaxis, the so-called directive axis.37,44,65–67 Functionalstudies in Nematostella revealed that the establish-ment and patterning of the directive axis depend onBMP signaling, but that this pathway is employed inan unconventional manner. Nvbmp2/4, Nvbmp5/8,and the extracellular BMP regulator Nvchordin(Nvchd) are expressed radially around the blastoporeat early gastrula stage, but their expression thenquickly becomes restricted to one side of the mid-gastrula embryo. Surprisingly, all three genes areexpressed on the same side, in partially overlappingdomains in the ectoderm. The BMPs are in addition



expressed in an elongated endodermal domain on thesame side65,68 and another BMP-related molecule,Nvgdf5-like, and a BMP inhibitor, Nvgremlin, startbeing expressed in similar endodermal domains onthe opposite side,44,68 leading to a complex arrange-ment of BMPs and BMP inhibitors along the direc-tive axis. Knockdown of the Nvbmps or of Nvchdabolishes all asymmetric gene expression, but in con-trast to the situation in many bilaterians, it revealeda negative feedback of the NvBMPs on their ownexpression, i.e., knockdown led to strong radialexpression around the blastopore.69 This suggestedthat the domain of Nvbmp transcription is actuallycharacterized by low BMP signaling activity, whichwas later confirmed by immunohistochemical detec-tion of the activated form of the intracellular BMPsignal transducer Smad1/5/8.15,70 Interestingly, hoxgenes are expressed along the directive axis in a pat-tern reminiscent of bilaterian anterior–posterior pat-terning and their expression is controlled by BMPsignaling, the major bilaterian dorso-ventral pattern-ing system.15,45,70 As for the patterning of the oral–aboral axis, these findings identify an interesting mixof conserved and divergent features. While a role ofBMP/Chordin signaling in secondary axis patterningis common to Nematostella and bilaterians, the rolesof individual players and their positions in this sig-naling network vary considerably. These studies haverevealed a similar molecular basis for the patterningof the secondary body axis; however, because thestructures that are arranged in a bilateral manner inanthozoans have no counterpart in bilaterians, itremains unclear whether the directive axis ofanthozoans is homologous to the dorso-ventral axisof bilaterians.

Axial patterning in Nematostella is directed byconserved developmental regulators with overall sim-ilar functions compared to bilaterian model systems,but there are plenty of differences in the exactdeployment of these regulators. Expression profilingof animals with manipulated axial patterning anddetection of direct targets of key transcription factorsby ChIP-seq will likely lead to a detailed picture ofthe transcriptional control of axial patterning inNematostella, and this will continue to provideinsights into the principles of axial patterning systemsand their evolution.

OVERVIEW OF NEMATOSTELLAGERM LAYER SPECIFICATION

Triploblastic animals are characterized by the pres-ence of three distinct germ layers called the endo-,

meso-, and the ectoderm, which form duringembryogenesis. During bilaterian gastrulation, theectoderm first separates from the endomesoderm(or mesendoderm), which subsequently segregatesinto endoderm and mesoderm.71–73 These tissues arearranged such that ectoderm surrounds the exteriorof the animal and interfaces with the environment,endoderm is the innermost germ layer, and the meso-derm lies between the endoderm and ectoderm. Theposition of each germ layer is critical because eachgerm layer generates a specific set of specialized tis-sues during development. For example, the epidermisderives from ectoderm, the gut arises from endoderm,and blood and musculature arise from mesoderm.

Most non-bilaterian animals, with exception toctenophores and medusa stages of a sub-group ofhydrozoan cnidarians are diploblastic animals thatpossess only two germ layers, the inner endodermand outer ectoderm. As a result, the evolutionary ori-gin of bilaterian mesoderm and its derivatives isintensely debated and investigated (reviewed in Ref74–80). As the closest diploblastic relative to the bila-terians, cnidarians can offer key insights as to the ori-gin of the bilaterian mesoderm.

Nematostella (and cnidarians in general) gener-ate an epidermis derived from the ectoderm and agastrodermis derived from the endoderm. There areno mesenchymal muscle cells or other mesodermallyderived tissues in cnidarians.75,81 However, the gas-trodermis is bifunctional in that it possesses absorp-tive cells associated with nutrient uptake and variousmyoepthelial cells with contractile functions.82 Thus,the cnidarian endoderm has both properties and celltypes that segregate into distinct endodermal andmesodermal tissues in bilaterians. Consistent, withhaving traditional functions associated with both thebilaterian endoderm and mesoderm, the Nematos-tella endoderm expresses a large number of genesthat are historically associated with either bilaterianendodermal or mesodermal formation in triploblasticorganisms such as otx and gata, respectively.74,83

These observations suggest that the precursor cells ofthe cnidarian gastrodermis form an endomesodermalgerm layer that fails to segregate into formal endo-derm and mesoderm. One prediction that arises fromthis is that the ancestral gene regulatory network(GRN) that describes the development of cnidarianendoderm/gastrodermis gave rise to the specific regu-latory network that we associate with specification ofendomesoderm in bilaterian animals. Testing ahypothesis as complex as the Nematostella endoder-mal GRN potentially being homologous to the bila-terian endomesodermal GRN would have been moredifficult in the pre-‘omics’ era. However, by



exploiting tools developed for studying Nematostelladevelopment and the ability to rapidly establish noveltools for ‘omics’ level studies in non-traditionalmodel systems, researchers are building an endoder-mal GRN in Nematostella to determine if and howthe cnidarian endodermal GRN gave rise to theendomesodermal GRNs of bilaterians. Addressingthis question will allow researchers to better infer theorigins and possible mechanisms that gave rise to thebilaterian mesodermal layer.

The Endodermal GRN of NematostellaBilaterian presumptive endomesoderm cells aredemarcated by nuclear β-catenin, which is in cooper-ation with the transcription factor TCF, the effectorof canonical Wnt signaling.84–88 Because previousreports implicated canonical Wnt (cWnt) signalingand nuclear β-catenin in Nematostella endodermformation,8,39 researchers sought to identify compo-nents of the Nematostella endodermal specificationpathway by pharmacologically expanding nuclearβ-catenin and thus the presumptive endoderm. Ani-mals with increased nuclear β-catenin were subjectedto microarray analysis to identify likely targets ofβ-catenin.40 This study identified over 100 transcrip-tion factors and signaling molecules potentiallydownstream of nuclear β-catenin.40 Many key genesassociated with bilaterian endoderm and mesodermformation that were previously shown to beexpressed in presumptive endoderm and/or bodywallendoderm in Nematostella such as snailA,B, otx,foxA,B, twist, wnts, tcf, and bmp were identified inthis array.22,36,68,74,83,89,90 Identified targets werefurther analyzed by an mRNA in situ screen to char-acterize their wild type spatiotemporal expressionpatterns. The genes were expressed in one ofthree distinct expression domains within the animalhemisphere prior to the onset of gastrulation(Figure 4(a)–(c)). The majority of the analyzed geneswere expressed in the ‘central domain’ that corre-sponds to cells around the animal pole of the blastula(Figure 4). Surrounding this central domain were tworings of expression, an inner ‘central ring’ that bor-ders or partially overlaps with edges of the centralring, and the ‘external ring’ surrounding the centralring.40 The gathered spatial expression patterns aredeposited in an online database91 where they aremined to predict elements of the gene regulatory net-work that govern endoderm formation and gastrula-tion movements.92,93 Current fate mappingexperiments clearly demonstrate that the animal polederivatives are internalized during gastrulation.8

However, it is not yet clear if cells in the central and

external ring domains are also internalized. Regard-less, the majority of genes identified in the microarrayrepresent genes expressed in presumptive endoderm.At first pass it is clear that (1) cells that internalizeduring gastrulation (cnidarian endoderm and bilater-ian endomesoderm) share characteristic nuclearβ-catenin, which is often linked to cWnt signalingand (2) the cnidarian endoderm and bilaterian meso-derm both express a highly overlapping suite of genesduring development.

By coupling the spatiotemporal gene expressionanalysis with known and additional functionalexperiments with the 100 genes identified aboveresearchers were able to generate a preliminary GRNdescribing endodermal specification at the blastulastage40 for Nematostella. Ectopic activation of cWntsignaling induced the formation of an excess of endo-derm and exogastrulation, the knockdown of NvTcf,the potential effector of this pathway, showed thatNvTcf is not required for gastrulation movementsbut for pharynx formation.40 The molecular analysisof the obtained phenotypes at the blastula stage,revealed that cWnt signaling is required for propergene expression in all three domains of the animalhemisphere and to restrict expression of aboral geneexpression.40 These results suggest that the canonicalWnt pathway in Nematostella plays a crucial role inlaunching a GRN in a broad domain within the ani-mal hemisphere (endodermal + presumptive ectoder-mal tissues). This is similar to what is observed inboth echinoderms and Xenopus.94 At later develop-mental stages, other pathways may be required tosegregate ectodermal from endodermal fates withinthe animal hemisphere. Interestingly, the Fgf/Erk andBmp/Smad pathways are expressed in the centraldomain and central ring respectively and could playa role in this process.40

While there are tremendous similarities betweenthe Nematostella endoderm and bilaterian mesendo-derm in terms of expression patterns and upstreamregulators, key differences exist that need to be con-sidered. Most of our understanding about bilaterianendomesodermal GRNs comes from work in echino-derms, where blimp1, otx, bra, foxA, and gataEform a so-called ‘kernel’ that is critical for the endo-mesoderm GRN.84,95–97 The ‘kernel’ genes are notonly expressed within the endomesodermal domainof the developing sea urchin embryo, but also crucialfor specification of the endo and mesodermal germlayer precursors.84,95–97 Thus, we would expect thesegenes to be expressed in the central domain in Nema-tostella. The current assumption is that the centraldomain will mainly give rise to endoderm derivatives/gastrodermis, while all other domains will give rise



to ectodermal derivatives, such as the pharynx.36

Two genes, Nvotx and Nvsnail, are expressed in thecentral domain. However, Nvwnt8, NvfoxA, andNvbrachyury are expressed in the central ring at theblastula stage. In Nematostella, a blimp-like geneexpressed in the animal hemisphere prior to gastrula-tion has yet to be identified, and Nvgata is expressedin individual cells throughout the ectoderm74 suggest-ing that neither of these critical ‘kernel’ genes areinvolved in cnidarian endodermal specification.Based on expression, the only ‘kernel’ gene defini-tively expressed in the correct region to be specifyingthe endoderm in Nematostella is Nvotx, but the cen-tral ring expression of NvfoxA and Nvbraychury atthe blastula stage does not exclude them as possibleendodermal GRN regulators during earlier develop-mental steps. Are these genes also part of the

ancestral ‘endomesodermal’ GRN or do they belongto a GRN that specifies ectodermal domains in thefuture mouth/pharynx? Functional studies in con-junction with modern cell or tissue tracking techni-ques (photoconversion, tissue-specific inducible GFPexpression) are required to determine the fate of theabove described expression domains and to link themto specific GRNs.

Taken together, it is reasonable to concludethat the bilaterian endomesodermal GRN is derivedfrom an ancestral GRN that also gave rise to the cni-darian endodermal GRN. Nuclear β-catenin and/orcanonical Wnt activity is a critical upstream regulatorin both GRNs, and they have a highly overlappingsuite of genes that define or are functionally requiredfor formation of the endoderm or endomesodermaltissue. It is not yet clear if differences between the

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hd147, bicaudalC-like1, wnt3, bmp2/4,bra, foxA, foxB, wnt8, wntA, tcf, lmx

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FIGURE 4 | (GRN) Co-expression domains within the animal hemisphere of the Nematostella blastula. (a, b) Diagram illustrating four co-expression domains within the animal hemisphere defined by differential spatial expression of the genes indicated in (b). (c) Examples of genesexpression in either the central ring (NvsnailA), the central domain (NvfoxB), the central domain + ring (NvashB), or the external ring (Nvwnt4). Insitu hybridization expression profiles are shown in either lateral view (a, top row of c) or animal view (b, bottom row of c) to distinguishexpression domains. (Reprinted with permission from Ref 40. Copyright 2012 PLoS Genetics)



bilaterian endomesodermal GRN and cnidarian pre-liminary endodermal GRN are going to be sufficientto explain why endomesoderm separates into twodistinct tissues and the cnidarian endoderm does not.To address this question in more detail, gene-specificfunctional studies of theses pathways as well as ofother transcription factors such as Nvbrachury andNvfoxA are required to better understand the archi-tecture and the molecular mechanism underlying thiscnidarian endodermal GRN and its developmentaloutputs. For instance do subsets of the later formingbilaterian mesodermal GRN regulate myoepithelialcells. While we begin to get a basic understanding ofthe wiring of this GRN, future work needs to takeinto account the dynamics of the regulatory stateover time as well as the cis-regulation that underliesthis process. This will enable the community to com-pare these findings with described GRNs from echi-noderms and Xenopus and determine themechanistic (i.e., presence of positive/negative feed-back loops, lock-down mechanisms of given regula-tory states, etc.) similarities or differences that led tothe evolution of distinct endodermal and mesodermalgerm layers in bilaterians.

OVERVIEW OF NEMATOSTELLANERVOUS SYSTEM

Most agree that the nervous systems of bilaterians(the central and peripheral nervous systems) arederived from a nerve net-like ancestral nervous sys-tem.98,99 Thus, better understanding development ofnerve nets in Nematostella and other cnidarians willinform us about the structure and molecular makeup of the ancestral nervous system that gave rise tothe cnidarian nerve net and the bilaterian nervoussystems. Here we will discuss the architecture anddevelopment of the Nematostella nervous system(Figure 5).

Neurons in Nematostella are born in both ecto-derm and endoderm,19,21 and neural soma are inter-mixed with other cell types. Although, cnidariannervous systems are described as being a nerve net,there are clear examples of neural condensations andneuropil-like structures in multiple cnidarian species.For example, longitudinal tracts of neurites run alongthe mesenteries in Nematostella18,21 (Figure 5(a)–(c)).In addition, oral and pharyngeal nerve rings havebeen reported18 (Figure 5(c)). It should be noted thatwhile cross reactive antibodies used to identify thepharyngeal and oral nerve rings showed a slightlyhigher concentration of neurons in a location consist-ent with that of a putative oral and pharyngeal nerve

ring18 recent data with transgenic lines, such as Nve-lav::morange, find relatively uniform neural concen-trations along the length of the oral–aboral axis, andthus it is not yet definitive that oral and pharyngealnerve rings are present. Nematostella possesses sen-sory and interneurons as well as myoepithelialcells18,21 suggesting that dedicated inter, sensory, andmuscle innervating neurons are present in the Nema-tostella nerve net. In addition, Nematostella possessesneurosecretory-like gland cells,100 and a cnidarian-specific neural cell type the cnidocyte.17 Cnidocytesare considered neural cells because they displaymechanosensory and calcium dependent ultrafastexocytosis neural-like properties.101 Cnidocytes areunique to cnidarians and offer an opportunity to

FIGURE 5 | Morphology of Nematostella nervous system injuvenile polyps. (a, b) 3D reconstruction of confocal Z-series acquiredfor two Nematostella transgenic lines that label the nervous system(a) NvElav::mOrange and (b) NvLWamide::mCherry. (c) Schematic ofjuvenile polyp nervous system highlights key structures and providesexamples of neuronal morphologies observed in transgenic animals.



investigate the evolution novel cell types (Box 1).Identification and characterization of the neuronalclasses in Nematostella will provide better under-standing as to which neurons are interneurons, sen-sory, motoneurons, and what distinctive propertieseach class has (neurotransmitter type, receptors,molecular markers, etc.).

Mechanisms of Neurogenesis

Neural InductionUnderstanding neural induction in Nematostella ischallenging, because no distinct neural structure likea central nervous system (CNS) exists, and we do notknow what the earliest definitively neural geneexpression is yet. As a result, how to assay inductionphenotypes is unclear. Current data argue thatNematostella and bilaterian neural induction occursvia distinct mechanisms. Inhibition of bmp2/4 (alsocalled Dpp) in the neural ectoderm via Nog and Chdis the most conserved neural induction mechanism inbilaterians. Thus bmp2/4 homologs suppress centralnervous system formation in bilaterians. Nvbmp2/4patterns the directive axis and together withNvbmp5/8 patterns endoderm. However, loss or gainof Nvbmp2/4 doesn’t disrupt embryonic neuralexpression in the ectoderm.69,102 On the other hand,loss or gain of Nvbmp2/4 activity results in loss of atleast a subset of endodermal neurons in planu-lae.69,102 In Nematostella, it appears NvBmp2/4 isrequired for endodermal neural development, butcan act as an inhibitor at high doses. One caveat tothese studies is that loss of later neural expression ismore likely due to broader defects in patterningresulting from loss of Nvbmp2/4 at earlier stages.Truly understanding neural induction will requireidentification of the earliest markers that distinguishcommitted neural cell types followed by a systematicassessment of potential upstream inputs that regulatetheir expression.

Embryonic NeurogenesisMany of the core neurogenic programs acting down-stream of induction in bilaterians function duringNematostella neurogenesis. Neurons arise in the ecto-derm at early gastrula stages.18,19,21 NvashA andNvsoxB(2), homologs of the bilaterian neural tran-scription factors, promote development of embryonicectodermal neurogenesis.19,103 NvashA is a homologof achaete-scute family bHLH proneural transcrip-tion factors. bHLH proneural genes encompass bothatonal and achaete-scute family transcription factors.Bilaterian proneural genes are necessary and

sufficient to induce neural development (summarizedin the study of Bertrand, 2002).104 Nematostella hasfour achaete-scute genes and at least seven atonal-likegenes encoded in its genome.105 NvsoxB(2) is aHMG-box sox family transcription factor related tothe bilaterian soxB1 and soxB2 families. sox genesare expressed in developing neurons inbilaterians.106–108 In particular, sox gene expressionin neural stem and progenitor cells is well known inneurogenesis of multiple bilaterian species as well asin other cnidarians.109 There are multiple sox genefamilies that function in neurogenesis as well asdevelopment of other tissues. Nematostella has14 sox genes, two of which have reported roles in

BOX 1

CNIDOCYTES AND THE EVOLUTION OFNOVEL CELL TYPES

Cnidocytes are stinging cells found only withinthe cnidarians. They represent one of the fewexamples of an unequivocal evolutionary nov-elty, and thus investigating their compositionand development can inform us about themechanisms by which novel cell types arise. Cni-docytes possess a unique organelle called a cni-docyst that is comprised of a harpoon woundup inside of a capsule. The capsule is generatedby a number of cnidarian-specific minicollagengenes. To date functional dissection of themolecular programs that generate cnidocytes islimited. Disruption of Notch signaling inhibitsproper differentiation of cnidocytes in Hydra126

and reduces the number of mature cnidocytesin Nematostella.111 In Nematostella the neuraltranscription factors Nvsoxb(2) and a uniquesplice isoform of the mesodermal transcriptionfactor Nvmef2 are required for proper specifica-tion of cnidocytes.30,103 From these limited stud-ies, we can infer that the evolution of thisnovel cell type likely required the emergence ofa novel gene family (the minicollagens) and co-option of pre-existing developmental transcrip-tion factors. Interestingly, there are threeclasses of cnidocytes and some studies suggestspecies-specific cnidocyte molecular compo-nents. This cell type is ripe for further studiesfocused on understanding the make up anddevelopment of cnidocytes which will provideinsight into the how novel proteins emerge,and how pre-existing developmental programsinteract with new genes to generate novel celltypes.



neural development.102,103,110 Morpholino knock-down of either NvashA or NvsoxB(2) results in areduction of NvanthoRFamide and Nvelav1expres-sion, and NvsoxB(2) morphant animals also showreduced numbers of cnidocytes.19,103 Global misex-pression of NvashA induces ectopic expression ofneural subtype markers, but interestingly only withintheir respective domains.19 Additional work hasshown that Notch activity can act to suppress neuraldevelopment by suppressing NvashA, and that Notchactivity also suppresses expression of NvsoxB(2).111,112 These data broadly suggest that neurogenictranscription factors in Nematostella function simi-larly to their bilaterian homologs. Teasing outdetailed regulatory networks that promote generalneuronal properties and neural subtype developmentwill improve our understanding of the level of con-servation between molecular mechanisms governingneurogenesis in cnidarians and bilaterians.

Analysis of NvsoxB(2) reporter transgenesdetermined that as many as 30% NvsoxB(2) expres-sing cells incorporate the thymidine analog EdU inpulse labeling experiments and form clones suggest-ing that a subset of the NvsoxB(2) + cells are prolif-erative neural progenitor cells.103 However, it is notentirely clear how widespread deployment of neuralprogenitor cells is, or how many divisions progeni-tors are capable of undergoing. Further advances intechniques to label and track individual clonesin vivo will provide greater detail in future studies.Still missing from our understanding is the origin ofthe neural progenitor cells. Are neural progenitorcells derived from a dedicated neural stem cell, a mul-tipotent stem cell, or from epithelial cells (like Dro-sophila sensory organ precursors)? Regardless,the presence of progenitor cells in Nematostella sug-gests that not only are the molecular mechanisms ofneurogenesis conserved between bilaterians andNematostella, but also the cellular dynamics are alsoconserved between these groups.

Larval NeurogenesisBeginning at late gastrula and early larval stages neu-ral marker gene expression is detected in the endo-derm. Throughout larval stages neurogenesis occursin endoderm and ectoderm. Here too sox and bHLHproneural genes have demonstrated roles in neuraldevelopment. Morpholino mediated knockdown ofNvsoxB(2) also reduced the number of endodermalneurons labeled by the Nvelav1 transgenicreporter.21,103 A more recent study identified an oralpopulation of ectodermal NvanthoRFamide labeledneurons and a small population of asymmetricallypositioned NvGLWamide oral endodermal

neurons.102 These neurons are patterned byNvsoxB(2a) activating expression of NvashB, whichin turn activates expression of an atonal-like bHLHproneural gene. The NvGLWamide neurons specifi-cally require additional positive inputs from Nvarp6,which is asymmetrically expressed in the domain thatgives rise to the NvGLWamide positive cells. Notchactivity was also shown to suppress endodermalexpression of NvashA suggesting that Notchalso acts upstream of neurogenic transcriptionfactors during larval endodermal neurogenesis.112

NvsoxB(2) + precursors were identified in the larvalendoderm and ectoderm, suggesting that larval neu-rogenesis occurs via specification of neural progeni-tor cells.103 In total the current data argue thatmolecular mechanisms responsible for neurogenesisare the same in both endoderm and ectoderm. Thusunderstanding neural development in either tissuewill inform about the ancestral neurogenic programthat gave rise to the bilaterian nervous systems.

Mechanisms of Neural PatterningNeural subtype patterning likely occurs via the com-binatorial action of regional patterning and neuraldifferentiation pathways.18–21,37,41,52 This is mostapparent in specification of the apical tuft sensoryorgan.20,37,41,113,114 Additional evidence comes fromthe regional distribution of distinct neural subtypes.Cnidocyte subtypes and molecular markers for ecto-dermal neurons are differentially distributed alongthe oral–aboral axis suggesting their identity is tiedto regional patterning cues.17–20,102 This hypothesisis supported by the observation that ubiquitous mis-expression of NvashA increases the number of neu-rons born throughout the embryo, but individualneural subtype markers only expand within the limitsof their normal domain boundaries.19 Lastly, pattern-ing of the directive axis restricts Nvarp6 to one sideof the endoderm where it specifies NvGLWamide +neurons.102 Taken together, the current model isthat neurogenic programs are induced more or lessglobally, but that the combinatorial action ofregional patterning and neurogenic transcription fac-tors act to produce distinct fates. This is similar tobilaterian CNS development in that a broad neuralprogram is initiated to neuralize a subset of the ecto-derm, which is followed by subdivision by regionalpatterning information to generate distinct domainsthat produce specific fates. To date our understand-ing about how regional patterning genes act to gener-ate specific fates is relatively poor in Nematostellaand further work is needed before we can compare



details of neural patterning between Nematostellaand bilaterians to each other.

The conserved activity of neurogenic transcrip-tion factors between Nematostella and bilaterianneural programs is clear, suggesting that the cnidar-ian nerve net and bilaterian nervous systems arederived from a common ancestor. There is a tremen-dous amount of speculation about how central nerv-ous systems evolved from something that resembleda cnidarian nerve net. Ideas range from the oral nervering in cnidarians being a precursor to bilateriannerve cords, the idea that the brain originated fromthe ectodermal nervous system in the aboral regionof cnidarian embryos (see above),115 and possibly themost speculative that bilaterian nervous systems mayhave a common origin with the endodermal nervenet.116 To resolve these hypotheses, current researchmust build on the foundational studies and identifyindividual neuronal cell types within cnidarians anddetermine if definitive homology can be drawnbetween cnidarian and bilaterian neuronal classes.Also, a better characterization of the molecular basisof neural induction in cnidarians must be completedand compared in detail to the bilaterian neural induc-tive programs. Regardless, of the answers investigatingthe cnidarian nerve net will improve our understand-ing of how ancestral nervous systems were patternedand functioned during animal evolution.

OVERVIEW OF REGENERATION

Nematostella has the potential to distinguish itself asa model to investigate the relationship between devel-opment and regeneration. Increasing evidence sug-gests that regeneration does not faithfullyrecapitulate development.117–119 Not to say com-pletely novel molecular programs are deployed dur-ing regeneration, but rather subtle differences thatlikely reflect the unique challenge of regeneration are

involved. Being able to directly investigate and com-pare development and regeneration in the same spe-cies is critical to better understand why and howthese processes vary from one another. One reasoncomparisons between development and regenerationare not more widespread is that most traditionalmodel systems are ideal for studying development orregeneration, but not both. Hydra, planarians, andaxolotls are the most well known regenerative animalmodels, but they lack the ability to reliably and costeffectively obtain large numbers of embryos or theylack critical tools to manipulate and/or visualize genefunction. Traditional model systems (mouse, Caenor-habditis elegans, Drosophila, chicks, Xenopus, etc.)display little or no regenerative capacity. Zebrafishdoes offer a powerful developmental system that alsodisplays impressive regenerative capacity.120 How-ever, most vertebrates are not as regenerative as zeb-rafish, which suggests that they may haveindependently derived their regenerative ability.Thus, developing model systems that complement thecurrent regenerative model animals including zebra-fish will allow conserved mechanisms of regenerationin animals to be identified. Moreover, by comparinghow regeneration and differentiation vary from oneanother in multiple species, it may be possible to bet-ter understand why regeneration does not recapitu-late development and use that knowledge to improvedesign and deployment of regenerative medical thera-pies. The power of Nematostella as a developmentalsystem has been discussed above, but they are alsohighly regenerative capable of whole body axisregeneration in less than 1 week (Figure 6) suggestingthat it is a good choice as a model system to comparedevelopment and regeneration.34,121

Nematostella RegenerationNematostella regeneration is either experimentally ornaturally induced by bisection of the animal into two

FIGURE 6 | Oral regeneration of juvenile Nematostella takes place in 6 days. After sub-pharyngeal bisection, missing oral part of the polypsregenerate within 6 days to reform a fully functional organism. Animals were fixed and stained with phalloidin (green) to show f-actin filamentsand propidium iodide (red) to visualize the nuclei. All images are lateral views with the oral part to the top.



halves or by physal pinching, respectively. The latterphenomenon is normally initiated by animals under-going asexual reproduction via transverse fission ofthe most aboral region (the physa). Unlike Hydra,cell proliferation is required for Nematostellaregeneration,121 and preliminary studies show thatafter bisection or physal pinching characteristicevents occur during regenerative growth.34,121

Although recent efforts have focused on characteriza-tion of morphological, cellular, and molecular eventsthat occur during Nematostella wound-healing andregeneration, these studies remain sparse and are het-erogeneous in their experimental approaches (differ-ent ages of animals used, different sites of bisections,variable culture temperature, etc.).33,121–124 A univer-sal protocol or normalized staging series would allowfor direct and more accurate comparisons to bedrawn between Nematostella regeneration, asexualreproduction, and regeneration in other animals. Afirst attempt to define a normalized staging series foradult Nematostella regeneration after supra-physalamputation has been recently presented by Bossertand colleagues.34 While this study is an importantresource, the chosen amputation site is unusual andadult tissue is more difficult to analyze due to the sizeand opacity of tissue compared to juveniles. In an

effort to have a common foundation for comparingexperimental results for scientists working on Nema-tostella regeneration, Amiel and colleagues have com-pared sub-pharyngeal oral regeneration in juvenilesand adults and shown that there are no age buttemperature-specific variations in the timing of thisprocess. The reassessment of cell proliferationrevealed that mitotic cells are detected as soon as12 hpa at the amputation site in juveniles and thatcellular proliferation is also required for oral regener-ation in adults suggesting that the cellular mechan-isms underlying this process may be shared betweenjuveniles and adults.32 In addition, the authors havedeveloped an in vivo assay to analyze the wound-healing success and showed that after amputation thewound is completely healed 6 hpa. A detailed mor-phological analysis of regenerating juveniles revealeda characteristic four-step process (Figure 7).32 Afteramputation (1) the mesenteries fuse to each otherand enter in contact to the amputation site. Whenthe tentacle buds become visible, (2) the mesenteriesare pulled/pushed down to create a gap between theamputation site and the mesenteries where high pro-liferation is detected and where (3) the pharyngeal lipstarts to form, followed by (4) the completion ofpharynx formation. Interestingly, the beginning of

wound open, mesenteries neither in contact with one another, nor with the amputation siteStep 0

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FIGURE 7 | Diagram summarizing the morphological and cellular events underlying Nematostella oral regeneration. The table below theillustration provides definitions for the various regeneration steps. *, mouth opening; ten, tentacles; ten bud, tentacle bud; pha, pharynx; pha lip,pharyngeal lip; mes, mesenteries. (Reprinted with permission from Ref 32. Copyright 2015 International Journal of Molecular Sciences)



pharynx formation between 60 and 72 hpa is accom-panied by the appearance of green autofluorescencethat can be used as an in vivo assay to determine suc-cessful pharynx formation/regeneration. Takentogether, this analysis laid down a basic frameworkto study oral regeneration after sub-pharyngealamputation in Nematostella juveniles by developingassays and defining specific landmarks of thisprocess that can be used to analyze the effects of vari-ous perturbation experiments.32 While both adultor juvenile staging systems allow improved compari-son of results obtained from different researchgroups investigating Nematostella regeneration,they are first drafts that need to be improved inthe future with additional details (e.g., molecularmarkers). Importantly, the present informationcan also serve to define (or to compare with)different regeneration types, such as aboral regenera-tion that will provide additional information on theconservation/divergence of the overall mechanismsunderlying the reformation of missing body parts inNematostella at the molecular, cellular, and tissuelevel.

Patterning Mechanisms DuringNematostella RegenerationOne of the main questions that can be addressed inNematostella is how patterning during regenerationrelates to patterning during development. To dateonly two papers have compared developmental andregenerative patterning in Nematostella. Briefly,Nematostella shows that major patterning moleculesare similarly deployed during development andregeneration, but that subtle differences in regenera-tion are observed. In Nematostella, high Wnt activityis used to specify oral fates during development (dis-cussed above). Hyperactivation of Wnt activity dur-ing regeneration is sufficient to induce completeectopic oral regeneration in aboral territories.125 Thisobservation argues that high Wnt activity also speci-fies oral fates during regeneration. A second studyspecifically assayed expression of a number of tran-scription factors with distinct patterns during devel-opment.33,124 The authors of that work identifiedtwo classes of genes. One class displayed regenerativeexpression patterns that more or less resembled thespatial expression pattern each gene displayed duringdevelopment. A second class, however, showed noexpression or distinct expression patterns duringregeneration that varied from their pattern duringdevelopment. Although, studies of Nematostellaregeneration are still preliminary, they do highlightthat a common observation in animals is that

regeneration varies albeit slightly from development.Given the fact that embryonic development is trig-gered by fertilization of the totipotent oocyte andregeneration is initiated by physical amputation ofvariable amounts of differentiated and stem cells, it isnot surprising that regeneration does not simply reca-pitulate development. Regeneration requires identifi-cation of what is missing and what of the adultbody remains, enacting a program to replace themissing cells/tissues, stopping regrowth at the appro-priate time, and finally reintegration of new tissuesand cell types into an already existing adultbody. Identification and characterization of differ-ences between development and regeneration willimprove our understanding of what is required forsuccessful regeneration and possibly aid in designingand deploying effective regenerative medicaltherapies.

CONCLUSION

Nematostella was originally developed to betterunderstand the origin and evolution of bilateriancharacteristics and animals in general. Those studiesyielded an understanding of the mechanisms that pat-tern the oral–aboral and directive axes of Nematos-tella, a preliminary GRN for formation of thebifunctional endoderm that likely gave rise to theendomesoderm of bilaterians, and the identificationand understanding of conserved molecular programsthat specify neurons in cnidarians and bilaterians.These three areas of research have been the primaryfocus of Nematostella research, and they provide aninitial understanding of how the cnidarian body plan,tissues, and cell types are patterned during develop-ment. What we have learned is that while many ofthe most upstream components necessary to patternsimilar structures and cell types in bilaterians areenacted in Nematostella, key differences are present.By further investigating these differences, we will notonly gain a better understanding of the ancestralstate from which bilaterian evolution occurred, butalso gain critical insights into the mechanisms bywhich animals control patterning and cell specifica-tion during development to arise at the variety offorms present in extant taxa. In addition, with theemergence of Nematostella as a model system, it isnow possible to focus on new questions such asunderstanding the relationship between mechanismsdeployed during development and regeneration ofidentical structures. The future of Nematostella is abright one. Ease of culture and embryo manipulation



make Nematostella accessible to essentially any oneinterested in incorporating this exciting new modelinto their research program. New perspectives are

likely to open novel avenues into which Nematostellacan inform us about metazoan biology, evolution,and biomedical applications.

FURTHER READINGRigo-Watermeier T et al. Functional conservation of Nematostella Wnts in canonical and noncanonical Wnt-signaling. BiolOpen 2012, 1:43–51.

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Overview The rise of the starlet sea anemone Nematostella ...inbios/Faculty/Layden/PDF/...Overview The rise of the starlet sea anemone Nematostella vectensis as a model system to investigate