EVO-DEVO

An axial Hox code controls tissuesegmentation and body patterning inNematostella vectensisShuonan He1, Florencia del Viso1, Cheng-Yi Chen1, Aissam Ikmi1,2,Amanda E. Kroesen1, Matthew C. Gibson1,3*

Hox genes encode conserved developmental transcription factors that govern anterior-posterior (A-P) pattering in diverse bilaterian animals, which display bilateral symmetry.Although Hox genes are also present within Cnidaria, these simple animals lack a definitiveA-P axis, leaving it unclear how and when a functionally integrated Hox code arose duringevolution.We used short hairpin RNA (shRNA)–mediated knockdown and CRISPR-Cas9mutagenesis to demonstrate that a Hox-Gbx network controls radial segmentation of the larvalendoderm during development of the sea anemone Nematostella vectensis. Loss of Hox-Gbxactivity also elicitsmarked defects in tentacle patterningalong the directive (orthogonal) axis ofprimary polyps. On the basis of our results, we propose that an axial Hox code may havecontrolledbodypatterningand tissue segmentationbefore theevolutionof thebilaterianA-Paxis.

Cnidarians (corals, jellyfish, and sea anem-ones) occupy a key position within theanimal phylogeny, serving as an essentialout-group for understanding the evolu-tion of developmental processes throughout

Bilateria (1–3). The characteristic polyp bauplanis a unifying feature within Cnidaria, typified bya tubelike body column with a single oral open-ing surrounded by tentacles (4, 5). Bilaterian bodyplans are more diverse, exhibiting increasing levelsof complexity that correlate with the expansion ofgenomically clustered Hox genes (6, 7). In arthro-pods and chordates, for instance, Hox genes areexpressed along the anterior-posterior (A-P) axis

in staggered domains, setting up amolecular codethat determines body segment identity and directsthe formation of distinct appendages (8, 9). Amongearly-branching phyla, true Hox genes are foundonly within Cnidaria, indicating an ancient evolu-tionary origin predating the bilaterian-cnidariansplit approximately 600million years ago (1, 10, 11).Nevertheless, functional requirements for cnidar-ian Hox genes are unclear, leaving the possibleancestral role of this crucial developmental genecluster poorly understood (12).The sea anemone Nematostella vectensis is a

model anthozoan cnidarian that hasmultipleHoxgenes (13, 14). Under the control of bone mor-

phogenetic protein (BMP) signaling, Anthox1a,Anthox6a, andAnthox8, togetherwithGastrulationbrain homeobox (Gbx) (a Hox-linked subfamilygene), exhibit partially overlapping endodermalexpression patterns in planula larvae (10, 14, 15).During this stage, the developing endodermundergoes morphogenetic segmentation intoeight sectors along the directive axis (Fig. 1, A toC), generating internal anatomical subdivisionsthat further correlate with the positioning of thefirst four tentacles in metamorphosed polyps (fig.S1). To determine whether the larval Hox-Gbx ex-pression domains specifically define endodermalsegment boundaries, we performed fluores-cence in situ hybridization (FISH) onmid–planulastage larvae. Anthox1a, Anthox8, Anthox6a, andGbx exhibited sharp expression territories, cell-autonomously defining segment boundaries (Fig.1, D to K, and fig. S2). Further, each nested Hoxexpression domain formed in concert with thestepwise sequence of endodermal boundary mor-phogenesis (fig. S3), indicating a temporal cor-relation between Hox-Gbx expression and tissuesegmentation. These observations suggest that aHox-Gbx–dependent code controls the formationof the eight endodermal segments (segments s1to s8) and specifies distinct positional identitiesalong the directive axis (Fig. 2A).To test the developmental requirements for

the Hox-Gbx code during segmentation of thelarval endoderm, we took advantage of the dis-tinctive specificity of the cnidarian microRNA

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1Stowers Institute for Medical Research, Kansas City, MO64110, USA. 2Developmental Biology Unit, EuropeanMolecular Biology Laboratory, 69117 Heidelberg, Germany.3Department of Anatomy and Cell Biology, The University ofKansas School of Medicine, Kansas City, KS 66160, USA.*Corresponding author. Email: mg2@stowers.org

Fig. 1. Nematostella Hox-Gbx expressionpatterns cell-autonomously correlate withendodermal segment boundaries inplanula larvae. (A) Nematostella life cycle.Embryos enter a free-swimming planulalarva stage at approximately 48 hourspostfertilization. (B and C) Wild-typemid-planula larvae stained to label F-actin.(B) Side view with oral pole (asterisk) facingupward, showing the focal plane for oral viewimages. (C) Oral view depicting the formationof eight endodermal segments. Scale bars,50 mm. (D to G) Fluorescent in situhybridizations, stained with Hoechst (DNA)to demonstrate the expression patternsof Nematostella Anthox1a, Anthox8,Anthox6a, and Gbx in planula larvae. Scalebar, 50 mm. (H to K) Magnified imagesfrom (D) to (G) illustrating the sharpexpression boundaries for each gene,corresponding todistinct endodermalsegment boundaries. Scale bar, 25 mm.

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pathway (16) to establish a robust short hairpinRNA (shRNA)–based gene knockdown (KD) tech-nique (figs. S4 to S6 andmovies S1 and S2). KD ofAnthox1a, Anthox8, Anthox6a, and Gbx elicitedclear segmentation defects that directly corre-lated with the genes’ endogenous expression do-mains (Fig. 2, B to F). In Anthox1aKD larvae, theboundaries flanking segment s5 were abolished,resulting in the fusion of s4, s5, and s6 into asingle large segment (s4-6) (Fig. 2C). In Anthox8KD larvae, segment boundaries between s3 ands4 and between s6 and s7 were lost, resulting intwo enlarged endodermal segments flanking s5(s3-4 and s6-7) (Fig. 2D). In Anthox6a KD ani-mals, the boundaries between s2 and s3 andbetween s7 and s8 were lost, generating fusionsegments s2-3 and s7-8 flanking s1 (Fig. 2E).Lastly, inGbxKD larvae, the boundaries flankings1 were abolished, resulting in the fusion of s8, s1,and s2 into a single large segment (s8-2) (Fig. 2F).Similar phenotypes were observed for at leasttwo independent shRNAs targeting each gene(Fig. 2 and materials and methods). Quantitativepolymerase chain reaction (qPCR) validation fur-ther confirmed substantial reduction of the targetmRNA level for each shRNA (fig. S7). These resultsdemonstrate that a Hox-Gbx–dependent codedrives morphogenetic tissue segmentation in theendoderm of developing Nematostella larvae.To characterize later roles ofAnthox1a,Anthox8,

Anthox6a, and Gbx genes in body patterning,experimental larvae were reared through meta-morphosis to the polyp stage. KD of each geneelicited marked and highly penetrant tentacle-patterning defects, each of which correspondedto the position of larval segmentation abnormal-ities (Fig. 2, G to K). In wild-type controls, fourtentacles of equal size developed in stereotypedradial positions corresponding to the endodermalsegments s2, s4, s6, and s8 (tentacles t2, t4, t6, andt8) (Fig. 2G and figs. S1C and S8A). In contrast,Anthox1a KD polyps exhibited a single large ten-tacle replacing t4 and t6, resulting in animalswiththree tentacles. The tip of this enlarged tentaclewas frequently bifurcated, suggesting a possiblefusion of t4 and t6 (t4-6) (Fig. 2H and fig. S8B).Anthox8 KD polyps specifically lost tentacles t4and t6 (Fig. 2I and fig. S8C), resulting in animalswith only two tentacles.Anthox6aKDpolypsmain-tained four tentacles, although t2 and t8 wereenlarged and partially fused with the adjacenttentacles t4 and t6, respectively (Fig. 2J, fig. S8D).Lastly, Gbx KD polyps consistently lost tentaclest2 and t8, resulting in two-tentacled animals witha mirror-image phenotype to Anthox8 KD ani-mals (Fig. 2K and fig. S8E). Similar developmen-tal requirements were not observed for the otherNematostella homeodomain-containing genes,Anthox6, Anthox7, and Cdx (fig. S9). Collectively,these experiments demonstrate key roles forAnthox1a, Anthox8, Anthox6a, and Gbx in con-trolling tentacle patterning, revealing a link be-tween the process of endodermal segmentationand the specification of tentacle primordia.Previous studies have demonstrated a role for

BMP signaling in regulating both larval Hox ex-pression and the development of mesenteries, di-

gestive and reproductive organs that form duringlarval development at the boundaries between en-dodermal segments (fig. S6, A to C) (15). To exploreadditional roles for Hox genes in axial pattern-ing, we developed a method to image Anthox1a,Anthox8, and Anthox6a KD animals by usingselective plane illumination microscopy (SPIM)(17). We observed only six mesenteries in eachKD condition (movies S1 and S3 to S5), consistentwith the earlier defects in boundary formation.Taken together with earlier results, these find-ings confirm that Hox-dependent segmentation

of the larval endoderm establishes key elementsof the polyp bauplan, including the positioningof mesenteries and the patterning of tentacleprimordia.To validate the developmental function of the

Nematostella Hox-Gbx code with a technicallyindependent approach,wenextmutatedAnthox1a,Anthox6a, Anthox8, and Gbx by using CRISPR-Cas9–mediated genome editing (18–20). After theinjection of Cas9–guide RNA (gRNA) complexesinto embryos (with either single or paired gRNAs),F0 indel mutations were recovered in all four

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Fig. 2. Nematostella Hox-Gbx genes control tissue segmentation and tentacle patterning.(A) Color-coded expression pattern of individual genes in wild-type larvae. (B to F) Oral views ofwild-type versus Hox-Gbx KD planula-stage larvae. (B) Wild type [n = 24 larvae (image isrepresentative of 24 of 26 wild-type larvae)]; (C) Anthox1a KD (n = 31/32); (D) Anthox8 KD(n = 26/28); (E) Anthox6a KD (n = 30/36); (F) Gbx KD (n = 26/33). Scale bar, 50 mm. hpf, hourspostfertilization. (G to K) Oral views of wild-type versus Hox-Gbx KD polyps. (G) Wild type (n = 197of 264 polyps); (H) Anthox1a KD polyps (shRNA1, n = 214/253; shRNA2, n = 165/192); (I) Anthox8 KDpolyps (shRNA1, n = 144/162; shRNA2, n = 112/132); (J) Anthox6a KD polyps (shRNA1, n = 92/126;shRNA2, n = 123/162); (K) Gbx KD polyps (shRNA1, n = 84/181; shRNA2, n = 50/70; shRNA3, n =24/38). Arrowheads indicate missing tentacles. Scale bar, 100 mm. dpf, days postfertilization.

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loci, along with low frequencies of the expectedtentacle phenotypes (Fig. 3 and fig. S10). Throughsubsequent controlled crosses, we obtained genet-ically null mutants for Anthox1a, Anthox8, andAnthox6a. In each case, these animals exhibitedtentacle-patterning defects identical to those ofthe cognate shRNA KD animals (figs. S11 to S14).Putative Gbxmutants showed severe growth de-fects, and only heterozygous animals were recov-ered from F0 founder crosses, suggesting thatGbx has additional roles in later developmental

processes. Taken together, these complementaryCRISPR-Cas9– and shRNA-based methods illu-minate clear developmental requirements for aHox-Gbx network during cnidarian development(Figs. 2 and 3 and figs. S7 to S14).In bilaterian systems, Hox-dependent patterns

typically arise through extensive cross-regulatoryinteractions (21–24). We therefore performed aseries of double-KD experiments to determinewhether Nematostella Hox genes exhibit geneticinteractions during early development. Consistent

with independent requirements for each locus,strictly additive phenotypes were observed inAnthox1a-Anthox8 (loss of t2 and t4), Anthox1a-Gbx (t4-6 fusion and loss of t2 and t8), andAnthox8-Gbx (no tentacles) double-KD animals(fig. S8, F to J). To further interrogate the mo-lecular regulation of Hox-Gbx gene expression,we took advantage of a transgenic Anthox8>GFPreporter line that faithfully recapitulates endog-enous Anthox8 expression in endodermal seg-ments s4, s5, and s6 (Fig. 4, A to C). Notably, the

He et al., Science 361, 1377–1380 (2018) 28 September 2018 3 of 4

Fig. 3. CRISPR-Cas9–mediated mutagen-esis of Anthox1a confirms its functionin tentacle patterning. (A) gRNA strategyfor the Anthox1a locus. F0 animalsinjected with either gRNA carried bothframeshift and non-frameshift indel muta-tions (arrowheads indicate mutationpositions). HD, homeodomain; PAM, proto-spacer adjacent motif; D, deletion of thespecified number of bases. (B and C) Incontrast to GFP gRNA–injected controls,putative Anthox1a F0 founders displayed thecharacteristic t4-6 tentacle fusion phenotypeobserved in KD experiments. Scale bars,100 mm. (D) Quantification of t4-6 fusionphenotypes observed in uninjected controls[wild type (WT)], GFP gRNA controls(F0) (gGFP), Anthox1a gRNA–injectedanimals (F0) (g1, g2, and g1+g2), andtwo independent shRNA KD groups(sh1 and sh2).

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Fig. 4. Nematostella Hox-Gbx genes comprise a spatial code thatdirects axial patterning. (A) Design of the Anthox8>GFP reporterconstruct. (B and C) Although the Actin promoter alone drives ubiquitousGFP expression, addition of the Anthox8 upstream enhancer regionrestricts GFP expression to segments s4, s5, and s6 at the mid-planulastage (n = 10 of 10 larvae). (D) Pbx shRNA–injected transgenic animalslost all endodermal segmentation and failed to activate the Anthox8reporter (n = 6 of 6 animals). (E) shRNAs targeting Anthox8substantially decreased the GFP signal (n = 5 of 6 animals), restricting

it to segment s5. Scale bars in (B) to (E), 50 mm. (F) Comparisonbetween the functional Hox codes in a representative cnidarian(Nematostella) and a representative vertebrate (mouse). Althoughthe Hox codes respond to different upstream signaling pathways alongdistinct axes [BMP in Nematostella and retinoic acid and fibroblastgrowth factor (RA and FGF) in mouse], similar downstream molecularprograms drive segmental patterning. Data on potential homologiesbetween Nematostella and bilaterian Hox genes are from previouspublications (12, 14).

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enhancer region in this construct contains a pre-viously identifiedMeis/Pbx-Hox binding site (25).Because Pbx is a key binding partner for bilaterianHox genes that biochemically interacts with sev-eral Hox proteins in Nematostella (25–28), wetested whether Pbx regulates Anthox8 expres-sion. LarvalAnthox8 transcription was undetect-able after Pbx shRNA KD, which caused a severeand uniform loss of endodermal segmentationand subsequentmetamorphic failure (Fig. 4D andfig. S8, K and L). To identify the putative Hoxcofactor responsible for Pbx-dependent activa-tion of Anthox8, we knocked down all Hox-Gbxgenes and assayed Anthox8>GFP expression.Only shRNAs targeting Anthox8 substantiallyreduced green fluorescent protein (GFP) inten-sity (Fig. 4E and fig. S15). Further validated byFISH experiments (fig. S16), these results demon-strate Pbx-dependent autoregulation ofAnthox8.In parallel experiments to explore cross-regulatoryinteractions, we also found that Anthox1a uni-directionally repressed Gbx expression in devel-oping endodermal segment s5 (fig. S17). Combined,these findings hint at the possibility of similarregulatorymechanisms between cnidarian andbilaterian Hox networks.In summary, this work leverages both classical

genetics and a robust gene KD methodology todemonstrate the existence of a functional Hoxcode in a developing cnidarian. Reminiscent ofthe sophisticated Hox networks that operate inarthropods and chordates,NematostellaHox-Gbxgenes encode axial identities, thus governing thepatterning of secondary structures such as tentaclesand mesenteries (Fig. 4F). Hox-Gbx–dependentendodermal segments may be established in amanner analogous to bilaterian posterior prev-alence, whereby posteriorly expressed Hox genesgenerally override the effects of genes that aremore anterior (29–31). According to this logic,Anthox1a expression in Nematostella segment

s5would reflect a dominant pole of the anthozoandirective axis, with Anthox8, Anthox6a, and Gbxoperating to define successive segment bounda-ries toward the opposite end (fig. S18). Althoughunderstanding the direct or indirect nature ofthese Hox-Gbx interactions will be an importantarea for future studies, our findings further dem-onstrate the existence of a Pbx-dependent func-tional network and provide initial evidence forboth Hox auto- and cross-regulation (Fig. 4). De-spite limited data regarding other cnidarian species,the phylogenetically basal position of Anthozoa(32) permits speculation that the NematostellaHox code reflects a conserved gene regulatorymodule, one that could have been co-opted todirect A-P patterning in the ancient urbilaterian.

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ACKNOWLEDGMENTS

We thank R. Krumlauf (Stowers Institute), P. Cartwright (Universityof Kansas), and D. Lambert (University of Rochester) forsuggestions and critical reading of the manuscript. We also thankM. Kirkman and K. Delventhal for genotyping assistance and theStowers Institute Aquatics Core facility for animal husbandry.Funding: This study was supported by the Stowers Institute forMedical Research. Author contributions: S.H. and M.C.G.designed and analyzed the experiments. S.H. developed the shRNAapproach and performed all RNA interference experiments.A.I. generated the Actin>GFP transgenic line and tested shRNAperdurance. S.H. generated the Anthox8>GFP transgenic line. S.H.and F.D.V. performed CRISPR-Cas9 genome editing. C.-Y.C.optimized the FISH protocol and performed time-course reversetranscription–qPCR analysis. S.H. and C.-Y.C. performed theFISH experiments. A.E.K., A.I., and S.H. performed SPIM imagingand SPIM data analysis. S.H. and M.C.G. wrote the manuscript.All authors discussed the experiments and read and approved themanuscript. Competing interests: None declared. Data andmaterials availability: Original data underlying this manuscriptcan be accessed from the Stowers Original Data Repository athttp://www.stowers.org/research/publications/libpb-1247. Alldata needed to evaluate the conclusions in the paper are presentin the paper or the supplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/361/6409/1377/suppl/DC1Materials and MethodsFigs. S1 to S18Tables S1 and S2References (33–46)Movies S1 to S5

21 December 2017; accepted 9 August 201810.1126/science.aar8384

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vectensisNematostellaAn axial Hox code controls tissue segmentation and body patterning in

Shuonan He, Florencia del Viso, Cheng-Yi Chen, Aissam Ikmi, Amanda E. Kroesen and Matthew C. Gibson

DOI: 10.1126/science.aar8384 (6409), 1377-1380.361Science 

, this issue p. 1377; see also p. 1310Scienceancestor.may have evolved to regulate both tissue segmentation and body patterning in the bilaterian-cnidarian common

controls the morphogenesis of radial endodermal segments and the patterning of tentacles. Thus, an ancient Hox code(see the Perspective by Arendt). Four homeobox-containing genes constitute a molecular network that coordinately

Nematostella vectensis gene function in a cnidarian, the sea anemone Hoxbased gene knockdowns to interrogate −RNA used a combination of CRISPR mutagenesis and short hairpin et al.body patterning in diverse bilaterian animals. He

genes encode conserved transcription factors that are best known for their role in governing anterior-posteriorHoxHox code in segmentation and patterning

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