Dorsoventral Patterning in Hemichordates:Insights into Early Chordate EvolutionChristopher J. Lowe1, Mark Terasaki2, Michael Wu3, Robert M. Freeman Jr.4, Linda Runft5, Kristen Kwan4, Saori Haigo4,Jochanan Aronowicz1, Eric Lander4,6,7, Chris Gruber8, Mark Smith8, Marc Kirschner4, John Gerhart3*1 Department of Organismal Biology and Anatomy, University of Chicago, Chicago, Illinois, United States of America, 2 Department of Cell Biology, University of ConnecticutHealth Center, Farmington, Connecticut, United States of America, 3 Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, UnitedStates of America, 4 Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America, 5 Department of Molecular, Cellular, andDevelopmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America, 6 Broad Institute of MIT and Harvard, Cambridge,Massachusetts, United States of America, 7 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 8 ExpressGenomics, Frederick, Maryland, United States of AmericaWe have compared the dorsoventral development of hemichordates and chordates to deduce the organization of theircommon ancestor, and hence to identify the evolutionary modifications of the chordate body axis after the lineagessplit. In the hemichordate embryo, genes encoding bone morphogenetic proteins (Bmp) 2/4 and 5/8, as well as severalgenes for modulators of Bmp activity, are expressed in a thin stripe of ectoderm on one midline, historically called‘‘ dorsal.’’ On the opposite midline, the genes encoding Chordin and Anti-dorsalizing morphogenetic protein (Admp)are expressed. Thus, we find a Bmp-Chordin developmental axis preceding and underl ying the anatomicaldorsoventral axis of hemichordates, adding to the evidence from Drosophila and chordates that this axis may be atleast as ancient as the first bilateral animals. Numerous genes encoding transcription factors and signaling ligands areexpressed in the three germ layers of hemichordate embryos in distinct dorsoventral domains, such as pox neuro,pituitary homeobox, distalless, and tbx2/3 on the Bmp side and netrin, mnx, mox, and single-minded on the Chordin-Admp side. When we expose the embryo to excess Bmp protein, or when we deplete endogenous Bmp by smallinterfering RNA injections, these expression domains expand or contract, reflecting their activation or repression byBmp, and the embryos develop as dorsalized or ventralized limit forms. Dorsoventral patterning is independent ofanterior/posterior patterning, as in Drosophila but not chordates. Unlike both chordates and Drosophila, neural geneexpression in hemichordates is not repressed by high Bmp levels, consistent with their development of a diffuse ratherthan centralized nervous system. We suggest that the common ancestor of hemichordates and chordates did not useits Bmp-Chordin axis to segregate epidermal and neural ectoderm but to pattern many other dorsoventral aspects ofthe germ layers, including neural cell fates within a diffuse nervous system. Accordingly, centralization was added inthe chordate line by neural-epidermal segregation, mediated by the pre-existing Bmp-Chordin axis. Finally, sincehemichordates develop the mouth on the non-Bmp side, like arthropods but opposite to chordates, the mouth andBmp-Chordin axis may have rearranged in the chordate line, one relative to the other.Citation: Lowe CJ, Terasaki M, Wu M, Freeman RM Jr., Runft L, et al. (2006) Dorsoventral patterning in hemichordates: Insights into early chordate evolution. PLoS Biol 4(9):e291. DOI: 10.1371/journal.pbio.0040291IntroductionArthropods (especially Drosophila) and chordates (especiallymouse, fish, frogs, and birds) and presumably all bilaterians arefundamentally similar in the development of their body plans[1,2]. Suites of genes are arranged in conserved domain mapsin both the anteroposterior and dorsoventral dimensions ofdeveloping embryos. Paradoxically, this conservation of axialpatterning provides the developmental platform for astonish-ing anatomical and physiological diversification both withinand between phyla. Clearly a major step in the evolution ofbilateral animals was the origination of these domain mapsand the usage of transcription factors and signaling proteinsby which diverse target genes could be expressed at specifictimes and places in development at subsequent stages.The evolution of the dorsoventral axis (transverse to theanteroposterior axis) is not well understood. It may haveoriginated during the transition from radial to bilateralanimals [3], although recent evidence raises the possibility itwas already cryptically present in radial animals [4,5].Bilateral animals, at least back t o the Cambrian, havenumerous anatomical and physiological specializations lo-cated along this axis, in all three germ layers. The mesodermaltissues such as the heart, blood forming elements (and thedirection of blood flow), gonads, visceral muscle, and striatedmuscle are arranged in dorsoventral patterns. The gill slits (inchordates and hemichordates) and other endodermal organsAcademic Editor: Edward De Robertis, Howard Hughes Medical Institute, UnitedStates of AmericaReceived March 7, 2006; Accepted June 28, 2006; Published August 22, 2006DOI: 10.1371/journal.pbio.0040291Copyright: Ó 2006 Lowe et al. This is an open-access article distributed under theterms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original authorand source are credited.Abbreviations: Admp, anti-dorsalizing morphogenetic protein; Bm p, bonemorphogenetic protein; Cv, crossveinless; dlx, distalless; Hh, hedgehog; pitx,pituitary homeobox;PoxN,poxneuro;RNAi,RNAinterference;Shh,sonichedgehog; Sim, single-minded; siRNA, small interfer i ng RNA; Tsg, twistedgastrulation* To whom correspondence should be addressed. E-mail: [email protected] Biology | www.plosbiology.org September 2006 | Volume 4 | Issue 9 | e2911603PLoSBIOLOGYhave similarly conserved patterns in the dorsoventral domain.Ectodermal examples include the central nervous system andepidermal territory. The dorsal and ventral poles of the axisare usually assigned based on the orientation of the animalrelated to gravity (dorsal up; ventral down) or on the locationof the mouth