ARTICLE doi:10.1038/nature10838 Ancient deuterostome origins of vertebrate brain signalling centres Ariel M. Pani1,2, Erin E. Mullarkey3*, Jochanan Aronowicz4*, Stavroula Assimacopoulos5, Elizabeth A. Grove3,4,5 & Christopher J. Lowe1,2,4 Neuroectodermal signalling centres induce and pattern many novel vertebrate brain structures but are absent, or divergent, in invertebrate chordates. This has led to the idea that signalling-centre genetic programs were first assembled in stem vertebrates and potentially drove morphological innovations of the brain. However, this scenario presumes that extant cephalochordates accurately represent ancestral chordate characters, which has not been tested using close chordate outgroups. Here we report that genetic programs homologous to three vertebrate signalling centres—the anterior neural ridge, zona limitans intrathalamica and isthmic organizer—are present in the hemichordate Saccoglossus kowalevskii. Fgf8/17/18 (a single gene homologous to vertebrate Fgf8, Fgf17 and Fgf18), sfrp1/5, hh and wnt1 are expressed in vertebrate-like arrangements in hemichordate ectoderm, and homologous genetic mechanisms regulate ectodermal patterning in both animals. We propose that these genetic programs were components of an unexpectedly complex, ancient genetic regulatory scaffold for deuterostome body patterning that degenerated in amphioxus and ascidians, but was retained to pattern divergent structures in hemichordates and vertebrates. During vertebrate development,the brainarises from coarsely patterned were secondarily simplified or lost along the lineages leading to the planar neuroectoderm through successive refinement of regional invertebrate chordates. identities,resulting after morphogenesis and growth ina complex struc- Hemichordates are a deuterostome phylum closely related to ture composed of highly specialized areas1–3. In contrast, invertebrate chordates26 and are a promising outgroup for investigating chordate chordates have relatively simple nervous systems that lack unambigu- evolution. Previous studies established that despite substantial body- ous homologues of many vertebrate brain regions4–6. These clear dis- plan divergence between the two groups,hemichordates and vertebrates parities in nervous system complexity indicate that key innovations in share a broadly conserved transcriptional regulatory architecture patterning mechanisms have attended the evolution of the vertebrate during early body patterning7,27–29, which is demonstrated by the close brain from a simpler central nervous system (CNS). However, attempts similarities in spatial arrangements of expression domains of many to identify the presumed genetic regulatory novelties have been mainly transcription factors that are involved in early bilaterian anteroposterior inconclusive, and comparative studies have mostly revealed similarities, patterning. This combination of morphological divergence and develop- rather than differences, in the early transcriptional architectures of mental genetic similarity makes hemichordates an informative out- diverse bilaterian nervous systems7–12. Notable exceptions are CNS sig- group for testing the proposed coupling of vertebrate morphological nalling centres, which have emerged as strong candidates for vertebrate and developmental genetic innovations. Here, we present descriptive genetic regulatory novelties involved in early vertebrate brain evolu- and functional evidence that genetic programs homologous to the ANR, tion8–10. These centres act as secondary organizers that mediate regional ZLI and IsO are present in S. kowalevskii, indicating that they were patterning in the CNS and are often necessary and sufficient for the elements of an ancient developmental genetic toolkit for deuterostome establishment of vertebrate-specific brain structures1–3,13–18. body patterning that were subsequently modified and elaborated in The anterior neural ridge (ANR), zona limitans intrathalamica stem vertebrates to regulate brain development. (ZLI) and isthmic organizer (IsO) are the three primary signalling centres that direct anteroposterior patterning in the vertebrate An ANR-like regulatory program in hemichordates anterior neural plate and then later in the developing brain1–3. The ANR is located in the anterior neural plate of vertebrates and is a Homologous signalling centres and their molecular signatures are source of fibroblast growth factors (FGFs) and secreted frizzled-related absent, or divergent, in amphioxus and ascidians8–10,19–25, consistent proteins (SFRPs), which establish and pattern the telencepha- with the idea that they are vertebrate novelties whose origins could lon1,2,15,16,30,31. Topologically consistent with vertebrate CNS expression have driven CNS innovations in stem vertebrates8–10. However, this domains, sfrp1/5 and fgf8/17/18 are expressed in S. kowalevskii anterior idea depends on amphioxus adequately representing ancestral states proboscis ectoderm (Fig. 1a–d). In vertebrates, FGFs and antagonists for chordate developmental genetic characters, and does not account of the Wnt pathway mediate ANR function and telencephalon pattern- for the possibility of secondary losses in cephalochordates. Here we ing15,16,30–32. In mice, conditional Fgfr1, Fgfr2 and Fgfr3 knockout present developmental data from the hemichordate S. kowalevskii to abolishes the telencephalon16,whereasFgf8 mutants have a posteriorized test an alternative scenario for signalling-centre origins; namely, that neocortex33. Similarly, treating S. kowalevskii embryos with the FGF ANR, ZLI and IsO genetic programs pre-date chordate origins and receptor inhibitor SU5402 (ref. 34) from late gastrula through to 1Committee on Evolutionary Biology, The University of Chicago, 1025 East 57th Street, Chicago, Illinois 60637, USA. 2Hopkins Marine Station, Department of Biology, Stanford University, 120 Oceanview Boulevard, Pacific Grove, California 93950, USA. 3Committee on Neurobiology, The University of Chicago, 947 East 58th Street, Chicago, Illinois 60637, USA. 4Department of Organismal Biology and Anatomy, The University of Chicago, 1027 East 57th Street, Chicago, Illinois 60637, USA. 5Department of Neurobiology, The University of Chicago, 947 East 58th Street, Chicago, Illinois 60637, USA. *These authors contributed equally to this work. 15 MARCH 2012 | VOL 483 | NATURE | 289 ©2012 Macmillan Publishers Limited. All rights reserved RESEARCH ARTICLE a b c d e f g h hh sfrp1/5 Sfrp1 fgf8/17/18 Fgf8 hh fgf-Sk1 hh/fgf-Sk1 fgf-Sk1 fz5/8 β-catenin siRNA i foxg rx fgf-Sk1 rx Control animal blastomere fgf-Sk1 Sfrp1 Fgf8 Shh Foxg1 sfrp1/5 fgf8/17/18 hh foxg j k no rst x * siRNA pr tel siRNA fgf-Sk1 SU5402 hh fz5/8 a-rhodamine di l m p q uvw y col mbmb tr hb rx DMSO Hemichordate Vertebrate (mouse) a-rhodamine Scrambled siRNA Scrambled siRNA Figure 1 | An ANR-like signalling centre in S. kowalevskii.a–h, S. (m) expression in embryos treated with DMSO. n, Expanded apical fgf-Sk1 kowalevskii and mouse in situ hybridizations for markers of ANR and expression in an embryo injected with fz5/8 siRNA. o,Retractedrx expression in telencephalon. S. kowalevskii embryos are at the double-groove stage (36 h), and an embryo injected with fz5/8 siRNA. p, fgf-Sk1 expression in a siRNA control are shown in dorsal view with the anterior (proboscis) to the top left of the image, embryo. q, rx expression in a siRNA control embryo. Insets in o, q show frontal except where noted. Embryos are optically cleared except in o, q (insets) and views of uncleared embryos. r, Wild-typefgf-Sk1 expression. s, fgf-Sk1 expression r–w. Mouse embryos are at approximately embryonic day 8.5. a, S. kowalevskii in descendants of a blastomere injected with b-catenin siRNA. t, Merged sfrp1/5 expression. b, Frontal view of mouse Sfrp1 expression; arrowhead darkfield and fluorescence images showing clonal descendants of the injected denotes ANR. Inset shows lateral view. c, S. kowalevskii fgf8/17/18 expression. cell (green). u,Wild-typerx expression. v, rx is not expressed in descendants of a d,MouseFgf8 expression; arrowhead denotes ANR. e, S. kowalevskii hh blastomere injected with b-catenin siRNA. w, Merged darkfield and fluorescence expression. f, S. kowalevskii fgf-Sk1 expression. g, Frontal viewofdoubleFISHfor images showing the location of the b-catenin-deficient clone (green). x, fgf-Sk1 hh and fgf-Sk1. h, fz5/8 expression. i, Anteroposterior expression topologies in S. expression in an embryo injected with hh siRNA. y, fgf-Sk1 expression in a kowalevskii and mouse embryos. Anterior to top. Asterisk indicates Shh is siRNA control embryo. Scale bars, 100 mminS. kowalevskii, and 200 mm(b)and expressed in the medial ganglionic eminence, near the ANR. j–k, foxg (j)andrx 500 mm(d) in mice. col, collar; di, diencephalon; hb, hindbrain; mb, midbrain; (k) expression in embryos treated with SU5402. l, m, foxg (l)andrx pr, proboscis; tel, telencephalon; tr, trunk. double-groove stages (28–36 h) eliminated the morphological boundary raises the possibility that an anterior signalling domain including between the proboscis and collar, and resulted in altered gene expres- FGFs, Wnt antagonists and hh was present in stem deuterostomes sion domains, indicating loss and/or posteriorization of anterior and was spatially partitioned during vertebrate evolution. proboscis (Fig. 1j–m). Expression of foxg, the S. kowalevskii homologue of the telencephalon marker foxg1, was completely eliminated (Fig. 1j, A ZLI-like regulatory program in hemichordates l), whereas rx, which is