BACKGROUND: Nodal is an important determinant of the left-right (LR) body axis in bilaterians, specifying the right side in protostomes and non-chordate deuterostomes as opposed to the left side in chordates. Amphioxus represents an early-branching chordate group, rendering it especially useful for studying the character states that predate the origin of vertebrates. However, its anatomy, involving offset arrangement of axial structures, marked asymmetry of the oropharyngeal region, and, most notably, a mouth positioned on the left side, contrasts with the symmetric arrangement of the corresponding regions in other chordates. RESULTS: We show that the Nodal signaling pathway acts to specify the LR axis in the cephalochordate amphioxus in a similar way as in vertebrates. At early neurula stages, Nodal switches from initial bilateral to the left-sided expression and subsequently specifies the left embryonic side. Perturbation of Nodal signaling with small chemical inhibitors (SB505124 and SB431542) alters expression of other members of the pathway and of left/right-sided, organ-specific genes. Upon inhibition, larvae display loss of the innate alternation of both somites and axons of peripheral nerves and loss of left-sided pharyngeal structures, such as the mouth, the preoral pit, and the duct of the club-shaped gland. Concomitantly, the left side displays ectopic expression of otherwise right-sided genes, and the larvae exhibit bilaterally symmetrical morphology, with duplicated endostyle and club-shaped gland structures. CONCLUSIONS: We demonstrate that Nodal signaling is necessary for establishing the LR embryonic axis and for developing profound asymmetry in amphioxus. Our data suggest that initial symmetry breaking in amphioxus and propagation of the pathway on the left side correspond with the situation in vertebrates. However, the organs that become targets of the pathway differ between amphioxus and vertebrates, which may explain the pronounced asymmetry of its oropharyngeal and axial structures and the left-sided position of the mouth.

Institute of Cellular and Organismic Biology Academia Sinica 128 Academia Road Section 2 Nankang Taipei 11529 Taiwan

Institute of Cellular and Organismic Biology Academia Sinica 128 Academia Road Section 2 Nankang Taipei 11529 Taiwan ; Institute of Oceanography National Taiwan University 1 Roosevelt Road Section 4 Taipei 10617 Taiwan

Institute of Molecular Genetics Academy of Sciences of the Czech Republic Videnska 1083 Prague 14220 Czech Republic

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Blum M, Feistel K, Thumberger T, Schweickert A. The evolution and conservation of left-right patterning mechanisms. Development. 2014;141(8):1603–1613. PubMed

Namigai EK, Kenny NJ, Shimeld SM. Right across the tree of life: the evolution of left-right asymmetry in the Bilateria. Genesis. 2014;52(6):458–470. PubMed

Palmer AR. Animal asymmetry. Curr Biol. 2009;19(12):R473–R477. PubMed

Raya A, Izpisua Belmonte JC. Left-right asymmetry in the vertebrate embryo: from early information to higher-level integration. Nat Rev Genet. 2006;7(4):283–293. PubMed

Blum M, Schweickert A, Vick P, Wright CV, Danilchik MV. Symmetry breakage in the vertebrate embryo: when does it happen and how does it work? Dev Biol. 2014;393(1):109–123. PubMed PMC

Shiratori H, Hamada H. The left-right axis in the mouse: from origin to morphology. Development. 2006;133(11):2095–2104. PubMed

Vandenberg LN, Levin M. A unified model for left-right asymmetry? Comparison and synthesis of molecular models of embryonic laterality. Dev Biol. 2013;379(1):1–15. PubMed PMC

Su YH. Telling left from right: left-right asymmetric controls in sea urchins. Genesis. 2014;52(3):269–278. PubMed

Tanaka C, Sakuma R, Nakamura T, Hamada H, Saijoh Y. Long-range action of Nodal requires interaction with GDF1. Genes Dev. 2007;21(24):3272–3282. PubMed PMC

Brennan J, Norris DP, Robertson EJ. Nodal activity in the node governs left-right asymmetry. Genes Dev. 2002;16(18):2339–2344. PubMed PMC

Kawasumi A, Nakamura T, Iwai N, Yashiro K, Saijoh Y, Belo JA, et al. Left-right asymmetry in the level of active Nodal protein produced in the node is translated into left-right asymmetry in the lateral plate of mouse embryos. Dev Biol. 2011;353(2):321–330. PubMed PMC

Saijoh Y, Oki S, Ohishi S, Hamada H. Left-right patterning of the mouse lateral plate requires nodal produced in the node. Dev Biol. 2003;256(1):160–172. PubMed

Nakamura T, Mine N, Nakaguchi E, Mochizuki A, Yamamoto M, Yashiro K, et al. Generation of robust left-right asymmetry in the mouse embryo requires a self-enhancement and lateral-inhibition system. Dev Cell. 2006;11(4):495–504. PubMed

Nakamura T, Hamada H. Left-right patterning: conserved and divergent mechanisms. Development. 2012;139(18):3257–3262. PubMed

Duboc V, Rottinger E, Lapraz F, Besnardeau L, Lepage T. Left-right asymmetry in the sea urchin embryo is regulated by nodal signaling on the right side. Dev Cell. 2005;9(1):147–158. PubMed

Luo YJ, Su YH. Opposing nodal and BMP signals regulate left-right asymmetry in the sea urchin larva. PLoS Biol. 2012;10(10):e1001402. PubMed PMC

Morokuma J, Ueno M, Kawanishi H, Saiga H, Nishida H. HrNodal, the ascidian nodal-related gene, is expressed in the left side of the epidermis, and lies upstream of HrPitx. Dev Genes Evol. 2002;212(9):439–446. PubMed

Yoshida K, Saiga H. Left-right asymmetric expression of Pitx is regulated by the asymmetric Nodal signaling through an intronic enhancer in Ciona intestinalis. Dev Genes Evol. 2008;218(7):353–360. PubMed

Burdine RD, Caspary T. Left-right asymmetry: lessons from Cancun. Development. 2013;140(22):4465–4470. PubMed PMC

Grande C, Patel NH. Nodal signalling is involved in left-right asymmetry in snails. Nature. 2009;457(7232):1007–1011. PubMed PMC

Kuroda R, Endo B, Abe M, Shimizu M. Chiral blastomere arrangement dictates zygotic left-right asymmetry pathway in snails. Nature. 2009;462(7274):790–794. PubMed

Watanabe H, Schmidt HA, Kuhn A, Hoger SK, Kocagoz Y, Laumann-Lipp N, et al. Nodal signalling determines biradial asymmetry in Hydra. Nature. 2014;515(7525):112–115. PubMed

Arendt D, Nubler-Jung K. Inversion of dorsoventral axis? Nature. 1994;371(6492):26. PubMed

De Robertis EM, Sasai Y. A common plan for dorsoventral patterning in Bilateria. Nature. 1996;380(6569):37–40. PubMed

Holland LZ, Carvalho JE, Escriva H, Laudet V, Schubert M, Shimeld SM, et al. Evolution of bilaterian central nervous systems: a single origin? EvoDevo. 2013;4(1):27. PubMed PMC

De Robertis EM. Evo-devo: variations on ancestral themes. Cell. 2008;132(2):185–195. PubMed PMC

Holland LZ, Albalat R, Azumi K, Benito-Gutierrez E, Blow MJ, Bronner-Fraser M, et al. The amphioxus genome illuminates vertebrate origins and cephalochordate biology. Genome Res. 2008;18(7):1100–1111. PubMed PMC

Putnam NH, Butts T, Ferrier DE, Furlong RF, Hellsten U, Kawashima T, et al. The amphioxus genome and the evolution of the chordate karyotype. Nature. 2008;453(7198):1064–1071. PubMed

Bourlat SJ, Juliusdottir T, Lowe CJ, Freeman R, Aronowicz J, Kirschner M, et al. Deuterostome phylogeny reveals monophyletic chordates and the new phylum Xenoturbellida. Nature. 2006;444(7115):85–88. PubMed

Delsuc F, Tsagkogeorga G, Lartillot N, Philippe H. Additional molecular support for the new chordate phylogeny. Genesis. 2008;46(11):592–604. PubMed

Bertrand S, Escriva H. Evolutionary crossroads in developmental biology: amphioxus. Development. 2011;138(22):4819–4830. PubMed

Schubert M, Holland LZ, Stokes MD, Holland ND. Three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) associated with the tail bud: the evolution of somitogenesis in chordates. Dev Biol. 2001;240(1):262–273. PubMed

Lu TM, Luo YJ, Yu JK. BMP and Delta/Notch signaling control the development of amphioxus epidermal sensory neurons: insights into the evolution of the peripheral sensory system. Development. 2012;139(11):2020–2030. PubMed

Glardon S, Holland LZ, Gehring WJ, Holland ND. Isolation and developmental expression of the amphioxus Pax-6 gene (AmphiPax-6): insights into eye and photoreceptor evolution. Development. 1998;125(14):2701–2710. PubMed

Lacalli T. Mucus secretion and transport in amphioxus larvae: organization and ultrastructure of the food trapping system, and implications for head evolution. Acta Zool. 2008;89(3):219–230.

Boorman CJ, Shimeld SM. Pitx homeobox genes in Ciona and amphioxus show left-right asymmetry is a conserved chordate character and define the ascidian adenohypophysis. Evol Dev. 2002;4(5):354–365. PubMed

Le Petillon Y, Oulion S, Escande ML, Escriva H, Bertrand S. Identification and expression analysis of BMP signaling inhibitors genes of the DAN family in amphioxus. Gene Expr Patt. 2013;13(8):377–383. PubMed

Onai T, Yu JK, Blitz IL, Cho KW, Holland LZ. Opposing Nodal/Vg1 and BMP signals mediate axial patterning in embryos of the basal chordate amphioxus. Dev Biol. 2010;344(1):377–389. PubMed PMC

Yasui K, Zhang S, Uemura M, Saiga H. Left-right asymmetric expression of BbPtx, a Ptx-related gene, in a lancelet species and the developmental left-sidedness in deuterostomes. Development. 2000;127(1):187–195. PubMed

Yu JK, Holland LZ, Holland ND. An amphioxus nodal gene (AmphiNodal) with early symmetrical expression in the organizer and mesoderm and later asymmetrical expression associated with left-right axis formation. Evol Dev. 2002;4(6):418–425. PubMed

Yu JK, Satou Y, Holland ND, Shin IT, Kohara Y, Satoh N, et al. Axial patterning in cephalochordates and the evolution of the organizer. Nature. 2007;445(7128):613–617. PubMed

Venkatesh TV, Holland ND, Holland LZ, Su MT, Bodmer R. Sequence and developmental expression of amphioxus AmphiNk2-1: insights into the evolutionary origin of the vertebrate thyroid gland and forebrain. Dev Genes Evol. 1999;209(4):254–259. PubMed

Onimaru K, Shoguchi E, Kuratani S, Tanaka M. Development and evolution of the lateral plate mesoderm: comparative analysis of amphioxus and lamprey with implications for the acquisition of paired fins. Dev Biol. 2011;359(1):124–136. PubMed

Kusakabe R, Kusakabe T, Satoh N, Holland ND, Holland LZ. Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: Implications for conserved mechanisms of chordate development. Dev Genes Evol. 1997;207(4):203–215. PubMed

Satoh G, Wang Y, Zhang P, Satoh N. Early development of amphioxus nervous system with special reference to segmental cell organization and putative sensory cell precursors: a study based on the expression of pan-neuronal marker gene Hu/elav. J Exp Zool. 2001;291(4):354–364. PubMed

Yu JK, Wang MC, Shin IT, Kohara Y, Holland LZ, Satoh N, et al. A cDNA resource for the cephalochordate amphioxus Branchiostoma floridae. Dev Genes Evol. 2008;218(11–12):723–727. PubMed

Yu JK, Holland LZ, Jamrich M, Blitz IL, Hollan ND. AmphiFoxE4, an amphioxus winged helix/forkhead gene encoding a protein closely related to vertebrate thyroid transcription factor-2: expression during pharyngeal development. Evol Dev. 2002;4(1):9–15. PubMed

Langeland JA, Tomsa JM, Jackman WR, Jr, Kimmel CB. An amphioxus snail gene: expression in paraxial mesoderm and neural plate suggests a conserved role in patterning the chordate embryo. Dev Genes Evol. 1998;208(10):569–577. PubMed

Mazet F, Luke GN, Shimeld SM. The amphioxus FoxQ1 gene is expressed in the developing endostyle. Gene Expr Patt. 2005;5(3):313–315. PubMed

Ogasawara M. Overlapping expression of amphioxus homologs of the thyroid transcription factor-1 gene and thyroid peroxidase gene in the endostyle: insight into evolution of the thyroid gland. Dev Genes Evol. 2000;210(5):231–242. PubMed

Somorjai I, Bertrand S, Camasses A, Haguenauer A, Escriva H. Evidence for stasis and not genetic piracy in developmental expression patterns of Branchiostoma lanceolatum and Branchiostoma floridae, two amphioxus species that have evolved independently over the course of 200 Myr. Dev Genes Evol. 2008;218(11–12):703–713. PubMed

Wang Y, Zhang PJ, Yasui K, Saiga H. Expression of Bblhx3, a LIM-homeobox gene, in the development of amphioxus Branchiostoma belcheri tsingtauense. Mech Dev. 2002;117(1–2):315–319. PubMed

Zhang Y, Mao B. Embryonic expression and evolutionary analysis of the amphioxus Dickkopf and Kremen family genes. J Genet Genomics. 2010;37(9):637–645. PubMed

Oulion S, Bertrand S, Belgacem MR, Le Petillon Y, Escriva H. Sequencing and analysis of the Mediterranean amphioxus (Branchiostoma lanceolatum) transcriptome. PLoS One. 2012;7(5):e36554. PubMed PMC

Zhang QJ, Luo YJ, Wu HR, Chen YT, Yu JK. Expression of germline markers in three species of amphioxus supports a preformation mechanism of germ cell development in cephalochordates. EvoDevo. 2013;4(1):17. PubMed PMC

Yu JK, Holland LZ. Cold Spring Harbor Protocols. 2009. Amphioxus (Branchiostoma floridae) spawning and embryo collection. PubMed

Fuentes M, Benito E, Bertrand S, Paris M, Mignardot A, Godoy L, et al. Insights into spawning behavior and development of the European amphioxus (Branchiostoma lanceolatum) J Exp Zool B Mol Dev Evol. 2007;308(4):484–493. PubMed

Hirakow R, Kajita N. Electron microscopic study of the development of amphioxus, Branchiostoma belcheri tsingtauense: the gastrula. J Morphol. 1991;207:27–52. PubMed

Hirakow R, Kajita N. Electron microscopic study of the development of amphioxus, Branchiostoma belcheri tsingtauense: the neurula and larva. Acta Anat Nippon. 1994;69:1–13. PubMed

Wu HR, Chen YT, Su YH, Luo YJ, Holland LZ, Yu JK. Asymmetric localization of germline markers Vasa and Nanos during early development in the amphioxus Branchiostoma floridae. Dev Biol. 2011;353(1):147–159. PubMed

Holland LZ, Holland PWH, Holland ND. Revealing homologies between body parts of distantly related animals by in situ hybridization to developmental genes: amphioxus versus vertebrates. In: Ferraris JD, Palumbi SR, editors. Molecular zoology: advances, strategies, and protocols. New York: Wiley-Liss; 1996. pp. 267–282.

Hiruta J, Mazet F, Yasui K, Zhang P, Ogasawara M. Comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates. Dev Dyn. 2005;233(3):1031–1037. PubMed

Lacalli TC. The dorsal compartment locomotory control system in amphioxus larvae. J Morphol. 2002;252(3):227–237. PubMed

Lacalli TC, Kelly SJ. Somatic motoneurons in amphioxus larvae: cell types, cell position and innervation patterns. Acta Zool. 1999;80:113–124.

Wicht H, Lacalli TC. The nervous system of amphioxus: structure, development, and evolutionary significance. Can J Zool. 2005;83(1):122–150.

Bardet PL, Schubert M, Horard B, Holland LZ, Laudet V, Holland ND, et al. Expression of estrogen-receptor related receptors in amphioxus and zebrafish: implications for the evolution of posterior brain segmentation at the invertebrate-to-vertebrate transition. Evol Dev. 2005;7(3):223–233. PubMed

Jackman WR, Langeland JA, Kimmel CB. islet reveals segmentation in the amphioxus hindbrain homolog. Dev Biol. 2000;220(1):16–26. PubMed

Li KL, Lu TM, Yu JK. Genome-wide survey and expression analysis of the bHLH-PAS genes in the amphioxus Branchiostoma floridae reveal both conserved and diverged expression patterns between cephalochordates and vertebrates. EvoDevo. 2014;5:20. PubMed PMC

Mazet F, Shimeld SM. The evolution of chordate neural segmentation. Dev Biol. 2002;251(2):258–270. PubMed

Jackman WR, Kimmel CB. Coincident iterated gene expression in the amphioxus neural tube. Evol Dev. 2002;4(5):366–374. PubMed

Inacio JM, Marques S, Nakamura T, Shinohara K, Meno C, Hamada H, et al. The dynamic right-to-left translocation of Cerl2 is involved in the regulation and termination of Nodal activity in the mouse node. PLoS One. 2013;8(3):e60406. PubMed PMC

Marques S, Borges AC, Silva AC, Freitas S, Cordenonsi M, Belo JA. The activity of the Nodal antagonist Cerl-2 in the mouse node is required for correct L/R body axis. Genes Dev. 2004;18(19):2342–2347. PubMed PMC

Schweickert A, Vick P, Getwan M, Weber T, Schneider I, Eberhardt M, et al. The nodal inhibitor Coco is a critical target of leftward flow in Xenopus. Curr Biol. 2010;20(8):738–743. PubMed

Nakamura T, Saito D, Kawasumi A, Shinohara K, Asai Y, Takaoka K, et al. Fluid flow and interlinked feedback loops establish left-right asymmetric decay of Cerl2 mRNA. Nat Commun. 2012;3:1322. PubMed

Oki S, Kitajima K, Marques S, Belo JA, Yokoyama T, Hamada H, et al. Reversal of left-right asymmetry induced by aberrant Nodal signaling in the node of mouse embryos. Development. 2009;136(23):3917–3925. PubMed

Pagan-Westphal SM, Tabin CJ. The transfer of left-right positional information during chick embryogenesis. Cell. 1998;93(1):25–35. PubMed

Vonica A, Brivanlou AH. The left-right axis is regulated by the interplay of Coco, Xnr1 and derriere in Xenopus embryos. Dev Biol. 2007;303(1):281–294. PubMed

Marjoram L, Wright C. Rapid differential transport of Nodal and Lefty on sulfated proteoglycan-rich extracellular matrix regulates left-right asymmetry in Xenopus. Development. 2011;138(3):475–485. PubMed PMC

Meno C, Shimono A, Saijoh Y, Yashiro K, Mochida K, Ohishi S, et al. lefty-1 is required for left-right determination as a regulator of lefty-2 and nodal. Cell. 1998;94(3):287–297. PubMed

Meno C, Takeuchi J, Sakuma R, Koshiba-Takeuchi K, Ohishi S, Saijoh Y, et al. Diffusion of nodal signaling activity in the absence of the feedback inhibitor Lefty2. Dev Cell. 2001;1(1):127–138. PubMed

Yamamoto M, Mine N, Mochida K, Sakai Y, Saijoh Y, Meno C, et al. Nodal signaling induces the midline barrier by activating Nodal expression in the lateral plate. Development. 2003;130(9):1795–1804. PubMed

Campione M, Steinbeisser H, Schweickert A, Deissler K, van Bebber F, Lowe LA, et al. The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping. Development. 1999;126(6):1225–1234. PubMed

Kitamura K, Miura H, Miyagawa-Tomita S, Yanazawa M, Katoh-Fukui Y, Suzuki R, et al. Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism. Development. 1999;126(24):5749–5758. PubMed

Lin CR, Kioussi C, O'Connell S, Briata P, Szeto D, Liu F, et al. Pitx2 regulates lung asymmetry, cardiac positioning and pituitary and tooth morphogenesis. Nature. 1999;401(6750):279–282. PubMed

Lu MF, Pressman C, Dyer R, Johnson RL, Martin JF. Function of Rieger syndrome gene in left-right asymmetry and craniofacial development. Nature. 1999;401(6750):276–278. PubMed

Ryan AK, Blumberg B, Rodriguez-Esteban C, Yonei-Tamura S, Tamura K, Tsukui T, et al. Pitx2 determines left-right asymmetry of internal organs in vertebrates. Nature. 1998;394(6693):545–551. PubMed

Shiratori H, Yashiro K, Shen MM, Hamada H. Conserved regulation and role of Pitx2 in situs-specific morphogenesis of visceral organs. Development. 2006;133(15):3015–3025. PubMed

Lacalli TC. Head organization and the head/trunk relationship in protochordates: problems and prospects. Int Comp Biol. 2008;48(5):620–629. PubMed

Satoh N. An aboral-dorsalization hypothesis for chordate origin. Genesis. 2008;46(11):614–622. PubMed

Gerhart J, Lowe C, Kirschner M. Hemichordates and the origin of chordates. Curr Opin Gen Dev. 2005;15(4):461–467. PubMed

Yasui K, Kaji T. The lancelet and ammocoete mouths. Zool Sci. 2008;25(10):1012–1019. PubMed

Christiaen L, Jaszczyszyn Y, Kerfant M, Kano S, Thermes V, Joly JS. Evolutionary modification of mouth position in deuterostomes. Semin Cell Dev Biol. 2007;18(4):502–511. PubMed

Soukup V, Horacek I, Cerny R. Development and evolution of the vertebrate primary mouth. J Anat. 2013;222(1):79–99. PubMed PMC

Schlosser G. Evolutionary origins of vertebrate placodes: insights from developmental studies and from comparisons with other deuterostomes. J Exp Zool B Mol Dev Evol. 2005;304(4):347–399. PubMed

Dickinson A, Sive H. Positioning the extreme anterior in Xenopus: cement gland, primary mouth and anterior pituitary. Semin Cell Dev Biol. 2007;18(4):525–533. PubMed

Wardle FC, Sive HL. What’s your position? the Xenopus cement gland as a paradigm of regional specification. BioEssays. 2003;25(7):717–726. PubMed

Yoshida K, Ueno M, Niwano T, Saiga H. Transcription regulatory mechanism of Pitx in the papilla-forming region in the ascidian, Halocynthia roretzi, implies conserved involvement of Otx as the upstream gene in the adhesive organ development of chordates. Dev Growth Differ. 2012;54(6):649–659. PubMed

Goodyer CG, Tremblay JJ, Paradis FW, Marcil A, Lanctot C, Gauthier Y, et al. Pitx1 in vivo promoter activity and mechanisms of positive autoregulation. Neuroendocrinol. 2003;78(3):129–137. PubMed

Christiaen L, Bourrat F, Joly JS. A modular cis-regulatory system controls isoform-specific pitx expression in ascidian stomodaeum. Dev Biol. 2005;277(2):557–566. PubMed

Duboc V, Rottinger E, Besnardeau L, Lepage T. Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo. Dev Cell. 2004;6(3):397–410. PubMed

Molina MD, de Croze N, Haillot E, Lepage T. Nodal: master and commander of the dorsal-ventral and left-right axes in the sea urchin embryo. Curr Opin Gen Dev. 2013;23(4):445–453. PubMed

Saudemont A, Haillot E, Mekpoh F, Bessodes N, Quirin M, Lapraz F, et al. Ancestral regulatory circuits governing ectoderm patterning downstream of Nodal and BMP2/4 revealed by gene regulatory network analysis in an echinoderm. PLoS Genet. 2010;6(12):e1001259. PubMed PMC

Hibino T, Nishino A, Amemiya S. Phylogenetic correspondence of the body axes in bilaterians is revealed by the right-sided expression of Pitx genes in echinoderm larvae. Dev Growth Differ. 2006;48(9):587–595. PubMed

Lowe CJ, Terasaki M, Wu M, Freeman RM, Jr, Runft L, Kwan K, et al. Dorsoventral patterning in hemichordates: insights into early chordate evolution. PLoS Biol. 2006;4(9):e291. PubMed PMC

Goodrich ES. “Proboscis pores” in craniate vertebrates, a suggestion concerning the premandibular somites and hypophysis. Q J Micr Sci. 1917;62:539–553.

Schlosser G. How old genes make a new head: redeployment of Six and Eya genes during the evolution of vertebrate cranial placodes. Int Comp Biol. 2007;47(3):343–359. PubMed

Kozmik Z, Holland ND, Kreslova J, Oliveri D, Schubert M, Jonasova K, et al. Pax-Six-Eya-Dach network during amphioxus development: conservation in vitro but context specificity in vivo. Dev Biol. 2007;306(1):143–159. PubMed

Butts T, Holland PW, Ferrier DE. Ancient homeobox gene loss and the evolution of chordate brain and pharynx development: deductions from amphioxus gene expression. Proc Biol Sci. 2010;277(1699):3381–3389. PubMed PMC

van Wijhe JW. Ueber Amphioxus. Anat Anz. 1893;8:152–172.

van Wijhe JW. Die Homologisierung des Mundes des Amphioxus und primitive Leibesgliederung der Wirbelthiere. Petrus Camper. 1906;4:61–102.

van Wijhe JW. On the anatomy of the larva of Amphioxus lanceolatus and the explanation of its asymmetry. Proc Kon Akad Wetensch Amsterdam. 1919;21:1013–1023.

MacBride EW. The formation of the layers in Amphioxus and its bearing on the interpretation of the early ontogenetic processes in other vertebrates. Q J Micr Sci. 1909;54:279–345.

Willey A. Amphioxus and the ancestry of the vertebrates. New York: MacMillan and Co.; 1894.

Veeman MT, Newman-Smith E, El-Nachef D, Smith WC. The ascidian mouth opening is derived from the anterior neuropore: reassessing the mouth/neural tube relationship in chordate evolution. Dev Biol. 2010;344(1):138–149. PubMed

Holland ND, Holland LZ. Stage- and tissue-specific patterns of cell division in embryonic and larval tissues of amphioxus during normal development. Evol Dev. 2006;8(2):142–149. PubMed

Kioussi C, Briata P, Baek SH, Rose DW, Hamblet NS, Herman T, et al. Identification of a Wnt/Dvl/beta-Catenin Pitx2 pathway mediating cell-type-specific proliferation during development. Cell. 2002;111(5):673–685. PubMed

Suh H, Gage PJ, Drouin J, Camper SA. Pitx2 is required at multiple stages of pituitary organogenesis: pituitary primordium formation and cell specification. Development. 2002;129(2):329–337. PubMed

Tremblay JJ, Lanctot C, Drouin J. The pan-pituitary activator of transcription, Ptx1 (pituitary homeobox 1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3. Mol Endocrinol. 1998;12(3):428–441. PubMed

Candiani S, Holland ND, Oliveri D, Parodi M, Pestarino M. Expression of the amphioxus Pit-1 gene (AmphiPOU1F1/Pit-1) exclusively in the developing preoral organ, a putative homolog of the vertebrate adenohypophysis. Brain Res Bull. 2008;75(2–4):324–330. PubMed

Monaghan AP, Kioschis P, Wu W, Zuniga A, Bock D, Poustka A, et al. Dickkopf genes are co-ordinately expressed in mesodermal lineages. Mech Dev. 1999;87(1–2):45–56. PubMed

Schweickert A, Steinbeisser H, Blum M. Differential gene expression of Xenopus Pitx1, Pitx2b and Pitx2c during cement gland, stomodeum and pituitary development. Mech Dev. 2001;107(1–2):191–194. PubMed

Dickinson AJ, Sive HL. The Wnt antagonists Frzb-1 and Crescent locally regulate basement membrane dissolution in the developing primary mouth. Development. 2009;136(7):1071–1081. PubMed PMC

Gage PJ, Qian M, Wu D, Rosenberg KI. The canonical Wnt signaling antagonist DKK2 is an essential effector of PITX2 function during normal eye development. Dev Biol. 2008;317(1):310–324. PubMed PMC

Minguillon C, Garcia-Fernandez J. The single amphioxus Mox gene: insights into the functional evolution of Mox genes, somites, and the asymmetry of amphioxus somitogenesis. Dev Biol. 2002;246(2):455–465. PubMed

Yasui K, Tabata S, Ueki T, Uemura M, Zhang SC. Early development of the peripheral nervous system in a lancelet species. J Comp Neurol. 1998;393(4):415–425. PubMed

Kawakami Y, Raya A, Raya RM, Rodriguez-Esteban C, Izpisua Belmonte JC. Retinoic acid signalling links left-right asymmetric patterning and bilaterally symmetric somitogenesis in the zebrafish embryo. Nature. 2005;435(7039):165–171. PubMed

Vermot J, Gallego Llamas J, Fraulob V, Niederreither K, Chambon P, Dolle P. Retinoic acid controls the bilateral symmetry of somite formation in the mouse embryo. Science. 2005;308(5721):563–566. PubMed

Vermot J, Pourquie O. Retinoic acid coordinates somitogenesis and left-right patterning in vertebrate embryos. Nature. 2005;435(7039):215–220. PubMed

Yoshiba S, Hamada H. Roles of cilia, fluid flow, and Ca2+ signaling in breaking of left-right symmetry. Trends Genet. 2014;30(1):10–17. PubMed

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