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Cerberus–Nodal–Lefty–Pitx Signaling Cascade Controls Left–Right Asymmetry in Amphioxus

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Cerberus–Nodal–Lefty–Pitx Signaling Cascade Controls Left–Right Asymmetry in Amphioxus Cerberus–Nodal–Lefty–Pitx signaling cascade controls left–right asymmetry in amphioxus Guang Lia,1, Xian Liua,1, Chaofan Xinga, Huayang Zhanga, Sebastian M. Shimeldb,2, and Yiquan Wanga,2 aState Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China; and bDepartment of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom Edited by Marianne Bronner, California Institute of Technology, Pasadena, CA, and approved February 21, 2017 (received for review December 14, 2016) Many bilaterally symmetrical animals develop genetically pro- Several studies have sought to dissect the evolutionary history of grammed left–right asymmetries. In vertebrates, this process is un- Nodal signaling and its regulation of LR asymmetry. Notably, der the control of Nodal signaling, which is restricted to the left side asymmetric expression of Nodal and Pitx in gastropod mollusc by Nodal antagonists Cerberus and Lefty. Amphioxus, the earliest embryos plays a role in the development of LR asymmetry, in- diverging chordate lineage, has profound left–right asymmetry as cluding the coiling of the shell (5, 6). Asymmetric expression of alarva.WeshowthatCerberus, Nodal, Lefty, and their target Nodal and/or Pitx has also been reported in some other lopho- transcription factor Pitx are sequentially activated in amphioxus trochozoans, including Pitx in a brachiopod and an annelid and embryos. We then address their function by transcription activa- Nodal in a brachiopod (7, 8). These data can be interpreted to tor-like effector nucleases (TALEN)-based knockout and heat-shock suggest an ancestral role for Nodal and Pitx in regulating bilat- promoter (HSP)-driven overexpression. Knockout of Cerberus leads erian LR asymmetry, however the picture is complicated by data Nodal Lefty Pitx to ectopic right-sided expression of , ,and ,whereas from other lineages. First, in ecdysozoan lineages, Nodal appears Cerberus overexpression of represses their left-sided expression. to have been lost (7, 9), whereas Pitx is generally symmetrically Cerberus Overexpression of Nodal in turn represses and activates expressed (8) and, where studied, does not function in LR asym- Lefty and Pitx ectopically on the right side. We also show Lefty Nodal Pitx Nodal metry (10, 11). Second, some lophotrochozoans do not show represses ,whereas activates . These data combine asymmetric Pitx expression (8, 12, 13). Third, functional studies on in a model in which Cerberus determines whether the left-sided protostome Nodal signaling are currently restricted to molluscs gene expression cassette is activated or repressed. These regulatory (5), so it is unclear whether the regulatory connection between steps are essential for normal left–right asymmetry to develop, as Nodal and Pitx is conserved even in those species that show when they are disrupted embryos may instead form two pheno- asymmetric expression. An additional complication is that Cer and typic left sides or two phenotypic right sides. Our study shows Lefty the regulatory cassette controlling left–right asymmetry was in are also absent from many invertebrate bilaterian lineages (9, place in the ancestor of amphioxus and vertebrates. This includes 14). The extracellular inhibition of Nodal by the proteins encoded the Nodal inhibitors Cerberus and Lefty, both of which operate in by these two genes is a critical component of Nodal signaling in feedback loops with Nodal and combine to establish asymmetric vertebrates (4, 15) and is required for both the generation and Nodal Pitx2 Pitx expression. Cerberus and Lefty are missing from most inverte- maintenance of asymmetric and . brate lineages, marking this mechanism as an innovation in the To examine the evolutionary history of Nodal signaling and its lineage leading to modern chordates. regulation, we turned to amphioxus, the basal chordate lineage and one that forms a typical chordate body plan while also de- amphioxus | Nodal | left–right asymmetry | TALEN | embryonic veloping pronounced LR asymmetries (16). During amphioxus development embryogenesis, the mouth opens on the left side of the head, ilaterians share three primary developmental axes. The an- Significance Bterior–posterior (AP) and dorsal–ventral (DV) axes define bilaterally symmetrical organization. The third axis, orthogonal Some bilaterally symmetrical animals show genetically pro- to these, is known as the medial–lateral or left–right (LR) axis grammed differences between their left and right sides, for and displays mirror-image symmetry. However, many bilaterian example the placement of the heart and viscera in humans and species deviate consistently from true symmetry, raising funda- other vertebrates. We dissect the regulation of this in embryos mental questions of how symmetry is broken and how different of amphioxus, a close relative of vertebrates that develops developmental programs can unfold on the left and right sides of extraordinary asymmetries as a larva. We demonstrate a sys- an organism (1). In vertebrates, this includes asymmetric devel- tem in which asymmetric expression of the signal gene Nodal is opment of the heart and viscera, disruption of which during controlled by positive and negative feedback loops with its embryogenesis causes a range of human disorders (2). own inhibitors. When this system is disrupted, embryos de- Correct LR organization in vertebrates is regulated by a gene velop mirror-image symmetry with two “left” sides or two “ ” cassette in which right-sided Cerberus (Cer) and left-sided Nodal right sides. Comparison with other animals shows how this and Lefty regulate left-sided expression of the Pitx family gene complex regulatory mechanism has evolved by addition of new Pitx2 and hence morphological LR asymmetry (3). Cer expression feedback regulation to a more ancient signaling interaction. on the right of the embryonic node is required to repress Nodal Author contributions: G.L., X.L., S.M.S., and Y.W. designed research; G.L., X.L., C.X., and signaling, which happens by Cer protein binding directly to Nodal H.Z. performed research; G.L., X.L., C.X., H.Z., S.M.S., and Y.W. analyzed data; and G.L., protein. This restricts the ability of Nodal protein to activate the X.L., S.M.S., and Y.W. wrote the paper. expression of the Nodal gene, leading to an up-regulation of Nodal The authors declare no conflict of interest. Lefty on the left of the embryo. expression is also up-regulated by This article is a PNAS Direct Submission. Nodal and, like Cer, acts as an extracellular inhibitor of Nodal. 1 – G.L. and X.L. contributed equally to this work. Coexpression of Nodal and Lefty can act as an activator inhibitor 2To whom correspondence may be addressed. Email: [email protected] or sebastian. system in the sense originally described by Turing (4). Nodal on [email protected]. Pitx2 the left of the embryo activates expression of , which directs This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the development of left-sided morphology (3). 1073/pnas.1620519114/-/DCSupplemental. 3684–3689 | PNAS | April 4, 2017 | vol. 114 | no. 14 www.pnas.org/cgi/doi/10.1073/pnas.1620519114 Downloaded by guest on September 30, 2021 +/− −/− Cer ,andCer genotypes in the expected ratio (SI Appendix, Fig. S4), and we assessed the expression of Nodal, Lefty,andPitx in +/− these embryos. Although Cer and wild-type embryos main- −/− tained wild-type expression, Cer embryos showed bilateral ex- pression of Nodal, Lefty,andPitx (Fig. 1 A–F). This indicates Cer is necessary to restrict the expression of Nodal, Lefty,andPitx to the left side of amphioxus embryos. To test whether Cer was also sufficient to regulate these genes, we induced overexpression of Cer using a heat-regulated HSP::Cer construct (SI Appendix, Fig. S5). This generated widespread ectopic Cer expression in 100% (31/31) of embryos (Fig. 1H) and abolished left-sided expression of Nodal, Lefty,andPitx in 100% (84/84), 95% (80/88), and 66% J L N Fig. 1. Cer regulates Nodal, Lefty, and Pitx expression in amphioxus. (46/70) of embryos, respectively (Fig. 1 , ,and ). Combined, (A–F) Expression of Nodal, Lefty, and Pitx in Cer wild-type/heterozygous these data show Cer is necessary and sufficient for regulating the + + + − − − (Cer / , Cer / ) or homozygous mutant (Cer / ) embryos. (G–N) Expression lateral expression of Nodal, Lefty,andPitx. of Cer, Nodal, Lefty, and Pitx in Control (uninjected but heat-shocked) and Nodal signaling is transduced by Tgfβ receptors of the Alk4/5/7 Cer misexpressed (Hsp70::Cer-injected and heat-shocked) embryos. Numbers family (22). Pharmacological inhibition of Alk4/5/7 in amphioxus at the bottom right of each panel indicate the number of times the phe- has been reported to block left-sided Nodal, Lefty,andPitx ex- notype shown was observed out of the total number of manipulated em- pression (18), and we found the same in our experiments (Fig. 2 B, bryos. (Scale bar, 50 μm.) All embryos are in dorsal view with anterior to left F H as indicated in the sketch at the bottom left. ,and ). However, the Alk4/5/7 receptor may also mediate signaling by other Tgfβ family ligands, including Activin and Tgfβ (23), raising uncertainty as to the ligand involved in its activation while the gill slits, which initiate opening at the ventral midline, later in amphioxus in vivo. Nodal in amphioxus also has an earlier extend up the right side. Other endodermal organs are similarly developmental role (24), precluding its inhibition by the TALEN asymmetric, and somites also show pronounced LR asymmetry. Cer, method as embryos would not develop to the stage where LR Nodal, Lefty,andPitx are asymmetrically expressed in amphioxus, asymmetry can be addressed. We therefore tested the effect of and pharmacological manipulation of the Alk4/5/7 Tgfβ receptor inducible and ectopic Nodal on amphioxus LR asymmetry. disrupts LR development, suggesting a function for Nodal in am- Overexpression of Nodal using an HSP::Nodal construct (Fig.
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