Cerberus-Nodal-Lefty-Pitx signaling cascade controls left - right asymmetry in amphioxus

Abstract

Many bilaterally symmetrical animals develop genetically programmed left-right asymmetries. In vertebrates, this process is under the control of Nodal signaling, which is restricted to the left side by Nodal antagonists Cerberus and Lefty. Amphioxus, the earliest diverging chordate lineage, has profound left-right asymmetry as a larva. We show that Cerberus, Nodal, Lefty, and their target transcription factor Pitx are sequentially activated in amphioxus embryos. We then address their function by transcription activator-like effector nucleases (TALEN)-based knockout and heat-shock promoter (HSP)-driven overexpression. Knockout of Cerberus leads to ectopic right-sided expression of Nodal, Lefty, and Pitx, whereas overexpression of Cerberus represses their left-sided expression. Overexpression of Nodal in turn represses Cerberus and activates Lefty and Pitx ectopically on the right side. We also show Lefty represses Nodal, whereas Pitx activates Nodal These data combine in a model in which Cerberus determines whether the left-sided gene expression cassette is activated or repressed. These regulatory steps are essential for normal left-right asymmetry to develop, as when they are disrupted embryos may instead form two phenotypic left sides or two phenotypic right sides. Our study shows the regulatory cassette controlling left-right asymmetry was in place in the ancestor of amphioxus and vertebrates. This includes the Nodal inhibitors Cerberus and Lefty, both of which operate in feedback loops with Nodal and combine to establish asymmetric Pitx expression. Cerberus and Lefty are missing from most invertebrate lineages, marking this mechanism as an innovation in the lineage leading to modern chordates.

Keywords: Nodal; TALEN; amphioxus; embryonic development; left–right asymmetry.

Fig. 1.

Cer regulates Nodal, Lefty, and Pitx expression in amphioxus. (A–F) Expression of Nodal, Lefty, and Pitx in Cer wild-type/heterozygous (Cer^+/+, Cer^+/−) or homozygous mutant (Cer^−/−) embryos. (G–N) Expression of Cer, Nodal, Lefty, and Pitx in Control (uninjected but heat-shocked) and Cer misexpressed (Hsp70::Cer-injected and heat-shocked) embryos. Numbers at the bottom right of each panel indicate the number of times the phenotype shown was observed out of the total number of manipulated embryos. (Scale bar, 50 μm.) All embryos are in dorsal view with anterior to left as indicated in the sketch at the bottom left.

Fig. 2.

Nodal regulates Cer, Nodal, Lefty, and Pitx expression in amphioxus. (A–H) Expression patterns of Nodal, Cer, Lefty and Pitx in control (DMSO) and Nodal pathway inhibitor (SB505124)-treated embryos. Embryos were treated from the midgastrula stage until they were collected for gene expression analysis (the 3-somite stage for Cer and around the 8-somite stage for other genes). (I–P) Expression patterns of Nodal, Cer, Lefty, and Pitx in Control (untreated) and Nodal protein-treated embryos. Embryos were cultured with 8 μg/mL of mouse recombinant Nodal protein for the same times as above. (Q–V) Expression patterns of Nodal, Lefty, and Pitx in control (uninjected but heat-shocked) and Nodal misexpressed (Hsp70::Nodal-injected and heat-shocked) embryos. Numbers at the bottom right of each panel indicate the number of times the phenotype shown was observed, out of the total number of embryos examined. (Scale bar, 50 μm.) All images are dorsal views with anterior to left as indicated in the sketch at the bottom right.

Fig. 3.

Lefty inhibits Nodal, Lefty, and Pitx expression, and Pitx overexpression or knockdown induces bilateral expression of Nodal and Lefty. (A–H) Expression patterns of Cer, Nodal, Lefty, and Pitx in control (uninjected) and Lefty TALEN mRNA-injected embryos. (I–N) Expression patterns of Nodal, Lefty, and Pitx in control (uninjected but heat-shocked) and Lefty misexpressed (Hsp70::Lefty-injected and heat-shocked) embryos. (A’–D’) Expression patterns of Nodal and Lefty in control (uninjected) and Pitx mRNA-injected embryos. (E’–J’) Expression patterns of Nodal, Lefty, and Pitx in control (uninjected but heat-shocked) and Pitx misexpressed (Hsp70::Pitx-injected and heat-shocked) embryos. (K’–N’) Expression patterns of Nodal and Lefty in control (uninjected) and Pitx–En mRNA-injected embryos. Numbers at the bottom right of each panel indicate the number of times the phenotype shown was observed, out of the total number of embryos examined. (Scale bar, 50 μm.) All images are dorsal views with anterior to left as indicated in the sketch at the bottom right.

Fig. 4.

Cer mutation or misexpression affects development of amphioxus LR asymmetry and the expression of organ-specific marker genes. (A–C) Asymmetrical morphologies of control (wild-type, Cer^+/−, or Hsp70::Cer-uninjected but heat-hocked) larvae. (A) Left lateral view of the pharyngeal region focused on the right side. (C) Transverse section of larva from A (arrow). (D–F) Symmetrical morphologies of Cer mutant larvae. (D) Left lateral view of the pharyngeal region focused on the sagittal plane. (E) Dorsal view of the pharyngeal region focused on the ventral side. (F) Transverse section of larva from D (arrow). (G–I) Symmetrical morphologies of Cer misexpressed larvae. (G) Left lateral view of the pharyngeal region focused on the sagittal plane. (H) Dorsal view of the pharyngeal region focused on the ventral side. (I) Transverse section of larva from G (arrow). (A’–D’) Expression of Dkk1/2/4 in the region destined to develop into the mouth opening (arrows). The expression domain is detected on the left side of Cer^+/+ or Cer^+/− siblings or control embryos and on both sides of Cer^−/− mutant embryos but is lost in Cer misexpressed embryos. (E’–H’) Expression of Pit in the prospective preoral pit (arrows). Pit transcripts are detected on the left side of Cer^+/+ or Cer^+/− siblings or control embryos and on both sides of Cer^−/− mutants but are undetectable in Cer misexpressed embryos. (I’–L’) Expression of Nkx2.1 in the prospective endostyle. Expression of Nkx2.1 in the endostyle can be divided into the left-side part (arrows) and the right-side part (tandem arrows) along the sagittal plane (indicated by red dotted lines). Although its expression in the left side is duplicated in Cer^−/− mutants and lost in Cer misexpressed embryos, its expression in the right side is lost in Cer^−/− mutants and duplicated in Cer misexpressed embryos. (M’–P’) Expression of FoxE4 in the prospective csg. The csg comprises two parts: the left-sided duct (arrows) and the right-sided glandular region (tandem arrows). FoxE4 expression in the duct is duplicated in Cer^−/− mutants and lost in Cer misexpressed embryos, but its expression in the glandular region is duplicated in Cer misexpressed embryos and lost in Cer^−/− mutants. (Q’–T’) Expression of Krox in the duct of the prospective csg (arrows). Its expression is lost in Cer^−/− mutants but duplicated in Cer misexpressed embryos. Embryos in A’–T’ are in dorsal view with anterior to left. On A’, L marks the left side and R the right side, and this applies to all panels from A’ to T’. Numbers at the bottom right of each panel indicate the number of times the phenotype shown was observed, out of the total number of embryos examined. In some images, red dotted lines separate the left and right of the embryos along the midline. Genotypes as depicted in Fig. 1. Asterisks in C and F denote the external opening of the csg. csg, club-shaped gland; csg-g, right-sided glandular region of csg; en, endostyle; fgs, first gill slit; m, mouth; op, oral papillae; pp, preoral pit. (Scale bar, 50 μm.) The scale bar in A’ also applies to B’–T’.

Fig. 5.

A gene regulatory network model for the control of LR asymmetry in amphioxus. At the gastrula stage, bilateral expression of Cer, Nodal, and Lefty are observed, but Pitx is not expressed, indicating Nodal regulation of Pitx is blocked. During the late gastrula and early neurula stages, Cer expression is maintained on the right side of the embryo. Cer continues to represses Nodal expression, and as a consequence, downstream activation of Nodal and Lefty is lost, and Pitx is not activated. In the absence of Pitx, right-sided morphology develops. On the left of the embryo, Cer expression is lost during the late gastrula to early neurula stage. Nodal is therefore not inhibited and can activate the expression of itself, Pitx, and Lefty. Left-sided Pitx expression results, and hence left-sided morphology develops. An equivalent diagram summarizing the interactions in vertebrates is shown for comparison.