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.B ilaterians share three primary developmental axes. The anterior-posterior (AP) and dorsal-ventral (DV) axes define bilaterally symmetrical organization. The third axis, orthogonal to these, is known as the medial-lateral or left-right (LR) axis and displays mirror-image symmetry. However, many bilaterian species deviate consistently from true symmetry, raising fundamental questions of how symmetry is broken and how different developmental programs can unfold on the left and right sides of an organism (1). In vertebrates, this includes asymmetric development of the heart and viscera, disruption of which during embryogenesis causes a range of human disorders (2).Correct LR organization in vertebrates is regulated by a gene cassette in which right-sided Cerberus (Cer) and left-sided Nodal and Lefty regulate left-sided expression of the Pitx family gene Pitx2 and hence morphological LR asymmetry (3). Cer expression on the right of the embryonic node is required to repress Nodal signaling, which happens by Cer protein binding directly to Nodal protein. This restricts the ability of Nodal protein to activate the expression of the Nodal gene, leading to an up-regulation of Nodal on the left of the embryo. Lefty expression is also up-regulated by Nodal and, like Cer, acts as an extracellular inhibitor of Nodal. Coexpression o...

My Favorite Animal, Amphioxus: Unparalleled for Studying Early Vertebrate Evolution

“…Other techniques that have represented great advances are the induction of spawning by temperature change that I already mentioned above, which allows one to obtain embryos on a daily basis during the spawning season, or the development of husbandry techniques with artificial sea water, which allows laboratories inland to work with amphioxus. Obtaining the complete life cycle of amphioxus in captivity has also been a very important step, particularly to create knockout lines of amphioxus using transcription activator‐like effector nucleases, (TALEN)‐based knockout . This knockout technique allowed Li and collaborators to demonstrate the master role of the Nodal antagonist Cerberus in the control of left‐right asymmetries in amphioxus .…”

“…Obtaining the complete life cycle of amphioxus in captivity has also been a very important step, particularly to create knockout lines of amphioxus using transcription activator‐like effector nucleases, (TALEN)‐based knockout . This knockout technique allowed Li and collaborators to demonstrate the master role of the Nodal antagonist Cerberus in the control of left‐right asymmetries in amphioxus . Other techniques allowing functional studies developed by various laboratories include the use of morpholinos to knockdown gene expression or the overexpression of a given gene through microinjection of capped‐mRNA.…”

“…Other techniques allowing functional studies developed by various laboratories include the use of morpholinos to knockdown gene expression or the overexpression of a given gene through microinjection of capped‐mRNA. These studies include the overexpression of chimeric transcription factors acting as constitutive repressors as a result of the fusion of the Drosophila Engrailed repressor domain to the sequence of a given transcription factor . The first morpholino microinjected in amphioxus embryos was a morpholino blocking the expression of Hox1 .…”

Dorsal-ventral patterning in amphioxus: current understanding, unresolved issues, and future directions

“…In vertebrates, Lefty itself negatively regulates the Smad2/3-mediated signaling and the expression of Nodal (Bisgrove et al, 1999;Branford and Yost, 2002;Feldman et al, 2002). A recent study that used gene-specific knock-out experiments in amphioxus showed that Smad2/3-mediated signaling activates Nodal and Lefty, and that Lefty strongly represses Nodal expression during the late gastrula/neurula stage (Li et al, 2017), suggesting that this autoregulatory loop also occurs during the blastula/early gastrula stages in amphioxus embryos. These Nodal-Lefty regulatory interactions may represent a reaction-diffusion system that operates during the axis establishment in deuterostomes (Branford and Yost, 2002;Duboc et al, 2008).…”

Evolution of nodal and nodal‐related genes and the putative composition of the heterodimers that trigger the nodal pathway in vertebrates

“…Understanding the evolution of genes that are involved in early developmental processes represents an important challenge in biology (Gilbert, ). In most of the cases, the information that is available is restricted to the presence of a particular developmental pathway, and of the genes that participate in it, but much less is known regarding the evolutionary history of the genes and pathways (Jones, Kuehn, Hogan, Smith, & Wright, ; Joseph & Melton, ; Lagadec et al, ; Levin, Johnson, Stern, Kuehn, & Tabin, ; Li et al, ; Raya, Carlos, & Belmonte, ; Seleiro, Connolly, & Cooke, ; Tadjuidje et al, ).…”