The Nodal signaling pathway is a signal transduction pathway important in regional and cellular differentiation during embryonic development. The Nodal family of proteins, a subset of the transforming growth factor beta (TGFβ) superfamily, is responsible for mesoendoderm induction, patterning of the nervous system, and determination of dorsal- ventral axis in vertebrate embryos. Activation of the Nodal pathway involves nodal binding to activin and activin-like receptors which leads to phosphorylation of the Smad2. The P-Smad2/Smad4 complex translocates into the nucleus to interact with transcription factors such as FoxH1, p53 and Mixer (Xenopus mix-like endodermal regulator). This will, in turn, lead to induction of target genes such as NODAL, Lefty, the antagonist of nodal cerberus, and others. The activation of the Nodal pathway induces the transcription of many target genes including of its own, but at the same time, micro-RNAs and other proteins interfere with this positive feedback loop in a negative manner at different points of the pathway. This balance of activation and inhibition of the signal is necessary to achieve the precise location, concentration and duration of downstream target genes that have an important role early in development. This article will summarize the role of some of the components that participate positively and negatively in regulation the signaling pathway. Although all the major components of Nodal signaling are evolutionarily conserved in almost all vertebrates, the regulation of each component of the pathway sometimes varies according to the species.
History The nodal gene was originally discovered by Conlon et al. by retroviral mutation in mice which led to the isolation of a gene that interfered with normal mouse gastrulation and embryo development. Further study of this gene by Zhou et al. showed that the nodal genes encode a secreted signaling peptide that was sufficient to induce mesoderm cells in the mouse embryo. This was an important finding as many other factors had been implicated in the formation of mesoderm in Xenopus whereas the difficulty of removal of these factors due to embryonic lethality and maternal contribution of genes had kept the ability to assay the knock out phenotypes elusive. Further studies of nodal signaling in other vertebrates such as Cyclops and Squint in zebrafish proved that nodal signaling is adequate to induce mesoderm in all vertebrates.
Selected components of the pathway
Lefty The Lefty proteins, divergent members of the TGFβ superfamily of proteins, act as extracellular antagonists of Nodal signaling. Expression studies of the Lefty homologue, antivin, in zebrafish show that Lefty likely acts as a competitive inhibitor of Nodal signaling. Overexpression of Lefty leads to a phenotype similar to a Nodal knockout while overexpression of the activin (nodal-related protein) receptor or even the receptor extracellular domain can rescue the phenotype. As the induction of Lefty is dependent upon Nodal expression, lefty acts a classic feedback inhibitor for Nodal signaling. Like nodals, all vertebrates have at least one Lefty gene while many, such as zebrafish and mouse, have two unique Lefty genes.
DAN proteins DAN proteins, such as Cerberus and Coco in Xenopus and Cerberus-like in mouse, also act as antagonists of Nodal signaling. Unlike Lefty proteins, DAN proteins bind directly to extracellular Nodal proteins and prevent signaling. Further, not all DAN proteins are specific to Nodal signaling and will also block bone morphogenetic proteins (BMPs) and, in the case of Cerberus and Coco, Wnt signaling as well. This activity is important in neural development and left-right symmetry as will be discussed later.
BMPs Lefty and Cerberus are not the only ones to be able to interact in the extracellular space with Nodal, there is biochemical evidence that BMP3 and BMP7 form heterodimers with Nodal, causing mutual inhibition of the involved pathways.
Convertases: Furin and PACE4 Nodal mRNA produces an immature protein form of Nodal that is cleaved by proteins called convertases in order to generate a mature Nodal. The subtilisin-like proprotein convertases (SPC) Furin (Spc1) and PACE4 (Spc4) recognize a specific sequence of the precursor of Nodal protein and cleaves it to form the mature Nodal ligand. Conversely, the immature form of Nodal is still capable to activate the pathway. During Nodal transportation to the extracellular space, the Nodal co-receptor captures the Nodal precursor in lipid rafts and once in the cell surface, Cripto interacts with the convertases and forms a complex that facilitates the processing of Nodal.
EGF-CFC proteins EGF-CFC proteins are membrane bound extracellular factors that serve as essential cofactor in Nodal signaling and in vertebrate development as a whole. This family of cofactors includes One-eyed Pinhead (oep) in Zebrafish, FRL1 in Xenopus, and Cripto and Criptic in mouse and human. Genetic studies of oep in zebrafish have shown that the knockout of both maternal and zygotic oep leads to a phenotype similar to that of the squint/Cyclops (Nodals) knockout. Similarly, over-expression of either the Nodal (squint/Cyclops) or oep with the knockout of the other does not show phenotypical differences. This evidence coupled with the data that overexpression of oep shows no phenotype corroborates the role of EGF-CFC as an essential cofactor in Nodal signaling.
Dapper2 In mouse, frog and fish, Dapper2, is a negative regulator of mesoderm formation acting through the down-regulation of the Wnt and TGFβ / nodal signaling pathways. In zebrafish, nodal is known to activate the gene expression of dapper2. In the cell surface Dapper2 tightly binds to the active form of the activin type 1 receptors and targets the receptor for lysosomal degradation. Dapper2 overexpression mimics nodal co-receptor loss of function because nodal signal cannot be transduced and therefore it produces less mesoderm. In the mouse embryo, dpr2 mRNA is located across all the embryo 7.5 days post conception (dpc) however its location changes at 8.5-dpc where it is observed at the prospective somites and by 10-dpc, neural tube, otic vesicle and gut; because Dapper2 and Nodal are expressed in the same region, this suggests that Dapper antagonizes mesoderm induction signals derived from Nodal. Somehow the reduction of activin receptors would lead to the decrease in activity of different TGFb pathways.
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