Nodal signaling
Encyclopedia
Nodal signaling is a signal transduction
pathway that is important in pattern formation
and differentiation
during embryo 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 factor
s 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 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 evolutionary conserved in almost all vertebrates, the regulation of each component of the pathway sometimes varies according to the species.
and embryo development. Further study of this gene by Zhou et al. showed that the nodal genes acted as a secreted signaling peptide that was sufficient to induce mesoderm cells in the mouse embryo. This was an important find 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.
s (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.
and BMP7
form heterodimers with Nodal, causing mutual inhibition of the involved pathways.
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.
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.
. Smad2 will then associate with Smad4
and translocate into the nucleus thereby stimulating transcription of nodal target genes. Evidence has been shown that another Smad, Smad3, can be phosphorylated by activated receptors and may also function as an activator of nodal genes. However, knockout of Smad2 in mice leads to disruption of the formation of the primitive streak
. This is not sufficient to knockdown all mesoendodermal genes showing that Smad3 has some overlapping function with Smad2. However, the expression of these genes is ubiquitous in Smad2 KO embryos whereas it is limited in the wild type. Smad3 knockouts do not have a phenotype showing that expression overlap with Smad2 is sufficient normal development.
negatively regulates the nodal pathway by inhibiting the interaction of Smad4 with other Smads
inside the nucleus via the mono-ubiquitination Smad4, this modification allow it to be transported out of the cytoplasm where it can be deubiquitinated by FAM protein, allowing it to form complexes again with other Smads. Another negative regulator of the pathway intervening with Smads is PPM1A, a phosphatase that acts with Phospho-Smad2/3 making it inactive. Subsequently, Smad2/3 is transported outside the nucleus with the help of RanBP2.
There are some other transcription factors that compete for some of the components of the transcriptional machinery for the activation of Nodal target genes. For instance, Tgif1 and Tgif2 are negative co-regulators that compete for the active form of Smad2, reducing the relative concentration of active Smad2 in the nucleus. In Xenopus, the loss-of-function of Tgf1 and Tgf2 causes the up-regulation of Xnr5 and Xnr6. Another example of transcriptional repressors in frog is XFDL, that binds to p53 obstructing the interaction with the Smad2/3/4 complex.
and mesoderm
, neuroectoderm
formation requires blocking nodal signaling which is accomplished by the expression of nodal antagonist, Cerberus. The role of nodal signaling reemerges later in development when nodal signaling is required to specify ventral cell neural patterning. Loss of function of Cyclops or oep in zebrafish results in cyclopic embryos characterized by a lack of medial floor plate and ventral forebrain. Not all nodals result in the formation of mesoectoderm. Xenopus nodal related 3, (Xnr3) a divergent member of the TGFβ superfamily, induces the expression of the protein, Xbra. The Xbra expression pattern, in correlation the expression pattern another neuroinducer, Xlim-1, result in the patterning of the organizer in Xenopus. This signaling in conjucture with other nodals, noggin, chordin, follistatin and others results in the final patterning of vertebrate central nervous system.
Signal transduction
Signal transduction occurs when an extracellular signaling molecule activates a cell surface receptor. In turn, this receptor alters intracellular molecules creating a response...
pathway that is important in pattern formation
Regional specification
In the field of developmental biology, regional specification is the process by which different areas are identified in the development of the early embryo. The process by which the cells become specified differs between organisms.-Cell fate determination:...
and differentiation
Cellular differentiation
In developmental biology, cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type. Differentiation occurs numerous times during the development of a multicellular organism as the organism changes from a simple zygote to a complex system of...
during embryo development
Developmental biology
Developmental biology is the study of the process by which organisms grow and develop. Modern developmental biology studies the genetic control of cell growth, differentiation and "morphogenesis", which is the process that gives rise to tissues, organs and anatomy.- Related fields of study...
.
The nodal
Nodal
Nodal can refer to* Nodal, a protein encoded by the gene NODAL and responsible for left-right asymmetry* Nodal , a novel music composition program...
family of proteins, a subset of the transforming growth factor beta (TGFβ)
TGF beta signaling pathway
The Transforming growth factor beta signaling pathway is involved in many cellular processes in both the adult organism and the developing embryo including cell growth, cell differentiation, apoptosis, cellular homeostasis and other cellular functions. In spite of the wide range of cellular...
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
Mothers against decapentaplegic homolog 2
Mothers against decapentaplegic homolog 2 also known as SMAD family member 2 or SMAD2 is a protein that in humans is encoded by the SMAD2 gene. MAD homolog 2 belongs to the SMAD, a family of proteins similar to the gene products of the Drosophila gene 'mothers against decapentaplegic' and the C....
. The P-Smad2/Smad4
Mothers against decapentaplegic homolog 4
SMAD family member 4, also known as SMAD4, is a protein that in humans is encoded by the SMAD4 gene.SMAD4 is a 552-amino acid protein involved in cell signaling. It belongs to the Darfwin family of proteins that modulate members of the TGFβ protein superfamily...
complex translocates
Protein targeting
Protein targeting or protein sorting is the mechanism by which a cell transports proteins to the appropriate positions in the cell or outside of it. Sorting targets can be the inner space of an organelle, any of several interior membranes, the cell's outer membrane, or its exterior via secretion...
into the nucleus
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
to interact with transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
s such as FoxH1
FOXH1
Forkhead box protein H1 is a protein that in humans is encoded by the FOXH1 gene.-Interactions:FOXH1 has been shown to interact with DRAP1 and Mothers against decapentaplegic homolog 2....
, p53
P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
and Mixer (Xenopus
Xenopus
Xenopus is a genus of highly aquatic frogs native to Sub-Saharan Africa. There are 19 species in the Xenopus genus...
mix-like endodermal regulator). This will, in turn, lead to induction of target genes such as Nodal, Lefty
Lefty (protein)
Lefty are proteins that are closely related members of the TGF-beta family of growth factors. These proteins are secreted and play a role in left-right asymmetry determination of organ systems during development...
, the antagonist of nodal Cerberus, and others.
The activation of 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 evolutionary 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 gastrulationGastrulation
Gastrulation is a phase early in the embryonic development of most animals, during which the single-layered blastula is reorganized into a trilaminar structure known as the gastrula. These three germ layers are known as the ectoderm, mesoderm, and endoderm.Gastrulation takes place after cleavage...
and embryo development. Further study of this gene by Zhou et al. showed that the nodal genes acted as a secreted signaling peptide that was sufficient to induce mesoderm cells in the mouse embryo. This was an important find as many other factors had been implicated in the formation of mesoderm in Xenopus
Xenopus
Xenopus is a genus of highly aquatic frogs native to Sub-Saharan Africa. There are 19 species in the Xenopus genus...
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 proteinBone morphogenetic protein
Bone morphogenetic proteins are a group of growth factors also known as cytokines and as metabologens . Originally discovered by their ability to induce the formation of bone and cartilage, BMPs are now considered to constitute a group of pivotal morphogenetic signals, orchestrating tissue...
s (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 BMP3Bone morphogenetic protein 3
Bone morphogenetic protein 3 is a protein that in humans is encoded by the BMP3 gene.The protein encoded by this gene is a member of the transforming growth factor beta superfamily. It, like other bone morphogenetic proteins is known for its ability to induce bone and cartilage development. It is...
and BMP7
Bone morphogenetic protein 7
Bone morphogenetic protein 7 or BMP7 is a protein that in humans is encoded by the BMP7 gene.-Function:The protein encoded by this gene is a member of the TGF-β superfamily...
form heterodimers with Nodal, causing mutual inhibition of the involved pathways.
Convertases: furin and PACE4
Nodal mRNA produces an immature protein form of nodalNodal
Nodal can refer to* Nodal, a protein encoded by the gene NODAL and responsible for left-right asymmetry* Nodal , a novel music composition program...
that is cleaved by proteins called convertases in order to generate a mature nodal. The subtilisin
Subtilisin
Subtilisin is a non-specific protease initially obtained from Bacillus subtilis.Subtilisins belong to subtilases, a group of serine proteases that initiate the nucleophilic attack on the peptide bond through a serine residue at the active site. They are physically and chemically...
-like proprotein convertases (SPC) Furin
Furin
Furin is a protein that in humans is encoded by the FURIN gene. It was named furin because it was in the upstream region of an oncogene known as FES. The gene was known as FUR and therefore the protein was named furin...
(Spc1) and PACE4
PCSK6
Proprotein convertase subtilisin/kexin type 6 is an enzyme that in humans is encoded by the PCSK6 gene.-Further reading:...
(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 mesodermMesoderm
In all bilaterian animals, the mesoderm is one of the three primary germ cell layers in the very early embryo. The other two layers are the ectoderm and endoderm , with the mesoderm as the middle layer between them.The mesoderm forms mesenchyme , mesothelium, non-epithelial blood corpuscles and...
formation acting through the down-regulation of the Wnt
Wnt signaling pathway
The Wnt signaling pathway is a network of proteins best known for their roles in embryogenesis and cancer, but also involved in normal physiological processes in adult animals.-Discovery:...
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
Activin type 1 receptors
The Activin type I receptors transduce signals for a variety of members of the Transforming growth factor beta superfamily of ligands. This family of cytokines and hormones include activin, Anti-müllerian hormone , bone morphogenetic proteins , and Nodal...
and targets the receptor for lysosomal degradation. Dapper2 overexpression mimics nodal
Nodal
Nodal can refer to* Nodal, a protein encoded by the gene NODAL and responsible for left-right asymmetry* Nodal , a novel music composition program...
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.
Smad
Smad proteins are responsible for transducing nodal signals into the nucleus. The binding of Nodal proteins to activin or activin-like serine/threonine kinase receptors results in the phosphorylation of Smad2Mothers against decapentaplegic homolog 2
Mothers against decapentaplegic homolog 2 also known as SMAD family member 2 or SMAD2 is a protein that in humans is encoded by the SMAD2 gene. MAD homolog 2 belongs to the SMAD, a family of proteins similar to the gene products of the Drosophila gene 'mothers against decapentaplegic' and the C....
. Smad2 will then associate with Smad4
Mothers against decapentaplegic homolog 4
SMAD family member 4, also known as SMAD4, is a protein that in humans is encoded by the SMAD4 gene.SMAD4 is a 552-amino acid protein involved in cell signaling. It belongs to the Darfwin family of proteins that modulate members of the TGFβ protein superfamily...
and translocate into the nucleus thereby stimulating transcription of nodal target genes. Evidence has been shown that another Smad, Smad3, can be phosphorylated by activated receptors and may also function as an activator of nodal genes. However, knockout of Smad2 in mice leads to disruption of the formation of the primitive streak
Primitive streak
The primitive streak is a structure that forms during the early stages of avian, reptilian and mammalian embryonic development.-Introduction:...
. This is not sufficient to knockdown all mesoendodermal genes showing that Smad3 has some overlapping function with Smad2. However, the expression of these genes is ubiquitous in Smad2 KO embryos whereas it is limited in the wild type. Smad3 knockouts do not have a phenotype showing that expression overlap with Smad2 is sufficient normal development.
Molecules affecting nodal activation via smad
EctoderminEctoderm specification
In Xenopus laevis, the specification of the three germ layers occurs at the blastula stage. Great efforts have been made to determine the factors that specify the endoderm and mesoderm. On the other hand, only a few examples of genes that are required for ectoderm specification have been described...
negatively regulates the nodal pathway by inhibiting the interaction of Smad4 with other Smads
SMAD (protein)
SMADs are intracellular proteins that transduce extracellular signals from transforming growth factor beta ligands to the nucleus where they activate downstream TGF-β gene transcription....
inside the nucleus via the mono-ubiquitination Smad4, this modification allow it to be transported out of the cytoplasm where it can be deubiquitinated by FAM protein, allowing it to form complexes again with other Smads. Another negative regulator of the pathway intervening with Smads is PPM1A, a phosphatase that acts with Phospho-Smad2/3 making it inactive. Subsequently, Smad2/3 is transported outside the nucleus with the help of RanBP2.
Transcriptional factors controlling signaling
Smad2/3/4 can associate to different transcription factors such as p53, Mixer and FosH1 and recognize specific cis-regulatory elements to activate the expression of Nodal target genes at a precise time and location and activate genes required for mesoderm induction.There are some other transcription factors that compete for some of the components of the transcriptional machinery for the activation of Nodal target genes. For instance, Tgif1 and Tgif2 are negative co-regulators that compete for the active form of Smad2, reducing the relative concentration of active Smad2 in the nucleus. In Xenopus, the loss-of-function of Tgf1 and Tgf2 causes the up-regulation of Xnr5 and Xnr6. Another example of transcriptional repressors in frog is XFDL, that binds to p53 obstructing the interaction with the Smad2/3/4 complex.
miRNAs controlling signaling
In vertebrates, the evolutionary conserved family of microRNAs miR-430/427/302 is expressed early in development. It has important roles in controlling mesoderm and endoderm specification, and it does it by regulating the protein expression levels of some Nodal signaling components. This family is composed by the teleost miR-430, the amphibian miR-427 and the mammalian miR-302. In zebra fish the miR-430 inhibits translation of Sqt, Lefty1 and Lefty2, in frogs miR-427 regulate Xnr5, Xnr6b, LeftyA and LeftyB, however in humans embryonic stem cells it has been shown that miR-302 negative regulates the expression of only Lefty1 and Lefty2 but it does not seem down-regulate Nodal protein expression levels.Mesoendoderm induction
Multiple studies have established that Nodal signal is required for the induction of most mesodermal and endodermal cell types and Squint/Cyclops knockouts in Zebrafish do not develop notochord, heart, kidneys or even blood. The origin and expression pattern of the nodal signaling proteins differs in different species. Mammalian nodal signaling is initiated ubiquitously in epiblast cells and is maintained by autoregulatory signaling of Wnt3 and limited by the induction of antagonists such as Cerberus-like and lefty. Studies in Xenopus have found that xnr expression (the Xenopus nodal) is induced by VegT at the vegetal pole and nodals spread to the blastula. Xnr expression is stabilized by the presence of β-catenin. This information raises the question of how nodal signaling leads to the induction of both endoderm and mesoderm. The answer comes in form of a gradient of nodal protein. Temporal and special differences in nodal signaling will result in different cell fates. With the addition of antagonists and variable range of different nodals, a map of cell fates including both mesoderm and endoderm can be drawn for the embryo. However, it is unclear whether nodals signaling in summated or if cells respond to the amplitude of the signal.Determination of the dorsal-ventral axis
Human anatomy is asymmetric with the heart located on the left side and the liver on the right. Asymmetry is a feature common to all vertebrates and even paired-symmetric organs such as lungs display asymmetries in the number of lobes. Evidence that nodal signaling is responsible for left-right specification comes from genetic analysis of organisms deficient in left-right specification. These genetic studies led to identification of mutations in components in the nodal signaling pathway such as ActRIIB, Criptic, and FoxH1 in mouse. These studies found that the left-right symmetry is created as a result of nodal antagonist expression on the right side of the embryo which is balanced by nodal upregulating itself on the other half of the embryo. The result is a nodal gradient that is high on the ventral side of the embryo and, through antagonist action, declines as a gradient to the midline where only abberent expression is seen.Neural patterning
As nodal signaling give rise to ectodermEctoderm
The "ectoderm" is one of the three primary germ cell layers in the very early embryo. The other two layers are the mesoderm and endoderm , with the ectoderm as the most exterior layer...
and mesoderm
Mesoderm
In all bilaterian animals, the mesoderm is one of the three primary germ cell layers in the very early embryo. The other two layers are the ectoderm and endoderm , with the mesoderm as the middle layer between them.The mesoderm forms mesenchyme , mesothelium, non-epithelial blood corpuscles and...
, neuroectoderm
Neuroectoderm
Neuroectoderm is the term for ectoderm which receives Bone Morphogenetic Protein-inhibiting signals from proteins such as noggin, which leads to the development of the nervous system from this tissue....
formation requires blocking nodal signaling which is accomplished by the expression of nodal antagonist, Cerberus. The role of nodal signaling reemerges later in development when nodal signaling is required to specify ventral cell neural patterning. Loss of function of Cyclops or oep in zebrafish results in cyclopic embryos characterized by a lack of medial floor plate and ventral forebrain. Not all nodals result in the formation of mesoectoderm. Xenopus nodal related 3, (Xnr3) a divergent member of the TGFβ superfamily, induces the expression of the protein, Xbra. The Xbra expression pattern, in correlation the expression pattern another neuroinducer, Xlim-1, result in the patterning of the organizer in Xenopus. This signaling in conjucture with other nodals, noggin, chordin, follistatin and others results in the final patterning of vertebrate central nervous system.