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Results for “Nodal Signaling Ligands”

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At least 19 recordsLinked to original sources
PubMed2002

Dual roles of Cripto as a ligand and coreceptor in the nodal signaling pathway.

The EGF-CFC gene Cripto encodes an extracellular protein that has been implicated in the signaling pathway for the transforming growth factor beta (TGF beta) ligand Nodal. Although recent findings in frog and fish embryos have suggested that EGF-CFC proteins function as coreceptors for Nodal, studies in cell culture have implicated Cripto as a growth factor-like signaling molecule. Here we reconcile these apparently disparate models of Cripto function by using a mammalian cell culture assay to investigate the signaling activities of Nodal and EGF-CFC proteins. Using a luciferase reporter assay, we found that Cripto has activities consistent with its being a coreceptor for Nodal. However, Cripto can also function as a secreted signaling factor in cell coculture assays, suggesting that it may also act as a coligand for Nodal. Furthermore, we found that the ability of Cripto to bind to Nodal and mediate Nodal signaling requires the addition of an O-linked fucose monosaccharide to a conserved site within EGF-CFC proteins. We propose a model in which Cripto has dual roles as a coreceptor as well as a coligand for Nodal and that this signaling interaction with Nodal is regulated by an unusual form of glycosylation. Our findings highlight the significance of extracellular modulation of ligand activity as an important means of regulating TGF beta signaling pathways during vertebrate development.

Activin Receptors, Type I↗
PubMed2006

A signalling relay involving Nodal and Delta ligands acts during secondary notochord induction in Ciona embryos.

The notochord is one of the defining features of chordates. The ascidian notochord is a rod like structure consisting of a single row of 40 cells. The anterior 32 ;primary' notochord cells arise from the A-line (anterior vegetal) blastomeres of the eight-cell stage embryo, whereas the posterior 8 ;secondary' notochord cells arise from the B-line (posterior vegetal) blastomeres of the eight-cell stage embryo. Specification of notochord precursors within these two lineages occurs in a spatially and temporally distinct manner. We show that specification of the secondary but not the primary notochord in Ciona intestinalis requires a relay mechanism involving two signalling pathways. First, we show evidence that acquisition of secondary notochord fate is dependent upon lateral Nodal signalling sources, situated in the adjacent b-line animal cells. Expression of the notochord specific gene Ci-Brachyury in the secondary notochord precursor was downregulated following selective inhibition of Nodal signal reception in B-line derivatives and also, strikingly, following selective inhibition of Nodal signal reception in A-line cell derivatives. Within the A-line, Nodal signals are required for localised expression of Delta2, which encodes a divergent form of Delta ligand. Using four distinct reagents to inhibit Delta2/Notch signals, we showed that Delta2 signalling from A-line cells, which activates the Notch/Su(H) pathway in adjacent B-line cells, is required for specification of the secondary notochord precursor. We propose a model whereby laterally produced Nodal acts to specify the secondary notochord precursor both directly in the B-line cells and via Delta2 induction in adjacent A-line cells.

Amino Acid Sequence↗
PubMed2002

Lefty antagonism of Squint is essential for normal gastrulation.

Activities of a variety of signaling proteins that regulate embryogenesis are limited by endogenous antagonists. The zebrafish Nodal-related ligands, Squint and Cyclops, and their antagonists, Lefty1 and Lefty2, belong to the TGFbeta-related protein superfamily, whose members have widespread biological activities. Among other activities, Nodals direct the formation of most mesendoderm. By inducing their own transcription and that of the Lefties, Nodal signals establish positive and negative autoregulatory loops. To investigate how these autoregulatory pathways regulate development, we depleted zebrafish embryos of Lefty1 and/or Lefty2 by using antisense morpholino oligonucleotides. Loss of Lefty1 causes aberrations during somitogenesis stages, including left-right patterning defects, whereas Lefty2 depletion has no obvious consequences. Depletion of both Lefty1 and Lefty2, by contrast, causes unchecked Nodal signaling, expansion of mesendoderm, and loss of ectoderm. The expansion of mesendoderm correlates with an extended period of rapid cellular internalization and a failure of deep-cell epiboly. The gastrulation defects of embryos depleted of Lefty1 and Lefty2 result from the deregulation of Squint signaling. In contrast, deregulation of Cyclops does not affect morphology or the transcription of Nodal target genes during gastrulation. Furthermore, we find that Cyclops is specifically required for the maintenance of lefty1 and lefty2 transcription.

Animals↗
PubMed2002

Effects of heterodimerization and proteolytic processing on Derrière and Nodal activity: implications for mesoderm induction in Xenopus.

Derrière is a recently discovered member of the TGFbeta superfamily that can induce mesoderm in explant assays and is expressed at the right time and location to mediate mesoderm induction in response to VegT during Xenopus embryogenesis. We show that the ability of Derrière to induce dorsal or ventral mesoderm depends strictly on the location of expression and that a dominant-negative Derrière cleavage mutant completely blocks all mesoderm formation when ectopically expressed. This differs from the activity of similar Xnr2 cleavage mutant constructs, which are secreted and retain signaling activity. Additional analysis of mesoderm induction by Derrière and members of the Nodal family indicates that these molecules are involved in a mutual positive-feedback loop and antagonism of either one of the signals can reduce the other. Interaction between Derrière and members of the Nodal family is also shown to occur through the formation of heterodimeric ligands. Using an oocyte expression system we show direct interaction between the mature Derrière ligand and members of both the Nodal and BMP families. Taken together, these findings indicate that Derrière and Nodal proteins probably work cooperatively to induce mesoderm throughout the marginal zone during early Xenopus development.

Amino Acid Sequence↗
PubMed1998

The Xenopus dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities.

Using a Xenopus expression-cloning screen, we have isolated Gremlin, a novel antagonist of bone morphogenetic protein (BMP) signaling that is expressed in the neural crest. Gremlin belongs to a novel gene family that includes the head-inducing factor Cerberus and the tumor suppressor DAN. We show that all family members are secreted proteins and that they act as BMP antagonists in embryonic explants. We also provide support for the model that Gremlin, Cerberus, and DAN block BMP signaling by binding BMPs, preventing them from interacting with their receptors. In addition, Cerberus alone blocks signaling by Activin- and Nodal-like members of the TGF beta superfamily. Therefore, we propose that Gremlin, Cerberus, and DAN control diverse processes in growth and development by selectively antagonizing the activities of different subsets of the TGF beta ligands.

Activins↗
PubMed1998

Zebrafish nodal-related 2 encodes an early mesendodermal inducer signaling from the extraembryonic yolk syncytial layer.

Nodal-related factors have been implicated in mesodermal and neural patterning, and left-right asymmetry, in mouse, frog, and chicken embryos. We describe the isolation and characterization of zebrafish nodal-related 2 (znr2). znr2 is expressed at low levels maternally, and zygotic transcripts localize to dorsal blastomeres at MBT. Slightly later, znr2 is also expressed dorsally in the extraembryonic yolk syncytial layer (YSL). During early gastrulation, znr2 expression expands to include deep and superficial cells in the entire marginal zone and YSL. During shield stages, expression is primarily localized to superficial noninvoluting cells of the organizer called dorsal forerunners. Znr2 misexpression in whole fish embryos expands or duplicates dorsoanterior and axial cell fates. Furthermore, Znr2 overexpression exclusively in the YSL, a region implicated in endogenous mesodermal induction, causes broadened or duplicated gsc expression in the overlying blastoderm. Functional comparison of Znr2 and another recently identified zebrafish nodal-related factor, Znr1/Cyclops, reveals distinct inductive properties of each ligand. Znr2 efficiently induces organizer-type dorsoanterior mesodermal and endodermal markers, but only weakly, if at all, neural markers. In contrast, while Znr1/Cyclops reproducibly induces mesodermal and neural markers, it is an inefficient inducer of organizer-type mesoderm. Our results suggest that znr2 encodes a robust mesendodermal inducer that signals nonautonomously during the earliest stages of embryonic patterning, and that part of this activity arises from within the YSL.

Amino Acid Sequence↗
PubMed2003

Mouse models of holoprosencephaly.

PURPOSE OF REVIEW: Holoprosencephaly (HPE) is the most common anomaly of forebrain development in humans. The pathogenesis of HPE results in a failure of the brain hemispheres to separate during early development. Here we review experimental models of HPE in which some of the genes known to cause HPE in humans have been disrupted in the mouse. RECENT FINDINGS: To date, mutations that cause HPE have been identified in seven genes. Three of these genes encode members of the Sonic hedgehog (SHH) signaling pathway, which regulates the development of ventral structures throughout the neuraxis. Two other HPE mutations affect signaling by Nodal ligands, which also play important roles in neural patterning. The roles of the two other known HPE genes are not yet clear. Analysis of genetically altered mice has revealed that mutations in other members of the SHH and Nodal signaling pathways also result in HPE phenotypes. SUMMARY: Studies of HPE in the mouse have provided a framework for understanding key developmental events in human brain development and may provide new candidate genes for human HPE. Despite this progress, fundamental mysteries remain about how molecules that pattern ventral brain regions ultimately disrupt the formation of the cerebral hemispheres in dorsal regions.

Animals↗
PubMed2006

Mechanisms underlying long- and short-range nodal signaling in Zebrafish.

Precise regulation of the signaling range of secreted molecules is essential for proper pattern formation during development. The Nodal family of TGF-beta proteins has been shown to function as both short- and long-range signals. But the underlying mechanisms remain elusive. In this study, we investigated the regulation of the signaling range of zebrafish Nodal proteins Cyclops and Squint, which are short- and long-range signals, respectively. We show that (1) the stability of Cyclops and Squint correlates with the activity range but increasing the stability of the short-range Cyclops does not increase its signaling range; (2) structural differences in the N-terminus region of the mature peptides of Cyclops and Squint determine their differences in the signaling range and swapping the N-terminus region of the Squint mature ligand into that of Cyclops makes the latter function at a distance.

Amino Acid Sequence↗
PubMed2001

Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo.

The dorsal ectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be specified towards an anterior neural fate by an activation signal, with its subsequent regionalization along the anteroposterior (AP) axis regulated by a graded transforming activity, leading to a properly patterned forebrain, midbrain, hindbrain and spinal cord. The activation phase involves inhibition of BMP signals by dorsal antagonists, but the later caudalization process is much more poorly characterized. Explant and overexpression studies in chick, Xenopus, mouse and zebrafish implicate lateral/paraxial mesoderm in supplying the transforming influence, which is largely speculated to be a Wnt family member. We have analyzed the requirement for the specific ventrolaterally expressed Wnt8 ligand in the posteriorization of neural tissue in zebrafish wild-type and Nodal-deficient embryos (Antivin overexpressing or cyclops;squint double mutants), which show extensive AP brain patterning in the absence of dorsal mesoderm. In different genetic situations that vary the extent of mesodermal precursor formation, the presence of lateral wnt8-expressing cells correlates with the establishment of AP brain pattern. Cell tracing experiments show that the neuroectoderm of Nodal-deficient embryos undergoes a rapid anterior-to-posterior transformation in vivo during a short period at the end of the gastrula stage. Moreover, in both wild-type and Nodal-deficient embryos, inactivation of Wnt8 function by morpholino (MO(wnt8)) translational interference dose-dependently abrogates formation of spinal cord and posterior brain fates, without blocking ventrolateral mesoderm formation. MO(wnt8) also suppresses the forebrain deficiency in bozozok mutants, in which inactivation of a homeobox gene causes ectopic wnt8 expression. In addition, the bozozok forebrain reduction is suppressed in bozozok;squint;cyclops triple mutants, and is associated with reduced wnt8 expression, as seen in cyclops;squint mutants. Hence, whereas boz and Nodal signaling largely cooperate in gastrula organizer formation, they have opposing roles in regulating wnt8 expression and forebrain specification. Our findings provide strong support for a model of neural transformation in which a planar gastrula-stage Wnt8 signal, promoted by Nodal signaling and dorsally limited by Bozozok, acts on anterior neuroectoderm from the lateral mesoderm to produce the AP regional patterning of the CNS.

Animals↗
PubMed2006

Inhibitor-resistant type I receptors reveal specific requirements for TGF-beta signaling in vivo.

Activin/nodal-like TGF-beta superfamily ligands signal through the type I receptors Alk4, Alk5, and Alk7, and are responsible for mediating a number of essential processes in development. SB-431542, a chemical inhibitor of activin/nodal signaling, acts by specifically interfering with type I receptors. Here, we use inhibitor-resistant mutant receptors to examine the efficacy and specificity of SB-431542 in Xenopus and zebrafish embryos. Treatment with SB-431542 eliminates Smad2 phosphorylation in vivo and generates a phenotype very similar to those observed in genetic mutants in the nodal signaling pathway. Inhibitor-resistant Alk4 efficiently rescues Smad2 signaling, developmental phenotype, and marker gene expression after inhibitor treatment. This system was used to examine type I receptor specificity for several activin/nodal ligands. We find that Alk4 can efficiently rescue signaling by a wide range of ligands, while Alk7 can only weakly rescue signaling by the same ligands. In whole embryos, nodal signaling during gastrulation can be rescued with Alk4, but not Alk7, while Alk5 can only mediate signaling by ligands expressed later in development. The combination of the ALK inhibitor SB-431542 with inhibitor-resistant ALKs provides a powerful set of tools for examining nodal/activin signaling during embryogenesis.

Activin Receptors↗
PubMed2003

One-eyed pinhead regulates cell motility independent of Squint/Cyclops signaling.

In vertebrates, EGF-CFC factors are essential for Nodal signaling. Here, we show that the zygotic function of one-eyed pinhead, the zebrafish EGF-CFC factor, is necessary for cell movement throughout the blastoderm of the early embryo. During the blastula and gastrula stages, mutant cells are more cohesive and migrate slower than wild-type cells. Chimeric analysis reveals that these early motility defects are cell-autonomous; later, one-eyed pinhead mutant cells have a cell-autonomous tendency to acquire ectodermal rather than mesendodermal fates. Moreover, wild-type cells transplanted into the axial region of mutant hosts tend to form isolated aggregates of notochord tissue adjacent to the mutant notochord. Upon misexpressing the Nodal-like ligand Activin in whole embryos, which rescues aspects of the mutant phenotype, cell behavior retains the one-eyed pinhead motility phenotype. However, in squint;cyclops double mutants, which lack Nodal function and possess a more severe phenotype than zygotic one-eyed pinhead mutants, cells of the dorsal margin exhibit a marked tendency to widely disperse rather than cohere together. Elsewhere in the double mutants, for cells of the blastoderm and for rare cells of the gastrula that involute into the hypoblast, motility appears wild-type. Notably, cells at the animal pole, which are not under direct regulation by the Nodal pathway, behave normal in squint;cyclops mutants but exhibit defective motility in one-eyed pinhead mutants. We conclude that, in addition to a role in Nodal signaling, One-eyed pinhead is required for aspects of cell movement, possibly by regulating cell adhesion.

Animals↗
PubMed2002

Multiple nodal-related genes act coordinately in Xenopus embryogenesis.

Four nodal-related genes (Xnr1-4) have been isolated in Xenopus to date, and we recently further identified two more, Xnr5 and Xnr6. In the present functional study, we constructed cleavage mutants of Xnr5 (cmXnr5) and Xnr6 (cmXnr6) which were expected to act in a dominant-negative manner. Both cmXnr5 and cmXnr6 inhibited the activities of Xnr5 and Xnr6 in co-overexpression experiments. cmXnr5 also inhibited the activity of Xnr2, Xnr4, Xnr6, derrière, and BVg1, but did not inhibit the activity of Xnr1 or activin. Misexpression of cmXnr5 led to a severe delay in initiation of gastrulation and phenotypic changes, including defects in anterior structures, which were very similar to those seen in maternal VegT-depleted embryos. Further, although the expression of Xnr1, Xnr2, and Xnr4 was not delayed in these embryos, it was markedly reduced. Injection of cmXnr5 had no notable effect on expression of Xnr3, Xnr6, derrière, or siamois. Several mesodermal and endodermal markers also showed delayed and decreased expression during gastrulation in cmXnr5-injected embryos. These results suggest that, in early Xenopus embryogenesis, nodal-related genes may heterodimerize with other TGF-beta ligands, and further that one nodal-related gene alone is insufficient for mesendoderm formation, which may require the cooperative interaction of multiple nodal-related genes.

Amino Acid Sequence↗
PubMed2003

Nodal signaling and vertebrate germ layer formation.

The understanding of germ layer formation in vertebrates began with classical experimental embryology. Early in the 20th century, Spemann and Mangold (1924) identified a region of the early embryo capable of inducing an entire embryonic axis. Termed the dorsal organizer, the tissue and the activity have been shown to exist in all vertebrates examined. In mice, for example, the activity resides in a region of the gastrula embryo known as the node. Experiments by the Dutch embryologist Nieuwkoop (1967a, 1967b, 1973, 1977) showed that a signal derived from the vegetal half of the amphibian embryo is responsible for the formation of mesoderm. Nieuwkoop's results allowed the development of in vitro assays that led, in the late 1980s and early 1990s, to the identification of growth factors essential for germ layer formation. Through more recent genetic investigations in mice and zebrafish, we now know that one class of secreted growth factor, called Nodal because of its localized expression in the mouse node, is essential for formation of mesoderm and endoderm and for the morphological rearrangements that occur during gastrulation.

Activins↗
PubMed2005

Xnr2 and Xnr5 unprocessed proteins inhibit Wnt signaling upstream of dishevelled.

Nodal and Nodal-related proteins activate the Activin-like signal pathway and play a key role in the formation of mesoderm and endoderm in vertebrate development. Recent studies have shown additional activities of Nodal-related proteins apart from the canonical Activin-like signal pathway. Here we report a novel function of Nodal-related proteins using cleavage mutants of Xenopus nodal-related genes (cmXnr2 and cmXnr5), which are known to be dominant-negative inhibitors of nodal family signaling. cmXnr2 and cmXnr5 inhibited both BMP signaling and Wnt signaling without activating the Activin-like signal in animal cap assays. Pro region construct of Xnr2 and Xnr5 did not inhibit Xwnt8, and pro/mature region chimera mutant cmActivin-Xnr2 and cmActivin-Xnr5 also did not inhibit Xwnt8 activity. These results indicate that the pro domains of Xnr2 and Xnr5 are necessary, but not sufficient, for Wnt inhibition, by Xnr family proteins. In addition, Western blot analysis and immunohistochemistry analysis revealed that the unprocessed Xnr5 protein is stably produced and secreted as effectively as mature Xnr5 protein, and that the unprocessed Xnr5 protein diffused in the extracellular space. These results suggest that unprocessed Xnr2 and Xnr5 proteins may be involved in inhibiting both BMP and Wnt signaling and are able to be secreted to act on somewhat distant target cells, if these are highly produced.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Nodal-related gene Xnr5 is amplified in the Xenopus genome.

In Xenopus, six nodal-related genes (Xnrs) have been identified to date. We found numerous tandem duplications of Xnr5 in the Xenopus laevis and Xenopus tropicalis genomes that involve highly conserved copies of coding and regulatory regions. The duplicated versions of Xnr5 were expressed in both the superficial and deep layer of dorsal endoderm and in the deep layer of ventral endoderm, where the initial inducers of mesendoderm formation would be expected to be localized. Overexpression of secreted inhibitors of Xnrs led to a substantially enhanced transcription of the duplicated Xnr5 genes and Xnr6 in embryos. Therefore, Xnr5 and Xnr6 have a novel feedback loop to inhibit transcription of Xnr5 and Xnr6. These results suggest that the initialization of a strong Xnr5 and Xnr6 signal is enabled by the rapid transcription from multiple genes. The novel feedback loop may negatively regulate transcription of Xnr5s and Xnr6 to limit overproduction of these potent inducers, with the Xnr5/Xnr6 signal then activating positive (Xnrs) and negative (Xlefty) loops, which regulate the range of mesodermal tissues produced.

Animals↗
PubMed1999

Identification of new genes ndr2 and ndr3 which are related to Ndr1/RTP/Drg1 but show distinct tissue specificity and response to N-myc.

Ndr1 was isolated as a gene upregulated in N-myc mutant mouse embryos and is repressed by N-myc and c-myc. Consistent with Myc regulation, the same gene was also isolated as one sensitive to transformation (Drg1), and in addition as one induced under a few stress conditions (RTP). Two new genes, Ndr2 and Ndr3, were identified which encode proteins highly related to Ndr1/RTP/Drg1 and constitute the Ndr gene family. Ndr2 and Ndr3 are under spatio-temporal regulations distinct from Ndr1, and are not activated in N-myc mutants. When whole embryo RNA was analyzed, Ndr3 expression was already high at 9.5 days postcoitus (dpc), while expression of Ndr2 and Ndr1 became significant after 12.5 dpc and 13. 5 dpc, respectively. At 14.5 dpc, expression of these genes partially overlaps, but many tissues are unique to one of them. For instance, Ndr1 is strongly expressed in the liver and gut epithelium, Ndr2 in the ventricular zone throughout the CNS, and Ndr3 in the spinal cord and the thymus rudiment. Genes of the Ndr family probably have tissue-dependent allotments of the possibly related functions.

Adaptor Proteins, Signal Transducing↗