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.     

Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation.

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.     

Two modes by which Lefty proteins inhibit nodal signaling

During vertebrate embryogenesis, members of the Lefty subclass of Transforming Growth Factor-beta (TGFbeta) proteins act as extracellular antagonists of the signaling pathway for Nodal, a TGFbeta-related ligand essential for mesendoderm formation and left-right patterning. Genetic and biochemical analyses have shown that Nodal signaling is mediated by activin receptors but also requires EGF-CFC coreceptors, such as mammalian Cripto or Cryptic. Misexpression experiments in zebrafish and frogs have suggested that Lefty proteins can act as long-range inhibitors for Nodal, possibly through competition for binding to activin receptors. Here we demonstrate two distinct and unexpected mechanisms by which Lefty proteins can antagonize Nodal activity. In particular, using a novel assay for Lefty activity in mammalian cell culture, we find that Lefty can inhibit signaling by Nodal but not by Activin or TGFbeta1, which are EGF-CFC independent. We show that Lefty can interact with Nodal in solution and thereby block Nodal from binding to activin receptors. Furthermore, Lefty can also interact with EGF-CFC proteins and prevent their ability to form part of a Nodal receptor complex. Our results provide mechanistic insights into how Lefty proteins can achieve efficient and stringent regulation of a potent signaling factor.     

The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm

Nodal signals, a subclass of the TGFbeta superfamily of secreted factors, induce formation of mesoderm and endoderm in vertebrate embryos. We have examined the possible dorsoventral and animal-vegetal patterning roles for Nodal signals by using mutations in two zebrafish nodal-related genes, squint and cyclops, to manipulate genetically the levels and timing of Nodal activity. squint mutants lack dorsal mesendodermal gene expression at the late blastula stage, and fate mapping and gene expression studies in sqt(-/-); cyc(+/+) and sqt(-/-); cyc(+/-) mutants show that some dorsal marginal cells inappropriately form hindbrain and spinal cord instead of dorsal mesendodermal derivatives. The effects on ventrolateral mesendoderm are less severe, although the endoderm is reduced and muscle precursors are located nearer to the margin than in wild type. Our results support a role for Nodal signals in patterning the mesendoderm along the animal-vegetal axis and indicate that dorsal and ventrolateral mesoderm require different levels of squint and cyclops function. Dorsal marginal cells were not transformed toward more lateral fates in either sqt(-/-); cyc(+/-) or sqt(-/-); cyc(+/+) embryos, arguing against a role for the graded action of Nodal signals in dorsoventral patterning of the mesendoderm. Differential regulation of the cyclops gene in these cells contributes to the different requirements for nodal-related gene function in these cells. Dorsal expression of cyclops requires Nodal-dependent autoregulation, whereas other factors induce cyclops expression in ventrolateral cells. In addition, the differential timing of dorsal mesendoderm induction in squint and cyclops mutants suggests that dorsal marginal cells can respond to Nodal signals at stages ranging from the mid-blastula through the mid-gastrula.     

Rapid differential transport of Nodal and Lefty on sulfated proteoglycan-rich extracellular matrix regulates left-right asymmetry in Xenopus

The spatiotemporally dynamic distribution of instructive ligands within embryonic tissue, and their feedback antagonists, including inherent stabilities and rates of clearance, are affected by interactions with cell surfaces or extracellular matrix (ECM). Nodal (here, Xnr1 or Nodal1 in Xenopus) and Lefty interact in a cross-regulatory relationship in mesendoderm induction, and are the conserved instructors of left-right (LR) asymmetry in early somitogenesis stage embryos. By expressing Xnr1 and Lefty proproteins that produce mature functional epitope-tagged ligands in vivo, we found that ECM is a principal surface of Nodal and Lefty accumulation. We detected Lefty moving faster than Nodal, with evidence that intact sulfated proteoglycans in the ECM facilitate the remarkable long distance movement of Nodal. We propose that Nodal autoregulation substantially aided by rapid ligand transport underlies the anteriorward shift of Nodal expression in the left LPM (lateral plate mesoderm), and speculate that the higher levels of chondroitin-sulfate proteoglycan (CSPG) in more mature anterior regions provide directional transport cues. Immunodetection and biochemical analysis showed transfer of Lefty from left LPM to right LPM, providing direct evidence that left-side-derived Lefty is a significant influence in ensuring the continued suppression of right-sided expression of Nodal, maintaining unilateral expression of this conserved determinant of asymmetry.     

Wave pinning and spatial patterning in a mathematical model of Antivin/Lefty–Nodal signalling

Nodal signals are key regulators of mesoderm and endoderm development in vertebrate embryos. It has been observed experimentally that in Xenopus embryos the spatial range of Nodal signals is restricted by the signal Antivin (also known as Lefty). Nodal signals can activate both Nodal and Antivin, whereas Antivin is thought to antagonise Nodal by binding either directly to it or to its receptor. In this paper we develop a mathematical model of this signalling network in a line of cells. We consider the heterodimer and receptor-mediated inhibition mechanisms separately and find that, in both cases, the restriction by Antivin to the range of Nodal signals corresponds to wave pinning in the model. Our analysis indicates that, provided Antivin diffuses faster than Nodal, either mechanism can robustly account for the experimental data. We argue that, in the case of Xenopus development, it is wave pinning, rather than Turing-type patterning, that is underlying Nodal–Antivin dynamics. This leads to several experimentally testable predictions, which are discussed. Furthermore, for heterodimer-mediated inhibition to prevent waves of Nodal expression from propagating, the Nodal–Antivin complex must be turned over, and diffusivity of the complex must be negligible. In the absence of molecular mechanisms regulating these, we suggest that Antivin restricts Nodal signals via receptor-mediated, and not heterodimer-mediated, inhibition.     

Lefty blocks a subset of TGFbeta signals by antagonizing EGF-CFC coreceptors

Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFbeta signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFbeta signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFbeta agonists and antagonists and suggest that subtle sequence variations in TGFbeta signals result in greater ligand diversity.     

Lefty Blocks a Subset of TGFβ Signals by Antagonizing EGF-CFC Coreceptors

Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFβ signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFβ signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFβ agonists and antagonists and suggest that subtle sequence variations in TGFβ signals result in greater ligand diversity.     

Lefty Blocks a Subset of TGFβ Signals by Antagonizing EGF-CFC Coreceptors

Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFβ signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFβ signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFβ agonists and antagonists and suggest that subtle sequence variations in TGFβ signals result in greater ligand diversity.     

Lefty-dependent inhibition of Nodal- and Wnt-responsive organizer gene expression is essential for normal gastrulation

During gastrulation, diffusible "organizer" signals, including members of the TGFbeta Nodal subfamily, pattern dorsal mesoderm and the embryonic axes. Simultaneously, negative regulators of these signals, including the Nodal inhibitor Lefty, an atypical TGFbeta factor, are induced by Nodal. This suggests that Lefty-dependent modulation of organizer signaling might regulate dorsal mesoderm patterning and axial morphogenesis. Here, Xenopus Lefty (Xlefty) function was blocked by injection of anti-Xlefty morpholino oligonucleotides (MO). Xlefty-deficient embryos underwent exogastrulation, an aberrant morphogenetic process not predicted from deregulation of the Nodal pathway alone. In the absence of Xlefty, both Nodal- (Xnr2, gsc, cer, Xbra) and Wnt-responsive (gsc, Xnr3) organizer gene expression expanded away from the dorsal blastopore lip. Conversely, coexpression of Xlefty with Nodal or Wnt reduced the ectopic expression of Nodal- (Xbra) and Wnt-responsive (Xnr3) genes in a dose-dependent manner. Furthermore, Xlefty expression in the ectodermal animal pole inhibited endogenous Nodal- and Wnt-responsive gene expression in distant mesoderm cells, indicating that Xlefty inhibition can spread from its source. We hypothesize that Xlefty negatively regulates the spatial extent of Nodal- and Wnt-responsive gene expression in the organizer and that this Xlefty-dependent inhibition is essential for normal organizer patterning and gastrulation.     

The Cerberus/Dan-family protein Charon is a negative regulator of Nodal signaling during left-right patterning in zebrafish

We have isolated a novel gene, charon, that encodes a member of the Cerberus/Dan family of secreted factors. In zebrafish, Fugu and flounder, charon is expressed in regions embracing Kupffer's vesicle, which is considered to be the teleost fish equivalent to the region of the mouse definitive node that is required for left-right (L/R) patterning. Misexpression of Charon elicited phenotypes similar to those of mutant embryos defective in Nodal signaling or embryos overexpressing Antivin(Atv)/Lefty1, an inhibitor for Nodal and Activin. Charon also suppressed the dorsalizing activity of all three of the known zebrafish Nodal-related proteins (Cyclops, Squint and Southpaw), indicating that Charon can antagonize Nodal signaling. Because Southpaw functions in the L/R patterning of lateral plate mesoderm and the diencephalon, we asked whether Charon is involved in regulating L/R asymmetry. Inhibition of Charon's function by antisense morpholino oligonucleotides (MOs) led to a loss of L/R polarity, as evidenced by bilateral expression of the left side-specific genes in the lateral plate mesoderm (southpaw, cyclops, atv/lefty1, lefty2 and pitx2) and diencephalon (cyclops, atv/lefty1 and pitx2), and defects in early (heart jogging) and late (heart looping) asymmetric heart development, but did not disturb the notochord development or the atv/lefty1-mediated midline barrier function. MO-mediated inhibition of both Charon and Southpaw led to a reduction in or loss of the expression of the left side-specific genes, suggesting that Southpaw is epistatic to Charon in left-side formation. These data indicate that antagonistic interactions between Charon and Nodal (Southpaw), which take place in regions adjacent to Kupffer's vesicle, play an important role in L/R patterning in zebrafish.     

Chick CFC controls Lefty1 expression in the embryonic midline and nodal expression in the lateral plate

Members of the EGF-CFC family of proteins have recently been implicated as essential cofactors for Nodal signaling. Here we report the isolation of chick CFC and describe its expression pattern, which appears to be similar to Cfc1 in mouse. During early gastrulation, chick CFC was asymmetrically expressed on the left side of Hensen's node as well as in the emerging notochord, prechordal plate, and lateral plate mesoderm. Subsequently, its expression became confined to the heart fields, notochord, and posterior mesoderm. Implantation experiments suggest that chick CFC expression in the lateral plate mesoderm is dependent on BMP signaling, while in the midline its expression depends on an Activin-like signal. The asymmetric expression domain within Hensen's node was not affected by application of FGF8, Noggin, or Shh antibody. Implantation of cells expressing human or mouse CFC2, or chick CFC on the right side of Hensen's node randomized heart looping without affecting expression of genes involved in left-right axis formation, including SnR, Nodal, Car, or Pitx2. Application of antisense oligodeoxynucleotides to the midline of Hamburger-Hamilton stage 4-5 embryos also randomized heart looping, but in contrast to the overexpression experiments, antisense oligodeoxynucleotide treatment resulted in bilateral expression of Nodal, Car, Pitx2, and NKX3.2, whereas Lefty1 expression in the midline was transiently lost. Application of the antisense oligodeoxynucleotides to the lateral plate mesoderm abolished Nodal expression. Thus, chick CFC seems to have a dual function in left-right axis formation by maintaining Nodal expression in the lateral plate mesoderm and controlling expression of Lefty1 expression in the midline territory.     

The role of maternal Activin-like signals in zebrafish embryos

Maternal Activin-like proteins, a subgroup of the TGF-beta superfamily, play a key role in establishing the body axes in many vertebrates, but their role in teleosts is unclear. At least two maternal Activin-like proteins are expressed in zebrafish, including the Vg1 orthologue, zDVR-1, and the nodal-related gene, Squint. Our analysis of embryos lacking both maternal and zygotic squint function revealed that maternal squint is required in some genetic backgrounds for the formation of dorsal and anterior tissues. Conditional inactivation of the ALK4, 5 and 7 receptors by SB-505124 treatment during the cleavage stages ruled out a role for maternal Squint, zDVR-1, or other Activin-like ligands before the mid-blastula transition, when the dorsal axis is established. Furthermore, we show that maternal Squint and zDVR-1 are not required during the cleavage stages to induce zygotic nodal-related gene expression. nodal-related gene expression decreases when receptor inhibition continues past the mid-blastula transition, resulting in a progressive loss of mesoderm and endoderm. We conclude that maternally expressed Activin-like signals do not act before the mid-blastula transition in zebrafish, but do have a variably penetrant role in the later stages of axis formation. This contrasts with the early role for these signals during Xenopus development.     

Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm

Nodal, a member of the TGF-β family of signaling molecules, has been implicated in pluripotency in human embryonic stem cells (hESCs) [Vallier, L., Reynolds, D., Pedersen, R.A., 2004a. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev. Biol. 275, 403-421], a finding that seems paradoxical given Nodal's central role in mesoderm/endoderm specification during gastrulation. In this study, we sought to clarify the role of Nodal signaling during hESC differentiation by constitutive overexpression of the endogenous Nodal inhibitors Lefty2 (Lefty) and truncated Cerberus (Cerb-S) and by pharmacological interference using the Nodal receptor antagonist SB431542. Compared to wildtype (WT) controls, embryoid bodies (EBs) derived from either Lefty or Cerb-S overexpressing hESCs showed increased expression of neuroectoderm markers Sox1, Sox3, and Nestin. Conversely, they were negative for a definitive endoderm marker (Sox17) and did not generate beating cardiomyocyte structures in conditions that allowed mesendoderm differentiation from WT hESCs. EBs derived from either Lefty or Cerb-S expressing hESCs also contained a greater abundance of neural rosette structures as compared to controls. Differentiating EBs derived from Lefty expressing hESCs generated a dense network of β-tubulin III positive neurites, and when Lefty expressing hESCs were grown as a monolayer and allowed to differentiate, they generated significantly higher numbers of β-tubulin positive neurons as compared to wildtype hESCs. SB431542 treatments reproduced the neuralising effects of Lefty overexpression in hESCs. These results show that inhibition of Nodal signaling promotes neuronal specification, indicating a role for this pathway in controlling early neural development of pluripotent cells.     

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.