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.     

Transmembrane Protein 147 (TMEM147) Is a Novel Component of the Nicalin-NOMO Protein Complex

Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely γ-secretase and the Nicalin-NOMO (Nodal modulator) complex. The γ-secretase complex, which contains Presenilin, APH-1, and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the β-amyloid precursor protein and Notch. Nicalin and its binding partner NOMO form a complex that was shown to modulate Nodal signaling in developing zebrafish embryos. Because its experimentally determined native size (200–220 kDa) could not be satisfyingly explained by the molecular masses of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinity-purified complex preparations for additional components in the low molecular mass range. A ∼22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO, it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to γ-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex, further emphasizing its similarity with γ-secretase.     

Lefty proteins are long-range inhibitors of squint-mediated nodal signaling

The regulation of signaling pathways by feedback inhibitors has become an emerging theme in the control of pattern formation during development. Nodal and Lefty proteins belong to divergent subfamilies of the TGF-beta family. Nodal signals promote mesendoderm induction in vertebrates, and Lefty proteins antagonize it. In zebrafish, Squint functions as a long-range Nodal signal during mesoderm induction. We report that the range over which Squint induces mesoderm is reduced by Lefty proteins. In contrast, the activity range of the short-range Nodal signal Cyclops is not regulated by Lefty activity. We present three lines of evidence that Lefty proteins diminish the range of Squint signaling by acting not only as antagonists of Squint autoregulation but also as long-range inhibitors of Squint activity. First, Lefty can block Nodal signaling at a distance. Second, Lefty regulates the range of Squint signaling before regulating squint expression. Third, Lefty restricts the range of Squint activity in squint mutant embryos, in which the endogenous gene is not subject to autoregulation. We also find that Lefty restricts the response to both high and low levels of Nodal signaling. These results indicate that Lefty proteins restrict the activity range of Nodal signals by dampening Nodal signaling in surrounding cells.     

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.     

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.     

斑马鱼胚胎中受Nodal信号调控基因的鉴定

Nodal信号在脊椎动物胚胎发育的中内胚层诱导、左右不对称性的建立、神经外胚层沿前后轴线的分化等方面起着重要的作用.为鉴定受Nodal信号调控的基因,特别是那些转录因子基因,通过将来自squint过量表达、缺失Nodal信号的MZoep突变体或野生型30%外包期胚胎的RNA与Affymetrix斑马鱼寡核苷酸芯片杂交.发现与野生型样本相比,在squint过量表达的样本中,265个转录本的表达显著增强(log2ratio>1),111个转录本的表达显著减弱(log2ratio<-1);在MZoep样本中,表达显著增强的(log2ratio>1)转录本有1495个,表达显著减弱(log2ratio<-1)的有550个.squint过量表达使26个转录因子基因的表达增强,11个转录因子基因的表达减弱;另一方面,MZoep突变体中表达增强的转录因子基因为69个,表达减弱的转录因子基因为30个.这些结果为进一步研究Nodal信号的转导机理和生物学功能提供了有益的数据.     

The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity

The Nodal and Hedgehog signaling pathways influence dorsoventral patterning at all axial levels of the CNS, but it remains largely unclear how these pathways interact to mediate patterning. Here we show that, in zebrafish, Nodal signaling is required for induction of the homeobox genes nk2.1a in the ventral diencephalon and nk2.1b in the ventral telencephalon. Hedgehog signaling is also required for telencephalic nk2.1b expression but may not be essential to establish diencephalic nk2.1a expression. Furthermore, Shh does not restore ventral diencephalic development in embryos lacking Nodal activity. In contrast, Shh does restore telencephalic nk2.1b expression in the absence of Nodal activity, suggesting that Hedgehog signaling acts downstream of Nodal activity to pattern the ventral telencephalon. Thus, the Nodal pathway regulates ventral forebrain patterning through both Hedgehog signaling-dependent and -independent mechanisms.     

Ttrap is an essential modulator of Smad3-dependent Nodal signaling during zebrafish gastrulation and left-right axis determination

During vertebrate development, signaling by the TGFbeta ligand Nodal is critical for mesoderm formation, correct positioning of the anterior-posterior axis, normal anterior and midline patterning, and left-right asymmetric development of the heart and viscera. Stimulation of Alk4/EGF-CFC receptor complexes by Nodal activates Smad2/3, leading to left-sided expression of target genes that promote asymmetric placement of certain internal organs. We identified Ttrap as a novel Alk4- and Smad3-interacting protein that controls gastrulation movements and left-right axis determination in zebrafish. Morpholino-mediated Ttrap knockdown increases Smad3 activity, leading to ectopic expression of snail1a and apparent repression of e-cadherin, thereby perturbing cell movements during convergent extension, epiboly and node formation. Thus, although the role of Smad proteins in mediating Nodal signaling is well-documented, the functional characterization of Ttrap provides insight into a novel Smad partner that plays an essential role in the fine-tuning of this signal transduction cascade.     

ALK7, a receptor for nodal, is dispensable for embryogenesis and left-right patterning in the mouse

Mesendoderm formation and left-right patterning during vertebrate development depend upon selected members of the transforming growth factor beta superfamily, particularly Nodal and Nodal-related ligands. Two type I serine/threonine kinase receptors have been identified for Nodal, ALK4 and ALK7. Mouse embryos lacking ALK4 fail to produce mesendoderm and die shortly after gastrulation, resembling the phenotype of Nodal knockout mice. Whether ALK4 contributes to left-right patterning is still unknown. Here we report the generation and initial characterization of mice lacking ALK7. Homozygous mutant mice were born at the expected frequency and remained viable and fertile. Viability at weaning was not different from that of the wild type in ALK7(-/-); Nodal(+/-) and ALK7(-/-); ALK4(+/-) compound mutants. ALK7 and ALK4 were highly expressed in interdigital regions of the developing limb bud. However, ALK7 mutant mice displayed no skeletal abnormalities or limb malformations. None of the left-right patterning abnormalities and organogenesis defects identified in mice carrying mutations in Nodal or in genes encoding ActRIIA and ActRIIB coreceptors, including heart malformations, pulmonary isomerism, right-sided gut, and spleen hypoplasia, were observed in mice lacking ALK7. Finally, the histological organization of the cerebellum, cortex, and hippocampus, all sites of significant ALK7 expression in the rodent brain, appeared normal in ALK7 mutant mice. We conclude that ALK7 is not an essential mediator of Nodal signaling during mesendoderm formation and left-right patterning in the mouse but may instead mediate other activities of Nodal and related ligands in the development or function of particular tissues and organs.     

Nodal-related signals establish mesendodermal fate and trunk neural identity in zebrafish

The vertebrate body plan arises during gastrulation, when morphogenetic movements form the ectoderm, mesoderm, and endoderm. In zebrafish, mesoderm and endoderm derive from the marginal region of the late blastula, and cells located nearer the animal pole form the ectoderm [1]. Analysis in mouse, Xenopus, and zebrafish has demonstrated that Nodal-related proteins, a subclass of the TGF-beta superfamily, are essential for mesendoderm development [2], but previous mutational studies have not established whether Nodal-related signals control fate specification, morphogenetic movements, or survival of mesendodermal precursors. Here, we report that Nodal-related signals are required to allocate marginal cells to mesendodermal fates in the zebrafish embryo. In double mutants for the zebrafish nodal-related genes squint (sqt) and cyclops (cyc) [3] [4] [5], dorsal marginal cells adopt neural fates, whereas in wild-type embryos, cells at this position form endoderm and axial mesoderm. Involution movements characteristic of developing mesendoderm are also blocked in the absence of Nodal signaling. Because it has been proposed [6] that inhibition of Nodal-related signals promotes the development of anterior neural fates, we also examined anteroposterior organization of the neural tube in sqt;cyc mutants. Anterior trunk spinal cord is absent in sqt;cyc mutants, despite the presence of more anterior and posterior neural fates. These results demonstrate that nodal-related genes are required for the allocation of dorsal marginal cells to mesendodermal fates and for anteroposterior patterning of the neural tube.