3538275
Vg1-Nodal heterodimers are the endogenous inducers of mesendoderm
Nodal is considered the key inducer of mesendoderm in vertebrate embryos and embryonic stem cells. Other TGF-beta-related signals, such as Vg1/Dvr1/Gdf3, have also been implicated in this process but their roles have been unclear or controversial. Here we report that zebrafish embryos without maternally provided vg1 fail to form endoderm and head and trunk mesoderm, and closely resemble nodal loss-of-function mutants. Although Nodal is processed and secreted without Vg1, it requires Vg1 for its endogenous activity. Conversely, Vg1 is unprocessed and resides in the endoplasmic reticulum without Nodal, and is only secreted, processed and active in the presence of Nodal. Co-expression of Nodal and Vg1 results in heterodimer formation and mesendoderm induction. Thus, mesendoderm induction relies on the combination of two TGF-beta-related signals: maternal and ubiquitous Vg1, and zygotic and localized Nodal. Modeling reveals that the pool of maternal Vg1 enables rapid signaling at low concentrations of zygotic Nodal.
4356765
Nodal signalling in vertebrate development
Communication between cells during early embryogenesis establishes the basic organization of the vertebrate body plan. Recent work suggests that a signalling pathway centering on Nodal, a transforming growth factor β-related signal, is responsible for many of the events that configure the vertebrate embryo. The activity of Nodal signals is regulated extracellularly by EGF-CFC cofactors and antagonists of the Lefty and Cerberus families of proteins, allowing precise control of mesoderm and endoderm formation, the positioning of the anterior–posterior axis, neural patterning and left–right axis specification.
1801581
Nodal signaling: developmental roles and regulation
Nodal-related ligands of the transforming growth factor-beta (TGFβ) superfamily play central roles in patterning the early embryo during the induction of mesoderm and endoderm and the specification of left-right asymmetry. Additional roles for this pathway in the maintenance of embryonic stem cell pluripotency and in carcinogenesis have been uncovered more recently. Consistent with its crucial developmental functions, Nodal signaling is tightly regulated by diverse mechanisms including the control of ligand processing, utilization of co-receptors, expression of soluble antagonists, as well as positive- and negative-feedback activities.
10516226
A novel retrovirally induced embryonic lethal mutation in the mouse: assessment of the developmental fate of embryonic stem cells homozygous for the 413.d proviral integration.
A genetic screen of transgenic mouse strains, carrying multiple copies of an MPSV neo retroviral vector, has led to the identification of a recessive embryonic lethal mutation, termed 413.d. This mutation is associated with a single proviral insertion and when homozygous, results in the failure of the early postimplantation embryo at the gastrulation stage of development. Embryonic stem cell lines (ES cells) were derived from 413.d intercross embryos. Genotyping, with respect to the 413.d integration site, identified wild-type, heterozygous and homozygous ES cell lines. The differentiation abilities and developmental potential of the ES cell lines were assessed using a number of in vitro and in vivo assays. Results indicate that the ES cell lines, regardless of genotype, are pluripotent and can give rise to tissue and cell types derived from all three germ layers. Furthermore, analysis of midgestation conceptuses (10.5 p.c.) and adult chimeras generated by injecting mutant ES cells into host blastocysts, provides strong evidence that the mutant cells can contribute to all extraembryonic tissues and somatic tissues, as well as to functional germ cells. These results indicate that the homozygous mutant cells can be effectively 'rescued' by the presence of wild-type cells in a carrier embryo.
4417566
Zebrafish organizer development and germ-layer formation require nodal-related signals
The vertebrate body plan is established during gastrulation, when cells move inwards to form the mesodermal and endodermal germ layers. Signals from a region of dorsal mesoderm, which is termed the organizer, pattern the body axis by specifying the fates of neighbouring cells,. The organizer is itself induced by earlier signals. Although members of the transforming growth factor-β (TGF-β) and Wnt families have been implicated in the formation of the organizer, no endogenous signalling molecule is known to be required for this process. Here we report that the zebrafish squint (sqt) and cyclops (cyc) genes have essential, although partly redundant, functions in organizer development and also in the formation of mesoderm and endoderm. We show that the sqt gene encodes a member of the TGF-β superfamily that is related to mouse nodal. cyc encodes another nodal-related protein,, which is consistent with our genetic evidence that sqt and cyc have overlapping functions. The sqt gene is expressed in a dorsal region of the blastula that includes the extraembryonic yolk syncytial layer (YSL). The YSL has been implicated as a source of signals that induce organizer development and mesendoderm formation,. Misexpression of sqt RNA within the embryo or specifically in the YSL induces expanded or ectopic dorsal mesoderm. These results establish an essential role for nodal-related signals in organizer development and mesendoderm formation.
34157248
Nodal is a novel TGF-beta-like gene expressed in the mouse node during gastrulation.
During gastrulation, the three germ layers of the embryo are formed and organized along the anterior-posterior body axis. In the mouse, gastrulation involves the delamination of ectodermal cells through the primitive streak and their differentiation into mesoderm. These processes do not occur in embryos homozygous for a retrovirally induced recessive prenatal lethal mutation, the strain 413-d insertional mutation. Instead of giving rise to mesoderm, embryonic ectoderm in 413-d mutants overproliferates and then rapidly degenerates, although extraembryonic lineages remain viable. Here we isolate a candidate for the mutated gene which encodes a new member of the transforming growth factor-beta (TGF-beta) superfamily. Expression is first detected in primitive streak-stage embryos at about the time of mesoderm formation. It then becomes highly localized in the node at the anterior of the primitive streak. This region is analogous to chick Hensen's node and Xenopus dorsal lip (Spemann's organizer), which can induce secondary body axes when grafted into host embryos (reviewed in refs 5 and 6). Our findings suggest that this gene, named nodal, encodes a signalling molecule essential for mesoderm formation and subsequent organization of axial structures in early mouse development.
27764390
Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation
Human embryonic stem (hES) cells hold promise for generating an unlimited supply of cells for replacement therapies. To characterize hES cells at the molecular level, we obtained 148,453 expressed sequence tags (ESTs) from undifferentiated hES cells and three differentiated derivative subpopulations. Over 32,000 different transcripts expressed in hES cells were identified, of which more than 16,000 do not match closely any gene in the UniGene public database. Queries to this EST database revealed 532 significantly upregulated and 140 significantly downregulated genes in undifferentiated hES cells. These data highlight changes in the transcriptional network that occur when hES cells differentiate. Among the differentially regulated genes are several components of signaling pathways and transcriptional regulators that likely play key roles in hES cell growth and differentiation. The genomic data presented here may facilitate the derivation of clinically useful cell types from hES cells.
9880166
Absence of Nodal signaling promotes precocious neural differentiation in the mouse embryo.
After implantation, mouse embryos deficient for the activity of the transforming growth factor-beta member Nodal fail to form both the mesoderm and the definitive endoderm. They also fail to specify the anterior visceral endoderm, a specialized signaling center which has been shown to be required for the establishment of anterior identity in the epiblast. Our study reveals that Nodal-/- epiblast cells nevertheless express prematurely and ectopically molecular markers specific of anterior fate. Our analysis shows that neural specification occurs and regional identities characteristic of the forebrain are established precociously in the Nodal-/- mutant with a sequential progression equivalent to that of wild-type embryo. When explanted and cultured in vitro, Nodal-/- epiblast cells readily differentiate into neurons. Genes normally transcribed in organizer-derived tissues, such as Gsc and Foxa2, are also expressed in Nodal-/- epiblast. The analysis of Nodal-/-;Gsc-/- compound mutant embryos shows that Gsc activity plays no critical role in the acquisition of forebrain characters by Nodal-deficient cells. This study suggests that the initial steps of neural specification and forebrain development may take place well before gastrulation in the mouse and highlights a possible role for Nodal, at pregastrula stages, in the inhibition of anterior and neural fate determination.
23206845
Efficient differentiation of human embryonic stem cells to definitive endoderm
The potential of human embryonic stem (hES) cells to differentiate into cell types of a variety of organs has generated much excitement over the possible use of hES cells in therapeutic applications. Of great interest are organs derived from definitive endoderm, such as the pancreas. We have focused on directing hES cells to the definitive endoderm lineage as this step is a prerequisite for efficient differentiation to mature endoderm derivatives. Differentiation of hES cells in the presence of activin A and low serum produced cultures consisting of up to 80% definitive endoderm cells. This population was further enriched to near homogeneity using the cell-surface receptor CXCR4. The process of definitive endoderm formation in differentiating hES cell cultures includes an apparent epithelial-to-mesenchymal transition and a dynamic gene expression profile that are reminiscent of vertebrate gastrulation. These findings may facilitate the use of hES cells for therapeutic purposes and as in vitro models of development.
21642344
Definitive endoderm differentiation of human-induced pluripotent stem cells using signaling molecules and IDE1 in three-dimensional polymer scaffold.
Human-induced pluripotent stem cells (hiPSCs) are considered to be potentially able to differentiate into all human cell lineages and thus hold promise as an unlimited source for cell replacement therapies in clinical applications. Definitive endoderm (DE) formation is the first and crucial step in the development of visceral organs such as liver, lung, pancreas and so forth. Therefore, efficient generation of DE cells ensures the efficient generation of eventual target cells used in cell therapy. In the present study, Matrigel-coated poly(lactic acid)/gelatin (PLA/gelatin) nanofibrous scaffolds were utilized to investigate the proliferation and differentiation of hiPSCs into DE cells. Analyses of DE-specific markers including Sox17, FoxA2, and Gooscoid (Gsc) genes revealed higher levels of mRNA and protein expression in the differentiated hiPSCs cells cultured on PLA/gelatin scaffolds than cells differentiated in two-dimensional (2D) culture. Our results showed that three-dimensional (3D) cultures could significantly promote DE differentiation in comparison with 2D culture. Also using small molecules such as inducer of definitive endoderm 1 (IDE1) and signaling molecules such as Activin A and Wnt3a could enhance the DE differentiation of hiPSCs with Activin A/Wnt3a being significantly more potent in both 2D and 3D cultures compared to IDE1. The results of this study may have impact in tissue engineering and cell replacement therapy of visceral organs-related diseases.
10426533
Development of definitive endoderm from embryonic stem cells in culture
The cellular and molecular events regulating the induction and tissue-specific differentiation of endoderm are central to our understanding of the development and function of many organ systems. To define and characterize key components in this process, we have investigated the potential of embryonic stem (ES) cells to generate endoderm following their differentiation to embryoid bodies (EBs) in culture. We found that endoderm can be induced in EBs, either by limited exposure to serum or by culturing in the presence of activin A (activin) under serum-free conditions. By using an ES cell line with the green fluorescent protein (GFP) cDNA targeted to the brachyury locus, we demonstrate that endoderm develops from a brachyury+ population that also displays mesoderm potential. Transplantation of cells generated from activin-induced brachyury+ cells to the kidney capsule of recipient mice resulted in the development of endoderm-derived structures. These findings demonstrate that ES cells can generate endoderm in culture and, as such, establish this differentiation system as a unique murine model for studying the development and specification of this germ layer.
12146184
Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells
The molecular mechanisms controlling inductive events leading to the specification and terminal differentiation of cardiomyocytes are still largely unknown. We have investigated the role of Cripto, an EGF-CFC factor, in the earliest stages of cardiomyogenesis. We find that both the timing of initiation and the duration of Cripto signaling are crucial for priming differentiation of embryonic stem (ES) cells into cardiomyocytes, indicating that Cripto acts early to determine the cardiac fate. Furthermore, we show that failure to activate Cripto signaling in this early window of time results in a direct conversion of ES cells into a neural fate. Moreover, the induction of Cripto activates the Smad2 pathway, and overexpression of activated forms of type I receptor ActRIB compensates for the lack of Cripto signaling in promoting cardiomyogenesis. Finally, we show that Nodal antagonists inhibit Cripto-regulated cardiomyocyte induction and differentiation in ES cells. All together our findings provide evidence for a novel role of the Nodal/Cripto/Alk4 pathway in this process.
5848430
Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm.
Nodal, a member of the TGF-beta 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 beta-tubulin III positive neurites, and when Lefty expressing hESCs were grown as a monolayer and allowed to differentiate, they generated significantly higher numbers of beta-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.
20194042
Regulated Nodal signaling promotes differentiation of the definitive endoderm and mesoderm from ES cells
Nodal signaling induces the formation of the endoderm and mesoderm during gastrulation. Nodal expression persists until the definitive endoderm progenitor has completely formed, and disappears thereafter. A tightly regulated Nodal expression system is essential for the differentiation of embryonic stem (ES) cells into distinct tissue lineages. On this basis, we established an ES cell differentiation system with the tetracycline-regulated expression of Nodal. The upregulated Nodal signaling pathway and its downstream transcriptional targets induced the specification of ES cells into definitive endoderm and mesoderm derivatives, and the subsequent downregulation of Nodal signaling promoted further maturation of the gut tube both in vitro and in vivo. Sustained expression of the Nodal gene inhibited the maturation of the definitive endoderm owing to persistent Oct3 and/or Oct4 expression and teratoma formation. Furthermore, quantitative single cell analysis by flow cytometry using CXCR4, VEGFR2 and PDGFR-α indicated that this protocol for definitive endoderm and mesoderm differentiation is superior to any other available protocol. Our findings also indicated that the Nodal or Nodal-related molecules secreted from Nodal-expressing ES cells could cause genetically unmanipulated ES cells to induce the expression of the Nodal signaling pathway and its downstream targets, which consequently leads to the differentiation of the ES cells into definitive endoderm and mesoderm. Our differentiation system, using tightly regulated Nodal expression, enabled us to investigate the mechanism of ES cell differentiation into definitive endoderm or mesoderm derivatives. Our findings also demonstrate that Nodal-expressing ES cells might be a source of highly active proteins that could be used for developing endoderm or mesoderm tissues in regenerative medicine.
9286546
Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway.
Genetic studies in fish, amphibia, and mice have shown that deficiency of Nodal signaling blocks differentiation into mesoderm and endoderm. Thus, Nodal is considered as a major inducer of mesendoderm during gastrulation. On this basis, Nodal is a candidate for controlling differentiation of pluripotent human embryonic stem cells (hESCs) into tissue lineages with potential clinical value. We have investigated the effect of Nodal, both as a recombinant protein and as a constitutively expressed transgene, on differentiation of hESCs. When control hESCs were grown in chemically defined medium, their expression of markers of pluripotency progressively decreased, while expression of neuroectoderm markers was strongly upregulated, thus revealing a neuroectodermal default mechanism for differentiation in this system. hESCs cultured in recombinant Nodal, by contrast, showed prolonged expression of pluripotency marker genes and reduced induction of neuroectoderm markers. These Nodal effects were accentuated in hESCs expressing a Nodal transgene, with striking morphogenetic consequences. Nodal-expressing hESCs developing as embryoid bodies contained an outer layer of visceral endoderm-like cells surrounding an inner layer of epiblast-like cells, each layer having distinct gene expression patterns. Markers of neuroectoderm were not upregulated during development of Nodal-expressing embryoid bodies, nor was there induction of markers for definitive mesoderm or endoderm differentiation. Moreover, the inner layer expressed markers of pluripotency, characteristic of undifferentiated hESCs and of epiblast in mouse embryos. These results could be accounted for by an inhibitory effect of Nodal-induced visceral endoderm on pluripotent cell differentiation into mesoderm and endoderm, with a concomitant inhibition of neuroectoderm differentiation by Nodal itself. There could also be a direct effect of Nodal in the maintenance of pluripotency. In summary, analysis of the Nodal-expressing phenotype suggests a function for the transforming growth factor-beta (TGF-beta) growth factor superfamily in pluripotency and in early cell fate decisions leading to primary tissue layers during in vitro development of pluripotent human stem cells. The effects of Nodal on early differentiation illustrate how hESCs can augment mouse embryos as a model for analyzing mechanisms of early mammalian development.
45108145
Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells
Preparation of specific lineages at high purities from embryonic stem (ES) cells requires both selective culture conditions and markers to guide and monitor the differentiation. In this study, we distinguished definitive and visceral endoderm by using a mouse ES cell line that bears the gfp and human IL2Rα (also known as CD25) marker genes in the goosecoid (Gsc) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc+Sox17+ definitive endoderm and Gsc−Sox17+ visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, we identified seven surface molecules that are expressed differentially in the two populations. One of the seven markers, Cxcr4, to which a monoclonal antibody is available allowed us to monitor and purify the Gsc+ population from genetically unmanipulated ES cells under the condition that selects definitive endoderm.
4233925
Relationship between asymmetric nodal expression and the direction of embryonic turning
GROWTH factors related to TGF-β provide important signals for patterning the vertebrate body plan1–3. One such family member, nodal, is required for formation of the primitive streak during mouse gastrulation4–6. Here we have used a nodal–lacZ reporter allele to demonstrate asymmetric nodal expression in the mouse node, a structure thought to be the functional equivalent of the frog and chick 'organizer'7, and in lateral plate mesoderm cells. We have also identified two additional genes acting with nodal in a pathway determining the left–right body axis. Thus we observe in inv mutant embryos8 that the sidedness of nodal expression correlates with the direction of heart looping and embryonic turning. In contrast, HNF3-β+/- nodallacZ/+ double-heterozygous embryos display LacZ staining on both left and right sides, and frequently exhibit defects in body situs. Taken together, these experiments, along with similar findings in chick9, demonstrate that elements of the genetic pathway that establish the left–right body axis are conserved in vertebrates.
9103898
A molecular pathway determining left-right asymmetry in chick embryogenesis
While significant progress has been made in understanding the molecular events underlying the early specification of the antero-posterior and dorso-ventral axes, little information is available regarding the cellular or molecular basis for left-right (LR) differences in animal morphogenesis. We describe the expression patterns of three genes involved in LR determination in chick embryos: activin receptor IIa, Sonic hedgehog (Shh), and cNR-1 (related to the mouse gene nodal). These genes are expressed asymmetrically during and after gastrulation and regulate the expression of one another in a sequential pathway. Moreover, manipulation of the sidedness of either activin protein or Shh expression alters heart situs. Together, these observations identify a cascade of molecular asymmetry in that determines morphological LR asymmetry in the chick embryo.
7236924
The zebrafish nodal-related gene southpaw is required for visceral and diencephalic left-right asymmetry
We have identified and characterized a new zebrafish gene, southpaw, that is required for visceral and diencephalic left-right asymmetry. southpaw encodes a new member of the nodal-related class of proteins, a subfamily within the transforming growth factorβsuperfamily of secreted factors. southpaw is expressed bilaterally in paraxial mesoderm precursors and then within the left lateral plate mesoderm. At late somite stages, left-sided southpaw expression transiently overlaps the left-sided expression domains of other genes that mark the developing heart, such as lefty2. We have injected morpholinos to block the translation of the southpaw mRNA or to block splicing of the southpaw pre-mRNA. These morpholinos cause a severe disruption of early (cardiac jogging) and late (cardiac looping) aspects of cardiac left-right asymmetry. As the left-right asymmetry of the pancreas is also affected, southpaw appears to regulate left-right asymmetry throughout a large part of the embryo. Consistent with the morphological changes, the left-sided expression domains of downstream genes (cyclops, pitx2, lefty1 and lefty2) are severely downregulated or abolished within the lateral plate mesoderm of Southpaw-deficient embryos. Surprisingly, despite the absence of southpaw expression in the brain, we find that early diencephalic left-right asymmetry also requires Southpaw activity. These observations lead to a model of how visceral organ and brain left-right asymmetry are coordinated during embryogenesis.
4316534
Conserved left–right asymmetry of nodal expression and alterations in murine situs inversus
VERTEBRATES have characteristic and conserved left–right (L–R) visceral asymmetries, for example the left-sided heart. In humans, alterations of L–R development can have serious clinical implications, including cardiac defects1. Although little is known about how the embryonic L–R axis is established, a recent study in the chick embryo revealed L–R asymmetric expression of several previously cloned genes, including Cnr-1 (for chicken nodal-related-1), and indicated how this L–R molecular asymmetry might be important for subsequent visceral morphogenesis2. Here we show that nodal3 is asymmetrically expressed in mice at similar stages, as is Xnr-1 (for Xenopus nodal related-1)4 in frogs. We also examine nodal expression in two mouse mutations that perturb L–R development, namely situs inversus viscerum (iv)5, in which assignment of L–R asymmetry is apparently random and individuals develop either normally or are mirror-image-reversed (situs inversus), and inversion of embryonic turning (inv)6, in which all individuals develop with situs inversus. In both, nodal expression is strikingly affected, being reversed or converted to symmetry. These results further support a key role for nodal and nodal-related genes in interpreting and relaying L–R patterning information in vertebrates. To our knowledge, our results provide the first direct evidence that iv and inv normally function well before the appearance of morphological L–R asymmetry.
19184774
Nodal activity in the node governs left-right asymmetry.
Nodal is expressed at the lateral edges of the mouse node, but its function in this "organizer" tissue remains unknown due to the early lethality of Nodal mutant embryos. Here we used a genetic strategy to selectively remove Nodal activity from the node. Embryos lacking Nodal in the node fail to initiate molecular asymmetry in the left lateral plate mesoderm and exhibit multiple left-right patterning defects. Nodal may also act as a short-range signal to establish a functional midline barrier. Our findings confirm that the mouse node is instrumental in initiating left-right axis specification and identify Nodal as the key morphogen regulating this process.
5396019
Broad mesodermal and endodermal deletion of Nodal at postgastrulation stages results solely in left/right axial defects
Nodal signaling is a critical regulator of multiple aspects of early vertebrate development including asymmetry along the left/right (LR) axis. To study Nodal function occurring specifically in the postgastrulation embryo, we have used Cre/loxP based conditional mutagenesis. A floxed allele of Nodal was generated and shown to have wild‐type function. This allele was then used in conjunction with the T‐Cre line, which expresses Cre recombinase broadly in the mesodermal and definitive endodermal lineages posterior to the cranial region. T‐Cre activity leads to complete deletion of Nodal before its normal transient expression in the early somite stage lateral plate mesoderm, thereby causing severe LR developmental defects. No other abnormalities were found, suggesting that Nodal signaling has no additional essential functions in developmental patterning within the extensive mesodermal and endodermal domains marked by T‐Cre activity. Developmental Dynamics 237:3591–3601, 2008. Published 2008 Wiley‐Liss, Inc.
29178641
Left-right patterning of the mouse lateral plate requires nodal produced in the node.
Initial determination of left-right (L-R) polarity in mammalian embryos takes place in the node. However, it is not known how asymmetric signals are generated in the node and transferred to the lateral plate mesoderm (LPM). Mice homozygous for a hypomorphic Nodal allele (Nodal(neo)) were generated and found to exhibit L-R defects, including right isomerism. Although the mutant embryos express Nodal at gastrulation stages, the subsequent expression of this gene in the node and left LPM is lost. A transgene that conferred Nodal expression specifically in the node rescued the L-R defects of the Nodal(neo/neo) embryos. Conversely, ectopic expression of the Nodal inhibitor Lefty2 in the node of Nodal(neo/+) embryos resulted in a phenotype similar to that of the Nodal(neo/neo) mutant. These results indicate that Nodal produced in the node is required for expression of Nodal and other left side-specific genes in the LPM.
35618553
Conserved requirement for EGF-CFC genes in vertebrate left-right axis formation.
Specification of the left-right (L-R) axis in the vertebrate embryo requires transfer of positional information from the node to the periphery, resulting in asymmetric gene expression in the lateral plate mesoderm. We show that this activation of L-R lateral asymmetry requires the evolutionarily conserved activity of members of the EGF-CFC family of extracellular factors. Targeted disruption of murine Cryptic results in L-R laterality defects including randomization of abdominal situs, hyposplenia, and pulmonary right isomerism, as well as randomized embryo turning and cardiac looping. Similarly, zebrafish one-eyed pinhead (oep) mutants that have been rescued partially by mRNA injection display heterotaxia, including randomization of heart looping and pancreas location. In both Cryptic and oep mutant embryos, L-R asymmetric expression of Nodal/cyclops, Lefty2/antivin, and Pitx2 does not occur in the lateral plate mesoderm, while in Cryptic mutants Lefty1 expression is absent from the prospective floor plate. Notably, L-R asymmetric expression of Nodal at the lateral edges of the node is still observed in Cryptic mutants, indicating that L-R specification has occurred in the node but not the lateral plate. Combined with the previous finding that oep is required for nodal signaling in zebrafish, we propose that a signaling pathway mediated by Nodal and EGF-CFC activities is essential for transfer of L-R positional information from the node.
6787053
Regulation of left-right patterning in mice by growth/differentiation factor-1
The transforming growth factor-β (TGF-β) superfamily encompasses a large group of structurally related polypeptides that are capable of regulating cell growth and differentiation in a wide range of embryonic and adult tissues. Growth/differentiation factor-1 (Gdf-1, encoded by Gdf1 ) is a TGF-β family member of unknown function that was originally isolated from an early mouse embryo cDNA library and is expressed specifically in the nervous system in late-stage embryos and adult mice. Here we show that at early stages of mouse development, Gdf1 is expressed initially throughout the embryo proper and then most prominently in the primitive node, ventral neural tube, and intermediate and lateral plate mesoderm. To examine its biological function, we generated a mouse line carrying a targeted mutation in Gdf1. Gdf1−/− mice exhibited a spectrum of defects related to left-right axis formation, including visceral situs inversus, right pulmonary isomerism and a range of cardiac anomalies. In most Gdf1−/− embryos, the expression of Ebaf (formerly lefty-1) in the left side of the floor plate and Leftb (formerly lefty-2),nodal and Pitx2 in the left lateral plate mesoderm was absent, suggesting that Gdf1 acts upstream of these genes either directly or indirectly to activate their expression. Our findings suggest that Gdf1 acts early in the pathway of gene activation that leads to the establishment of left-right asymmetry.
22388129
Dvr1 transfers left-right asymmetric signals from Kupffer's vesicle to lateral plate mesoderm in zebrafish.
An early step in establishing left-right (LR) symmetry in zebrafish is the generation of asymmetric fluid flow by Kupffer's vesicle (KV). As a result of fluid flow, a signal is generated and propagated from the KV to the left lateral plate mesoderm, activating a transcriptional response of Nodal expression in the left lateral plate mesoderm (LPM). The mechanisms and molecules that aid in this transfer of information from the KV to the left LPM are still not clear. Here we provide several lines of evidence demonstrating a role for a member of the TGFβ family member, Dvr1, a zebrafish Vg1 ortholog. Dvr1 is expressed bilaterally between the KV and the LPM. Knockdown of Dvr1 by morpholino causes dramatically reduced or absent expression of southpaw (spaw, a Nodal homolog), in LPM, and corresponding loss of downstream Lefty (lft1 and lft) expression, and aberrant brain and heart LR patterning. Dvr1 morphant embryos have normal KV morphology and function, normal expression of southpaw (spaw) and charon (cha) in the peri-KV region and normal expression of a variety of LPM markers in LPM. Additionally, Dvr1 knockdown does not alter the capability of LPM to respond to signals that initiate and propagate spaw expression. Co-injection experiments in Xenopus and zebrafish indicate that Dvr1 and Spaw can enhance each other's ability to activate the Nodal response pathway and co-immunoprecipitation experiments reveal differential relationships among activators and inhibitors in this pathway. These results indicate that Dvr1 is responsible for enabling the transfer of a left-right signal from KV to the LPM.
24168467
Distinct and cooperative roles of mammalian Vg1 homologs GDF1 and GDF3 during early embryonic development.
Vg1, a member of the TGF-beta superfamily of ligands, has been implicated in the induction of mesoderm, formation of primitive streak, and left-right patterning in Xenopus and chick embryos. In mice, GDF1 and GDF3 - two TGF-beta superfamily ligands that share high sequence identity with Vg1 - have been shown to independently mimic distinct aspects of Vg1's functions. However, the extent to which the developmental processes controlled by GDF1 and GDF3 and the underlying signaling mechanisms are evolutionarily conserved remains unclear. Here we show that phylogenetic and genomic analyses indicate that Gdf1 is the true Vg1 ortholog in mammals. In addition, and similar to GDF1, we find that GDF3 signaling can be mediated by the type I receptor ALK4, type II receptors ActRIIA and ActRIIB, and the co-receptor Cripto to activate Smad-dependent reporter genes. When expressed in heterologous cells, the native forms of either GDF1 or GDF3 were incapable of inducing downstream signaling. This could be circumvented by using chimeric constructs carrying heterologous prodomains, or by co-expression with the Furin pro-protein convertase, indicating poor processing of the native GDF1 and GDF3 precursors. Unexpectedly, co-expression with Nodal - another TGF-beta superfamily ligand involved in mesoderm formation - could also expose the activities of native GDF1 and GDF3, suggesting a potentially novel mode of cooperation between these ligands. Functional complementarity between GDF1 and GDF3 during embryonic development was investigated by analyzing genetic interactions between their corresponding genes. This analysis showed that Gdf1(-/-);Gdf3(-/-) compound mutants are more severely affected than either Gdf1(-/-) or Gdf3(-/-) single mutants, with defects in the formation of anterior visceral endoderm and mesoderm that recapitulate Vg1 loss of function, suggesting that GDF1 and GDF3 together represent the functional mammalian homologs of Vg1.
23144806
Identification and cloning of localized maternal RNAs from xenopus eggs
A central question in developmental biology is to explain how cells in different regions of an embryo acquire different developmental fates. We have begun to address this question by investigating whether specific RNAs are localized within a frog egg. Differential screening of a cDNA library shows that most maternal RNAs are uniformly distributed along the animal-vegetal axis. However, we find that a rare class of maternal RNAs is localized. cDNA clones of four localized RNAs have been characterized. Three of these cDNAs are derived from maternal RNAs that are concentrated in the animal hemisphere of unfertilized eggs and remain localized through the early blastula stage. One cDNA is derived from a maternal RNA found almost exclusively in the vegetal hemisphere at both stages. These studies show that some informational molecules, specifically RNAs, are localized in eggs and are inherited by particular blastomeres.
40022353
A maternal mRNA localized to the vegetal hemisphere in xenopus eggs codes for a growth factor related to TGF-β
We report that Vg1, a maternal mRNA localized to the vegetal hemisphere of frog eggs, encodes a member of the transforming growth factor-beta (TGF-beta) family of proteins. Furthermore, we show that Vg1 mRNA is distributed to presumptive endodermal cells after fertilization. Previous studies had shown that the vegetal end of a frog egg produces a signal that induces the overlying animal pole cells to form mesodermal tissue. More recently it has been shown that fibroblast growth factor (FGF) and TGF-beta can participate in the induction of muscle. Together, these results lead us to propose that the formation of mesoderm during frog development is specified by the products of localized maternal mRNAs, including Vg1.
30723975
Bucky ball functions in Balbiani body assembly and animal-vegetal polarity in the oocyte and follicle cell layer in zebrafish.
The Balbiani body is an evolutionarily conserved asymmetric aggregate of organelles that is present in early oocytes of all animals examined, including humans. Although first identified more than 150 years ago, genes acting in the assembly of the Balbiani body have not been identified in a vertebrate. Here we show that the bucky ball gene in the zebrafish is required to assemble this universal aggregate of organelles. In the absence of bucky ball the Balbiani body fails to form, and vegetal mRNAs are not localized in oocytes. In contrast, animal pole localized oocyte markers are expanded into vegetal regions in bucky ball mutants, but patterning within the expanded animal pole remains intact. Interestingly, in bucky ball mutants an excessive number of cells within the somatic follicle cell layer surrounding the oocyte develop as micropylar cells, an animal pole specific cell fate. The single micropyle permits sperm to fertilize the egg in zebrafish. In bucky ball mutants, excess micropyles cause polyspermy. Thus bucky ball provides the first genetic access to Balbiani body formation in a vertebrate. We demonstrate that bucky ball functions during early oogenesis to regulate polarity of the oocyte, future egg and embryo. Finally, the expansion of animal identity in oocytes and somatic follicle cells suggests that somatic cell fate and oocyte polarity are interdependent.
849583
The DVR-1 (Vg1) transcript of zebrafish is maternally supplied and distributed throughout the embryo.
It is not known how region- or tissue-specific differences are generated in the zebrafish embryo. To look at the potential role of maternal transcripts in generating cell diversity, we have isolated and characterized the zebrafish homologue of Xenopus DVR-1 (Vg1), a maternally supplied RNA that encodes a member of the transforming growth factor-beta superfamily. The zebrafish DVR-1 RNA is maternally supplied and its protein product shares a high degree of sequence identity with Xenopus DVR-1. These conserved features indicate that DVR-1 is likely to have an essential function in early embryogenesis. However, unlike the frog transcript, which is restricted to vegetal cells, DVR-1 RNA is distributed equally among all zebrafish blastomeres. We suggest that the ubiquitous distribution of DVR-1 RNA reflects a significant aspect of the developmental strategy of the zebrafish in which each blastomere retains an equivalent developmental potential throughout the cleavage period.
14505029
The Vg1-related protein Gdf3 acts in a Nodal signaling pathway in the pre-gastrulation mouse embryo
The formation of the anterior visceral endoderm (AVE) in the pre-gastrulation mouse embryo represents a crucial event in patterning of the anterior-posterior axis. Here, we show that the transforming growth factorβ (Tgfβ) family member Gdf3 (growth-differentiation factor 3), a close relative of Xenopus Vg1, resembles the Tgfβ ligand Nodal in both its signaling activity and its role in AVE formation in vivo. Thus, in cell culture, Gdf3 signaling requires the EGF-CFC co-receptor Cripto and can be inhibited by Lefty antagonists. In Xenopus embryos, Gdf3 misexpression results in secondary axis formation, and induces morphogenetic elongation and mesendoderm formation in animal caps. In mouse embryos, Gdf3 is expressed in the inner cell mass and epiblast, and null mutants frequently exhibit abnormal formation or positioning of the AVE. This phenotype correlates with defects in mesoderm and definitive endoderm formation, as well as abnormal Nodal expression levels. Our findings indicate that Gdf3 acts in a Nodal-like signaling pathway in pre-gastrulation development, and provide evidence for the functional conservation of Vg1 activity in mice.
9619639
Mesendoderm induction and reversal of left-right pattern by mouse Gdf1, a Vg1-related gene.
TGFbeta signals play important roles in establishing the body axes and germ layers in the vertebrate embryo. Vg1 is a TGFbeta-related gene that, due to its maternal expression and vegetal localization in Xenopus, has received close examination as a potential regulator of development in Xenopus, zebrafish, and chick. However, a mammalian Vg1 ortholog has not been identified. To isolate mammalian Vg1 we screened a mouse expression library with a Vg1-specific monoclonal antibody and identified a single cross-reactive clone encoding mouse Gdf1. Gdf1 is expressed uniformly throughout the embryonic region at 5.5-6.5 days postcoitum and later in the node, midbrain, spinal cord, paraxial mesoderm, lateral plate mesoderm, and limb bud. When expressed in Xenopus embryos, native GDF1 is not processed, similar to Vg1. In contrast, a chimeric protein containing the prodomain of Xenopus BMP2 fused to the GDF1 mature domain is efficiently processed and signals via Smad2 to induce mesendoderm and axial duplication. Finally, right-sided expression of chimeric GDF1, but not native GDF1, reverses laterality and results in right-sided Xnr1 expression and reversal of intestinal and heart looping. Therefore, GDF1 can regulate left-right patterning, consistent with the Gdf1 loss-of-function analysis in the mouse (C. T. Rankin, T. Bunton, A. M. Lawler, and S. J. Lee, 2000, Nature Genet. 24, 262-265) and a proposed role for Vg1 in Xenopus. Our results establish that Gdf1 is posttranslationally regulated, that mature GDF1 activates a Smad2-dependent signaling pathway, and that mature GDF1 is sufficient to reverse the left-right axis. Moreover, these findings demonstrate that GDF1 and Vg1 are equivalent in biochemical and functional assays, suggesting that Gdf1 provides a Vg1-like function in the mammalian embryo.
471204
Synergistic interaction between Gdf1 and Nodal during anterior axis development.
Growth and Differentiation Factor 1 (GDF-1) has been implicated in left-right patterning of the mouse embryo but has no other known function. Here, we demonstrate a genetic interaction between Gdf1 and Nodal during anterior axis development. Gdf1-/-;Nodal+/- mutants displayed several abnormalities that were not present in either Gdf1-/- or Nodal+/- single mutants, including absence of notochord and prechordal plate, and malformation of the foregut; organizing centers implicated in the development of the anterior head and branchial arches, respectively. Consistent with these deficits, Gdf1-/-;Nodal+/- mutant embryos displayed a number of axial midline abnormalities, including holoprosencephaly, anterior head truncation, cleft lip, fused nasal cavity, and lack of jaws and tongue. The absence of these defects in single mutants indicated a synergistic interaction between Nodal and GDF-1 in the node, from which the axial mesendoderm that gives rise to the notochord, prechordal plate, and foregut endoderm originates, and where the two factors are co-expressed. This notion was supported by a severe downregulation of FoxA2 and goosecoid in the anterior primitive streak of double mutant embryos. Unlike that in the lateral plate mesoderm, Nodal expression in the node was independent of GDF-1, indicating that both factors act in parallel to control the development of mesendodermal precursors. Receptor reconstitution experiments indicated that GDF-1, like Nodal, can signal through the type I receptors ALK4 and ALK7. However, analysis of compound mutants indicated that ALK4, but not ALK7, was responsible for the effects of GDF-1 and Nodal during anterior axis development. These results indicate that GDF-1 and Nodal converge on ALK4 in the anterior primitive streak to control the formation of organizing centers that are necessary for normal forebrain and branchial arch development.
9657293
Genetic dissection of nodal function in patterning the mouse embryo.
Loss-of-function analysis has shown that the transforming growth factor-like signaling molecule nodal is essential for mouse mesoderm development. However, definitive proof of nodal function in other developmental processes in the mouse embryo has been lacking because the null mutation blocks gastrulation. We describe the generation and analysis of a hypomorphic nodal allele. Mouse embryos heterozygous for the hypomorphic allele and a null allele undergo gastrulation but then display abnormalities that fall into three distinct mutant phenotypic classes, which may result from expression levels falling below critical thresholds in one or more domains of nodal expression. Our analysis of each of these classes provides conclusive evidence for nodal-mediated regulation of several developmental processes in the mouse embryo, beyond its role in mesoderm formation. We find that nodal signaling is required for correct positioning of the anteroposterior axis, normal anterior and midline patterning, and the left-right asymmetric development of the heart, vasculature, lungs and stomach.
14975915
Misexpression of chick Vg1 in the marginal zone induces primitive streak formation.
In the chick embryo, the primitive streak is the first axial structure to develop. The initiation of primitive streak formation in the posterior area pellucida is influenced by the adjacent posterior marginal zone (PMZ). We show here that chick Vg1 (cVg1), a member of the TGFbeta family of signalling molecules whose homolog in Xenopus is implicated in mesoderm induction, is expressed in the PMZ of prestreak embryos. Ectopic expression of cVg1 protein in the marginal zone chick blastoderms directs the formation of a secondary primitive streak, which subsequently develops into an ectopic embryo. We have used cell marking techniques to show that cells that contribute to the ectopic primitive streak change fate, acquiring two distinct properties of primitive streak cells, defined by gene expression and cell movements. Furthermore, naive epiblast explants exposed to cVg1 protein in vitro acquire axial mesodermal properties. Together, these results show that cVg1 can mediate ectopic axis formation in the chick by inducing new cell fates and they permit the analysis of distinct events that occur during primitive streak formation.
17621306
Interactions between Wnt and Vg1 signalling pathways initiate primitive streak formation in the chick embryo.
The posterior marginal zone (PMZ) of the chick embryo has Nieuwkoop centre-like properties: when transplanted to another part of the marginal zone, it induces a complete embryonic axis, without making a cellular contribution to the induced structures. However, when the PMZ is removed, the embryo can initiate axis formation from another part of the remaining marginal zone. Chick Vg1 can mimic the axis-inducing ability of the PMZ, but only when misexpressed somewhere within the marginal zone. We have investigated the properties that define the marginal zone as a distinct region. We show that the competence of the marginal zone to initiate ectopic primitive streak formation in response to cVg1 is dependent on Wnt activity. First, within the Wnt family, only Wnt8C is expressed in the marginal zone, in a gradient decreasing from posterior to anterior. Second, misexpression of Wnt1 in the area pellucida enables this region to form a primitive streak in response to cVg1. Third, the Wnt antagonists Crescent and Dkk-1 block the primitive streak-inducing ability of cVg1 in the marginal zone. These findings suggest that Wnt activity defines the marginal zone and allows cVg1 to induce an axis. We also present data suggesting some additional complexity: first, the Vg1 and Wnt pathways appear to regulate the expression of downstream components of each other's pathway; and second, misexpression of different Wnt antagonists suggests that different classes of Wnts may cooperate with each other to regulate axis formation in the normal embryo.
23751713
Vg1 is an essential signaling molecule in Xenopus development
Xenopus Vg1, a transforming growth factor β (Tgfβ) family member, was one of the first maternally localized mRNAs identified in vertebrates. Its restriction to the vegetal pole of the egg made it the ideal candidate to be the mesoderm-inducing signal released by vegetal cells, but its function in vivo has never been resolved. We show that Vg1 is essential for Xenopus embryonic development, and is required for mesoderm induction and for the expression of several key Bmp antagonists. Although the original Vg1 transcript does not rescue Vg1-depleted embryos, we report that a second allele is effective. This work resolves the mystery of Vg1 function, and shows it to be an essential maternal regulator of embryonic patterning.
18406302
Regulation of TGFβ and related signals by precursor processing.
Secreted cytokines of the TGFβ family are found in all multicellular organisms and implicated in regulating fundamental cell behaviors such as proliferation, differentiation, migration and survival. Signal transduction involves complexes of specific type I and II receptor kinases that induce the nuclear translocation of Smad transcription factors to regulate target genes. Ligands of the BMP and Nodal subgroups act at a distance to specify distinct cell fates in a concentration-dependent manner. These signaling gradients are shaped by multiple factors, including proteases of the proprotein convertase (PC) family that hydrolyze one or several peptide bonds between an N-terminal prodomain and the C-terminal domain that forms the mature ligand. This review summarizes information on the proteolytic processing of TGFβ and related precursors, and its spatiotemporal regulation by PCs during development and various diseases, including cancer. Available evidence suggests that the unmasking of receptor binding epitopes of TGFβ is only one (and in some cases a non-essential) function of precursor processing. Future studies should consider the impact of proteolytic maturation on protein localization, trafficking and turnover in cells and in the extracellular space.
22624408
Secretion and mesoderm‐inducing activity of the TGF‐beta‐related domain of Xenopus Vg1.
Vg1 is a maternal mRNA localized to the vegetal hemisphere of Xenopus embryos during blastula stages, a region responsible for the induction of mesoderm in the adjacent marginal zone. Its homology to the transforming growth factor‐beta family, which includes several proteins with mesoderm‐inducing activity, suggests a role for Vg1 as an endogenous mesoderm‐inducing factor. However, expression of Vg1 protein in the animal hemisphere, following injection of synthetic mRNA, has no effect on development, and isolated animal caps are not mesodermalized. It is shown that Vg1 protein fails to form dimers and is not processed to release the putative bioactive domain. Furthermore it is shown that the N‐terminal signal peptide of Vg1 is not cleaved following translocation into the ER, which may explain the failure of this protein to dimerize. To explore the role of Vg1 in amphibian development, a fusion protein has been made of the preproregion of Xenopus bone morphogenetic protein‐4 and the putative bioactive C‐terminal domain of Vg1. This fusion protein forms dimers and the C‐terminal domain of Vg1 is secreted. Injection of this construct into Xenopus embryos induces the formation of a second dorsal axis and isolated animal caps are mesodermalized. The results are consistent with a role for Vg1 in mesoderm induction during Xenopus development.
23934271
Developmental expression of the protein product of Vg1, a localized maternal mRNA in the frog Xenopus laevis.
Vg1 is a maternal mRNA localized in the vegetal cortex of Xenopus laevis oocytes, that encodes a protein homologous to the mammalian growth factor TGF‐beta. Using a polyclonal antibody to a T7‐Vg1 fusion protein, we have identified the native protein. We find that a single protein of Mr 40 kd is immunoprecipitated following in vitro translation of oocyte poly(A)+ RNA, whilst two proteins of Mr 45 and 43.5 kd are immunoprecipitated from oocyte and embryo extracts. Synthesis of at least the 40 kd, in vitro, and 45 kd, in vivo, proteins is specifically inhibited following treatment of the respective systems with antisense Vg1 (but not histone H4) oligodeoxynucleotides. Tunicamycin treatment reveals the in vivo proteins to be glycosylated versions of a 40 kd protein, modified by the addition of either two or three N‐linked oligosaccharide side chains. Both proteins are sensitive to digestion by the enzyme endoglycosidase‐H, and are segregated within a membrane fraction from which they can be released by high pH treatment. Their synthesis is first detectable in stage IV oocytes and continues throughout early embryogenesis until the late gastrula. During embryogenesis the relative proportions of the two proteins change, the 45 kd protein being predominant in early embryogenesis and the 43.5 kd protein in late embryogenesis. Synthesis only occurs in the vegetal hemisphere at all stages; however, in the large oocyte diffusion of both proteins into the animal hemisphere occurs.
2502031
Localized synthesis of the Vg1 protein during early Xenopus development.
The Xenopus Vg1 gene encodes a maternal mRNA that is localized to the vegetal hemisphere of both oocytes and embryos and encodes a protein related to the TGF-beta family of small secreted growth factors. We have raised antibodies to recombinant Vg1 protein and used them to show that Vg1 protein is first detected in stage IV oocytes and reaches maximal levels in stage VI oocytes and eggs. During embryogenesis, Vg1 protein is synthesized until the gastrula stage. The embryonically synthesized Vg1 protein is present only in vegetal cells of an early blastula. We find that Vg1 protein is glycosylated and associated with membranes in the early embryo. Our results also suggest that a small proportion of the full-length Vg1 protein is cleaved to give a small peptide of M(r) = approximately 17 x 10(3). These results support the proposal that the Vg1 protein is an endogenous growth-factor-like molecule involved in mesoderm induction within the amphibian embryo.
21413522
Processed Vg1 protein is an axial mesoderm inducer in xenopus
Vg1 is a TGF beta-related growth factor encoded by a maternal mRNA localized to vegetal blastomeres in Xenopus embryos. Vg1 precursor protein is abundant in vegetal cells, but the processed mature form has not been readily detected and no activity has been demonstrated for the putative Vg1 mature protein. We have engineered a BMP2-Vg1 fusion (BVg1) that promotes formation of mature Vg1 protein in vivo. Injection of BVg1 mRNA induces dorsal mesoderm in animal cap cells, and BVg1 expression in ultraviolet-ventralized embryos fully restores a normal dorsal axis. Blastomeres expressing BVg1 act as a Nieuwkoop center, the region that induces the Spemann organizer. our results lead us to suggest that localized posttranslational processing of Vg1 precursor protein on the future dorsal side of the embryo is a key step in generating dorsal mesoderm and the body axis in Xenopus.
2697106
Induction of dorsal mesoderm by soluble, mature Vg1 protein.
Mesoderm induction during Xenopus development has been extensively studied, and two members of the transforming growth factor-beta family, activin beta B and Vg1, have emerged as candidates for a natural inducer of dorsal mesoderm. Heretofore, analysis of Vg1 activity has relied on injection of hybrid Vg1 mRNAs, which have not been shown to direct efficient secretion of ligand and, therefore, the mechanism of mesoderm induction by processed Vg1 protein is unclear. This report describes injection of Xenopus oocytes with a chimeric activin-Vg1 mRNA, encoding the pro-region of activin beta B fused to the mature region of Vg1, resulting in the processing and secretion of mature Vg1. Treatment of animal pole explants with mature Vg1 protein resulted in differentiation of dorsal, but not ventral, mesodermal tissues and dose-dependent activation of both dorsal and ventrolateral mesodermal markers. At high doses, mature Vg1 induced formation of 'embryoids' with a rudimentary axial pattern, head structures including eyes and a functional neuromuscular system. Furthermore, truncated forms of the activin and FGF receptors, which block mesoderm induction in the intact embryo, fully inhibited mature Vg1 activity. To examine the mechanism of inhibition, we have performed receptor-binding assays with radiolabeled Vg1. Finally, follistatin, a specific inhibitor of activin beta B which is shown not to block endogenous dorsal mesoderm induction, failed to inhibit Vg1. The results support a role for endogenous Vg1 in dorsal mesoderm induction during Xenopus development.
17894136
Plasticity underlies tumor progression: role of Nodal signaling
The transforming growth factor beta (TGFβ) superfamily member Nodal is an established regulator of early embryonic development, with primary roles in endoderm induction, left-right asymmetry, and primitive streak formation. Nodal signals through TGFβ family receptors at the plasma membrane and induces signaling cascades leading to diverse transcriptional regulation. While conceptually simple, the regulation of Nodal and its molecular effects are profoundly complex and context dependent. Pioneering work by developmental biologists has characterized the signaling pathways, regulatory components, and provided detailed insight into the mechanisms by which Nodal mediates changes at the cellular and organismal levels. Nodal is also an important factor in maintaining pluripotency of embryonic stem cells through regulation of core transcriptional programs. Collectively, this work has led to an appreciation for Nodal as a powerful morphogen capable of orchestrating multiple cellular phenotypes. Although Nodal is not active in most adult tissues, its reexpression and signaling have been linked to multiple types of human cancer, and Nodal has emerged as a driver of tumor growth and cellular plasticity. In vitro and in vivo experimental evidence has demonstrated that inhibition of Nodal signaling reduces cancer cell aggressive characteristics, while clinical data have established associations with Nodal expression and patient outcomes. As a result, there is great interest in the potential targeting of Nodal activity in a therapeutic setting for cancer patients that may provide new avenues for suppressing tumor growth and metastasis. In this review, we evaluate our current understanding of the complexities of Nodal function in cancer and highlight recent experimental evidence that sheds light on the therapeutic potential of its inhibition.
24917807
Fluid dynamics of nodal flow and left–right patterning in development
The manner in which the nodal flow determines the breaking of left–right symmetry during development is a beautiful example of the application of fluid dynamics to developmental biology. Detailed understanding of this crucial developmental process has greatly advanced by the transfer of ideas between these two disciplines. In this article, we review our and others' work on applying fluid dynamics and dynamical systems to the problem of left–right symmetry breaking in vertebrates. Developmental Dynamics 237:3477–3490, 2008. © 2008 Wiley‐Liss, Inc.
24982316
Nodal signaling and the evolution of deuterostome gastrulation
Chordates, including vertebrates, evolved within a group of animals called the deuterostomes. All holoblastic deuterostomes gastrulate at the vegetal pole and the blastopore becomes the anus, while a mouth is formed at the anterior or to the oral side. Nodal is a member of the TGF‐β superfamily of signaling molecules that are important in signaling between cells during many embryonic processes in vertebrate embryos. Nodal has also been found in other invertebrate deuterostomes, such as ascidians and sea urchins, but, so far, is missing in protostomes. Nodal has been shown to be particularly important in determining left‐right asymmetries in vertebrate embryos, but less information is available for its developmental role in the invertebrate deuterostomes. We review gastrulation in the deuterostomes, then examine nodal expression early during mesoderm formation and later during the establishment of asymmetries in both vertebrates and invertebrates. Nodal is expressed asymmetrically on the left side in chordates and on the presumptive oral side of the embryo in echinoid echinoderms. The expression of nodal is in different germ layers in embryos of different phyla. Expression is in the ectoderm in most of the invertebrate deuterostomes, and in the mesoderm in vertebrates. We summarize the work that has been published to date, especially nodal expression in the invertebrate deuterostomes, and suggest future experiments to better understand the evolution of nodal signaling and deuterostome gastrulation. Developmental Dynamics 234:269–278, 2005. © 2005 Wiley‐Liss, Inc.
5992429
Running the gauntlet: an overview of the modalities of travel employed by the putative morphogen Nodal.
A fundamental challenge in biology is to explain how progenitor cells in developing tissues acquire distinct fates according to their position. In vertebrates, the positional information that specifies the germ layers and the primary body axes is mediated by Nodal. Nodal meets the criteria of a morphogen since it can diffuse through tissues and signal far away from its source to directly specify multiple cell fates in a dosage-dependent manner. To consider the relationship between trafficking and graded Nodal signaling, this review summarizes recent findings how the spreading of Nodal activity is regulated by factors that promote long range signaling during left-right axis formation, and by structural features that affect Nodal protein stability and endosomal sorting.
18007532
Nodal Flow and the Generation of Left-Right Asymmetry
The establishment of left-right asymmetry in mammals is a good example of how multiple cell biological processes coordinate in the formation of a basic body plan. The leftward movement of fluid at the ventral node, called nodal flow, is the central process in symmetry breaking on the left-right axis. Nodal flow is autonomously generated by the rotation of cilia that are tilted toward the posterior on cells of the ventral node. These cilia are built by transport via the KIF3 motor complex. How nodal flow is interpreted to create left-right asymmetry has been a matter of debate. Recent evidence suggests that the leftward movement of membrane-sheathed particles, called nodal vesicular parcels (NVPs), may result in the activation of the non-canonical Hedgehog signaling pathway, an asymmetric elevation in intracellular Ca(2+) and changes in gene expression.
41811302
Roles of nodal‐lefty regulatory loops in embryonic patterning of vertebrates
Nodal is a signalling molecule that belongs to the transforming growth factor–β superfamily of proteins, and Lefty proteins are antagonists of Nodal signalling. The nodal and lefty genes form positive and negative regulatory loops that resemble the reaction‐diffusion system. As a pair, these genes control various events of vertebrate embryonic patterning, including left‐right specification and mesoderm formation. In this review, we will focus on recent studies that have addressed the roles of nodal and lefty in mouse development.
40129420
Patterning of the zebrafish embryo by nodal signals.
Publisher Summary This chapter summarizes the significant contribution that research on zebrafish has made toward identifying components of the nodal signaling pathway and defining the functions of nodal proteins during development. Nodal signals are essential for establishing the body plan of vertebrate embryos. Nodal proteins are secreted signaling factors in the transforming growth factor β (TGF β) family that have been found in every vertebrate. In zebrafish, two Nodal-related proteins, Cyclops (Cyc) and Squint (Sqt), have been characterized. Cyc and Sqt are synthesized as proproteins, which are cleaved to make the mature form. The chapter reveals that the cyc and sqt genes have distinct, dynamic expression patterns in the embryo. These expression patterns reflect the complex roles of nodal signals during development and are important for understanding when and where patterning is taking place. In zebrafish and other vertebrates nodal proteins are involved in multiple patterning events during development, including initial specification of the endoderm and mesoderm, formation of the ventral neural tube, and regulation of left–right asymmetry.
6310239
Activin/Nodal signalling before implantation: setting the stage for embryo patterning
Activins and Nodal are members of the transforming growth factor beta (TGF-β) family of growth factors. Their Smad2/3-dependent signalling pathway is well known for its implication in the patterning of the embryo after implantation. Although this pathway is active early on at preimplantation stages, embryonic phenotypes for loss-of-function mutations of prominent components of the pathway are not detected before implantation. It is only fairly recently that an understanding of the role of the Activin/Nodal signalling pathway at these stages has started to emerge, notably from studies detailing how it controls the expression of target genes in embryonic stem cells. We review here what is currently known of the TGF-β-related ligands that determine the activity of Activin/Nodal signalling at preimplantation stages, and recent advances in the elucidation of the Smad2/3-dependent mechanisms underlying developmental progression.
226488
Activin/Nodal signalling in stem cells
Activin/Nodal growth factors control a broad range of biological processes, including early cell fate decisions, organogenesis and adult tissue homeostasis. Here, we provide an overview of the mechanisms by which the Activin/Nodal signalling pathway governs stem cell function in these different stages of development. We describe recent findings that associate Activin/Nodal signalling to pathological conditions, focusing on cancer stem cells in tumorigenesis and its potential as a target for therapies. Moreover, we will discuss future directions and questions that currently remain unanswered on the role of Activin/Nodal signalling in stem cell self-renewal, differentiation and proliferation. Summary: This Review discusses how Activin/Nodal signalling controls the self-renewal and differentiation of stem cells during organ development and homeostasis.
205989382
Nodal signalling in embryogenesis and tumourigenesis.
With few exceptions, most cells in adult organisms have lost the expression of stem cell-associated proteins and are instead characterized by tissue-specific gene expression and function. This cell fate specification is dictated spatially and temporally during embryogenesis. It has become increasingly apparent that the elegant and complicated process of cell specification is "undone" in cancer. This may be because cancer cells respond to their microenvironment and mutations by acquiring a more permissive, plastic epigenome, or because cancer cells arise from mutated stem cells. Regardless, these advanced cancer cells must use stem cell-associated proteins to sustain their phenotype. One such protein is Nodal, an embryonic morphogen belonging to the transforming growth factor-β (TGF-β) superfamily. First described in early developmental models, Nodal orchestrates embryogenesis by regulating a myriad of processes, including mesendoderm induction, left-right asymmetry and embryo implantation. Nodal is relatively restricted to embryonic and reproductive cell types and is thus absent from most normal adult tissues. However, recent studies focusing on a variety of malignancies have demonstrated that Nodal expression re-emerges during cancer progression. Moreover, in almost every cancer studied thus far, the acquisition of Nodal expression is associated with increased tumourigenesis, invasion and metastasis. As the list of cancers that express Nodal grows, it is essential that the scientific and medical communities fully understand how this morphogen is regulated in both normal and neoplastic conditions. Herein, we review the literature relating to normal and pathological Nodal signalling. In particular, we emphasize the role that this secreted protein plays during morphogenic events and how it signals to support stem cell maintenance and tumour progression.
7330734
Dose-dependent Nodal/Smad signals pattern the early mouse embryo.
Nodal signals in the early post-implantation stage embryo are essential to establish initial proximal-distal (P-D) polarity and generate the final anterior-posterior (A-P) body axis. Nodal signaling in the epiblast results in the phosphorylation of Smad2 in the overlying visceral endoderm necessary to induce the AVE, in part via Smad2-dependent activation of the T-box gene Eomesodermin. Slightly later following mesoderm induction a continuum of dose-dependent Nodal signaling during the process of gastrulation underlies specification of mesodermal and definitive endoderm progenitors. Dynamic Nodal expression during the critical 72 h time window immediately following implantation, accomplished by a series of feed-back and feed-forward mechanisms serves to provide key positional cues required for establishment of the body plan and controls cell fate decisions in the early mammalian embryo.
24608054
Nodal signalling and asymmetry of the nervous system
The role of Nodal signalling in nervous system asymmetry is still poorly understood. Here, we review and discuss how asymmetric Nodal signalling controls the ontogeny of nervous system asymmetry using a comparative developmental perspective. A detailed analysis of asymmetry in ascidians and fishes reveals a critical context-dependency of Nodal function and emphasizes that bilaterally paired and midline-unpaired structures/organs behave as different entities. We propose a conceptual framework to dissect the developmental function of Nodal as asymmetry inducer and laterality modulator in the nervous system, which can be used to study other types of body and visceral organ asymmetries. Using insights from developmental biology, we also present novel evolutionary hypotheses on how Nodal led the evolution of directional asymmetry in the brain, with a particular focus on the epithalamus. We intend this paper to provide a synthesis on how Nodal signalling controls left–right asymmetry of the nervous system. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’.
6002456
Nodal expression and detection in cancer: experience and challenges.
Nodal is a TGF-β-related embryonic morphogen that is expressed in multiple human cancers. Detection of Nodal expression in these tissues can be challenging if issues related to Nodal transcription and protein processing are not considered. Here, we discuss certain characteristics related to Nodal expression and function and how these can facilitate acquisition and interpretation of expression data, contributing to our understanding of the potential role of Nodal in human cancer. We also discuss how Nodal could be exploited clinically as a novel biomarker for cancer progression and therapeutic target.
6940991
Nodal signals pattern vertebrate embryos
Abstract.Vertebrate embryonic patterning requires several conserved inductive signals–including Nodal, Bmp, Wnt and Fgf signals. Nodal, which is a member of the transforming growth factor β (TGFβ) superfamily, activates a signal transduction pathway that is similar to that of other TGFβ members. Nodal genes, which have been identified in numerous vertebrate species, are expressed in specific cell types and tissues during embryonic development. Nodal signal transduction has been shown to play a pivotal role in inducing and patterning mesoderm and endoderm, and in regulating neurogenesis and left-right axis asymmetry. Antagonists, which act at different steps in the Nodal signal transduction pathway, have been shown to tightly modulate the inductive activity of Nodal.
34478095
Nodal signalling and apoptosis.
Nodal, a member of the transforming growth factor beta family, was first cloned from a 7.5 day post-coitum mouse embryo cDNA library. Nodal exerts its biological effects by signalling through its types I and II serine/threonine kinase receptor complex and intracellular Smad proteins. The type II receptors for Nodal are Activin type II receptors ActRIIA and ActRIIB, whereas the putative type I receptors are Activin receptor like kinase (ALK) 4 and ALK7. The main Smad proteins involved in Nodal signalling are Smad2 and Smad3. Studies of Nodal in adult tissues indicate that it is pro-apoptotic in rat ovarian granulosa cells, human trophoblast cells and human ovarian epithelial cancer cells and is growth inhibitory in the latter two cell types. This review summarises the progress made on the functions of Nodal in the apoptosis of adult tissues, especially in the ovary and placenta.
6909277
Response to Nodal morphogen gradient is determined by the kinetics of target gene induction
Morphogen gradients expose cells to different signal concentrations and induce target genes with different ranges of expression. To determine how the Nodal morphogen gradient induces distinct gene expression patterns during zebrafish embryogenesis, we measured the activation dynamics of the signal transducer Smad2 and the expression kinetics of long- and short-range target genes. We found that threshold models based on ligand concentration are insufficient to predict the response of target genes. Instead, morphogen interpretation is shaped by the kinetics of target gene induction: the higher the rate of transcription and the earlier the onset of induction, the greater the spatial range of expression. Thus, the timing and magnitude of target gene expression can be used to modulate the range of expression and diversify the response to morphogen gradients. DOI: http://dx.doi.org/10.7554/eLife.05042.001
9040823
The EGF-CFC Protein One-Eyed Pinhead Is Essential for Nodal Signaling
The zebrafish EGF-CFC gene one-eyed pinhead (oep) is required zygotically for the formation of the ventral neuroectoderm, endoderm, and prechordal plate. Here we report that embryos lacking both maternal and zygotic Oep activity are defective in germ layer formation, organizer development, and the positioning of the anterior-posterior axis. An identical phenotype is displayed by double mutants for the nodal-related genes squint and cyclops. Mutations in oep eliminate the response to Squint and Cyclops overexpression but are suppressed by expression of Activin and activated forms of the type I receptor ActRIB and Smad2. Expression of the murine EGF-CFC gene cripto rescues oep mutants. These results suggest a conserved role for EGF-CFC proteins as essential extracellular cofactors for Nodal signaling during vertebrate development.
18573004
Regulation of midline development by antagonism of lefty and nodal signaling.
The embryonic midline is crucial for the development of embryonic pattern including bilateral symmetry and left-right asymmetry. In zebrafish, lefty1 (lft1) and lefty2 (lft2) have distinct midline expression domains along the anteroposterior axis that overlap with the expression patterns of the nodal-related genes cyclops and squint. Altered expression patterns of lft1 and lft2 in zebrafish mutants that affect midline development suggests different upstream pathways regulate each expression domain. Ectopic expression analysis demonstrates that a balance of lefty and cyclops signaling is required for normal mesendoderm patterning and goosecoid, no tail and pitx2 expression. In late somite-stage embryos, lft1 and lft2 are expressed asymmetrically in the left diencephalon and left lateral plate respectively, suggesting an additional role in laterality development. A model is proposed by which the vertebrate midline, and thus bilateral symmetry, is established and maintained by antagonistic interactions among co-expressed members of the lefty and nodal subfamilies of TGF-beta signaling molecules.
15628968
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.
4346617
Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling
Zebrafish cyclops (cyc) mutations cause deficiencies in the dorsal mesendoderm, and ventral neural tube,, leading to neural defects and cyclopia,. Here we report that cyc encodes a transforming growth factor-β (TGF-β)-related intercellular signalling molecule that is similar to mouse nodal. cyc is expressed in dorsal mesendoderm at gastrulation and in the prechordal plate until early somitogenesis. Expression reappears transiently in the left lateral-plate mesoderm, and in an unprecedented asymmetric pattern in the left forebrain. Injection of cyc RNA non-autonomously restores sonic hedgehog -expressing cells of the ventral brain and floorplate that are absent in cyc mutants, whereas inducing activities are abolished by cycm294, a mutation of a conserved cysteine in the mature ligand. Our results indicate that cyc provides an essential non-cell-autonomous signal at gastrulation, leading to induction of the floorplate and ventral brain.
15525247
Protein tyrosine phosphatase hPTPN20a is targeted to sites of actin polymerization.
The human genome encodes 38 classical tyrosine-specific PTPs (protein tyrosine phosphatases). Many PTPs have been shown to regulate fundamental cellular processes and several are mutated in human diseases. We report that the product of the PTPN20 gene at the chromosome locus 10q11.2 is alternatively spliced to generate 16 possible variants of the classical human non-transmembrane PTP 20 (hPTPN20). One of these variants, hPTPN20a, was expressed in a wide range of both normal and transformed cell lines. The catalytic domain of hPTPN20 exhibited catalytic activity towards tyrosyl phosphorylated substrates, confirming that it is a bona fide PTP. In serum-starved COS1 cells, hPTPN20a was targeted to the nucleus and the microtubule network, colocalizing with the microtubule-organizing centre and intracellular membrane compartments, including the endoplasmic reticulum and the Golgi apparatus. Stimulation of cells with epidermal growth factor, osmotic shock, pervanadate, or integrin ligation targeted hPTPN20a to actin-rich structures that included membrane ruffles. The present study identifies hPTPN20a as a novel and widely expressed phosphatase with a dynamic subcellular distribution that is targeted to sites of actin polymerization.
26902459
Novel subcellular locations and functions for secretory pathway Ca2+/Mn2+-ATPases.
Secretory pathway Ca2+/Mn2+-ATPases (SPCAs) are important for maintenance of cellular Ca2+ and Mn2+ homeostasis, and, to date, all SPCAs have been found to localize to the Golgi apparatus. The single Drosophila SPCA gene (SPoCk) was identified by an in silico screen for novel Ca2+-ATPases. It encoded three SPoCk isoforms with novel, distinct subcellular specificities in the endoplasmic reticulum (ER) and peroxisomes in addition to the Golgi. Furthermore, expression of the peroxisome-associated SPoCk isoform was sexually dimorphic. Overexpression of organelle-specific SPoCk isoforms impacted on cytosolic Ca2+ handling in both cultured Drosophila cells and a transporting epithelium, the Drosophila Malpighian (renal) tubule. Specifically, the ER isoform impacted on inositol-trisphosphate-mediated Ca2+ signaling and the Golgi isoform impacted on diuresis, whereas the peroxisome isoform colocalized with Ca2+ "spherites" and impacted on calcium storage and transport. Interfering RNA directed against the common exons of the three SPoCk isoforms resulted in aberrant Ca2+ signaling and abolished neuropeptide-stimulated diuresis by the tubule. SPoCk thus contributed to both of the contrasting requirements for Ca2+ in transporting epithelia: to transport or store Ca2+ in bulk without compromising its use as a signal.
37449749
COPII and COPI traffic at the ER-Golgi interface.
Protein traffic is necessary to maintain homeostasis in all eukaryotic organisms. All newly synthesized secretory proteins destined to the secretory and endolysosmal systems are transported from the endoplasmic reticulum to the Golgi before delivery to their final destinations. Here, we describe the COPII and COPI coating machineries that generate carrier vesicles and the tethers and SNAREs that mediate COPII and COPI vesicle fusion at the ER-Golgi interface.
8561524
Specific heterodimer formation is a prerequisite for uroplakins to exit from the endoplasmic reticulum.
Much of the lower urinary tract, including the bladder, is lined by a stratified urothelium forming a highly differentiated, superficial umbrella cell layer. The apical plasma membrane as well as abundant cytoplasmic fusiform vesicles of the umbrella cells is covered by two-dimensional crystals that are formed by four membrane proteins named uroplakins (UPs) Ia, Ib, II, and III. UPs are synthesized on membrane-bound polysomes, and after several co- and posttranslational modifications they assemble into planar crystals in a post-Golgi vesicular compartment. Distension of the bladder may cause fusiform vesicles to fuse with the apical plasma membrane. We have investigated the early stages of uroplakin assembly by expressing the four uroplakins in 293T cells. Transfection experiments showed that, when expressed individually, only UPIb can exit from the endoplasmic reticulum (ER) and move to the plasma membrane, whereas UPII and UPIII reach the plasma membrane only when they form heterodimeric complexes with UPIa and UPIb, respectively. Heterodimer formation in the ER was confirmed by pulse-chase experiment followed by coimmunoprecipitation. Our results indicate that the initial building blocks for the assembly of crystalline uroplakin plaques are heterodimeric uroplakin complexes that form in the ER.
17832635
pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein.
Green florescent protein (GFP) variants that are sensitive to changes in pH are invaluable reagents for the analysis of protein dynamics associated with both endo- and exocytotic vesicular trafficking. Ratiometric pHluorin is a GFP variant that displays a bimodal excitation spectrum with peaks at 395 and 475 nm and an emission maximum at 509 nm. Upon acidification, pHluorin excitation at 395 nm decreases with a corresponding increase in the excitation at 475 nm. GFP2, a GFP variant that contains mammalianized codons and the folding enhancing mutation F64L, displays ~8-fold higher florescence compared to pHluorin upon excitation at 395 nm. Using GFP2 as a template, an enhanced ratiometric pHluorin (pHluorin2) construct was developed to contain fully mammalianized codons, the F64L mutation and ten of the thirteen pHluorin-specific mutations. As a result, pHluorin2 displays markedly higher florescence when compared to pHluorin while maintaining the ratiometric pH-sensitivity. Unlike native pHluorin, pHluorin2 expressed in the ligand-binding domain of the parathyroid hormone 1 receptor is readily detectable by confocal microscopy and displays a marked increase in florescence upon ligand-induced endocytosis to intracellular vesicles. Thus, pHluorin2's enhanced florescence while sustaining ratiometric pH-sensitivity represents a significant improvement for this methodological approach.
12967200
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.
36573578
The signaling and functions of heterodimeric bone morphogenetic proteins.
Heterodimeric bone morphogenetic proteins (BMPs) consist of disulfide-linked dimeric monomers derived from different BMP members. Owing to this specific constitution pattern, they bear high affinity to both type I and type II BMP receptors simultaneously. Meanwhile, the antagonism efficiency of extracellular antagonists to heterodimeric BMPs is also significantly lower than that to homodimeric ones. All these specific properties confer heterodimeric BMPs with distinct signaling and bio-functions that are characterized by more speediness, lower concentration/dose threshold and higher efficiency than homodimeric BMPs. Consequently, heterodimeric BMPs bear promising application potential in inducing osteogenesis. In addition, they may play indispensible roles in organogenesis. In this review, we summarize the current knowledge of heterodimeric BMPs in their signaling pathways and bio-functions.
6738355
Efficient expression of a heterodimer of bone morphogenetic protein subunits using a baculovirus expression system.
Recombinant baculoviruses as expression vectors for Xenopus bone morphogenetic protein (xBMP)-2, 4 and 7 were generated. The conditioned medium of insect cells infected with the virus for xBMP-2 or 4 showed strong alkaline phosphatase-inducing activity in a mouse osteoblastic cell line, MC3T3-E1, although a large portion of the activity remained in the infected cells. In contrast, xBMP-7 was preferentially secreted into the medium, but had only weak activity. Conditioned media following simultaneous inoculation with the viruses for xBMP-7 and for xBMP-2 or 4 showed a remarkably increased level of activity. The increased activity was clearly separated from other peaks derived from single infection on a cation-exchange column and was found to arise from the disulfide-linked heterodimer consisting of xBMP-4 and 7 subunits by immunoblot analysis. The heterodimer also augmented osteocalcin production and parathyroid hormone-sensitivity more strongly than the homodimers. These results suggest that our expression system provides a convenient source of heterodimeric BMP with high osteogenic differentiation-inducing activity.
13159229
Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo.
The bone morphogenetic proteins (BMPs), a subgroup of the TGF-beta gene super-family, are dimeric molecules involved in the growth, differentiation and repair of a wide variety of tissues. Based on the observation that several of the BMPs co-purify when isolated from bovine bone and that a pattern of co-localization exists during mouse embryogenesis, we co-expressed various combinations of BMPs in Chinese hamster ovary cells to test for possible heterodimer formation and activity. Transient co-expression of BMP-2 with either BMP-5, BMP-6 or BMP-7, or BMP-4 transiently co-expressed with BMP-7, resulted in more BMP activity than expression of any single BMP. Stable cell lines were then made in order to purify and characterize co-expressed BMPs in more detail. Co-expression of BMP-2 with BMP-7 yielded heterodimeric BMP-2/7 with a specific activity about 20-fold higher than BMP homodimers in an in vitro alkaline phosphatase induction assay. These heterodimers were also 5- to 10-fold more potent than BMP-2 in inducing cartilage and bone in an in vivo assay. Similar results were obtained with BMP-2/6 heterodimer. These experiments demonstrate the increased potency of several BMP heterodimers relative to BMP homodimers and support the hypothesis that such heterodimeric forms are likely to have natural biological functions.
10604576
BMP heterodimers assemble hetero-type I receptor complexes that pattern the DV axis
Patterning the embryonic dorsoventral axis of both vertebrates and invertebrates requires signalling through bone morphogenetic proteins (BMPs). Although a well-studied process, the identity of the physiologically relevant BMP signalling complex in the Drosophila melanogaster embryo is controversial, is generally inferred from cell culture studies and has not been investigated in vertebrates. Here, we demonstrate that dorsoventral patterning in zebrafish, Danio rerio, requires two classes of non-redundant type I BMP receptors, Alk3/6 and Alk8 (activin-like kinases 3/6 and 8). We show, under physiological conditions in the embryo, that these two type I receptor classes form a complex in a manner that depends on Bmp2 and Bmp7. We found that both Bmp2–7 heterodimers, as well as Bmp2 and Bmp7 homodimers, form in the embryo. However, only recombinant ligand heterodimers can activate BMP signalling in the early embryo, whereas a combination of Bmp2 and Bmp7 homodimers cannot. We propose that only heterodimers, signalling through two distinct classes of type I receptor, possess sufficient receptor affinity in an environment of extracellular antagonists to elicit the signalling response required for dorsoventral patterning.
17113093
Ventral mesoderm induction and patterning by bone morphogenetic protein heterodimers in Xenopus embryos
Bone morphogenetic proteins (BMPs) perform diverse functions in vertebrate development. Here we demonstrate that the heterodimeric BMP-4/7 protein directly induces ventral mesoderm and blood in Xenopus animal caps, and BMP-2/7 heterodimers may function similarly. We also provide indirect evidence that BMP heterodimers function in embryos, using assays with dominant-negative BMP ligands. Homodimeric BMP-2 and BMP-4 proteins do not induce mesoderm, but they ventralize mesoderm induction by activin. In contrast, BMP-7 protein interferes with mesoderm induction by activin, but BMP-7 stimulates ventral mesoderm induction by the heterodimer, BMP-4/7. This novel property of BMP-7 distinguishes it from other BMPs. BMP-7 may therefore function in early embryogenesis to antagonize activin signals and potentiate BMP signals. We propose that BMP heterodimers convey signals for ventral mesoderm induction and patterning in Xenopus development.
20743716
Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in dorsoventral pattern formation.
A bone morphogenetic protein (BMP) signaling pathway acts in the establishment of the dorsoventral axis of the vertebrate embryo. Here we demonstrate the genetic requirement for two different Bmp ligand subclass genes for dorsoventral pattern formation of the zebrafish embryo. From the relative efficiencies observed in Bmp ligand rescue experiments, conserved chromosomal synteny, and isolation of the zebrafish bmp7 gene, we determined that the strongly dorsalized snailhouse mutant phenotype is caused by a mutation in the bmp7 gene. We show that the original snailhouse allele is a hypomorphic mutation and we identify a snailhouse/bmp7 null mutant. We demonstrate that the snailhouse/bmp7 null mutant phenotype is identical to the presumptive null mutant phenotype of the strongest dorsalized zebrafish mutant swirl/bmp2b, revealing equivalent genetic roles for these two Bmp ligands. Double mutant snailhouse/bmp7; swirl/bmp2b embryos do not exhibit additional or stronger dorsalized phenotypes, indicating that these Bmp ligands do not function redundantly in early embryonic development. Furthermore, overexpression experiments reveal that Bmp2b and Bmp7 synergize in the ventralization of wild-type embryos through a cell-autonomous mechanism, suggesting that Bmp2b/Bmp7 heterodimers may act in vivo to specify ventral cell fates in the zebrafish embryo.
83703605
Facilitated Transport of a Dpp/Scw Heterodimer by Sog/Tsg Leads to Robust Patterning of the Drosophila Blastoderm Embryo
Patterning the dorsal surface of the Drosophila blastoderm embryo requires Decapentaplegic (Dpp) and Screw (Scw), two BMP family members. Signaling by these ligands is regulated at the extracellular level by the BMP binding proteins Sog and Tsg. We demonstrate that Tsg and Sog play essential roles in transporting Dpp to the dorsal-most cells. Furthermore, we provide biochemical and genetic evidence that a heterodimer of Dpp and Scw, but not the Dpp homodimer, is the primary transported ligand and that the heterodimer signals synergistically through the two type I BMP receptors Tkv and Sax. We propose that the use of broadly distributed Dpp homodimers and spatially restricted Dpp/Scw heterodimers produces the biphasic signal that is responsible for specifying the two dorsal tissue types. Finally, we demonstrate mathematically that heterodimer levels can be less sensitive to changes in gene dosage than homodimers, thereby providing further selective advantage for using heterodimers as morphogens.
42108140
Mesoderm induction by BMP-4 and -7 heterodimers.
Bone morphogenetic proteins (BMPs) are peptide growth factors belonging to the TGF-beta superfamily. A large number of these ligands, including BMP-2, -4 and -7 is expressed during early embryogenesis in the vertebrate embryo. In this study, we demonstrate that BMP-7 has ventralizing activity both in ectodermal explants as well as in whole embryos. As it was the case for BMP-2 and BMP-4, BMP-7 is a very poor inducer when provided as a homodimer protein. Because of this weak mesoderm inducing activity, it has been suggested that mesoderm induction by BMPs might represent an artifact of overexpression. We provide evidence demonstrating that unlike the homodimers of BMP-4 or BMP-7, the purified recombinant heterodimer of Xenopus BMP-4 and BMP-7 (BMP-4/7) has a potent mesoderm inducing activity at physiological concentrations. These results provide the first evidence for an embryonic function of BMP-4/7 heterodimers in the vertebrate embryo.
42339292
derrière: a TGF-beta family member required for posterior development in Xenopus.
TGF-beta signaling plays a key role in induction of the Xenopus mesoderm and endoderm. Using a yeast-based selection scheme, we isolated derrière, a novel TGF-beta family member that is closely related to Vg1 and that is required for normal mesodermal patterning, particularly in posterior regions of the embryo. Unlike Vg1, derrière is expressed zygotically, with RNA localized to the future endoderm and mesoderm by late blastula, and to the posterior mesoderm by mid-gastrula. The derrière expression pattern appears to be identical to the zygotic expression domain of VegT (Xombi, Brat, Antipodean), and can be activated by VegT as well as fibroblast growth factor (FGF). In turn, derrière activates expression of itself, VegT and eFGF, suggesting that a regulatory loop exists between these genes. derrière is a potent mesoderm and endoderm inducer, acting in a dose-dependent fashion. When misexpressed ventrally, derrière induces a secondary axis lacking a head, an effect that is due to dorsalization of the ventral marginal zone. When misexpressed dorsally, derrière suppresses head formation. derrière can also posteriorize neurectoderm, but appears to do so indirectly. Together, these data suggest that derrière expression is compatible only with posterior fates. In order to assess the in vivo function of derrière, we constructed a dominant interfering Derrière protein (Cm-Derrière), which preferentially blocks Derrière activity relative to that of other TGFbeta family members. Cm-derrière expression in embryos leads to posterior truncation, including defects in blastopore lip formation, gastrulation and neural tube closure. Normal expression of anterior and hindbrain markers is observed; however, paraxial mesodermal gene expression is ablated. This phenotype can be rescued by wild-type derrière and by VegT. Our findings indicate that derrière plays a crucial role in mesodermal patterning and development of posterior regions in Xenopus.
23672160
Endodermal Nodal-related signals and mesoderm induction in Xenopus.
In Xenopus, mesoderm induction by endoderm at the blastula stage is well documented, but the molecular nature of the endogenous inductive signals remains unknown. The carboxy-terminal fragment of Cerberus, designated Cer-S, provides a specific secreted antagonist of mesoderm-inducing Xenopus Nodal-Related (Xnr) factors. Cer-S does not inhibit signalling by other mesoderm inducers such as Activin, Derrière, Vg1 and BMP4, nor by the neural inducer Xnr3. In the present study we show that Cer-S blocks the induction of both dorsal and ventral mesoderm in animal-vegetal Nieuwkoop-type recombinants. During blastula stages Xnr1, Xnr2 and Xnr4 are expressed in a dorsal to ventral gradient in endodermal cells. Dose-response experiments using cer-S mRNA injections support the existence of an endogenous activity gradient of Xnrs. Xnr expression at blastula can be activated by the vegetal determinants VegT and Vg1 acting in synergy with dorsal (beta)-catenin. The data support a modified model for mesoderm induction in Xenopus, in which mesoderm induction is mediated by a gradient of multiple Nodal-related signals released by endoderm at the blastula stage.
35077229
Maternal Oct1/2 is required for Nodal and Vg1/Univin expression during dorsal-ventral axis specification in the sea urchin embryo.
The TGFβ family member Nodal is expressed early in the presumptive ventral ectoderm of the early sea urchin embryo and its activity is crucial for dorsal-ventral (D/V) axis specification. Analysis of the nodal promoter identified a number of critical binding sites for transcription factors of different families including Sox, Oct, TCF and bZIP, but in most cases the specific factors that regulate nodal expression are not known. In this study, we report that the maternal factor Oct1/2 functions as a positive regulator of nodal and that its activity is essential for the initiation of nodal expression. Inhibition of Oct1/2 mRNA translation produced embryos with severe axial defects similar to those observed following inhibition of Nodal function. We show that perturbing Oct1/2 function specifically disrupted specification of the ventral and dorsal ectodermal regions and that these effects were caused by the failure of nodal to be expressed early in development. Furthermore, we identified the key gene vg1/univin, which is also necessary for nodal expression, as an additional factor that was completely dependent on Oct1/2 for its zygotic expression. These data demonstrate that the maternal Oct1/2 protein plays an early and essential role in D/V axis specification by initiating the expression of nodal and vg1/univin, two genes that act at the top of the D/V ectoderm gene regulatory network.
41393274
Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by Univin, a TGF-β related to Vg1
The TGF-β family member Nodal is essential for specification of the dorsal-ventral axis of the sea urchin embryo, but the molecular factors regulating its expression are not known. Analysis of the nodal promoter is an excellent entry point to identify these factors and to dissect the regulatory logic driving dorsal-ventral axis specification. Using phylogenetic footprinting, we delineated two regulatory regions located in the 5′ region of the nodal promoter and in the intron that are required for correct spatial expression and for autoregulation. The 5′ regulatory region contains essential binding sites for homeodomain, bZIP, Oct, Tcf/Lef, Sox and Smad transcription factors, and a binding site for an unidentified spatial repressor possibly related to Myb. Soon after its initiation, nodal expression critically requires autoregulation by Nodal and signaling by the maternal TGF-β Univin. We show that Univin is related to Vg1, that both Nodal and Univin signal through Alk4/5/7, and that zygotic expression of univin, like that of nodal, is dependent on SoxB1 function and Tcf/β-catenin signaling. This work shows that Tcf, SoxB1 and Univin play essential roles in the regulation of nodal expression in the sea urchin and suggests that some of the regulatory interactions controlling nodal expression predate the chordates. The data are consistent with a model of nodal regulation in which a maternal TGF-β acts in synergy with maternal transcription factors and with spatial repressors to establish the dorsal-ventral axis of the sea urchin embryo.
16324413
Early developmental expression and experimental axis determination by the chicken Vg1 gene
BACKGROUND
Genes of the transforming growth factor beta (TGF beta) superfamily have been implicated in the earliest steps of developmental patterning in vertebrates. In Xenopus, the Vg1 gene is a candidate for the initiator of axis formation: its RNA and protein are broadly but appropriately localized at the start of development, and processed Vg1 protein is a powerful inducer of organized axial tissue in blastular animal caps in vitro and when locally produced in vivo after injection of Vg1 mRNA into blastomeres. Site-specific proteolytic processing occurs ubiquitously for most TGF beta members, producing the active peptide ligand, but is tightly restricted, by unknown mechanisms, for endogenous Vg protein in Xenopus and zebrafish embryos. This restriction may be involved in the spatial localization of activity required for an organizing role.


RESULTS
We have characterized an amniote (chick) orthologue of Vg1, cVg1, and examined its developmental expression. The early expression of cVg1 includes a phase broadly related to the known time and site of axis (primitive streak) initiation; the initial transcription of cVg1 is centred in the posterior marginal zone (PMZ), a region of the blastoderm known to contain the axial organizing activity at this stage. We also observed later neural and paraxial mesodermal expression of cVg1, which has not been described previously for Vg homologues in other vertebrates. We have grafted transfected COS cells, producing processed cVg1 protein, to peripheral positions around the chick early blastoderm. Such grafts initiate formation of morphologically complete primitive streaks, simulating the properties of grafts from the PMZ.


CONCLUSIONS
In vertebrate development, Vg genes may be required for an evolutionarily conserved early step in positioning or induction of the axis.
27067375
EGF-CFC proteins are essential coreceptors for the TGF-beta signals Vg1 and GDF1.
The TGF-beta signals Nodal, Activin, GDF1, and Vg1 have been implicated in mesoderm induction and left-right patterning. Nodal and Activin both activate Activin receptors, but only Nodal requires EGF-CFC coreceptors for signaling. We report that Vg1 and GDF1 signaling in zebrafish also depends on EGF-CFC proteins, but not on Nodal signals. Correspondingly, we find that in Xenopus Vg1 and GDF1 bind to and signal through Activin receptors only in the presence of EGF-CFC proteins. These results establish that multiple TGF-beta signals converge on Activin receptor/EGF-CFC complexes and suggest a more widespread requirement for coreceptors in TGF-beta signaling than anticipated previously.
18620337
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.
9431219
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.
17534423
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.
4431903
Activin- and Nodal-related factors control antero–posterior patterning of the zebrafish embryo
Definition of cell fates along the dorso–ventral axis depends on an antagonistic relationship between ventralizing transforming growth factor-β superfamily members, the bone morphogenetic proteins and factors secreted from the dorsal organizer, such as Noggin and Chordin. The extracellular binding of the last group to the bone morphogenetic proteins prevents them from activating their receptors, and the relative ventralizer:antagonist ratio is thought to specify different dorso–ventral cell fates. Here, by taking advantage of a non-genetic interference method using a specific competitive inhibitor, the Lefty-related gene product Antivin, we provide evidence that cell fate along the antero–posterior axis of the zebrafish embryo is controlled by the morphogenetic activity of another transforming growth factor-β superfamily subgroup—the Activin and Nodal-related factors. Increasing antivin doses progressively deleted posterior fates within the ectoderm, eventually resulting in the removal of all fates except forebrain and eyes. In contrast, overexpression of activin or nodal-related factors converted ectoderm that was fated to be forebrain into more posterior ectodermal or mesendodermal fates. We propose that modulation of intercellular signalling by Antivin/Activin and Nodal-related factors provides a mechanism for the graded establishment of cell fates along the antero–posterior axis of the zebrafish embryo.
30360070
Antivin, a novel and divergent member of the TGFbeta superfamily, negatively regulates mesoderm induction.
Mesoderm induction and patterning are mediated by members of the TGFbeta superfamily. We have isolated a novel zebrafish member, antivin, that structurally is highly related to mouse lefty. Overexpression of antivin completely abolishes mesoderm induction at blastula stage, yet resultant embryos develop well-patterned epidermal and neural derivatives. The mesoderm-inhibiting activity of antivin can be mimicked by lefty and is suppressed by increasing levels of the mesodermal inducer Activin or its receptors. On the basis of its expression and activity, we propose that Antivin normally functions as a competitive inhibitor of Activin to limit mesoderm induction in the early embryo.
24504592
How a Cytokine Is Chaperoned through the Secretory Pathway by Complexing with Its Own Receptor: Lessons from Interleukin-15 (IL-15)/IL-15 Receptor α
ABSTRACT While it is well appreciated that receptors for secreted cytokines transmit ligand-induced signals, little is known about additional roles for cytokine receptor components in the control of ligand transport and secretion. Here, we show that interleukin-15 (IL-15) translocation into the endoplasmic reticulum occurs independently of the presence of IL-15 receptor α (IL-15Rα). Subsequently, however, IL-15 is transported through the Golgi apparatus only in association with IL-15Rα and then is secreted. This intracellular IL-15/IL-15Rα complex already is formed in the endoplasmic reticulum and, thus, enables the further trafficking of complexed IL-15 through the secretory pathway. Just transfecting IL-15Rα in cells, which transcribe but normally do not secrete IL-15, suffices to induce IL-15 secretion. Thus, we provide the first evidence of how a cytokine is chaperoned through the secretory pathway by complexing with its own high-affinity receptor and show that IL-15/IL-15Rα offers an excellent model system for the further exploration of this novel mechanism for the control of cytokine secretion.
21613749
Folding and Dimerization of Tick-Borne Encephalitis Virus Envelope Proteins prM and E in the Endoplasmic Reticulum
ABSTRACT Flavivirus envelope proteins are synthesized as part of large polyproteins that are co- and posttranslationally cleaved into their individual chains. To investigate whether the interaction of neighboring proteins within the precursor protein is required to ensure proper maturation of the individual components, we have analyzed the folding of the flavivirus tick-borne encephalitis (TBE) virus envelope glycoproteins prM and E by using a recombinant plasmid expression system and virus-infected cells. When expressed in their polyprotein context, prM and E achieved their native folded structures with half-times of approximately 4 min for prM and about 15 min for E. They formed heterodimeric complexes within a few minutes after synthesis that were required for the final folding of E but not for that of prM. Heterodimers could also be formed in trans when these proteins were coexpressed from separate constructs. When expressed without prM, E could form disulfide bonds but did not express a specific conformational epitope and remained sensitive to reduction by dithiothreitol. This is consistent with a chaperone-like role for prM in the folding of E. PrM was able to achieve its native folded structure without coexpression of E, but signal sequence cleavage at the N terminus was delayed. Our results show that prM is an especially rapidly folding viral glycoprotein, that polyprotein cleavage and folding of the TBE virus envelope proteins occurs in a coordinated sequence of processing steps, and that proper and efficient maturation of prM and E can only be achieved by cosynthesis of these two proteins.
14762687
Formation and intracellular transport of a heterodimeric viral spike protein complex
We have analyzed the heterodimerization and intracellular transport from the ER to the Golgi complex (GC) of two membrane glycoproteins of a bunyavirus (Uukuniemi virus) that matures by a budding process in the GC. The glycoproteins G1 and G2, which form the viral spikes, are cotranslationally cleaved in the ER from a 110,000-D precursor. Newly synthesized G1 was transported to the GC and incorporated into virus particles about 30-45 min faster than newly synthesized G2. Analysis of the kinetics of intrachain disulfide bond formation showed that G1 acquired its mature form within 10 min, while completion of disulfide bond formation of G2 required a considerably longer time (up to 60 min). During the maturation process, G2 was transiently associated with the IgG heavy chain binding protein for a longer time than G1. Protein disulfide isomerase also coprecipitated with antibodies against G1 and G2. In virus particles, G1 and G2 were present exclusively as heterodimers. Immunoprecipitation with monoclonal antibodies showed that heterodimerization occurred rapidly, probably in the ER, between newly made G1 and mature, dimerization competent G2. Taken together, our results show that these two viral glycoproteins have different maturation kinetics in the ER. We conclude that the apparent different kinetics of ER to GC transport of G1 and G2 is due to the different rates by which these proteins fold and become competent to enter into heterodimeric complexes prior to exit from the ER.
25434224
Expression of activin mRNA during early development in Xenopus laevis.
Activins are members of the transforming growth factor-beta superfamily, a class of peptide growth factors that can regulate the growth and differentiation of a variety of cell types. In mesoderm induction assays, activins A and B were shown to be very potent inducers and it was only recently demonstrated that they are crucial for initial mesoderm induction in Xenopus embryos. To determine the source of activin protein for initial mesoderm induction and to investigate whether activins may play further roles in embryonic development we have examined the localization of the mRNAs encoding the activin beta A and beta B subunits in Xenopus embryos. Activin beta A and beta B mRNAs are found in the follicle cells surrounding oocytes but not in oocytes themselves or fertilized eggs. During embryogenesis activin mRNA is first detected after the midblastula transition and expression increases as development proceeds. Activin beta B mRNA is homogeneously distributed during blastula and early gastrula stages but restricted to the dorso-anterior region in neurula stage embryos. At the early tailbud stage activin expression becomes confined to the brain, eye analgen, visceral pouches, otic vesicles, and the anterior notochord.
35068018
Activins are expressed early in Xenopus embryogenesis and can induce axial mesoderm and anterior structures
We show that mammalian and Xenopus activins induce dorsal axial mesoderm and anterior structures in explants of Xenopus blastula cells that would otherwise form epidermis. The induced explants of animal cap cells can form notochord, muscle, neural tissue, and eyes all arranged in a rudimentary axial pattern. Activin A shares inductive properties and antigenic determinants with PIF, an inducing factor recently isolated from mouse macrophage culture supernatants. Genes encoding Xenopus activin beta A and beta B chains were cloned. Activin beta B transcripts are first detected in Xenopus blastula, whereas activin beta A transcripts do not appear until the late gastrula stage. Recombinant Xenopus activin beta B protein induces mesodermal and neural tissues similar to those induced by mammalian activin A and PIF. Furthermore, ectopic expression of Xenopus activin beta B produces a second body axis in embryos injected with synthetic mRNA. Our results suggest that early induction and axial patterning are accomplished by endogenous activin B, not activin A, in Xenopus.
39517375
Disruption of BMP signals in embryonic Xenopus ectoderm leads to direct neural induction.
Bone morphogenetic proteins (BMPs), which have been implicated in the patterning of mesoderm, are members of the transforming growth factor-beta (TGF-beta) superfamily. We have investigated the roles of Xenopus BMP-7 (XBMP-7) and BMP-4 (XBMP-4), and activin (another TGF-beta-related molecule) in early development by generating dominant-negative versions of these growth factors. Mutations were generated by altering the cleavage sites that are required for maturation of the active dimeric forms of XBMP-7, XBMP-4, and activin. These mutant constructs, designated Cm-XBMP-7, Cm-XBMP-4, and Cm-activin, result in polypeptides that allow for dimerization of the subunits, but are incapable of maturation. Expression of Cm-XBMP-7 and Cm-XBMP-4, but not Cm-activin, in the ventral marginal zone of the Xenopus embryo results in the development of a secondary axis, similar to that seen by ectopic expression of the truncated BMP receptor. These results suggest that the cleavage mutants interfere with BMP signaling during mesodermal patterning. We also found that expression of Cm-XBMP-7 or Cm-XBMP-4 in animal cap ectoderm directly induces neuroectoderm. The neural induction was specific for Cm-XBMP-7 and Cm-XBMP-4 because ectopic expression of Cm-activin or Vg-1 did not mimic the same phenotype. Molecular study of neural patterning by Cm-XBMP-7 and Cm-XBMP-4 revealed that only anterior neuroectodermal markers are expressed in response to these Cm-XBMPs. These results suggest that the BMPs are involved in the specification of ectoderm in Xenopus development, and that neural induction requires the removal of BMP signals in the ectoderm. We propose that neural induction occurs by a default mechanism, whereby the inhibition of BMP signaling is required for the conversion of ectoderm to neuroectoderm in the developing Xenopus embryo.
4333831
Functional analysis of activins during mammalian development
ACTIVINS are dimeric (βAβA; βBβB; βAβB) members of the transforming growth factor-β superfamily1. They are widely expressed during murine development1–6, are highly conserved during vertebrate evolution1,7–11, and may be involved in mesoderm induction and neurulation in Xenopus laevis and Oryzias latipes10–17 . To investigate the function of mammalian activins in vivo, we generated mice with mutations either in activin-βA or in both activin-βA and activin-βB. Activin-βA-deficient mice develop to term but die within 24 h of birth. They lack whiskers and lower incisors and have defects in their secondary palates, including cleft palate, demonstrating that activin-βA must have a role during craniofacial development. Mice lacking both activin subunits show the defects of both individual mutants but no additional defects, indicating that there is no functional redundancy between these proteins during embryogenesis. In contrast to observations in lower vertebrates10–17, zygotic expression of activins is not essential for mesoderm formation in mice.
1513079
Effects of truncated activin and FGF receptors and of follistatin on the inducing activities of BVg1 and activin: does activin play a role in mesoderm induction?
Activin and Vg1, two members of the TGF‐beta family, are believed to play roles in mesoderm induction and axis formation in the amphibian embryo. Both molecules are provided maternally, either as protein (activin) or as RNA and protein (Vg1), and experiments with a truncated form of a type IIB activin receptor have led to the conclusion that activin is required for induction of mesoderm in vivo. In this paper we first show that truncated versions of two different Xenopus activin receptors also have severe effects on the activity of the mature region of Vg1, suggesting that such receptors may block the function of several members of the TGF‐beta family. We go on to demonstrate that follistatin, a secreted protein which binds activin and blocks its activity, does not interfere with Vg1 signalling. Furthermore, overexpression of follistatin mRNA in Xenopus embryos does not perturb mesoderm formation. Taken together, our data show that the effects of truncated activin receptors on Xenopus development can be explained by the inhibition of Vg1 activity, while the lack of effect of follistatin argues against a function for activin in mesoderm induction.
22254087
A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse.
The 413.d insertional mutation arrests mouse development shortly after gastrulation. nodal, a novel TGF beta-related gene, is closely associated with the locus. The present study provides direct evidence that the proviral insertion causes a loss of function mutation. nodal RNA is initially detected at day 5.5 in the primitive ectoderm. Concomitant with gastrulation, expression becomes restricted to the proximal posterior regions of the embryonic ectoderm. nodal RNA is also expressed in the primitive endoderm overlying the primitive streak. A few hours later, expression is strictly confined to the periphery of the mature node. Interestingly 413.d mutant embryos show no morphological evidence for the formation of a primitive streak. Nonetheless, about 25% of mutant embryos do form randomly positioned patches of cells of a posterior mesodermal character. Data presented in this report demonstrate the involvement of a TGF beta-related molecule in axis formation in mammals.
3740410
CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering
In just 3 years CRISPR genome editing has transformed biology, and its popularity and potency continue to grow. New CRISPR effectors and rules for locating optimum targets continue to be reported, highlighting the need for computational CRISPR targeting tools to compile these rules and facilitate target selection and design. CHOPCHOP is one of the most widely used web tools for CRISPR- and TALEN-based genome editing. Its overarching principle is to provide an intuitive and powerful tool that can serve both novice and experienced users. In this major update we introduce tools for the next generation of CRISPR advances, including Cpf1 and Cas9 nickases. We support a number of new features that improve the targeting power, usability and efficiency of CHOPCHOP. To increase targeting range and specificity we provide support for custom length sgRNAs, and we evaluate the sequence composition of the whole sgRNA and its surrounding region using models compiled from multiple large-scale studies. These and other new features, coupled with an updated interface for increased usability and support for a continually growing list of organisms, maintain CHOPCHOP as one of the leading tools for CRISPR genome editing. CHOPCHOP v2 can be found at http://chopchop.cbu.uib.no
3664235
CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing
Major advances in genome editing have recently been made possible with the development of the TALEN and CRISPR/Cas9 methods. The speed and ease of implementing these technologies has led to an explosion of mutant and transgenic organisms. A rate-limiting step in efficiently applying TALEN and CRISPR/Cas9 methods is the selection and design of targeting constructs. We have developed an online tool, CHOPCHOP (https://chopchop.rc.fas.harvard.edu), to expedite the design process. CHOPCHOP accepts a wide range of inputs (gene identifiers, genomic regions or pasted sequences) and provides an array of advanced options for target selection. It uses efficient sequence alignment algorithms to minimize search times, and rigorously predicts off-target binding of single-guide RNAs (sgRNAs) and TALENs. Each query produces an interactive visualization of the gene with candidate target sites displayed at their genomic positions and color-coded according to quality scores. In addition, for each possible target site, restriction sites and primer candidates are visualized, facilitating a streamlined pipeline of mutant generation and validation. The ease-of-use and speed of CHOPCHOP make it a valuable tool for genome engineering.
23087203
Method for isolation of PCR-ready genomic DNA from zebrafish tissues.
Here we describe a method for the isolation of PCR-ready genomic DNA from various zebrafish tissues that is based on a previously published murine protocol. The DNA solutions are of sufficient quality to allow PCR detection of transgenes from all commonly used zebrafish tissues. In sperm, transgene amplification was successful even when diluted 1000-fold, allowing easy identification of transgenic founders. Given its speed and low cost, we anticipate that the adoption of this method will streamline DNA isolation for zebrafish research.
1351008
Enzymatic assembly of DNA molecules up to several hundred kilobases
We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
19327966
Stages of embryonic development of the zebrafish
We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis—the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts. ©1995 Wiley‐Liss, Inc.
205420589
Fiji: an open-source platform for biological-image analysis
Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
23471047
High-resolution in situ hybridization to whole-mount zebrafish embryos
The in situ hybridization (ISH) technique allows the sites of expression of particular genes to be detected. This protocol describes ISH of digoxigenin-labeled antisense RNA probes to whole-mount zebrafish embryos. In our method, PCR-amplified sequence of a gene of interest is used as a template for the synthesis of an antisense RNA probe, which is labeled with digoxigenin-linked nucleotides. Embryos are fixed and permeabilized before being soaked in the digoxigenin-labeled probe. We use conditions that favor specific hybridization to complementary mRNA sequences in the tissue(s) expressing the corresponding gene. After washing away excess probe, hybrids are detected by immunohistochemistry using an alkaline phosphatase-conjugated antibody against digoxigenin and a chromogenic substrate. The whole procedure takes only 3 days and, because ISH conditions are the same for each probe tested, allows high throughput analysis of zebrafish gene expression during embryogenesis.
39848083
Measurement of binding kinetics using the gel electrophoresis mobility shift assay
The gel electrophoresis mobility shift assay is a technique for the qualitative and quantitative analysis of protein‐DNA complexes. The ability to resolve reactants, reaction intermediates and products makes this method particularly well‐suited for the measurement of the assembly and dissociation rates of protein‐nucleic acid complexes. Here we identify conditions that must be met and variations of the technique that are useful for the measurement of reaction rates.
25195795
Kinetics of protein-protein association explained by Brownian dynamics computer simulation.
Protein-protein bond formations, such as antibody-antigen complexation or aggregation of protein monomers into dimers and larger aggregates, occur with bimolecular rate constants on the order of 10(6) M-1.s-1, which is only 3 orders of magnitude slower than the diffusion-limited Smoluchowski rate. However, since the protein-protein bond requires rotational alignment to within a few angstroms of tolerance, purely geometric estimates would suggest that the observed rates might be 6 orders of magnitude below the Smoluchowski rate. Previous theoretical treatments have not been solved for the highly specific docking criteria of protein-protein association--the entire subunit interface must be aligned within 2 A of the correct position. Several studies have suggested that diffusion alone could not produce the rapid association kinetics and have postulated "lengthy collisions" and/or the operation of electrostatic or hydrophobic steering forces to accelerate the association. In the present study, the Brownian dynamics simulation method is used to compute the rate of association of neutral spherical model proteins with the stated docking criteria. The Brownian simulation predicts a rate of 2 x 10(6) M-1.s-1 for this generic protein-protein association, a rate that is 2000 times faster than that predicted by the simplest geometric calculation and is essentially equal to the rates observed for protein-protein association in aqueous solution. This high rate is obtained by simple diffusive processes and does not require any attractive or steering forces beyond those achieved for a partially formed bond. The rate enhancement is attributed to a diffusive entrapment effect, in which a protein pair surrounded and trapped by water undergoes multiple collisions with rotational reorientation during each encounter.