Nodal Signal Research Articles

Zili Inhibits Transforming Growth Factor-β Signaling by Interacting with Smad4

Piwi proteins are required for germ cell proliferation, differentiation, and germ line stem cell maintenance. In normal tissues, human and mouse Piwil2 are primarily expressed in testis but widely expressed in tumors. However, the underlying mechanism remains largely unknown. In vertebrates, transforming growth factor (TGF)-β signaling plays an important role in patterning embryo and control of cell growth and differentiation. A previous study has shown a role for Zili, a Piwil2 gene in zebrafish, in germ cells in zebrafish. Here we report that zili functions in patterning the early embryo and inhibits TGF-β signaling. Whole mount expression analysis shows that zili expresses not only in PGCs but also in axis. Ectopic expression of zili causes fusion of the eyes and reduction of mesodermal marker genes expression, suggesting that zili functions to inhibit Nodal signaling and mesoderm formation. Genetic interaction shows that zili inhibits Nodal and bone morphogenetic protein signaling. The results of protein interaction assays identify that Zili binds to Smad4 via its N-terminal domain and prevents the formation of Smad2/3/4 and Smad1/5/9/4 complexes to antagonize TGF-β signaling. This work shows that zili plays a role in early embryogenesis beyond germ line as a novel negative regulator of TGF-β signaling, extending the function of Piwi proteins in vertebrates.

Reversal of left-right asymmetry induced by aberrant Nodal signaling in the node of mouse embryos

The node at the anterior tip of the primitive streak serves as an initial generator of the left-right (L-R) axis in mammalian embryos. We now show that a small disturbance in molecular signaling at the node is responsible for the L-R reversal of visceral organs in the inv mutant mouse. In the node of wild-type embryos, the expression of Nodal and Cerl2 (Dand5), which encodes an inhibitor of Nodal, is asymmetric, with the level of Nodal expression being higher on the left side and that of Cerl2 expression higher on the right. In inv/inv embryos, however, a localized reduction in the level of Cerl2 expression results in upregulation of the Nodal signal and a consequent induction of Lefty expression in the node. The ectopic expression of Lefty1 delays the onset of Nodal expression in the lateral plate mesoderm. L-R asymmetry of Cerl2 expression in the node also becomes reversed in a manner dependent on the Nodal signal. Nodal expression in the lateral plate mesoderm then appears on the right side, probably reflecting the balance between Nodal and Cerl2 in the node. The inhibition of Cerl2 expression by the Nodal signal suggests a mechanism for amplification of the cue for L-R asymmetry provided by nodal flow and for stabilization of asymmetric gene expression around the node. In inv/inv embryos, this system may function in reverse as a result of ectopic production of Lefty, which inhibits the Nodal signal on the left side in a manner dependent on leftward nodal flow.

Colorectal Carcinoma: Local Tumor Staging and Assessment of Lymph Node Metastasis by High‐Resolution MR Imaging in Surgical Specimens

Purpose. To assess the accuracy of high-resolution MR imaging as a means of evaluating mural invasion and lymph node metastasis by colorectal carcinoma in surgical specimens. Materials and Methods. High-resolution T1-weighted and T2-weighted MR images were obtained in 92 surgical specimens containing 96 colorectal carcinomas. Results. T2-weighted MR images clearly depicted the normal colorectal wall as consisting of seven layers. In 90 (94%) of the 96 carcinomas the depth of mural invasion depicted by MR imaging correlated well with the histopathologic stage. Nodal signal intensity on T2-weighted images (93%) and nodal border contour (93%) were more accurate than nodal size (89%) as indicators of lymph node metastasis, and MR imaging provided the highest accuracy (94%–96%) when they were combined. Conclusion. High-resolution MR imaging is a very accurate method for evaluating both mural invasion and lymph node metastasis by colorectal carcinoma in surgical specimens.

Comparing a Novel PET Agent, Yttrium-86 Oxine, with Biopsy and FACS Analysis for Tracking in Vivo Migration Patterns of Different Preparations of DC from a Single Donor

Aim: The objective of this study was compare the in vivo distribution of different preparations of dendritic cells (DC) from a single donor after intradermal (ID) administration and correlate FACS analysis of excised tissue and imaging findings.Method: DC were generated from a single healthy donor apheresis product. The DC types were GM-CSF + IL-13 (IL-13 imDC), then further matured with FMKp+IFNg (IL-13mDC), GM-CSF + IL-4 (IL-4 imDC) and matured (IL-4 mDC) and GM-CSF+IFN-α (IFN-α DC). Antigen naive DC preparations were labelled with Yttrium-86 oxine (prepared on site) by incubation at room temperature for 30 minutes followed by centrifugation and resuspension. 5 × 105 radio-labelled cells in 50_L was injected ID into the footpad and the volar aspect of the foreleg of Balb/c nude mice (n=5 per group). A cohort (n=5) of control mice were similarly injected with Yttrium-86 oxine only. Images were acquired on a small animal PET (SAPET) (Mosaic, Philips Medical Systems) at 2, 24 and 45 hours post administration. Mice were then euthanized and popliteal, axillary and inguinal lymph nodes (LN) harvested. LN were processed into single cell suspension, stained with anti-human HLA-DR and CD11c and analysed by flow cytometry. SAPET images were graded: 0 – no signal, 1 – marginal, 2 – definite nodal signal, 3 – strong signal. DC detection by FACS was graded: 0 – no detection, 1 – <0.5% DC, 2 – 0.5.⋄1.5% DC, 3 – >1.5% DC of all viable cellular events. Results: Pre-injection analysis of DC showed expression of HLA-DR, HLA-ABC, CD11c, CD80 and CD86, low levels of CD83, CCR5 and CCR7 along with down regulation of CD14. Matured DC populations showed increased levels of CD83 and CD86. Similar phenotypic expression on the DC populations were observed prior to cryopreservation and upon thawing. Mean DC radiolabelling efficiency of 72% (54% – 88%). There was a minor decrease in cell numbers and viability after labelling most likely due to oxine related toxicity. There were no substantial differences in tracking between cell types. In 28 of 43 nodes assessed, FACS and SAPET results were congruent (27 FACS and SAPET positive and 1 FACS and SAPET negative). Of the 15 discrepant samples, 13 were SAPET positive and FACS negative, while 2 were FACS positive and PET negative. Average FACS and SAPET grades at 45 hours were higher for popliteal nodes than axillary nodes. No false positive nodal accumulation was visualised at 45 hours in control mice. Conclusion: This novel PET technique provides a robust, reproducible method for quantification of in vivo functional DC tracking to LN. Detection of DC migration using this novel PET agent is a more reliable technique than biopsy and FACS analysis, which has an inherent sampling error.

Cis‐regulatory control of the nodal gene, initiator of the sea urchin oral ectoderm gene network

Expression of the nodal gene initiates the gene regulatory network which establishes the transcriptional specification of the oral ectoderm in the sea urchin embryo. The cis‐regulatory system of the nodal gene transduces anisotropically distributed cytoplasmic cues that distinguish the future oral and aboral domains of the early embryo. Here we establish the genomic basis for the initiation and maintenance of nodal gene expression in the oral ectoderm. A 5′ cis‐regulatory module functions both to initiate expression of the nodal gene and to maintain its expression by means of feedback input from the Nodal signal transduction system. These functions are mediated respectively by target sites for bZIP transcription factors, and by SMAD target sites. At least one SMAD site is also needed for the initiation of expression. An intron module also contains SMAD sites which respond to Nodal feedback, and in addition acts to repress vegetal expression. These observations explain the main features of nodal expression in the oral ectoderm: since the activity of bZIP factors is redox sensitive, and the initial polarization of oral vs aboral fate is manifested in a redox differential, the bZIP sites account for the activation of nodal on the oral side; and since the immediate early signal transduction response factors for Nodal are SMAD factors, the SMAD sites account for the feedback maintenance of nodal gene expression.

Long-range action of Nodal requires interaction with GDF1

GDF1 (growth/differentiation factor 1), a Vg1-related member of the transforming growth factor-beta superfamily, is required for left-right patterning in the mouse, but the precise function of GDF1 has remained largely unknown. In contrast to previous observations, we now show that GDF1 itself is not an effective ligand but rather functions as a coligand for Nodal. GDF1 directly interacts with Nodal and thereby greatly increases its specific activity. Gdf1 expression in the node was found necessary and sufficient for initiation of asymmetric Nodal expression in the lateral plate of mouse embryos. Coexpression of GDF1 with Nodal in frog embryos increased the range of the Nodal signal. Introduction of Nodal alone into the lateral plate of Gdf1 knockout mouse embryos did not induce Lefty1 expression at the midline, whereas introduction of both Nodal and GDF1 did, showing that GDF1 is required for long-range Nodal signaling from the lateral plate to the midline. These results suggest that GDF1 regulates the activity and signaling range of Nodal through direct interaction.

Utility of a New Bolus-injectable Nanoparticle for Clinical Cancer Staging

BACKGROUND: In this study, we report on the use of a new, bolus-injectable, carboxymethyl dextran-based magnetic nanoparticle (MNP), ferumoxytol, to improve detection in loco-regional lymph nodes by magnetic resonance imaging (MRI). Methods: This preliminary study was performed as a prospective, single-center, open label pilot study to determine the magnitude of nodal MRI signal changes and to determine the optimal time points for imaging following intravenous (IV) bolus injection of the MNP. The study group consisted of 10 patients, all of whom were diagnosed with prostate cancer before any systemic therapy. RESULTS: All 10 patients had lymph nodes evaluated by histopathology. Of the evaluated 26 lymph nodes, 20 were benign and 6 were malignant. The mean short-axis diameter of benign lymph nodes was 6 mm and the mean short-axis diameter of malignant lymph nodes was 7 mm. Following IV administration, there was a significant change in mean signal-to-noise ratio (SNR) of benign lymph nodes (P < .0001) whereas there was little change in the mean SNR of malignant nodes (P = .1624). No adverse events were encountered. CONCLUSION: Ferumoxytol is safe and, at the appropriate circulation interval, modulates nodal signal intensity, allowing for identification of malignant nodal involvement by MRI.

Requirement of Glycosylphosphatidylinositol Anchor of Cripto-1 for trans Activity as a Nodal Co-receptor

Cripto-1 (CR-1) has an indispensable role as a Nodal co-receptor for patterning of body axis in embryonic development. CR-1 is reported to have a paracrine activity as a Nodal co-receptor, although CR-1 is primarily produced as a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Regulation of cis and trans function of CR-1 should be important to establish the precise body patterning. However, the mechanism by which GPI-anchored CR-1 can act in trans is not well known. Here we confirmed the paracrine activity of CR-1 by fluorescent cell-labeling and immunofluorescent staining. We generated COOH-terminal-truncated soluble forms of CR-1 based on the attachment site for the GPI moiety (omega-site), which we identified in the present study. GPI-anchored CR-1 has a significantly higher activity than COOH-terminal-truncated soluble forms to induce Nodal signal in trans as well as in cis. Moreover, transmembrane forms of CR-1 partially retained their ability to induce Nodal signaling only when type I receptor Activin-like kinase 4 was overexpressed. NTERA2/D1 cells, which express endogenous CR-1, lost the cell-surface expression of CR-1 after phosphatidylinositol-phospholipase C treatment and became refractory to stimulation of Nodal. These observations suggest that GPI attachment of CR-1 is required for the paracrine activity as a Nodal co-receptor.

Maternal nodal and zebrafish embryogenesis

In fish and amphibians, the dorsal axis is specified by the asymmetric localization of maternally provided components of the Wnt signalling pathway. Gore et al. suggest that the Nodal signal Squint (Sqt) is required as a maternally provided dorsal determinant in zebrafish. Here we test their proposal and show that the maternal activities of sqt and the related Nodal gene cyclops (cyc) are not required for dorsoventral patterning.

Haemodynamics determined by a genetic programme govern asymmetric development of the aortic arch

Laterality of the internal organs of vertebrates is determined by asymmetric Nodal signalling in the lateral plate mesoderm. A deficiency of such signalling results in heterotaxia syndrome, characterized by anomalous laterality of visceral organs and complex congenital heart conditions. Pitx2, the transcription factor induced by the Nodal signal, regulates left-right asymmetric morphogenesis. The cellular and molecular bases of asymmetric morphogenesis remain largely unknown, however. Here we show that ablation of unilateral Pitx2 expression in mice impairs asymmetric remodelling of the branchial arch artery (BAA) system, resulting in randomized laterality of the aortic arch. Pitx2-positive cells were found not to contribute to asymmetrically remodelled arteries. Instead, Pitx2 functions in the secondary heart field and induces a dynamic morphological change in the outflow tract of the heart, which results in the provision of an asymmetric blood supply to the sixth BAA. This uneven distribution of blood flow results in differential signalling by both the platelet-derived growth factor receptor and vascular endothelial growth factor receptor 2. The consequent stabilization of the left sixth BAA and regression of its right counterpart underlie left-sided formation of the aortic arch. Our results therefore indicate that haemodynamics, generated by a Pitx2-induced morphological change in the outflow tract, is responsible for the asymmetric remodelling of the great arteries.

Sulfated glycosaminoglycans are necessary for Nodal signal transmission from the node to the left lateral plate in the mouse embryo

Situs-specific organogenesis in the mouse results from leftward fluid flow in the node cavity and subsequent left-sided expression of Nodal in the lateral plate mesoderm (LPM). Nodal expression at the node is essential for the subsequent asymmetric Nodal expression in the left LPM, but the precise role of Nodal produced at the node has remained unknown. We have now investigated how the Nodal signal is transferred from the node to the LPM. Externally supplied Nodal protein failed to signal to the LPM, suggesting that the Nodal signal is transferred to the LPM via an internal route rather than an external one. Transgenic rescue experiments showed that the Nodal co-receptor Cryptic (Cfc1) is required only in the LPM, not at the node, for asymmetric Nodal expression in the LPM, indicating that the Nodal signal is not relayed indirectly between the node and LPM. Nodal interacts in vitro with sulfated glycosaminoglycans (GAGs), which are specifically localized to the basement membrane-like structure between the node and LPM in the mouse embryo. Inhibition of sulfated GAG biosynthesis prevents Nodal expression in the LPM. These data suggest that Nodal produced at the node might travel directly to the LPM via interaction with sulfated GAGs.

Cis-regulatory analysis ofnodaland maternal control of dorsal-ventral axis formation by Univin, a TGF-β related to Vg1

The TGF-beta 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-beta 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/beta-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-beta acts in synergy with maternal transcription factors and with spatial repressors to establish the dorsal-ventral axis of the sea urchin embryo.

Cerberus is a feedback inhibitor of Nodal asymmetric signaling in the chick embryo

The TGF-beta-related molecule Nodal plays an essential and conserved role in left-right patterning of the vertebrate embryo. Previous reports have shown that the zebrafish and mouse Cerberus-related proteins Charon and Cerberus-like-2 (Cerl-2), respectively, act in the node region to prevent the Nodal signal from crossing to the right side, whereas chick Cerberus (cCer) has an unclear function in the left-side mesoderm. In this study, we investigate the transcriptional regulation and function of cCer in left-right development. By analyzing the enhancer activity of cCer 5' genomic sequences in electroporated chick embryos, we identified a cCer left-side enhancer that contains two FoxH1 and one SMAD binding site. We show that these Nodal-responsive elements are necessary and sufficient for the activation of transcription in the left-side mesoderm. In transgenic mouse embryos, cCer regulatory sequences behave as in chick embryos, suggesting that the cis-regulatory sequences of Cerberus-related genes have diverged during vertebrate evolution. Moreover, our findings from cCer overexpression and knockdown experiments indicate that cCer is a negative-feedback regulator of Nodal asymmetric signaling. We propose that cCer and mouse Cerl-2 have evolved distinct regulatory mechanisms but retained a conserved function in left-right development, which is to restrict Nodal activity to the left side of the embryo.

FGF9/16/20 and Wnt-5α signals are involved in specification of secondary muscle fate in embryos of the ascidian, Halocynthia roretzi

The tail muscle cells of the ascidian tadpole larva originate from two different lineages, the B- (primary) and A- and b- (secondary) line blastomeres of the eight-cell stage embryo. The primary muscle cells assume muscle fate cell-autonomously with the involvement of a localized muscle determinant, macho-1. On the other hand, fate determination of secondary muscle cells is a non-cell-autonomous process that depends on cellular interactions. In this paper, we investigated the mechanisms underlying fate specification of secondary muscle cells in Halocynthia roretzi. We found that FGF and Wnt5 signals were required. In contrast, the Nodal signal, which is required for specification of A-line muscle cells in another ascidian, Ciona intestinalis, was not necessary for the formation of any secondary muscle cells in Halocynthia embryo. Therefore, Halocynthia and Ciona show distinctly different mechanisms for generation of the secondary lineages, despite the fact that embryogenesis appears very similar between these species. We also found that the mechanisms involved in specification of A- and b-line muscle cells were distinct in that the required timing of the FGF signal for the A-line muscle cells preceded that for the b-line. Moreover, the inducer blastomeres for specification of these two lineages were different.

Cis-regulatory control of the nodal gene, initiator of the sea urchin oral ectoderm gene network

Expression of the nodal gene initiates the gene regulatory network which establishes the transcriptional specification of the oral ectoderm in the sea urchin embryo. This gene encodes a TGFβ ligand, and in Strongylocentrotus purpuratus its transcription is activated in the presumptive oral ectoderm at about the 30-cell stage. Thereafter Nodal signaling occurs among all cells of the oral ectoderm territory, and nodal expression is required for expression of oral ectoderm regulatory genes. The cis-regulatory system of the nodal gene transduces anisotropically distributed cytoplasmic cues that distinguish the future oral and aboral domains of the early embryo. Here we establish the genomic basis for the initiation and maintenance of nodal gene expression in the oral ectoderm. Functional cis-regulatory control modules of the nodal gene were identified by interspecific sequence conservation. A 5′ cis-regulatory module functions both to initiate expression of the nodal gene and to maintain its expression by means of feedback input from the Nodal signal transduction system. These functions are mediated respectively by target sites for bZIP transcription factors, and by SMAD target sites. At least one SMAD site is also needed for the initiation of expression. An intron module also contains SMAD sites which respond to Nodal feedback, and in addition acts to repress vegetal expression. These observations explain the main features of nodal expression in the oral ectoderm: since the activity of bZIP factors is redox sensitive, and the initial polarization of oral vs. aboral fate is manifested in a redox differential, the bZIP sites account for the activation of nodal on the oral side; and since the immediate early signal transduction response factors for Nodal are SMAD factors, the SMAD sites account for the feedback maintenance of nodal gene expression.

A signalling relay involving Nodal and Delta ligands acts during secondary notochord induction inCionaembryos

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

Nodal Pricing for Distribution Networks: Efficient Pricing for Efficiency Enhancing DG

As distributed generation (DG) becomes more widely deployed, distribution networks become more active and take on many of the same characteristics as transmission. We propose the use of nodal pricing that is often used in the pricing of short-term operations in transmission. As an economically efficient mechanism, nodal pricing would properly reward DG for reducing line losses through increased revenues at nodal prices and signal prospective DG where it ought to connect with the distribution network. Applying nodal pricing to a model distribution network, we show significant price differences between buses reflecting high marginal losses. Moreover, we show the contribution of a DG resource located at the end of the network to significant reductions in losses and line loading. We also show the DG resource has significantly greater revenue under nodal pricing, reflecting its contribution to reduced line losses and loading.

A mutation in the zebrafish Na,K‐ATPase subunit atp1a1a.1 provides genetic evidence that the sodium potassium pump contributes to left‐right asymmetry downstream or in parallel to nodal flow

While there is a good conceptual framework of dorsoventral and anterioposterior axes formation in most vertebrate groups, understanding of left-right axis initiation is fragmentary. Diverse mechanisms have been implied to contribute to the earliest steps of left-right asymmetry, including small molecule signals, gap junctional communication, membrane potential, and directional flow of extracellular liquid generated by monocilia in the node region. Here we demonstrate that a mutation in the zebrafish Na,K-ATPase subunit atp1a1a causes left-right defects including isomerism of internal organs at the anatomical level. The normally left-sided Nodal signal spaw as well as its inhibitor lefty are expressed bilaterally, while pitx2 may appear random or bilateral. Monocilia movement and fluid circulation in Kupffer's vesicle are normal in atp1a1a(m883) mutant embryos. Therefore, the Na,K-ATPase is required downstream or in parallel to monocilia function during initiation of left-right asymmetry in zebrafish.

Nodal signals pattern vertebrate embryos.

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 beta (TGFbeta) superfamily, activates a signal transduction pathway that is similar to that of other TGFbeta 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.

Abstracts of papers presented at the fifteenth Genetics Society's Mammalian Genetics and Development Workshop held at the Institute of Child Health, University College London on 22 and 23 November 2004

The Transforming growth factor-β (TGF-β) family control diverse cellular processes and specify cell-fate/differentiation during embryogenesis in vertebrates and invertebrates. Mutations disrupting TGF-β signalling lead to developmental abnormalities and a range of diseases such as cancer. Nodal is a major TGF-β signal, responsible for gastrulation in embryogenesis. Arkadia (Akd) was discovered by mouse gene-trap mutagenesis and encodes a nuclear E3 ubiquitin ligase. Akd allows the Nodal signal to reach its maximum level and Akd-null mice lack mammalian organiser (MO) and mesendodermal tissues. Although Akd RNA is ubiquitously expressed, Akd-null mice lose a subset of Nodal-dependent functions. The specificity of Akd function is therefore most likely to be regulated post-transcriptionally or by co-factors. Akd possesses differentially spliced 5′ untranslated regions (UTRs) and large 3′ UTR. We have employed bioinformatics and developed a reporter system to address Akd post-transcriptional regulation. Akd RNA may initiate from different promoters and 5′ UTR differential splicing, upstream AUGs (uAUGs) and open-reading frames upstream (uORFs) may regulate protein translation. 5′ and 3′ UTRs can interact to either destabilise or decrease translational efficiency of RNA. The nature of this interaction is cell-type and signal level dependent. These data may represent mechanisms by which translational control of Arkadia is achieved and ultimately how TGF-β/Nodal signalling is regulated during embryogenesis.