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Nodal Morphogens

Alexander F. Schier

Department of Molecular and Cellular Biology, Harvard Stem Cell Institute, Center for Brain Science, Broad Institute, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138 Correspondence: [email protected]

Nodal signals belong to the TGF-b superfamily and are essential for the induction of meso- derm and endoderm and the determination of the left–right axis. Nodal signals can act as morphogens—they have concentration-dependent effects and can act at a distance from their source of production. Nodal and its feedback inhibitor Lefty form an activator/inhibitor pair that behaves similarly to postulated reaction–diffusion models of tissue patterning. Nodal morphogen activity is also regulated by microRNAs, convertases, TGF-b signals, co- receptors, and trafficking factors. This article describes how Nodal morphogens pattern embryonic fields and discusses how Nodal morphogen signaling is modulated.

n his 1901 book “Regeneration,” Thomas signals in mesendoderm induction and left– IHunt Morgan speculated that “if we suppose right axis specification. I then discuss how the materials or structures that are characteristic Nodal morphogen gradients are thought to be of the vegetative half are gradually distributed generated, modulated, and interpreted. from the vegetative to the animal half in decreas- ing amounts, then any piece of the egg will contain more of these things at one pole than the THE NODAL SIGNALING PATHWAY other” and “gastrulation depends on the relative Like most TGF-b signals, Nodal ligands activate amounts of the materials in the different parts serine/threonine kinase receptors that phos- of the blastula” (Morgan 1901). Although phorylate Smad proteins to regulate gene Morgan’s speculations referred to the sea expression (reviewed in Schier 2003; Shen urchin embryo, they foretold our current 2007; Wu and Hill 2009). In the case of Nodal, understanding of morphogen gradients in frog the signal is received by type I and II Activin and fish development. Morgan’s “materials,” receptors and EGF-CFC coreceptors. Receptor “structures,” and “things” are the Nodal signals activation results in the phosphorylation of that create a vegetal-to-animal activity gradient the transcription factors Smad2 and Smad3. to regulate germ layer formation and pattern- This leads to their binding to Smad4, nuclear ing. This article discusses how Nodal signaling translocation, and association with additional provides positional information to fields of transcription factors to regulate target genes. cells. I first portray the components of the sig- Several extracellular proteins, including pro- naling pathway and describe the role of Nodal cessing enzymes and antagonists, regulate this

Editors: James Briscoe, Peter Lawrence, and Jean-Paul Vincent Additional Perspectives on Generation and Interpretation of Morphogen Gradients available at www.cshperspectives.org Copyright # 2009 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a003459 Cite this article as Cold Spring Harb Perspect Biol 2009;1:a003459

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A.F. Schier

MiR-430

Convertases Processing Lefty Nodal

Cerberus MiR-15/16 EGF-CFC

Activin receptors Dapper 2 Recycling Rap2 Lysosomal degradation

Ubiquitination P Ectodermin Smad2 Smad4 Dephosphorylation PPM1A FAM/Usp9x Deubiquitination Nuclear export RanBP3

FoxH1 Mixer p53

Figure 1. The Nodal signaling pathway. After processing of the Nodal precursor by convertases, Nodal ligands signal through activin receptors and EGF-CFC coreceptors. Cerberus and Lefty act as extracellular inhibitors. MicroRNAs belonging to the miR-430 family target Nodal (zebrafish squint) and Lefty mRNAs for degradation and repression. Mir-15/16 represses the type II activin receptor. Rap2 is involved in the recycling of activin receptors whereas Dapper 2 targets activin receptor complexes for degradation in the lysosome. Pathway activation is mediated by phosphorylation of Smad2 and its association with Smad4 and transcription factors such as FoxH1, Mixer, and p53. Phospho-Smad2 is dephosphorylated by PPM1A and exported from the nucleus by RanBP3. Smad4 activity and stability is regulated by the ubiquitinase Ectodermin and the deubiquitinase FAM/Usp9x. See text for details.

core pathway. In addition, intracellular mol- Drosophila or Caenorhabditis elegans. They are ecules such astranscriptional cofactors, proteins now also known to be present in deuterostomes involved in receptor trafficking, and miRNAs (e.g., sea urchin) and in the protostome group regulate Nodal signaling. To understand how of Lophotrochozoa (e.g., snails) where, similar Nodal morphogen activity is regulated, one to chordates, they control left–right asymmetry first needs to understand the molecular basis of and chirality (Duboc et al. 2004; Chea et al. Nodal signal transduction (Fig. 1). 2005; Grande and Patel 2009). The absence of Nodal in Drosophila or C. elegans might reflect the different, more derived modes of mesendo- Nodal Signals, Convertases, and derm and left–right specification in these Extracellular Antagonists systems. Although there is only one Nodal TGF-b signals belonging to the Nodal sub- gene in mouse (Zhou et al. 1993), there are family were initially found in chordates but three in zebrafish (Cyclops, Squint, and were notably absent from Ecdysozoa such as Southpaw) (Erter et al. 1998; Feldman et al.

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Nodal Morphogens

1998; Rebagliati et al. 1998a; Rebagliati et al. binding to receptors, and thus regulate embry- 1998b; Sampath et al. 1998; Long et al. 2003) onic patterning (Bouwmeester et al. 1996; and five in Xenopus (Xnr1, 2, 4, 5, and 6) Piccolo et al. 1999; Bertocchini and Stern (Jones et al. 1995; Joseph and Melton 1997; 2002; Silva et al. 2003; Hashimoto et al. 2004; Takahashi et al. 2000). Marques et al. 2004; Yamamoto et al. 2004; Nodal proteins are translated as propro- Tavares et al. 2007; Belo et al. 2008). Loss of teins, consisting of a prodomain and a mature Lefty antagonists results in ectopic and pro- ligand domain. Studies in mouse have shown longed activity of Nodal signaling, leading to that the convertases Spc1 and Spc4 (also ectopic mesendoderm formation and abnormal known as Furin and Pace4, respectively) cleave left–right patterning (Meno et al. 1998; Meno Nodal precursors at R-X-(K/R/X)-R consensus et al. 1999; Agathon et al. 2001; Meno et al. sequences (Beck et al. 2002). The Nodal precur- 2001; Chen and Schier 2002; Feldman et al. sor can be secreted and processed extracellularly 2002; Yamamoto et al. 2004). by Spc1 and Spc4. Processing is essential for activation of the Nodal signaling pathway in Receptors and Signal Transducers zebrafish and mouse embryonic tissues (Beck et al. 2002; Le Good et al. 2005), but a non- Nodal signals assemble receptor complexes con- processable Nodal precursor can activate the sisting of type I and type II activin receptors pathway in mouse extraembryonic ectoderm (ActRIB; ActRIIA/B) that function as serine/ (Ben-Haim et al. 2006). threonine kinases (Reissmann et al. 2001; Yeo Additional signaling complexity is intro- and Whitman 2001; Yan et al. 2002). Assembly duced by the finding that several related results in the phosphorylation and activation TGF-b ligands act through the same pathway of the type I receptor by the type II receptor. as Nodal. For example, mouse GDF1 and EGF-CFC proteins are extracellular GPI-linked GDF3, and the related frog and zebrafish Vg1, factors that are required for Nodal signaling activate the pathway via Activin receptors and and embryogenesis (Schier et al. 1997; Shen EGF-CFC coreceptors (Thomsen and Melton et al. 1997; Ding et al. 1998; Zhang et al. 1998; 1993; Cheng et al. 2003; Chen et al. 2006; Gritsman et al. 1999; Yan et al. 1999; Bamford Andersson et al. 2007; Karkera et al. 2007). et al. 2000; Shen and Schier 2000; de la Cruz Double mutant analysis has shown that Nodal et al. 2002; Dorey and Hill 2006; Onuma et al. can have both overlapping and nonredundant 2006). For example, absence of the EGF-CFC roles with GDF1. GDF1 and Nodal are both protein one-eyed pinhead inactivates the required for left–right specification, suggesting pathway and renders embryos resistant to interdependent or synergistic roles (Rankin Nodal (Gritsman et al. 1999). It is thought et al. 2000; Andersson et al. 2006). Indeed, that EGF-CFC proteins act as coreceptors by GDF1 can heterodimerize with Nodal to gener- binding to Nodal and the type I activin receptor ate a more active ligand than the corresponding (Reissmann et al. 2001; Yeoand Whitman 2001; homodimers (Tanaka et al. 2007). Yan et al. 2002; Cheng et al. 2003; Cheng et al. Extracellular inhibitors such as Lefty and 2004; Chu et al. 2005; Minchiotti 2005). Cerberus antagonize Nodal signaling. Leftys Recent tissue culture studies have high- are divergent members of the TGF-b family lighted the importance of ligand and receptor and block Nodal signaling by binding to trafficking in Nodal signaling. For example, Nodal itself and to EGF-CFC coreceptors the mammalian EGF-CFC protein Cripto can (Meno et al. 1996; Bisgrove et al. 1999; Meno promote Nodal signaling by linking the pro- et al. 1999; Thisse and Thisse 1999; Thisse cessing and trafficking of Nodal (Constam et al. 2000; Agathon et al. 2001; Chen and 2009). Cripto forms a complex with Nodal Schier 2002; Chen and Shen 2004; Cheng et al. precursors and convertases at the surface of 2004). Cerberus and Cerberus-like proteins responding cells and facilitates Nodal pro- like Charon bind directly to Nodal, inhibit its cessing and translocation to early endosomes

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A.F. Schier

(Blanchet et al. 2008a; Blanchet et al. 2008b). Smad4 and other transcription factors Moreover, on internalization, Cripto facilitates (Massague et al. 2005; Ross and Hill 2008). the interaction with Activin receptors by attenu- These activated Smad complexes accumulate ating the sorting of Nodal into intraluminal ves- in the nucleus as a result of decreased export icles that are destined for lysosomal degradation rate and increased import rate compared with (Blanchet et al. 2008b). Although the in vivo rel- monomeric unphosphorylated Smads. Smads evance of these findings remains to be tested, shuttle between nucleus and cytoplasm and they suggest important roles of trafficking thus can continuously monitor and respond during Nodal signaling. Indeed, previous to receptor activity (Bourillot et al. 2002; studies have shown that receptor trafficking Inman et al. 2002; Xu et al. 2002; Nicolas et al. plays an important role in TGF-b signaling. 2004; Schmierer and Hill 2005; Schmierer Receptors can either be recycled or targeted et al. 2008). for degradation depending on the trafficking Smad4 mutant phenotypes are less severe route (Constam 2009). Signaling is thought to than Smad2/3 mutant phenotypes, indicating occur in endosomes generated by clathrin- Smad4-independent gene regulation by Nodal mediated internalization, whereas degradation (Chu et al. 2005). Moreover, Smad3/4 have rela- is thought to be mediated by a lipid-raft/caveo- tively poor affinity for DNA, whereas Smad2 has lar internalization pathway and trafficking to no DNA binding activity. Thus, Smad proteins lysosomes (Di Guglielmo et al. 2003). It must form complexes with specific transcrip- remains to be determined whether this model tion factors to recognize and regulate Nodal- applies to Nodal signaling. For example, the responsive cis-elements (Massague et al. degradative uptake of Nodal does not involve 2005; Ross and Hill 2008). Proteins such as caveolin-positive carriers in tissue culture FoxH1, Mixer, and p53 form complexes with (Blanchet et al. 2008b), and signaling is Smad proteins and contribute to the specific mediated by interaction with EGF-CFC pro- recognition and regulation of subsets of teins localized to flotillin-positive lipid rafts Nodal target genes (Chen et al. 1996; Germain (Blanchet et al. 2008b). It is clear, however, et al. 2000; Hoodless et al. 2001; Yamamoto that the regulation of Activin receptor traffick- et al. 2001; Hart et al. 2002; Cordenonsi et al. ing can modulate signaling (Jullien and 2003; Takebayashi-Suzuki et al. 2003). For Gurdon 2005). The Ras GTPase Rap2 promotes example, loss of FoxH1 or Mixer leads to recycling of nonbound Activin Receptors and distinct phenotypes in zebrafish (Kikuchi et al. delays degradation of ligand-receptor com- 2000; Pogoda et al. 2000; Sirotkin et al. 2000), plexes and thus up-regulates signaling (Choi whereas FoxH1; Mixer double mutants show et al. 2008). In contrast, Dapper 2 is induced more than additive phenotypes (Kunwar et al. by Nodal signaling and recruited to late endo- 2003). These results have led to the model that somes where it binds the type I activin receptor Smad2/3/4 cofactors regulate distinct but par- and enhances the lysosomal degradation of tially overlapping sets of Nodal downstream Nodal receptors (Zhang et al. 2004). In addi- genes (Ross and Hill 2008). tion, subunits of the PP2A phosphatase influ- The activity of Smad complexes is regulated ence activin receptor levels and signaling post-translationally (Lin et al. 2006; Episkopou (Batut et al. 2008). These studies indicate that et al. 2001; Niederlander et al. 2001; Iratni et al. ligand and receptor trafficking and stability 2002; Dupont et al. 2005; Cordenonsi et al. have important roles in regulating the strength 2007; Levy et al. 2007; Mavrakis et al. 2007; and duration of Nodal signaling. Nagano et al. 2007; Yun et al. 2007; Sasai et al. 2008; Dai et al. 2009; Dupont et al. 2009). Smad2/3 is activated by receptor-mediated Transcription Factors and Target Genes phosphorylation and inhibited by phospha- Nodal receptor activation results in phos- tases such as PPM1A (Lin et al. 2006). De- phorylation of Smad2/3 and association with phosphorylated Smad2/3 is then recognized

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Nodal Morphogens

by RanBP3 and exported from the nucleus (Dai that although the core Nodal signaling path- et al. 2009). Interference with PPM1A or way has been conserved across evolution, the RanBP3 activity leads to an increase of TGF-b downstream targets are divergent between dif- signaling. Additionally, ubiquitination and ferent tissues. de-ubiquitination of Smad4 contribute to Nodal signaling. Smad4 monoubiquitination THE ROLES OF NODAL SIGNALS IN by Ectodermin lowers pathway activity, whereas DEVELOPMENT de-ubiquitination by FAM/Usp9x allows inter- action with P-Smad2 and pathway activation To discuss how Nodal proteins provide posi- (Dupont et al. 2005; Dupont et al. 2009). In tional information, one first needs to under- contrast, the ubiquitin ligase Arkadia increases stand how the pathway is used during pathway activity, but its exact mechanism of mesendoderm induction and left–right pat- action is controversial (Episkopou et al. 2001; terning. For a discussion of Nodal signals in Niederlander et al. 2001; Koinuma et al. 2003; other developmental contexts, see Shen 2007. Levy et al. 2007; Mavrakis et al. 2007; Nagano et al. 2007). Cofactor activity is also regulated. Mesendoderm Induction For example, Drap1 can bind to FoxH1 and attenuate Nodal signaling, and Serum response The formation of the three germ layers—endo- factor interacts with Smad2 and FoxH1 to derm, mesoderm, and ectoderm—is a prerequi- inhibit the formation of Smad2-FoxH1 site for the formation of vertebrate organs complexes (Iratni et al. 2002; Yun et al. 2007). (Kimelman and Schier 2002; Stern 2004; The Zn-finger factor XFDL156 binds p53, Schier and Talbot 2005; Solnica-Krezel 2005; blocking its interaction with P-Smad2 (Sasai Heasman 2006; Tam and Loebel 2007; Arnold et al. 2008). Conversely, Ras/MAPK signaling and Robertson 2009). Nodal signals are essen- promotes p53 phosphorylation and enhances tial for the induction and patterning of meso- its interaction with P-Smad2 (Cordenonsi derm and endoderm (Conlon et al. 1991; et al. 2007). Conlon et al. 1994; Feldman et al. 1998; A plethora of Nodal target genes have been Gritsman et al. 1999; Agius et al. 2000; Schier identified (Dickmeis et al. 2001; Bennett et al. and Shen 2000; Brennan et al. 2001; Schier 2007a; Guzman-Ayala et al. 2009). For exam- 2003; Bennett et al. 2007b; Shen 2007). For ple, genomic analyses in zebrafish show that example, in the absence of Nodal signaling, Nodal initiates a cascade of regulatory events mouse embryos do not form the primitive by inducing the expression of transcription streak and zebrafish embryos lack all endoderm factors and additional signaling molecules and head and trunk mesoderm, including noto- (Dickmeis et al. 2001; Bennett et al. 2007a). In chord, heart, kidney, blood, liver, pancreas, and addition, Nodal also has direct effects on cell gut (Conlon et al. 1991; Conlon et al. 1994; behavior by regulating cytoskeletal components Feldman et al. 1998; Gritsman et al. 1999; and other differentiation genes. With the excep- Bennett et al. 2007b). Nodal signaling is active tion of Nodal itself, the feedback inhibitor Lefty during blastula stages, when cells are pluripo- and the transcription factor pitx2, no down- tent. During normal embryogenesis, spatially stream genes have been found that are shared localized activity of the Nodal pathway between Nodal signaling in mesendoderm induces and patterns mesendoderm at the and left–right specification in all vertebrates appropriate position (Fig. 2). Conversely, (Adachi et al. 1999; Bisgrove et al. 1999; Norris ectopic activation of the pathway induces and Robertson 1999; Osada et al. 2000; Saijoh ectopic mesendoderm (Jones et al. 1995; et al. 2000; Faucourt et al. 2001; Shiratori et al. Toyama et al. 1995; Schier 2003; Shen 2007). 2001; Norris et al. 2002; Vincent et al. 2004; The in vitro differentiation of embryonic stem Saijoh et al. 2005; Shiratori et al. 2006; cells uses this inducing property of Nodal in Guzman-Ayala et al. 2009). This suggests which activation of Nodal signaling helps to

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A.F. Schier

Animal the epiblast. Nodal precursor protein produced Ectoderm by the epiblast induces the transcription of the convertases Furin and Pace4 in extraembryonic ectoderm (Ben-Haim et al. 2006). These con- vertases are secreted and process Nodal in the Mesoderm adjacent epiblast, initiating an autoregulatory feedback that enhances Nodal transcription in Endoderm a subset of epiblast cells that include mesendo- Mesoderm derm precursors (Beck et al. 2002). In addition Cyclops Lefty Squint to the local generation of Nodal mRNA and protein, inhibition of the pathway by extra- Yolk cellular antagonists further restricts mesen- Vegetal doderm formation. For example, loss of the inhibitor Lefty leads to the transformation of Figure 2. Mesoderm and endoderm induction in presumptive ectoderm into mesendoderm zebrafish. Ectoderm, mesoderm, and mesendoderm (Meno et al. 1999; Agathon et al. 2001; Chen precursors are arranged from animal to vegetal and Schier 2002; Feldman et al. 2002; Perea- positions at the blastula stage. The Nodal signals Squint and Cyclops and the inhibitor Lefty are Gomez et al. 2002). expressed at the blastula margin. Genetic studies suggest that Cyclops has short-range activity whereas Squint and Lefty have long-range activity. See text for details. Left–Right Patterning Vertebrate organs are positioned asymmetri- cally not only along the anterior–posterior maintain the pluripotent state and drive devel- and dorsal–ventral axes but also the left–right opment along the mesendodermal pathway axis (Shiratori and Hamada 2006). Nodal (Parisi et al. 2003; Brandenberger et al. 2004; genes are expressed in the left lateral plate meso- Kubo et al. 2004; Vallier et al. 2004; D’Amour derm and required for left–right axis specifica- et al. 2005; James et al. 2005; Pfendler et al. tion (Levin et al. 1995; Collignon et al. 1996; 2005; Vallier et al. 2005; Camus et al. 2006; Lowe et al. 1996; Yan et al. 1999; Lowe et al. Takenaga et al. 2007; Smith et al. 2008; Vallier 2001; Long et al. 2003; Kumar et al. 2008). et al. 2009). Asymmetric activation of the pathway induces Several mechanisms spatially restrict acti- asymmetric organ morphogenesis (Yan et al. vation of Nodal signaling. In zebrafish and 1999; Bamford et al. 2000; Concha et al. 2000; frog, Nodal genes are transcribed in the Concha et al. 2003; Halpern et al. 2003; vegetal region, which overlaps with endoderm Yashiro et al. 2007; Davis et al. 2008; de precursors and is adjacent to presumptive Campos-Baptista et al. 2008; Kurpios et al. mesoderm. Thus, local transcription generates 2008; Bakkers et al. 2009; Roussigne et al. a local source of Nodal signals. It appears that 2009). For example, activation of Nodal signal- high levels of Nodal signaling in and close to ing in left heart progenitors allows leftward the source induce endoderm, whereas lower movement and enhances the speed of cardio- levels induce mesoderm in neighboring myocytes (Baker et al. 2008; de Campos- cells (Schier 2003; Schier and Talbot 2005; Baptista et al. 2008; Bakkers et al. 2009). In Shen 2007). the zebrafish diencephalon, left-sided Nodal In contrast to the transcriptional restriction expression promotes neurogenesis in the left of Nodal gene expression in zebrafish and frog, habenula and the movement of the parapineal the local activation of mouse Nodal signaling organ to the left (Concha et al. 2000; Concha is initially achieved post-transcriptionally. et al. 2003; Halpern et al. 2003; Roussigne Nodal transcription is initiated throughout et al. 2009). During mouse development,

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Nodal Morphogens

Nodal signaling is required for asymmetric left versus right. However, similar to the re- organogenesis, ranging from lung lobe for- striction of Nodal signaling in mesendoderm mation (Yan et al. 1999) to gut morphogenesis development, extracellular antagonists belong- (Davis et al. 2008; Kurpios et al. 2008). In the ing to the Lefty family restrict Nodal signaling absence of Nodal signaling, organ asymmetry to only the left side (Meno et al. 1998; is lost, randomized, or isomeric. Feldman et al. 2002; Nakamura et al. 2006). Nodal expression in the left lateral plate mesoderm is dependent on Nodal expression in cells in or next to the embryonic midline NODAL SIGNALS AS MORPHOGENS (Fig. 3). For example, mouse Nodal is expressed Nodal signals can act as morphogens—they in the node and induces Nodal in the left lateral directly act at a distance from their site of pro- plate mesoderm. This process requires intact duction and induce concentration-dependent cilia and fluid flow in the node (Nonaka et al. responses in target cells. 1998; Nonaka et al. 2002) and appears to be mediated by the movement of Nodal protein to the lateral plate mesoderm (Brennan et al. Direct Long-Range Effects of Nodal Signals 2002; Nonaka et al. 2002; Saijoh et al. 2003; Nakamura et al. 2006; Oki et al. 2007). In con- The range of vertebrate TGF-b signals was a trast to mesendodermal patterning, graded contentious issue in the 1990s. It was unclear Nodal signaling does not appear to be required whether such signals can act directly at a dis- for left–right patterning. Thus, Nodal signaling tance or depend on relay signals that indirectly might simply control the binary decision of mediate their effects (Gurdon et al. 1994; Jones et al. 1996; Reilly and Melton 1996; McDowell et al. 1997). Studies of Activin revealed long-range effects, whereas studies of Midline TGF-b1 suggested short-range activity; how- Left Right ever, because the tested ligands are neither LPM LPM expressed in the blastula nor required for normal mesendoderm induction, the in vivo

Lefty relevance of these experiments was unclear (Schier and Shen 2000). The isolation of

Lefty Nodal genes allowed the analyses of endoge- nously expressed and functionally essential Nodal Nodal TGF-bs. This revealed both short- and long- range activity of Nodal signals during mesendo- derm induction (Chen and Schier 2001). Nodal/ GDF1 In zebrafish, several lines of evidence suggest cilia that the Nodal signals Cyclops and Squint have flow short- and long-range effects, respectively (Chen and Schier 2001). First, misexpression Node of Squint from a localized source induces long- Figure 3. Model for left–right patterning by Nodal range activation of Nodal downstream genes in and Lefty. Nodal/GDF1 heterodimers form in the surrounding tissue, whereas Cyclops only has region of the node and activate Nodal signaling in short-range activity (Fig. 2). Second, in vivo the lateral plate mesoderm (LPM). Cilia-induced target gene induction in Squint mutants is fluid flow biases Nodal activation toward the left. Subsequent Nodal autoregulation, induction of only short-range and originates from Cyclops. Lefty expression, and long-range inhibition by Lefty Conversely, Squint-mediated gene induction lead to robust pathway activation on the left and is still long-range in Cyclops mutants. Third, inhibition on the right. See text for details. the long-range effect of Squint appears to be

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A.F. Schier

direct, because Squint generated in coreceptor prechordal plate mesoderm (Schier et al. 1997; mutant (i.e., nonresponding) cells can traverse Gritsman et al. 2000; Thisse et al. 2000; a field of nonresponsive, coreceptor mutant Dougan et al. 2003; Vincent et al. 2003). cells and activate Nodal target genes in distant Togetherwith the local expression and long- wild-type cells. These experiments rule out a range activity of Nodal ligands, these findings Squint-induced relay signal and provided evi- have suggested a model wherein Nodal ligands dence that Squint can act directly at a long such as Squint form a concentration gradient range. Further support for direct long-range from the vegetal to the animal pole, much as Nodal action during mesendoderm formation T.H. Morgan had predicted (Schier 2003). has been provided by the distribution of This gradient provides positional information Xenopus Nodal protein Xnr2 (Williams et al. so that cells acquire fates according to their 2004). Local expression of Xnr2-GFP results in location in the gradient. A gradient of Nodal the extracellular movement of the fluorescent signals has not yet been visualized, but an protein from the source into nonexpressing activity gradient can be visualized by the tissue. domains of target gene expression, P-smad2 Nodal signals also have long-range activity levels, and nuclear accumulation of Smad2- during left–right specification. For example, fluorescent protein fusions (Faure et al. 2000; mouse Nodal can be generated in nonrespon- Gritsman et al. 2000; Chen and Schier 2001; sive coreceptor mutant node cells, traverse non- Lee et al. 2001; Harvey and Smith 2009). For responsive mesoderm, and activate Nodal example, zebrafish Smad2 accumulates in a target genes in distant wild-type cells in the vegetal-to-animal gradient in zebrafish blastula LPM (Oki et al. 2007). Similarly, Nodal ex- nuclei (Harvey and Smith 2009). These obser- pression in the LPM is required for expression vations support the idea that an activity gradi- of lefty1 in the midline (Yamamoto et al. ent of Nodal signaling specifies different cell 2003). Taken together, these studies clearly types along the vegetal-animal axis. establish that Nodal ligands can have direct long-range effects. Time-dependent Effects of Nodal Signals The spatial concentration gradient model is Concentration-dependent Effects of consistent with the gene expression domains Nodal Signals of Nodal downstream genes and the blastula Nodal signals can induce dose-dependent fate map, but it has several limitations. First, effects in responding cells as seen by varying zebrafish mesendoderm can eventually form the concentration of Nodal ligands and deter- normally even in the absence of the long-range mining downstream gene expression. Such signal Squint, i.e., solely by the action of the experiments have revealed at least two thresh- short-range ligand Cyclops (Feldman et al. olds for Nodal-dependent gene activation. For 1998; Chen and Schier 2001; Dougan et al. example, low levels of Nodal are sufficient to 2003). Second, downstream responses are not induce targets such as ntl/T/Brachyury and only determined by the concentration of the floating head/Xnot, whereas the targets goose- signal at a given time but also the duration of coid and casanova/sox32 are activated only by pathway activity. For example, expression of high levels of Nodal (Gurdon and Bourillot high-threshold targets is absent on premature 2001; Schier and Talbot 2005). Further evidence block of the pathway by receptor inhibitors for dose-dependent effects of Nodal signaling or loss of coreceptors (Gritsman et al. 2000; comes from mutants that partially decrease Aoki et al. 2002; Hagos and Dougan 2007). A Nodal activity. For example, partial reduction similar loss of high-threshold target gene of Nodal, Smad2/3, or EGF-CFC coreceptor expression is observed on delayed pathway activity leads to loss of high threshold target activation caused by late expression of corecep- gene expression and absence of endoderm and tors (Gritsman et al. 2000). Moreover, cells

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Nodal Morphogens

exposed to a uniform dose of Nodal mRNA pro- A Reaction–Diffusion System: Mid-range gressively move from low- to high-threshold Activation by Nodal and Long-range fates with increasing time of exposure (Hagos Inhibition by Lefty and Dougan 2007). Analogously, nuclear The noncanonical TGF-b signal Lefty is a accumulation of Smad2 increases within a potent feedback inhibitor of Nodal signaling one-hour time window in the late zebrafish (Meno et al. 1999). Several studies have blastula (Harvey and Smith 2009). These exper- suggested that Nodal and Lefty constitute an iments reveal a clear role for prolonged activator/inhibitor pair as postulated in reac- exposure to Nodal ligands and suggest that the tion–diffusion models of pattern formation cumulative dose (concentration and time) of (Saijoh et al. 2000; Chen and Schier 2002; Nodal determines the fate of responding cells. Hamada et al. 2002; Schier 2003; Nakamura The cumulative dose model might also explain et al. 2006). In such models, a locally acting acti- why short-range Cyclops can induce all mesen- vator induces both its own synthesis and the dodermal cell types in the absence of long-range synthesis of a long-range inhibitor (Fig. 4) Squint. Different times of exposure of blasto- (Turing 1952; Meinhardt and Gierer 2000). meres to Cyclops might allow the differential In the classic reaction–diffusion system, such activation of Nodal signaling and the generation interactions can result in self-organization that of different cell types. generates patterns in an initially homogenous The duration of Nodal signaling might be field of cells. Nodal/Lefty share the activator/ measured at several points in the pathway. For inhibitor and self-enhancement features of example, to elicit a particular response, suffi- this system. For example, in the zebrafish blas- cient ligand has to accumulate over time. tula, Nodal activates Nodal and Lefty transcrip- Thus, in Squint mutants, induction of cell tion at the margin, and Lefty is required to fates is delayed, whereas overexpression of restrict the range of Nodal signaling by blocking Nodal ligands can accelerate fate specification both the generation of Nodal locally and the (Hagos and Dougan 2007). Timing is also response to Nodal at a distance (Meno et al. likely to influence events at the receptor level. 1999; Chen and Schier 2002). The Nodal/Lefty For example, blocking receptor activity after interaction occurs in three steps: (1) Nodal exposure to high doses of Nodal attenuates the expression is activated, resulting in pathway response and leads to absence of high-threshold activation. (2) Pathway activation results in target gene expression (Hagos and Dougan Lefty expression. (3) Lefty inhibits the pathway. 2007). This result argues against the idea that a given concentration of Nodal is sufficient to induce a long-lasting response and suggests Nodal [c] that cumulative dose is measured at the receptor level or downstream. Indeed, studies on Activin suggest that one mechanism of Lefty memory of previous exposure to ligand might be the high stability and residence of signal- receptor complexes in intracellular vesicles after endocytosis and before lysosomal Source Target field x degradation (Dyson and Gurdon 1998; Figure 4. Reaction/Diffusion Model for Patterning by Gurdon and Bourillot 2001; Jullien and Nodal and Lefty. Nodal induces its own expression Gurdon 2005). Taken together, these studies and forms a concentration gradient (c, concen- suggest that the cumulative dose of Nodal sig- trations of Nodal and Lefty; x, distance). Lefty is induced by Nodal and blocks Nodal signaling. The naling determines cells fates. It is unclear, model postulates that Lefty has a longer range and however, how concentration and duration are more shallow distribution than Nodal. Thus, Nodal translated into positional identities (Ashe and signaling is active close to the source but inhibited Briscoe 2006). at a distance. See text for details.

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The exact pattern of pathway activation depends not been visualized in vivo, and it remains on the local concentrations of Nodal and Lefty. unclear how exactly their interaction provides In the blastula, genetic experiments indicate positional information. that Squint and Cyclops are in excess close to the source, whereas Lefty is present at higher Modulation of Nodal Morphogen Activity levels at a distance from the source (Chen and Schier 2002). In the absence of Lefty, more The distribution and activity of morphogens are Nodal is generated, and cells at a distance are controlled by multiple factors. Prominent roles no longer inhibited by Lefty. On excess of are played by the rate and level of morphogen Lefty, Nodal production and signaling are production at the source, the rate of morphogen inhibited. Thus, Nodal/Lefty constitute a movement from the source into surrounding reaction–diffusion activator/inhibitor pair tissues, and the availability and stability of the but they do not generate pattern de novo in an morphogen. For example, the higher the pro- initially homogenous field of cells (Fig. 2). duction, diffusion, and stability of a morpho- Rather, localized maternal determinants acti- gen, the longer is its range. Conversely, the vate Nodal expression, which then induces effects of morphogens are determined by the Lefty. The Nodal/Lefty interaction is also an responsiveness of target cells. For example, example of how a morphogen, Nodal, can receptor levels and inhibitors can influence modulate its effects by inducing a secondary cellular responses. In the following section, signal, Lefty. Thus, target cells are not merely some of the molecular mechanisms that modu- passive responders but change the activity late the range and activity of Nodal morphogens gradient by their response. is discussed. It is worth emphasizing, however, The Nodal/Lefty activator/inhibitor pair that this analysis is still in its infancy. For also plays a role during left–right specification example, when experimental manipulations (Hamada et al. 2002; Nakamura et al. 2006). change the Nodal signaling range or response, In this system, Nodal/Lefty interactions appear it is often unclear whether this change is to amplify small differences between left and caused by alterations in morphogen produc- right lateral plate mesoderm (Fig. 3). Both left tion, mobility, trafficking or stability, changes and right can initially express low levels of in regulatory feedback interactions, or differ- Nodal but by an unknown mechanism, cilia- ences in target cell responsiveness. induced flow in the node is thought to gener- ate a slightly higher accumulation of Nodal in Expression left lateral plate mesoderm. This initial asym- metry is amplified by Nodal autoregulation The concentrations and ratio of Nodal and and the induction of Lefty on the left and in Lefty signals determine the range of Nodal the midline. The long-range activity of left- signaling—the more Nodal and the less Lefty, sided and midline Lefty then suppresses Nodal the higher the activation of the pathway. The amplification on the right. Mathematical mod- expression levels of these genes must therefore eling supports this self-enhancement lateral be exquisitely regulated. For instance, in frog inhibition model (Nakamura et al. 2006). As and zebrafish, Nodal genes are first activated in the blastula, this system is not entirely self- on the future dorsal side and then are expressed organizing but biased by a prepattern. In con- in the vegetal and marginal region of the blas- trast to the blastula, however, the output is not tula (Schier 2003). This expression pattern is graded but discrete: On (left) versus off (right). reflected in the activation of the pathway. In Taken together, these studies have provided frogs, P-Smad2 is first detected on the dorsal strong genetic evidence that Nodal/Lefty are side, and in zebrafish, nuclear Smad2 levels are part of a two-component reaction–diffusion highest in dorsal blastomeres (Faure et al. system. However, the presumptive long-range 2000; Lee et al. 2001; Harvey and Smith 2009). distributions of Nodal and Lefty proteins have Correspondingly, genetic studies have shown

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Nodal Morphogens

that the induction of dorsal mesodermal cell zebrafish miR-430 dampens the mRNA levels fates requires higher levels of Nodal signaling and translation of Squint, lefty1, and lefty2 than ventral and lateral mesoderm, and misex- (Choi et al. 2007), one of the many roles of pression studies have revealed that the induc- this microRNA (Giraldez et al. 2005; Giraldez tion of dorsal mesoderm markers such as gsc et al. 2006; Mishima et al. 2006). In the requires higher levels of Nodal signaling than absence of miR-430, both agonist and an- the induction of pan-mesodermal genes such tagonist are up-regulated. Intriguingly, this as ntl (Schier et al. 1997; Gritsman et al. 2000; up-regulation leads to an imbalance of Nodal Dougan et al. 2003). Moreover, fate mapping versus Lefty inputs, so that Lefty activity pre- studies have shown that partial reduction of vails and Nodal signaling is reduced. It is Nodal signaling leads to the loss of dorsal meso- unclear why Lefty derepression dominates dermal cell fates (Gritsman et al. 2000; Dougan Squint derepression. It is conceivable that et al. 2003). Instead, these cells acquire more Lefty is misexpressed at higher levels than animal cell fates and form neural structures. Squint on loss of miR-430 repression. Alter- Importantly, no transformation into more natively, it is possible that nonlinear steps in ventral mesodermal cell fates is observed on Nodal/Lefty regulation favor inhibition of the inhibition of Nodal signaling. Thus, despite pathway in the absence of miR-430. A similar higher nuclear accumulation of Smad2 on the inhibition of Nodal antagonism is observed dorsal side and the suggestion that Nodal sig- in human ES cells, where the miR-430 ortho- naling patterns mesoderm along the dorsal– logue miR-302 is required to repress Lefty and ventral axis (Harvey and Smith 2009), there is promote mesendoderm development (Rosa no evidence for a requirement of graded et al. 2009). Nodal signaling in specifying dorsal versus MicroRNAs have also been implicated in the ventral mesodermal fates. Rather, graded regulation of type II activin receptor expression. Nodal signaling patterns the vegetal-animal Xenopus miR-15/16 is expressed in ventral- axis and blocks the formation of neural struc- lateral regions and inhibits type II Activin re- tures at the dorsal margin (Feldman et al. ceptor expression (Martello et al. 2007). This 2000; Carmany-Rampey and Schier 2001; results in higher activin receptor levels in Dougan et al. 2003). dorsal compared with ventral blastomeres, Following the induction of Nodal gene resulting in higher responsiveness to Nodal expression, regulatory interactions between signals on the dorsal side. This effect is further Nodal and Lefty generate an intricate system augmented by Rap2, which is involved in recy- to modulate Nodal signaling. Nodal activates cling of Activin receptors (Choi et al. 2008). its own transcription, which if unchecked Rap2 is initially enriched dorsally and thus results in a positive feedback that dramatically also contributes to higher receptor levels and increases Nodal gene expression. However, thus the earlier and higher induction of Nodal because Nodal also activates Lefty expression signaling in dorsal blastomeres. These studies and Lefty inhibits pathway activity, a negative demonstrate that microRNAs are important feedback dampens, spatially restricts, and tem- modulators of Nodal morphogen signaling. porally attenuates Nodal signaling (Meno et al. 1999; Chen and Schier 2002; Feldman Stability and Movement et al. 2002; Dougan et al. 2003). It is still unclear how exactly these auto- and cross- Nodal pathway activation in a field of cells is not regulatory interactions contribute to the only dependent on the levels of Nodal and Lefty robustness and precision of patterning, but at the source, but also on the levels that reach recent evidence suggests that the balance of target cells. These levels are determined by the Nodal/Lefty is carefully regulated not only movement and stability of Nodal and Lefty— at the level of transcription but also post- the more stable and mobile, the longer the transcriptionally by microRNAs. In particular, range of the signal.

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The movement of Nodal proteins is poorly region in the Cyclops prodomain restricts its understood. It has been shown in Xenopus range (Tian et al. 2008). Expression of the explants that the Nodal protein Xnr2 is distrib- mature domain of Squint or mouse Nodal uted through extracellular routes and not results in an unstable signal that only acts at a through uptake and release from cells, a short range, whereas the mature domain of process called transcytosis (Williams et al. Cyclops does not have any activity by itself and 2004). It is unclear, however, if this extracellular requires the prodomain (Le Good et al. 2005; movement is purely diffusive or if active trans- Tian et al. 2008). In contrast, insertion of an port is involved. Studies of the TGF-b signals N-glycosylation site increases the stability of Activin and Dpp favor this possibility, but mouse Nodal in cell culture and extends its detailed biophysical studies are required to range in zebrafish embryos (Le Good et al. directly test Nodal diffusion (McDowell et al. 2005). These results have suggested that 1997; Kinoshita et al. 2006; Kicheva et al. increased proteolytic maturation of Nodal 2007; Kicheva and Gonzalez-Gaitan 2008). potentiates local signaling, whereas increased Such studies are also crucial to determine Nodal stability extends long-range signaling. In whether the different activity ranges of Squint, tissue culture, Nodal precursor is more stable Cyclops, and Lefty are because of different dif- than processed Nodal, indicating that the site fusibilities. For example, studies of Dpp have and timing of processing might influence the measured a diffusion coefficient of 0.1 mm2/s range of signaling (Blanchet et al. 2008a; in imaginal discs (Kicheva et al. 2007; Kicheva Constam 2009). Interaction with EGF-CFC and Gonzalez-Gaitan 2008). Modeling studies proteins can target mouse Nodal into specific indicate that this value would only allow short- endocytic compartments and might thus also range signaling within the 2–3 hour time frame contribute to the stability and intracellular during which the zebrafish blastula is patterned movement of Nodal ligands (Le Good et al. by Nodal signals (Lee, Robson, and Schier, 2005; Blanchet et al. 2008a; Blanchet et al. unpublished results). Hence, it is unclear how 2008b). These studies suggest that the location diffusibility contributes to the range of Nodal and regulation of Nodal processing plays a signaling. This is a particularly important major role in determining stability and range. issue because one of the tenets of reaction– The range of Nodal signaling is modulated diffusion models is that the inhibitor (Lefty) by additional extracellular proteins. For has a longer range than the activator (Nodal). example, Nodal secreted in the node can acti- It is also unclear how processing, stability, vate signaling in the distant LPM only in the post-translationalmodifications,andtrafficking presence of the glycosaminoglycan chondroitin contribute to the range of Nodal signals. Differ- sulfate (Oki et al. 2007). Because ectopic Nodal ent Nodal signals can have different signaling can still activate the pathway on chondroitin ranges. For instance, Cyclops has a shorter sig- sulfate perturbation, it is likely that the inter- naling range than Squint (Chen and Schier action of Nodal with sulfated glycosamino- 2001). The differences in range are thought to glycans increases ligand movement or stability be caused by differences in both the prodomains and not receptor binding or activation. and mature domains (Chen and Schier 2001; Further complexity arises from the poten- Jing et al. 2006; Tian et al. 2008). For example, tial for TGF-b heterodimer formation. For a chimeric protein consisting of the Cyclops example, the related TGB-b signal GDF1 is prodomain and Squint mature domain has a also involved in left–right specification longer range than Cyclops protein but a (Rankin et al. 2000). Similar to Nodal, it is shorter range than Squint. An acidic region in expressed in the node and LPM. Node ex- the amino terminus of the Squint mature pression is required for left-sided activation of ligand is required for the longer range of the Nodal target genes, and expression in the LPM Cyclops-Squint chimera (Jing et al. 2006). is required for the expansion of Nodal signaling Conversely, a presumptive lysosomal targeting in the LPM. Native GDF1 homodimers appear

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Nodal Morphogens

largely inactive but GDF1/Nodal heterodimers wherein a naı¨ve group of cells homogeneously are active and even more potent than Nodal responds to different morphogen concentra- homodimers in activating Nodal reporter tions. For example, ectodermin is maternally genes (Tanaka et al. 2007). These heterodimers expressed and localized to the animal pole in also have a longer activity range but it is Xenopus, thus creating a zone that is less respon- unclear if this is only because of increased sive to Nodal and BMP signals (Dupont et al. specific activity in pathway activation or also 2005; Dupont et al. 2009). Additional tran- caused by increased diffusion or stability. scription factors such as Sox3, Xema/FoxI1e, Serum response factor, and XFDL156, and the Cerberus-like protein Coco are also expressed Interpretation in presumptive ectoderm and protect ectoderm The mechanisms underlying the interpretation from Nodal inducers (Bell et al. 2003; Suri et al. of Nodal signals in target cells are poorly under- 2005; Mir et al. 2007; Yun et al. 2007; Zhang and stood. Studies of Activin in Xenopus have Klymkowsky 2007; Sasai et al. 2008). Thus, pre- revealed a linear relationship of TGF-b concen- patterning will modulate the generation of and tration, receptor activation, Smad complex response to Nodal signaling gradients. formation, and downstream gene activation (Gurdon and Bourillot 2001). Moreover, CONCLUDING REMARKS Activin can act in a ratchet mechanism, i.e., cells have a memory of the highest levels of Studies of Nodal signaling led to the discovery Activin they have been exposed to and of the bona fide mesendoderm inducers and respond accordingly. This memory resides at left–right determinants, identified the first the level of receptor-ligand complexes and is endogenous vertebrate morphogen, established maintained by the intracellular localization of the existence of an activator/inhibitor pair signaling complexes in endosomes (Jullien as postulated in reaction–diffusion models, and Gurdon 2005). It is unclear whether revealed roles for microRNAs in morphogen Nodal signals behave similarly to Activin. For signaling and highlighted the complex roles of example, Activin is not dependent on processing, trafficking, and post-translational EGF-CFC proteins for pathway activation and modifications during morphogen signaling. cannot be inhibited by Leftys (Gritsman et al. Despite this progress, a quantitative or bio- 1999; Cheng et al. 2004). Thus, it is possible physical understanding of Nodal morphogens that Nodal–receptor complexes are disrupted is largely elusive. We do not know the shape of by Lefty, undermining potential ratchet mech- the putative Nodal morphogen gradient, the anisms and pathway linearity. in vivo diffusion, processing, trafficking, and Finally, it is not clear how Nodal target genes degradation properties of Nodal and Lefty, the respond to different levels of Nodal signaling. binding and reaction kinetics of the pathway Based on studies of other morphogens, it is components, the mechanisms by which signal assumed that the differential affinity for cis- concentration and duration are translated into regulatory elements determines the threshold positional information, or the robustness and at which a downstream gene is activated. The precision encoded by the Nodal morphogen responses of known downstream genes to dif- gradient. Filling these gaps will be necessary to ferent concentrations of Nodal are consistent truly understand how the Nodal morphogen with this model, but direct evidence is lacking. patterns developing tissues. The situation is further complicated by the existence of maternally generated prepatterns ACKOWLEDGMENTS in early fish and frog embryos. The localization of pathway antagonists in the presumptive I thank Daniel Constam, Susan Mango, Michael ectoderm changes the response properties of Shen, Will Talbot, and members of my lab for target tissues and complicates simple models comments on the manuscript, and the NIH,

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A.F. Schier

factor FoxH1 (FAST) mediates Nodal signaling during Yeo C, Whitman M. 2001. Nodal signals to Smads through anterior-posterior patterning and node formation in Cripto-dependent and Cripto-independent mechan- the mouse. Genes Dev 15: 1242–1256. isms. Mol Cell 7: 949–957. Yamamoto M, Mine N, Mochida K, Sakai Y,Saijoh Y,Meno Yun CH, Choi SC, Park E, Kim SJ, Chung AS, Lee HK, Lee C, Hamada H. 2003. Nodal signaling induces the midline HJ, Han JK. 2007. Negative regulation of Activin/Nodal barrier by activating Nodal expression in the lateral plate. signaling by SRF during Xenopus gastrulation. Development 130: 1795–1804. Development 134: 769–777. Yamamoto M, Saijoh Y, Perea-Gomez A, Shawlot W, Zhang C, Klymkowsky MW. 2007. The Sox axis, Nodal Behringer RR, Ang SL, Hamada H, Meno C. 2004. signaling, and germ layer specification. Differentiation Nodal antagonists regulate formation of the anteropos- 75: 536–545. terior axis of the mouse embryo. Nature 428: 387–392. Zhang J, Talbot WS, Schier AF. 1998. Positional cloning Yan YT, Gritsman K, Ding J, Burdine RD, Corrales JD, Price identifies zebrafish one-eyed pinhead as a permissive SM, Talbot WS, Schier AF, Shen MM. 1999. Conserved EGF-related ligand required during gastrulation. Cell requirement for EGF-CFC genes in vertebrate left–right 92: 241–251. axis formation. Genes Dev 13: 2527–2537. Zhang L, Zhou H, Su Y,Sun Z, Zhang H, Zhang L, Zhang Y, Yan YT, Liu JJ, Luo YEC, Haltiwanger RS, Abate-Shen C, Ning Y,Chen YG, Meng A. 2004. Zebrafish Dpr2 inhibits Shen MM. 2002. Dual roles of Cripto as a ligand and mesoderm induction by promoting degradation of nodal coreceptor in the nodal signaling pathway. Mol Cell receptors. Science 306: 114–117. Biol 22: 4439–4449. Zhou X, Sasaki H, Lowe L, Hogan BL, Kuehn MR. Yashiro K, Shiratori H, Hamada H. 2007. Haemodynamics 1993. Nodal is a novel TGF-b-like gene expressed in determined by a genetic programme govern asymmetric the mouse node during gastrulation. Nature 361: development of the aortic arch. Nature 450: 285–288. 543–547.

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Nodal Morphogens

Alexander F. Schier

Cold Spring Harb Perspect Biol 2009; doi: 10.1101/cshperspect.a003459 originally published online August 26, 2009

Subject Collection Generation and Interpretation of Morphogen Gradients

Regulation of Organ Growth by Morphogen Gradients in Planarian Regeneration and Gradients Homeostasis Gerald Schwank and Konrad Basler Teresa Adell, Francesc Cebrià and Emili Saló Signaling Gradients during Paraxial Mesoderm Shaping Morphogen Gradients by Proteoglycans Development Dong Yan and Xinhua Lin Alexander Aulehla and Olivier Pourquié Morphogen Gradient Formation Forming Patterns in Development without Ortrud Wartlick, Anna Kicheva and Marcos Morphogen Gradients: Scattered Differentiation González-Gaitán and Sorting Out Robert R. Kay and Christopher R.L. Thompson Nodal Morphogens Robust Generation and Decoding of Morphogen Alexander F. Schier Gradients Naama Barkai and Ben-Zion Shilo Gradients and the Specification of Planar Polarity Models for the Generation and Interpretation of in the Insect Cuticle Gradients David Strutt Hans Meinhardt Vertebrate Limb Development: Moving from Graded Dorsal and Differential Gene Regulation in Classical Morphogen Gradients to an Integrated the Drosophila Embryo 4-Dimensional Patterning System Gregory T. Reeves and Angelike Stathopoulos Jean-Denis Bénazet and Rolf Zeller Establishing and Interpreting Graded Sonic Chemical Gradients and Chemotropism in Yeast Hedgehog Signaling during Vertebrate Neural Robert A. Arkowitz Tube Patterning: The Role of Negative Feedback Vanessa Ribes and James Briscoe Systems Biology of the Self-regulating Gradients in the Brain: The Control of the Morphogenetic Gradient of the Xenopus Gastrula Development of Form and Function in the Jean-Louis Plouhinec and E. M. De Robertis Cerebral Cortex Stephen N. Sansom and Frederick J. Livesey For additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/

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Copyright © 2009 Cold Spring Harbor Laboratory Press; all rights reserved