Molina MD , Quirin M , Haillot E , De Crozé N , Range R , Rouel M , Jimenez F , Amrouche R , Chessel A , Lepage T .

	Fig 1. Repression of the proximal promoter region of nodal by ETS factors and expression pattern of Yan/Tel.A, Alignment of the proximal cis-regulatory module of nodal (R module) of the sea urchin species Paracentrotus lividus and Strongylocentrotus purpuratus showing the conservation of ETS binding sites (highlighted in light green). Two extra ETS binding sites are present in the cis-regulatory module of P. lividus (highlighted in light brown). B, Luciferase assays showing that mutations of putative ETS sites increase the transcriptional activity of the nodal promoter. The data are presented as the ratio of luciferase expression between each construct and the wild-type R module (green). The bars on the graphs represent the standard error. C, Expression pattern of yan/tel during normal development. After a phase of maternal ubiquitous expression, yan/tel is strongly expressed in the precursors of the skeletogenic mesoderm (arrowheads at HB stage). EB, early blastula; HB, hatching blastula; vv, vegetal view; lv, lateral view. In the lateral view, animal is to the top.
	Fig 2. Yan/Tel controls the spatial restriction of nodal expression in the ectoderm.A, Inhibition of maternal Yan/Tel function disrupts dorsal-ventral axis formation. Note the radial arrangement of the primary mesenchymal cells (PMCs, arrowheads) at gastrula stage (24hpf) and the rounded shape of the embryo at prism stage (36hpf). (hpf), hours post-fertilization. B-C, Inhibition of yan/tel mRNA translation causes a massive ectopic expression of nodal and chordin at blastula (B) and gastrula (C) stages. Note that the expression has expanded throughout most of the ectoderm but is excluded from the animal pole region. apv, animal pole view. D, Inhibition of yan/tel mRNA translation expands the expression of the ventral ectodermal markers bmp2/4 and lefty and suppresses the expression of the dorsal ectodermal markers 29D and tbx2/3. E, Inhibition of yan/tel mRNA translation expands the expression of the ventral mesodermal marker gata1/2/3 and suppresses the expression of dorsal mesodermal marker gcm. F, Random local inhibition of Yan/Tel function orients the dorsal-ventral axis. In all embryos injected randomly with the yan/tel morpholino into one blastomere at the 2 or 4-cell stage, nodal expression (blue) overlaps with the progeny of the injected blastomere (red). G, Ectopic expression of the Nodal downstream target gene chordin following inhibition of yan/tel mRNA translation depends on Nodal pathway activity. Note that inhibition of yan/tel function followed by treatment with the Nodal receptor inhibitor SB431542 blocks the ectopic expression of chordin observed in yan/tel morphants. SB, swimming blastula stage; LG, late gastrula stage. MB, mesenchyme blastula stage; vv, vegetal view; lv, lateral view. In the lateral views, animal is to the top, and ventral to the left.
	Fig 3. Yan/Tel is required before Lefty to restrict nodal expression and acts redundantly with FoxQ2 in the animal pole region.A, Time-course of nodal expression in control, lefty morpholino and yan/tel morpholino injected embryos. Note that ectopic expression of nodal is detected in the yan/tel morphants earlier than in the lefty morphants. (EB), early blastula; (PHB), pre-hatching blastula. B, Note that nodal is ectopically expressed in the animal pole region (asterisks) of embryos co-injected with foxQ2 and tel or panda morpholinos, but absent from this region after co-injection of the lefty and panda or yan/tel morpholinos. All views are lateral. Animal is to the top, and ventral to the left.
	Fig 4. Phosphorylation of Yan/Tel by MAPKs regulates nodal expression.A, Structure of wild-type Yan/Tel and of phosphorylation site mutant forms of Yan/Tel used in this study. The Yan/Tel protein contains a SAM domain, an ETS binding site domain and several MAPK consensus phosphorylation sites indicated as S or T. The 3 canonical consensus MAPK phosphorylation sites are highlighted in yellow and the cluster of 4 putative phosphorylation sites is highlighted in blue. B, Overexpression of a wild-type form of Yan/Tel causes little effects on development of the embryos, while overexpression of phosphorylation mutant forms of Yan/Tel progressively radializes the embryos. The phosphorylation mutant yan/tel10A produces a phenotype similar to the nodal loss-of-function phenotype: note the radial arrangement of the PMCs, the abundance of pigment cells, the straight archenteron and the thickened ectoderm. Overexpression of the phosphorylation mutant form yan/tel13A fully radializes the embryo and disrupts gastrulation. All images are vegetal views at pluteus stage. Lateral views of the same embryo are shown in the upper corner. C, Overexpression of the phosphorylation mutant forms of yan/tel increase the percentage of embryos that fail to express nodal compared to overexpression of wild-type yan/tel mRNA. Embryos are shown at swimming blastula stage (SB) and are vegetal views. D, Western blot against HA-tagged versions of wild-type and mutant Yan/Tel. Mutation of the β-TRCP degradation motif does not change significantly the migration of Yan/Tel but results in a marked stabilization of the protein. In contrast, MAPK, cluster and yan/tel13A phosphorylation mutants migrate predominantly as a fast migrating isoform. HA-Tagged GFP mRNA was co-injected as a control. E, Random injection of a phosphorylation mutant form of Yan/Tel efficiently orients the dorsal-ventral axis. DIC and fluorescent images of an embryo injected randomly into one blastomere at the 2-cell stage with the yan/tel13A mRNA. In all embryos injected, nodal expression (blue) at swimming blastula stage is systematically found on the side opposite to the injection clone (red). vv, vegetal view; lv, lateral view, with animal to the top, and ventral to the left.
	Fig 5. MAP kinases phosphorylate sea urchin Yan/Tel.A, Western blot against a wild type HA-tagged version of yan/tel in the presence or absence of the p38 inhibitor BIRB-796, the ERK inhibitor U0126, the JNK inhibitor Sp600125 or a combination of all three drugs. Wild-type Yan/Tel typically migrates as multiple bands with three to four migrating isoforms. Slower migrating isoforms are absent after treatment with the BIRB-796, U0126 or SP600125 drugs while wild type Yan/Tel migrates as a unique faster migrating isoform after simultaneous inhibition of all three MAP Kinases. B, p38, JNK and ERK are required for strong expression of nodal. Treatment with the JNK inhibitor SP600125 or combined inhibition of p38, ERK and JNK reduce although do not abolish the expression of nodal. Lv, lateral view. HB, hatching blastula, EB, early blastula. C, Western blot against p38, JNK and ERK phosphorylated forms during development. Note that these MAP kinases are upregulated between the 60-cell stage and the EB stage. 2, 16 and 60 refers to the number of cells; VEB, Very Early Blastula; EB, Early Blastula; PHB, Prehatching Blastula; HB, Hatching blastula. D, Fluorescent immunostaining against phosphorylated forms of ERK and p38. At early blastula stage, nuclear P-ERK is detected in ectodermal cells (arrowheads) and in the precursors of the skeletogenic mesoderm located at the vegetal pole, while nuclear P-p38 shows a broader distribution. The dorsal clearance of nuclear P-p38 staining is pointed by arrowheads. EB, Early Blastula; PHB, Prehatching Blastula. E, Epistasis experiments with Yan/Tel, p38, JNK and ERK. Similar to the inhibition of yan/tel function, simultaneous inhibition of p38, ERK or JNK and Yan/Tel results in a massive ectopic expression of nodal. SB, Swimming blastula stage. F, Western blot against the HA-tagged form of wild type Yan/Tel. Overexpression of Nodal mRNA enriches the slowest migrating isoform of wild type Yan/Tel suggesting that Nodal can promote the phosphorylation of Yan/Tel. This effect can be reversed by the addition of the MAP kinase inhibitors of p38, ERK or JNK. vv, vegetal view. lv, lateral view. In lateral views, animal is to the top. G, Western blot against the HA-tagged form of wild type Yan/Tel. Treatment with the Nodal receptor inhibitor SB431542 enriches the fast migrating Yan/Tel isoform. Reciprocally, overexpression of the mRNA encoding the activated Nodal receptor Alk4QD or treatment with nickel chloride (a treatment that phenocopies nodal overexpression) promotes phosphorylation of Yan/Tel as indicated by the enrichment of the slowest migrating isoform of Yan/Tel.
	Fig 6. GSK3β phosphorylates sea urchin Yan/Tel and regulates its stability.A, Structure of Yan/Tel protein. B, Alignment showing conservation of the β-TRCP motif of sea urchin Yan/Tel and human β-catenin, Emi, IKβ, p100 and Snail proteins. Conserved phosphorylation sites are highlighted in orange. C, Western blot against the HA-tagged form of wild type or the phosphorylation mutant Yan/Tel (Yan/Tel10A). Wild-type Yan/Tel typically migrates as multiple bands with three to four migrating isoforms while the phosphorylation mutant Yan/Tel10A migrates as a unique and faster migrating isoform. Slower migrating isoforms of wild type Yan/Tel are absent after treatment with lithium, a GSK3β inhibitor. Note the enrichment of wild type Yan/Tel protein after treatment with the GSK3β inhibitor and the proteasome inhibitor MG132. Reciprocally, overexpression of GSK3β mRNA causes a strong reduction of the intensity of the signal of wild type Yan/Tel. The abundance of the unique and faster isoform observed after overexpression of the phosphorylation mutant Yan/Tel10A it is not altered by the presence of lithium or GSK3β mRNA. D, Western blot against the HA-tagged form of wild type Yan/Tel. Overexpression of mRNA encoding the β-TRCP ubiquitin ligase eliminates the slower phosphorylation isoforms of wild type Yan/Tel. HA-Tagged GFP mRNA was co-injected as a control. E, Western blot against GSK3-Tyr216 phosphorylated form during development. Note that GSK3 phosphorylated on Tyr216, which is the active form of this kinase, is upregulated during cleavage and peaks at the early blastula stage. 2, 16 and 60 refers to the number of cells; VEB, Very Early Blastula; EB, Early Blastula; PHB, Prehatching Blastula; HB, Hatching blastula. F, Short treatments with lithium abolish nodal expression. A short treatment with lithium at early blastula stage is sufficient to completely eliminate nodal expression and to dramatically reduce foxq2 expression, without expanding the expression of the endodermal marker foxa. vv, vegetal view. lv, lateral view. The upper panels show vegetal views of the same embryo. In lateral views, animal is to the top. G, Epistasis experiments with Yan/Tel and short treatment with lithium. In this experiment, a short treatment with lithium eliminated nodal expression in 50% of the treated embryos. In contrast, similar to the ectopic expression of nodal observed after inhibition of yan/tel function, simultaneous inhibition of GSK3β and Yan/Tel resulted in a massive ectopic expression of nodal in 100% of the observed embryos. HB, Hatching blastula stage. lv, lateral view. Animal is to the top. H, GSK3β phosphorylates Yan/Tel in vitro. Alignment of the sea urchin Yan/Tel β-TRCP and cluster domains and the β-TRCP domain of human β-catenin. Conserved positions of putative phosphorylation sites are highlighted in brown. Western blot of a GSK3β-CK1 in vitro kinase assay shows phosphorylation by the Kinase GSK3β of the wild type β-TRCP and cluster sea urchin Yan/Tel GST–fused peptides but not of the alanine mutated GST peptides. A GST-peptide of the β-TRCP domain of human β-catenin was used as a control.
	Fig 7. Epistasis experiments with Panda and Yan/Tel.A, Overexpression of yan/tel mRNA or panda mRNA into one cell at the two-cell stage orients the axis even in the absence of Panda or Tel/Yan, respectively. B, panda mRNA overexpression into one cell at the two-cell stage (FLDX clone developed in red) does not restrict early nodal expression (blue) to the ventral side in the absence of Yan/Tel. Instead, Yan/Tel mRNA overexpression into one cell at the two-cell stage (FLDX clone developed in red) confine nodal expression (blue) to the ventral side even in the absence of Panda. Vegetal views, ventral to the left. C, Western blot against the HA-tagged form of wild type Yan/Tel. Overexpression of the mRNA encoding Panda eliminates the slower phosphorylation isoforms of wild type Yan/Tel. D, Embryos injected with suboptimal doses of Panda or Yan/Tel morpholinos develop into pluteus larvae, while double Panda + Yan/Tel morphants appear partially ventralized and show radial chordin expression at late gastrula stage (LG). lv, Lateral view, ventral to the left. vv, vegetal view.
	Fig 8. Model of the regulation of nodal expression by GSK3, Yan/Tel and Panda.Model of the regulation of nodal expression by the maternal factors Yan/Tel and Panda. Starting at the 32-cell stage GSK3 activity in the animal hemisphere starts to target Yan/Tel for degradation, thereby releasing the repression of Yan/Tel on nodal expression and allowing nodal expression to be initiated. On the dorsal side of the embryo, Panda creates the asymmetry of nodal expression by antagonizing nodal expression by a mechanism that is not elucidated but that may rely on the function of Yan/Tel. At early blastula stage, nodal expression in the animal pole is repressed by the presence of FoxQ2. In the rest of the ectoderm, MAP kinases, GSK3 and possibly Nodal signaling contribute to promote nodal expression by promoting phosphorylation and degradation of Yan/Tel. On the contrary, Panda signaling on the dorsal side may prevent degradation of Yan/Tel contributing to repress nodal expression on the dorsal side.

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