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The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo.

Haillot E, Molina MD, Lapraz F, Lepage T - PLoS Biol. (2015)

Bottom Line: We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression.Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates.Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-β may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Valrose, iBV, UMR 7277 CNRS, Inserm U1091, UNS, University of Nice Sophia Antipolis, Nice, France.

ABSTRACT
Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-β may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8. We provide evidence that, although panda mRNA is broadly distributed in the early embryo, local expression of panda mRNA efficiently orients the dorsal-ventral axis and that Panda activity is required locally in the early embryo to specify this axis. Taken together, these findings demonstrate that maternal panda mRNA is both necessary and sufficient to orient the dorsal-ventral axis. These results therefore provide evidence that in the highly regulative sea urchin embryo, the activity of spatially restricted maternal factors regulates patterning along the dorsal-ventral axis.

No MeSH data available.


Related in: MedlinePlus

The BMP type I receptor Alk1/2 is essential for D/V patterning.(A) Morphology of embryos at 72 hours after fertilization (hpf) injected with morpholinos targeting either the alk3/6, alk1/2, or bmp2/4 transcripts. Note the striking similarity of the phenotypes of alk1/2 and bmp2/4 morphants that both develop with a ciliary band on the dorsal side (black arrowheads) compared to the less severe phenotype of alk3/6 morphants that is evidenced by the presence of pigment cells (black arrows) and of a less well-developed ciliary band on the dorsal side. (B) Expansion of the ventral and ciliary band fates at the expense of the dorsal ectoderm in alk1/2 morphants was revealed by the analysis of marker genes. Controls and alk1/2 morphants embryos were stained by in situ hybridization with the indicated probes. In alk1/2 morphants at mesenchyme blastula, the territory expressing the ventral marker genes, nodal, chordin, and foxA is largely normal, but consistently, a slight broadening of nodal expression is observed (white arrowheads), while expression of the dorsal gene hox7 is suppressed. At the gastrula stage, however, this ventralization is patent with chordin and foxA expression extending towards the dorsal side in alk1/2 morphants (black arrowheads). Also note the dramatic dorsal expansion of the ciliary band genes foxG and onecut in the alk1/2 morphants. (C) Injection of high doses (2 mM) of alk1/2 morpholino caused a massive ectopic expression of nodal in about 50% of the embryos at the mesenchyme blastula stage. (D) Phospho-Smad1/5/8 immunostaining in control or alk1/2 morphants. p.Smad1/5/8 in the ectoderm and in the dorsal chain of primary mesenchyme cells (PMCs) (white arrowheads) of alk1/2 morphants is largely abolished. LV, lateral view; VV, vegetal pole view; AV, animal pole view; D, dorsal; V, ventral.
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pbio.1002247.g001: The BMP type I receptor Alk1/2 is essential for D/V patterning.(A) Morphology of embryos at 72 hours after fertilization (hpf) injected with morpholinos targeting either the alk3/6, alk1/2, or bmp2/4 transcripts. Note the striking similarity of the phenotypes of alk1/2 and bmp2/4 morphants that both develop with a ciliary band on the dorsal side (black arrowheads) compared to the less severe phenotype of alk3/6 morphants that is evidenced by the presence of pigment cells (black arrows) and of a less well-developed ciliary band on the dorsal side. (B) Expansion of the ventral and ciliary band fates at the expense of the dorsal ectoderm in alk1/2 morphants was revealed by the analysis of marker genes. Controls and alk1/2 morphants embryos were stained by in situ hybridization with the indicated probes. In alk1/2 morphants at mesenchyme blastula, the territory expressing the ventral marker genes, nodal, chordin, and foxA is largely normal, but consistently, a slight broadening of nodal expression is observed (white arrowheads), while expression of the dorsal gene hox7 is suppressed. At the gastrula stage, however, this ventralization is patent with chordin and foxA expression extending towards the dorsal side in alk1/2 morphants (black arrowheads). Also note the dramatic dorsal expansion of the ciliary band genes foxG and onecut in the alk1/2 morphants. (C) Injection of high doses (2 mM) of alk1/2 morpholino caused a massive ectopic expression of nodal in about 50% of the embryos at the mesenchyme blastula stage. (D) Phospho-Smad1/5/8 immunostaining in control or alk1/2 morphants. p.Smad1/5/8 in the ectoderm and in the dorsal chain of primary mesenchyme cells (PMCs) (white arrowheads) of alk1/2 morphants is largely abolished. LV, lateral view; VV, vegetal pole view; AV, animal pole view; D, dorsal; V, ventral.

Mentions: We showed previously that during D/V patterning in the sea urchin embryo, transduction of the BMP2/4 signals requires the activity of the type-I BMP receptor Alk3/6, the functional orthologue of Thickveins, which transduces Dpp signals in Drosophila. We noticed, however, that blocking Alk3/6 consistently produced a phenotype much less severe than the BMP2/4 loss-of-function phenotype. For example, while bmp2/4 morphants typically lack a population of immunocytes called pigment cells that requires BMP signaling, alk3/6 morphants always develop with numerous pigments cells (arrows in Fig 1A). This suggested that residual BMP signaling in alk3/6 morphants allows formation of pigment cells and/or that additional BMP type I receptors may contribute to transduction of BMP2/4 signals in the absence of Alk3/6. Indeed, in addition to alk3/6, the sea urchin genome contains a second gene encoding a BMP type I receptor named Alk1/2, which is mostly similar to Alk1 and Alk2 from vertebrates and to Saxophone from Drosophila. Like alk3/6, alk1/2 is expressed maternally and ubiquitously during the cleavage and blastula stages (S1 Fig). To evaluate the contribution of Alk1/2 in BMP2/4 signaling, we knocked it down with antisense morpholinos. Interestingly, blocking alk1/2 mRNA translation disrupted D/V axis formation and produced a phenotype stronger than that resulting from inhibition of Alk3/6 (Fig 1A). When the alk1/2 morpholino was injected at 1.2 mM, most alk1/2 morphants failed to develop their ventral arms and dorsal apex and appeared rounded. Alk1/2 morphants also lacked most pigment cells and developed with an ectopic ciliary band and ectopic spicules on the dorsal side, a phenotype largely identical to the bmp2/4 morphant phenotype. These phenotypes could be suppressed by coinjection of a modified wild-type alk1/2 mRNA immune against the morpholino (see S1 Fig). As shown previously in the case of Alk3/6 and of BMP2/4, blocking Alk1/2 caused a dramatic expansion of the ciliary band territory at the expense of the dorsal ectoderm, as evidenced by the massive ectopic expression of foxG and onecut on the presumptive dorsal side and the lack of expression of dorsal marker genes such as hox7 (Fig 1B). Unexpectedly, blocking Alk1/2 function, unlike blocking BMP2/4 or Alk3/6, caused a weak but consistent ventralization, as evidenced by the expression of chordin or foxA that extended to the dorsal side at the gastrula stage (black arrowheads in Fig 1B). Consistent with this ventralization, we found that at blastula stages, embryos injected with high doses of the alk1/2 morpholino displayed a massive ectopic expression of nodal similar to that observed in lefty morphants (Fig 1C). This phenotype, which is not observed in bmp2/4 or alk3/6 morphants, suggests that, in addition to BMP2/4, Alk1/2 may also be required to transduce an unidentified dorsalizing signal. Finally, consistent with the absence of expression of dorsal marker genes, inhibition of alk1/2 mRNA translation, like inhibition of bmp2/4 or alk3/6, drastically reduced phospho-Smad1/5/8 signaling in the dorsal ectoderm (Fig 1D). We conclude that Alk1/2 plays a pivotal role in transduction of BMP2/4 in the sea urchin and that the activities of Alk1/2 and Alk3/6 are nonredundant, both being functionally required during D/V patterning to transduce BMP2/4 signals and to activate Smad1/5/8 signaling in the dorsal ectoderm. Furthermore, these results suggest that in addition to BMP2/4, Alk1/2 may be required for transduction of (a) still unidentified signal(s) that regulate(s) D/V patterning.


The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo.

Haillot E, Molina MD, Lapraz F, Lepage T - PLoS Biol. (2015)

The BMP type I receptor Alk1/2 is essential for D/V patterning.(A) Morphology of embryos at 72 hours after fertilization (hpf) injected with morpholinos targeting either the alk3/6, alk1/2, or bmp2/4 transcripts. Note the striking similarity of the phenotypes of alk1/2 and bmp2/4 morphants that both develop with a ciliary band on the dorsal side (black arrowheads) compared to the less severe phenotype of alk3/6 morphants that is evidenced by the presence of pigment cells (black arrows) and of a less well-developed ciliary band on the dorsal side. (B) Expansion of the ventral and ciliary band fates at the expense of the dorsal ectoderm in alk1/2 morphants was revealed by the analysis of marker genes. Controls and alk1/2 morphants embryos were stained by in situ hybridization with the indicated probes. In alk1/2 morphants at mesenchyme blastula, the territory expressing the ventral marker genes, nodal, chordin, and foxA is largely normal, but consistently, a slight broadening of nodal expression is observed (white arrowheads), while expression of the dorsal gene hox7 is suppressed. At the gastrula stage, however, this ventralization is patent with chordin and foxA expression extending towards the dorsal side in alk1/2 morphants (black arrowheads). Also note the dramatic dorsal expansion of the ciliary band genes foxG and onecut in the alk1/2 morphants. (C) Injection of high doses (2 mM) of alk1/2 morpholino caused a massive ectopic expression of nodal in about 50% of the embryos at the mesenchyme blastula stage. (D) Phospho-Smad1/5/8 immunostaining in control or alk1/2 morphants. p.Smad1/5/8 in the ectoderm and in the dorsal chain of primary mesenchyme cells (PMCs) (white arrowheads) of alk1/2 morphants is largely abolished. LV, lateral view; VV, vegetal pole view; AV, animal pole view; D, dorsal; V, ventral.
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Related In: Results  -  Collection

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Show All Figures
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pbio.1002247.g001: The BMP type I receptor Alk1/2 is essential for D/V patterning.(A) Morphology of embryos at 72 hours after fertilization (hpf) injected with morpholinos targeting either the alk3/6, alk1/2, or bmp2/4 transcripts. Note the striking similarity of the phenotypes of alk1/2 and bmp2/4 morphants that both develop with a ciliary band on the dorsal side (black arrowheads) compared to the less severe phenotype of alk3/6 morphants that is evidenced by the presence of pigment cells (black arrows) and of a less well-developed ciliary band on the dorsal side. (B) Expansion of the ventral and ciliary band fates at the expense of the dorsal ectoderm in alk1/2 morphants was revealed by the analysis of marker genes. Controls and alk1/2 morphants embryos were stained by in situ hybridization with the indicated probes. In alk1/2 morphants at mesenchyme blastula, the territory expressing the ventral marker genes, nodal, chordin, and foxA is largely normal, but consistently, a slight broadening of nodal expression is observed (white arrowheads), while expression of the dorsal gene hox7 is suppressed. At the gastrula stage, however, this ventralization is patent with chordin and foxA expression extending towards the dorsal side in alk1/2 morphants (black arrowheads). Also note the dramatic dorsal expansion of the ciliary band genes foxG and onecut in the alk1/2 morphants. (C) Injection of high doses (2 mM) of alk1/2 morpholino caused a massive ectopic expression of nodal in about 50% of the embryos at the mesenchyme blastula stage. (D) Phospho-Smad1/5/8 immunostaining in control or alk1/2 morphants. p.Smad1/5/8 in the ectoderm and in the dorsal chain of primary mesenchyme cells (PMCs) (white arrowheads) of alk1/2 morphants is largely abolished. LV, lateral view; VV, vegetal pole view; AV, animal pole view; D, dorsal; V, ventral.
Mentions: We showed previously that during D/V patterning in the sea urchin embryo, transduction of the BMP2/4 signals requires the activity of the type-I BMP receptor Alk3/6, the functional orthologue of Thickveins, which transduces Dpp signals in Drosophila. We noticed, however, that blocking Alk3/6 consistently produced a phenotype much less severe than the BMP2/4 loss-of-function phenotype. For example, while bmp2/4 morphants typically lack a population of immunocytes called pigment cells that requires BMP signaling, alk3/6 morphants always develop with numerous pigments cells (arrows in Fig 1A). This suggested that residual BMP signaling in alk3/6 morphants allows formation of pigment cells and/or that additional BMP type I receptors may contribute to transduction of BMP2/4 signals in the absence of Alk3/6. Indeed, in addition to alk3/6, the sea urchin genome contains a second gene encoding a BMP type I receptor named Alk1/2, which is mostly similar to Alk1 and Alk2 from vertebrates and to Saxophone from Drosophila. Like alk3/6, alk1/2 is expressed maternally and ubiquitously during the cleavage and blastula stages (S1 Fig). To evaluate the contribution of Alk1/2 in BMP2/4 signaling, we knocked it down with antisense morpholinos. Interestingly, blocking alk1/2 mRNA translation disrupted D/V axis formation and produced a phenotype stronger than that resulting from inhibition of Alk3/6 (Fig 1A). When the alk1/2 morpholino was injected at 1.2 mM, most alk1/2 morphants failed to develop their ventral arms and dorsal apex and appeared rounded. Alk1/2 morphants also lacked most pigment cells and developed with an ectopic ciliary band and ectopic spicules on the dorsal side, a phenotype largely identical to the bmp2/4 morphant phenotype. These phenotypes could be suppressed by coinjection of a modified wild-type alk1/2 mRNA immune against the morpholino (see S1 Fig). As shown previously in the case of Alk3/6 and of BMP2/4, blocking Alk1/2 caused a dramatic expansion of the ciliary band territory at the expense of the dorsal ectoderm, as evidenced by the massive ectopic expression of foxG and onecut on the presumptive dorsal side and the lack of expression of dorsal marker genes such as hox7 (Fig 1B). Unexpectedly, blocking Alk1/2 function, unlike blocking BMP2/4 or Alk3/6, caused a weak but consistent ventralization, as evidenced by the expression of chordin or foxA that extended to the dorsal side at the gastrula stage (black arrowheads in Fig 1B). Consistent with this ventralization, we found that at blastula stages, embryos injected with high doses of the alk1/2 morpholino displayed a massive ectopic expression of nodal similar to that observed in lefty morphants (Fig 1C). This phenotype, which is not observed in bmp2/4 or alk3/6 morphants, suggests that, in addition to BMP2/4, Alk1/2 may also be required to transduce an unidentified dorsalizing signal. Finally, consistent with the absence of expression of dorsal marker genes, inhibition of alk1/2 mRNA translation, like inhibition of bmp2/4 or alk3/6, drastically reduced phospho-Smad1/5/8 signaling in the dorsal ectoderm (Fig 1D). We conclude that Alk1/2 plays a pivotal role in transduction of BMP2/4 in the sea urchin and that the activities of Alk1/2 and Alk3/6 are nonredundant, both being functionally required during D/V patterning to transduce BMP2/4 signals and to activate Smad1/5/8 signaling in the dorsal ectoderm. Furthermore, these results suggest that in addition to BMP2/4, Alk1/2 may be required for transduction of (a) still unidentified signal(s) that regulate(s) D/V patterning.

Bottom Line: We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression.Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates.Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-β may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Valrose, iBV, UMR 7277 CNRS, Inserm U1091, UNS, University of Nice Sophia Antipolis, Nice, France.

ABSTRACT
Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-β may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8. We provide evidence that, although panda mRNA is broadly distributed in the early embryo, local expression of panda mRNA efficiently orients the dorsal-ventral axis and that Panda activity is required locally in the early embryo to specify this axis. Taken together, these findings demonstrate that maternal panda mRNA is both necessary and sufficient to orient the dorsal-ventral axis. These results therefore provide evidence that in the highly regulative sea urchin embryo, the activity of spatially restricted maternal factors regulates patterning along the dorsal-ventral axis.

No MeSH data available.


Related in: MedlinePlus