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

International audienceSpecification 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

p38 MAPK as an essential regulator of dorsal-ventral axis specification and skeletogenesis during sea urchin development: a re-evaluation

International audienceDorsal-ventral axis formation in the sea urchin embryo relies on the asymmetrical expression of the TGFβ Nodal. The p38-MAPK pathway has been proposed to be essential for dorsal-ventral axis formation by acting upstream of nodal expression. Here, we report that, in contrast to previous studies that used pharmacological inhibitors of p38, manipulating the activity of p38 by genetic means has no obvious impact on morphogenesis. Instead, we discovered that p38 inhibitors strongly disrupt specification of all germ layers by blocking signalling from the Nodal receptor and by interfering with the ERK pathway. Strikingly, while expression of a mutant p38 that is resistant to SB203580 did not rescue dorsal-ventral axis formation or skeletogenesis in embryos treated with this inhibitor, expression of mutant Nodal receptors that are resistant to SB203580 fully restored nodal expression in SB203580-treated embryos. Taken together, these results establish that p38 activity is not required for dorsal-ventral axis formation through nodal expression nor for skeletogenesis. Our results prompt a re-evaluation of the conclusions of several recent studies that linked p38 activity to dorsal-ventral axis formation and to patterning of the skeleton

Deciphering and modelling the TGF-β signalling interplays specifying the dorsal-ventral axis of the sea urchin embryo

International audienceDuring sea urchin development, secretion of Nodal and BMP2/4 ligands and their antagonists Lefty and Chordin from a ventral organizer region specifies the ventral and dorsal territories. This process relies on a complex interplay between the Nodal and BMP pathways through numerous regulatory circuits. To decipher the interplay between these pathways, we used a combination of treatments with recombinant Nodal and BMP2/4 proteins and a computational modelling approach. We assembled a logical model focusing on cell responses to signalling inputs along the dorsal-ventral axis, which was extended to cover ligand diffusion and enable multicellular simulations. Our model simulations accurately recapitulate gene expression in wild type embryos, accounting for the specification of ventral ectoderm, ciliary band and dorsal ectoderm. Our model simulations further recapitulate various morphant phenotypes, reveals a dominance of the BMP pathway over the Nodal pathway, and stresses the crucial impact of the rate of Smad activation in D/V patterning. These results emphasise the key role of the mutual antagonism between the Nodal and BMP2/4 pathways in driving early dorsal-ventral patterning of the sea urchin embryo

Maternal but not zygotic Panda function is required for the spatial restriction of <i>nodal</i> expression.

<p>(A) Injection of a morpholino oligonucleotide targeting the translation start site of <i>panda</i> mRNA, but not of a morpholino targeting a splice junction, disrupts the establishment of D/V polarity. The lineage tracer Fluoresceinated Lysine-Fixable Dextran (FLDX) was coinjected with the morpholino. (B) In situ hybridizations against the <i>nodal</i> transcript at early stages. In the absence of maternal, but not of zygotic, Panda, a massive ectopic expression of <i>nodal</i> is observed starting at the 60-cell stage. Note that <i>nodal</i> expression remains radially expressed up to the mesenchyme blastula stage. Massive and early ectopic expression of <i>nodal</i> is also observed in the double <i>alk1/2</i>+<i>alk3/6</i> morphants. VEB, very early blastula (about 120 cells); EB, early blastula (about 220 cells); PHB, prehatching blastula (about 300 cells); HB, hatching blastula (about 400 cells); SB, swimming blastula; LB, late blastula; MB, mesenchyme blastula. (C) Scheme summarizing the perturbations of <i>nodal</i> expression caused by blocking Panda or Alk1/2+Alk3/6. VV, vegetal pole view; V, ventral; D, dorsal.</p

Panda plays a pivotal role during D/V axis formation.

<p>(A) Morphological phenotypes resulting from injection into the egg of antisense morpholino oligonucleotide targeting the translation start site of the <i>panda</i> transcript. Down-regulation of <i>panda</i> completely radializes the embryos during the first 48 h, but a partial recovery of D/V polarity occurs afterwards, as evidenced by the formation of a short dorsal apex. This phenotype is similar to that caused by treatments with recombinant Nodal protein at 1 μg/ml. (B) In situ hybridization on control embryos and <i>panda</i> morphants at the blastula and gastrula stages with ventral, ciliary band, and dorsal marker genes. Note the dramatic ectopic expression of <i>nodal</i>, <i>chordin</i>, and <i>foxA</i> at the blastula stage in <i>panda</i> morphants. At the late gastrula stage, despite their radialized morphology, <i>panda</i> morphants are patterned along the D/V axis, as evidenced by the restricted expression of <i>nodal</i>, <i>foxA</i>, <i>foxG</i>, <i>onecut</i>, and <i>hox7</i>. Note, however, the extended expression of these ventral markers compared to control embryos. (C) A second morpholino oligonucleotide targeting the 5' UTR of the <i>panda</i> transcript produces similar phenotypes and radializes the expression of <i>nodal</i> and <i>chordin</i>. (D) Scheme describing the changes in fate maps caused by the single inactivation of <i>panda</i> or <i>bmp2/4</i> or by the double inactivation of <i>panda</i> and <i>bmp2/4</i>. LV, lateral view; VV, vegetal pole view; AV, animal pole view; V, ventral; D, dorsal.</p

The BMP type I receptor Alk1/2 is essential for D/V patterning.

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

Panda, like BMP2/4, requires Alk1/2 and Alk3/6 to pattern the D/V axis, but Panda, unlike BMP2/4, does not appear to activate phosphorylation of Smad1/5/8.

<p>(A) Coinjection of the <i>panda</i> and <i>alk1/2</i> morpholinos or of the <i>panda</i> and <i>alk3/6</i> morpholinos causes a strong ventralization, as does the double inactivation of <i>bmp2/4</i> + <i>alk1/2</i> or of <i>bmp2/4</i> and <i>alk3/6</i>. (B) While the simple knockdown of <i>bmp2/4</i> is not sufficient to cause ectopic expression of <i>nodal</i>, the double knockdown of <i>bmp2/4</i> + <i>alk1/2</i> or <i>bmp2/4</i> + <i>alk3/6</i> causes massive ectopic expression of <i>nodal</i>. (C) Panda requires Alk3/6 to orient the D/V axis when misexpressed. Local overexpression of <i>panda</i> orients the D/V axis in nearly 100% of the injected embryos. However, if the eggs are first injected with the <i>alk3/6</i> morpholino, panda is no longer able to orient the D/V axis. (D) Western blot of phospho-Smad1/5/8 in control embryos at the indicated stages or in embryos overexpressing <i>panda</i> or injected with a <i>panda</i> morpholino. Note that phosphoSmad1/5/8 is undetectable before the late blastula stage and that overexpression of <i>panda</i> does not appear to cause phosphorylation of Smad1/5/8. B1, very early blastula; B2, early blastula. (E) Phospho-Smad1/5/8 immunostaining at very early and early blastula stages or mesenchyme blastula stages in control embryos and in embryos overexpressing <i>panda</i>, <i>bmp2/4</i>, <i>alk3/6QD</i> mRNA, or <i>alk1/2QD</i>. The highly sensitive alkaline phosphatase-based detection of pSmad1/5/8 does not allow detection of Smad1/5/8 signaling at early stages in control embryos. In contrast, following overexpression of <i>bmp2/4</i>, <i>alk3/6QD</i>, or <i>alk1/2QD</i> into the egg or into one blastomere at the two-cell stage, strong nuclear phosphoSmad1/5/8 immunostaining is easily detected at early blastula. This pSmad1/5/8 immunoreactivity is not detected following injection of <i>panda</i> mRNA. Nevertheless, overexpression of <i>panda</i> induces weak ectopic Smad1/5/8 signaling at mesenchyme blastula. (F) Overexpression of <i>panda</i> expands Smad1/5/8 signaling at the mesenchyme blastula stage. pSmad1/5/8 is normally restricted to the dorsal ectoderm and dorsal PMCs at mesenchyme blastula. Overexpression of <i>panda</i> expands the territory in which pSmad1/5/8 is detected toward the ventral side. LV, lateral view; VV, vegetal view; SVV, surface and vegetal view; V, ventral; D, dorsal.</p

Panda activity has to be provided locally to efficiently rescue <i>panda</i> morphants.

<p>Differential interference contrast (DIC) and fluorescence images of embryos injected with a <i>panda</i> morpholino into the egg and then with <i>panda</i> mRNA either into the egg or into one blastomere at the two-cell stage. While providing Panda activity into the egg does not rescue D/V polarity, injection of <i>panda</i> mRNA or of low doses (50 μg/ml) of mRNA encoding the activated form of Alk3/6 (Alk3/6QD) into one blastomere at the two-cell stage fully rescues D/V polarity of <i>panda</i> morphants. LV, lateral view; V, ventral; D, dorsal.</p

Panda belongs to a subfamily of TGF-β that includes <i>Drosophila</i> Maverick and vertebrate GDF15.

<p>Phylogenetic analysis of TGF-β ligands using the maximum likelihood method. The analysis was performed using the full-length proteins. Representative taxa from deuterostomes, protostomes, and cnidarians were used (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002247#pbio.1002247.s011" target="_blank">S1 Text</a> for a list of these taxa). These 162 sequences were collected from diverse databases using the National Center for Biotechnology Information (NCBI) research tool (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>). Full-length sequences were aligned using ClustalOmega with default parameters (<a href="http://www.ebi.ac.uk/clustalw/" target="_blank">http://www.ebi.ac.uk/clustalw/</a>), and gap optimisation and obvious alignment error correction were made using Bioedit 7.0.5.3 (<a href="http://www.mbio.ncsu.edu/BioEdit/bioedit.html" target="_blank">http://www.mbio.ncsu.edu/BioEdit/bioedit.html</a>). The tree was calculated using the maximum likelihood method with PhyML with substitution model WAG (<a href="http://atgc.lirmm.fr/phyml/" target="_blank">http://atgc.lirmm.fr/phyml/</a>). A consensus tree with a 45% cutoff value was derived from 500 bootstrap analysis using Mega 3.1 (<a href="http://www.megasoftware.net/" target="_blank">http://www.megasoftware.net/</a>). Numbers above nodes represent a percentage of bootstrap values supporting this node. The original tree is presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002247#pbio.1002247.s006" target="_blank">S5 Fig</a>.</p

Ability of various components of the Nodal or BMP pathways to orient the D/V axis following misexpression into one blastomere at the two-cell stage.

<p>Mo, morpholino antisense oligonucleotide.</p><p>Ability of various components of the Nodal or BMP pathways to orient the D/V axis following misexpression into one blastomere at the two-cell stage.</p