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 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

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 double inactivation of <i>alk1/2</i> and <i>alk3/6</i> causes massive ectopic expression of <i>nodal</i>, resulting in extreme ventralization.

<p>(A) Morphology of embryos at 72 hpf injected with morpholinos targeting either the <i>alk3/6</i>, <i>alk1/2</i>, or <i>bmp2/4</i> transcripts or injected with a mixture of the <i>alk1/2</i> and <i>alk3/6</i> morpholinos. Simultaneous down-regulation of Alk1/2 and Alk3/6 caused a strong ventralization similar to that resulting from overexpression of <i>nodal</i> or from treatment with nickel chloride (a treatment that ventralizes sea urchin embryos by causing massive ectopic expression of <i>nodal</i>). Note the presence of a ciliary band in the vegetal pole region (black arrowheads) and the prominent proboscis (white arrowheads) in the animal pole region in the double <i>alk1/2</i> + <i>alk3/6</i> morphants and in <i>nodal</i>-overexpressing or nickel-treated embryos. (B) In situ hybridization on controls and <i>alk1/2</i> + <i>alk3/6</i> morphants at the blastula and gastrula stages with ventral, ciliary band, and dorsal marker genes. The strong ventralization of <i>alk1/2</i> + <i>alk3/6</i> morphants is presaged by the massive ectopic expression of <i>nodal</i> at blastula stages. Note the radial expression of the ciliary band markers <i>foxG</i> and <i>onecut</i> in the vegetal pole region of <i>alk1/2</i> + <i>alk3/6</i> morphants at the gastrula stage. (C) Scheme describing the changes in fate maps caused by the single or double inactivation of type I BMP receptors. In the simple <i>alk3/6</i> knockdown, the ventral ectoderm remains unaffected and the dorsal ectoderm is converted into ciliary band, while in the <i>alk1/2</i> morphants, the ventral ectoderm is expanded, giving rise to a partial ventralization. In contrast, in the double <i>alk1/2</i> + <i>alk3/6</i> morphants, the whole ectoderm is converted into ventral ectoderm. LV, lateral view; VV, vegetal pole view; AV, animal pole view; FV, frontal view; V, ventral; D, dorsal.</p

Model of D/V axis formation: The sequential activities of Panda, Lefty, and BMP2/4 establish the D/V axis.

<p>(A) Expression and functional requirements of the main ligands and receptors involved in D/V patterning. (B,C) The sequential activities of Panda, Lefty, and BMP2/4 progressively define the D/V axis. (1) During cleavage, a gradient of maternal Panda activity, acting through Alk3/6 and Alk1/2, first antagonizes <i>nodal</i> expression and breaks the radial symmetry of the embryo. (2) At the early blastula stage (7 h 30 hpf), following this initial symmetry breaking, spatially restricted Nodal induces Lefty, and Lefty, which diffuses more than Nodal, prevents Nodal autoregulation outside the presumptive ventral ectoderm. This second phase of Nodal antagonism is required to maintain the spatial restriction of <i>nodal</i> expression. (3) Starting at the hatching blastula stage (9 hpf), Nodal induces BMP2/4, and BMP2/4 signaling synergizes with Lefty to antagonize Nodal on the dorsal side. V, ventral; D, dorsal.</p

Total codon usage across the protein-coding genes of the <i>Strigamia</i> mitochondrial genome.

<p>Standard one-letter abbreviations for amino acids (Aa) using invertebrate mitochondrial genetic code.</p><p>Total codon usage across the protein-coding genes of the <i>Strigamia</i> mitochondrial genome.</p

Maximum Likelihood phylogenetic analysis of mitochondrial protein-coding genes from Arthropoda species including <i>S</i>. <i>maritima</i>.

<p>Support at nodes are SH-like support values. A monophyletic Chilopoda is resolved as sister group to Pancrustacea (SH-like support = 0.99) and Pauropoda (represented by <i>Pauropus longiramus</i>) placed as sister group to Chelicerata (SH-like support = 0.97). Within the Chilopoda, Scutigeromorpha (<i>Scutigera coleoptrata</i>) are resolved as sister group to the three other chilopod orders represented in our phylogeny (Lithobiomorpha, (<i>Bothropolys sp</i>., <i>Lithobius forficatus</i> and <i>Cermatobius longicornis</i>); Scolopendromorpha (<i>Scolopocryptos sp</i>.) and Geophilomorpha (<i>Strigamia maritima</i>) with SH-like support = 0.99.</p