Simplified synteny of <i>NANOG</i> loci in osteichthyans.

<p>The synteny of the loci where <i>NANOG</i> orthologues are found in actinopterygians (<i>TMSF9</i> - <i>IPO4</i> region) and sarcopterygians (<i>SLC2A3</i> - <i>AICDA</i> region) are shown on the left-hand and right-hand sides, respectively. The relevant chromosomes or gene scaffolds are given. The figure is not drawn to scale, “empty” spaces along the chromosomes (e.g. between <i>TM9SF1</i> and <i>IPO4</i> in sarcopterygians) do not reflect actual distances but are meant to facilitate comparisons. Double slashes (//) denote that intervening genes were omitted for simplicity (e.g. between <i>FEN1</i> and <i>IPO4</i> in <i>Danio rerio</i>). In species in which two <i>NANOG</i> paralogues were found, numbers indicate which paralogue was named “<i>NANOG1</i>” or “<i>NANOG2</i>” in this work (note that these names do not imply orthologous relationships). This region contains multiple paralogues of <i>NANOG</i>, <i>AICDA</i> and <i>SLC2A3</i> in Guinea pig; and of both <i>NANOG</i> (<i>NANOGP1</i>, <i>P1</i> on the figure) and <i>SLC2A3</i> (named <i>SLC2A14</i>, <i>A14</i> on the figure) in Hominidae. More detailed information regarding these two regions is listed in File S2, including coordinates for the genes presented on this figure.</p

Phylogenetic analysis of NANOG paralogues in osteichthyans.

<p>Translated sequences of exons 2 and 3 were analyzed by Maximum Likelihood and Bayesian inference. Here we show a strict consensus tree, using the scaffold of the ML tree. Branch support was assessed and is given next to the relevant branches (top number: aLRT for the ML analysis and bottom number PP for the Bayesian analysis). The actinopterygian sequences form a monophyletic group that was used to root the tree. Duplicates are highlighted in red. Note that two paralogues are found in coelacanth (<i>Latimeria chalumnae</i>), Archosaurs (<i>Alligator mississipiensis</i>; <i>Gallus gallus</i>; <i>Meleagris gallopavo</i>; <i>Taeniopygia guttata</i>; <i>Melopsittacus undulates</i>; <i>Anas platyrhynchos</i>; <i>Geospiza fortis</i>; <i>Ficedula albicollis</i>), Testudines (<i>Pelodiscus sinensis</i>, <i>Chrysemys picta belli</i>), platypus (<i>Ornithorhynchus anatinus</i>), Tasmanian devil (<i>Sarcophilus harrisii</i>), Guinea pig (<i>Cavia porcellus</i>), Hominidae (<i>Pan troglodytes</i>; <i>Homo sapiens</i>) and spotted gar (<i>Lepisosteus oculatus</i>). The topology suggests that independent duplication events occurred in the three latter clades.</p

ventx1/2 activity is necessary to maintain an uncommitted cell population in early gastrulae.

<p>(<b><i>A</i></b>) NF2-embryos were injected radially twice in both blastomeres with control MO (30 ng/blastomere), or a 1∶1 mix of <i>ventx1/2</i> MOs (30 ng/blastomere). Variations of gene expression at 516-, 1000-, 2000-, 4000-cell and NF10.5 stages were assessed by RT-QPCR as in <b>Fig. 2.</b> Dorsal (<i>siamois</i>, <i>gsc</i>, <i>hhex</i>), and ventral (<i>wnt8</i>) mesendoderm, endoderm (<i>xnr5</i>, <i>mixer</i>) and ectoderm (<i>tfap2a</i>, <i>k81a1</i>) markers were monitored. Kinetic graphs represent means of fold-change relative to NF10.5 controls +/− s.e.m, and significance was assessed using paired t-test (*p≤0.05, **p≤0.005, ***p≤0.0005), and undetectable levels of transcript noted as Φ. (<b><i>B</i></b>) Animal injections were performed twice in a single blastomere NF2-embryos, using MO conditions described in (<b><i>A</i></b>); fldx was used as a lineage tracer. WISH with an <i>oct91</i> probe (left panel) were performed at stage NF10.5 and the progeny of the injected blastomere was revealed by fluorescence (right panel). Embryos are positioned with the animal side upwards; white arrows indicate the injected side. (<b><i>C</i></b>) Injections were performed using mRNA and MO conditions described in <b>Fig. 2</b>. All embryos were collected at stage NF10.5 and processed for RT-QPCRs using the pluripotency marker <i>oct91</i>. Data and graphs are presented as in <b>Fig. 2</b>.</p

<i>mNanog</i>, <i>ventx1/2,</i> and <i>msx1</i> cause distinct effects on early patterning gene expression.

<p>For gain-of-function experiments, NF3-embryos were injected radially in all blastomeres with water, <i>msx1</i> mRNA (0.3 ng/blastomere, red), <i>mNanog</i> mRNA (0.15 ng/blastomere, blue) or <i>ventx1/2</i> mRNAs (0.5 ng/blastomere, green); For loss-of-function experiments, NF-2 embryos were injected twice radially in both blastomeres with control MO (30 ng/blastomere), or a 1∶1 mix of <i>ventx1/2</i> MOs (30 ng/blastomere, purple). All embryos were collected at stage NF10.5 and processed for RT-QPCRs. Ectodermal, mesodermal and endodermal markers were assayed (each quantification was performed at least 3 times independently). For all RT-QPCR, graphs represent means of the fold-change calculated versus the appropriate control (fldx injected embryos in cases of overexpression and control MO for <i>ventx1/2</i> knock-down) +/− s.e.m, and significance was assessed using paired t-test (*p≤0.05, **p≤0.005, ***p≤0.0005).</p

<i>mNanog</i> and <i>ventx1/2</i> overexpression cause similar effects in <i>Xenopus</i> embryos.

<p>(<b><i>A</i></b>) Four-cell stage embryos (NF3) were injected in both dorsal blastomeres, with a 1∶3 mix of <i>ventx1.2</i> and <i>ventx2.1-b</i> mRNAs (<i>ventx1/2</i>; 0.5 ng per blastomere), with mouse <i>Nanog</i> mRNA (<i>mNanog</i>; 0.15 ng/blastomere), or with water for control. Representative phenotypes observed at tailbud stage (NF28) are shown (lateral views, anterior to the left, dorsal to the top). (<b><i>B</i></b>) Percentages of observed phenotypes in three independent experiments for mock (n = 14), <i>ventx1/2</i> (n = 31) and <i>mNanog</i> (n = 36) mRNAs injections. (<b><i>C</i></b>) Embryos injected as in (<b><i>A</i></b>) were collected at early gastrulae (NF10.5; whole embryos: ventral view, dorsal side to the top; hemisected embryos: lateral view, dorsal to the left, animal side to the top) and tailbud (NF28; ventral view, anterior to the left) stages and processed for whole-mount <i>in situ</i> hybridization (WISH) with a <i>gsc</i> or <i>hhex</i> probe, or with <i>hba4</i> (black arrowheads) and <i>egr2</i> (white arrowheads), respectively. The number of embryos showing staining similar to the one photographed over the total number of embryos assayed is indicated.</p

<i>ventx1/2</i> overexpression prevents multiple lineage commitment.

<p>(<b><i>A</i></b>) NF2-embryos were injected twice in one blastomere, either with <i>msx1</i> mRNAs (0.6 ng/blastomere), <i>ventx1/2</i> mRNAs (1 ng/blastomere), <i>mNanog</i> mRNA (0,3 ng/blastomere), or with water for control; fldx was used as a lineage tracer. WISH with a <i>k81a1</i> probe were performed at stage NF10.5 (left panels, animal views, dorsal side to the top). The progeny of the injected blastomere was revealed by fluorescence; white arrows indicate the injected side (right panels). (<b><i>B</i></b>) Sixteen-cell stage embryos (NF5) were injected in one AB4 blastomere with <i>msx1</i> mRNA (0.15 ng), <i>ventx1/2</i> mRNAs (0.5 ng), <i>mNanog</i> mRNA (0.15 ng), or water, collected at stage NF10.5 and processed for WISH with a <i>k81a1</i> probe (animal views). Black stripped lines mark the border between injected and uninjected domains. (<b><i>C</i></b>) NF2-embryos were injected twice in one blastomere with <i>msx1</i> mRNA (5 ng/blastomere), <i>ventx1/2</i> mRNAs (5 ng/blastomere), <i>ventx1/2</i>+<i>msx1</i> mRNAs (5 ng +5 ng/blastomere), or with water. WISH with a <i>myf5</i> probe were performed at stage NF10.5; black arrowheads point to <i>myf5</i>-expressing territories (left panels, ventral views, dorsal side to the top). The progeny of the injected blastomere was revealed by fldx fluorescence; white arrows point to the injected side (right panels).</p

Interest of heterogeneous methods in pathway completion and filling thanks to tracking of process metadata.

<p>Completion of the 6-hydroxymethyl-dihydropterin diphosphate biosynthesis I and the tetrahydrofolate biosynthesis pathways in <i>E</i>. <i>siliculosus</i> via the combination of annotation (yellow), orthology (green) and gap-filling (blue). The dihydrofolate compound with the dotted line is an instance of the dihydrofolate-glu-n class, following MetaCyc classes ontology structure. The class compound is the original reactant of the dihydrofolatereduct-rxn reaction retrieved with annotation, whereas the previous reaction of the pathway (dihydrofolatesynth-rxn) produces the instance dihydrofolate. Hence the gap-filling step that, using an extended version of MetaCyc, selects an instantiated version of dihydrofolatesynth-rxn that consumes the instance dihydrofolate.</p

AuReMe workspace.

<p><i>Overview of the AuReMe workspace</i>. Admissible inputs include standard formats in genomics and metabolic model fields that can be outputs of major reconstruction platforms. <i>AuReMe</i> acts as a workflow controller to administer the reconstruction or modification of the GSM performed by heterogeneous and independent tools. The latter are part of the services of <i>AuReMe</i> (reconstruction tools, analyses, manual curation) and can be chained together, either in a pre-set pipeline or in a customized one. In any case the <i>PADMet</i> data manager stores adequate information regarding the model and its metadata, most importantly the process ones, that keeps track of the modifications performed (at what step a reaction was added, by which tool etc.). At any time, the reconstruction can be monitored locally via an automatically-generated wiki that informs the user about the state of the model. Outputs of <i>AuReMe</i> can be self-sufficient or be integrated again in many existing platforms.</p

Survey of 19 GSM reconstruction procedures.

<p>Variability of reference ID-databases, available annotations and metadata about reactions and heterogeneity of the methods used in each GSM reconstruction pipeline.</p

<i>Tisochrysis lutea</i> metabolic model exploration: Origin of reactions according to the reconstruction pipeline.

<p>(A) Comparison of the numbers of EC numbers introduced in the network either by the annotation pipeline or by the orthology-based analysis 898 enzymes were identified via annotation-based information and 790 enzymes through orthology-based data, among which 524 were already identified via annotation information. (B) <i>Number of T-Iso ortholog enzymes according to their origin in template models</i>. For each of the 790 T-Iso ortholog enzymes, the figure depicts in which of the four template models an ortholog of the enzyme had been identified. The four templates used were: <i>A</i>. <i>thaliana</i>, <i>C</i>. <i>reinhardtii</i>, <i>E</i>. <i>siliculosus</i> and <i>Synechocystis</i> sp. PCC 6803 to decipher ortholog enzymes in <i>T</i>. <i>lutea</i>. (C) <i>T-Iso carnosine biosynthesis</i>. Reconstruction of T-Iso carnosine synthesis pathway was performed using three sources of data (i) T-Iso genome annotations (cyan star); (ii) template metabolic models (stars) of four organisms: <i>A</i>. <i>thaliana</i> (blue), <i>C</i>. <i>reinhardtii</i> (green), <i>E</i>. <i>siliculosus</i> (red), and <i>Synechocystis</i> sp. (yellow) with orthology-based information; (iii) complete proteomes of the four organisms (squares) with sequence alignment information (best reciprocal hit in blasts). All reactions of the T-Iso carnosine biosynthesis are common to the four organisms except for three of them: ASPDECARBOX-RXN, HISTIDPHOS-RXN, and CARNOSINE-SYNTHASE-RXN. The first seems to belong to an alternative pathway to produce β-alanine, also found in <i>C</i>. <i>reinhardtii</i>, <i>Synechocystis</i> sp and <i>Candidatus Phaeomarinobacter ectocarpi</i>, a symbiotic bacterium to <i>E</i>. <i>siliculosus</i>. HISTIDPHOS-RXN was not found in <i>E</i>. <i>siliculosus</i> but was identified in its symbiotic bacterium <i>Candidatus</i> Phaeomarinobacter ectocarpi. CARNOSINE-SYNTHASE-RXN was only identified in algae (<i>C</i>. <i>reinhardtii</i>, <i>E</i>. <i>siliculosus</i> and <i>T</i>. <i>lutea</i>).</p