Ciona robusta adults were obtained from the National Bio-Resource Project for Ciona (NBRP, Japan). 50 unperturbed and Foxd-morphant embryos were collected at the 32-, 64-, and 112-cell stages. RNA was extracted using a Dynabeads mRNA DIRECT Purification Kit (Thermo Fischer Scientific) and libraries were made with an Ion Total RNA-Seq kit ver 2 (Thermo Fischer Scientific). The libraries were sequenced with an Ion PGM instrument (Thermo Fischer Scientific).

The purpose of this project is to identify genes positively and negatively regulated by signaling molecules that belongs to the TGFbeta superfamily.

Vegetal view of a 64-cells stage embryo probed for Chordin expression following egg micro-injection of a FGF9/16/20 MO. Chordin is down-regulated in A-line notochord and b-line neural cells.

Aniseed annotation : no signal is detected in B-line cells.

Vegetal view of a 64-cell stage embryo probed for Chordin expression following egg micro-injection of a FoxD-a/b MO. Chordin is down-regulated in A-line notochord.

Vegetal view of a 64-cell stage embryo probed for Chordin expression following egg micro-injection of a FoxA-a MO. Chordin expression was down-regulated in A-line notochord.

Vegetal view of a 110-cell stage embryo probed for Chordin expression following egg micro-injection of a ZicL MO. ZicL is down-regulated in A-line notochord.

Aniseed annotation : staining is not detected in B-line cells on the picture.

Vegetal view of a 110-cell stage embryo probed for Chordin expression following egg micro-injection of a FoxD-a/b MO. Chordin expression was down-regulated in A-line nerve cord cells and TLCs.

Animal view with anterior to the top of a 112-cell stage embryo (stage 10) stained with Chordin after electroporation with the construct pFOG::FGF9/16/20 at the 16-cell stage. In the panel, gene expression is presented in blue.
Original annotation: Expression was observed in the posterior ectoderm (b4.2 lineage or b-line ectoderm) as well as part of the anterior ectoderm.
Aniseed annotation: Expression was observed in the a8.23, a8.24, a8.26, a8.27, a8.28, a8.29, a8.30, a8.31 and a8.32 cell pairs of the anterior ectoderm.

Animal view with anterior to the top of a 112-cell stage embryo (stage 10) stained with Chrodin after electroporation with the construct pFOG::Lefty, a Nodal antagonist, at the 16-cell stage. In the panel, posterior ectoderm is marked in yellow and anterior ectoderm in white. Expression was lost in the b-line neural lineage.

Animal view with anterior to the top of a 112-cell stage embryo (stage 10) stained with Chordin after electroporation with the pFOG::Lefty (Nodal antagonist) and pFOG::FGF9/16/20 constructs at the 16-cell stage. In the panel, posterior ectoderm is marked in yellow and anterior ectoderm in white. Expression was lost in the b-line neural lineage.

Animal view with anterior to the top (neural plate view with vegetal side to the left) of a 112-cell stage embryo (stage 10) stained with Chordin after electroporation with the construct pFOG>Nodal at the 16-cell satge. In the panel, gene expression is marked in blue. Ectopic expression of Chordin was observed throughout the ectoderm.

Animal view with anterior to the top (neural plate view with vegetal side to the left)of a 112-cell stage embryo (stage 10) stained with Chordin after electroporation with the pFOG>Nodal and pFOG::FGF9/16/20 constructs at the 16-cell satge. In the panel, gene expression is marked in blue. Ectopic expression of Chordin was observed throughout the ectoderm.

Vegetal view with anterior to the top of a 10-stage embryo stained with Chordin afetr U0126 treatment at the 8-cell stage (MEK inhibitor). Black arrowhead indicates the b-line neural precursors. In In the panel, anterior ectoderm in white and posterior ectoderm in yellow. Expression of Chordin was lost in the four daughter cells of the b6.5 lineage.

Vegetal view of a 10-stage embryo stained with Chordin after SB431542 (Nodal signaling pathway inhibitor) treatment at the 16-cell stage. In the panel, anterior ectoderm is marked in white and posterior ectoderm in yellow. Black arrowheads indicate b-line neural precursors. Chordin expression was abolished in the four daughter cells of the b6.5 lineage.

Expression of Chordin at the neural plate stage, following the injection of FOG::BMP2/4 construct.
Graphs show the percentage of embryos with strong, weak or ectopic expression, as indicated in the key. Embryos were scored 'ectopic' if at least one additional cell showed expression. The number of embryos analysed is indicated above the graphs. cont, control; BMP, FOG::BMP2/4.

Original annotation:
We found that overexpression of BMP2/4 had effects very similar to overexpression of Nodal, with expression of lateral markers expanded into medial neural plate cells at the expense of medial marker expression.

Expression of Chordin at the neural plate stage, following the injection of FOG::Nodal construct.
Graphs show the percentage of embryos with strong, weak or ectopic expression, as indicated in the key. Embryos were scored 'ectopic' if at least one additional cell showed expression. The number of embryos analysed is indicated above the graphs. cont, control; Nodal, FOG::Nodal.

Original annotation:
We found that overexpression of BMP2/4 had effects very similar to overexpression of Nodal, with expression of lateral markers expanded into medial neural plate cells at the expense of medial marker expression.

Expression of Chordin at the neural plate stage, following the injection of FOG::Nog construct.
Graphs show the percentage of embryos with strong, weak or ectopic expression, as indicated in the key. Embryos were scored 'ectopic' if at least one additional cell showed expression. The number of embryos analysed is indicated above the graphs. cont, control; Nog, FOG::Noggin.

Expression of Chordin at the neural plate stage, following the injection of Xenopus Chordin RNA.
Graphs show the percentage of embryos with strong, weak or ectopic expression, as indicated in the key. Embryos were scored 'ectopic' if at least one additional cell showed expression. The number of embryos analysed is indicated above the graphs. cont, control; XChd, Xenopus Chordin RNA injection.

A vegetal view of a mid gastrula embryo probed for Ci-Chordin expression following the injection of Delta2-MO.
Graph shows the percentage of embryo with ectopic, strong or weak Ci-chordin expression.'Weak' means at least one cell with downregulated expression, 'strong' means at least one A-line precursor cell with strong expression.

Original Annotation : Ci-Chordin expression was not affected by the inhibition of Delta2/Notch signaling.

Vegetal view of a mid gastrula embryo probed for Ci-Chordin expression following the injection of DAPT.
Graph shows the percentage of embryo with ectopic, strong or weak Ci-Chordin expression following the injection of DAPT.'Weak' means at least one cell with downregulated expression, and 'strong' means at least one A-line precursor cell with strong expression.

Original Annotation : Ci-Chordin expression was not affected by the inhibition of Delta2/Notch signaling.

Graph shows the percentage of mid gastrula embryo with ectopic, strong or weak Ci-Chrodin expression following the injection of dnDel2, a dominant negative of Delta2.
'Weak' means at least one cell with downregulated expression, and 'strong' means at least one A-line precursor cell with strong expression.

Original Annotation : In the majority of embryos Ci-Chordin expression was not affected by the inhibition of Delta2/Notch signalling.

Vegetative view of a mid gastrula probed for Chordin expression, following injection of Snail morpholino (Snail-MO (Imai's 25mer MO)).
Original annotation : Expression of Chordin was unaffected by Snail knockdown.

Vegetative view of a mid gastrula probed for Chordin expression, following injection of Snail morpholino (Snail-MO_2 (Hudson 2015)).
Original annotation : Expression of Chordin was unaffected by Snail knockdown.

Vegetative view of a mid gastrula probed for Chordin expression, where Snail is overexpressed in medial lateral neural plate (electroporation of pETR>Snail). Chordin expression pattern is unaffected.

Vegetative view of a mid gastrula probed for Chordin expression and treated with SB431542 (Nodal inhibitor) until early gastrula stage. Chordin expression is lost in lateral cells of A- and a-neural plate.

Vegetative view of a mid gastrula probed for Chordin expression and treated with SB431542 (Nodal inhibitor) until early gastrula stage, where Snail is overexpressed in medial lateral neural plate (electroporation of pETR>Snail). Chordin neural plate expression is lost.

Vegetative view of a mid gastrula probed for Chordin expression, where Nodal is overexpressed in animal territories (electroporation of pFOG>Nodal).
Original annotation : Nodal overexpression induces ectopic expression of Chordin within its correct row of cells (all A- and a-neural plate)

Neural plate view of a Late gastrula stage embryo probed for Ci-Chordin expression in which Nodal signalling has been blocked. Essentially all of the neural plate expression was lost and only notochord expression remained. Control sibling embryo shows the wild type pattern of the gene.

Neural plate view of a Late gastrula stage embryo probed for Ci-Chordin expression in which Nodal signalling has been blocked by Nodal MO injection. Essentially all of the neural plate expression was lost and only notochord expression remained. Control sibling embryo shows the wild type pattern of the gene.

Vegetal view a late gastrula stage embryo. The expression of Ci-chordin was examined in embryos that expressed Ci-FoxD/Ci-nodal trangene.

Original annotation:
Expression of ci-chordin was activated by Ci-FoxD/Ci-nodal in all the neural plate.

Aniseed annotation:
According to the picture, it seems that there is also expression in the epidermis.
Asterisk indicates Ci-chordin expression in the presumptive notochord cells, which was not dependent of the nodal signal. Red arrowhead indicates ectopic expression.

Vegetal view of a late gastrula stage embryo. The expression of Ci-chordin was examined in SB431542-treated embryos.

Original annotation:
Expression of ci-chordin was repressed by SB431542 in all the neural plate. Asterisk indicates Ci-chordin expression in the presumptive notochord cells, which was not dependent of the nodal signal. Blue arrowheads indicate blastomeres in which the expression was diminished.

Vegetal view a late gastrula stage embryo. The expression of Ci-chordin was examined in embryos that expressed Ci-FoxD/Ci-lefty transgene.

Original annotation:
Expression of ci-chordin was repressed by Ci-FoxD/Ci-lefty in all the neural plate.
Asterisk indicates Ci-chordin expression in the presumptive notochord cells, which was not dependent of the nodal signal. Blue arrowheads indicate blastomeres in which the expression was diminished.