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-cell stage embryo probed for Delta-like expression and injected with ephrin-Ad MO.

Original annotation:
Delta-like is the most resistant to MO inhibition with only 40% loss of expression.

Vegetal view of a 64-cell stage embryo probed for Ci-delta2 expression in which Ci-FoxAa has been blocked by MO injection. Control picture: sibling embryo shows the wild type expression pattern of the gene.

Animal view of a 64-cell stage embryo probed for Delta-like expression following egg micro-injection of a FoxA-a MO. Delta-like was ectopically expressed in a-line neural cells, the expression in TLCs and b-line neural cells were unaffected.

Vegetal view of a 64-cell stage embryo probed for Ci-Delta2 expression in which Nodal signalling has been blocked with SB431542. No signal can be identified.

Vegetal view of a 64-cell stage embryo probed for Ci-Delta2 expression in which Nodal signalling has been inhibited with Nodal-Mo. No signal can be identified.

Vegetal view of a 64-cell stage embryo probed for Ci-Delta2 expression in which Nodal signalling has been blocked with tALK4/5/7 mRNA. mRNA were injected into A4.1 on the right hand side at 8-cell stage and then analysed for Ci-delta2 expression at 64-cell stage (left side is used as a control). There is no staining on the right side compared to the control side.

Vegetal view of a 64-cell stage embryo probed for Ci-delta2 expression following electroporation with pSP1.72-FOG::Ci-FoxA-a overexpression construct. Control: sibling embryo shows the wild type expression pattern of the gene. Expression is lost after FoxA-a electroporation.

Vegetal and lateral views of a 110-cell stage embryo probed for Ci-Delta2 expression following egg micro-injection of a mRNA Nodal. Ci-Delta2 was expressed in up to all eight A-line neural cells in 59% of cases. Control sibling embryo shows the wild type pattern of the gene.

Vegetal view of a 110-cell stage embryo probed for Ci-Delta2 expression in which Nodal signalling has been blocked by incubation into the SB431542 inhibitor of Alk4/5/7. The expression was lost in b-line, A8.15 and A8.16. Control sibling embryo shows the wild type pattern of the gene.

Vegetal view of a 110-cell stage embryo probed for Ci-Delta2 expression in which Nodal signalling has been blocked by injection of a Nodal MO. The expression was lost in b-line, A8.15 and A8.16. Control sibling embryo shows the wild type pattern of the gene.

Vegetal view of a 110-cell stage embryo probed for Ci-Snail expression in which Nodal signalling has been blocked by injection of mRNA for a dominat negative version of the Alk4/5/7 type I nodal receptor. The expression was lost in b-line, A8.15 and A8.16. Control sibling embryo shows the wild-type pattern of the gene.

Vegetal view of a 110-cell stage embryo probed for Delta-like expression following egg micro-injection of a Neurogenin MO. Delta-like is down-regulated in A7.6.

Vegetal view of a 110-cell stage embryo probed for Delta-like expression following egg micro-injection of a Nodal MO.

Vegetal view of a 110-cell stage embryo probed for Delta-like expression following egg micro-injection of a ZicL MO.

Vegetal view of a 110-cell stage embryo probed for Delta-like expression following egg micro-injection of a FoxD-a/b MO. Delta-like expresssion was down-regulated in A-line nerve cord cells and not affected in b-line.

Vegetal view of a 110-cell stage embryo showing expression of Delta-like. Embryos were treated with U0126 at the late 32-cell stage.

Original annotation
A7.6 group genes are ectopically expressed in the anterior endoderm blastomeres upon MEK inhibition, whereas A7.6 expression is unaffected.

Vegetal views of 110-cell stage embryos co-injected with ephrin-Ad MO and torso-FGFR mRNA into 1-cell stage and probed for Delta-like.

Original annotation:
Delta-like expression was now repressed in the A7.6 blastomere in 80% of the embryos (arrows). Ectopic activation of Delta-like in b-line blastomeres is often observed (arrowheads).

Vegetal view of a 110-cell stage embryo probed for Delta-like and after injection of FoxD::ephrin-Ad. This construct was used to drive Ci-ephrin-Ad throughout the vegetal hemisphere at the 16-cell stage (thanks to FoxD enhancer).

Original annotation:
Delta-like expression is observed in A7.6 blastomeres and in anterior endoderm.

Vegetal and lateral views of 110-cell stage embryos injected at the 1-cell stage with torso-FGFR mRNA and probed for delta.

Original annotation:
Delta-like expression was only sporadically reduced.

Vegetal view of a 110-cell stage embryo probed for Delta-like following injection of FoxD::Nodal. This construct was used to drive Nodal throughout the vegetal hemisphere (thanks to FoxD enhancer).

Original Annotation: Delta-like sometimes displayed ectopic expression in the A-line neural precursors, indicating by arrowheads.

Aniseed Annotation: Ectopic expression of Hand-like is showing in A8.7 and A8.8 cells.

Vegetal view of a 110-cell stage embryo showing expression of Delta-like. Embryos were treated with U0126 at the 16-cell stage (picture and graph) and at the early 32-cell stage, late 32-cell stage and 64-cell stage (graph).

Original annotation:
These embryos often displayed normal expression in A7.6 cell pair but additional patterns were also detected, including expression in A7.6 along with ectopic expression in the endoderm, loss of A7.6 expression along with gain of expression in the endoderm, or complete loss of expression.

Vegetal view of 110-cell stage embryo injected with ephrin-Ad MO and treated with the MEK inhibitor U0126 at the 32-cell stage and probed for Delta-like expression.

Original annotation :
Delta-like is expressed in both the A7.6 blastomere and the anterior endoderm.

Aniseed annotation :
According to the picture, we can't determine if there is expression in b7.9 and b7.10 cell pairs (compare to the control).

Vegetal view of a 110-cell stage embryo. The expression of Ci-delta-like was examined in embryos that expressed Ci-FoxD/Ci-nodal.

Original annotation:
The Ci-FoxD/Ci-nodal transgene mediated ectopic expression of Ci-delta-like in the ventral nerve cord lineage (red arrowheads).

Vegetal view of a 110-cell stage embryo. The expression of Ci-delta-like was examined in SB431542-treated embryos.

Original annotation:
SB431542 induced repression of Ci-delta-like (blue arrowheads).

Aniseed annotation:
Very weak expression seems to be detected in A7.6 cell pair.

Vegetal view of a 110-cell stage embryo. The expression of Ci-delta-like was examined in embryos that expressed Ci-FoxD/Ci-lefty.

Original annotation: The Ci-FoxD/Ci-lefty transgene mediated repression of Ci-delta-like (blue arrowheads).

Vegetal and lateral views of an early gastrula stage embryo probed for Ci-Delta2, following injection of a morpholino against Delta2 (Delta2-MO bis). Numbers indicate any expression over the total number of embryos analyzed.

Original Annotation :
Expression is not affected by the inhibition of Delta2/Notch signaling.

Vegetal and lateral views of an early gastrula stage embryo probed for Ci-Delta2, following injection of DAPT. Numbers indicate any expression over the total number of embryos analyzed.

Original Annotation :
Expression is not affected by the inhibition of Delta2/Notch signaling due to DAPT.

Animal view with anterior to the top of a 112-cell stage embryo (stage 10) stained with Delta2 after electroporation with the construct pFOG::FGF9/16/20 at the 16-cell stage. In the panel, gene expression is presented in blue. Expression was observed in the posterior ectoderm (b4.2 lineage or b-line ectoderm).

Animal view with anterior to the top of a 112-cell stage embryo (stage 10) stained with Delta2 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 Delta2 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 Delta2 after electroporation with the construct pFOG>Nodal at the 16-cell satge. In the panel, posterior ectoderme is marked in yellow and gene expression in blue.
Original annotation: There was no ectopic expression of Delta2 in the posterior (b-line) ectoderm. But ectopic expression of Delta2 was observed in the anterior neural tissue precursors (a6.5 lineage).

Animal view with anterior to the top of a 112-cell stage embryo (stage 10) stained with Delta2 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.
Original annotation: Induction of posterior neural tissue in anterior ectoderm.

Vegetal view with anterior to the top of a 10-stage embryo stained with Delta2 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 Delta2 was lost in the four daughter cells of the b6.5 lineage.

Vegetal view of a 10-stage embryo stained with Delta2 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. Delta2 expression was abolished in the four daughter cells of the b6.5 lineage.

Animal view with anterior to the top of a 112-cell stage embryo (stage 10) stained with Delta2 after injection of a Otx morpholino (Otx MO). In the panel, anterior ectoderm in white, posterior ectoderm in yellow and gene expression in blue. Expression was lost in the b8.17, b8.18, b8.19 and b8.20 cell pairs.

Animal view with anterior to the top of a 112-cell stage embryo (stage 10) stained with Delta2 after injection of a Otx overconstruct (pFOG-Otx. In the panel, anterior ectoderm in white, posterior ectoderm in yellow and gene expression in blue. Otx overexpression was sufficient to activate throughout the ectoderm.

Animal view, anterior to the top, of a 10-stage embryo stained with Delta2 after OtxHDenR injection, a dominant negative form of Otx, at the 16-cell stage. Expression was lost as indicated by the black arrow.

Animal view with anterior to the top of an early gastrula stage embryo stained with Delta2 after injection of pFOG::Lefty at the 16-cell stage. Expression was lost in the whole embryo.

Animal view with anterior to the top of an early gastrula stage embryo stained with Delta2 after overexpression of pFOG>Otx at the 16-cell stage. Ectopic expression was observed in the embryo.

Animal view with anterior to the top of an early gastrula stage embryo stained with Delta2 after overexpression of pFOG>Otx and pFOG::Lefty at the 16-cell stage. Ectopic expression was observed in the embryo.

Tilted view of an early gastrula stage embryo stained with Delta2 after overexpression of pFOG>Nodal at the 16-cell stage. Delta2 expressing cells are colored in blue and ectopic expression is circled in red.

Vegetal view of a late gastrula stage embryo probed for Delta-like expression, following the injection of Neurogenin-MO.

Original annotation:
Expression in A9.32 cell pair was lost (white arrow).

Vegetal view of a Ciona robusta 6-row neural plate stage embryo probed for Delta-like expression and injected with a morpholino against Nodal.
Delta-like expression is lost when Nodal is inhibited.

Vegetal view of a Ciona robusta 6-row neural plate stage embryo probed for Delta-like expression and treated with a pharmacological inhibitor of TGFβ type I receptors ALK4, ALK5 and ALK6 (SB431542) that inhibits Nodal activity.
Delta-like expression is lost when Nodal is inhibited.

Vegetal view of a Ciona robusta 6-row neural plate stage embryo probed for Delta-like expression and electroporated with pFOG>Nodal to drive expression of Nodal throughout the animal hemisphere from the 16-cell stage of development.
Delta-like expression is enlarged throughout the neural plate when Nodal is overexpressed. Strong Delta-like expression is observed in neural plate rows I, II and III.

Dorsal view of the motor ganglion of a Ciona robusta initial tailbud embryo probed for Delta2 expression (red) and electroporated with FGF8/17/18>GFP reporter construct (green nuclei) and FGF8/17/18>Eph3ΔC overexpression construct.
Original comment: Conversion of A10.59 to A10.60 fate with FGF8/17/18>Eph3ΔC results in loss of Delta2 expression in A10.59 in electroporated side but not in unelectroporated side.
The A9.30 lineage is visualized using the FGF8/17/18 reporter construct (green nuclei).

Dorsal view of an early tailbud stage embryo probed for Delta like expression, following the injection of Neurogenin-MO.
Red and yellow arrowheads indicate boundaries between the neck and the visceral ganglion, and between the visceral ganglion and the caudal nerve cord, respectively.

Original Annotation :
Expression was lost (white arrow).

Dorsal view of an early tailbud stage embryo probed for Delta-like expression following the injection of Delta-like-MO.
Red and yellow arrowheads indicate boundaries between the neck and the visceral ganglion, and between the visceral ganglion and the caudal nerve cord, respectively.

Original Annotation :
Ectopic expression was observed (black arrow).

Lateral view of the motor ganglion of a Ciona robusta early tailbud embryo probed for Delta2 expression (red) and electroporated with FGF8/17/18>GFP reporter construct (green nuclei) and FGF8/17/18>Eph3ΔC overexpression construct.
Delta2 is expressed in all the A9.30 progeny (ie. A11.120, A11.119, A11.118 and A11.117).
Original comment: Ectopic Delta2 expression is seen in A11.119 upon loss of A10.59 fate (n56/71) and is interpreted as loss of Notch activation in A11.119 according to the model of lateral inhibition.
The A9.30 lineage is visualized using the FGF8/17/18 reporter construct (green nuclei, diagram).

Original annotation: expression lost compared to the control.
Aniseed annotation: weak expression in the visceral ganglion.

Original annotation : expression down regulated in embryos injected with Ascl.b MO.

Original annotation : expression down regulated in embryos injected with Tox MO. Lateral view of the embryo is shown.