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.

Ciona robusta 2-cell stage embryo (anterior to the left) probed for Foxd.b expression and simultaneously injected with MOs targeting Hes.a, Pem and Ccnb3.
No Foxd.b expression is observed.

Ciona robusta 4-cell stage embryo (anterior to the left) probed for Foxd.b expression, treated with the GSK3-beta inhibitor BIO.
No Foxd.b expression is observed, as in the WT control.

Ciona robusta 4-cell stage embryo (anterior to the left) probed for Foxd.b expression and injected with Ccnb3-targeting MO.
No Foxd.b expression is observed, as in the WT control.

Ciona robusta 4-cell stage embryo (anterior to the left) probed for Foxd.b expression, treated with the GSK3-beta inhibitor BIO and injected with Ccnb3-targeting MO.
No Foxd.b expression is observed, as in the WT control.

Ciona robusta 4-cell stage embryo (anterior to the left) probed for Foxd.b expression and injected with Ccnb3 and Pem-targeting MOs.
No Foxd.b expression is observed, as in the WT control.

Ciona robusta 4-cell stage embryo (anterior to the left) probed for Foxd.b expression, treated with the GSK3-beta inhibitor BIO and injected with Ccnb3 and Pem-targeting MOs.
No Foxd.b expression is observed, as in the WT control.

Ciona robusta 4-cell stage embryo (anterior to the left) probed for Foxd.b expression and simultaneously injected with MOs targeting Hes.a, Pem and Ccnb3.
No Foxd.b expression is observed.

Ciona robusta 8-cell stage embryo (anterior to the left) probed for Foxd.b expression and treated with the GSK3-beta inhibitor BIO.
No Foxd.b expression is observed, as in the WT control.

Ciona robusta 8-cell stage embryo (anterior to the left) probed for Foxd.b expression and injected with Ccnb3-directed morpholino.
No Foxd.b expression is observed, as in the WT control.

Ciona robusta 8-cell stage embryo (anterior to the left) probed for Foxd.b expression, treated with the GSK3-beta inhibitor BIO and injected with Ccnb3-targeting MO.
Precocious Foxd.b expression is observed in a4.2, A4.1 and b4.2 blastomers (red arrowheads).
Original annotation: Precocious expression of Foxd.b was observed at the 8-cell stage with the combination of Ccnb3 MO knockdown and treatment with the GSK3-beta inhibitor BIO, which accelerates nuclear accumulation of beta-catenin.
Second image is the drawing of a 8-cell stage embryo where each cell is labelled to indicate its identity.

Ciona robusta 8-cell stage embryo (anterior to the left) probed for Foxd.b expression and injected with Ccnb3 and Pem-targeting MOs.
No Foxd.b expression is observed, as in the WT control.

Ciona robusta 8-cell stage embryo (anterior to the left) probed for Foxd.b expression, treated with the GSK3-beta inhibitor BIO and injected with Ccnb3 and Pem-targeting MOs.
Precocious Foxd.b expression is observed in a4.2, A4.1, B4.1 and b4.2 blastomers (red arrowheads).
Original annotation: Precocious expression of Foxd.b was observed at the 8-cell stage with the combination of Ccnb3 MO knockdown and treatment with the GSK3-beta inhibitor BIO, which accelerates nuclear accumulation of beta-catenin.
Second image is the drawing of a 8-cell stage embryo where each cell is labelled to indicate its identity.

Ciona robusta 8-cell stage embryo (anterior to the left) probed for Foxd.b expression and simultaneously injected with MOs targeting Hes.a, Pem and Ccnb3.
No Foxd.b expression is observed.

Vegetal view of a Ciona robusta 16-cell stage embryo probed for Foxd.b expression and injected with Ccnb3 mRNA.
No Foxd.b expression is observed.
Original annotation: Ccnb3 overexpression resulted in the failure to detect any expression of early zygotic genes and a loss of cell adhesion.

Vegetal view of a Ciona robusta 16-cell stage embryo probed for Foxd.b expression and injected with Ccnb3 mRNA with mutations in the cdk-binding domain (Ccnb3 AAA mRNA).
Foxd.b expression is observed in A5.1, A5.2 and B5.1 blastomers, as in the WT control.
Original annotation: Expression was not lost in embryos injected with a mutant form of the Ccnb3 mRNA harboring inactivating amino acid substitutions in the cdk-binding site (Ccnb3 AAA).

Vegetal view of a Ciona robusta 16-cell stage embryo probed for Foxd.b expression and injected with Ccnb1 mRNA.
Foxd.b expression is observed in A5.1, A5.2 and B5.1 blastomers, as in the WT control.
Original annotation: Expression was not lost in embryos injected with Ccnb1 mRNA.

Vegetal view of a Ciona robusta 16-cell stage embryo probed for Foxd expression and injected with β-catenin MO.
Foxd expression is lost.

Lateral view, vegetal pole to the right of a Ciona robusta 16-cell stage embryo probed for Foxd expression and treated with BIO from the eight-cell stage. BIO is a chemical inhibitor of the upstream inhibitory regulator of β-catenin, GSK-3.
Foxd is expressed in A5.1, A5.2, B5.1, a5.3, a5.4, b5.3 and b5.4 blastomers.
Original comment: Ectopic stabilisation of β-catenin resulted in activation of Foxd in ectoderm lineages at the 16-cell stage.

Lateral view of a 16-cell stage embryo probed for Ci-foxD expression following injection of TCF-MO.

Lateral view of a 16-cell satge embryo. Ci-foxD expression was visualized at the 16-cell stage in beta-catenin-MO injected embryos..

Vegetal view of a Ciona robusta 32-cell stage embryo probed for Foxd expression and treated with U0126 from the 16-cell stage.
Foxd is expressed in A6.1, A6.3, B6.2 and B6.1 blastomer pairs, as in WT conditions.

Vegetal view of a Ciona robusta 32-cell stage embryo probed for Foxd expression and injected with morpholinos against Foxa.a.
Foxd is expressed in A6.1, A6.3, B6.2 and B6.1 blastomer pairs, as in WT conditions.

Batch of Ciona robusta 32-cell stages embryos probed for FoxD expression and electroporated with pFT>FoxD. In this overexpression construct, the upstream regulatory sequences of the Fucosyltransferase-like (FT) gene drives Foxd expression in ectoderm cells from the 64-cell stage.
No Foxd signal can be observed.

Vegetal view of a late 32-cell stage embryo showing expression of Ci-FoxD, following injection of Ci-PGAP1 MO.

Original annotation:
Expression of Ci-FoxD became undetectable in embryos injected with morpholino against Ci-PGAP1.

Vegetal view of a late 32-cell stage embryo showing expression of Ci-FoxD, following injection of Ci-ZF278 MO.

Original annotation:
Expression of Ci-FGF9/16/20 became undetectable in embryos injected with morpholino against Ci-ZF278.

Vegetal view of a late 32-cell stage embryo showing expression of Ci-FoxD, following injection of Ci-Spatial/C4orf17 MO.

Original annotation:
Expression of Ci-FGF9/16/20 became undetectable in embryos injected with morpholino against Ci-Spatial/C4orf17.

Vegetal view of a late 32-cell stage embryo showing expression of Ci-FoxD, following injection of Ci-FLJ10634 MO.

Original annotation:
Expression of Ci-FGF9/16/20 became undetectable in embryos injected with morpholino against Ci-FLJ10634.

Vegetal view of a late 32-cell stage embryo showing expression of Ci-FoxD, following injection of Ci-beta-catenin Mo.

Original annotation:
Expression of Ci-FGF9/16/20 became undetectable in embryos injected with morpholino against Ci-beta-catenin.

Vegetal view of a late 32-cell stage embryo showing expression of Ci-FoxD, following the injection of Ci-C10orf11.

Original annotation:
Expression of Ci-FGF9/16/20 became undetectable in embryos injected with morpholino against Ci-C10orf11.

Vegetal view of a late 32-cell stage embryo probed for Ci-FoxD, in which eggs were injected with a mixture of DeltaN Ci-Beta Catenin mRNA and morpholino against Ci-ZF278.

Original annotation:
Injection of DeltaN Ci-Beta Catenin mRNA could restore the early expression of Ci-FoxD in embryos knocked down for Ci-ZF278.

Vegetal view of a late 32-cell stage embryo probed for Ci-FoxD, in which eggs were injected with a mixture of DeltaN Ci-Beta Catenin mRNA and morpholino against Ci-Beta Catenin.

Original annotation:
Injection of DeltaN Ci-Beta Catenin mRNA could restore the early expression of Ci-FoxD in embryos knocked down for Ci-Beta Catenin.

Vegetal view of a late 32-cell stage embryo probed for Ci-FoxD, in which eggs were injected with a mixture of DeltaN Ci-Beta Catenin mRNA and morpholino against Ci-PGAP1.

Original annotation:
Injection of DeltaN Ci-Beta Catenin mRNA could restore the early expression of Ci-FoxD in embryos knocked down for Ci-PGAP1.

Vegetal view of a late 32-cell stage embryo probed for Ci-FoxD, in which eggs were injected with a mixture of DeltaN Ci-Beta Catenin mRNA and morpholino against Ci-C10orf11.

Original annotation:
Injection of DeltaN Ci-Beta Catenin mRNA could restore the early expression of Ci-FoxD in embryos knocked down for Ci-C10orf11.

Vegetal view of a late 32-cell stage embryo probed for Ci-FoxD, in which eggs were injected with a mixture of DeltaN Ci-Beta Catenin mRNA and morpholino against Ci-Spatial/C4orf17.

Original annotation:
Injection of DeltaN Ci-Beta Catenin mRNA could restore the early expression of Ci-FoxD in embryos knocked down for Ci-Spatial/C4orf17.

Vegetal view of a late 32-cell stage embryo probed for Ci-FoxD, in which eggs were injected with a mixture of DeltaN Ci-Beta Catenin mRNA and morpholino against ci-FLJ10634.

Original annotation:
Original annotation: Injection of DeltaN Ci-Beta Catenin mRNA could restore the early expression of Ci-FoxD in embryos knocked down for Ci-FLJ10634.

Vegetal and lateral view of a late-32 cell stage embryo stained with foxD expression after BIO treatment at the late 16-cell stage. Red arrow point to the NN cells, blue arrow point to the E cell. The green dashed line marks the animalvegetal boundary of embryos oriented along the A-V axis.
Original annotation: Expression in NN cell. No expression in animal cells.
Aniseed annotation: Expression in the whole embryon.
(+HD photo)

Lateral view of a late-32 cell stage embryo stained with foxD expression after BIO treatment at the late 16-cell stage. The green dashed line marks the animalvegetal boundary of embryos oriented along the A-V axis.
Original annotation: Expression in NN cell. Expression in animal cells.
Aniseed annotation: Expression in the whole embryon.
(+HD photo)

Vegetal view of a late 32-cell stage embryo marked with foxD after TCF∆C treatment at the 8-cell stage in A4.1 cell. The red arrow points the NN cells and the blue arrow the E cells. The green dashed line marks the region containing the cells from the A4.1 cell lineage.
Original annotation: Loss of expression in A6.1 and A6.3 cells (E cells).
Aniseed annotation: Normal expression in B6.1 cell pair.
(+HD photo)

Vegetal view of a late 32-cell stage embryo marked with foxD after TCF∆C treatment at the 16-cell stage in A5.1 cell. The red arrow points the NN cells and the blue arrow the E cells. The green dashed line marks the region containing the cells from the A5.1 cell lineage.
Original annotation: Loss of expression in A6.1 (E cell).
Aniseed annotation: Normal expression in the other A6.1 as well as the A6.3 and B6.1 cell pairs.
(+HD photo)

Vegetal view of a late-32 cell stage embryo stained with foxD expression after TCF∆C treatment at the 16-cell stage in A5.3 cell and BIO treatment at the late 8-cell stage. The green dashed line marks the region containing the cells from the A5.3 cell lineage.
Original annotation: Expression of foxD in animal cells after BIO treatmen but no expression in the A5.3 cell lineage.
Aniseed annotation: Expression is observed in the whole embryo except for a6.5 and a6.6.
(+HD photo)

Vegetal and tilted view of a late-32 cell stage embryo stained with foxD expression after Wnt5-MO treatment on the unfertelized egg. Red arrow point to the NN cells and the blue arrow to the E cells.
Original annotation: Expression in E cells but not in NN cells.
Aniseed annotation: Expression is observed in A6.1, A6.3 and B6.1 cell pairs. (+HD photo)

Vegetal view of a late-32 cell stage embryo stained with foxD expression after NotRNA treatment. Original annotation: No expression either in NN or E cells. Aniseed annotation: Expression is not observed in the entire embryo.

Vegetal view of late 32-cell stage embryos, showing expression of Ci-FoxD following the injection of Ci-Rga-MO.

Ci-FoxD was still expressed in the vegetal hemisphere for 80% of embryos.

Batch of Ciona robusta 76-cell stages embryos probed for FoxD expression and electroporated with pFT>FoxD. In this overexpression construct, the upstream regulatory sequences of the Fucosyltransferase-like (FT) gene drive Foxd expression in ectoderm cells from the 64-cell stage.
Around 90% of the embryos showed any level of Foxd expression in ectoderm.

Batch of Ciona robusta 110-cell stages embryos probed for FoxD expression and electroporated with pFT>FoxD. In this overexpression construct, the upstream regulatory sequences of the Fucosyltransferase-like (FT) gene drive Foxd expression in ectoderm cells from the 64-cell stage.
Around 90% of the embryos showed any level of Foxd expression in ectoderm.

Batch of Ciona robusta early gastrulas probed for FoxD expression and electroporated with pFT>FoxD. In this overexpression construct, the upstream regulatory sequences of the Fucosyltransferase-like (FT) gene drive Foxd expression in ectoderm cells from the 64-cell stage.
Around 90% of the embryos showed weak Foxd expression in ectoderm.

Mid tailbud stage embryo probed for FoxD expression following the injection of msxb-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 in the nerve cord (white arrow).

Original annotation: expression lost in the presumptive ocellus (arrow) Aniseed annotation: expression lost in ocellus but not other cells surrounding (unidentifiable region).