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 late-32 cell stage embryo stained with ZicL expression after BIO treatment at the late 16-cell stage. Red arrow point to the NN cells, blue arrow point to the E cell.
Original annotation: No expression either in NN or E cells.
Aniseed annotation: Expression is observed in B6.4 cell pair.

Vegetal view of a late 32-cell stage embryo marked with ZicL after injection of TCF∆C 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 A6.2 and A6.4 cells (NN cells).
Aniseed annotation:Normal expression in B6.2 and B6.4 cell pairs.
(+HD photo)

Vegetal view of a late 32-cell stage embryo marked with ZicL after injection of TCF∆C 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: Expression in A6.1 cell (E cell) as well as in A6.2 and A6.4 cells (NN cells).
Aniseed annotation:No expression in the other A6.1 cell and normal expression in the other A6.2 and A6.4 cell as well as the B6.2 and B6.4 cell pairs.
(+HD photo)

Vegetal view of a late-32 cell stage embryo stained with ZicL expression after Wnt5-MO treatment on the unfertelized egg. Red arrow point to the NN cells.
Original annotation: No expression either in NN.
Aniseed annotation: Expression is observed in B6.2 and B6.4 cell pairs.
(+HD photo)

Vegetal view of a late-32 cell stage embryo stained with zicL expression after injection of TCF∆C 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 zicL in cells from the a5.3 cell lineage.
Aniseed annotation: Expression is observed in a6.5 and a6.6 cells as well as in B6.4 cell pair.
(+HD photo)

Vegetal view of a late-32 cell stage embryo stained with ZicL 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 a late 32-cell stage embryo. Effects of overexpression is revealed by whole-mount in situ hydridization. Ci-ZicL expression was detected in B6.2, B6.4, A6.2 and A6.4 blastomeres in Ci-macho1-overexpressing embryos. However, the intensity of signal is weaker than the control.

Vegetal view of a late 32-cell stage embryo. Effects of functionnal suppression of Ci-macho1 is revealed by whole-mount in situ hydridization. Ci-ZicL expression was detected in B6.2, B6.4, A6.2 and A6.4 blastomeres in Ci-macho1-suppressed embryos. However, the intensity of signal is weaker than the control.

Animal and vegetal view of a 32-cell stage embryo, showing expression of Ci-ZicL in Ci-Tbx6b overexpressing embryo.

Animal and vegetal view of 32-cell stage embryo, showing expression of Ci-ZicL in Ci-Tbx6c overexpressing embryo.

Vegetal view of a 32-cell stage embryo, showing expression of Ci-ZicL in Ci-Tbx6b knockdown embryo.

Vegetal view of a 32-cell stage embryo, showing expression of Ci-ZicL in Ci-Tbx6c knockdown embryo.

Vegetal view of 32-cell stage embryos, showing expression of Ci-ZicL in Ci-Tbx6b/Ci-Tbx6c knockdown embryo.

Original annotation : ZicL expression at the 32-cell stage in a BZ1/2 morphant embryo (shown in animal view). Black arrowheads indicate the WT expression of ZicL. No precocious ZicL expression was detected in BZ1/2 morphants at the 32-cell stage.

Original annotation : ZicL expression at the 32-cell stage in a BZ1/2/Hes-a morphant embryo (shown in animal view). Ectopic expression is shown by yellow arrowheads. Black arrowheads indicate the WT expression of ZicL. We found ectopic and precocious ZicL expression in the anterior animal cells of BZ1, BZ2 and Hes-a triple morphants

Original annotation : ZicL expression at the 32-cell stage in a Hes-a morphant embryo (shown in animal view). Ectopic expression is shown by yellow arrowheads. Black arrowheads indicate the expression of ZicL. In embryos injected with only the Hes-a MO, ectopic expression is observed only weakly in a small number of cells. Ectopic expression of ZicL in BZ1/2/Hes-a morphants at the 32-cell stage is dependent on FGF signaling, because this ectopic expression is lost in BZ1/2/Hes-a morphants treated with U0126 (Fig. 3L) and because bFGF treatment evokes ectopic expression in all anterior ectodermal cells of BZ1/2/Hes-a morphants (Fig. 3M,N).

Original annotation : ZicL expression at the 32-cell stage in BZ1/2/Hes-a morphant embryos treated with U0126 from early 16-cell stage to 32-cell stage (shown in animal views).
Ectopic expression of ZicL in BZ1/2/Hes-a morphants at the 32-cell stage is dependent on FGF signaling, because this ectopic expression is lost in BZ1/2/Hes-a morphants treated with U0126.

bFGF treatment do not affect ZicL expression at the 32-cell stage.
Original annotation : ZicL expression at the 32-cell stage in an embryos treated with bFGF (shown in animal view).

Original annotation : ZicL expression at the 32-cell stage in BZ1/2/Hes-a morphant embryos treated with bFGF (shown in animal views). Ectopic expression is shown by yellow arrowheads. bFGF treatment evokes ectopic expression in all anterior ectodermal cells of BZ1/2/Hes-a morphants.

Vegetal view of a 64-cell stage embryo. Ci-ZicL expression was examined in embryos that expressed Ci-FoxD/Ci-nodal transgene.

Original annotation:
The Ci-FoxD/Ci-nodal transgene did not affect Ci-ZicL expression.

Vegetal view of a 64-cell stage embryo. Ci-ZicL expression was examined in SB431542-treated embryos.

Original annotation:
SB431542 treatment did not affect Ci-ZicL expression.

Vegetal view of a 64-cell stage embryo. Ci-ZicL expression was examined in embryos that expressed Ci-FoxD/Ci-lefty transgene.

Original annotation:
The Ci-FoxD/Ci-lefty transgene did not affect Ci-ZicL expression.

Vegetal view of a late-64 cell stage embryo stained with ZicL expression after BIO treatment at the late 16-cell stage. Red arrow point to the NN cells, blue arrow point to the E cell.
Original annotation: No expression either in NN or E cells.
Aniseed annotation: Expression is observed in B7.7 and B7.8 cell pairs.

Vegetal view of a late-64 cell stage embryo stained with ZicL expression after BIO treatment at the late 8-cell stage.
Original annotation: No expression in NN.
Aniseed annotation: Expression is observed in B7.7 and B7.8 cell pairs.

Original annotation : ZicL expression at the 64-cell stage in a BZ1/2 morphant embryo (shown in anterior views; animal pole up). In BZ1/2 morphants, ZicL is precociously expressed at the 64-cell stage in the bi-potential progenitors a7.9 and a7.10.

Original annotation : ZicL expression at the 64-cell stage in BZ1/2 morphant embryos (shown in anterior views; animal pole up). The embryo is treated with U0126. Ectopic ZicL expression is rarely observed in BZ1/2 morphants treated with the MEK inhibitor U0126 at the 44-cell stage.

Original annotation : ZicL expression at the 64-cell stage in embryos treated with bFGF at the 44-cell stage (shown in anterior views; animal pole up). These embryos do not show ectopic ZicL expression.

Original annotation : ZicL expression at the 64-cell stage in BZ1/2 morphant embryos treated with bFGF at 44-cell stage (shown in anterior views; animal pole up).
BZ1/2 morphant embryos treated with recombinant human basic fibroblast growth factor (bFGF) at the 44-cell stage ectopically express ZicL in a7.9/a7.10 and in neighboring epidermal/brain progenitor cells (a7.13/a7.14).

Vegetal view of a 110-cell stage embryo probed for Zicl expression following egg micro-injection of a FGF9/16/20 MO. ZicL was down-regulated in a-line neural cells and b-line neural cells.

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

Vegetal view of a 110-cell stage embryo probed for ZicL expression following egg micro-injection of a FoxA-a MO. ZicL was down-regulated in A-line nerve cord cells and a-line neural cells.

Original annotation : ZicL expression at the early gastrula stage in embryos expressing BZ1 under the DMRT1 upstream sequence (shown in anterior views; animal pole up). The overexpression constructs were injected into one anterior animal cell of eight-cell embryos. Descendants of the blastomere injected with the constructs are encircled by pink dashed lines. The expression is illustrated schematically at the bottom right in blue (brain). Following injection of these constructs into an anterior animal cell at the eight-cell stage, ZicL expression is partially lost in the injected octant.

Original annotation : ZicL expression at the early gastrula stage in embryos expressing BZ2 under the DMRT1 upstream sequence (shown in anterior views; animal pole up). The overexpression constructs were injected into one anterior animal cell of eight-cell embryos. Descendants of the blastomere injected with the constructs are encircled by pink dashed lines. The expression is illustrated schematically at the bottom right in blue (brain). Following injection of this construct into an anterior animal cell at the eight-cell stage, ZicL expression is completely lost in the injected octant.

Original annotation : The expression of ZicL in morphant embryos at the early gastrula stage (shown in animal view). The expression is illustrated schematically in the lower right by red (palps) and blue (brain).
ZicL, which is normally expressed in the brain lineage at the early gastrula stage, is ectopically expressed in the palp lineage in BZ1/2 morphants.

Vegetal view of a mid gastrula stage embryo showing Ci-ZicL expression. Embryos were treated with SB431542 from the 16-cell stage.

Original annotation:
Expression of ci-ZicL diminished in the A-line nerve cord lineage-cells.
Blue arrowhead indicates blastomeres in which the expression was diminished.

Vegetal view of a mid gastrula stage embryo showing Ci-ZicL expression. Embryos were treated with SB431542 from the 64-cell stage.

Original annotation:
SB431542 treatment from the 64-cell stage did not affect the expression pattern of Ci-ZicL.

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

Original annotation:
Ci-FoxD/Ci-nodal transgene mediated ectopic activation of Ci-ZicL expression in all A-line nerve cord-lineage cells.
Red arrowheads indicates ectopic expression.

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

Original annotation:
The Ci-FoxD/Ci-lefty transgene diminished the Ci-ZicL expression in A-line cells. Expression in a-line and b-line cells were not affected.
Blue arrowhead indicates blastomeres in which the expression was diminished.

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

Original annotation:
The SB431542 treatment diminished the Ci-ZicL expression in A-line cells. Expression in a-line and b-line cells were not affected.
Blue arrowhead indicates blastomeres in which the expression was diminished.

Vegetal view of a late gastrula stage embryo probed for ZicL expression following egg micro-injection of a FoxD-a/b MO. ZicL is down-regulated in a-line neural cells and A-line nerve cord cells.

Aniseed annotation : Weak staining is detected in B-line cells in this picture. b-line cells are not observed in the picture.

Vegetal view of a late gastrula stage embryo probed for ZicL expression following egg micro-injection of a FGF9/16/20-MO.
Origina Annotation : ZicL is down-regulated in a-line neural cells and b-line neural cells.