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 an early tailbud embryo probed for Ci-Noto1 expression following egg micro-injection of a FGF9 MO.

Early tailbud stage probed for Ci-Noto1 expression following injection of SB431542 inhibitor. This picture shows that Ci-Noto1 expression in the secondary notochord precursor remained expressed after treatment with SB431542.

Vegetal view of an early tailbud stage embryo probed for Ci-Noto1 expression in which nodal signalling has been blocked with SB431542. Cell division was blocked by cytochalasin-B. Treatment with SB431542 selectively abolished expression of Ci-Noto1 in the secondary notochord precursors (B8.6 cell pair).

Vegetal view of an early tailbud stage embryo probed for Ci-Noto1 expression in which Nodal signalling has been inhibited by injection of Nodal-Mo. Cell division was blocked by cytochalasin-B. Injection of Nodal-Mo causes a severe downregulation of Ci-Noto1 expression in B8.6 cell pair.

Dorsal view of an early tailbud embryo probed for Ci-Noto1 expression. Embryos were traited with a pharmacological inhibitor U0126 from the 44 cell stage. Expression is lost.

Dorsal view of an early tailbud embryo probed for Ci-Noto1 expression. Embryos were traited with a pharmacological inhibitor U0126 from the 32 cell stage. Expression is lost.

Dorsal view of an early tailbud embryo probed for Ci-Noto1 expression. Embryos were traited with a pharmacological inhibitor U0126 from the 64 cell stage. A majority of embryos expressed a reduced level of Ci-Noto1 in the notochord.

Dorsal view of an early tailbud embryo probed for Ci-Noto1 expression. Embryos were traited with a pharmacological inhibitor U0126 from the 110 cell stage. A majority of embryos expressed a reduced level of Ci-Noto1 in the notochord.

Expression of Ci-Noto1 at the early tailbud stage in embryos traited with cytochalasin from the 64 cell stage, with a pharmacological inhibitor U0126 from the 32 cell stage and injected with FGF9 MO.

Expression of Ci-Noto1 at the early tailbud stage in embryos traited with cytochalasin from the 64 cell stage and injected with FGF9 MO.

Expression of Ci-Noto1 at the early tailbud stage in embryos traited with cytochalasin from the 64 cell stage. Embryos were traited with a pharmacological inhibitor U0126 from the 32 cell stage.

Lateral views of early tailbud embryos probed for Ci-Noto1 expression following injection of FGF9-MO. Staining observed in the notochord is perturbed compare to the control.

Lateral views of early tailbud embryos probed for Ci-Noto1 expression following injection of FGF9-MO, FGF8-MO and bFGF. Staining observed in the notochord is perturbed compare to the control.

Lateral view of an early tailbud embryo probed for Ci-Noto1 expression following injection of FGF9-MO and FGF8-MO. Staining observed in the notochord in the control is lost.

Vegetal view of an early tailbud stage embryo probed for Ci-Noto1 expression in which Delta signalling has been blocked with Del2-Mo. Cell division has been blocked with CytochalasinB. Treatement with Del2-Mo selectively abolished Ci-noto1 expression in the secondary notochord precursors.

Vegetal view of an early tailbud stage embryo probed for Ci-Noto1 expression in which gamma-secretase has been inhibited with a pharmacological reagent DAPT. Cell division has been blocked with CytochalasinB. Treatment with DAPT selectively abolished Ci-noto1 expression in the secondary notochord precursor.

Lateral view of a Ciona robusta early tailbud embryo probed for Noto1 expression and injected into the pair of anterior animal cells at the 8-cell stage with morpholino oligos against Prdm1-r.a, Prdm1-r.b and Hes.a.
Noto1 expression is observed in the notochord and in the trunk.
Original annotation: Noto1, which is a marker for notochord, was expressed ectopically in the trunk region of Prdm1-r.a/b/Hes.a morphant tailbud embryos.
n, number of embryos examined. % : percentage of embryos with ectopic expression (cyan arrowhead).

Lateral view of the head of a Ciona robusta early tailbud embryo probed for Noto1 (green) and LacZ (magenta) expressions and injected into the pair of anterior animal cells at the 8-cell stage with Prdm1-r.a/ Prdm1-r.b/Hes.a MOs concomitantly with lacZ mRNA.
Noto1 expression is observed in the notochord and in the trunk.
Original annotation: Cells that ectopically expressed Noto1 were derived from the anterior animal blastomeres of 8-cell embryos, as indicated by our finding that lacZ mRNA, injected as a tracer together with Prdm1-r.a, Prdm1-r.b and Hes.a MOs, was detected in the same cells that expressed Noto1.
n, number of embryos examined. % : Percentage of embryos with simultaneous expression of Noto1 and lacZ.

Lateral view of a Ciona robusta early tailbud embryo probed for Noto1 expression and injected into the pair of anterior animal cells at the 8-cell stage with morpholino oligos against Prdm1-r.a and Prdm1-r.b.
Noto1 expression is observed in the notochord.
Original annotation: Prdm1-r.a/b double morphants rarely expressed Noto1 ectopically.
n, number of embryos examined. % : percentage of embryos with ectopic expression.

Lateral view of a Ciona robusta early tailbud embryo probed for Noto1 expression and injected into the pair of anterior animal cells at the 8-cell stage with morpholino oligos against Prdm1-r.a, Prdm1-r.b and Hes.a.
Noto1 expression is observed in the notochord and in the trunk.
Original annotation: Noto1, which is a marker for notochord, was expressed ectopically in the trunk region of Prdm1-r.a/b/Hes.a morphant tailbud embryos.
n, number of embryos examined. % : percentage of embryos with ectopic expression (cyan arrowhead).

Lateral view of the head of a Ciona robusta early tailbud embryo probed for Noto1 (green) and LacZ (magenta) expressions and injected into the pair of anterior animal cells at the 8-cell stage with Prdm1-r.a/ Prdm1-r.b/Hes.a MOs concomitantly with lacZ mRNA.
Noto1 expression is observed in the notochord and in the trunk.
Original annotation: Cells that ectopically expressed Noto1 were derived from the anterior animal blastomeres of 8-cell embryos, as indicated by our finding that lacZ mRNA, injected as a tracer together with Prdm1-r.a, Prdm1-r.b and Hes.a MOs, was detected in the same cells that expressed Noto1.
n, number of embryos examined. % : Percentage of embryos with simultaneous expression of Noto1 and lacZ.

Mid tailbud stage embryo probed for Ci-Noto1 expression following injection of of Ci-fkh/Ci-Bra.

Original annotation:
The Ciona Noto1 gene is expressed ectopically in the mutant as compared with the control embryo. In particular, staining is observed both in the endogenous notochord and transformed endoderm (no picture available in the paper).

Lateral view of a mid tailbud embryo stained for Ci-Noto1 mRNA after electroporation of Ci-IkappaB MO. The expression in the notochord was not altered (arrowhead).

Lateral view of a late tailbud embryo stained for Ci-Noto1 mRNA after electroporation of Ci-IkappaB MO. The expression in the notochord was not altered (arrowhead).