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 Mnx expression following egg micro-injection of a FoxA-a MO. Mnx was down-regulated in A-lin and B-line notochord.

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

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

Vegetal view of a 64-cell stage embryo probed for Mnx expression following egg micro-injection of a Ets/Pointed2 MO. Mnx was down-regulated in A-line and B-line notochord.

Vegetal view of a 110-cells stage embryo probed for Mnx expression following egg micro-injection of a FGF9/16/20 MO. Mnx was down-regulated in A-line and B-line notochord.

Vegetative view of a mid gastrula probed for Mnx expression, following injection of Snail morpholino (Snail-MO (Imai's 25mer MO)).
Original annotation : Red arrowheads mark ectopic expression. Mnx showed a marked upregulation following Snail knockdown, with strong ectopic expression observed in the lateral neural plate of most embryos.

Vegetative view of a mid gastrula probed for Mnx expression, following injection of Snail morpholino (Snail-MO 2 (Hudson 2015)).
Original annotation : Red arrowheads mark ectopic expression. Mnx showed a marked upregulation following Snail knockdown, with strong ectopic expression observed in the lateral neural plate of most embryos.

Vegetative view of a mid gastrula probed for Mnx expression, where Snail is overexpressed in medial lateral neural plate (electroporation of pETR>Snail). Mnx WT expression in the neural plate is lost.

Vegetative view of a mid gastrula probed for Mnx expression and treated with SB431542 (Nodal inhibitor) until early gastrula stage.
Original annotation : SB431542 treatment results in ectopic expression of Mnx in the lateral neural plate (red arrowheads).
Aniseed annotation : It seems that the observed stage is earlier than the control embryo.

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

Vegetative view of a mid gastrula probed for Mnx expression, where Nodal is overexpressed in animal territories (electroporation of pFOG>Nodal). Mnx WT expression in the neural plate is lost, and signal in the primary muscle lineage is strongly reduced.

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

Original annotation:
The Ci-FoxD/Ci-nodal transgene suppressed the Ci-mnx expression.
Blue arrowhead indicates blastomeres in which the expression was diminished.

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

Original annotation:
Ci-FoxD/Ci-lefty transgene mediated ectopic activation of Ci-Mnx expression.
Red arrowheads indicates ectopic expression.

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

Original annotation:
SB431542 mediated ectopic activation of Ci-Mnx expression.
Red arrowheads indicates ectopic expression.

Vegetal view of a late gastrula stage embryo probed for Mnx expression following egg micro-injection of a Snail MO. Mnx was ectopically expressed in lateral part of A-line nerve cord cells.

Vegetal view of a late gastrula stage embryo probed for Mnx expression following egg micro-injection of a FoxB MO. Mnx expression was down-regulated in A-line nerve cord cells.

Vegetal view of a late gastrula stage embryo probed for Mnx expression following egg micro-injection of a ZicL MO.

Vegetal view of a late gastrula embryo probed for Ci-Mnx expression following the injection of DAPT.

Original Annotation : Ci-Mnx expression was not affected by inhibition of Delta2/Notch signaling due to DAPT.
The numbers indicate the mean number of cells (c) per embryo expressing a given gene, n=total number of embryos analysed.

Dorsal view of an early tailbud stage embryo probed for Mnx 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 :
Mnx expression was lost (white arrow).

Lateral view of a mid tailbud stage embryo probed for Mnx expression, following the injection of Mnx MO.

Original Annotation :
Ectopic expression was observed in the nerve cord (black arrow)

Aniseed Annotation :
According to the picture, it seems that muscle cells are also stained.

Lateral view of the motor ganglion of a Ciona robusta mid tailbud embryo probed for Mnx (green) and Vsx (red) expressions and treated with the MEK inhibitor U0126 from 6hpf.
Mnx is expressed in A10.57, A10.58, A10.64 and A19.63.
Original comment: : U0126 at 6 hpf results in loss of Mnx from A11.118 and expansion of Mnx in the A9.29 lineage (asterisks), and ectopic Vsx in all four descendants of A9.30 (arrowheads).

Lateral view of the motor ganglion of a Ciona robusta mid tailbud embryo probed for Mnx (green) and Vsx (red) expressions and treated with the MEK inhibitor U0126 from 6hpf.
Vsx is expressed in A11.117, A11.118, A11.119 and A11.120.
Original comment: : U0126 at 6 hpf results in loss of Mnx from A11.118 and expansion of Mnx in the A9.29 lineage (asterisks), and ectopic Vsx in all four descendants of A9.30 (arrowheads).

Lateral view of the motor ganglion of a Ciona robusta mid tailbud embryo probed for Mnx (green) and Vsx (red) expressions and treated with the MEK inhibitor U0126 from 8hpf.
Mnx is expressed in A10.57 and A10.58.
Original comment: U0126 at 8 hpf results in loss of Mnx from A11.118 and expansion of Mnx in the A9.29 lineage (asterisks), and ectopic Vsx in A11.118 (arrowheads).

Lateral view of the motor ganglion of a Ciona robusta mid tailbud embryo probed for Mnx (green) and Vsx (red) expressions and treated with the MEK inhibitor U0126 from 8hpf.
Vsx is expressed in A11.117 and A11.118.
Original comment: U0126 at 8 hpf results in loss of Mnx from A11.118 and expansion of Mnx in the A9.29 lineage (asterisks), and ectopic Vsx in A11.118 (arrowheads).

Lateral view of the motor ganglion of a Ciona robusta mid tailbud embryo probed for Mnx (green) and Vsx (red) expressions and treated with the MEK inhibitor U0126 from 9hpf.
Mnx is expressed in A10.57 and A11.118 (asterik and double arrowhead respectively).
Original comment: U0126 at 9 hpf does not alter Mnx or Vsx expression

Lateral view of the motor ganglion of a Ciona robusta mid tailbud embryo probed for Mnx (green) and Vsx (red) expressions and treated with the MEK inhibitor U0126 from 9hpf.
Vsx is expressed in A11.117 (arrowhead).
Original comment: U0126 at 9 hpf does not alter Mnx or Vsx expression