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 Hand-like expression and injected with ephrin-Ad MO.

Original annotation: Hand-like displays an almost complete loss of expression.

Vegetal view of a 64-cell stage embryo showing expression of Hand-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 a 110-cell stage embryo probed for NoTrlc expression following egg micro-injection of a FoxD-a/b MO.

Original annotation: expression is not affected.

Vegetal view of a 110-cell stage embryo probed for NoTrlc expression following egg micro-injection of a Nodal MO. NoTrlc was down-regulated in TLCs.

Vegetal view of a 110-cells stage embryo probed for NoTrlc expression following egg micro-injection of a FoxA-a MO. NoTrlc was down-regulated in TLCs.

Vegetal view of a 110-cell stage embryo showing expression of Hand-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.

Vetetal views of embryos injected at the 1-cell stage with torso-FGFR mRNA and probed for Hand-like expression.

Original annotation: expression of Hand-like is modestly reduced.

Vegetal view of a 110 cell stage embryo probed for Hand-like expression following 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). Hand-like expression is observed in A7.6 blastomeres and in anterior endoderm (arrows).

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

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

Aniseed Annotation: Ectopic expression of Hand-like is showing in A8.7*.

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

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

Lateral view of a tailbud stage embryo probed for NoTrlc expression following egg micro-injection of a FGF9/16/20 MO. NoTrlc was down-regulated in TVCs.

Lateral view of a tailbud stage embryo probed for NoTrlc expression following egg micro-injection of a Mesp MO. NoTrlc is down-regulated in TVCs.

Lateral view of a mid tailbud stage embryo co-electroporated with Mesp-lacZ (red) and Ttf-GATAa-WRPW and probed for Handlike (green) expression.
The construct Ttf-GATAa-DBD was used as a control.

Original annotation:
There was no detectable difference in the TVC expression of Handlike between Ttf-GATAa-WRPW and control embryos.

Control: sagital view of WT mid tailbud embryos showing expression of Hand-like by fluorescent ISH (A), the position of the B7.5 lineage (A') and the overlay (A"). Note that Hand-like is expressed in the anterior B7.5-derived heart progenitors.

Experimental embryos: sagital view of late tailbud embryos electroporated with mesp::FoxF-WRPW, showing expression of Hand-like by fluorescent ISH (C), the position of the B7.5 lineage (C') and the overlay (C"). Note that Hand-like is expressed in the anterior B7.5-derived heart progenitors although they do not migrate into the trunk.

Control: sagital view of WT mid tailbud embryos showing expression of Hand-like by fluorescent ISH (A), the position of the B7.5 lineage (A') and the overlay (A"). Note that Hand-like is expressed in the anterior B7.5-derived heart progenitors.

Experimental embryos: sagital view of late tailbud embryos derived from FoxF MO injected eggs, showing expression of Hand-like by fluorescent ISH (B), the position of the B7.5 lineage (B') and the overlay (B"). Note that Hand-like is expressed in the anterior B7.5-derived heart progenitors although they do not migrate into the trunk.

Control: sagital view of WT mid tailbud embryos showing expression of Hand-like by fluorescent ISH (A), the position of the B7.5 lineage (A') and the overlay (A"). Note that Hand-like is expressed in the anterior B7.5-derived heart progenitors.

Experimental embryos: sagital view of late tailbud embryos co-electroporated with mesp::FoxF-WRPW and mesp:ETS1/2-VP16, showing expression of Hand-like by fluorescent ISH (D), the position of the B7.5 lineage (D') and the overlay (D"). Note that Hand-like is expressed in all B7.5-derived cells although they do not migrate into the trunk. In this experiment, Ets1/2-VP16 transforms all B7.5 progeny into heart precursors, and FoxF-WRPW prevents their migration but not their differentiation.

Lateral view of a Ciona robusta mid tailbud embryo probed for Hand-like expression and electroporated with Mesp>FGFR-DN overexpression construct. Staining is lost in the trunk ventral cells but remains in a few cells at the tip of the tail.
Original annotation: Targeted expression of the dominant negative FGFR in the B7.5 lineage prevent the B7.5 lineage from expressing the heart lineage marker, Hand-like. Note Hand-like expression in the tip of the tail despite loss in the heart lineage.

Lateral view of a Ciona robusta late tailbud embryo probed for Hand-like expression and electroporated with Mesp>Ets1/2-VP16 overexpression construct. Staining is observed in the whole B7.5 lineage.
Original annotation: Targeted expression of the constitutively active Ets1/2 in the B7.5 lineage causes both lineages (Trunk ventral cells and anterior ventral primary muscle lineage)to express the heart lineage marker Hand-like.