Oocytes of a single individual of Ciona intestinalis (Roscoff, France) were fertilized by a mixture of sperm of two individuals and development was followed microscopically. Embryos were snapfrozen in liquid nitrogen at the appropriate stage and kept at -80°C. Samples at stage 0, 8, 12, 15, 21, 26 were collected from the same fertilzation, while sample from stage 11 was independently collected from a different individual. Total RNA was prepared with the RNA II kit of Magerey Nagel and the quality was checked with the Bioanalyzer, all samples had a RIN value of higher than 9. Strand specific libraries were constructed and the sequencing (50PE) was performed with Illumina HiSeq 2000 technology (BGI, Hong Kong, China). Reads were aligned onto C.robusta genome and considered to reflect the expression of the C.robusta orthologous genes.

Dorsal view of a mid tailbud embryo (10 hpf) probed for ci-Nodal (red) and Ci-Rx (green) expression (focus on the sensory vesicle region).
Image of DAPI staining is additionally merged (blue). Dorsal views are shown with anterior to the top. Dashed lines indicate the midline.

Original annotation:
Ci-Rx is expressed bilaterally in the SV until 10 hpf and then down regulated on the left side by 11 hpf when the expression of Ci-Nodal starts to be detectable in the left SV. At 12 hpf, Ci-Nodal expression in the left SV expands, while the expression of Ci-Rx is no longer detectable on the left side of the SV.

Dorsal view of a mid tailbud embryo (11 hpf) probed for ci-Nodal (red) and Ci-Rx (green) expression (focus on the sensory vesicle region).
Image of DAPI staining is additionally merged (blue). Dorsal views are shown with anterior to the top. Dashed lines indicate the midline.

Original annotation:
Ci-Rx is expressed bilaterally in the SV until 10 hpf and then down regulated on the left side by 11 hpf when the expression of Ci-Nodal starts to be detectable in the left SV. At 12 hpf, Ci-Nodal expression in the left SV expands, while the expression of Ci-Rx is no longer detectable on the left side of the SV.

Dorsal view of an early tailbud DMSO-treated embryo probed for ci-Rx expression.

Original annotation:
During the early tail bud stage, Ci-Rx was bilaterally expressed in the SV in control embryos.

Electroporation experiments with a 3.0 kb genomic DNA fragment starting immediatly upstream from the Ci-Rx coding region linked to LacZ reporter genes. The construct is named Rx-LacZ in the paper. Lateral and dorsal views of electroporated embryos, showing three cells in the anterior region of the developing nervous system.

The last picture shows a semi-thin transverse section at the level indicated by the picture above, showing expression in two groups of brain precursor cells.

No embryo picture in the article.
At the tailbud stage, INTR0.12 (the 125bp fragment located in the 3rd intron of Rx (KH.C12.152) cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the tailbud stage, B1.6 (the 1647bp fragment located upstream (-2952/-1299bp)of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the tailbud stage, D0.48 (the 481bp fragment located upstream (-1318/-838bp)of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the tailbud stage, G0.14 (the 138bp fragment located upstream (-897/-708bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the tailbud stage, H0.14 (the 137bp fragment located upstream (-729/-593bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

Dorsal view of a WT Ciona robusta tailbud probed for Rx (KH.C12.152) expression. Rx is expressed in the anteriot sensory vesicle.
Original comment: Expression is in three groups of cells in the anterior brain (cns, central nervous system).
Scale bar: 50 μm

Dorsal view of a WT Ciona robusta tailbud probed for Rx (KH.C12.152, red) and Onecut (green) expressions.
E: Onecut is expressed in the sensory vesicle and in the visceral ganglion.
D: DAPI staining for nuclei (blue).
F: Rx is expressed in the sensory vesicle.
G: Merge image of D, E, and F. Orange color in G indicates co-expression territory of Ci-Rx and Ci-Onecut.
Original comment: The most anterior cells expressing Ci-Rx consistently also co-express Ci-Onecut. Furthermore, we observed Ci-Onecut co-localization in the two more anterior groups of cells that express Ci-Rx. Unfortunately, it was not been possible to establish if Ci-Onecut is expressed in the more posterior cell marked by Ci-Rx, because Ci-Rx expression in this cell is too faint and transient to be detected with this technique.

Dorsal view of a WT Ciona robusta tailbud probed for Rx (KH.C12.152, red) and Onecut (green) expressions.
F: Rx is expressed in the sensory vesicle.
E: Onecut is expressed in the sensory vesicle and in the visceral ganglion.
D: DAPI staining for nuclei (blue).
G: Merge image of D, E, and F. Orange color in G indicates co-expression territory of Ci-Rx and Ci-Onecut.
Original comment: The most anterior cells expressing Ci-Rx consistently also co-express Ci-Onecut. Furthermore, we observed Ci-Onecut co-localization in the two more anterior groups of cells that express Ci-Rx. Unfortunately, it was not been possible to establish if Ci-Onecut is expressed in the more posterior cell marked by Ci-Rx, because Ci-Rx expression in this cell is too faint and transient to be detected with this technique.

No embryo picture in the article.
At the tailbud stage, M0.1 (the 96bp fragment located upstream (-2559/-2440bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the tailbud stage, J-B (the 35bp fragment located upstream (-577/-543bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the tailbud stage, J-C (the 30bp fragment located upstream (-547/-518bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the tailbud stage, J-D (the 34bp fragment located upstream (-531/-498bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the tailbud stage, J-E (the 50bp fragment located upstream (-502/-453bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

Lateral view of a WT Ciona robusta tailbud electroporated with J-A>LacZ reporter construct. The 31bp element located upstream of Rx TSS drives LacZ expression in the central nervous system (cns) of 22% of the tested embryos.
Original comment: The J-A construct was able to closely recapitulate the endogenous Ci-Rx expression in the sensory vesicle at both the tailbud and larva stages.

Dorsal view of a late tailbud embryo (12 hpf) probed for ci-Nodal (red) and Ci-Rx (green) expression (focus on the sensory vesicle region).
Image of DAPI staining is additionally merged (blue). Dorsal views are shown with anterior to the top. Dashed lines indicate the midline.

Original annotation:
Ci-Rx is expressed bilaterally in the SV until 10 hpf and then down regulated on the left side by 11 hpf when the expression of Ci-Nodal starts to be detectable in the left SV. At 12 hpf, Ci-Nodal expression in the left SV expands, while the expression of Ci-Rx is no longer detectable on the left side of the SV.

Dorsal view of a late tailbud DMSO-treated embryo probed for ci-Rx expression.

Original annotation:
In the control late tail bud embryos, expression of Ci-Rx was detected on the right side of the SV as previously reported.

Dorsal view of a late tailbud embryo probed for ci-Rx expression.

Conversion to aniseed format of DBTGR database entry from Kusakabe team.

Original annotation:
Brain vesicle.

Dorsal view of a WT Ciona robusta tailbud electroporated with A3.0>GFP reporter construct. The 3kb region located upstream of Rx TSS recapitulates Rx endogenous expression in the sensory vesicle.
Original comment: Expression of the reporter gene is visible in the same three groups of cells of the endogenous transcript in the anterior brain (cns, central nervous system).
Scale bar indicates 50 μm

Lateral view of a WT Ciona robusta tailbud electroporated with L0.33>LacZ reporter construct. The 333bb region located upstream of Rx TSS only drives LacZ ectopic expression in the mesenchyme (me).

Lateral view of a Ciona robusta tailbud probed for Rx (KH.C12.152) expression and electroporated with ETR>GFP (control experiment).
Rx is expressed in the sensory vesicle (sv).

Lateral view of a WT Ciona robusta tailbud electroporated with C1.3>LacZ reporter construct. The 1288bb region located upstream of Rx drives LacZ expression in the central nervous system (cns) and ectopic expression in the mesenchyme (me) and in some epidermal cells of the tail .
Scale bar: 100 μm

Lateral view of a WT Ciona robusta tailbud electroporated with J0.2>LacZ reporter construct. The 197bb region located upstream of Rx TSS drives LacZ expression in the central nervous system (cns).

Lateral view of a WT Ciona robusta tailbud electroporated with K0.27>LacZ reporter construct. The 274bb region located upstream of Rx TSS drives LacZ expression in the central nervous system (cns), and ectopically in the mesenchyme and in the tail epidermis.

Lateral view of a WT Ciona robusta tailbud electroporated with J-F>LacZ reporter construct. This 50bp element located upstream of Rx TSS drives LacZ expression in the central nervous system (cns) and in the mesenchyme of 30% of the tested embryos.
Original comment: The J-F construct was able to closely recapitulate the endogenous Ci-Rx expression in the sensory vesicle at both the tailbud and larva stages.

Dorsal view of a WT Ciona robusta tailbud electroporated with E0.49>LacZ reporter construct. The 492bb region located upstream of Rx TSS drives LacZ expression in the central nervous systme (cns).
Scale bar: 100 μm

Lateral view of a WT Ciona robusta tailbud electroporated with F0.49>LacZ reporter construct. The 487bp region located upstream of Rx TSS drives LacZ reporter expression in the sensory vesicle (cns, central nervous system) and in the mesenchyme (me).
Original comment: The F0.49 construct was able to induce the expression of LacZ in the same territories as the endogenous Ci-Rx at tailbud and larva stages. Moreover, it is evident that the F0.49 fragment also showed ectopic expression in the trunk mesenchyme and in the tail epidermis in a high percentage of transgenic embryos.

Dorsal view of a tadpole embryo probed for ci-Rx (red) and opsin (green) expression.

Lateral view of a WT Ciona robusta larva probed for Rx (KH.C12.152) expression. Rx is expressed in the sensory vesicle (sv).
Original comment: Expression is in the sensory vesicle in the cells surrounding the ocellus.
Scale bar: 50 μm

Electroporation experiments with a 3.0 kb genomic DNA fragment starting immediatly upstream from the Ci-Rx coding region linked to LacZ reporter genes. The construct is named Rx-LacZ in the paper. Electroporated larva showing the signal in the sensory vesicle around the ocellus.

The last picture shows a semi-thin transverse section at the level indicated by the picture above, showing expression in the posterior wall of the sensory vesicle.

Electroporation experiments with a 3.0 kb genomic DNA fragment starting immediatly upstream from the Ci-Rx coding region linked to GFP reporter genes. The construct is named Rx-GFP in the paper.

Fluorescence images of electroporated larvae. GFP expression is shown in almost the entire posterior half of the sensory vesicle, in the sensory neurons of the palps, in a mass of neuronal fibers around the sensory vesicle and in a tract of fibers extending from the sensory vesicle into the visceral ganglion.

Electroporation experiments with a 3.0 kb genomic DNA fragment starting immediatly upstream from the Ci-Rx coding region linked to GFP reporter genes. The construct is named Rx-GFP in the paper.

Bright-fiels images of electroporated larvae. GFP expression is shown in almost the entire posterior half of the sensory vesicle, in the sensory neurons of the palps, in a mass of neuronal fibers around the sensory vesicle and in a tract of fibers extending from the sensory vesicle into the visceral ganglion.

No embryo picture in the article.
At the larval stage, INTR0.12 (the 125bp fragment located in the 3rd intron of Rx (KH.C12.152) cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

Lateral view of the head of a WT Ciona robusta larva electroporated with A3.0>GFP reporter construct. The 3kb region located upstream of Rx TSS drives GFP expression in the sensory vesicle (sv) and in the mesenchyme (me).
Original comment:Expression of the reporter gene is visible in the sensory vesicle in the cells surrounding the ocellus in the same territory as the endogenous transcript and in some ectopic cells of the mesenchyme.
Scale bar indicates 50 μm

No embryo picture in the article.
At the larva stage, B1.6 (the 1647bp fragment located upstream (-2952/-1299bp)of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the larva stage, D0.48 (the 481bp fragment located upstream (-1318/-838bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the larva stage, G0.14 (the 138bp fragment located upstream (-897/-708bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the larva stage, H0.14 (the 137bp fragment located upstream (-729/-593bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the larva stage, M0.1 (the 96bp fragment located upstream (-2559/-2440bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the larva stage, J-B (the 35bp fragment located upstream (-577/-543bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the larva stage, J-C (the 30bp fragment located upstream (-547/-518bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the larva stage, J-D (the 34bp fragment located upstream (-531/-498bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

No embryo picture in the article.
At the larva stage, J-E (the 50bp fragment located upstream (-502/-453bp) of Rx TSS, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.

Lateral view of a WT Ciona robusta larva electroporated with C1.3>LacZ reporter construct. The 1288bb region located upstream of Rx TSS drives LacZ expression in the sensory vesicle (sv)
Original comment:This Ci-Rx enhancer also induces ectopic expression in the trunk mesenchyme (me) and in some epidermal cells of the tail.
Scale bar: 100 μm

Lateral view of a WT Ciona robusta larva electroporated with E0.49>LacZ reporter construct. The 492bb region located upstream of Rx TSS drives LacZ in the sensory vesicle.

Lateral view of a WT Ciona robusta larva electroporated with F0.49>LacZ reporter construct. The 487bp region located upstream of Rx TSS drives LacZ reporter expression in the sensory vesicle (cns, central nervous system), in the tail epidermis (e) and in the mesenchyme (me).
Original comment: The F0.49 construct was able to induce the expression of LacZ in the same territories as the endogenous Ci-Rx at tailbud and larva stages. Moreover, it is evident that the F0.49 fragment also showed ectopic expression in the trunk mesenchyme and in the tail epidermis in a high percentage of transgenic embryos.

Lateral view of a WT Ciona robusta larva electroporated with J0.2>LacZ reporter construct. The 197bb region located upstream of Rx TSS drives LacZ expression in the sensory vesicle (sv).

Lateral view of a WT Ciona robusta larva electroporated with K0.27>LacZ reporter construct. The 274bb region located upstream of Rx TSS drives LacZ expression in the sensory ve (sv), and ectopically in the mesenchyme (me), in tail muscles (m) and in the tail epidermis.

Lateral view of a WT Ciona robusta larva electroporated with L0.33>LacZ reporter construct. The 333bb region located upstream of Rx TSS only drives LacZ ectopic expression in the mesenchyme (me) and in some tail muscles (m).

Lateral view of a WT Ciona robusta larva electroporated with J-A>LacZ reporter construct. The 31bb element located upstream of Rx TSS drives LacZ expression in the sensory vesicle (sv) of 22% of the tested embryos.
Original comment: The J-A construct was able to closely recapitulate the endogenous Ci-Rx expression in the sensory vesicle at both the tailbud and larva stages.

Lateral view of a WT Ciona robusta larva electroporated with J-F>LacZ reporter construct. This 50bp element located upstream of Rx TSS drives LacZ expression in the sensory vesicle (sv) of 30% of the tested embryos.
Original comment: The J-F construct was able to closely recapitulate the endogenous Ci-Rx expression in the sensory vesicle at both the tailbud and larva stages.

No embryo picture in the article.
Electroporation experiments of the J0.2 MUT construct, carrying the 5 point mutations (of the Onecut conserved binding site) showed only 29% of the embryos with reporter gene expression in contrast to the 65% of positive embryos electroporated with the wild type J0.2 construct.

No embryo picture in the article.
At the larva stage, J-A-MUT (the 31bp Rx enhancer with a mutated Clox binding site, cloned upstream the human β-globin basal promoter and LacZ reporter gene) has no enhancer activity.