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.

Late neurula stage embryo (7.5hpf) electroporated with a construct containing region upstream Ci-RhoD and a LacZ reporter gene.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -3472/+982

Original annotation:
LacZ transcipts could be detected in the TVCs (arrows) as early 8.5hpf, while notochord expression (n) started earlier.

Early tailbud stage embryo (8.5hpf) electroporated with a construct containing region upstream Ci-RhoD and reporter LacZ. Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference. Coordinates in the paper : -3472/+982

Original annotation:
LacZ transcripts could be detected in the TVCs (arrows) as early 8.5hpf, while notochord expression started earlier.

Original annotation: expression in the notochord was stronger in the anterior-most cells.
Gene name: RhoF/D (Rif)

Aniseed annotation:
Data and annotation provided by the author.

Lateral view of a mid tailbud stage embryo electroprated with a construct containing the second exon and the most of the second intron of the RhoD/F transcript, in front of FOG basal promoter et LacZ reporter gene.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +498/+922

Original annotation :
This construct produced significant expression in the TVCs.

Mid tailbud stage embryos electroporated with a construct containing region upstream Ci-RhoD and reporter LacZ. Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference. Coordinates in the paper : -3472/+982.

Original annotation:
LacZ transcripts could be detected in the TVCs (arrows) as early 8.5hpf, while notochord expression started earlier.

Mid tailbud stage embryo electroporated with a construct containing the region upstream Ci-RhoD. Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference. Coordinates in the paper : -3472/+982

Original annotation:
X-gal staining in muscle and mesenchyme is weak.

Latreal view of a mid tailbud embryo electroporated with a construct containing region -3406bp/83bp upstream Ci-RhoD.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -3472/+17

Original annotation:
RhoD expression was detected in notochord (strong staining) and mesenchyme (weak staining). The loss of EtS1/2 and FoxF sites is sufficient to abolish Ci-RhoD expression in TVCs and muscles.

Mid tailbud stage embryos electroporated with a construct containing region -3406bp/601bp upstream Ci-RhoD. No picture available in the article.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -3472/+982

Original annotation:
RhoD expression was detected in notochord (strong staining) and mesenchyme (weak staining). The loss of EtS1/2 and FoxF sites is sufficient to abolish Ci-RhoD expression in TVCs and muscles.

Mid tailbud embryos electroporated with a construct containing region -2606bp/82bp upstream Ci-RhoD. No picture available in the article.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -2672/+17

Original annotation:
RhoD expression was detected in notochord (strong staining) and mesenchyme (weak staining). The loss of EtS1/2 and FoxF sites is sufficient to abolish Ci-RhoD expression in TVCs and muscles.

Mid tailbud embryos electroporated with a construct containing region -1299p/83bp upstream Ci-RhoD. No picture available in the article.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -1365/+17

Original annotation:
RhoD expression was detected in muscles and mesenchyme (strong staining).

Mid tailbud stage embryos electroporated with a construct containing region -2606bp/1049bp upstream Ci-RhoD. No picture available in the article.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -2672/+982

Original annotation:
RhoD expression was detected in notochord (strong staining), TVC (strong staining), muscles (weak staining) and mesenchyme (weak staining).

Mid tailbud stage embryos electroporated with a construct containing region -1299/1049 upstream Ci-RhoD. No picture available in the article.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference. Coordinates in the paper : -1365/+982

Original annotation:
RhoD expression was detected in TVCs, muscles and mesenchyme (strong staining).

Lateral view of a mid tailbud stage embryo electroporated with a construct containing region -1005bp/1048bp upstream Ci-RhoD.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -1071/+982

Original annotation:
RhoD expression was detected in TVCs, muscles and mesenchyme (strong staining). The loss of region from -3472 to -1071 is sufficient to abolish Ci-RhoD expression in notochord.

Mid tailbud embryos electroporated with a construct containing region -645bp/1049bp upstream Ci-RhoD. No picture available in the article.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -1365/+982

Original annotation:
RhoD expression was detected in TVCs, muscles and mesenchyme (weak staining). The loss of the region from -3472 to -711 is sufficient to abolish Ci-RhoD expression in notochord.

Mid tailbud embryos electroporated with a construct containing region -441bp/1049bp upstream Ci-RhoD. No picture available in the article.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -507/982

Original annotation:
RhoD expression was detected in TVCs, muscles and mesenchyme. The loss of the region from -3472bp to -507bp is sufficient to abolish RhoD expression in notochord.

Mid tailbud embryos electroporated with a construct containing RhoD regulatory region from 228bp to 989bp, in front of FOG basal promoter and a LacZ reporter gene.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -1365/+982
No picture available in the article.

Original annotation:
About 38% of embryos shows a strong staining, and 25% a weak staining.

Mid tailbud embryos electroporated with a construct containing RhoD regulatory region from 228bp to 835bp, in front of FOG basal promoter and a LacZ reporter gene.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -161/+768
No picture available in the article.

Mid tailbud embryos electroporated with a construct containing RhoD regulatory region from 228bp to 575bp, in front of FOG basal promoter and a LacZ reporter gene.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +161/+508
No picture available in the article.

Mid tailbud embryos electroporated with a construct containing RhoD regulatory region from 448bp to 990bp, in front of FOG basal promoter and a LacZ reporter gene.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +380/+922
No picture available in the article.

Original annotation:
About 10% of embryos shows a strong staining, and 25% a weak staining.

Mid tailbud embryos electroporated with a construct containing RhoD regulatory region from 448bp to 835bp, in front of FOG basal promoter and a LacZ reporter gene.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +380/+768
No picture available in the article.

Original annotation:
About 20% of embryos shows a weak staining, and a few, a strong staining.

Mid tailbud embryos electroporated with a construct containing RhoD regulatory region from 448bp to 576bp, in front of FOG basal promoter and a LacZ reporter gene.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +380/+508
No picture available in the article.

Mid tailbud embryos electroporated with a construct containing RhoD regulatory region from 819bp to 989bp, in front of FOG basal promoter and a LacZ reporter gene.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +752/+922
No picture available in the article.

Region from +565bp to +989 upstream the RhoD/F gene, containing the second exon. The first Ets1/2 site was mutated (indicated by "X").
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +498/+922
In red and green ellipses, putative Ets1/2 and FoxF sites are represented respectively.
Bars depict the proportion of embryos showing weak (light blue) or strong (dark blue) X-gal staining in TVC, muscle (and mesenchyme?).

Mid tailbud embryos electroporated with a construct containing RhoD regulatory region from 647bp to 989bp, in front of FOG basal promoter and a LacZ reporter gene.
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +580/+922

Original annotation:
Staining in notochord, TVC, muscle and mesenchyme is lost.

Aniseed annotation:
Staining is observed in the dorsal midline epidermis, secondary muscle lineage and maybe in the palps.

Region from +565bp to +989 upstream the RhoD/F gene, containing the second exon. The first FoxF site was mutated (indicated by "X").
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +498/+922
In red and green ellipses, putative Ets1/2 and FoxF sites are represented respectively.
Bars depict the proportion of embryos showing weak (light blue) or strong (dark blue) X-gal staining in TVC, muscle (and mesenchyme?).

Region from +565bp to +989 upstream the RhoD/F gene, containing the second exon. The second FoxF site was mutated (indicated by "X").
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +498/+922
In red and green ellipses, putative Ets1/2 and FoxF sites are represented respectively.
Bars depict the proportion of embryos showing weak (light blue) or strong (dark blue) X-gal staining in TVC, muscle (and mesenchyme?).

Region from +565bp to +989 upstream the RhoD/F gene, containing the second exon. The third FoxF site was mutated (indicated by "X").
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +498/+922
In red and green ellipses, putative Ets1/2 and FoxF sites are represented respectively.
Bars depict the proportion of embryos showing weak (light blue) or strong (dark blue) X-gal staining in TVC, muscle (and mesenchyme?).

Region from +565bp to +989 upstream the RhoD/F gene, containing the second exon. The fourth FoxF site was mutated (indicated by "X").
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +498/+922
In red and green ellipses, putative Ets1/2 and FoxF sites are represented respectively.
Bars depict the proportion of embryos showing weak (light blue) or strong (dark blue) X-gal staining in TVC, muscle (and mesenchyme?).

Region from +565bp to +989 upstream the RhoD/F gene, containing the second exon. The second and the third FoxF site were mutated (indicated by "X").
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +498/+922
In red and green ellipses, putative Ets1/2 and FoxF sites are represented respectively.
Bars depict the proportion of embryos showing weak (light blue) or strong (dark blue) X-gal staining in TVC, muscle (and mesenchyme?).

Region from +565bp to +989 upstream the RhoD/F gene, containing the second exon. The second and fourth FoxF site were mutated (indicated by "X").
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +498/+922
In red and green ellipses, putative Ets1/2 and FoxF sites are represented respectively.
Bars depict the proportion of embryos showing weak (light blue) or strong (dark blue) X-gal staining in TVC, muscle (and mesenchyme?).

Region from +565bp to +989 upstream the RhoD/F gene, containing the second exon. The third and the fourth FoxF site were mutated (indicated by "X").
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +498/+922
In red and green ellipses, putative Ets1/2 and FoxF sites are represented respectively.
Bars depict the proportion of embryos showing weak (light blue) or strong (dark blue) X-gal staining in TVC, muscle (and mesenchyme?).

Region from +565bp to +989 upstream the RhoD/F gene, containing the second exon. The second, third and fourth FoxF site were mutated (indicated by "X").
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +498/+922
In red and green ellipses, putative Ets1/2 and FoxF sites are represented respectively.
Bars depict the proportion of embryos showing weak (light blue) or strong (dark blue) X-gal staining in TVC, muscle (and mesenchyme?).

Region from +565bp to +989 upstream the RhoD/F gene, containing the second exon. The second Ets1/2 site was mutated (indicated by "X").
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +498/+922
In red and green ellipses, putative Ets1/2 and FoxF sites are represented respectively.
Bars depict the proportion of embryos showing weak (light blue) or strong (dark blue) X-gal staining in TVC, muscle (and mesenchyme?).

Region from +565bp to +989 upstream the RhoD/F gene, containing the second exon. The first and second Ets1/2 sites were mutated (indicated by "X").
Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : +498/+922
In red and green ellipses, putative Ets1/2 and FoxF sites are represented respectively.
Bars depict the proportion of embryos showing weak (light blue) or strong (dark blue) X-gal staining in TVC, muscle (and mesenchyme?).

Close-up views of the tail-trunk boundary of mid-tailbud embryos electroporated with RhoDF>lacZ (E) or Mesp>GFP (F). Β-galactosidase and GFP were detected by immunohistochemistry. Blue staining (F) : phalloidin staining.

Original annotation:
RhoD expression was detected in notochord (n), mesenchyme and B7.5-lineage cells.