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.

64-cell stage wild type embryo probed for citb3f17 clone. Expression was detected in the notochord from the initial tailbud stage (data not shown in the article).

Mid gastrula stage wild type embryo probed for citb3f17 clone. Expression was detected in the notochord from the initial tailbud stage (data not shown in the article).

Mid neurula stage wild type embryo probed for citb3f17 clone. Expression was detected in the notochord from the initial tailbud stage (data not shown in the article).

Initial tailbud wild type embryo probed for citb3f17 clone. Expression was detected in the notochord from the initial tailbud stage (data not shown in the article).

Original annotation: somewhat stronger in trunk ventral cells (B7.5 line) and notochord

Aniseed annotation:
Data and annotation provided by the author, not available in the article.

Mid tailbud wild type embryo probed for citb3f17 clone. Expression was detected in the notochord from the initial tailbud stage (data not shown in the article).

Tailbud stage embryos probed for Ci-tensin expression. No picture available in the paper.

Original Annotation :
Expression in notochord, muscle, palps, mesenchyme and other territories.

Region of Ci-tensin from +2942bp to +6022bp in front of a LacZ reporter gene expression. This construct is named p(-3.0 k)tensin/lacZ in the paper.

Original Annotation :
The p(-3.0k)tensin/lacZ construct exhibited reporter expression in notochord cells, although expression was also observed in cells of the epidermis and mesenchyme.

Region of Ci-tensin from +3545bp to +6022bp in front of a LacZ reporter gene expression. This construct is named p(-2.5 k)tensin/lacZ in the paper.

Original Annotation :
The p(-2.5k)tensin/lacZ construct exhibited reporter expression in notochord cells, although expression was also observed in cells of the epidermis and mesenchyme.

Region of Ci-tensin from +3974bp to +6022bp in front of a LacZ reporter. This construct is named p(-2.0 k)tensin/lacZ in the paper.

Original Annotation :
The p(-2.0k)tensin/lacZ construct exhibited reporter expression in notochord cells, although expression was also observed in cells of the epidermis and mesenchyme.

Region of Ci-tensin from +4620bp to +6022bp in front of a LacZ reporter gene. This construct is named p(-1.5 k)tensin/lacZ in the paper.
The orange box indicates anterior A-lineage notochord specific regulatory region of the gene.

Original Annotation :
The p(-1.5k)tensin/lacZ construct exhibited reporter expression in notochord cells, although expression was also observed in cells of the epidermis and mesenchyme.

Region of Ci-tensin from +5581bp to +6022bp in front of a LacZ reporter gene. This construct is named p(-0.5 k)tensin/lacZ in the paper.

Original Annotation :
The p(-0.5 k)tensin/lacZ failed to drive reporter expression in the notochord.
According to the paper, there is also ectopic expression in epidermis and mesenchyme.