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.

Vegetal pole view of a 16-cell stage embryo probed with Ci-Tbx6c expression.

Expression of Ci-Tbx6c was detected by whole-mount in situ hybridization. Vegetal view of a 16-cell stage embryo.

Vegetal view of a 44-cell stage embryo stained for Ci-Tbx6c mRNA by in situ hybridization. Expression is observed in the B6.3-Cells.

Expression of Ci-Tbx6c was detected by whole-mount in situ hybridization. Vegetal view of a 32-cell stage embryo.

Vegetal view of a late 32-cell stage embryo.Tbx6c expression was detected in B6.1, B6.2, B6.4 blastomeres at the 32-cell stage (signal in B6.2 and B6.1 cell pairs is scarcely visible).

Vegetal pole view of a late 32-cell stage embryo probed with Ci-Tbx6c. According to the authors, expression of Ci-Tbx6c is detected in both B6.2 and B6.4 cell pairs. However, the B6.2 cell pair seems to be unstained.

32-cell stage embryo electroporated with the proximal region of Ci-FCol1 and probed for reporter expression.
The proximal region contains four putative Ci-Tbx6b/c-binding sites (red ovals) and one putative Ci-Macho1 site (green square).
The percentages of transgenic embryos displaying the patterns shown, and the total number of embryos scored, are indicated at the bottom of each panel.

Original Annotation : Starting at the 32-cell stage, over one third of the embryos electroporated with the proximal 230 bp CiFCol1 muscle CRM showed lacZ expression in primary muscle precursors (orange arrowheads).

Expression of Ci-Tbx6c was detected by whole-mount in situ hybridization. Vegetal view of a 64-cell stage embryo.

Vegetal pole view of a 64-cell stage embryo probed with Ci-Tbx6c expression. According to the authors, the expression of Tbx6c is detected in all B7.4, B7.5, B7.7 and B7.8 cell pairs (signal in B7.4 cell pair is scarcely visible).

Vegetative pole view of a 110-cell stage embryo probed with Ci-Tbx6c.

Vegetal view of a 110-cell stage embryo stained for Ci-Tbx6c mRNA by in situ hybridization. Expression is observed in the B7.5-Cells.

Expression of Ci-Tbx6c was detected by whole-mount in situ hybridization. Vegetal view of a 110-cell stage embryo.

Aniseed annotation: according to the picture, it seems that the staining is localized in B7.5 cell pair.

112-cell stage embryos probed for Ci- Tbx6c expression.

Original Annotation : Expression of Ci-Tbx6c in control embryos is detected in a subset of muscle precursors (orange arrowheads).

112-cell stage embryo electroporated with the proximal region of Ci-FCol1 and probed for reporter expression.
The proximal region contains four putative Ci-Tbx6b/c-binding sites and one putative Ci-Macho1 site.

Original Annotation : By the 112-cell stage, high levels of lacZ transcripts were observed in most pairs of muscle precursors (orange arrowheads), except for the A8.16 pair.

ANISEED Annotation : According to the picture it seems that expression was localized in the B8.15 and B8.16 and not in B8.15 cell pair and B8.16 cell pair.

Vegetal view of a mid-gastrula stage embryo stained for Ci-Tbx6c mRNA by in situ hybridization. Arrows indicate B7.5 lineage cells.

Expression of Ci-Tbx6c was detected by whole-mount in situ hybridization. View of a late gastrula stage embryo.

Early neurula stage embryo electroporated with the proximal region of Ci-FCol1 and probed for reporter expression.
The proximal region contains four putative Ci-Tbx6b/c-binding sites and one putative Ci-Macho1 site.

Original Annotation : At the early neurula stage, this pattern translated into a homogeneous accumulation of beta-galactosidase in the majority of muscle cells (orange arrowheads).

Expression of Ci-Tbx6c was detected by whole-mount in situ hybridization. View of a mid neurula stage embryo.

Early tailbud stage embryo electroporated with the proximal region of Ci-FCol1 and probed for reporter expression.
The proximal region contains four putative Ci-Tbx6b/c-binding sites and one putative Ci-Macho1 site.

Original Annotation : At the early tailbud stage, this CRM was still active in muscle (orange arrowheads) and mesenchyme (pink arrowhead).

Mid tailbud electroporated with a a proximal region (230bp) of the regulatory region of Ci-FCol1 cloned upstream of the FoxA-a basal promoter (pink box in the schematics).
The proximal region contains four putative Ci- Tbx6b/c-binding sites (red ovals) and one putative Ci-Macho1 site (green square).

Reporter expression was detected in mesenchyme (pink arrowhead) and muscle (orange arrowhead).

Mid tailbud stage embryo electroporated with the proximal region of Ci-FCol1 and probed for reporter expression.
The proximal region contains four putative Ci-Tbx6b/c-binding sites and one putative Ci-Macho1 site.

Original Annotation : At the mid-tailbud stage, transcriptional activity had faded almost completely from the muscle in the majority of the embryos, whereas a residual signal was occasionally seen in mesenchyme cells (pink arrowhead).

Mid tailbud stage embryo electroporated with the proximal region of Ci-FCol1 and probed for reporter expression.
The proximal region contains four putative Ci-Tbx6b/c-binding sites and one putative Ci-Macho1 site.

ANISEED Annotation : At the mid-tailbud stage, the reporter expression was detected in the muscles (orange arrowheads) and in the msenchyme (pink arrowhead).