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.

Animal view of a WT 16-cell stage embryo probed for BZ1 expression .
Original annotation : In situ hybridisation for BZ1 : embryos are shown in animal view. BZ1 is expressed in all anterior animal cells (a-line cells) at the 16-cell stage.

Vegetal, animal and lateral (from left to right) views of a WT Ciona robusta probed for Prdm1-r.a expression at the early 32-cell stage.
Original comment: Prdm1-r.a is expressed in five pairs of animal cells (a6.5 to a6.8 and b6.5) and a pair of vegetal cells (B6.4) at the 32-cell stage.
Scale bar: 100 μm.

Animal view of a Ciona robusta 32-cell stage embryo probed for Prdm1-r.b expression. This control embryo was injected with a control MO and treated with DMSO.
Prdm1.b expression is observed in B6.4, b6.5 and in a-line blastomers.
n, number of embryos examined. % : percentages of embryos that showed the wild-type expression pattern.

Animal view of a WT 32-cell stage embryo probed for BZ1 expression .
Original annotation : In situ hybridisation for BZ1 : embryos are shown in animal view. Prior to the 64-cell stage, BZ1 expression diminishes in all but one descendant, a7.13. This gene is also transiently expressed in b6.5 and in a pair of posterior vegetal cells.

Animal view of a WT 64-cell stage embryo probed for BZ1 expression .
Original annotation : In situ hybridisation for BZ1 : embryos are shown in animal view. Prior to the 64-cell stage, expression of this gene diminishes in all but one descendant, a7.13. It is also expressed in B7.5 at the 64-cell stage.

Animal view of a WT early gastrula probed for BZ1 expression. BZ1 is no longer expressed in a neural lineage at early gastrula.
Original annotation : In situ hybridisation for BZ1 : embryos are shown in animal view. This gene is transiently expressed in a pair of posterior vegetal cells.
Aniseed annotation : it seems that expression is localized in B7.7 and B8.15 cell pairs.

Lateral view of a WT early gastrula probed for BZ1 expression .
Original annotation : In situ Expression of BZ1 at the early gastrula stage in normal embryos. Embryos in are shown in lateral views (animal pole right). This gene is transiently expressed in a pair of posterior vegetal cells.
Aniseed annotation : it seems that expression is localized in B7.7 and/or B8.15 cell pairs.

Latteral view of a WT early tailbud probed for BZ1 expression .
Original annotation : In situ hybridisation for BZ1 : BZ1 (KH.C12.493) and BZ2 (KH.C12.105) were expressed in non-overlapping tissues of tailbud embryos.