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 view of a 32-cell stage embryo probed for Ci-AChE expression.

Vegetal view of a 112-cell stage embryo probed for Ci-AChE expression.

Vegetal view of a gastrula stage embryo probed for Ci-AChE expression. Cells where mRNA is first detected are indicated by arrowheads.

Dorsal view of a neurula stage embryo probed for Ci-AChE expression.

View of early tailbud stage embryos showing histochemical detection of AchE activity in muscle. Embryo cleavage was arrested by cytochalasin B at 110 cell stage.

Expression of AChE at the early tailbud stage in embryos traited with cytochalasin from the 64 cell stage.

Vegetal view of a cleavage-arrested 64-cell Ciona robustaembryo at 14 hours post fertilization electroporated with pTLacZ (Bra>LacZ) and assayed by histochemical methods for acetylcholinesterase (brown-stained cells), a highly specific marker of muscle differentiation in Ciona intestinalis and β-galactosidase (blue-stained cells).
Ache is expressed in the B-line muscle precursors, B7.6, B7.5, B7.8 and B7.7. LacZ staining is observed in the notochord precursors A7.3 and A7.7 and in a mesenchyme precursor B7.3.
Scale bar: 50μm.

Tailbud stage embryo probed for Ci-AChE expression. Cell division was blocked by cytochalasin D at the 32-cell stage, gene expression profile corresponds to the stage observed.
Arrowheads indicate the B-line blastomeres expressing Ci-AChE.

Original annotation:
When embryos were treated with CytD from the 32-cell stage, expression of the Ci-AChE mRNA was detected in two to four cells (presumably B6.2 and B6.4 blastomere pairs).

Tailbud stage embryo probed for Ci-AChE expression. Cell division was blocked by cytochalasin D at the 64-cell stage, gene expression profile corresponds to the stage observed.
Arrowheads and arrows indicate the B-line and A-line blastomeres expressing Ci-AChE, respectively.

Original annotation:
Treatment from the 64-stage resulted in the expression of Ci-AChE in up to six cells (presumably B7.4, B7.5 and B7.8).

Tailbud stage embryo probed for Ci-AChE expression. Cell division was blocked by cytochalasin D at the 110-cell stage, gene expression profile corresponds to the stage observed.
Arrowheads indicate the B-line blastomeres expressing Ci-AChE.

Original annotation:
Treatment from 110-cell stage resulted in the expression of Ci-AChE in 6-10 cells (presumably B7.5, B8.7, B8.8, B8.15, and B8.16).

Lateral view of an early tailbud stage embryo showing the expression of Ci-AChE/LacZ in tail muscle cells.

Lateral view of an early tailbud stage embryo showing the expression of Ci-AChE (delta)/LacZ.

Tailbud stage embryo probed for Ci-AChE expression. Cell division was blocked by cytochalasin D at the 32-cell stage, gene expression profile corresponds to the stage observed. Arrowheads indicate the B-line blastomeres expressing Ci-AChE.

Original annotation:
Ci-AChE ⁄ lacZ was expressed in most of the embryos treated with CytD from the 32-cell stage.

Tailbud stage embryo probed for Ci-AChE expression. Cell division was blocked by cytochalasin D at the 64-cell stage, gene expression profile corresponds to the stage observed. The embryo seems to have been proceeding to the 76-cell stage, since the B7.4 blastomere on the right side divided into B8.7 and B8.8 (red arrowheads).
Arrowheads and arrows indicate the B-line and A-line blastomeres expressing Ci-AChE, respectively.

Original annotation:
Patterns of expression of Ci-AChE ⁄ lacZ in embryos treated with CytD from the 64-cell stage were similar to those of the endogenous Ci-AChE.

Tailbud stage embryo probed for Ci-AChE expression. Cell division was blocked by cytochalasin D at the 110-cell stage, gene expression profile corresponds to the stage observed. Arrowheads indicate the B-line blastomeres expressing Ci-AChE.

Original annotation:
Patterns of expression of Ci-AChE ⁄ lacZ in embryos treated with CytD from the 110-cell stage were also similar to those of the endogenous Ci-AChE.

Lateral view of a mid tailbud stage embryo probed with AchE expression.

Lateral view of a tail muscles in a mid tailbud stage embryo probed with AchE expression. Expression of tissue-specific differenciation markers in morpholino oligonucleotide-injected embryos at the stage corresponding to the mid tailbud stage.

Original annotation Embryos injected with MO against cilv006h21 as classified as " ball" phenotype: they developped into spherical-shaped cell masses, like balls. In these embryos, detection of the tissue specific markers indicated thaht almost the major tissues were lost or reduced.

View of mid tailbud stage embryos showing histochemical detection of AchE activity in muscle.

View of mid tailbud stage embryos showing histochemical detection of AchE activity in muscle.

Lateral view of a mid tailbud stage embryo showing expression of acetylcholine esterase in muscle cells.

View of a mid tailbud stage embryo, showing expression of acetylcholine esterase in muscle cells. Embryos were treated with cytochalasin B from the 110-cell stage.

Lateral view of a late tailbud wild type embryo probed for Ci-AchE (acetylcholinesterase) expression. This picture shows expression in the primary and secondary muscle lineage.

Dorsal view of a late tailbud wild type embryo probed for AchE expression.The picture shows expression in muscle cells. Aniseed anotation: we can see expression in mesenchyme cells.

Lateral view of a late tailbud wild type embryo probed for Ci-AchE (acetylcholinesterase) expression. This picture shows expression in the primary and secondary muscle lineage.