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.

Zygote probed for Ci-delta1-protocadherin-like expression. No picture available in the article.

Original Annotation :
Unlocalized signal detected.

Animal and vegetal views of a 16-cell stage embryo probed for Ci-delta1-protocadherin-like expression.

Original Annotation :
The Ci-delta1-protocadherin-like expression was detected in a5.3, a5.4 and in b5.3, b5.4 cell pairs.

Animal end vegetal views of a 32-cell stage embryo probed for Ci-delta1-protocadherin-like expression.

Original Annotation :
The Ci-delta1-protocadherin-like expression was detected in a6.5, a6.6, a6.7, a6.8, b6.5, b6.6, b6.7, b6.8 cell pairs.

Animal and vegetal views of a 64-cell stage embryo probed for Ci-delta1-protocadherin-like expression.

Original Annotation :
The Ci-delta1-protocadherin-like expression was detected in all a line and b line cells. (a7.9, a7.10, a7.11, a7.12, a7.13, a7.14, a7.15, a7.16, b7.9, b7.10, b7.11, b7.12, b7.13, b7.14, b7.15, b7.16 cell pairs).

Animal and vegetal views of an early gastrula stage embryo probed for Ci-delta1-protocadherin-like expression.

Original Annotation :
The Ci-delta1-protocadherin-like expression was detected in a8.17, a8.18, a8.19, a8.20, a8.25 cell pairs.
Comment :
Signals derived from mRNA expressed before early gastrula stages might be observed in animal hemisphere.

Vegetal view of a gastrula stage embryo showing Delta1-protocadherin expression. Expression was examined by whole-mount in-situ hybridization in embryos that carried Ci-FoxD/lacZ.

Original annotation:
The anterior neural plate cells strongly expressed δ 1-protocadherin-like at the late gastrula stage.

Animal and vegetal views of a late gastrula stage embryo probed for Ci-delta1-protocadherin-like expression.

Original Annotation :
The Ci-delta1-protocadherin-like expression was detected in nervous system (a9.33, a9.34, a9.35, a9.36, a9.37, a9.38, a9.39, a9.40 cell pairs).
Comment :
Expression in a9.34 and a9.38 was weaker than the gene expression in the other neural plate cells.

Dorsal view of a late neurula embryo probed for Ci-delta1-protocadherin-like expression.

Original Annotation :
The Ci-delta1-protocadherin-like expression was detected in nervous system.
Comment :
Signals were observed around the blastopore and those anterior to them were signals of nervous system.

Dorsal view of an initial tailbud stage embryo probed for Protocadherin 9 expression.

Original annotation :
Protocadherin 9 (a putative ortholog of vertebrates notochord genes) is expressed in the nerve cord (blue arrowhead).

Lateral view of a tailbud stage embryo showing Delta1-protocadherin expression. Expression was examined by whole-mount in-situ hybridization in embryos that carried Ci-FoxD/lacZ. nc nerve cord; pa, presumptive palp region.

Original annotation:
Expression was then observed in the nerve cord and the palp region at the initial tailbud stage.

Aniseed annotation:
According to the picture and to the developmental table established in Hotta et al. 2007, the developmental stage of this embryo seems to be "early tailbud". Moreover, it seems that there is also expression in the tip of the tail.

A dorsal view of an early tailbud embryo probed for Ci-delta1-protocadherin-like expression.

Original Annotation :
The Ci-delta1-protocadherin-like expression was detected in the nerve cord.
Comment :
The nerve cord expression could be in the bottom row of nerve cord.

Dorsal view of a late tailbud embryo probed for Ci-delta1-protocadherin-like expression.

Original Annotation :
The Ci-delta1-protocadherin-like expression was detected in the nerve cord.
Comment :
The nerve cord expression could be in the bottom row of nerve cord.