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-delta-protocadherin-4 expression. No picture available on the article.

Original Annotation :
No signal detected.

Animal and vegetal views of a 16-cell stage embryo probed for Ci-delta-protocadherin-4 expression.

Original Annotation :
The expression of Ci-delta-protocadherin-4 was detected in a5.3, a5.4, b5.3, b5.4 cell pairs.

Vegetal view of a WT Ciona robusta late 32-cell stage embryo probed for δ-protocadherin-4 expression.
No expression is observed.

Animal and vegetal views of a 32-cell stage embryo probed for Ci-delta-protocadherin-4 expression.

Original Annotation :
The expression of Ci-delta-protocadherin-4 was detected in A6.1, A6.2, A6.3, A6.4, B6.1 cell pairs.
Comment :
Signals observed in the animal hemisphere could be those for the mRNA expressed at the 16-cell stage.

Vegetal view of a WT Ciona robusta 64-cell stage embryo probed for δ-protocadherin-4 expression.
Expression is observed in A7.1, A7.2, A7.4, A7.5, A7.6, A7.8, B7.1, B7.2, B7.3 and B7.4 blastomere pairs.
In schematic representation of gene expression, notochord precursors are shown in red and nerve cord precursors in blue. Blastomeres with gene expression are marked by dots.

Animal and vegetal view of a 64-cell stage embryo probed for Ci-delta-protocadherin-4 expression.

Original Annotation :
The expression of Ci-delta-protocadherin-4 was detected in A7.1, A7.2, A7.4, A7.5, A7.8, B7.1, B7.2, B7.4 cell pairs.

Vegetal view of a WT Ciona robusta 112-cell stage embryo probed for δ-protocadherin-4 expression.
Expression is observed in A8.7, A8.8, A8.15, A8.16, A7.1, A7.2, A7.5, B7.1, B7.2, B8.7 and B8.8 blastomere pairs.
In schematic representation of gene expression, notochord precursors are shown in red and nerve cord precursors in blue. Blastomeres with gene expression are marked by dots.

An early gastrula stage embryo probed for Ci-delta-protocadherin-4 expression.

Original Annotation :
The expression of Ci-delta-protocadherin-4 was detected in A7.1, A7.2, A7.5, A8.7, A8.8, A8.15, A8.16, B7.1, B7.2, B7.5, B8.7, B8.8, B8.15, B8.16 cell pairs.
Comment :
Signals in A8.7, A8.8, B8.15 and B8.16 cells were weak.

Animal and vegetal views of a late gastrula stage embryo probed for Ci-delta-protocadherin-4 expression.

Original Annotation :
The expression of Ci-delta-protocadherin-4 was detected in the epidermis, muscle and in the nervous system (A9.14, A9.16, A9.30 cell pairs).
Comment :
expression started in a9.35, a9.36, a9.39 and a9.40 at later gastrula stage. Weak gene expression was observed in the anterior epidermis.

Dorsal view of a late neurula embryo probed for Ci-delta-protocadherin-4 expression.

Original Annotation :
The expression of Ci-delta-protocadherin-4 was detected in muscle and nervous system.
Comment :
Signals observed around the blastopore and anterior to them were signals of nervous system.

Dorsal view of an early tailbud embryo probed for Ci-delta-protocadherin-4 expression.

Original Annotation :
The expression of Ci-delta-protocadherin-4 was detected in brain, mesenchyme, muscle, nerve cord, palp.
Comment :
Expression in the mesenchyme was weak. The nerve cord expression was in the lateral rows of nerve cord.

Dorsal view of a late tailbud embryo probed for Ci-delta-protocadherin-4 expression.

Original Annotation :
The expression of Ci-delta-protocadherin-4 was detected in mesenchyme, muscle, nerve cord, palp, sensory vesicle, visceral ganglion.
Comment :
Expression in the mesenchyme and the muscle was weak. The nerve cord expression was in the lateral rows of nerve cord. Expression in the anterior part of sensory vesicle was weaker than the posterior part .