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.

Early tailbud stage wild type embryo probed for cilv24m17 expression (no picture available in the article).

Original annotation:
No expression was detected in the brain at this stage.

Mid tailbud stage wild type embryo probed for cilv24m17 expression (no picture available in the article).

Original annotation:
No expression was detected in the brain at this stage.

Expression of CLSTR14373 at tailbud stage.
Black arrows indicate hybridization signals in the brain.

Original annotation :
Ci-gnrh-1, was expressed in the posterior brain at the late tailbud stage and was broadly expressed in the brain at the larval stage.

Late tailbud stage wild type embryo probed for cilv24m17 expression (no picture available in the article).

Original annotation:
No expression was detected in the brain at this stage.

Expression of CLSTR14373 at tailbud stage.
Black arrows indicate hybridization signals in the brain.

Original annotation :
Ci-gnrh-1, was expressed in the posterior brain at the late tailbud stage and was broadly expressed in the brain at the larval stage.

Aniseed annotation :
According to the picture, it seems that the staining is located in the dorsal anterior brain.

Conversion to aniseed format of DBTGR database entry from Kusakabe team.

Original annotation:
Brain vesicle; visceral ganglion; nerve cord.

Lateral view of a WT Ciona robusta larva probed for gnrh1 expression. Second image is the magnified head and trunk region.
Original annotation:Ci-gnrh1 is strongly expressed in a population of cells in the brain vesicle, the anterior portion of the central nervous system. Transcripts of Ci-gnrh1 were also detected in the motor ganglion (mg) but its expression level was much lower than that in the brain vesicle. In addition to the expression in the central nervous system, Ci-gnrh1 is also expressed in parts of the peripheral nervous system: it is expressed in a few cells in the adhesive organ (pn, papillar neurons) and a subpopulation of rostral epidermal neurons (RTENs).
Scale bars: 100 mm in (A); 50 mm in (C).

Larvae probed for cilv24m17 expression (no picture available in the article).

Original annotation:
Expression was detected in the brain.

Expression of CLSTR14373 at larval stage.

Original annotation :
The gene was expressed in the brain. Arrows indicate hybridization signals in the brain.

Expression of CLSTR14373 at tadpole stage.
Black arrows indicate hybridization signals in the brain.

Original annotation :
Ci-gnrh-1, was expressed in the posterior brain at the late tailbud stage and was broadly expressed in the brain at the larval stage.

Lateral view of the head and trunk of a WT Ciona robusta larva electroporated with gnrh1>GFP reporter construct.
GFP is localized in the brain vesicle (bv), in the motor ganglion (mg) and in the papillar neurons (pn) whose axons are indicated by white arrowheads.
Original annotation: The cells expressing Ci-gnrh1 in the adhesive organ are likely to be papillar neurons, as they possess long neurites (probably axons) as visualized by GFP under the control of the cis-regulatory region of Ci-gnrh1.