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.

Original annotation:
Staining ubiquitous.

Original annotation:
Staining ubiquitous.

Original annotation:
Staining ubiquitous.

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

Original annotation:
Visceral ganglion.

Aniseed annotation:
According to the pictures, it seems that there is a staining in the entire central nervous system.

Lateral view of a WT Ciona robusta larva probed for gnrh2 expression. The second image is the magnified head and trunk region.
Original annotation: Ci-gnrh2 is expressed throughout the entire nerve cord (nc) from the posterior trunk to the tail tip. The brain vesicle and the motor ganglion also contained cells expressing Ci-gnrh2. In the brain vesicle, there are at least two populations of Ci-gnrh2-expressing cells: one is located close to the ocellus pigment cell, while the other is located in the posterior brain region.

Anterior lateral view of a hatched Ciona larva probed for GnRH2 (KH.C9.484) expression.
Original annotation : Bright field anterior lateral views. Animal hybridized with a GnRH2 mRNA probe. GnRH is expressed near the oral opening. White arrowhead indicates the position of the oral opening. Red arrow marks area in presumed PPE-derived neurons.

GnRH regulatory sequence drives expression near the oral opening.

Original annotation : Lateral anterior view of a larva co-electroporated with GnRH>GFP (yellow) and Six1/2>mCherry (magenta), and counterstained with phalloidin (grey).
Insets show Six1/2 expression (left) and GnRH expression (right) surrounding the oral opening (dotted line). The GnRH1 neuron is marked with an asterisk. Brackets indicate PPE-derived GnRH neurons.

Original annotation : Lateral view of a larva electroporated with GnRH>mCherry (yellow), stained with an acetylated a-tubulin antibody (blue) and stained with phalloidin (grey). Arrowhead indicates PPE-derived GnRH neuron (PPE proto-placodal ectoderm).

Lateral view of a WT Ciona robusta larva electroporated with gnrh2>GFP reporter construct.
GFP expression is observed in the brain vesicle (bv), in the motor ganglion (mg) and in the entire nerve cord (nc).
Original annotation: Expression of GFP under the control of the cis-regulatory region of Ci-gnrh2 recapitulated the endogenous gene expression pattern

Lateral view of the head of a WT Ciona robusta larva electroporated with gnrh2>GFP reporter construct. GFP expression is observed in the brain vesicle (bv), in the motor ganglion (mg) and in the nerve cord (nc). The white arrowhead indicates a GFP positive neurite.
Original annotation: In some specimens, Ci-gnrh2::gfp visualized a neuron in the motor ganglion, which had a neurite (presumably an axon) extending into the tail along the nerve cord.

Lateral view of a WT Ciona robusta larva electroporated with gnrh2>GFP reporter construct.
GFP expression is observed in the brain vesicle (bv), in the motor ganglion (mg) and in the entire nerve cord (nc).
Original annotation: Expression of GFP under the control of the cis-regulatory region of Ci-gnrh2 recapitulated the endogenous gene expression pattern

Lateral view of the head of a WT Ciona robusta larva electroporated with gnrh2>GFP reporter construct. GFP expression is observed in the brain vesicle (bv), in the motor ganglion (mg) and in the nerve cord (nc). The white arrowhead indicates a GFP positive neurite.
Original annotation: In some specimens, Ci-gnrh2::gfp visualized a neuron in the motor ganglion, which had a neurite (presumably an axon) extending into the tail along the nerve cord.