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.

Differents views of a tailbud stage embryo probed for Ci-GAD expression.
Original Annotation : Picture A : Lateral view demonstrates that Ci-GAD is expressed in the posterior sensory vesicle precursor cells (asterisk) and in the palp precursors (arrowhead) located in front of the vesicle cavity primordium (arrow). Only one cell in the tail expressed Ci-GAD.
Picture B : View of he trunk from above depicts that Ci-GAD is expressed in three cells of the Central Nervous System (CNS) : two ventral and one dorsal (asterisks). The Ci-GAD expression was also detected in the palp precursors (arrowhead, arrow : vesicle cavity primordium).
Picture C : Transverse histological section of hybridized embryo shows that Ci-GAD was expressed in the neural tube (nt).
Picture D : A ventral view shows that Ci-GAD was expressed in two epidermal cells of the tail (no : notochord).
Picture E : Dorsal view of an early tailbud shows that Ci-GAD expression was detected in three cells in the tail (arrowheads).
Picture F : Tranverse section shows that Ci-GAD transcripts were detected in the tail epidermis. (no = notochord)

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

Original annotation:
Expression was detected.

Differents views of C.inestinalis tadpole probed for Ci-GAD expression. Anterior in on the left.
Original Annotation :
Picture A : Lateral view of a larva shows that Ci-GAD expression was detected in the CNS, precisely in the sensory vesicle (sv), the neck (ne) and the visceral ganglion (vg) as well in the adhesive papillae (ap) and in two epidermal neurons in the tail.
Picture B : Enlarged dorsal view of the same larva shows that Ci-GAD is expressed in neurons in the tail.
Picture C : In this dorsal view we can see that Ci-GAD expression was detected in three cells in the tail and in fibers (arrowhead) connecting the posterior sensory vesicle to the visceral ganglion.
Picture D : Lateral, high-magnification view of Ci-GAD-positive tail bipolar neuron (no:notochord).
Picture E : Transverse section of the tail (depicted in D) shows that Ci-GAD expression was detected in bipolar epidermal neuron (arrowhead : nerve cord, e: epidermis, m : muscle cell).
Picture F : Lateral view of a larva trunk demonstrates that Ci-GAD transcripts were detected in the sensory vesicle, the neck and the visceral ganglion. Although the entire posterior sensory vesicle shows Ci-GAD expression, only the most anterior part of the dorsal wall of the vesicle cavity displays Ci-GAD signals (asterisk). Ci-GAD is also expressed in fibers that extend ventrally from the neck to the visceral ganglion (arrowhead).
Picture G : Signals of Ci-GAD expression were also present in two sensory neurons in the adhesive papillae (asterisks).
Picture H : Dorsal view of a larval CNS shows that the asymmetrical pattern of expression in the sensory vesicle, specially in the photoreceptor complex (pc), in the coronet cell (cc) area on the left side, and in neurons of the dorsal cluster (dc) situated in the middle of these two areas. Rostrally Ci-GAD is also expressed in the dorsal wall of the sensory vesicle cavity (asterisk).
Picture I : Enlarged dorsolateral view of larval CNS shows that Ci-GAD is expressed in two somata in the posterior sensory vesicle and ventrally in the nerve fibers (arrowhead; oc = ocellus).
Picture J : Dorsal view of the trunk showing the asymmetrical pathway of Ci-GAD positive fibers connecting the vesicle to the ganglion (arrowhead).
Picture K : A dorsolateral view of the visceral ganglion shows that Ci-GAD expression was detected in several neurons located dorsally in the ganglion. Moreover Ci-GAD was expressed ventrally (arrowhead).
Picture L : Dorsal view at the level of the visceral ganglion of a specimen in which the reaction was blocked early : Ci-GAD was expressed in two cells protruding from the anterior margin (asterisks).
Picture M : Sagittal section of the trunk that demonstrates Ci-GAD expression is detected in the dorsal portion of the visceral ganglion(asterisk). A positive signal is also present ventrally in nerve fibers.
Picture N : Transverse, slightly oblique section shows that Ci-GAD is expressed in the dorsal (asterisk) and ventral part (white arrowhead) of the visceral ganglion.
Picture B2 : A sagittal view of the anterior sensory vesicle section. Ci-GAD was expressed below the pharynx (ph).
Picture C2 : A sagittal view of the anterior sensory vesicle section. Ci-GAD was strongly expressed in otolith and in the middorsal wall of the anterior sensory vesicle cavity.
Picture D2 : A sagittal view of the anterior sensory vesicle section. Ci-GAD expression was detected in the photoreceptor complex(pc), the ocellus (oc) and in several coronet cells(arrowhead).
Picture E2 : A sagittal view of the posterior sensory vesicle section. Ci-GAD is expressed in a group of neurons, ventral an dorsal clusters and in coronet cells.
Picture F2 : A sagittal view of the posterior sensory vesicle section. The expression of Ci-GAD was detected in ventral and dorsal cluster, in coronet cells, and in ocellus.
Picture G2 : A sagittal view of the posterior sensory vesicle section. Along the caudal extent of the posterior sensory vesicle, Ci-GAD is expressed only on the left side of the neural tube.
Picture H2 : A sagittal view of the neck section. Ci-GAD expression is detected ventrally in the neck.
Picture I2 : A sagittal view of the visceral ganglion section. Ci-GAD is expressed dorsally and ventrally in the visceral ganglion.
Picture J2 : A sagittal view of the visceral ganglion section. Ci-GAD expression was detected at the level of dorsal neurons.

Lateral view of a larvae probed for Ci-GAD expression.

Original Annotation : Ci-GAD is expressed mainly in the posterior sensory vesicle. Other expression was seen in the dorsal visceral ganglion (white arrowhead).

Ciona intestinalis juveniles probed for Gad expression, specimen was opened along the endostyle in order to better visualize the cerebral ganglion.
Gad is recognizable in neurons located in anterior and posterior ends of cerebral ganglion (cg).
Scale bars: 100 μm