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.

Views of immunostainings of a transverse section through the anterior region of adult Ciona (first picture) and of a longitudinal section in the region of the atrial siphon (AS) for the second picture.

Original Annotation : Pic B : The staining reveals intense expression in the neuropile of the cerebral ganglion (CG).
The brown/gray structures indicated by the asterisks are fecal material. Some areas of tissue appear orange because of the presence of natural pigments.
"LM" means ongitudinal muscles, "Tu" tunic, "CM" circular muscles, "BB" the branchial basket and "ES" endostyle.

Pic C : Intense immunostaining can be seen in the neuropile of the cerebral ganglion (CG). Immunostained fibers are also present in branches of the posterior nerve (PNB) associated with the atrial siphon but are not easy to see at this magnification. As in B, cells that appear orange are naturally pigmented.

Differents views of CiCBR immunoreactivity in a sequential series of transverse sections of the Ciona cerebral ganglion, which lies close to the body wall epithelium (BWE) between the oral and the atrial siphons.

Original Annotation : A: Immunostained axon profiles in the anterior nerves (AN1, AN2) and surrounding the ciliated funnel (CF).
B: Immunostained meshwork of fibers in the neuropile of the anterior pole of the cerebral ganglion (CG). Immunostained axons are also evident around the ciliated duct (CD) and in close association with a band of circular muscle (CM; arrow).
C,D: Immunostained meshwork of fibers in the neuropile in the central region of the cerebral ganglion.
E: Immunostained meshwork of fibers in the neuropile of the posterior pole of the cerebral ganglion (CG). An intensely stained axon process (arrow) can be seen close to the unstained neural gland (NG).
F: Immunostained axon profiles in the posterior nerves (PN1, PN2); immunostained axons (arrow) can also be seen in branches of the posterior nerves associated with the atrial siphon.

Differents views of CiCBR immunoreactivity in the central and peripheral nervous system of adult Ciona at high magnification.

Original Annotation : A: CiCBR expression is concentrated in a dense meshwork of fibers in the central neuropile (N) region of the cerebral ganglion. The cortical layer (CL) of the ganglion is unstained.
B: High-magnification image of the cerebral ganglion showing CiCBR immunoreactivity associated with fibers and axon varicosities in the neuropile (N) of the ganglion. The cortical layer (CL) is largely unstained, but a single immunostained cell body can be seen (arrowhead). A single immunostained axon with intensely stained varicosities can be seen emanating from the surface of the ganglion and projecting locally (arrow).
C: CiCBR-immunoreactive fibers in a branch of one of the posterior nerves associated with the atrial siphon.
D: A single, varicose CiCBR-immunoreactive fiber (arrow) can be seen here associated with one of the languets (La), which are located anteriorly along the dorsal margin of the branchial basket. The languets collect food-laden sheets of mucus from the branchial basket and move these as a rolled-up cord toward the esophagus. In this image, a mucus cord (MC) can be seen in contact with cilia (Ci) emanating from the epithelium of the languets.

Differents views of CiCBR immunoreactivity in the central and peripheral nervous system of adult Ciona at high magnification.

Original Annotation : Pics A and B CiCBR-immunoreactive fibers can be seen located near the surface of the cerebral ganglion.
Pic C a high-magnification image shows CiCBR-immunoreactive fibers projecting from the surface of the anterior pole of the cerebral ganglion.
Pic D CiCBR positive fibers can be seen projecting over and around the ciliated funnel, which is located anterior to the cerebral ganglion.
Pic E CiCBR-immunoreactive fibers can be seen in the anterior medial nerve 1 (AMN1), projecting over the circularly orientated muscle strands in the prebranchial zone of the pharynx at the base of the oral siphon. A single immunostained fiber can be seen projecting from the nerve and terminating with an intensely stained bulbous varicosity (arrowhead).
F Fibers expressing Ci-CBR can be seen in the anterior medial nerve 2 (AMN2) at high magnification.
G CiCBRimmunoreactive fibers can be seen in the ALN2a branch (uppermost) and the ALN2b branch (lowermost) of anterior lateral nerve. Note the immunostained terminal varicosity of one of the axons derived from ALN2A (arrow).
In H CiCBR-immunoreactive fibers can be seen in the posterior nerves; branches of the posterior lateral nerves, PLN1a, PLN1b, PLN2a, and PLN2b, which are not visible in Figure 4, can be seen in this image.
In I CiCBR-positive fibers can be seen in a high-magnification image of posterior lateral nerve 2 (PLN2); note the intensely stained varicosities and the single immunostained fiber projecting from the nerve (arrow).