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.

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

Original annotation:
Brain vesicle; papilla; epidermal neurons.

Whole mount view and schematic representation of a late tailbud stage embryo electroporated with the pVGluT::EGFP reporter construct and immunostained with an anti-GFP antibody.
Original annotation: Expression was observed in the caudal epidermal neurons (CENs) constituting the posterior larval peripheral nervous system (PNS). The posterior ventral CENs are indicated by the red arrowhead, the the dorsal CENs by the yellow arrowhead and the anterior ventral CENs by the gap between the blue arrows.

Lateral view of a WT Ciona robusta late tailbud embryo probed for VGLUT expression. Second picture is a HD image of the same embryo.
Original annotation: Arrowheads indicate cells expressing Ci-VGLUT in anterior trunk epidermis. A white arrow indicates Ci-VGLUT expression in the developing brain vesicle. Black arrows indicate prospective caudal epidermal neuron precursors.
Scale bar : 100µm

Lateral view of a WT Ciona robusta tadpole larva probed for VGLUT expression. Second image is a magnification of the head and trunk region. Colored picture are HD unannotated images of the same embryo.
Original annotation: Staining is observed in the following locations : ATEN, apical trunk epider-mal neuron; bv, brain vesicle; DCEN, dorsocaudal epidermal neuron;oc, ocellus; ot, otolith; PN, papillar neuron; RTEN, rostral epidermalneuron; VCEN, ventrocaudal epidermal neuron.
Scale bar : 100µm

Lateral view of a WT Ciona robusta tadpole larva probed for VGLUT expression. Staining is observed in the sensory vesicle including otolith and ocellus, in palps neurons and in epidermal neurons in the tail and at the tip of the tail.
Original annotation: A larva showing Ci-VGLUT expression at the tip of the tail(asterisk). Because two cells comprising a single pair are adjacent to each other, in situ hybridization signals of the two cells are not seen as two separate dots in the photograph.
Scale bar : 100µm

Immunofluorescent staining with an anti-Ci-VGLUT antibody in a WT Ciona robusta tadpole larva.
A: A whole tadpole larva.
B: Right lateral view of the trunk of a larva.
C: Left lateral view of the trunk of a larva.
Original annotation: White arrowheads indicate afferent axon bundles from rostral trunk epidermal neurons. Black arrowheads indicate axons of caudal epidermal neurons. White arrows indicate axons of posterior apical trunk epidermal neurons. Conspicuous staining of Ci-VGLUT-immunoreactive axons and nerve terminals were observed in the posterior brain vesicle(asterisks). Glutamatergic axons were also found along the anteroposterior axis of the visceral ganglion (black arrows). bv, brain vesicle; oc,ocellus; ot, otolith; PN, papillar neurons; vg, visceral ganglion. Scalebars : 100µm

Left lateral view of a whole WT Ciona robusta larva incubated with the anti-Ci-VGLUT antibody.
Fluorescence is observed in the sensory vesicle and in the visceral ganglion. In other experiements whith higher magnification, fluorescence is also observed in the epidermal neurons.

Ciona robusta larva that developed from a fertilized egg electroporated with pCi-VGLUT>EGFP.
Original annotation: Localization of GFP was visualized byimmunofluorescent staining with an anti-GFP antibody. GFP localization was observed in the cell bodies of neurons located at the sites where the endogenous gene expression was observed by in situ hybridization. In neurons expressing GFP, axons were also labeled. An asterisk indicates a GFP-positive cell at the tip of the tail. ATENa, anterior apical trunk epidermal neuron; ATENp, posterior apicaltrunk epidermal neuron; DCEN, dorsocaudal epidermal neuron; oc,ocellus; ot, otolith; PN, papillar neuron; RTEN, rostoral trunk epider-mal neuron; VCEN, ventrocaudal epidermal neuron. Scale bar 100µm

Lateral view of the head of a WT Ciona robusta tadpole larva electroporated with VGLUT>eGFP at the 1-cell stage.
Original annotation:
A: A combined confocal microscopeimage of 46 optical sections (each 0.9µm in depth) of the trunk of alarva.
B–D: Confocal microscope images at different depths of the same series of optical sections as shown in A.
In B (a combined image of optical sections #8–10) the axonal pathway from a papillar neuron connecting to one of the rostral trunk epidermal neurons is indicated by arrowheads.
In C (a combined image of optical sections #23–46), afferent axons from rostral trunk epidermal neurons are indicated by white arrows. In D, nerve terminals from both rostral trunk epidermal neurons (white arrow) and otolith-associated neurons (yellow arrow) can be seen in one optical section (optical section #25).
Scale bar : 50µm

Lateral view of the head of WT Ciona robusta tadpole larvae electroporated with VGLUT>eGFP at the 1-cell stage.
Original annotation: Examples of peripheral and central prospective glutamatergic neurons in the trunk region. White arrows indicate an axon of a rostral trunk epidermal neuron projecting to the posterior brain vesicle. A group of prospective glutamatergic neurons located in the posterior brain vesicle sends axons posteriorly to the visceral ganglion (yellow arrowheads). An axon from a caudal epidermal neuron runs along the visceral ganglion and projects onto the posterior brainvesicle (white arrowheads in E). A group of prospective glutamatergic neurons adjacent to the sensory pigment cells sends axons to the posterior brain vesicle (yellow arrow in G).
Scale bar : 50µm

Lateral view of the head of a WT Ciona robusta tadpole larva electroporated with VGLUT>eGFP at the 1-cell stage. Fluorescence is observed in anterior and posterior apical trunk epidermal neurons (ATENa and ATENp), and in the sensory vesicle.
Original annotation: A bilaterally aligned pair of two epidermal neurons is located dorsal to the sensory pigment cells. These cells are different from and located anterior to the previously identified apical trunk epidermal neurons (ATENp).

Scale bar : 50µm

Lateral view of the head of a WT Ciona robusta tadpole larva electroporated with VGLUT>eGFP at the 1-cell stage. Fluorescence is observed in the anterior apical trunk epidermal neuron (ATENa), in the sensory vesicle and in the palps.
Original annotation: Confocal images at different depths of the same specimen as shown in B.
Although the axon terminal of ATENa appears to occur close to or within the posterior sensory vesicle in the combined confocal image (B), the optical sections indicate that the axon actually ends at the dorso-lateral surface of the trunk (C). Note that the axon terminal of ATENa is not seen in optical sections in which prospective glutamatergic neurons at the posterior brain vesicle are clearly seen (D).
oc, ocellus; ot, otolith
Scale bar : 50µm

Lateral view of the head of a WT Ciona robusta tadpole larva electroporated with VGLUT>eGFP at the 1-cell stage. Fluorescence is observed in anterior and posterior apical trunk epidermal neurons (ATENa and ATENp), and in the sensory vesicle.
Original annotation: A bilaterally aligned pair of two epidermal neurons is located dorsal to the sensory pigment cells. These cells are different from and located anterior to the previously identified apical trunk epidermal neurons (ATENp).

Scale bar : 50µm

Lateral view of the head of a WT Ciona robusta tadpole larva electroporated with VGLUT>eGFP at the 1-cell stage. Fluorescence is observed in the anterior apical trunk epidermal neuron (ATENa), in the sensory vesicle and in the palps.
Original annotation: Confocal images at different depths of the same specimen as shown in B.
Although the axon terminal of ATENa appears to occur close to or within the posterior sensory vesicle in the combined confocal image (B), the optical sections indicate that the axon actually ends at the dorso-lateral surface of the trunk (C). Note that the axon terminal of ATENa is not seen in optical sections in which prospective glutamatergic neurons at the posterior brain vesicle are clearly seen (D).
oc, ocellus; ot, otolith
Scale bar : 50µm

Lateral view of a WT Ciona robusta larva electroporated with VGLUT>GFP (green) and assayed for Arrestin immunolocalization (magenta).
Original annotation: Double immunofluorescent staining of GFP (green) with a photoreceptor-specific marker, arrestin (magenta). Some of the photoreceptor cells (PRC) express Ci-VGLUT>GFP (yellow arrows). Photoreceptor cells are labeled with Ci-VGLUT-GFP, as clearly shown by double staining with Ci-arr.
Scale bars:50µm.

Ciona intestinalis juveniles probed for Vglut immunolocalization. Specimen was opened along the endostyle in order to better visualize the cerebral ganglion.
Original comment: Cerebral ganglion (cg) and anterior nerves (arrowheads) marked by the antibody anti-Ci-Vglut. Note the thin nerve (arrow) at velum (v) level. (t) is for tentacles.
Scale bar: 200 μm

c, d e: The same area of velum (v) and tentacles (t) in bright field (panel c), labelled with DAPI (panel d), and with antibody anti-Ci-Vglut (panel e). Note in panel e coronal nerves (arrowheads).

Scale bar: 100 μm