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.

Lateral and dorsal views of a WT Ciona robusta neurula probed for ASIC (KH.C1.215) expression. Staining is observed in the posterior epidermis of the tail.
Original annotation: Whole-mount embryos hybridized with a digoxigenin-labeled antisense probe to the unique sequence of the CiASICa transcript (shorter splicing variant of ASIC).

Lateral view of an early tailbud stage wild type embryo probed for ciad68f24 expression.

Original annotation:
Expression was detected in the ventral part of the brain.

Aniseed Annotation :
According to the picture, it seems there is no expression at this stage in the brain.

Lateral view of a mid tailbud wild type embryo probed for ciad68f24 expression.

Original annotation:
Expression was detected in the ventral part of the brain.

Lateral view of a WT Ciona robusta mid tailbud embryo probed for ASIC (KH.C1.215) expression.
Original annotation: Staining is detected primarily in presumptive bipolar neurons in the tail.
Staining of a middle tailbud stage embryo with a probe unique to the CiASICb transcript (longer splicing variant of ASIC).

Lateral and dorsal views of a WT Ciona robusta late tailud embryo probed for ASIC (KH.C1.215) expression.
Original annotation: Late tailbud stage embryo showing staining of bipolar neurons of the tail and the developing sensory vesicle in the trunk.
Whole-mount embryos hybridized with a digoxigenin-labeled antisense probe to the unique sequence of the CiASICa transcript (shorter splicing variant of ASIC).

Lateral view of a late tailbud stage wild type embryo probed for ciad68f24 expression.

Original annotation:
Expression was detected in the ventral part of the brain.

Lateral view of the posterior tail of a WT Ciona robusta late tailbud embryo co-electroporated with Asic>unc76::eGFP (green) and Neurog b-line>nls::LacZ (red).
eGFP is fused to UNC-76 tags to ensure even labelling of axons.
eGFP signalling is observed in the bipolar tail neurons (BTNs).
Original annotation : BTN expressing Asic>unc-76::eGFP reporter in embryo electroporated with Neurog b-line>nls::lacZ as a control.

Lateral view of a Ciona robusta late tailbud embryo co-electroporated with Neurog b-line>H2B::mCherry (magenta nuclei) and Asic>unc76::eGFP (green). Neurog b-line cis-regulatory element drives mCherry expression in the b-line tail epidermis including the bipolar tail neurons.
Scale bars 25 μm.

Lateral view of a Ciona robusta late tailbud embryo co-electroporated with Neurog b-line>H2B::mCherry (magenta nuclei) and Asic>unc76::eGFP (green). eGFP signalling is observed in the bipolar tail neurons (BTNs).
Scale bars 25 μm.

Lateral view of the tail of a WT Ciona robusta late tailbud embryo processed for immunofluorescence with anti-CiASIC antibody.
Original annotation: Tail of a late tailbud embryo showing staining in somata and processes of presumptive bipolar neurons in the tail.
Arrows indicate somata of presumptive bipolar neurons.

Head and tail of a WT Ciona robusta hatched larva processed for immunofluorescence with anti-CiASIC antibody.
Original annotation:
1st picture: Staining of cell bodies of neurons in sensory vesicle and visceral ganglion of a hatched larva.
2nd picture: Tail of a hatched larva with staining in cell bodies and processes of bipolar neurons.
Arrows indicate somata of presumptive bipolar neurons.
Brightly fluorescent small round cells under the tunic are test cells trapping the antibody.

Tail region of a WT Ciona robusta hatched larva processed for immunofluorescence with anti-CiASIC (green) and anti-acetylated tubulin (red) antibodies.
Original annotation: Somata of bipolar neurons and their processes run parallel to the nerve cord. Nerve cord and sensory cilia are labeled with acetylated tubulin antibody.
Arrows indicate somata of presumptive bipolar neurons; arrowheads indicate sensory ciliae.

Lateral view af a WT Ciona robusta larva co-electroporated with ASICa>RFP and ASICb>GFP constructs. These cis-regulatory regions drive isoforms of the same gene (KH.C1.215).
Original annotation: Both enhancer elements direct reporter gene expression in the sensory vesicle and visceral ganglion in the trunk and in bipolar neurons of the tail.
1st picture: Fluorescence micrograph of RFP expression driven by the CiASICa enhancer.
2nd picture: Bright-field image of a C. intestinalis larva coelectroporated at the one-cell stage with the CiASICa enhancer driving RFP and the CiASICb enhancer driving GFP.
3rd picture: Fluorescence micrograph of GFP expression driven by the CiASICb enhancer.

Lateral view af a WT Ciona robusta larva co-electroporated with ASICa>RFP and ASICb>GFP constructs. These cis-regulatory regions drive isoforms of the same gene (KH.C1.215).
Original annotation: Both enhancer elements direct reporter gene expression in the sensory vesicle and visceral ganglion in the trunk and in bipolar neurons of the tail.
1st picture: Fluorescence micrograph of GFP expression driven by the CiASICb enhancer.
2nd picture: Bright-field image of a C. intestinalis larva coelectroporated at the one-cell stage with the CiASICa enhancer driving RFP and the CiASICb enhancer driving GFP.
3rd picture: Fluorescence micrograph of RFP expression driven by the CiASICa enhancer.

Lateral view af a WT Ciona robusta larva co-electroporated with ASICa>RFP and ASICb>GFP constructs. These cis-regulatory regions drive isoforms of the same gene (KH.C1.215).
Original annotation: Both enhancer elements direct reporter gene expression in the sensory vesicle and visceral ganglion in the trunk and in bipolar neurons of the tail.
1st picture: Fluorescence micrograph of RFP expression driven by the CiASICa enhancer.
2nd picture: Bright-field image of a C. intestinalis larva coelectroporated at the one-cell stage with the CiASICa enhancer driving RFP and the CiASICb enhancer driving GFP.
3rd picture: Fluorescence micrograph of GFP expression driven by the CiASICb enhancer.

Lateral view af a WT Ciona robusta larva co-electroporated with ASICa>RFP and ASICb>GFP constructs. These cis-regulatory regions drive isoforms of the same gene (KH.C1.215).
Original annotation: Both enhancer elements direct reporter gene expression in the sensory vesicle and visceral ganglion in the trunk and in bipolar neurons of the tail.
1st picture: Fluorescence micrograph of GFP expression driven by the CiASICb enhancer.
2nd picture: Bright-field image of a C. intestinalis larva coelectroporated at the one-cell stage with the CiASICa enhancer driving RFP and the CiASICb enhancer driving GFP.
3rd picture: Fluorescence micrograph of RFP expression driven by the CiASICa enhancer.

Dorasl view of a Ciona robusta larva micro-injected with Asic1b>CFP(green) and Atonal >Kaede (red) reporter constructs.
Asic1 cis-regulatory region drives CFP expression in the sensory vesicle and visceral ganglion in the trunk and in bipolar neurons of the tail (BTNs).
Scale bars = 100μm.

Dorasl view of a Ciona robusta larva micro-injected with Asic1b>CFP(green) and Atonal >Kaede (red) reporter constructs.
Atonal cis-regulatory region drives Kaede expression in palps neurons, rostral trunk epidermal neurons (RTENs), anterior and posterior apical trunk epidermal neurons (aATENs and pATENs), Caudal epidermal neurons (CESNs) and bipolar tail neurons (BTNs).
Original annotation: This Atoh1/Atonal/lin‐32 reporter is active in nearly all sensory neurons, including CESNs, consistent with previous in situ hybridization results (Joyce Tang, Chen, & Zeller,2013). Interestingly, Atonal is expressed in BTNs.
Scale bars = 100μm.