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.

Vegetal view of a mid tailbud probed for Onecut and Arrestin expression (respectively labelled in red and green).
Original annotation : Arrestin probe is a specific marker for photoreceptor cells.
Onecut shows a clear co-localization with the Arrestin probe at larva stage thus demonstrating that at level of the sensory vesicle the Onecut gene is expressed in the photoreceptor cells.
First image : Arrestin ISH channel only.
Second image : Yellow color in the merged image represents co-expression territories, edge of the embryo is highlighted with a thin white line.

Dorsal view of a late tailbud DMSO-treated embryo probed for ci-opsin1 (green) and ci-arrestin (red) expression.

Original annotation:
In control embryos, expression of Ci-opsin1 and Ci-arrestin was detected in a few cells on the right side of the SV at the late tail bud stage.

Lateral view of a ate tailbud stage wild type embryo probed for Ci-arr expression. This picture shows expression in the photosensory organs.

Immusostaining of a late tailbud stage embryo for Arrestin. Expression observed of sensory vesicle (SV) and presence of palps.

Immunostaining of a late tailbud stage embryo for Arrestin. Expression of sensory vesicle.

Late tailbud stage embryo after injection of a control morpholino. Palps, pigmented cells and Arrestin staining can be observed.

Lateral views of WT Ciona robusta late tailbud stage embryos probed for Arrestin1 expression.
Arrestin1 is expressed in the sensory vesicle.
"n" is the number of scored embryos ; % corresponds to the proportion of embryos showing similar staining patterns. Arrowhead indicates expression in photoreceptor cells and their precursors.

Double whole-mount in situ hybridization experiments of Ci-prom1/2 (red) and Ci-arrestin (green) at early larval stage.

Original annotation:
From double in situ hybridization assays, we detected a perfect match of Ci-prom1/2 expression with Ci-arrestin in photoreceptors cells associated with the pigmented ocellus that are evident in the right lateral view of the early larval stage.

Expression of the Ci-Gai1-GFP fusion gene in photoreceptor cells of the ocellus. Combined confocal microscope images of 28 optical sections of the brain vesicle of a larva.
Localization of green fluorescent protein (GFP, in green) and arrestin (in magenta),a photoreceptor marker, was examined by immunofluorescent staining.

Original annotation: as shown in the merged picture (picture3), GFP driven by the Ci-Gai1 promoter was expressed in photoreceptor cells of the ocellus (arrowhead).
The last picture is an optical section showing colocalization of GFP and arrestin in photoreceptor cells (OC, ocellus; OT, otolith).

Vegetal view of a mid tailbud probed for Neurogenin and Arrestin expression (respectively labelled in red and green). Arrestin probe is a specific marker for photoreceptor cells, and is also expressed in part of the visceral ganglion.
First image : Arrestin ISH channel only.
Second image : Yellow color in the merged image represents co-expression territories, edge of the embryo is highlighted with a thin white line.

Experimental conditions description : Lateral view of a Ciona robusta larva electroporated with VGLUT>GFP (green) and assayed for Arrestin immunolocalization (magenta). Arrestin is localized in the photoreceptor cells of the ocellus.
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.

Dorsal view of the head of a WT Ciona robusta larva electroporated with ciGluA>GFP (green) with arrestin immunostaining (pink).
arrestin is expressed in the photoreceptor cells.
B is the 3D reconstruction images showing merged DIC and GFP staining. The yellow arrowhead indicates the otolith, and the red arrowhead indicates the ocellus. The bracket indicates part of the tail. B'' is a magnification.

Dorsal view of the head of a WT Ciona robusta larva electroporated with ciGluA>GFP (green) with arrestin immunostaining (red).
arrestin is expressed in the photoreceptor cells.
The white box is a higher magnification image of the photoreceptor region with arrows indicating cells that coexpress GFP and arrestin.

Dorsal view of the head of a WT Ciona robusta larva electroporated with ciGluA>GFP (green) with arrestin immunostaining (pink).
arrestin is expressed in the photoreceptor cells.
B is the 3D reconstruction images showing merged DIC and GFP staining. The yellow arrowhead indicates the otolith, and the red arrowhead indicates the ocellus. The bracket indicates part of the tail. B'' is a magnification.

Dorsal view of the head of a WT Ciona robusta larva electroporated with ciGluA>GFP (green) with arrestin immunostaining (red).
arrestin is expressed in the photoreceptor cells.
The white box is a higher magnification image of the photoreceptor region with arrows indicating cells that coexpress GFP and arrestin.

Dorsal view of the head of a WT Ciona robusta larva with arrestin immunostaining (red).
arrestin is expressed in the photoreceptor cells.

Dorsal view of the head of a WT Ciona robusta larva electroporated with ciGluA>GFP (green), with arrestin immunostaining (red).
arrestin is expressed in the photoreceptor cells.

Dorsal view of a tadpole DMSO-treated embryo probed for ci-opsin1 (green) and ci-arrestin (red) expression.

Original annotation:
In control embryos, expression of Ci-opsin1 and Ci-arrestin was detected in a few cells on the right side of the SV at the late tail bud stage, and in the photoreceptor cells in the larva.

Lateral views of WT Ciona robusta larvae probed for Arrestin expression. Pictures are confocal merged images of Arrestin hybridized larvae. Blue and green embryos are DAPI controls.
Arrestin is expressed in the sensory vesicle.
"n" is the number of scored embryos ; % corresponds to the proportion of embryos showing similar staining patterns.

Lateral view of a wild type tadpole embryo stained for Ci-arr mRNA by in situ hybridization. Staining is observed in the ocellus (oc) containing in the brain vesicle.

Expression of GFP in larvae that developed from fertilized eggs electroporated with the pCi-arr-EGFP construct.

Original annotation:
Picture a (lateral view): GFP fluorescence was observed only in the subsets of cells in the brain vesicle of a larva.
Picture b (dorsal view): a trunk region of a larva showing GFP fluorescence both in photoreceptor cells (arrows) of the ocellus (oc) and in the left side of the brain vesicle (arrowheads).
Picture c (dorsal view): a trunk region of a larva showing GFP fluorescence only in photoreceptor cells of the ocellus.
Picure d (dorsal view): a trunk region of a larva showing GFP fluorescence only in left side cells in the brain vesicle (arrowheads).

Lateral view of a larvae co-electroporated with Dmrt>H2B:mCherry (magenta) and Arrestin>H2B:CFP (blue).
Original annotation : Clusters of Arrestin cells are consistently observed posterior to the Dmrt expression domain. Arrestin reporter gene is specifically expressed in photoreceptor cells.

Lateral view of a larvae co-electroporated with Six3/6>H2B:mCherry (magenta) and Arrestin>H2B:CFP (blue).
Original annotation : Arrestin reporter labels a cluster of cells surrounding the ocellus, being specifically expressed in photoreceptor cells
Aniseed annotation : it seems that there is also expression in the visceral ganglion.