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 data from Yutaka Satou's gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :Six12.
Expression pattern : No signal was detected

Conversion to Aniseed format of data from Yutaka Satou's gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :Six12.
Expression pattern : No distinct zygotic signal was detected

Conversion to Aniseed format of data from Yutaka Satou's gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :Six12.
Expression pattern : No distinct zygotic signal was detected

Conversion to Aniseed format of data from Yutaka Satou's gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :Six12.
Expression pattern : No distinct zygotic signal was detected

Conversion to Aniseed format of data from Yutaka Satou's gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :Six12.
Expression pattern : No distinct zygotic signal was detected

Conversion to Aniseed format of data from Yutaka Satou's gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :Six12.
Expression pattern : No distinct zygotic signal was detected

Early neurula probed for Six1/2 (pink) and Eya (blue) expression, and electroporated with ZicL>GFP (yellow). Dorsal view of an embryo at early neurula stage electroporated with ZicL>GFP and hybridized with Six1/2 and Eya mRNA probes.
Original annotation : We found that the Ciona homologue of Six1, Six1/2, as well as its co-factor Eya, is initially expressed in a row of eight cells (a10.77, a10.69, a10.101, a10.103) located immediately anterior to the neural plate, as marked by ZicL.

Neurula stage wild type embryo probed for Ci-Six1/2 expression. The signal is confined to the anterior border of the neural plate (arrow).

Conversion to Aniseed format of data from Yutaka Satou's gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :Six12.
Expression pattern : nervous system

Dorsal view of an embryo at mid-neurula stage co-electroporated with Six1/2>mCherry (magenta) and Chordin>GFP (blue).
Six1/2 enhancer drives expression in 16 cells located immediately anterior to the neural plate.

Dorsal view of an initial tailbud embryo co-electroporated with Six1/2GFP (green) and ZicL>CFP (yellow). At this stage, the cells initially expressing Six1/2 have divided once, giving rise to 16 cells in total. Brackets indicate the derivatives of the annotated lineages. Anterior is to the left.

Original annotation : Dorsal view of initial tailbud embryo electroporated with Six1/2>mCherry (magenta) and counterstained with phalloidin (blue). Activity of Six1/2 enhancer is observed in cells arranged in a U shape, anterior to the neural plate.
Schematic indicates that the U shape is composed of 16 cells in total at the 11th generation (that is, a11.154, a11.138, etc.). There are no further divisions of these cells until after the late tailbud I stage. The green cells indicate a11.205, which are fated to become PPE-derived GnRH neurons.

Original annotation : Dorsal view of a tailbud embryo electroporated with Six1/2>mCherry (magenta) and counterstained with phalloidin (blue).
At the initial tailbud stage II, the lateral edges of the Six1/2+ cells begin to intercalate towards the midline.
Schematic indicates that the U shape is composed of 16 cells in total at the 11th generation (that is, a11.154, a11.138, etc.). The green cells indicate a11.205, which are fated to become PPE-derived GnRH neurons. The schematic shows a dotted circle where the future opening of the oral siphon forms.

Dorsal view of an early tailbud stage wild type embryo probed for Six1/2 expression.

Conversion to Aniseed format of data from Yutaka Satous gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :Six12.
Expression pattern : nervous system.
Comment : anterior region of the brain

Mid tailbud stage wild type embryo probed for Ci-Six1/2 expression. Expression is confined to the buccal cavity (arrow).

Conversion to Aniseed format of data from Yutaka Satous gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :Six12.
Expression pattern : nervous system, epidermis.
Comment : anterior region of the brain, epidermis in the lateral part of the trunk

Ventral, lateral and dorsal (view of the head) views of a mid tailbud stage wild type embryo probed for Ci-Six4/5 (red) and Ci-Six1/2 (blue) expression.

Original annotation: the arrow indicates the buccal cavity expression of Ci-Six1/2. Ci-Six4/5 occupies a more anterior position than Ci-Six1/2. However many cell co-express both genes.

Lateral view of a late tailbud stage wild type embryo probed for Ci-Six1/2 expression. Second picture is a magnification of the left side of a section through the head of an embryo.

Original annotation:
Expression has initiated in the endoderm (en) of the head and in several bilateral patches of ectoderm (e) (nt, neural tube).

Lateral view of a late tailbud wild type embryo probed for Ci-Six1/2 and Ci-FoxIc expression.

Original Annotation : Double in situs with Ci-Six1/2 (red) and Ci-FoxIc (blue). The buccal cavity domain of Ci-Six1/2 is marked by an arrow. The insitus showed that the lateral epidermis Ci-Six1/2 was entirely enclosed within the domain of Ci-FoxIc.

Lateral view of a late tailbud wild type embryo probed for Ci-Six1/2 and Ci-FoxIb expression.

Original Annotation : Double in situs with Ci-Six1/2 (red) and Ci-FoxIb (blue). The buccal cavity domain of Ci-Six1/2 is marked by an arrow. The insitus showed that in the lateral epidermis Ci-Six1/2 was entirely enclosed within the domain of Ci-foxIb.

Lateral view of a late tailbud embryo probed for Ci-Six1/2 and Ci-Pax3/7 expression.

Original Annotation : Double in situs with Ci-Six1/2 (red) and Ci-Pax3/7 (blue). The picture shows that the lateral ectoderm domain expressing Ci-Six1/2 to be adjacent to the posterior domain of Ci-Pax3/7. Ci-Six1/2 expression was detected in the buccal cavity.

Lateral view of a late tailbud embryo probed for Ci-Six1/2 and Ci-Pax2/5/8 expression.

Original Annotation : Double in situs with Ci-Six1/2 (red) and Ci-Pax2/5/8 (blue). The picture shows that the lateral ectoderm domain expressing Ci-Six1/2 to be adjacent to the region of the brain that expressed Ci-Pax2/5/8. Ci-Six1/2 expression was detected in the buccal cavity.

Original annotation : Dorsal view of a late tailbud embryo electroporated with Six1/2>mCherry (magenta) and counterstained with phalloidin (blue).
At this stage, the Six1/2+ cells are positioned on top of the anterior sensory vesicle over the ocellus.
Schematic indicates that 16 cells in total at the 11th generation (that is, a11.154, a11.138, etc.) where six1/2 enhancer is active.

Original annotation : Anterior lateral view of late tailbud I stage embryo electroporated with Six1/2>GFP (magenta) and CNGA>mCherry (green). Asterisks indicate the ocellus. The arrowheads indicate a PPE-derived cell fated to become a GnRH neuron.
First image shows the Six1/2>GFP channel, and the second shows the merged image of Six1/2>GFP and CNGA>mCherry

At the tailbud stage, six1/2 enhancer is active in cells positioned on top of the anterior sensory vesicle, over the ocellus.
Original Annotation : row V neural plate and a10.103 cell pair
Aniseed Annotation : fates of row V / a10.103 cell pair that could correspond to the original annotation is neurohypophysis primordium, siphon primordia and part of palps.

Original annotation : Lateral anterior view of a tailbud electroporated with Six1/2>mCherry and counterstained with phalloidin (grey).
Arrowhead indicates the posterior-most Six1/2+ cell that undergoes neurogenesis.
Aniseed Annotation : fates of row V / a10.103 cell pair that could correspond to the original annotation is neurohypophysis primordium, siphon primordia and part of palps.

Dorsal view of a tadpole stage wild type embryo probed for Ci-Six1/2 expression.

Original annotation:
Expression was apparent in the buccal cavity and future atrial siphon primordia (arrowhead) and was also detected in mesenchyme posterior to the brain (arrow).

Original annotation : Lateral anterior view of a larva co-electroporated with GnRH>GFP (yellow) and Six1/2>mCherry (magenta), and counterstained with phalloidin (grey).
Insets show Six1/2 expression (left) and GnRH expression (right) surrounding the oral opening (dotted line). The GnRH1 neuron is marked with an asterisk. Brackets indicate PPE-derived GnRH neurons.