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 64-cell stage wild type embryo probed for cieg40d16.

Original Annotation :
Expression was detected in a-line.
Gene name: RorB

Animal and vegetal views of a 110-cell stage wild type embryo probed for cieg40d16 expression.

Original Annotation :
Expression was detected in a-line, A7.1, A7.2, A7.5, B7.1 and B7.2 cell pairs.
Gene name: RorB

110 cell-stage embryo electroporated with Ci-Rorb cis-regulatory region from -330bp to 113bp (construct named BS2/3) in front of LacZ reporter. Data not shown in the article.

Original annotation:
BS2/3 in front of a LacZ reporter gene did not drive any expression, indicating that this element does not contain the information required to drive Ci-Rorb expression. We suggest that the BS2/3 element is a basal promoter.

A 112-cell stage embryo electroporated with Rorb_BS1 enhancer construct.

Original Annotation :
BS1 drive a strong expression in all a-line blastomeres (neural and epidermal cells).

A 112-cell stage embryo electoporated with Rorb_BS4 intron-enhancer.

Original Annotation :
Rorb_BS4 intron-enhancer drive the expression in neural a-line blastomeres (and not in epidermal blastomeres).

Gastrula stage wild type embryo probed for Ci-Rorb expression.

Original Annotation :
At all the gastrula stages, Ci-Rorb is expressed in the neural and epidermal a-line. We also observed at these stages two bilaterally symmetrical spots of Ci-Rorb expression corresponding to the endodermal progenitors

Late gastrula stage wild type embryo probed for Ci-Rorb expression.

Original Annotation :
At all the gastrula stages, Ci-Rorb is expressed in the neural and epidermal a-line. We also observed at these stages two bilaterally symmetrical spots of Ci-Rorb expression corresponding to the endodermal progenitors.

Late gastrula stage embryo electroporated in fertilized eggs with Rorb cis-regulatory region from -593bp to 113bp in front of a GFP reporter gene (construct named BS1/2/3 in the paper).

Original Annotation :
Although no expression was detected in endodermal cells, where Ci-Rorb is expressed, BS1/2/3 drove EGFP expression in several anterior domains of the electroporated embryo trunks. BS1/2/3 is indeed active in the a-line blastomeres from the 110-cell stage to the larval stage.
We can observe a strong autofluorescence in the tail muscles progenitors, as seen in (B1) on a negative embryo at early neural plate stage.

Mid neurula stage wild type embryo probed for Ci-Rorb expression.

Original Annotation :
During neurulation, Ci-Rorb is expressed in the precursors of the anterior epidermis, in the most rostral rows of the neural plate, and in the invaginated precursors of the anterior endoderm.

Lateral view of an early tailbud stage wild type embryo probed for Ci-Rorb expression.

Original Annotation :
In late neurula early-/mid-tailbud stage, we observed that Ci-Rorb remains strongly expressed throughout the head epidermis as well as in the developing nervous system (whole sensory vesicle and stomodaeum). Again, Ci-Rorb is expressed in anterior endodermal tissues that have reached the anterior region of the head.

Latreal view of an early tailbud stage embryo electroporated in fertilized eggs with Rorb cis-regulatory region from -593bp to 113bp in front of a GFP reporter gene (construct named BS1/2/3 in the paper).

Original Annotation :
However, at early- and mid-tailbud stages, EGFP was restricted to the posterior part of the head epidermis in a consistent way with the weak epidermal expression during late neurula stages.

A tailbud stage embryo electroporated with Rorb_BS1 enhancer construct.

Original Annotation :
BS1 doesn't drive expression at this stage (in 98% of embryos).

Lateral view of a mid tailbud stage wild type embryo probed for Ci-Rorb expression.

Original Annotation :
In late neurula early-/mid-tailbud stage, we observed that Ci-Rorb remains strongly expressed throughout the head epidermis as well as in the developing nervous system (whole sensory vesicle and stomodaeum). Again, Ci-Rorb is expressed in anterior endodermal tissues that have reached the anterior region of the head.

A tailbud stage embryo electoporated with Rorb_BS4 intron-enhancer.

Original Annotation :
Rorb_BS4 intron-enhancer drive the expression in head epidermis (70% of embryos were stained as here).

Late tailbud stage wild type embryo probed for Ci-Rorb expression.

Original Annotation :
At the late tail-bud stage, the distribution of Ci-Rorb transcripts showed a rostral restriction within the anterior epidermis and nervous system, as well as in the anterior endoderm.

Lateral view of a late tailbud stage embryo electroporated in fertilized eggs with Rorb cis-regulatory region from -593bp to 113bp in front of a GFP reporter gene (construct named BS1/2/3 in the paper).

Original Annotation : At late-tailbud stage, BS1/2/3 was found to be active at least in the head epidermis, in the sensory vesicle and in the presumptive domain of the palps, like the endogenous Ci-Rorb.

Lateral view of a wild type tadpole probed for Ci-Rorb expression.

Original Annotation :
At larval stage, the expression of Ci-Rorb is restricted to the rostral part of the sensory vesicle, the deep domain of the stomodaeum (=oral siphon primordia in ANISEED) and a week expression is seen in the most anterior area of the head endoderm.

Lateral views of a larval stage embryo electroporated with Rorb cis-regulatory region from -593bp to 113bp in front of a GFP reporter gene (construct named BS1/2/3 in the paper).

A young adult showing a transient expression of the BL::Venus construct.
The second picture is a high magnification of the primordium of the dorsal tubercle.

Original Annotation :
The expression is detected in the primordium of the dorsal tubercule (yellow arrowhead) and at the tip of the endostyle (red arrowhead).

An adult showing a transient expression of the BL::Venus construct.
The second picture is a section of the neural complex of the adult.

Original Annotation :
The expression is found in the ciliated funnel (next to the dorsal tubercle, DT), neural gland (NG) and the cerebral ganglion (Gg).