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.

64-cell stage wild type embryo probed for Ci-trop expression. Expression was detected from the late gastrula stage.

110-cell stage wild type embryo probed for Ci-trop expression. Expression was detected from the late gastrula stage.

Aniseed annotation: according to the picture, it seems that there is a staining B8.5 and B7.7 cell pairs.

Mid gastrula stage wild type embryo probed for Ci-trop expression.

Aniseed annotation: according to the picture, it seems that there is a staining in A-line notochord precursors.

Vegetal view of a late-gastrula wild type embryo stained for Ci-trop mRNA by in situ hybridization. Staining is detected in all the notochord precursor cells. The blastopore (bp) is indicated by an arrow.

Neural plate view of a Late gastrula stage wild type embryo probed for Ci-trop expression. Expression was detected from the late gastrula stage.

Side view (anterior is to the left, dorsal is up)of a mid-neurula wild type embryo stained for Ci-trop mRNA by in situ hybridization. Staining is detected in each of the invaginating notochord precursor cells.

Neural plate view of a Mid neurula stage wild type embryo probed for Ci-trop expression. Expression was detected from the late gastrula stage.

Dorsal view of a late neurula wild type embryo stained for Ci-trop mRNA by in situ hybridization. Staining is detected in the two rows of notochord precursor cells.

Conversion to Aniseed format of Ghost database entry from Satou Y et al., Development 2001; 128:2893-2904.

Original annotation: expression pattern: Notochord

Dorsal view of a initial tailbud stage wild type embryo probed for Ci-trop expression. Expression was detected from the late gastrula stage.

Conversion to Aniseed format of Ghost database entry from Satou Y et al., Development 2001; 128:2893-2904.

Original annotation: expression pattern: Notochord

Lateral view of an early tailbud wild type embryo stained for Ci-trop mRNA by in situ hybridization. Staining is detected in the two rows of notochord precursor cells.

Conversion to Aniseed format of Ghost database entry from Satou Y et al., Development 2001; 128:2893-2904.

Original annotation: expression pattern: Notochord

Lateral view of a mid tailbud stage wild type embryo probed for Ci-trop expression. We can see expression in the notochord.

Lateral view of a Mid tailbud stage wild type embryo probed for Ci-trop expression. Expression was detected from the late gastrula stage.

Conversion to Aniseed format of Ghost database entry from Satou Y et al., Development 2001; 128:2893-2904.

Original annotation: expression pattern: Notochord

Lateral view of an late tail-bud wild type embryo stained for Ci-trop mRNA by in situ hybridization. Staining is detected in the two rows of notochord precursor cells.

Lateral view of a late tailbud electroporated with a lacZ transgene which contains the -3 kb of the 5' flanking Ci-trop cis regulatory region sequence. Staining is observed in the notochord (red arrowhead). Staining is also detected in the tail epidermis (green arrowhead) of ~50% of the electroporated embryos.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -3000bp/-1bp

Lateral view of a late tailbud electroporated with a lacZ transgene which contains the -3kb of the 5' flanking Ci-trop promoter sequence, with an internal deletion of 300bp between -1.4 kb and -1.1 kb. There is a loss of staining in the notochord, and persistence of expression in the epidermis and mesenchyme.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -3000bp/-1bp delta -1400bp/-1100bp

Lateral view of a late tailbud electroporated with a Ci-fkh/lacZ (the -0,45 kb upstream ci-fkh) fusion gene containing a ~500 bp region of the Ci-trop 5'-flanking sequence (from -1.8 kb to -1.3 kb). Staining is observed in the notochord.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -1800bp/-1300bp

Lateral view of a late tailbud electroporated with a Ci-fkh/lacZ (the -0,45kb upstream ci-fkh) fusion genes containing a ~400 bp region of the Ci-trop 5'-flanking sequence (from -1.8 kb to -1.4 kb). Expression in the notochord is lost, ectopic expression is observed in the mesenchyme.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -1800bp/-1400bp

Lateral view of a late tailbud embryo electroporated with a lacZ transgene which contains the -1,4 kb of the 5' flanking Ci-trop promoter sequence. Strong staining is detected in the notochord, similar to that obtained with the full-length fusion gene.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -1400bp/-1bp

Lateral view of a late tailbud embryo electroporated with a Ci-fkh/lacZ (the -0,45kb upstream ci-fkh) fusion genes containing a ~115 bp region of the Ci-trop 5'-flanking sequence (from -1.4 kb to -1.3 kb). Expression is observed in the notochord.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -1400bp/-1285bp

Lateral view of a late tailbud embryo electroporated with a lacZ transgene which contains the -1377 bp of the 5' flanking Ci-trop cis regulatory sequence. Strong expression is detected in the notochord.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -1384bp/-1bp

Lateral view of a late tailbud embryo electroporated with a lacZ transgene which contains the -1355 bp of the 5' flanking Ci-trop cis-regulatory region. Expression in the notochord is lost, but ectopic expression is detected in the tail epidermis and trunk mesenchyme.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -1362bp/-1bp

Lateral view of a late tailbud embryo electroporated with a lacZ transgene which contains the -1,3 kb of the 5' flanking Ci-trop cis-regulatory sequence. Expression in the notochord is lost, staining persists in the tail epidermis and trunk mesenchyme.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -1300bp/-1bp

Lateral view of a late tailbud electroporated with a lacZ transgene which contains the -3kb of the 5' flanking Ci-trop promoter sequence and co-electroporated with a Ci-bra fusion gene containing -2.6 kb of 5'-flanking sequence from the Ci-forkhead (Ci-fkh) gene. The transgene results in the misexpression of Ci-bra in endodermal and neuronal tissues. There is at least a partial transformation of endoderm into notochord. The Ci-trop/lacZ fusion gene is expressed in the notochord (red arrowhead), transformed endoderm (yellow arrowhead), and regions of the tail epidermis (green arrowhead).

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -3000bp/-1bp

Conversion to Aniseed format of Ghost database entry from Kusakabe T et al., Dev Biol. 2002; 242:188-203.

Original annotation: expression pattern: not clear

Conversion to Aniseed format of Ghost database entry from Ogasawara et al., Dev Genes Evol. 2002; 212:173-185.

Original annotation: expression pattern: Endostyle Pharyngeal gill Heart Intestine Neural gland Epidermis Stomach Esophagus