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 110-cell stage wild type embryo probed for Ci-MLC (Myosin Light Chain) expression. This picture shows expression in the muscle lineage, particularly in the B8.15 and B8.7 cell pairs.

View of a 110-cell stage embryo showing expression of Ci-MLC in muscle precursor cells.

Vegetal pole view of a 110-cell stage wild typeembryo probe for Ci-MLC (Myosin Light Chain) expression.This picture shows expression in the muscle cells lineage.

Lateral view of a mid-tailbud stage embryo probed with Ci-MLC (Myosin Light Chain) expression.

View of a mid tailbud stage embryo showing expression of Ci-MLC in muscle. Embryos are allowed to develop to the 110-cell stage and then cleavage was arrested by cytochalasin.

Lateral view of a mid tailbud stage embryo, showing expression of Ci-MLC in muscle cells.

Lateral view of a late tailbud stage wild type embryo probed for Ci-MLC (Myosin like chain) expression. This picture shows expression in the muscle lineage cells.

Dorsal pole view of a late tailbud stage wild type embryo probed for Ci-MLC (Myosin Light Chain) expression. This picture shows expression in the tail muscle cell lineage.

Sagittal view of a late tail-bud stage embryo, electroporated with a GFP fusion gene containing the 643-bp Ci-MLC2 enhancer (construct named Ci-MLC2-EGFP(WT) in the paper). GFP expression is observed in tail muscles cells.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -502bp/+141bp

Views of a late tail-bud stage embryos, electroporated with a GFP fusion gene containing the 643-bp Ci-MLC2 enhancer (construct named Ci-MLC2-EGFP(WT) in the paper). GFP expression is observed in tail muscles cells.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -502bp/+141bp

No picture available in the paper. Late tailbud stage embryos electroporated with a GFP fusion gene containing the 643-bp Ci-MLC2 in which the 18 pb between the two motifs M2 had been mutated (construct named Ci-MLC2-EGFP (uM2i) in the paper). Expression in tail muscles is kept.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -502bp/+141bp

No picture available in the paper. Late tailbud stage embryos electroporated with a GFP fusion gene containing the 643-bp Ci-MLC2(deltaM2p/d) which the two motifs M2 mutated. Expression in tail muscles is lost.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -502bp/+141bp

No picture avaible in the paper. Late tailbud stage embryos electroporated with a GFP fusion gene containing the 643-bp Ci-MLC2 which the first of the two motifs M2 had been mutated (construct named Ci-MLC2-EGFP (uM2d) in the paper). Expression in tail muscles is lost.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -502bp/+141bp

No picture available in the paper. Late tailbud stage embryos electroporated with a GFP fusion gene containing the 643-bp Ci-MLC2 in which the second of the two motifs M2 had been mutated (construct named Ci-MLC2-EGFP (uM2p) in the paper). Expression in tail muscles is kept.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -502bp/+141bp

Control:
Fluorescence image of late-tail bud stage embryos electroporated with a GFP fusion gene containing the 643-bp Ci-MLC2 enhancer. GFP expression is observed in tail muscles cells.

Picture:
Fluorescence image of late-tail bud stage embryos electroporated with a GFP fusion gene containing the 643-bp Ci-MLC2(deltaM2p/d) which the two motifs M2 and the 18-bp sequence between them had been mutated
Expression in tail muscles is lost

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -502bp/+141bp

Sagittal view of late tail-bud stage embryos, electroporated with a GFP fusion gene containing the 115-bp Ci-MLC2 basal enhancer (construct named bCi-MLC2-EGFP in the paper). No GFP expression is observed.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -115bp/-1bp

Sagittal view of late tail-bud stage embryos, electroporated with a GFP fusion gene containing a 115-bp Ci-MLC2 basal enhancer which 4 M2d sites (AGTGACGTGAGAA) were added in 5prime (construct named bCi-MLC2-EGFP+4M2d in the paper). Expression in tail muscle cells was detected.

Be careful, coordinates in the paper are different from ANISEED coordinates. We took the KH transcript model as reference.
Coordinates in the paper : -115bp/-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