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.

Ventral view of a late 16-cell stage embryo probed for cdc25 expression.
Original annotation : Ci-cdc25 expression in the precursor blastomeres of epidermis was examined by WISH. The embryos were imaged from the ventral (animal) side. The anterior end is toward the top. The number and percentage of embryos with normal Ci-cdc25 expression in the epidermis are shown. The denominators represent the total number of embryos examined

Animal view of a 32-cell stage embryo probed for cdc25 expression.
Original annotation : Ci-cdc25 expression in the precursor blastomeres of epidermis was examined by WISH. The embryos were imaged from the ventral (animal) side. The anterior end is toward the top. The number and percentage of embryos with normal Ci-cdc25 expression in the epidermis are shown. The denominators represent the total number of embryos examined.

Animal view of a 64-cell stage embryo probed for cdc25 expression.
Original annotation : Ci-cdc25 expression in the precursor blastomeres of epidermis was examined by WISH. The embryos were imaged from the ventral (animal) side. The anterior end is toward the top. The number and percentage of embryos with normal Ci-cdc25 expression in the epidermis are shown. The denominators represent the total number of embryos examined

Animal view of a 112-cell stage embryo probed for cdc25 expression.
Original annotation : Ci-cdc25 expression was examined by WISH in wild-type conditions. Embryos were imaged from the ventral side with the anterior end toward the top. Scale bars, 50 mm

Expression of cdc25 at 5.0 hour after fertilization, animal view.

Expression of cdc25 at 5.5 hour after fertilization.

Animal view of an early gastrula embryo probed for cdc25 expression (pseudocolored in magenta). Nuclei are shown by DAPI (green)
Original annotation : Ci-cdc25 expression (pseudocolored in magenta) during gastrulation examined by FISH. Nuclei are shown by DAPI (green). The images show the ventral side of the embryos, and anterior is toward the right. The expression of Ci-cdc25 along the A-P axis was quantified from the boxed portions of the image. Normalized fluorescence intensities averaged along the D-V axis are shown for different embryos as line plots of different colors. The black-line plots represent the intensity quantified in the picture. a.u., arbitrary units.

Animal view of an early gastrula embryo probed for cdc25 expression.
Original annotation : Ci-cdc25 expression was examined by WISH in wild-type controls. Embryos were imaged from the ventral side with the anterior end toward the top. Scale bars, 50 mm.

Animal view of an early gastrula embryo probed for cdc25 expression.
Original annotation :Ci-cdc25 expression was examined by WISH in the wild-type controls. Embryos were imaged from the ventral side with the anterior end toward the top.

Animal view of an early gastrula embryo probed for cdc25 expression (pseudocolored in magenta). Nuclei are shown by DAPI (green).
Original annotation : Expression of cdc25 was examined at 5hpf by FISH. The embryos were imaged from the ventral side with the anterior end toward the right.

Animal view of a mid gastrula embryo probed for cdc25 expression (pseudocolored in magenta). Nuclei are shown by DAPI (green)
Original annotation : : Ci-cdc25 expression (pseudocolored in magenta) during gastrulation examined by FISH. Nuclei are shown by DAPI (green). The images show the ventral side of the embryos, and anterior is toward the right. The expression of Ci-cdc25 along the A-P axis was quantified from the boxed portions of the image. Normalized fluorescence intensities averaged along the D-V axis are shown for different embryos as line plots of different colors. The black-line plots represent the intensity quantified in the picture. a.u., arbitrary units.

Animal view of a mid gastrula embryo probed for cdc25 expression.
Original annotation : Ci-cdc25 expression was examined by WISH in wild-type controls. Embryos were imaged from the ventral side with the anterior end toward the top. Scale bars, 50 mm.

Animal view of a mid gastrula embryo probed for cdc25 expression.
Original annotation :Ci-cdc25 expression was examined by WISH in the wild-type controls. Embryos were imaged from the ventral side with the anterior end toward the top.

Animal view of a mid gastrula embryo probed for cdc25 expression (pseudocolored in magenta).
Original annotation : Expression of cdc25 was examined at 5.5 hpf by FISH. The embryos were imaged from the ventral side with the anterior end toward the right.
The graph shows quantified expression level : The expression of cdc25 along the A-P axis was quantified from the image. Normalized fluorescence intensities averaged along the D-V axis are shown for different embryos as line plots of different colors. The black-line plots represent the intensity quantified in the FISH image (a.u., arbitrary units).

The 5kpb region located upstream the transcription start site of cdc25 (KHC5.12) is strongly active in the epidermis of gastrula embryos (region called -4976 in the article supplementary data).
Original Comment : Deletion analysis of the Ci-cdc25 upstream region. Embryos were electroporated with the various deletion constructs shown on the left. Expression of the reporter Kaede was detected by WISH. The right graph shows the percentage of embryos expressing Kaede in 0%, within 50%, and > 50% of the epidermal territory.

The 2.3kpb region located upstream the transcription start site of cdc25 (KHC5.12) is strongly active in the epidermis of gastrula embryos (region called -2295 in the article).
Original Comment :
Deletion analysis of the Ci-cdc25 upstream region. Embryos were electroporated with the various deletion constructs shown on the left. Expression of the reporter Kaede was detected by WISH. The right graph shows the percentage of embryos expressing Kaede in 0%, within 50%, and > 50% of the epidermal territory.
The embryos were imaged from the ventral side with the anterior side toward the top. Kaede mRNA was detected by WISH at the gastrula stage (5.5 hpf). Scale bar, 50 μm

The 5kpb region located upstream the transcription start site of cdc25 (KHC5.12) and lacking the cis-regulatory modules (-2109/-1983) is strongly active in the epidermis of gastrula embryos. Region called -4976 Delta 1984-2109 in the article.
Original Comment :
Deletion analysis of the Ci-cdc25 upstream region. Embryos were electroporated with the various deletion constructs shown on the left. Expression of the reporter Kaede was detected by WISH. The right graph shows the percentage of embryos expressing Kaede in 0%, within 50%, and > 50% of the epidermal territory.

The 2.3kpb region located upstream the transcription start site of cdc25 (KHC5.12) and lacking the cis-regulatory modules CRM1 and 2 (-2109/-1983) is active in the muscle and in the epidermis of gastrula embryos (region called -2295 Δ1984-2109 in the article).
Original Comment :
Deletion analysis of the Ci-cdc25 upstream region. Embryos were electroporated with the various deletion constructs shown on the left. Expression of the reporter Kaede was detected by WISH. The right graph shows the percentage of embryos expressing Kaede in 0%, within 50%, and > 50% of the epidermal territory. The embryos were imaged from the ventral side with the anterior side toward the top. Kaede mRNA was detected by WISH at the gastrula stage (5.5 hpf). Scale bar, 50 μm Note that Kaede expression was markedly reduced in the epidermis (Ep), while its expression remained in the muscle (Mu) (when compared to the whole 2.3kb region).

The 1983pb region located upstream the transcription start site of cdc25 (KHC5.12) is not active in the epidermis of gastrula embryos (region called -1983 in the article).
Original Comment :
Deletion analysis of the Ci-cdc25 upstream region. Embryos were electroporated with the various deletion constructs shown on the left. Expression of the reporter Kaede was detected by WISH. The right graph shows the percentage of embryos expressing Kaede in 0%, within 50%, and > 50% of the epidermal territory.

The 2.1kpb region located upstream the transcription start site of cdc25 (KHC5.12) is strongly active in the epidermis of gastrula embryos (region called -2109 in the article).
Original Comment : Deletion analysis of the Ci-cdc25 upstream region. Embryos were electroporated with the various deletion constructs shown on the left. Expression of the reporter Kaede was detected by WISH. The right graph shows the percentage of embryos expressing Kaede in 0%, within 50%, and > 50% of the epidermal territory.

The 2kpb region located upstream the transcription start site of cdc25 (KHC5.12) is active in the epidermis of gastrula embryos (region called -2010 in the article).
Original annotation :Deletion analysis of the Ci-cdc25 upstream region. Embryos were electroporated with the various deletion constructs shown on the left. Expression of the reporter Kaede was detected by WISH. The right graph shows the percentage of embryos expressing Kaede in 0%, within 50%, and > 50% of the epidermal territory

The 2.1kpb region located upstream the transcription start site of cdc25 (KHC5.12) is strongly active in the epidermis of gastrula embryos.
Original annotation :: Expression of Kaede is observed in the epidermis (Ep) in an embryo electroporated with pSP-cdc25( 2109 bp)>Kaede. The embryos were imaged from the ventral side with the anterior end toward the top.
This construct corresponds to the upper line of the chart, where GATA, AP2-2 and Sox binding sites are respectively symbolized by red, blue and brown squares.

The 2kpb region located upstream the transcription start site of cdc25 (KHC5.12) is strongly active in the epidermis of gastrula embryos.
Original annotation : Kaede expression in an embryo expressing pSP-cdc25( 2010 bp) was examined by WISH. Kaede expression in the epidermis was observed. The embryos were imaged from the ventral side with the anterior end toward the top.
This construct corresponds to the second line of the chart, where GATA, AP2-2 and Sox binding sites are respectively symbolized by red, blue and brown squares.

The 2kpb region located upstream the transcription start site of cdc25 (KHC5.12) is weakly active in the epidermis of gastrula embryos when the first GATA binding site is mutated.
Original annotation ::
This construct corresponds to the third line of the chart, where GATA, AP2-2 and Sox binding sites are respectively symbolized by red, blue and brown squares. Effects of mutating the GATA-binding sites in CRM1 were examined by whole-mount in situ hybridization (WISH) of Kaede.

The 2kpb region located upstream the transcription start site of cdc25 (KHC5.12) is still strongly active in the epidermis of gastrula embryos when the second GATA binding site is mutated.
Original annotation : This construct corresponds to the fourth line of the chart, where GATA, AP2-2 and Sox binding sites are respectively symbolized by red, blue and brown squares. Effects of mutating the GATA-binding sites in CRM1 were examined by whole-mount in situ hybridization (WISH) of Kaede.

The 2kpb region located upstream the transcription start site of cdc25 (KHC5.12) is no longer active in the epidermis of gastrula embryos when both GATA binding sites are mutated.
Original annotation : Kaede expression in an embryo expressing pSP-cdc25( 2010 bp)>Kaede with mutations in the two GATA-binding sites was examined by WISH. Note that Kaede expression in the epidermis (Ep) was reduced in mGATA BS12. Ne, neural ectoderm; Mu, muscle.
This construct corresponds to the last line of the chart, where GATA, AP2-2 and Sox binding sites are respectively symbolized by red, blue and brown squares.

The 2.1kpb region located upstream the transcription start site of cdc25 (KHC5.12) is still active in the epidermis of gastrula embryos when lacking both GATA binding sites.
Original annotation : In the background with mutations in the two GATA-binding sites in CRM1, effects of mutating or deleting the AP-2-
This construct corresponds to the upper line of the chart, where GATA, AP2-2 and Sox binding sites are respectively symbolized by red, blue and brown squares.sites in the CRM2 were examined by WISH of Kaede.

At mid gastrula stage, epidermis activity of the cis-regulatory module CRM2 of cdc25 (KHC5.12) is strongly reduced when the first AP-2 binding site is deleted.
Original annotation : In the background with mutations in the two GATA-binding sites in CRM1, effects of deleting the AP-2- sites in the CRM2 were examined by WISH of Kaede.
This construct corresponds to the 5th line of the chart, where GATA, AP2-2 and Sox binding sites are respectively symbolized by red, blue and brown squares.

At mid gastrula stage, epidermis activity of the cis-regulatory module CRM2 of cdc25 (KHC5.12) is strongly reduced when the second AP-2 binding site is mutated.
Original annotation : In the background with mutations in the two GATA-binding sites in CRM1, effects of deleting the AP-2 binding sites in the CRM2 were examined by WISH of Kaede.
This construct corresponds to the 6th line of the chart, where GATA, AP2-2 and Sox binding sites are respectively symbolized by red, blue and brown squares.

At mid gastrula stage, epidermis activity of the cis-regulatory module CRM2 of cdc25 (KHC5.12) is no longer observed when all Sox binding sites are mutated.
Original annotation : In the background with mutations in the two GATA-binding sites in CRM1, effects of deleting the Sox-binding sites in the CRM2 were examined by WISH of Kaede. Kaede expression was not observed when the three Sox-binding sites in the CRM2 were mutated. Ep, epidermis; Ne, neural ectoderm; Mu, muscle. The embryos were imaged from the ventral side with the anterior end toward the top.
This construct corresponds to the last line of the chart, where GATA, AP2-2 and Sox binding sites are respectively symbolized by red, blue and brown squares.

At mid gastrula stage, epidermis activity of the cis-regulatory module CRM2 of cdc25 (KHC5.12) is strongly reduced when the first AP-2 binding site is mutated.
Original annotation : In the background with mutations in the two GATA-binding sites in CRM1, effects of mutating the AP-2- sites in the CRM2 were examined by WISH of Kaede.
This construct corresponds to the 2nd line of the chart, where GATA, AP2-2 and Sox binding sites are respectively symbolized by red, blue and brown squares.

At mid gastrula stage, epidermis activity of the cis-regulatory module CRM2 of cdc25 (KHC5.12) is reduced when the second AP-2 binding site is deleted.
Original annotation : In the background with mutations in the two GATA-binding sites in CRM1, effects of mutating the AP-2- sites in the CRM2 were examined by WISH of Kaede.
This construct corresponds to the 3rd line of the chart, where GATA, AP2-2 and Sox binding sites are respectively symbolized by red, blue and brown squares.

At mid gastrula stage, epidermis activity of the cis-regulatory module CRM2 of cdc25 (KHC5.12) is strongly reduced when both AP-2 binding site are mutated.
Original annotation : In the background with mutations in the two GATA-binding sites in CRM1, effects of mutating the AP-2- sites in the CRM2 were examined by WISH of Kaede.
This construct corresponds to the 4th line of the chart, where GATA, AP2-2 and Sox binding sites are respectively symbolized by red, blue and brown squares.

Expression of cdc25 at 6.0 hour after fertilization, animal view.

Conversion to Aniseed format of Ghost database entry from Fujiwara S et al., Mech Dev. 2002; 112Gene name: 115-127. Original annotation: . Expression pattern: Epidermis

Animal view of a late gastrula embryo probed for cdc25 expression (pseudocolored in magenta). Nuclei are shown by DAPI (green)
Original annotation : Ci-cdc25 expression (pseudocolored in magenta) during gastrulation examined by FISH. Nuclei are shown by DAPI (green). The images show the ventral side of the embryos, and anterior is toward the right. The dotted box represents the region projected along the D-V axis to obtain the cross section (lower panel). In the graph, he expression of Ci-cdc25 along the A-P axis was quantified from the boxed portions of the images. Normalized fluorescence intensities averaged along the D-V axis are shown for different embryos as line plots of different colors. The black-line plots represent the intensity quantified in the picture. a.u., arbitrary units. (D00) The fluorescence intensity in the picture is represented as a heatmap (3rd image) : in the lower images are the sagittal sections at the midline of the upper images.

Animal view of a late gastrula embryo probed for cdc25 expression.
Original annotation : Ci-cdc25 expression was examined by WISH in wild-type controls. Embryos were imaged from the ventral side with the anterior end toward the top. Scale bars, 50 mm.

Animal view of a late gastrula embryo probed for cdc25 expression.
Original annotation :Ci-cdc25 expression was examined by WISH in the wild-type controls. Embryos were imaged from the ventral side with the anterior end toward the top.

Animal view of a late gastrula embryo probed for cdc25 expression (pseudocolored in magenta). Nuclei are shown by DAPI (green).
Original annotation : Expression of cdc25 was examined at 6.0 hpf by FISH. The embryos were imaged from the ventral side with the anterior end toward the right.
The graph shows quantified expression level : The expression of cdc25 along the A-P axis was quantified from the image. Normalized fluorescence intensities averaged along the D-V axis are shown for different embryos as line plots of different colors. The black-line plots represent the intensity quantified in the FISH image (a.u., arbitrary units). The encart shows the sagittal section at the midline of the image.

Expression of cdc25 at 6.5 hour after fertilization, lateral view.

Expression of cdc25 at 7.0 hour after fertilization, lateral view. Expression was very weak.

Expression of cdc25 at 7.5 hour after fertilization, lateral view. Me, mesenchyme; np, neural plate.

Expression of cdc25 at 8.0 hour after fertilization, lateral view. Me, mesenchyme; np, neural plate.

Conversion to Aniseed format of Ghost database entry from Satou Y et al., Development 2001; 128:2893-2904. Original annotation: the entire body was stained very weakly.. Expression pattern: not clear

Expression of cdc25 at 8.5 hour after fertilization, lateral view. Weak expression was detected in the epidermis.

Expression of cdc25 at 9.0 hour after fertilization, lateral view. Weak expression was detected in the epidermis.

Conversion to Aniseed format of Ghost database entry from Satou Y et al., Development 2001; 128:2893-2904. Original annotation: the entire body was stained very weakly.. Expression pattern: not clear

Conversion to Aniseed format of Ghost database entry from Satou Y et al., Development 2001; 128:2893-2904. Original annotation: the entire body was stained very weakly.. Expression pattern: not clear

Conversion to Aniseed format of Ghost database entry from Satou Y et al., Development 2001; 128:2893-2904. Original annotation: the entire body was stained very weakly.. 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: not detected