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 :ZicL.
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 :ZicL.
Expression pattern : No distinct zygotic signal was detected

Vegetal view (anterior is up) of a 32-cell stage embryo stained for Ci-ZicL-B mRNA by in situ hybridization.

Vegetal view of a late-32 cell stage embryo stained with ZicL expression. Red arrow point to the NN cells, blue arrow point to the E cell.
Original annotation: NN cell formation at the 32-cell stage is marked by expression of zicL.
Aniseed annotation: Expression is also observed in B6.2 and B6.4 cell pairs.

Vegetal view of a 32-cell stage embryo, electroporated with a lacZ transgene containing the -658bp of the 5' flanking Ci-ZicLB promoter sequence with mutations of the putative Fox2, Fox4 and Fox5 binding sites. The expression of lacZ in A6.2 and A6.4 was diminished but not in B6.2 and B6.4. The expression was not completely abolished even in A-line cells (arrowhead).

Vegetal view of a 32-cell stage embryo, electroporated with a lacZ transgene containing the -658bp of the 5' flanking Ci-ZicLB promoter sequence within an internal deletion was made between the -295bp to -205bp. This transgene was expressed in B6.2 and B6.4, but not in A6.2 and A6.4. This transgene lacks the two putative Fox-binding site Fox-2 and Fox-3. The region between the deletion is needed to the expression in A6.2 and A6.4 cells at the 32-cell stage.

Vegetal view of a 32-cell stage embryo, electroporated with a lacZ transgene containing the -127bp of the 5' flanking Ci-ZicLB promoter sequence (-127Z construct). The expression is detected only in the B6.4 cells.

Vegetal view of a 32-cell stage embryo, electroporated with a lacZ transgene containing the -658bp of the 5' flanking Ci-ZicLB promoter sequence (-658Z construct). The expression is detected in the A6.2, A6.4, B6.2, B6.4 cells like in the endogenous expression pattern (see control).

Vegetal view of a 32-cell stage embryo, electroporated with a lacZ transgene containing the -658bp of the 5' flanking Ci-ZicLB promoter sequence with mutation of the putative Fox-2 binding site (Fox2-mut construct). This transgene did not diminish the expression in the B6.2, B6.4 , A6.2 and A6.4 cells.

Vegetal view of a 32-cell stage embryo, electroporated with a lacZ transgene containing the -658bp of the 5' flanking Ci-ZicLB promoter sequence which contains an internal deletion between -127bp and -89bp (DeltaNP construct). The strong expression was comparable to -658bp transgene without deletion.

Vegetal view of a 32-cell stage embryo, electroporated with a lacZ transgene containing the -658bp of the 5' flanking Ci-ZicLB promoter sequence which contains an internal deletion between -205bp and -89bp (DeltaSnP construct). The expression was faint in B6.4 and not observed in B6.2. These results suggest that the region between nucleotide positions -205 and -89 is an enhancer module necessary for activation in B6.2 and B6.4.

Vegetal view of a 32-cell stage embryo, electroporated with a lac Z transgene containing the -295bp of the 5' flanking Ci-ZicLB promoter sequence (-295Z construct).

Vegetal view of a 32-cell stage embryo, electroporated with a lacZ transgene containing the -658bp of the 5' flanking Ci-ZicLB promoter sequence which contains an internal deletion between -205bp and -127bp (DeltaSnN). The expression was weak but detected not only on A6.2 and A6.4 but also in B6.2 and B6.4.

Vegetal view of a 32-cell stage embryo, electroporated with a lacZ transgene containing the -658bp of the 5' flanking Ci-ZicLB promoter sequence containing a mutation of Ets4 binding site (CGATCGGAAGTCA into CGATCAAGAGTCA)(Ets-mut construct). This transgene did not diminish the expression in the notochord/mesenchyme lineage, but resulted in an unexpected ectopic activation in the a-line presumptive ectoderm blastomeres. The ectopic expression was observed in a6.5,a6.6,a6.7, and a6.8, which give rise to the epidermis and central nervous system.

Vegetal view of a 32-cell stage embryo, electroporated with a lacZ transgene containing the -205bp of the 5' flanking Ci-ZicLB promoter sequence (-205Z construct). This transgene was expressed in B6.2 and B6.4, but not in A6.2 and A6.4. This truncated transgene lacks the two putative Fox-binding site Fox2 and Fox3.

Vegetal view of a 32-cell stage embryo, electroporated with a lacZ transgene containing the -658bp of the 5' flanking Ci-ZicLB promoter sequence (-658Z construct). The expression is detected in the A6.2, A6.4, B6.2, B6.4 cells like in the endogenous expression pattern (see control).

Vegetal view of a 32-cell stage embryo, electroporated with a lacZ transgene containing the -259bp of the 5' flanking Ci-ZicLB promoter sequence (-259Z construct). This transgene gave similar expression pattern at the 32-cell stage :A6.2, A6.4, B6.2, B6.4, as the -658Z although the enhancer activity gradually decreased as the enhancer was shortened. This transgene lacks the two putative Fox-binding sites Fox2 and Fox3.

Vegetal view of a WT Ciona robusta 32-cell stage embryo probed for Zic-r.b expression.
Zic-r.b is expressed in A6.2, A6.4, B6.2 and B6.4 blastomers.

Animal view of a WT Ciona robusta 32-cell stage embryo probed for Zic-r.b expression.
Zic-r.b is expressed in A6.2, A6.4, B6.2 and B6.4 blastomer pairs.

Animal view of a WT Ciona robusta 32-cell stage embryo probed for Zic-r.b expression.
Zic-r.b is expressed in A6.2, A6.4, B6.2 and B6.4 blastomer pairs.

Vegetal view of a 32-cell stage wild type embryo stained for Ci-ZicL mRNA by in situ hybridization.

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 :ZicL.
Expression pattern : A6.2, A6.4, B6.2, B6.4

Vegetal view of a late 32-cell stage embryo. Ci-ZicL is expressed in B6.2, B6.4, A6.2 and A6.4 blastomeres.

View of a 32-cell stage embryo, showing expression of Ci-ZicL in B6.2, B6.4, A6.2 and A6.4 blastomeres.

Original annotation : ZicL expression at the 32-cell stage in control embryos (shown in 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: not clear

Vegetal view (anterior is up)of a 44-cell stage embryo stained for Ci-ZicL-B mRNA by in situ hybridization.

Vegetal view of a 44-cell stage embryo, electroporated with a lacZ transgene containing the -658bp of the 5' flanking Ci-ZicLB promoter sequence (658Z construct). The expression is detected in the A7.4, A7.3, A7.7, A7.8, B7.3, B7.4 cells like in the endogenous expression pattern (see control). Howewer a delicate spatiotemporal difference in the amount of lacZ transcript was not detected, this was probably because lacZ mRNA was relatively stable, and because the transgene was unevenly distributed between blastomeres.

Vegetal view of a 44-cell stage embryo, electroporated with a lacZ transgene containing the -205bp of the 5' flanking Ci-ZicLB promoter sequence (-205Z construct). The expression is detected in all the daughter cells of A6.2, A6.4, B6.2 and B6.4.

Vegetal view of a WT Ciona robusta 64-cell stage embryo probed for Zic-r.b expression.
Zic-r.b is expressed in A7.4, A7.8, A7.3, A7.7, B7.3 and B7.4 blastomer pairs.

Vegetal view of a 64-cell stage wild type embryo stained for Ci-ZicL mRNA by in situ hybridization.

Vegetal view of a 64-cell stage wild type embryo stained for Ci-ZicL mRNA by in situ hybridization.

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 :ZicL.
Expression pattern : A7.3, A7.4, A7.7, A7.8, B7.3, B7.4, B7.5, B7.7, B7.8

Vegetal view of a 64-cell stage embryo showing Ci-ZicL expression.

Vegetal view of a late-64 cell stage embryo stained with ZicL expression. Red arrow point to the NN cells, blue arrow point to the E cell.
Original annotation: Expression in NN cells.
Aniseed annotation: Expression is also observed in B7.3, B7.4, B7.5, B7.7 and B7.8 cell pairs.
(+HD photo)

ZicL expression at the 64-cell stage in a control embryo (shown in anterior view, animal pole up).

Vegetal view of a 64-cell stage embryo, electroporated with a lacZ transgene containing the -205bp of the 5' flanking Ci-ZicLB promoter sequence which contains mutations of Fox4 and Fox5 (Fox 4/5-mut construct). The expression was only in B-line cells. The result suggests that Fox transcription factor(s) are necessary in the enhancer module in A-line cells but not in B-line cells.

Vegetal view (anterior is up) of a 76-cell stage embryo stained for Ci-ZicL-B mRNA by in situ hybridization (Nc, nerve cord; Mu, muscle, No, notochord, Me, mesenchyme).

Vegetal view of a 110-cell stage wild type embryo stained for Ci-ZicL mRNA by in situ hybridization.

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 :ZicL.
Expression pattern : A8.5, A8.6, A8.7, A8.8, A8.13, A8.14, A8.15, A8.16, B8.5, B8.7, B8.8, B7.5, B7.7, B8.15, B8.16, a8.17, a8.18, a8.19, a8.20, a8.21, a8.22, a8.23, a8.25, a8.26, a8.27, a8.28.
Comment : The expression in B8.5, B8.7 and B8.8 becomes weaker.

Aniseed annotation :
Expression is also seen in a8.23.

Vegetal view (anterior is up) of a 32-cell stage embryo stained for Ci-ZicL-B mRNA by in situ hybridization (Nc, nerve cord; Mu, muscle, No, notochord).

Vegetal view (anterior is up) of a 32-cell stage embryo stained for Ci-ZicL-B mRNA by in situ hybridization. Green arrowheads show the a-line presumptive CNS cells.

Vegetal view of a 110-Cell stage embryo stained for Ci-ZicL mRNA by in situ hybridization.

Aniseed Comment : update of annotation of (Imai et al.,2004), based on (Imai et al.,2006).

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

Conversion to Aniseed format of Ghost database entry from Fujiwara S et al., Mech Dev. 2002; 112Gene name: 115-127. Original annotation: . Expression pattern: not clear.

Aniseed annotation :
Annotation was completed.

Vegetal view of a 110-cell stage embryo, electroporated with a lacZ transgene containing the -658bp of the 5' flanking Ci-ZicLB promoter sequence (-658Z construct). The expression is detected in the notochord, mesenchyme, muscles and nerve cord like in the endogenous expression pattern (see control). Howewer a delicate spatiotemporal difference in the amount of lacZ transcript was not detected, this was probably because lacZ mRNA was relatively stable, and because the transgene was unevenly distributed between blastomeres (Nc, nerve cord; Mu, muscle; Me, mesenchyme; No, notochord).

Vegetal view of a 110-cell stage embryo, electroporated with a lacZ transgene containing the -205bp of the 5' flanking Ci-ZicLB promoter sequence. The expression is detected in all the daughter cells of A6.2, A6.4, B6.2 and B6.4 (control injected with -658Z construct).

Dorso-animal view of a Ciona robusta early gastrula embryo probed for Zic-r.b expression and injected into the pair of anterior animal cells of 8-cell embryos with a control MO.
Zic-r.b expression is observed in neural plate precursors (of rows I to IV) and in tail muscles.
Cyan arrowheads indicate expression in a-line cells. n, number of embryos examined. % : Percentages of embryos with loss of Zic-r.b expression in the a-line cells are shown.

At the early gastrula stage, ZicL is expressed in the precursors of rows III and IV of the a-neural plate, in the muscles and in the A-neural plate precursors (rows I and II).

At the early gastrula stage, ZicL is expressed in the precursors of rows III and IV of the neural plate, and in the muscles.
Original annotation : The expression of ZicL in control embryos at the early gastrula stage (shown in animal view). The expression is illustrated schematically in the lower right by red (palps) and blue (brain).

Batch of WT Ciona robusta mid gastrula stage embryos probed for Zic-r.b expression.
Zic-r.b is expressed in the ectoderm (neural plate rows I, III IV and b- neural plate).

Batch of control Ciona robusta mid gastrula stage embryos probed for Zic-r.b expression and electroporated with pFT>Tomato reporter construct.
Zic-r.b is expressed in the ectoderm (neural plate rows I, III IV and b- neural plate).

Batch of WT Ciona robusta mid gastrula stage embryos probed for Zic-r.b expression.
Zic-r.b is expressed in the ectoderm (neural plate rows I, III IV and b- neural plate).

Batch of control Ciona robusta mid gastrula stage embryos probed for Zic-r.b expression and electroporated with pFT>Tomato reporter construct.
Zic-r.b is expressed in the ectoderm (neural plate rows I, III IV and b- neural plate).

Late gastrula stage wild type embryo probed for ZicL expression. No picture available in the article.

Original Annotation :
Ci-ets/pointed2 was expressed in A9.15, A9.16, A9.29, b9.37 and b9.38 cell pairs.

Vegetal view of a mid gastrula stage embryo showing Ci-ZicL expression. Embryos were treated with DMSO from the 16-cell stage.

Vegetal view (anterior is up) of a mid-gastrula embryo stained for Ci-ZicL-B mRNA by in situ hybridization.

Vegetal view of a mid gastrula WT embryo co-electroporated with Dmrt>H2B:CFP (blue, nuclei) and ZicL>mCherry:caax (magenta, plasma membrane).
Original annotation : Rows V and VI express Dmrt exclusively and contribute to the palps, OSP, and peripheral nervous system. ZicL and Dmrt reporters co-localize in rows III and IV, which give rise to the anterior sensory vesicle. Rows I and II express ZicL exclusively and contribute to posterior brain, motor ganglion, and tail nerve cord. Scale bars in (C, D)1⁄450 μm.

Vegetal view of a mid gastrula WT embryo probed for six3/6 expression (red) and electroporated with ZicL>YFP-caax (white, plasma membrane-localized). Signal is detected in neural plate rows I, II, III and IV.

Vegetal view of a mid gastrula WT embryo probed for Myt1L (red) and Tyrp1A (green) expression and electroporated with ZicL>YFP-caax (white, plasma membrane-localized). Signal is detected in neural plate rows I, II, III and IV.

ZicL>YFP:caax is active in neural plate rows I, II, III and IV at the mid gastrula stage.

ZicL>YFP:caax is active in neural plate rows I, II, III and IV at the mid gastrula stage.

Vegetal view of a mid gastrula embryo probed for Six3/6 expression (red) and co-electroporated with Dmrt>H2B:YFP (green) and ZicL>LacZ (white). LacZ signal is detected in neural plate rows I, II, III and IV, and in the primary muscle lineage.

Vegetal view of a late gastrula embryo probed for Elk1/3/4 expression (red), co-electroporated with Dmrt>YFP:caax (green) and ZicL>LacZ (white).
LacZ signal is detected in neural plate rows I, II, III and IV, and in the primary muscle lineage.

Aniseed Comment : update of annotation of (Imai et al.,2004), based on (Imai et al.,2005).

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 :ZicL.
Expression pattern : nervous system.
Comment : A9.13, A9.14, A9.15, A9.16, A9.29, A9.30, A9.32, a9.33, a9.34, a9.37, a9.38, a9.49, a9.50, b9.34, b9.37, b9.38.

Vegetal view of a late gastrula stage embryo showing Ci-ZicL expression.

Vegetal view of a late gastrula stage wild type embryo probed for ZicL expression. The expression of Ci-ZicL was detected is b9.33, b9.37, b9.38, A9.29, A9.15, a9.49, a9.33, a9.37, a9.50, a9.4, a9.38 cells.

Aniseed Comment : update of annotation of (Imai et al.,2004), based on (Imai et al.,2006).

Aniseed Comment : update of annotation of (Imai et al.,2004), based on (Imai et al.,2006).

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

Vegetal view of a late gastrula embryo probed for Ets1/2 expression (red), co-electroporated with Dmrt>YFP:caax (green) and ZicL>LacZ (white).
LacZ signal is detected in neural plate rows I, II, III and IV, and in the primary muscle lineage.

Early neurula electroporated with ZicL>GFP (yellow) and probed for Six1/2 (pink) and Eya (blue) expression.
Original annotation : Dorsal view of an embryo at early neurula stage electroporated with ZicL>GFP and hybridized with Six1/2 and Eya mRNA probes. The neural plate is marked by ZicL.

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 :ZicL.
Expression pattern : nervous system

Aniseed annotation :
It seems that expression is localized in a-line neural plate.

Original annotation : Dorsal view of an initial tailbud embryo co-electroporated with Six1/2GFP (green) and ZicL>CFP (yellow). Brackets indicate the derivatives of the annotated lineages. Anterior is to the left.

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 :ZicL.
Expression pattern : No distinct zygotic signal was detected

Views of mid tailbud stage embryos showing reporter expression of the region upstream of PTFb, drived by the fog promoter in mesenchyme.

Views of mid tailbud stage embryos showing reporter expression of the region upstream of PTFb in notochord.

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 :ZicL.
Expression pattern : No distinct zygotic signal was detected

Lateral view of a late tailbud embryo co-electroporated with Dmrt>H2B:CFP (blue, nuclei) and ZicL>mCherry:caax (magenta, plasma membrane).
Original annotation : Colored linesindicate territories derived from neural plate rows. Rows V and VI express Dmrt exclusively and contribute to the palps, OSP, and peripheral nervous system. ZicL and Dmrt reporters co-localize in rows III and IV, which give rise to the anterior sensory vesicle. Rows I and II express ZicL exclusively and contribute to posterior brain, motor ganglion, and tail nerve cord. Scale bar 1⁄450 μm.

Views of larvae showing reporter expression of the region upstream of PTFb drived by the fog promoter in mesenchyme, muscle and endoderm.

Views of larvae showing reporter expression of the region upstream of PTFb in notochord.