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

Vegetal view (dorsal is up) of a 32-cell stage wild type embryo stained for Ci-Bra 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 :Brachyury.
Expression pattern : No distinct zygotic signal was detected

Vegetal view of a WT Ciona robusta late 32-cell stage embryo probed for brachyury expression.
No expression is observed.

Anterior view (animal to the top) of a WT Ciona robusta 64-cell stage embryo probed for Brachyury expression and injected with a control MO.
Brachyury expression is observed in the notochord progenitors A7.3 and A7.7 blastomers (black arrowheads).
n, number of embryos examined. % : Percentages of embryos with ectopic expression

Vegetal view of a WT Ciona robusta 64-cell stage embryo probed for Brachyury expression.
Brachyury is expressed in A7.3 and A7.7 blastomers.

Vegetal view of a WT Ciona robusta 64-cell stage embryo probed for Brachyury expression.
Brachyury is expressed in B7.3, A7.3 and A7.7 blastomers.

Anterior view, animal pole up of a WT Ciona robusta 32-cell stage embryo probed for Brachyury expression.
Brachyury is expressed in A7.3, B7.3 and A7.7 blastomer pairs.

64-cell stage wild type embryo probed for Ci-brachyury expression.

Ci-Bra was expressed in the notochord precursors.

Vegetal view of a 64-cell stage wild type probed for Ci-brachyury expression.

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 :Brachyury.
Expression pattern : A7.3, A7.7

Vegetal view of a 64-cell stage embryo probed for Ci-Bra expression. Expression was detected in the both early and late markers of notochord.

Vegetal view of a 64 cell stage embryo probed for Ci-Bra expression. Staining is observed in the notochord precursors.

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

Vegetal view of a 64-cell stage embryo stained with bra expression. Expression observed in A7.3 and A7.7 cell pairs.

Vegetal view of a WT Ciona robusta 64-cell stage embryo probed for brachyury expression.
Expression is observed in A7.3, A7.7 and B7.7 blastomere pairs.
In schematic representation of gene expression, notochord precursors are shown in red and nerve cord precursors in blue. Blastomeres with gene expression are marked by dots.

Vegetal view of a 64 cell stage embryo probed for ci-bra expression.

Vegetal view anterior is up of 64 cell-stage embryos electroporated with the Brachyury cis-regulatory region (3.5 Kb upstream the TATA box of brachyury gene ci0100134430). The reporter reproduced the spatial expression pattern of endogenous Ci-Bra at the 64-cell stage. Some mosaicism is apparent on the pictures.

Vegetal view of 64 cell-stage embryos electroporated with the Brachyury cis-regulatory region (483bp upstream the TSS of brachyury gene ci0100134430). The 483bp region possesses minimal notochord enhancer activity.

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

Vegetal view (dorsal is up) of a 110-cell stage wild type embryo stained for Ci-Bra mRNA by in situ hybridization.

Original annotation :
Gene name :Brachyury.
Expression pattern : A8.5, A8.6, A8.13, A8.14, B8.6

Vegetal view of a 110-cell stage wild type embryo probed for Ci-Bra expression. This picture shows expression in the primary and secondary notochord precursors.

Vegetal view of a 110-cell stage wild type probed for Ci-brachyury expression.

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 :Brachyury.
Expression pattern : A8.5, A8.6, A8.13, A8.14, B8.6

Vegetal pole view of a 110-cell stage wild type embryo probed for Ci-Bra expression. This picture shows expression in the primary and secondary notochord precursors.

Vegetal view of a 110 cell stage embryo probed for Ci-Bra expression. Staining is observed in the notochord precursors.

110-cell stage wild type embryo probed for Ci-Bra expression. GFP mRNA was injected as a control in A4.1 right hand side.

110-cell stage wild type embryo probed for Ci-Bra expression. GFP mRNA was injected as a control in B4.1 on the right hand side.

Vegetal view of a 110 cell stage embryo probed for Ci-Bra expression. Staining is observed in the notochord precursors.

Vegetal view of a 110 cell stage embryo probed for Ci-bra expression. Staining is detected in A8.5, A8.6, A8.13, A8.14 and B8.6 cell pairs.

Vegetal view of a 110-cell stage embryo showing Brachyury expression.

Vegetal pole view of a 110-cell stage wild type embryo probed for Ci-Bra expression. Ablation of A6.3 on the left-hand side leads to the loss of Ci-Bra expression in the secondary notochord precursor on the ablated side.

Vegetal ple view of a 110-cell stage wild type embryo probed for Ci-Bra expresion. Ablation of b5.3 on the right hand side leads to an inhibition of Ci-Bra expression in the secondary notochord precursor on the ablated side.

Expression of Brachyury in WT stage 10 embryos

Vegetal view of a WT Ciona robusta 112-cell stage embryo probed for brachyury expression.
Expression is observed in A8.5, A8.6, A8.13, A8.14 and B8.6 blastomere pairs.
In schematic representation of gene expression, notochord precursors are shown in red and nerve cord precursors in blue. Blastomeres with gene expression are marked by dots.

Vegetal view (anterior is up) of 110-cell stage embryo stained for Brachyury mRNA (AV955122).

Expression of Brachyury in an uninjected sibbling embryo. No picture available in the article.

Vegetal view of a 110-cell stage embryo probed for Ci-bra expression.

Expression of a notochord-marker, Ci-Bra, analysed at the 110-cell stage by in situ hybridisation in partial embryo derived from isolated cells (A6.2 and A6.4 blastomeres).

Original annotation : according to the cell lineage, each of A6.2 and A6.4 blastomeres should generate two notochord and two neural precursors at the 110-cell stage. When isolated shortly before their division (at the late 32-cell stage), these cells showed equivalent fate attribution, expressing Ci-Bra in an average of 2.0. By contrast, when they were isolated early in the cell cycle (at the 24-cell stage), the resultant partial embryos had an average of 3.2 cells positive for Ci-Bra.

Expression of a notochord-marker, Ci-Bra, analysed at the 110-cell stage by in situ hybridisation in partial embryo derived from isolated cells (A6.2 and A6.4 blastomeres).

Original annotation : According to the cell lineage, each of A6.2 and A6.4 blastomeres should generate two notochord and two neural precursors at the 110-cell stage. When isolated shortly before their division (at the late 32-cell stage), these cells showed equivalent fate attribution, expressing Ci-Bra in an average of 2.0. By contrast, when they were isolated early in the cell cycle (at the 24-cell stage), the resultant partial embryos had an average of 3.2 cells positive for Ci-Bra.

Expression of Ci-Bra, analysed at the 110-cell stage by in situ hybridisation in partial embryo derived from isolated cells (A6.2 and A6.4 blastomeres).

Original annotation : according to the cell lineage, each of A6.2 and A6.4 blastomeres should generate two notochord and two neural precursors at the 110-cell stage. When isolated at the late 32-cell stage, these cells showed equivalent fate attribution, expressing Ci-Bra in an average of 1.6 cells (42 samples analyzed).

Expression of Ci-Bra, analysed at the 110-cell stage by in situ hybridisation in partial embryo derived from isolated cells (A6.2 and A6.4 blastomeres) following injection of ephrinAd-MO.

Original annotation : according to the cell lineage, each of A6.2 and A6.4 blastomeres should generate two notochord and two neural precursors at the 110-cell stage. When isolated at the late 32-cell stage and after injection of ephrinAd-MO, the resultant partial embryos produce more notochord cells (2.9 cells) at the expense of neural cells (0.5 cells).

Expression of a notochord-marker, Ci-Bra analysed at the 110-cell stage by in situ hybridisation in partial embryo derived from isolated cells (A4.1 blastomeres).

Original annotation: The number of cells expressing Ci-Bra in the A4.1-derived partial embryos was significantly higher than that predicted from the cell lineage (6.4 versus 4).

Expression of a notochord-marker, Ci-Bra, analysed at the 110-cell stage by in situ hybridisation in partial embryo derived from co-isolated cells (A4.1 and a4.2 blastomeres).

Original annotation : the average number of Ci-Bra positive cells at the equivalent of the 110-cell stage was reduced to 3.2 cells.

Vegetal view (anterior is up) of 110 cell-stage embryos electroporated with a Ci-Brachyury cis-regulatory region (from 3.5 Kb upstream the TATA box of brachyury gene ci0100134430 to the 18th codon) driving LacZ, and processed by ISH. The reporter reproduces the notochord-specific spatial expression pattern of endogenous Ci-Bra at the 110-cell stage.

Vegetal view (anterior is up) of 110-cell stage embryos electroporated with the Brachyury cis-regulatory region (3.5 Kb upstream the TSS of brachyury gene ci0100134430) that is mutated in the two potential ZicL DNA binding motifs (B1: CACAGCTGG and B2: CCAGCTGTG). Mutation in both ZicL DNA-binding motifs abolished completely reporter expression.

Vegetal view of 110 cell-stage Ciona intestinalis embryos electroporated in fertilized eggs with a short Brachyury cis-regulatory region (starting 483 bp upstream the TATA box of brachyury gene ci0100134430 and extending up the first aa). This construct has a weaked notochord enhancer activity than the sequence starting -3.5kb upstream of the TATA box, shown here as control. Expression in B-line notochord is unclear.

Vegetal view of 110 cell-stage Ciona intestinalis embryos injected with a Brachyury cis-regulatory region (483 bp upstream the TATA box of brachyury gene ci0100134430, driving LacZ) mutated for the two ZicL DNA-binding motifs. The mutations abolish reporter expression. The control picture shows the expression of the wt construct in sibbling embryos.

Embryos electroporated with a construct (ebra290 construct in the paper) containing the entire Ci-fog cis regulatory region from -290bp to the ATG. The Ci-bra enhancer covering position -470 to -62 was inserted upstream of pfog. Electroporation of this construct led to robust notochord expression.

Vegetal (left) and lateral (right) views of a Ciona robusta early gastrula embryo probed for Brachyury expression and injected with a control MO.
Brachyury expression is observed in the notochord progenitors (A8.13, A8.14, A8.6, A8.5 and B8.6 blastomers).
n, number of embryos examined. % : Percentages of embryos with ectopic expression

Vegetal view of a WT Ciona robusta early gastrula stage embryo probed for Brachyury expression.
Brachyury, a notochord marker, is expressed in A8.5, A8.6, A8.13, A8.14 and B8.6 blastomere pairs.

Vegetal view of an early gastrula embryo stained with bra expression. Red arrow points to the notochord (NN cell originally). Expression observed in A8.5, A8.6, A8.13, A8.14 and B8.6 cell pairs.
(+HD photo)

Neural plate view of a Mid gastrula stage wild type probed for Ci-brachyury expression.

Dorsal view of a Ciona robusta late gastrula embryo probed for Brachyury expression and injected with a control MO.
Brachyury expression is observed in the notochord.
n, number of embryos examined. % : Percentages of embryos with ectopic expression.

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

Neural plate view of a Late gastrula stage wild type probed for Ci-brachyury expression.

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 :Brachyury.
Expression pattern : notochord

Vegetal view of a late gastrula cell stage embryo probed for Ci-Bra expression. Staining is observed in the notochord.

Vegetal view of a late gastrula cell stage embryo probed for Ci-Bra expression. Staining is observed in the notochord.

vegetal view (dorsal is up) of a late-gastrula stage embryo stained for Ci-Bra mRNA by in situ hybridization.

WT expression of brachyury in late gastrulae, dorsal view. Expression is seen in the notochord.

Vegetal view of a late gastrula stage embryo electroporated with a construct that contains the Ci-Bra cis regulatory region from -3.5kb to the first 17 codons. Both primary and secondary notochord lineage cells are stained in the right half of the embryo. The expression in 10 of the 20 notochord precursors suggesting that the transgene was retained in 1 of the 2 blastomeres at the 2-cell stage. The stained cells are beginning to invagine into the blastocoel.

Neural plate view of a Mid neurula stage wild type probed for Ci-brachyury expression.

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 :Brachyury.
Expression pattern : notochord

Lateral view of a neurula wild type embryo probed for Ci-bra expression.

Dorsal view of a mid neurula stage embryo, electroporated with a construct containing the Ci-Bra cis regulatory region from -3.5kb to the first 17 codons. Staining is detected in the notochord lineage cells derived from one side of the embryo.

Horizontal view of a mid-neurula stage embryo stained for Ci-Bra mRNA by in situ hybridization.

WT expression of brachyury in late neurulae, dorsal view. Expression is seen in the notochord.

Dorsal view of a initial tailbud stage wild type probed for Ci-brachyury expression.

Latreal view (pic C) of an early tailbud wild type embryo probed for Ci-Bra with the GFP like reporter. The second picture is a enlarged screen of the notochord.
The Ci-Brachyury expression was in green due to GFP.

Original Annotation : Ci-Brachyury was expressed 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 :Brachyury.
Expression pattern : notochord

Sagittal view of an early tailbud stage embryo, electroporated with a construct containing Ci-Bra cis regulatory region from -3.5kp to the first 17 codons. The notochord cells are undergoing the of intercalation, which appears to proceed in an anteroposterior wave. The anterior cells are already arrayed in a single column of cells, while those in more posterior regions have not completed intercalation.

Picture 1 : Sagittal view of a early-tail bud stage embryo, electroporated with a construct containing the 3.5kb Ci-Bra promoter driving the expression of a lacZ reporter gene containing a nuclear localization signal. Intercalation is complete, the stained notochord cells are arrayed in a single column cells that run along the length of the tail.

Picture 2 : Similar to picture 1, except that there is "ectopic" staining in the mesenchyme. This expression might reflect perdurance of Ci-Bra-lacZ products activated in the B7.3 secondary lineage precursor cell, wich specifies both notochord and mesenchyme cells.

View of cleavage arrested (from 110-cell stage) of early tailbud stage embryos electroporated with the Brachyury cis-regulatory region (3.5 Kb upstream the TSS of brachyury gene ci0100134430). The reporter contruct is detected in some cases in the notochord, in other cases in notochord, muscle and mesenchyme precursors.

Lateral view of early tailbud stage embryos electroporated with the Brachyury cis-regulatory region mutated for the ZicLb2 DNA binding motif (483 Kb upstream the TSS of brachyury gene ci0100134430). Mutation in ZicLb2 DNA Binding motif reduces reporter expression.

Lateral view of an early tailbud stage embryo, electroporated with a construct containing the ci-Bra regulatory region from -300bp to the first 17 codons. The removal of the distal sna1 site results in a derepression of the reporter in tail muscle (orange arrowhead), staining is conserved in notochord (red arrowhead).

Sagittal view of an early tailbud stage embryo, electroporated with a truncated 300-bp Ci-Bra/lacz transgene which is mutated in the Su(H)1 binding site (GTGGGAA to GTGGcAA). This substitution results in reduced binding of a GST/Ci-Su(H) fusion protein and a weak staining in the tail muscles and trunk mesenchyme, but this mutagenized transgene is essentially inactive in the notochord.

Vegetal view of a cleavage-arrested 64-cell Ciona robustaembryo at 14 hours post fertilization electroporated with pTLacZ (Bra>LacZ) and assayed by histochemical methods for acetylcholinesterase (brown-stained cells), a highly specific marker of muscle differentiation in Ciona intestinalis and β-galactosidase (blue-stained cells).
Ache is expressed in the B-line muscle precursors, B7.6, B7.5, B7.8 and B7.7. LacZ staining is observed in the notochord precursors A7.3 and A7.7 and in a mesenchyme precursor B7.3.
Scale bar: 50μm.

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

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 :Brachyury.
Expression pattern : notochord

Sagittal view of a mid tailbud stage embryo stained for Ci-Bra mRNA by in situ hybridization.

Lateral view of a mid tailbud, electroporated with a construct containing the 3.5 kb Ci-Bra promoter driving the expression of a lacZ reporter gene. Half of the primary lineage and secondary lineage notochord cells are stained, although the posterior secondary lineage cells are more lightly stained than the primary lineage cells.

Lateral view of a mid tailbud, electroporated with a construct containing the 3.5 kb Ci-Bra promoter driving the expression of a lacZ reporter gene. The reporter reproduced the spatial expression pattern of endogenous Ci-Bra at tailbud.

Lateral view of mid tailbud embryos electroporated in fertilized eggs with the Brachyury cis-regulatory region (3.5 Kb upstream the TATA box of brachyury gene ci0100134430) that is mutated in the two potential ZicL DNA binding motifs (B1: CACAGCTGG (seq complementaire: CCAGCTGTG) go to CAgAcgTcG (CgAcgTcTG) and B2: CCAGCTGTG go to CgAcgTcTG ). Mutation in both ZicL DNA binding motif reduce the reporter expression compare to the non mutated 3.5Kb construct or with the two constructs mutated only in one ZicL DNA binding motif (B1 or B2). By opposition this constuct didn't abolish reporter expression as the 483 bp construct mutated in the two ZicL DNA binding motifs.

Lateral view of mid-tailbud embryos electroporated in fertilized eggs with the Brachyury cis-regulatory region (3.5 Kb upstream the TSS of brachyury gene ci0100134430) that is mutated in the ZicL-b1 DNA binding motifs (B1: CACAGCTGG (seq complementaire: CCAGCTGTG) go to CAgAcgTcG (CgAcgTcTG) ). Mutation in ZicL-b1 DNA binding site reduces expression of the reporter in notochord cells. About 53% of stained embryos still exhibit the reporter expression in most notochord cells.

Lateral view of mid tailbud embryos electroporated in fertilized eggs with the Brachyury cis-regulatory region (3.5 Kb upstream the TATA box of brachyury gene ci0100134430) that is mutated in ZicLb2 DNA binding motifs (B2: CCAGCTGTG go to CgAcgTcTG ). Mutation in ZicLb2 DNA binding motif did not induce a significante expression level decrease of reporter in notochord cells compare to the same construct mutated in ZicLb1 DNA binding motif.

Mid tailbud stage embryo electroporated with a lacZ transgene containing the -839bp Ci-Bra regulatory sequence (construct named -790bp in the publication). Staining is observed in notochord. No picture available in the paper.

Sagittal view of a mid tailbud stage embryo, electroporated with a construct containing the minimal 483bp Ci-Bra promoter driving the expression of a lacZ reporter gene. Intense staining is observed in the notochord cells (red arrow) and weak expression is also detected in the trunk mesenchyme.

Sagittal view of mid tailbud stage embryo electroporated with a construct containing the cis- regulatory region of Ci-Bra gene (-483pb upstream the TSS to the 17 first codons. LacZ is detectable in notochord (red arrowhead) and mesenchyme.

Lateral view of mid tailbud stage embryos electroporated with the Brachyury cis-regulatory region mutated for the two ZicL DNA binding motif (483 Kb upstream the TSS of brachyury gene ci0100134430). Mutation in the two ZicL DNA Binding motif abolishes reporter expression.

Lateral view of mid tailbud stage embryos electroporated in fertilized eggs with the Brachyury cis-regulatory region (483 kb upstream the TSS of brachyury gene ci0100134430). The 3,5 kb genomic DNA fragment upstream from the CiBra transcription start site contains the cis-regulatory information required for authentic Ci-Bra expression, since it mediate a precise notochord-specific expression pattern of reporter gene in transgenic embryos, while the 483bp region appeared to possess minimal enhancer activity.

Lateral view of mid tailbud stage embryo electroporated in fertilized eggs with the Brachyury cis-regulatory region (483bp upstream the TSS of brachyury gene ci0100134430). Staining is detected in notochord (black arrow) and sometimes in mesenchyme (white arrow).

Sagittal view of a mid tailbud stage embryo, electroporated with a construct containing the minimal 483bp Ci-Bra promoter driving the expression of a lacZ reporter gene containing a nuclear localization signal. Expression is restricted to the notochord.

Lateral view of a mid tailbud stage embryo electroporated with the Brachyury cis-regulatory region in which there is a spacer inserted between the sna1 site (-350/-322) and Su(H)1 site (-261/-253) (483bp upstream the TSS of brachyury gene ci0100134430). Staining is detected in all mesodermal lineage, including notochord (arrow) and tail muscles (arrowhead) and in cerebral vesicle of the CNS (open arrowhead).

Lateral view of a mid tailbud stage embryo electroporated with the Brachyury cis-regulatory region in which there is a spacer inserted between the Su(H)2 site (-246/-248) and the sna2 site (-222/-214) (483bp upstream the TSS of brachyury gene ci0100134430). Staining is detected in all mesodermal lineage and in some ependymal cells of the spinal cord (open arrowhead).

Lateral view of mid tailbud stage embryo electroporated in fertilized eggs with the Brachyury cis-regulatory region deleted for the Snail DNA binding located between -222/-214 upstream the transcription start site in which there insert the drosophila sna DNA binding site (483bp upstream the TSS of brachyury gene ci0100134430). Staining is detected in notochord that reveal cis complementation between drosophila and Ciona sna DNA binding sites.

Sagittal view of a mid tailbud stage embryo electroporated with a construct containing the minimal 483bp Ci-Bra/lacZ enhancer which contains nucleotides changes just outside of each Ci-Su(H) recognition sequence. A normal staining pattern is observed, with the levels of expression slightly higher than normal.

Lateral view of a mid tailbud stage embryo electroporated with Brachyury cis-regulatory region deleted for the Snail DNA bindind located between -330/-322 upstream the start of transcription (483bp upstream the TSS of brachyury gene ci0100134430). Staining is detected in notochord and tail muscle, sometimes in mesenchyme. Disrupting Snail DNA binding site results in lost of repression of the transgene in tail muscle.

Lateral view of a mid tailbud stage embryo electroporated with Brachyury cis-regulatory region deleted for the Snail DNA bindind site, located between -222/-214 upstream the start of transcription (483bp upstream the TSS of brachyury gene ci0100134430). Staining is detected in notochord and tail muscle, sometimes in mesenchyme. Disrupting Snail DNA binding site results in the lost of repression of the transgene in tail muscle. Comparing the two deletion in sna1 and Sna2 DNA binding sites suggest that there are both required for efficient repression.

Lateral view of mid tailbud stage embryos electroporated with the Brachyury cis-regulatory region mutated for the ZicLb1 DNA binding motif (483 Kb upstream the TSS of brachyury gene ci0100134430) . Mutation in ZicLb1 DNA Binding motif abolishes reporter expression.

mid tailbud stage embryo electroporated with a lacZ transgene containing the -363bp Ci-Bra regulatory sequence (construct named -299bp in the publication). Ectopic staining is observed in tail muscles. No picture available in the paper.

mid tailbud stage embryo electroporated with a lacZ transgene containing the -324bp Ci-Bra regulatory sequence (construct named -275bp in the publication). Ectopic staining is observed in tail muscles. No picture available in the paper.

Lateral view of a mid tailbud stage embryo electroporated with Brachyury cis-regulatory region (300bp upstream the TSS of brachyury gene ci0100134430). Staining is detected in notochord, mesenchyme and tail muscle (due to deletion of the first sna DNA binding site upstream -300bp).

Lateral view of mid-tailbud stage embryo electroporated with the Ci-Bra cis regulatory region from -300bp to the 17 first codons in which a drosophila Sna binding site (CACCTTGCTGGG) was inserted in 5 of the su(H)1 site. Staining is detected in notochord that reveal cis complementation between drosophila and Ciona sna DNA binding sites.

Picture 1 : Lateral view of a mid tailbud stage embryo electroporated with a lacZ transgene containing the -299bp Ci-Bra regulatory sequence (construct named -250bp in the publication). Strong staining is observed in the notochord. Ectopic staining is observed in tail muscles (arrow).

Picture 2 : The same transgene as the one used in the picture 1 sometimes shows ectopic staining in the mesenchyme and in the tail muscles (arrow).

Lateral view of mid-tailbud stage embryo, electroporated with a lacZ transgene containing the truncated -299bp Ci-Bra promoter gene (construct named -250bp in the publication). This Ci-Bra promoter fragment lacks the distal repression element, so ectopic staining is observed in the muscles and trunk mesenchyme. Strong staining is observed in both the notochord and ectopic mesodermal tissues.

Lateral view of mid-tailbud stage embryo electroporated with a lacZ transgene containing the truncated -299 bp Ci-Bra promoter fragment containing an internal 24 bp deletion that removes the putative Su(H) binding sites (construct named -250bp w/o Su(H) in the publication). Staining is selectively lost in the notochord, but ectopic expression persists in the other mesodermal lineages, the tail muscles and mesenchyme.

Picture 1 : Lateral view of a late tailbud stage embryo, electroporated with a lacZ transgene containing the Ci-Bra cis regulatory region from -191 bp to the first 17 codons. This construct lacks one of the three E-box sequences. Staining is essentially lost in the notochord, but ectopic expression continues to be observed in some tissues. In this embryo, the central group of 8 muscle cells expresses the transgene.

Picture 2 : The same transgene sometimes shows ectopic expression in both tail muscles and mesenchyme.

Ectopic expression may be due to the removal of repressor binding site(s).

A view (pic D) of a late tailbud wild type embryo probed for Ci-Bra after the electroporation of Bra::GFP. The second picture is a enlarged screen of the notochord.
The Ci-Brachyury expression was in green due to GFP.

Original Annotation : Ci-Brachyury was expressed in notochord. This gene is responsible of the right intercalation of the cells of notochord to form a single cell row.

Lateral view of a Ciona robusta late tailbud embryo immunostained with the Ci-Bra-specific antibody (red), electroporated with Ci-Bra>GFP (green) and counterstained with DAPI (blue nuclei)
Ci-Bra is observed in the nuclei of the 40 notochord cells.

Tailbud stage embryo, microinjected with a transgene containing the 483bp minimal Ci-Bra enhancer attached to a GFP reporter gene. The tail wraps around the central trunk since this living embryo is contained within an intact chorion. Staining is observed in half of the primary lineage notochord cells.

Tailbud stage embryo, microinjected with a transgene containing the 483bp minimal Ci-Bra enhancer attached to a GFP reporter gene. Staining is observed in 4 of the 8 secondary lineage notochord cells.

Tailbud stage embryo, microinjected with a transgene containing the 483bp minimal Ci-Bra enhancer attached to a GFP reporter gene. Staining persists in half the notochord cells. By this time the individual notochord cells have become irregular in shape and flattened against the surrounding sheath.

Picture 1 : Sagittal view of a late tailbud stage embryo, electroporated with a lacZ transgene containing the minimal 483pb Ci-Bra enhancer. Staining obtained with the intact minimal 483pb Ci-Bra is restricted to 25% of the primary lineage notochord cells.

Picture 2 : The same transgene as that used in the picture 1 shows ectopic staining in the mesenchyme. Mesenchyme staining is often associated with strong expression in secondary lineage notochord cells. It is possible the lacZ reporter mRNA lacks a signal sequences required for asymmetric localization.

Late tail bud stage embryo, electroporated with a lacZ transgene containing the minimal -483bp Ci-Bra promoter containing an internal 24bp deletion that removes the putative Su(H) binding sites. This deletion within this transgene results in a severe reduction in the expression of the transgene (no picture available).

Late tailbud stage embryo, electroporated with a lacZ transgene containing the minimal -483bp Ci-Bra promoter containing an internal deletion that removes the three tandem repeats of a 15bp sequence. This deletion within this transgene results in a reduction in the expression of the transgene in notochord (no picture available).

Late tailbud stage embryo, electroporated with a lacZ transgene containing the -143 bp Ci-Bra promoter sequence. This construct lacks 2 of the 3 E-box sequences. No staining in the notochord is detected and there is a severe reduction in the ectopic expression in the muscles and mesenchyme. No picture is available.

Late tailbud stage embryo, electroporated with a lacZ transgene containing the Ci-Bra cis regulatory region from -71bp to the first 17 codons. This construct lacks the 3 E-box sequences. No staining is detected in the notochord and the ectopic expression in the muscles and mesenchyme which is detected when the E-box sequences are present, is absent. No picture is available.

View of a tail from tabued embryos. Original annotation: expression of Brachyury>mCherry (red) int he notochord.

Dorso-lateral view of a larva electroporated with pBra>RFP. Signal is detected in 12 to 20 cells of the notochors in 95% of the electroporated embryos.