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 Satous gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :Otx.
Expression pattern : Unlocalized signal was detected.
Comment : Maternal weak signal was observed ubiquitously mainly in the early claeving stages.

Aniseed annotation :
No picture was detected on Ghost

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 :Otx.
Expression pattern : No distinct zygotic signal was detected.
Comment : Maternal weak signal was observed ubiquitously mainly in the early claeving stages.

Animal views with anterior to the top of a 32-cell stage embryo stained with Otx. Anterior ectoderm is marked in white, posterior ectoderm in yellow and gene expression in blue. Expression was observed in the a6.5, b6.5 and B6.4 cell pairs.

Vegetal (left) and animal (right) views of a WT Ciona robusta probed for Otx expression at the early 32-cell stage.
Original comment: Otx is expressed in three pairs of vegetal cells (B6.1, B6.2, and B6.4) and two pairs of animal cells (a6.5 and b6.5) at the 32-cell stage in normal embryos.

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 :Otx.
Expression pattern : B6.1, B6.2, B6.4.

Aniseed annotation :
This corresponds to the early 32-cell stage morphology and expression profile, hence the stage change compared to the original annotation.

Animal view of a 32 cell stage embryo showing Ci-otx expression in B6.2 and B6.4 cell pairs. B6.1 expression can be seen through the embryo due to its transparency.

Animal view of Otx expression at 32-cell stage.
Otx is expressed in a6.5 and b6.5 neural precursors (black arrowheads) and in B6.4 and B6.2 vegetal blastomeres (arrows).
All experiments from this article where performed on Type A Ciona intestinalis embryos.

Otx is expressed specifically in the neural lineage of embryos injected with a control morpholino oligonucleotide.

Animal view of a 32-cell stage embryo stained with Otx.
Expression was observed in the a6.5, a6.7 and b6.5 (black arrowheads) as well as in the B6.2 and B6.4 cell pairs (blue arrowheads: vegetal cells).

Vegetal view of a 32-cell stage embryo labeled with a-element of Otx>LacZ driven by Bachyury basal promoter. Expression was observed in a6.5 and b6.5 cell pairs (blach arrowheads: endogenous Otx expression), in a6.8, b6.6 and b6.7 cell pairs (red arrowheads: ectopic expression) as well in the B6.2 cell pair (blue arrowheads: vegetal cells).

Animal view of a WT Ciona robusta 32-cell stage embryo probed for Otx expression.
Otx is expressed in a6.5, B6.4 and b6.5 cell pairs.

Vegetal view of a late 32-cell stage embryo. Ci-Otx expression appears in B6.1, B6.2 and B6.4 blastomeres.

Vegetal view of a late 32-cell stage embryo, showing expression of Ci-Otx.

Animal view of a late 32 cell stage embryo showing Ci-otx expression in B6.2 and B6.4 cell pairs. B6.1 expression can be seen through the embryo due to its transparency. Expression also detected in a6.5 and b6.5 cell pairs.

Animal view (anterior is up) of an Xgal staining of late 32-cell stage Ciona intestinalis embryo electroporated with the indicated Otx cis regulatory region. No LacZ protein activity is detected eventhough lacZ mRNA is detected at the same stage in the accompanying picture.

Animal view (anterior is up) of a late 32-cell stage embryo stained for LacZ mRNA by in situ hybridization. Note that expression of the transgene is mosaic. Expression in B6.1 is driven by the construct but cannot be seen on this picture.
LacZ mRNA is present at this stage but LacZ protein has not yet been synthesized (see accompanying Xgal stained embryo)

Animal view of a 32-cell stage embryo stained LacZ reporter gene of the Otx[SBE-a]>LacZ construct containing two putative Smad binding element and one binding element for Smad4 as well as the a-element of Otx.
Expression was observed in the b6.5 cell pair (black arrowheads: endogenous Otx expression) as well as in the B6.2 and B6.4 cell pairs (blue arrowheads: vegetal cells).

A 44-cell stage wild type embryo probed for Ci-otx expression.

Original Annotation : Otx was expressed in a6.5 (arrowheads), b6.5 and B6.4.

Control picture shows the otx expression pattern in sibbling uninjected embryos.

The control picture shows otx expression in a sibbling uninjected embryo.

Animal explant (b4.2) of a 44-cell stage embryo, treated with 100ng/ml bFGF from the 8- to 44-cell stage and probed for Ci-otx expression in bFGF. Otx is activated in all cells.

Animal view of a 44 cell stage embryo showing Ci-otx expression in a6.5, b6.5 and B6.4 cell pairs.

Animal view of a 44-cell embryo stained for Otx mRNA by ISH. Expression is seen in the a6.5, b6.5, and B6.4 blastomeres.

View of 44-cell stage animal cap explants dissected at the 8-cell stage from embryos injected with FGF9/16/20 mRNA (AB086097) before fertilization. Satining was for Otx mRNA (AF305499). FGF9/16/20 drive expression of endogenous Otx throughout the cap. The control picture corresponds to an animal cap from a sibbling uninjected embryo.

View of 44-cell stage animal cap explants dissected at the 8-cell stage, cultured in sea water and stained for Otx mRNA (AF305499).

View of 44-cell stage animal cap explants dissected at 8-cell stage, treated with 200microg/ml of protein synthesis inhibitor (puromycin, Sigma P7255) from 8-cell stage and stained for Otx mRNA. The control corresponds to animal caps cultured in normal sea water.

a4.2 anterior animal explant of a 44-cell stage embryo probed for Ci-otx expression. The explant was cultured in sea water. Otx expression is lost, revealing a role for a vegetal inductive signal.

View of 44-cell stage animal cap explants dissected at 8-cell stage, treated with 200microg/ml of protein synthesis inhibitor (puromycin, Sigma P7255) and with 100microg/ml of bFGF (Sigma, F0291) to mimic FGF9/16/20 overexpression, from 16-cell stage and stained for Otx mRNA. Animal cap explants show puromycin independent expression of Otx mRNA. The controls show: 1) activation of otx by FGF in the absence of puromycin in sibbling caps . 2) lack of activation of otx by puromycin alone in sibbling caps. 3) lack of activation in sibbling caps cultured in normal sea water.

View of 44-cell stage animal cap explants dissected at 8-cell stage, treated with 100 ng/ml of bFGF (Sigma,F0291)to mimic FGF9/16/20 overexpression, from 16-cell and stained for Otx mRNA. The control picture corresponds to animal caps at the same stage not treated with FGF.

View of 44-cell stage animal cap explants dissected at 8-cell stage, and stained for Otx mRNA at the 44-cell stage.

b4.2 posterior animal explant of a 44-cell stage embryo probed for Ci-otx expression. The explant explant was cultured in sea water. Otx expression is lost, revealing a role for a vegetal inductive signal.

44 cell stage A4.1/B4.1 explant, Ci-otx expression is observed in the B line cells (81% explants), following the normal expression pattern observed in 44 cell stage embryos although this has not been formally confirmed. Wild type 44 cell stage embryo also included showing Ci-otx expression pattern.

44 cell stage a4.2/b4.2 explant showing loss of Ci-otx expression (94% explants). Wild type control of 44 cell stage embryo showing Ci-otx expression also included.

View of 44-cell stage animal cap explants dissected at 8-cell stage from embryos electroporated with the a-element from the cis-regulatory region of Ci-Otx (between-1541/-1417 bp upstream the first nucleotide of cDNA AF305499), injected with FGF9/16/20 mRNA (AB086097) before fertilization and stained for LacZ activity. FGF9/16/20 drives expression of the otx a-element cis-regulatory region in all animal cells. The control picture shows animal caps from sibbling embryos not injected with FGF9/16/20.

View of 44-cell stage animal cap explants taken at 8-cell stage from embryos electroporated with the a-element from the cis-regulatory region of Ci-Otx (between-1541/-1417 bp upstream the first nucleotide of Otx cDNA AF305499) and stained for LacZ activity.

View of 44-cell stage animal cap explants taken at 8-cell stage from embryos electroporated with the a-element from the cis-regulatory region of Otx (between-1541/-1417 bp upstream the first nucleotide of Otx cDNA AF305499, treated with 200 microg/ml of protein synthesis inhibitor (puromycin, Sigma P7255) from the 8-cell stage and stained for LacZ mRNA. The control picture corresponds to animal caps of similar embryos, which were not treated with FGF.

View of 44-cell stage animal cap explants dissected at 8-cell stage from embryos electroporated with the a-element from the cis-regulatory region of Ci-Otx (between-1541/-1417 bp upstream the first nucleotide of Otx cDNA AF305499), treated with 200microg/ml of protein synthesis inhibitor (puromycin, Sigma P7255) from 8-cell stage, and with 100 ng/ml of bFGF (Sigma, F0291) to mimic FGF9/16/20 from 16-cell stage and stained for LacZ mRNA. Animal cap explants show strong puromycin independent expression of LacZ mRNA. Three controls are provided: 1) animal caps treated with FGF without puromycin, 2) animal caps treated with puromycin alone, 3) animal caps cultured in plain sea water

View of 44-cell stage animal cap explants dissected at 8-cell stage from embryos electroporated with the a-element from the cis-regulatory region of Ci-Otx (between-1541/-1417 bp upstream the first nucleotide of Otx cDNA AF305499), treated with 100 ng/ml of bFGF (Sigma, F0291) to mimic FGF9/16/20 from 16-cell stage and stained for LacZ mRNA. Animal cap explants show strong expression of LacZ mRNA. Control: animal caps cultured in plain sea water

View of 44-cell stage animal cap explants taken at 8-cell stage from embryos electroporated with the a-element from the cis-regulatory region of Ci-Otx (between-1541/-1417 bp upstream the first nucleotide of Otx cDNA AF305499) and stained for LacZ mRNA.

Animal view with anterior to the top of a 64-cell stage embryo stained with Otx.

Animal view of a Ciona robusta 64-cell stage embryo probed for Otx expression and injected with a control morpholino oligo.
Otx expression is observed in a7.9, a7.10, b7.10, b7.9 and B7.7 blastomers.
n, number of embryos examined. % : Percentages of embryos with Foxa.a expression in the a-neural lineage (cyan arrowheads).

Vegetal view of a 64-cell stage wild type embryo probed for otx expression.

Original annotation :
Gene name :Otx.
Expression pattern : A7.1, A7.2, A7.5, A7.6, a7.9, a7.10, B7.1, B7.2, B7.3, B7.5, B7.7, B7.8, b7.9, b7.10

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 :Otx.
Expression pattern : A7.1, A7.2, A7.5, A7.6, a7.9, a7.10, B7.1, B7.2, B7.3, B7.5, B7.7, B7.8, b7.9, b7.10

Vgetal view of a 64-cell embryo stained for Otx mRNA by ISH. Expression is seen in the a6.5, b6.5 progeny (a7.9/10, b7.9/10), and several B-line blastomeres.

Vegetal view (anterior is up) of a 64-cell stage embryo electroporated with a construct that contains the cis-regulatory region of Ci-otx (-3541 bp upstream the first nucleotide of cDNA AF305499 to ATG in second exon) driving LacZ, and stained with Xgal. Staining in the a6.5, b6.5 and some B lineages, which do not express the transgene anymore, is due to the stability of LacZ. The weak nucler signal in the A-line indicates onset of expression. Unpublished data from V. Bertrand.

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 :Otx.
Expression pattern : A7.1, A7.2, A7.5, A7.6, B7.1, B7.2, B7.5, B7.7, B8.15, a8.17, a8.19

Vegetal view of an 110 cell stage embryo showing Ci-otx expression. Expression is found strongly throughout the endoderm precursors and weakly in mesenchyme, TVC and TLC precursors.

Vegetal view of an early gastrula stage embryo (shortly after the 110-cell stage) probed for Ci-otx expression. Anterior is up. Expression is strong throughout the endoderm, mesenchyme and brain/pharynx precursors.

Posterior animal explant (b4.2) at the early gastrula stage (shortly after 110-cell stage) probed for Ci-otx expression. This animal explant was cultured in sea water alone.

Animal explant (a4.2) at the mid gastrula stage probed for Ci-otx expression in bFGF. This animal explant was cultured in sea water alone.

Vegetal view (anterior is up) of an embryo electroporated with a construct that contains a cis-regulatory region of Ci-otx (from -271 bp upstream the first nucleotide of cDNA AF305499 to 161 bp donwstream) and stained with XGal at 110 cells stage.No staining was detected. This region may include the endogenous Ci-otx minimal promoter.

Vegetal view (anterior is up) of an embryo electroporated with a construct that contains the cis-regulatory region of Ci-otx (from -1417 bp upstream the first nucleotide of cDNA AF305499 to +161 pb downstream) and stained for Xgal protein at 110 cells stage. Expression is restricted to the vegetal and b6.5-line progeny of cells expressing otx at earlier stages.

Vegetal view (anterior is up) of a 110-cell stage embryo electroporated with a construct that contains a cis-regulatory region of Ci-otx (from -706 bp upstream the first nucleotide of cDNA AF305499 to +161 bp downstream) driving LacZ and stained with XGal. Expression is driven in the progeny of b- and B-line cells expressing Ci-otx at earlier stages. Same expression pattern as -1133+161 construct.

Vegetal view (anterior is up) of a 110-cell stage embryo electroporated with a construct that contains the cis-regulatory region of Ci-otx (-3541 bp upstream the first nucleotide of cDNA AF305499 to ATG in second exon) driving LacZ, and stained with Xgal. Staining in the a6.5, b6.5 and some B lineages, which do not express the transgene anymore, is due to the stability of LacZ.

Animal view (anterior is up) of a 110-cell stage embryo electroporated with a construct that contains the a-element of the cis regulatory region of Ci-otx (from -1541 to -1417 bp upstream of the first nucleotide of cDNA AF305499) driving the pBra minimal promoter and LacZ. Embryos were fixed at the 110-cell stage and and stained with XGal. Due to mosaic inheritance of the plasmid, embryos are stained on the right side only. Staining is restricted to the progeny of a6.5 and b6.5.

Vegetal view (anterior is up) of a 110-cell embryo electroporated with a construct that contains the cis-regulatory region of Ci-otx (from -3541 bp upstream of the first nucleotide of cDNA AF305499 to +161 bp downstream) driving LacZ, and stained with XGal. Some mosaicism is apparent. Staining in a, b and some A (notochord) and B (muscle) lines, in which the transgene is not expressed at this stage, is due to the stability of LacZ protein. Same expression pattern as -3541-ATG.

Vegetal view (anterior is up) of an embryo electroporated with a construct containing the cis-regulatory region of otx gene extending -1541 bp upstream the first nucleotide of cDNA AF305499 to +161 bp downstream. The embryo was stained with XGal at the 110-cell stage. This construct drives the same expression as -3541-ATG. Expression in the a-line most B-line and b-line is due to the remanence of the LacZ protein, the gene being off in these territories at this stage.

Vegetal view (anterior is up) of a 110-cell embryo electroporated with a construct that contains the cis-regulatory region of Ci-otx (from -2165 bp upstream of the first nucleotide of cDNA AF305499 to +161 bp downstream) driving LacZ, and stained with XGal. Some mosaicism is apparent. Staining in a, b and some A (notochord) and B (muscle) lines, in which the transgene is not expressed at this stage, is due to the stability of LacZ protein. Same expression pattern as -3541-ATG.

Vegetal view (anterior is up) of a 110-cell embryo electroporated with a construct that contains the cis-regulatory region of Ci-otx (from -1723 bp upstream of the first nucleotide of cDNA AF305499 to +161 bp downstream) driving LacZ, and stained with XGal. Some mosaicism is apparent. Staining in a, b and some A (notochord) and B (muscle) lines, in which the transgene is not expressed at this stage, is due to the stability of LacZ protein. Same expression pattern as -3541-ATG.

Vegetal view (anterior is up) of a 110-cell stage embryo electroporated with a construct that contains a cis-regulatory region of Ci-otx (from -1133 bp upstream the first nucleotide of cDNA AF305499 to +161 bp downstream) driving LacZ and stained with XGal. Expression is driven in the progeny of b- and B-line cells expressing Ci-otx at earlier stages. Same expression pattern as -1133+161 construct.

Vegetal view (anterior is up) of an embryo electroporated with a construct that contains a cis-regulatory region of Ci-otx (from -271 bp upstream the first nucleotide of cDNA AF305499 to exon 2) and stained with XGal at 110 cells stage. No staining was detected. This region may include the endogenous Ci-otx minimal promoter.

Vegetal view (anterior is up) of a 110-cell stage embryo electroporated with a construct that contains the a-pbra element of Ci-Otx (from -1541 to -1417 bp upstream the first nucleotide of cDNA AF305499 placed in front of the brachyury minimal promoter driving LacZ). Expression is detected in a6.5 and b6.5 line, plus some unclear and possibly artefactual endodermal staining.

No picture provided. Mutation of the two ETS sites in the a-element leads to a loss of a6.5-line expression as well as to a large reduction in b6.5 line activity (from 30% of embryos in a-element to less than 5% in the mutated element).

Vegetal view (anterior is up) of a 110-cell stage embryo electroporated with a construct that contains the mutated a-pbra element of Ci-Otx, with a mutated distal most ETS site, (from -1541 to -1417 bp upstream the first nucleotide of cDNA AF305499 placed in front of the brachyury minimal promoter driving LacZ). Expression is detected in a6.5 and b6.5 line, but at a much lower frequency than in the parental a-element (a6.5 line: 2% instead of 20%; b6.5 line: 15% instead of 30%).

Vegetal view (anterior is up) of a 110-cell stage embryo electroporated with a construct that contains the mutated a-pbra element of Ci-Otx, with a mutated distal most ETS site, (from -1541 to -1417 bp upstream the first nucleotide of cDNA AF305499 placed in front of the brachyury minimal promoter driving LacZ). Expression is detected in a6.5 and b6.5 line, but at a slightly lower frequency than in the parental a-element (a6.5 line: 6% instead of 20%; b6.5 line: 20% instead of 30%).

Vegetal view (anterior is up) of a 110-cell stage embryo electroporated with a construct that contains the mutated a-pbra element of Ci-Otx, in which the two proximal most GATA sites were mutated GATA to TATA, (from -1541 to -1417 bp upstream the first nucleotide of cDNA AF305499 placed in front of the brachyury minimal promoter driving LacZ). Expression is detected in a6.5 and b6.5 line, but at a much lower frequency than in the parental a-element (a6.5 line: 2% instead of 20%; b6.5 line: 20% instead of 30%).

No picture available. Embryos were electroporated with a construct that contains the mutated a-pbra element of Ci-Otx, in which the 5 distal most GATA sites were mutated GATA to TATA, placed in front of the brachyury minimal promoter driving LacZ). No Expression was detected at the 110-cell stage.

No picture available. Embryos were electroporated with a construct that contains the a-pbra element of Ci-Otx (from -1472 to -1417 bp upstream the first nucleotide of cDNA AF305499 placed in front of the brachyury minimal promoter driving NLS-LacZ. Expression was detected in a6.5 and b6.5 line at levels only slightly lower than the complete a-element (-1541/-1417).

No picture available. this construct contains a single copy of the 2 ETS sites of the otx a-lement, the minimal enhancer driving expression in a6.5, b6.5. Electroporation of the construct gives no LacZ staining.

No picture available. This construct contains a dimer of the 2 ETS sites of the otx a-element, the minimal enhancer driving expression in a6.5, b6.5. Electroporation of the construct gives LacZ staining in the a6.5 and b6.5 line as well as in most vegetal cells responsive to FGF9/16/20. Only 10-20% of embryos show staining in each tissue.

No picture available. Embryos electroporated with this construct, which harbors an internal deletion of the ETS sites in the minimal a-element (Otx -1472/-1417), show no LacZ staining.

No picture available. Embryos electroporated with this construct, which harbors a dimer of an internal deletion of the ETS sites in the minimal a-element (Otx -1472/-1417) show b6.5-line staining in around 10% of embryos.

Embryos electroporated with a construct that contains the Ci-fog cis-regulatory region from -314bp to +1bp lacking of the firs intron and mutated for these two GATA sites. GATA sites were replaced by two GATA sites from the neural a-element of Ci-otx (Bertrand et al., 2003). This permutation maintained early-animal-wide expression.

embryos electroporated with a construct (amEts-pfog-290 construct in the paper) containing the entire Ci-fog cis regulatory region from -290bp to the ATG. The otx neural a-element was inserted upstream of pfog with point mutation in the two Ets sites. Electroporation of this construct led to a strong animal-wide expression

Vegetal view of a Ciona robusta early gastrula stage embryo probed for Otx expression and injected with a control morpholino oligo.
Otx expression is observed in a8.17, a 8.19, b8.17 and b8.19 animal blastomers and in A7.1, A7.2, A7.5, A8.11, A8.12, B7.1, B7.2, B7.5, B7.7, B8.15 and B8.12 vegetal blastomers.
n, number of embryos examined. % : Percentages of embryos with Otx expression in the neural lineage (cyan arrowheads).

Vegetal view of a mid gastrula stage embryo probed for Ci-Otx expression.

Neural plate and lateral views of a mid gastrula stage wild type probed for Ci-OTX expression.

Dorsal view of a mid gastrula embryo showing Ci-otx expression. Figure B shows an embryo co-stained with nuclear dye syto11 to show the position of nuclei and identify cells.

Mid gastrula stage b4.2 explant, treated with bFGF, showing loss Ci-otx expression (75% explants). Wild type embryo and untreated BSA explant, assayed for Ci-otx expression, are included.

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 :Otx.
Expression pattern : nervous system.
Comment : weak expression in the muscle and other tissues, which are descendants of the blastomere expressing this gene at the early gastrula stage.

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

Dorsal view, anterior to the left, of an early neurula embryo showing Ci-otx expression. The embryo is co-stained with nuclear dye syto11 to identify cell position.

Neural plate view of a early neurula stage wild type probed for Ci-OTX expression. One picture shows the WT expression in cleaving embryos, the other, the expression in cytochalasin arrested embryos at the 64-cell stage. Expression is detected in the whole a-line neural plate as well as in the A7.4 (medial) derivatives of the neural plate.

Vegetal pole view of a cleavage arrested early neurula in which the b6-5 blastomere was ablated on the right side at the 32-cell stage and probed for Ci-OTX expression.

Control arrested sibling embryo shows the wild type pattern of the gene. Insert of the control sibling embryo shows a control cleaving embryos for Ci-OTX expression. Cell division was blocked by cytochalasin B, gene expression profile was at the equivalent of the late neurula to the stage observed.

Vegetal view of a late gastrula stage embryo probed for Ci-Otx expression in which Nodal signalling has been blocked by incubation in SB431542 which inhibits the Alk4/5/7 type 1 nodal receptor. Inhibition of Nodal signalling leads to a lateral extension of Otx which now covers the whole A-line neural plate including 47.8 derivatives. Control sibling embryo shows the wild type pattern of the gene. Insert of the control sibling embryo shows a control cleaving embryos for Ci-Otx expression. Cell division was blocked by cytochalasin B, gene expression profile corresponds to the stage observed.

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 :Otx.
Expression pattern : nervous system, epidermis.

Mid neurula stage wild type embryo probed for Ci-Otx expression.

Ci-Otx was expressed in the neural tissue precursors.

Dorsal view of a neurula stained for Otx mRNA by ISH. Expression is detected throughout the a-line neural plate and in the medial (A7.4-derived) rows of the A-line neural plate.

Dorsal view of a neurula stained for Otx mRNA by ISH. Expression is detected throughout the a-line neural plate and in the medial (A7.4-derived) rows of the A-line neural plate. The cytochalasin-treated cleavage arrested embryos demonstrate that only the progeny of A7.4 is labelled in the A-line neural plate. D: animal view; H: vegetal view.

Animal caps cultured until the mid neurula stage and probed for Otx expression. No expression was detected.

Control: Dorsal view of a neurula stained for Otx mRNA by ISH. Expression is detected throughout the a-line neural plate and in the medial (A7.4-derived) rows of the A-line neural plate.

Animal caps cultured until the mid neurula stage in the presence of 2 micromolar of the MEK inhibitor PD184352 and probed for Otx expression. No expression was detected.

Control 1: Animal caps cultured until the mid neurula stage and probed for Otx expression. No expression was detected.

Control 2: Dorsal view of a neurula stained for Otx mRNA by ISH. Expression is detected throughout the a-line neural plate and in the medial (A7.4-derived) rows of the A-line neural plate.

Animal caps cultured until the mid neurula stage in the presence of 100ng/ml bFGF and probed for Otx expression. expression was induced throughout the animal explant, indicating that otx is a target of FGF.

Control 1: Animal caps cultured until the mid neurula stage and probed for Otx expression. No expression was detected.

Control 2: Dorsal view of a neurula stained for Otx mRNA by ISH. Expression is detected throughout the a-line neural plate and in the medial (A7.4-derived) rows of the A-line neural plate.

Late neurula embryos showing Ci-otx expression in a line neural plate and anterior epidermis. Figures A. and B. dorsal view and figure C. lateral view.

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 :Otx.
Expression pattern : epidermis.

Two-color fluorescence WISH was performed at the early tailbud stage. Otx expression is shown in red, Pax2/5/8 in green.

Original annotation:
Ci-Otx is expressed in the sensory vesicle, neurohypophysis primordium, palps and pharynx. Ci-Pax2/5/8a is expressed just posterior to the Ci-Otx expression domain.

Two-color-fluorescence WISH was performed at the early tailbud stage. Otx expression is shown in red, Hox1 in green.

Original annotation:
Ci-Otx is expressed in sensory vesicle, palps, neurohypophysis primordium and pharynx. The first domain of Ci-Hox1 is also just behind the Ci-Otx domain without any gap.

Two-color-fluorescence WISH was performed at the early tailbud stage (named stage Tr:Ta=1:1 in the paper). Otx expression is shown in red, Ci-En in green.

Original annotation:
Ci-Otx is expressed in sensory vesicle, palps, neurohypophysis primordium and pharynx. The posterior boundaries of the first Ci-En domain and the Ci-Otx domain coincide exactly.

Dorsal view of an early tailbud wild type embryo probed for Otx expression.
Red and yellow arrowheads indicate boundaries between the neck and the visceral ganglion, and between the visceral ganglion and the caudal nerve cord, respectively.

Original Annotation :
Otx was expressed in A11.64 and A11.63 cell pairs ( posterior sensory vesicle).

Aniseed Annotation :
According to the picture, it seems that there is also expression in the anterior sensory vesicle.

Dorsal view of an early tailbud stage wild type embryo probed for Ci-otx expression.
The insert shows in red cells from the posterior sensory vesicle (PSV) and light-blue cells from the middle part of the visceral ganglion (VG). Broken white lines indicate the boundaries of the PSV/neck and the neck/VG.

Original Annotation :
Ci-otx expression was detected only in A11.64 and A11.63 cell pairs (posterior sensory vesicle) in addition to the anterior sensory vesicle.

Dorsal and lateral views, anterior to left, of mid tailbud embryo showing Ci-Otx expression in dorsal anterior epidermis, throughout the anterior sensory vesicle and part of the posterior sensory vesicle.

Lateral view of an early tailbud stage embryo probed for Otx and En expression.

Original annotation:
The anterior expression domain of Ci-En corresponds to the most posterior region of the brain that is marked by Ci-Otx.

Lateral view of an early tailbud stage embryo probed for Otx and FgfL expression.

Original annotation :
Ci-FgfL, whose product was not asigned to any vertebrate FGFs, is expressed in the middle of the Ci-Otx domain.

Posterior half (B4.1 and b4.2) explant grown to the late tailbud stage and probed for Ci-otx expression. The explants showed no expression of Ci-otx (98% negative, n=111).

Animal half (a 4.2 + b4.2) of a late tailbud stage embryo probed for Ci-otx expression in bFGF. In this case, this animal explant was cultured in sea water alone and have a rounded,disc-shape morphology.

Anterior half explant (A4.1 and a4.2) cultured to the early tailbud stage and probed for Ci-otx expression. 78% positive, n=45.The authors found that a large area was induced to express Ci-otx in a manner strikingly similar to that observed in whole embryo.

B4.1+a4.2 recombinate, grown to the early tailbud stage and probed for Ci-otx expression. Ci-otx was strongly induced in 11% (n=95) of cases with further 20% showing a small patch of expression.

This shows that the B-line has a lower anterior neural tissue inducing potential than the A-line.

A4.1 with b4.2 recombined at the 8-cell stage, left to develop until the early tailbud stage, and probed for Ci-otx expression. Strong Ci-otx was observed in 28% (n=86) of cases with a further 19% showing a small patch of expression.

This experiment shows that the b4.2 blastomere is less competent that the a4.2 one to form anterior neural tissue in response to A-line mediated neural induction.

A mid tailbud wild type probed for Ci-otx expression.

Original Annotation : Otx was expressed in the nervous system.

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

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 :Otx.
Expression pattern : epidermis.

Two-color-fluorescence WISH was performed at the mid tailbud stage. Otx expression is shown in red, Pax2/5/8a in green.

Original annotation:
Ci-Otx is expressed in sensory vesicle, neurohypophysis primordium, palps and pharynx. The expression domain of Ci-Pax2/5/8a is still adjoining the Ci-Otx domain.

Two-color-fluorescence WISH was performed at the mid tailbud stage. Otx expression is shown in red, Hox1 in green.

Original annotation:
Ci-Otx is expressed in sensory vesicle, neurohypophysis primordium, pharynx and Palps. There is a gap between the Ci-Otx domain and the first domain of Ci-Hox1.

Two-color-fluorescence WISH was performed at the mid tailbud stage (named stage Tr:Ta=1:2 in the paper). Otx expression is shown in red, En in green.

Original annotation:
Ci-Otx is expressed in sensory vesicle, neurohypophysis primordium, pharynx and palps. The expression pattern of Ci-En has dramatically altered by this stage. Ci-En is expressed in three domains aligned along A-P axis. Interestingly, none of these domains overlaps with the Otx domain.

Lateral view of a mid tailbud stage embryo probed for Ci-otx expression. expression is detected in the sensory vesicle, and anterior epidermis and oral siphon primorpdium (+palps??).

Mid tailbud stage A4.1/B4.1 explant showing weak Ci-otx expression (91% explants). Expression may represent residual expression in anterior endoderm from A4.1 derivatives as isolated B4.1 explants show no Ci-otx expression. Wild type embryo, assayed for Ci-otx expression, is included.

Animal explant (b4.2) grown to the mid tailbud stage and probed for Ci-otx expression.

Mid tailbud stage a4.2/b4.2 explant showing loss of Ci-otx expression (98% explants). Wild type embryo, assayed for Ci-otx expression, is included.

Mid tailbud stage b4.2 explant, treated with bFGF, showing absence of Ci-otx expression in 90% of explants.
Wild type embryo and untreated BSA explant controls, assayed for Ci-otx expression, are included.

Vegetal half explant at the late tailbud stage probed for otx expression. Rare expression in A4.1 derivatives (9%) may represent residual expression obscured by the neural expression in whole embryos

Animal explant (a4.2) cultured to the late tailbud stage embryo and probed for Ci-otx. No expression detected in 92% of explants.

lateral view of an embryo electroporated with a construct that contains a cis-regulatory region of Ci-otx (from -271 bp upstream the first nucleotide of cDNA AF305499 to 161 bp donwstream) and stained with XGal at the mid tailbud stage. weak staining in the mesenchyme and a few muscle cells is detected. Unpublished results from V. Bertrand.

Lateral view of a mid-tailbud embryo electroporated with pOtx -3541/+1333::NLS-LacZ and hybridised with a LacZ in situ probe. Expression is detected in part of the endoderm, in the anterior sensory vesicle, and in the palps and anterior epidermis and in part of the posterior endoderm. Unpublished data by V. Bertrand.

Lateral view of a mid-tailbud embryo electroporated with pOtx -3541/+161::NLS-LacZ and hybridised with a LacZ in situ probe. Expression is detected in the posterior endoderm and in the anterior sensory vesicle. Unpublished data by V. Bertrand.

The region extending from -271bp to exon 2 of Otx drives late expression in the palps, part of the anterior epidermis and sensory vesicle. Unpublished results from V. Bertrand.

Late tailbud embryo probed for Ci-Otx expression (in blue), Ci-Eph3 expression (in green) and Ci-En expression (in red).

Aniseed annotation:
Otx was expressed in the sensory vesicle, the visceral ganglion, the neurohypophysis primordium, and the pharynx.

Two-color-fluorescence WISH was performed at the late tailbud stage. Otx expression is shown in red, Hox1 in green.

Original annotation: Ci-Otx expression extends caudally along the ventral midline of the neural tube.

Two-color-fluorescence WISH was performed at the late tailbud stage. Otx expression is shown in red, Pax 2/5/8a in green.

Original annotation:
Ci-Otx is expressed in neurohypophysis primordium, pharynx, sensory vesicle and palps and extends caudally along the ventral midline of the neural tube. Ci-Pax 2/5/8a domain anteriorly abuts the Ci-Otx domain.

Two-color-fluorescence WISH was performed at the late tailbud stage. Otx expression is shown in red, en in green.

Original annotation:Ci-Otx is expressed in neurohypophysis primordium, pharynx, sensory vesicles and palps and extends caudally along the ventral midline of the neural tube. Expression of Ci-En was detected in three domains. The first domain is located immediately posterior to the Ci-Otx.

Lateral view, anterior to left, of late tailbud embryo showing Ci-otx expression in sensory vesicle and weaker expression in anterior epidermis

Lateral view of the anterior region of a late tailbud embryo showing otx expression in the sensory vesicle.

This reporter construct shows no activity in Ciona intestinalis late tailbuds.

Lateral view of the head of a WT Ciona robusta swimming larva probed for Otx expression.
Otx is only expressed in the sensory vesicle.

6 days post fertilization WT Ciona robusta juvenile probed for Otx expression. The second image is the magnified posterior tip of the endostyle of another embryo.
Otx is expressed in the anterior endostyle (black arrow).

WT Ciona robusta 6 days post fertilization juveniles probed for Otx expression. The second image is the magnified posterior tip of the endostyle of another embryo.
Otx is expressed in the anterior tip of the endostyle (black arrow).
Numbers indicate the proportion of juveniles showing the phenotype represented by the panel.
Scale bar: 50 µm.