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 :Tbx2/3.
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 :Tbx2/3.
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 :Tbx2/3.
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 :Tbx2/3.
Expression pattern : B6.2

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 :Tbx2/3.
Expression pattern : A7.6, B7.7

Expression of Ci-Tbx2/3 was detected by whole-mount in situ hybridization. Vegetal view of a 64-cell stage embryo (TL, Trunk Lateral cells).

Aniseed annotation : precursors of TLC is A7.6 cell pair at 64 cell stage.

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 :Tbx2/3.
Expression pattern : B7.7

Expression of Ci-Tbx2/3 was detected by whole-mount in situ hybridization. Vegetative view of a 110-cell stage embryo (TL, Trunk lateral cells; mes, mesenchyme).

Aniseed annotation : Precursors of TLC at 110 cell stage is A7.6 cell pair and precursors of mesenchyme are B7.7 and B8.5 cell pairs.

Vegetal view of a WT Ciona robusta late gastrula embryo probed for T-box containing transcription factor (Tbx2/3) expression.
Tbx2/3 is expressed in the notochord (red arrowhead), in the muscle (orange arrowhead) and in the neural plate (blue arrowhead).
Probe synthesized from an RT-PCR fragment.

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 :Tbx2/3.
Expression pattern : nervous system, epidermis

Original annotation: At the late-gastrula stage, Tbx2/3 was expressed in the entire ventral ectoderm. Note that Tbx2/3 is also expressed in the notochord. Ventral and lateral views are shown.

Expression of Ci-Tbx2/3 was detected by whole-mount in situ hybridization. Vegetative view of a late gastrula stage embryo. At the late gastrula stage, the signal was transiently seen in epidermal cells of the posterior embryonic region (epi, epidermis).

Vegetal view, anterior left, of a WT Ciona robusta late gastrula embryo electroporated with Tbx2/3(1937bp)>LacZ.
LacZ staining is observed in the notochord (red arrowhead), in tail muscles (orange arrowhead).
Original comment: Upon electroporation of a 1.9kb region spanning part of the second and third introns , the resulting embryos displayed robust notochord activity, recapitulating the onset and pattern of the endogenous gene in this domain.

Lateral view of a WT Ciona robusta mid neurula embryo probed for Tbx2/3 expression.
Tbx2/3 is expressed in the notochord (red arrowhead), in the tail nerve chord (blue arrowhead) and in the head epidermis (green arrowhead).

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 :Tbx2/3.
Expression pattern : notochord, nervous system, epidermis

Original annotation: At the neurula stage, expression of Tbx2/3 was diminished in the posterior half of the tail ectoderm, and became weaker in the rest of the ventral ectoderm. Note that Tbx2/3 is also expressed in the notochord and palps. Ventral and lateral views of the embryo are shown.
Aniseed annotation: Note that palps progenitors at mid neurula stage are: a10.69 cell pair; a10.70 cell pair; a10.77 cell pair; a10.78 cell pair; a9.36 cell pair a9.40 cell pair.

Vegetal view of a WT Ciona robusta mid neurula embryo electroporated with Tbx2/3(1937bp)>LacZ.
LacZ staining is observed in the notochord (red arrowhead) and weakly in tail muscles (orange arrowhead).
Original comment: Upon electroporation of a 1.9 kb region spanning part of the second and third introns, the resulting embryos displayed robust notochord activity, recapitulating the onset and pattern of the endogenous gene in this domain

Lateral view of a WT Ciona robusta initial tailbud embryo probed for Tbx2/3 expression.
Tbx2/3 is expressed in the notochord (red arrowhead), in the tail nerve chord (blue arrowhead) and in the head epidermis (green arrowhead).

Expression of Ci-Tbx2/3 was detected by whole-mount in situ hybridization. View of a late neurula stage embryo (br, brain; pap, palps; no, notochord).

Lateral view of a WT Ciona robusta initial tailbud embryo electroporated with Tbx2/3(1937bp)>LacZ.
LacZ staining is observed in the notochord (red arrowhead) and weakly in tail muscle (orange arrowhead).
Original comment: Upon electroporation of a 1.9 kb region spanning part of the second and third introns, the resulting embryos displayed robust notochord activity, recapitulating the onset and pattern of the endogenous gene in this domain.

Early tailbud stage wild type embryo probed for Tbx2/3 expression. No picture available in the article.

Original Annotation :
Tbx2/3 was expressed in A11.126-28 cells (lateral nerve cord).

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 :Tbx2/3.
Expression pattern : notochord, nervous system, nerve cord, epidermis

Original annotation: At the early-tailbud stage, expression of Tbx2/3 vanished completely in the ventral tail ectoderm. Note that Tbx2/3 is also expressed in the notochord, nerve cord, brain and palps. Ventral and lateral views of the embryo are shown.

Expression of Ci-Tbx2/3 was detected by whole-mount in situ hybridization. View of an early tailbud stage embryo (br, brain; nc, nerve cord; no, notochord; pap, palps).

Lateral view of a WT Ciona robusta early tailbud embryo probed for Tbx2/3 expression.
Tbx2/3 is expressed in the notochord (red arrowhead), in the anterior sensory vesicle and in the tail nerve chord (blue arrowheads) and in the head epidermis (green arrowheads).

Lateral view of a WT Ciona robusta early tailbud embryo electroporated with Tbx2/3(1937bp)>LacZ.
LacZ staining is observed in the notochord (red arrowhead)and in the head epidermis (green arrowhead).
Original comment: Upon electroporation of a 1.9 kb region spanning part of the second and third introns, the resulting embryos displayed robust notochord activity, recapitulating the onset and pattern of the endogenous gene in this domain.

Lateral view of a WT Ciona robusta mid tailbud embryo probed for Tbx2/3 expression.
Tbx2/3 is expressed in the notochord (red arrowhead), in the tail nerve chord and sensory vesicle (blue arrowheads) and in the head epidermis (green arrowheads).

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 :Tbx2/3.
Expression pattern : notochord, nervous system, nerve cord, epidermis

Expression of Ci-Tbx2/3 was detected by whole-mount in situ hybridization. View of a mid tailbud stage embryo (br, brain; pap, palps; nc, nerve cord; no, notochord).

Lateral view of a WT Ciona robusta mid tailbud embryo electroporated with Tbx2/3(2917bp updstream)>LacZ.
LacZ staining is observed in the anterior sensory vesicle (blue arrowhead), in the head epidermis (green arrowhead) and in the tail muscles (orange arrowhead).
Original comment: Constructs containing genomic fragments located 5' of the Ci-Tbx2/3 transcription start site exhibited staining only in Ci-Tbx2/3 expression domains outside of the notochord.

Lateral view of a WT Ciona robusta mid tailbud embryo electroporated with Tbx2/3(294bp)>LacZ.
LacZ staining is observed in the notochord mainly (red arrowhead), in the tail muscles (orange arrowhead) and in the head epidermis (green arrowhead).
Original comment: A 294 bp region drove consistent lacZ expression in the notochord and was enriched for T-box binding sites, containing nine sequence blocks (T1-T9) matching the generic TNNCAC Ci-Bra consensus binding site.
Schematic representation of the constructs tested to characterize the sequences controlling the activity of the Ci-Tbx2/3 notochord CRM: Red and gray boxes represent fragments exhibiting or lacking notochord expression of the lacZ reporter, respectively. Light-red boxes depict constructs with weakened notochord staining. T-box binding sites are illustrated as purple rectangles.

Lateral views of WT Ciona robusta mid tailbud embryos electroporated with Tbx2/3(294bp)>LacZ.
LacZ staining is observed in the notochord (red arrowhead) and in the tail muscles (orange arrowhead).

Lateral view of a WT Ciona robusta mid tailbud embryo electroporated with Tbx2/3(294bp)>LacZ.
LacZ staining is observed in the notochord mainly (red arrowhead), in the tail muscles (orange arrowhead) and in the head epidermis (green arrowhead).
Original comment: A 294 bp region drove consistent lacZ expression in the notochord and was enriched for T-box binding sites, containing nine sequence blocks (T1-T9) matching the generic TNNCAC Ci-Bra consensus binding site.
Schematic representation of the constructs tested to characterize the sequences controlling the activity of the Ci-Tbx2/3 notochord CRM: Red and gray boxes represent fragments exhibiting or lacking notochord expression of the lacZ reporter, respectively. Light-red boxes depict constructs with weakened notochord staining. T-box binding sites are illustrated as purple rectangles.

Lateral views of WT Ciona robusta mid tailbud embryos electroporated with Tbx2/3(294bp)>LacZ.
LacZ staining is observed in the notochord (red arrowhead) and in the tail muscles (orange arrowhead).

Lateral view of a WT Ciona robusta mid tailbud embryo probed for Tbx2/3 expression.
Tbx2/3 is expressed in the notochord (red arrowhead), in the sensory vesicle and in the tail nerve chord (blue arrowheads) and in the head epidermis (green arrowheads).

Vegetal view of a WT Ciona robusta mid tailbud embryo electroporated with Tbx2/3(1937bp)>LacZ.
LacZ staining is observed in the notochord (red arrowhead)and weakly in tail muscles (orange arrowhead).
Original comment: Upon electroporation of a 1.9 kb region spanning part of the second and third introns, the resulting embryos displayed robust notochord activity, recapitulating the onset and pattern of the endogenous gene in this domain.

Lateral view of a WT Ciona robusta mid tailbud embryo electroporated with Tbx2/3(294bp)>LacZ.
LacZ staining is observed in the notochord (red arrowhead), in tail muscle (orange arrowhead) and in the mesenchyme (purple arrowhead).
Original comment: A 294 bp region drove consistent lacZ expression in the notochord and was enriched for T-box binding sites, containing nine sequence blocks (T1-T9) matching the generic TNNCAC Ci-Bra consensus binding site.
Schematic representation of the 294 bp Ci-Tbx2/3 notochord CRM: Red boxes represent constructs with notochord activity; configurations exhibiting weak or lack of notochord activity are depicted by light-red and gray boxes, respectively. Dark purple rectangles depict T-box binding sites. Right: percentage of embryos exhibiting notochord staining when electroporated with the wild-type (WT) or T-box (T) mutant plasmids to the left of each bar.

Lateral view of a WT Ciona robusta mid tailbud embryo electroporated with Tbx2/3(294bp T-box5mut)>LacZ.
LacZ staining is observed in the notochord (red arrowhead) and in the mesenchyme (purple arrowhead).
Original comment: Obliterating either of the T-box binding sites individually did not result in a decrease in notochord activity.
Schematic representation of the 294 bp Ci-Tbx2/3 notochord CRM: Red boxes represent constructs with notochord activity; configurations exhibiting weak or lack of notochord activity are depicted by light-red and gray boxes, respectively. Dark purple rectangles depict T-box binding sites. Right: percentage of embryos exhibiting notochord staining when electroporated with the wild-type (WT) or T-box (T) mutant plasmids to the left of each bar.

Lateral view of a WT Ciona robusta mid tailbud embryo electroporated with Tbx2/3(294bp T-box6mut)>LacZ.
LacZ staining is observed in the notochord (red arrowhead) and in tail muscles (orange arrowhead).
Original comment: Obliterating either of the T-box binding sites individually did not result in a decrease in notochord activity.
Schematic representation of the 294 bp Ci-Tbx2/3 notochord CRM: Red boxes represent constructs with notochord activity; configurations exhibiting weak or lack of notochord activity are depicted by light-red and gray boxes, respectively. Dark purple rectangles depict T-box binding sites. Right: percentage of embryos exhibiting notochord staining when electroporated with the wild-type (WT) or T-box (T) mutant plasmids to the left of each bar.

Lateral view of a WT Ciona robusta mid tailbud embryo electroporated with Tbx2/3(294bp T-box7mut)>LacZ.
LacZ staining is observed in the notochord (red arrowhead), weakly in tail muscles (orange arrowhead) and in the head epidermis (green arrowhead).
Original comment: Obliterating either of the T-box binding sites individually did not result in a decrease in notochord activity.
Schematic representation of the 294 bp Ci-Tbx2/3 notochord CRM: Red boxes represent constructs with notochord activity; configurations exhibiting weak or lack of notochord activity are depicted by light-red and gray boxes, respectively. Dark purple rectangles depict T-box binding sites. Right: percentage of embryos exhibiting notochord staining when electroporated with the wild-type (WT) or T-box (T) mutant plasmids to the left of each bar.

Lateral view of a WT Ciona robusta mid tailbud embryo electroporated with Tbx2/3(294bp T-box5-6mut)>LacZ.
Weak LacZ staining is observed in tail muscles (orange arrowhead) and in the mesenchyme (purple arrowhead).
Original comment: Disruption of T6 in combination with either T5 or T7 resulted in a drastic abrogation of notochord activity.
Schematic representation of the 294 bp Ci-Tbx2/3 notochord CRM: Red boxes represent constructs with notochord activity; configurations exhibiting weak or lack of notochord activity are depicted by light-red and gray boxes, respectively. Dark purple rectangles depict T-box binding sites. Right: percentage of embryos exhibiting notochord staining when electroporated with the wild-type (WT) or T-box (T) mutant plasmids to the left of each bar.

Lateral view of a WT Ciona robusta mid tailbud embryo electroporated with Tbx2/3(294bp T-box6-7mut)>LacZ.
Strong LacZ staining is observed in the tail muscles (orange arrowhead) and in the mesenchyme (purple arrowhead).
Original comment: Disruption of T6 in combination with either T5 or T7 resulted in a drastic abrogation of notochord activity.
Schematic representation of the 294 bp Ci-Tbx2/3 notochord CRM: Red boxes represent constructs with notochord activity; configurations exhibiting weak or lack of notochord activity are depicted by light-red and gray boxes, respectively. Dark purple rectangles depict T-box binding sites. Right: percentage of embryos exhibiting notochord staining when electroporated with the wild-type (WT) or T-box (T) mutant plasmids to the left of each bar.

Lateral view of a WT Ciona robusta mid tailbud embryo electroporated with Tbx2/3(294bp T-box5-7mut)>LacZ.
Weak LacZ staining is observed in the notochord (red arrowhead) and in the tail muscles (orange arrowhead).
Original comment: Simultaneous disruption of T5 and T7 was less effective (than T6 plus T5 or T7 mutation), with 6% of embryos still exhibiting notochord staining, suggesting that the individual T-box sites are mostly, although not completely, redundant.
Schematic representation of the 294 bp Ci-Tbx2/3 notochord CRM: Red boxes represent constructs with notochord activity; configurations exhibiting weak or lack of notochord activity are depicted by light-red and gray boxes, respectively. Dark purple rectangles depict T-box binding sites. Right: percentage of embryos exhibiting notochord staining when electroporated with the wild-type (WT) or T-box (T) mutant plasmids to the left of each bar.

Lateral view of a WT Ciona robusta mid tailbud embryo electroporated with Tbx2/3(294bp T-box5-6-7mut)>LacZ.
LacZ staining is observed in the tail muscles (orange arrowhead) and in the mesenchyme (purple arrowhead).
Original comment: Any residual notochord activity was lost upon simultaneous mutation of T5, T6 and T7.
Schematic representation of the 294 bp Ci-Tbx2/3 notochord CRM: Red boxes represent constructs with notochord activity; configurations exhibiting weak or lack of notochord activity are depicted by light-red and gray boxes, respectively. Dark purple rectangles depict T-box binding sites. Right: percentage of embryos exhibiting notochord staining when electroporated with the wild-type (WT) or T-box (T) mutant plasmids to the left of each bar.

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