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 :Pitx.
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 :Pitx.
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 :Pitx.
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 :Pitx.
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 :Pitx.
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 :Pitx.
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 :Pitx.
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 :Pitx.
Expression pattern : nervous system.
Comment : the most anterior part of the brain.

Aniseed annotation :
A couple of labelled cells in the stomodeum.

Lateral view of an early tailbud embryo probed for the minimal enhancer upstream of Pitx gene from -2751 to -2540, called D1abcde in the paper.

Original annotation:
Expression in the anterior neural boundary (ANB).

Aniseed annotation:
The anterior neural boundary corresponds to the neurohypophysis primordium in the ANISEED anatomical dictionary.

Lateral view of a mid-tailbud wild type embryo stained for Ci-Pitxc mRNA (which is a isoform of Ci-Pitx composed of exons3-5 and either exon2, see the first picture for probe mapping) by in situ hybridization.

Original annotation:
Ci-pitxc-specific expression in posterior neural cells (pnc, black arrowheads), left epidermis (epi, black asterisks). (anb, anterior neural boundary).

Aniseed annotation:
According to the ANISEED anatomical dictionary, the posterior neural cells corresponds to the dorsal posterior sensory vesicle.

Lateral view of a mid-tailbud wild type embryo stained for Ci-Pitx3' mRNA by in situ hybridization (see the first picture for probe mapping).

Original annotation:
Staining is observed in posterior neural cells (pnc, black arrowheads), left epidermis (epi, black asterisks) and in anterior neural boundary (anb, black arrows).

Aniseed annotation:
According to the ANISEED anatomical dictionary, the posterior neural cells corresponds to the dorsal posterior sensory vesicle and the anterior neural boundary corresponds to the neurohypophysis primordium.

Lateral view of a mid tailbud stage wild type embryo probed for Ci-pitx expression. The second picture is a detail of the cerebral vesicle region. We can see expression in three cells of the dorsal anterior sensory vesicle. One seems to belong to the ventral neural cell layer, and two other cells define the most anterior limit between the neuroectoderm and the epidermis (ep, epidermis; ne, neck; no, notochord; cv, cerebral vesicle; en, endoderm).

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 :Pitx.
Expression pattern : nervous system, epidermis.
Comment : the most anterior part of the brain; epidermis except cells along the dorsal midline

Lateral view of a mid tailbud stage probed for Pitx expression. The embryo developed within the chorion.

Original annotation :
Pitx is expressed only in the left epidermis.
Closed and open arrowheads indicate the expression of Pitx in the anterior neural boundary and the posterior sensory vesicle, respectively.

Lateral view of a mid tailbud stage probed for Pitx expression in embryos injected with control morpholino.

Original annotation :
In control MO-injected embryos, ci-Pitx expression was detected in the left epidermis as wall as anterior neural boundary (closed arrowheads) and posterior sensory vesicle (open arrowheads).

Lateral view of a mid-tailbud wild type embryo stained for Ci-Pitxa/b mRNA (which is an isoform of Ci-Pitx composed of exons3-5 and either exon1, see the first picture for probe mapping) by in situ hybridization.

Original annotation:
Ci-pitxa/b-specific expression in anterior neural boundary (anb, black arrows)(pnc, posterior neural cells ; epi, left epidermis).

Aniseed annotation:
According to the ANISEED anatomical dictionary, the anterior neural boundary corresponds to the neurohypophysis primordium.

Expression of artificial enhancers in the anterior neuroectoderm of mid-tailbud embryos
The diagram of the construct represents the regulatory region with the binding sites (yellow : Pax site, white : Otx site, Green : Forkhead site, Red : Smad site).

Original annotation:
Two (D1(abd), D1(ab)) copies of the 54bp D1(ab) drive expression in most of the embryos (88% (n=167)).

Expression of artificial enhancers in the anterior neuroectoderm of mid-tailbud embryos
The diagram of the construct represents the regulatory region with the binding sites (yellow : Pax site, white : Otx site, Green : Forkhead site, Red : Smad site).
In this construct (named D1(ab)5 in the paper) the fragments a and b(in the paper) were repeat 5 times: the first pax forkhead, otx and smad sites were repeated 5 times.

Original annotation:
Five (D1(ab)x5) copies of the 54bp D1(ab) drive expression in most of the embryos (77% (n=72)).

Schematic representation of mutation introduced into the pD1bcde:CES2 construct. On the right is shown for each construct the rate of mid-tailbud embryos expressing the lacZ reporter in the anterior neural boundary (anb), the anterior epidermis (ae), the ventro-anterior sensory vesicle (vasv) and the mesenchyme (mes) (m1 n=56).

Original annotation:
Most mutations led to a general reduction of the expression level in all of aforementioned domains, while mesenchyme expression remained high. Only construct m3 (block M1 and M2) retained the activity in the anterior neural boundary.

Schematic representation of mutation introduced into the pD1bcde:CES2 construct. On the right is shown for each construct the rate of mid-tailbud embryos expressing the lacZ reporter in the anterior neural boundary (anb), the anterior epidermis (ae), the ventro-anterior sensory vesicle (vasv) and the mesenchyme (mes) (m4 n=58).

Original annotation:
Most mutations led to a general reduction of the expression level in all of aforementioned domains, while mesenchyme expression remained high. Only construct m3 (block M1 and M2) retained the activity in the anterior neural boundary.

Schematic representation of mutation introduced into the pD1bcde:CES2 construct. On the right is shown for each construct the rate of mid-tailbud embryos expressing the lacZ reporter in the anterior neural boundary (anb), the anterior epidermis (ae), the ventro-anterior sensory vesicle (vasv) and the mesenchyme (mes) (m0 n=198).

Original annotation:
Most mutations led to a general reduction of the expression level in all of aforementioned domains, while mesenchyme expression remained high. Only construct m3 (block M1 and M2) retained the activity in the anterior neural boundary.

Schematic representation of mutation introduced into the pD1bcde:CES2 construct. On the right is shown for each construct the rate of mid-tailbud embryos expressing the lacZ reporter in the anterior neural boundary (anb), the anterior epidermis (ae), the ventro-anterior sensory vesicle (vasv) and the mesenchyme (mes) ( m2 n=348).

Original annotation:
Most mutations led to a general reduction of the expression level in all of aforementioned domains, while mesenchyme expression remained high. Only construct m3 (block M1 and M2) retained the activity in the anterior neural boundary.

Schematic representation of mutation introduced into the pD1bcde:CES2 construct. On the right is shown for each construct the rate of mid-tailbud embryos expressing the lacZ reporter in the anterior neural boundary (anb), the anterior epidermis (ae), the ventro-anterior sensory vesicle (vasv) and the mesenchyme (mes) (m5/6 n=182).

Original annotation:
Most mutations led to a general reduction of the expression level in all of aforementioned domains, while mesenchyme expression remained high. Only construct m3 (block M1 and M2) retained the activity in the anterior neural boundary.

Schematic representation of mutation introduced into the pD1bcde:CES2 construct. On the right is shown for each construct the rate of mid-tailbud embryos expressing the lacZ reporter in the anterior neural boundary (anb), the anterior epidermis (ae), the ventro-anterior sensory vesicle (vasv) and the mesenchyme (mes) (m7/8/9 n=29).

Original annotation:
Most mutations led to a general reduction of the expression level in all of aforementioned domains, while mesenchyme expression remained high. Only construct m3 (block M1 and M2) retained the activity in the anterior neural boundary.

Schematic representation of mutation introduced into the pD1bcde:CES2 construct. On the right is shown for each construct the rate of mid-tailbud embryos expressing the lacZ reporter in the anterior neural boundary (anb), the anterior epidermis (ae), the ventro-anterior sensory vesicle (vasv) and the mesenchyme (mes) (m3 n=40).

Original annotation:
Most mutations led to a general reduction of the expression level in all of aforementioned domains, while mesenchyme expression remained high. Only construct m3 (block M1 and M2) retained the activity in the anterior neural boundary.

Expression of artificial enhancers in the anterior neuroectoderm of mid-tailbud embryos
The diagram of the construct represents the regulatory region with the binding sites (yellow : Pax site, white : Otx site, Green : Forkhead site, Red : Smad site).
In this construct (named D1(ab)-75-(ab) in the paper) the fragments a and b(in the paper) were repeated 2 times. There is a 75 bp spacer region between the two fragments.

Original annotation:
Introduction of spacer regions of 75 bp between the two D1(ab) fragments strongly decreased the activity of the tandem constructs 2.5% (n=79). Scores were obtained after one week of LacZ revelation.

Expression of artificial enhancers in the anterior neuroectoderm of mid-tailbud embryos
The diagram of the construct represents the regulatory region with the binding sites (yellow : Pax site, white : Otx site, Green : Forkhead site, Red : Smad site).
In this construct (named D1(ab)(ab-Omut) in the paper) the fragments a and b(in the paper) were repeat 2 times but the second otx site was mutated.

Original annotation:
Mutations of O site in the second copy of (ab) strongly decrease lacZ expression. (D1(ab)(ab-Omut): 0% (n=137)).

Expression of artificial enhancers in the anterior neuroectoderm of mid-tailbud embryos
The diagram of the construct represents the regulatory region with the binding sites (yellow : Pax site, white : Otx site, Green : Forkhead site, Red : Smad site).
In this construct (named D1(ab)-50-(ab) in the paper) the fragments a and b(in the paper) were repeated 2 times. There is a 50 bp spacer region between the two fragments.

Original annotation:
Introduction of spacer regions of 50 bp between the two D1(ab) fragments strongly decreased the activity of the tandem constructs (16.9% (n=154)). Scores were obtained after one week of LacZ revelation.

Expression of artificial enhancers in the anterior neuroectoderm of mid-tailbud embryos
The diagram of the construct represents the regulatory region with the binding sites (yellow : Pax site, white : Otx site, Green : Forkhead site, Red : Smad site).
In this construct (named D1(ab)-25-(ab) in the paper) the fragments a and b(in the paper) were repeated 2 times. There is a 25 bp spacer region between the two fragments.

Original annotation:
Introduction of spacer regions of 25 bp between the two D1(ab) fragments strongly decreased the activity of the tandem constructs (31.6% (n=76)). Scores were obtained after one week of LacZ revelation.

Expression of artificial enhancers in the anterior neuroectoderm of mid-tailbud embryos
The diagram of the construct represents the regulatory region with the binding sites (yellow : Pax site, white : Otx site, Green : Forkhead site, Red : Smad site).
In this construct (named D1(ab)(ab-Gmut) in the paper) the fragments a and b(in the paper) were repeated 2 times but the second smad site was mutated.

Original annotation:
Mutations of G site in the second copy of (ab) strongly decrease lacZ expression. (D1(ab)(ab-Gmut): 0% (n=118)).

Expression of artificial enhancers in the anterior neuroectoderm of mid-tailbud embryos
The diagram of the construct represents the regulatory region with the binding sites (yellow : Pax site, white : Otx site, Green : Forkhead site, Red : Smad site).
In this construct (named D1(ab)(ab-Tmut) in the paper) the fragments a and b(in the paper) were repeat 2 times but the second forkhead site was mutated.

Original annotation:
Mutations of T site in the second copy of (ab) strongly decrease lacZ expression. (D1(ab)(ab-Tmut): 7% (n=84)).

Expression of artificial enhancers in the anterior neuroectoderm of mid-tailbud embryos
The diagram of the construct represents the regulatory region with the binding sites (yellow : Pax site, white : Otx site, Green : Forkhead site, Red : Smad site).
In this construct (named D1(ab)(ab-Pdel) in the paper) the fragments a and b(in the paper) were repeat 2 times and the second pax site was deleted.

Original annotation:
Only 17% of the embryos express lacZ following the deletion of the second P site (D1(ab)(ab-Pdel), n=90).

This region is unable to drive expression in the neurohypophysis primordium due to the deletion. Expression of artificial enhancers in the anterior neuroectoderm of mid-tailbud embryos
The diagram of the construct represents the regulatory region with the binding sites (yellow : Pax site, white : Otx site, Green : Forkhead site, Red : Smad site).
In this construct (named D1ab in the paper) the fragments c, d and e (defined in the paper) were deleted.

Original annotation:
Deletions of (de) abolish LacZ expression.

Side view of an embryo co-electroporated with pDistalPitx:lacZ pDistalPitx:lacZ, 5.3kb, containing D1) and pSix3:Venus (control).
The graph shows the number of lacZ expressing cells.

Original annotation:
Three positive cells can be detected in the ANB.

Aniseed annotation:
ANB (anterior neural boundary) corresponds to neurohypophysis primordium in the ANISEED anatomical dictionary.

Expression of artificial enhancers in the anterior neuroectoderm of mid-tailbud embryos
The diagram of the construct represents the regulatory region with the binding sites (yellow : Pax site, white : Otx site, Green : Forkhead site, Red : Smad site).
In this construct (named D1abd in the paper) there is a deletion from -2693 to -2646, so the three last Smad sites are absents.

Original annotation:
Deletions abolish LacZ expression.

Expression of artificial enhancers in the anterior neuroectoderm of mid-tailbud embryos
The diagram of the construct represents the regulatory region with the binding sites (yellow : Pax site, white : Otx site, Green : Forkhead site, Red : Smad site).
In this construct (named D1(ab)-150-(ab) in the paper) the fragments a and b(in the paper) were repeated 2 times. There is a 150 bp spacer region between the two fragments.

Original annotation:
Introduction of spacer regions of 150 bp between the two D1(ab) fragments strongly decreased the activity of the tandem constructs (1.9% (n=106)) Scores were obtained after one week of LacZ revelation.

Expression of artificial enhancers in the anterior neuroectoderm of mid-tailbud embryos
The diagram of the construct represents the regulatory region with the binding sites (yellow : Pax site, white : Otx site, Green : Forkhead site, Red : Smad site).
In this construct (named D1abde in the paper) there is two deletions from -2693 to -2646 and from -2606 to -2588.

Original annotation:
D1(abde) construct drives lacZ expression in the anterior neuroectoderm (ANB), ventro-anterior sensory vesicle (vasv) and epineural epidermis (ene) in 77.9% of developed embryos (n=57).

Schematic representation of D1(bcde). On the right is shown for the construct the rate of mid-tailbud embryos expressing the lacZ reporter in the anterior neural boundary (anb), the anterior epidermis (ae), the ventro-anterior sensory vesicle (vasv) and the mesenchyme (mes) (wt n=444).

Lateral view of a mid-tail bud stage embryo electroporated with a lacZ transgene containing the proximal fragment (fragment upstream exon2) of Ci-Pitx regulatory region.

Original annotation:
Proximal construct activity in posterior neural cells and derivatives (pnc, black arrowheads), anterior stomodeum (ast, black arrow; inset: close-up, dorsal view), ocellus (oc, white arrows), and visceral ganglion (white arrowhead). Ectopic expression in mesenchyme (mes), tail tissues (tt), and ventral cerebral vesicle (vCV).

Distal construct activity in ANB and posterior stomodeum (black arrows, lateral views, inset: close-up, dorsal view). Note the ectopic expression in dorsoposterior stomodeal cells (squared bracket).

Dorsal view of a mid tailbud stage electroporated with a construct containing the Pitx regulatory region from -3853 bp to 147 bp driving the expression of a lacZ reporter gene.

Original annotation :
Expression of LacZ was detected bilaterally in the epidermis. Examples of embryos representing the left-sided and right-sidedexpressions are shown in the insets.

Tailbud stage embryo (no picture available) electroporated with a construct containing the Pitx regulatory region from -413 bp to -303 bp (called del_2 in the picture) driving the expression of a lacZ reporter gene. This construct doesn't direct any expression.

Tailbud stage embryo (no picture available) electroporated with the Pitx regulatory region from -13 bp to 147 bp (called del_6 in the picture) driving the expression of a lacZ reporter gene.

Dorsal view of a mid tailbud stage electroporated with a construct containing a Pitx regulatory region from -3853 bp to -553 pb driving the expression of a lacZ reporter gene.

Original annotation :
Only ectopic expression in muscle and in mesenchyme was detected.

Lateral view of a mid tailbud stage electroporated with a construct containing the Pitx regulatory region from -553 bp to 147 bp driving the expression of a lacZ reporter gene. The embryos developed in presence of DMSO.

Original annotation :
Expression of LacZ was billateraly detected in the epidermis. Examples of embryos representing the left-sided and right sided expression are shown in the insets.

Dorsal view of a mid tailbud stage electroporated with a construct containing the Pitx regulatory region from -553 bp to 147 bp driving the expression of a lacZ reporter gene (construct named i4 3prime-553 in the paper).

Original annotation :
Epidermal expression of LacZ was restricted in the left side.

Tailbud stage embryo (no picture available) electroporated with a construct containing Pitx regulatory region from -353 bp to -383 pb (called del_1 in the picture/paper) driving the expression of a lacZ reporter gene. This construct doesn't direct any expression.

Tailbud stage embryo (no picture available) electroporated with a construct containing the Pitx regulatory region from -323 bp to -163 bp (called del_3 in the picture) driving the expression of a lacZ reporter gene. This construct directs expression in epidermis.

Tailbud stage embryo (no picture available) electroporated with a construct containing Pitx regulatory regiong from -233 bp to -53 bp (called del_4 in the picture) driving the expression of a lacZ reporter gene.
This construct doesn't direct any expression.

Tailbud stage embryo (no picture available) electroporated with a construct containing the Pitx regulatory region from -133 bp to 33 bp (called del_5 in the picture) driving the expression of a lacZ reporter gene.
This construct doesn't direct any expression.

Late tailbud stage wild type embryo probed for Ci-Six3/6 (red) and Ci-Pitx (blue) expression. The arrow indicates Ci-Six3/6 staining in the pigment cell.

Dorsal view of the head of a Ciona robusta tailbud embryo probed for Pitx expression. This control embryo was treated with DMSO and dechorionated shortly before fixation.
Pitx is asymetrically expressed in the head epidermis, in the internal germ layers and in the anterior neural boundary.

Tadpole stage embryo (No picture available in the article) electroporated with a lacZ transgene containing two fragments of Ci-Pitxa/b regulatory region from -3815 to -2733 and from -2546 to 56 bp (contruct named #9 delta (-3164/-2043)).
The gene is still expressed in the tail muscles, but the expression in the stomodeum is lost. Morever, there is an ectopic expression in the mesenchyme.

Tadpole stage embryo (no picture available in the paper) electroporated with a lacZ transgene containing two fragments of Ci-Pitxa/b regulatory region from -3815 to -3164 and from -2043 to 56 bp (construct named #8 delta(-3164/-2043) in the paper).
The gene is still expressed in the tail muscles, but the expression in the stomodeum is lost. Morever, there is an ectopic expression in the mesenchyme.

Lateral view of a tadpole stage embryo electroporated with a lacZ transgene containing the ci-Pitx regulatory region from -3164bp to -2043bp and from -1652bp to -1328bp (construct named #10 ine the paper).
Staining is observed only in the stomodeum.

Sagittal view of a tadpole stage wild type embryo probed for Ci-Pitx expression. Staining is observed in the invaginating pharynx (anterior to the melanized otolith and ocelle) and appears in the posterior endoderm and visceral ganglion.

Ventral view (picture 1, schema L) and lateral view (picture 2, schema M) of a tadpole embryo stained for Ci-Pitx3' mRNA (see the first picture for probe mapping) by in situ hybridization.

Original annotation:
Staining is observed in stomodeum (st, black arrows), ocellus (oc, white arrows), left visceral ganglion (vg, white arrowheads), and left posterior trunk endoderm (pte, white asterisks) (tm, tail muscles).

Aniseed annotation:
The stomodeum corresponds to the oral siphon primordium in the ANISEED anatomical dictionary.

Ventral view (picture 1, schema L) and lateral view (picture 2, schema M) of a tadpole embryo stained for Pitxc mRNA (which is a isoform of Ci-Pitx composed of exons3-5 and either exon2, see the first picture for probe mapping) by in situ hybridization.

Original annotation:
Staining is observed in ocellus (oc, white arrows), left visceral ganglion (vg, white arrowheads), and left posterior trunk endoderm (pte, white asterisks) (tm, tail muscles ; st, stomodeum).

Tadpole stage embryo (no picture available in the paper) electroporated with a lacZ transgene containing two fragments of Cis-Pitxa/b regulatory region from -2733 to -2520 and from -1652 to -1328 bp (construct named #11 in the paper).
The gene is still expressed in the stomodeum (oral siphon primordium), but the expression in the tail muscles is lost.

Ventral view (picture 1, schema L) and lateral view (picture 2, schema M) of a tadpole embryo stained for Ci-Pitxa/b mRNA (which is a isoform of Ci-Pitx composed of exons3-5 and either exon1, see the first picture for probe mapping) by in situ hybridization.

Original annotation:
Staining is observed stomodeum (st, black arrows) and tail muscles (tm) (oc, ocellus ; vg, left visceral ganglion ; pte, left posterior trunk endoderm).

Aniseed annotation:
The stomodeum corresponds to the oral siphon primordium in the ANISEED anatomical dictionary.

Molecular dissection of the proximal construct. Expression is scored both qualitatively (+, +/−, −) and semiquantitatively (%exp: percentage of expression, n: number of embryos).
P1+ construct leads to no activity.

Tadpole stage embryo (no picture available in the paper) electroporated with a lacZ transgene containing two fragments of Ci-Pitxa/b regulatory region from -5500bp to -1328bp (construct named #2 in the paper). The gene is expressed in the tail muscles and in the stomodeum.

Tadpole stage embryo (no picture available in the paper) electroporated with a lacZ transgene containing two fragments of Ci-Pitxa/b regulatory region from -3164bp to 56bp (construct named #4 in the paper).
The gene is still expressed in the tail muscles, and in the stomodeum.

Tadpole stage embryo (no picture available in the paper) electroporated with a lacZ transgene containing two fragments of Ci-Pitxa/b regulatory region from -5500bp to 56bp (construct named #1 in the paper).
The gene is expressed in the tail muscles and in the stomodeum.

Molecular dissection of the proximal construct. Expression is scored both qualitatively (+, +/−, −) and semiquantitatively (%exp: percentage of expression, n: number of embryos).
P3P2:pdist construct is expressed in the anterior stomodeum.

Molecular dissection of the proximal construct. Expression is scored both qualitatively (+, +/−, −) and semiquantitatively (%exp: percentage of expression, n: number of embryos).
pCES:P3 construct leads to no activity.

Molecular dissection of the proximal construct. Expression is scored both qualitatively (+, +/−, −) and semiquantitatively (%exp: percentage of expression, n: number of embryos).
pCES:P1 construct leads to no activity.

Detailed stomodeal activities of distal construct using EYFP (green), lateral (Picture 1) and dorsal (Picture 2) views. Confocal sections (Picture 1, 2) show cell cortices (F-actin, red), nuclei (DAPI, blue); note the antero-ventral stomodeal flexure and endodermal actin-rich area (open arrowheads). Open arrows point to the tip of anterior-most expressing cells. Scale bar: 10 Am.

Detailed stomodeal activities of proximal construct using EYFP (green), lateral (picture 2) and dorsal (picture 1) views. Arrows point to the ocellus. Scale bar: 10 Am.

Detailed stomodeal activities proximal constructs using EYFP (green), dorsal views.
Confocal sections show cell cortices (F-actin, red), nuclei (DAPI, blue); note the antero-ventral stomodeal flexure and endodermal actin-rich area (open arrowheads). Open arrows point to the tip of anterior-most expressing cells and white arrows point to the ocellus.

Lateral view of a tadpole stage embryo electroporated with a lacZ transgene containing the Ci-Pitx regulatory region from -3815bp to +55bp (construct named #3 in the paper).
Staining is observed in the stomodeum and in the tail muscles.

Lateral view of a tadpole stage embryo electroporated with a lacZ transgene containing the Ci-Pitx regulatory region from -2760bp to +140bp.

Original annotation :
Anterior stomodeal expression. Note the ectopic staining.

Detailed stomodeal activities of distal construct using EYFP (green), lateral (Picture 1) and dorsal (Picture 2) views.

Lateral view of a tadpole stage embryo electroporated with a lacZ transgene containing the Ci-Pitx regulatory region from -1776bp to +55bp (construct named #7 in the paper).
Staining is lost in the stomodeum, partially in the tail muscles. Also ectopic expression is observed in the mesenchyme.

Lateral view of a tadpole stage embryo electroporated with a lacZ transgene containing proximal fragment (fragment upstream exons2) of Ci-Pitx regulatory region which contains -7865bp to +140bp (construct named P3+ in the paper).

Original annotation:
Anterior stomodeal expression.

Lateral view of a tadpole stage embryo electroporated with a lacZ transgene containing the Ci-Pitx regulatory region from -2430bp to +55bp (construct named #5 in the paper).
Staining is observed in the tail muscles but lost in the stomodeum. Ectopic expression is also observed in mesenchyme.

Lateral view of a tadpole stage embryo electroporated with a lacZ transgene containing the Ci-Pitx regulatory region from -2750bp to -2250bp which fused with the forkhead promoter (construct named PCES:2 in the paper).
Mosaic embryos were scored positive for stomodeal expression.

Lateral view of a tadpole stage embryo electroporated with a lacZ transgene containing the Ci-Pitx regulatory region from -2750bp to -2250bp which fused with the distal promoter construct named P2:pdist in the paper).
Mosaic embryos were scored positive for stomodeal expression.

Tadpole stage embryo (no picture available in the paper) electroporated with a lacZ transgene containing the Ci-Pitxa/b regulatory region from -2043bp to 56 bp.
The gene is still expressed in the tail muscles, but the expression in the stomodeum is lost. Morever, there is an ectopic expression in the mesenchyme.

Dorsal view of the visceral ganglion of a WT Ciona robusta swimming tadpole co-electroporated with Vsx>unc76::GFP (green) and Pitx>unc76::mCherry (red).
Original annotation: Vsx>GFP labels A13.474 and A11.117 pairs (green).
(F) Pitx>mCherry, by contrast, labels only A11.117 (red).
(G) Merged image of E and F. A13.474 does not appear to have dendrites.
(H) Magnified view of inset in F, showing putative dendrites belonging to A11.117 (arrows).

Dorsal view of the visceral ganglion of a WT Ciona robusta swimming tadpole co-electroporated with Vsx>unc76::GFP (green) and Pitx>unc76::mCherry (red).
Original annotation: Pitx>mCherry labels only A11.117 (red).
(E) Vsx>GFP labels A13.474 and A11.117 pairs (green).
(G) Merged image of E and F. A13.474 does not appear to have dendrites.
(H) Magnified view of inset in F, showing putative dendrites belonging to A11.117 (arrows).

Dorsal view of the visceral ganglion of a WT Ciona robusta swimming tadpole electroporated with Pitx>VenusYFP showing labeling of both left and right cells in the A11.117 pair. The dendrites are oriented towards the midline, and the axons do not project contralaterally.

Lateral view of the motor ganglion of a WT Ciona robusta larva co-electroporated with Vsx>GFP (green) and Pitx>mCherry (red) reporter constructs.
Original comment: Visualization of A13.474 and A11.117 interneurons by co-electroporation of Vsx>GFP (green) and Pitx>mCherry (red). Vsx is expressed in both neurons, whereas Pitx is expressed only in A11.117 (yellow).

Lateral view of the motor ganglion of a WT Ciona robusta larva co-electroporated with Vsx>GFP (green) and Pitx>mCherry (red) reporter constructs.
Original comment: Visualization of A13.474 and A11.117 interneurons by co-electroporation of Vsx>GFP (green) and Pitx>mCherry (red). Vsx is expressed in both neurons, whereas Pitx is expressed only in A11.117 (yellow).