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.

Assay platform	Illumina HiSeq 2000
Layout	Paired-end
Stranded	Yes
Read length	49
Normalized data types	FPKM RLE
Wild type - Replica 1
	Territories	whole embryo -
	Biomaterial(s)	C. robusta formely Ciona int. type A, Stage 0 (Unfertilized egg)
		C. robusta formely Ciona int. type A, Stage 8 (64-cell)
		C. robusta formely Ciona int. type A, Stage 11 (early gastrula)
		C. robusta formely Ciona int. type A, Stage 12 (mid gastrula)
		C. robusta formely Ciona int. type A, Stage 15 (mid neurula)
		C. robusta formely Ciona int. type A, Stage 21 (mid tailbud I)
		C. robusta formely Ciona int. type A, Stage 26 (hatching larva)
Wild type - Replica 2
	Territories	whole embryo -
	Biomaterial(s)	C. robusta formely Ciona int. type A, Stage 0 (Unfertilized egg)
		C. robusta formely Ciona int. type A, Stage 8 (64-cell)
		C. robusta formely Ciona int. type A, Stage 11 (early gastrula)
		C. robusta formely Ciona int. type A, Stage 12 (mid gastrula)
		C. robusta formely Ciona int. type A, Stage 15 (mid neurula)
		C. robusta formely Ciona int. type A, Stage 21 (mid tailbud I)
		C. robusta formely Ciona int. type A, Stage 26 (hatching larva)

Vegetal view of an 110-cell stage embryo probed for Onecut (HNF6) expression.
Original annotation : No signal was detected in embryos at the 110-cell stage.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	No expression -

Vegetal view of a gastrula stage probed for Onecut (HNF6) and neurogenin expression (respectively labelled in green and red).
Original annotation : In Ciona embryos, Neurogenin is expressed in the visceral ganglion precursors at gastrula stage, while no Onecut expression is observed.
First image : Onecut ISH channel only.
Second image : Yellow color in the merged image represents co-expression territories.
Third image : Embryo used for imaging.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	No expression -

Vegetal view of a late gastrula probed for Onecut (HNF6) expression.
Original annotation : The expression of Onecut starts at the late gastrula stage along the antero- posterior axis of the neural plate. In particular, the signal is localized in four cells in the most anterior region of the neural plate, that will give rise to the sensory vesicle (red arrow), and into two pairs of bilateral cells, arranged in two rows along the neural folds in the posterior half of the developing nervous system (black arrows). Among these cells, Onecut expression seems to be stronger in the first pair of cells representing precursors of the visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	A9.32 cell pair - a9.36 cell pair - a9.40 cell pair - a9.52 cell pair -

Vegetal view of a late gastrula stage embryo probed for Onecut (HNF6) and neurogenin expression (respectively labelled in red and green).
Original annotation : At the late gastrula stage it is possible to detect Onecut expression in the most anterior neural plate rows, corresponding to the brain precursors (green arrowhead) and a new signal appears in the visceral ganglion precursors, in the same cells expressing Neurogenin (white arrowheads)
First image : Onecut ISH channel only.
Second image : Yellow color in the merged image represents co-expression territories.
Third image : Embryo used for imaging.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	A9.32 cell pair - a9.36 cell pair - a9.40 cell pair - a9.52 cell pair -

Dorso-lateral view (anterior is on the left) of a tailbud probed for Onecut (HNF6) expression.
Original annotation : At the neurula stage the Onecut gene is expressed in the more anterior precursors of the sensory vesicle (red arrowheads) and in the developping neural plate.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	neural plate -

Vegetal view of a mid neurula probed for Onecut (HNF6) expression.
Original annotation : At neurula stage, the transcript continues to be present in the same territories observed in the previous stage (ie. in precursors of the visceral ganglion and the sensory vesicle), although the signal in the most anterior part of the neural plate becomes wider and more intense (red arrow).

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	neural plate -

Vegetal view of a neurula-stage embryo probed for Onecut (HNF6) and neurogenin expression (respectively labelled in green and red).
Original annotation : At neurula stage Onecut is expressed in the tail neurons (blue arrowheads) and in the visceral ganglion precursors in the same cells also expressing Neurogenin (yellow arrowheads). At level of the anterior neural plate, Onecut is restricted to the central region, between the Neurogenin expressing cells.
First image : Onecut ISH channel only.
Second image : Yellow color in the merged image represents co-expression territories.
Third image : Embryo used for imaging.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	A10.63 cell pair - A10.64 cell pair - a line neural plate -

Dorsal view (anterior is on the left) of a neurula electroporated with Onecut/HNF6(-2590/-4)>LacZ (K construct in Pezzotti et al. 2014).
Original annotation : At neurula stage the LacZ expression has been detected by in situ hybridization in some cells of the anterior neural plate and in two pairs of bilateral cells localized in the posterior half of the developing neural plate.
Aniseed annotation : expression is also observed in mesenchyme and tail muscles.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	neural plate - tail muscles -

Dorsal view of a neurula electroporated with Onecut/HNF6(-935/-674)>LacZ (FG construct in Pezzotti et al. 2014).
Original annotation : The FG construct is the only one able to reproduce the Onecut endogenous expression. Red arrowhead indicates the signal at level of the sensory vesicle, black arrowhead the intermediate signal at level of the visceral ganglion and white arrowhead the more posterior one in the caudal nerve cord.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	neural plate - tail muscles -

Dorsal view of a neurula electroporated with Onecut/HNF6(-935/-674)NgnMut>LacZ (FG(NgnMut) construct in Pezzotti et al. 2014).
Original annotation : Electroporation experiments with the FG(NgnMut) construct demonstrated that at neurula stage the transgenic embryos do not show any specific signal, thus suggesting that Neurogenin binding site is necessary for FG activation in the Onecut territories.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

Vegetal view of a late-gastrula stage embryo probed for Onecut (HNF6) and neurogenin expression (respectively labelled in green and red).
Original annotation : At late neurula stage the two genes are perfectly co-expressed in the visceral ganglion and caudal nerve cord precursors while in the future sensory vesicle they still present different regions of expression and there are only few cells that co-express both genes that seem to represent the photoreceptor cell precursors (pink arrowheads).
First image : Onecut ISH channel only.
Second image : Yellow color in the merged image represents co-expression territories.
Third image : Embryo used for imaging.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	neural plate -

Conversion to Aniseed format of Ghost database entry from Satou Y et al., Development 2001; 128:2893-2904.

Original annotation:
Expression pattern: Brain Nerve cord.
Comment : brain and visceral ganglion.

Aniseed annotation : A staining has been detected in the nervous sytem but also in the visceral ganglion and the tail nerve cord.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	nervous system -

Conversion to Aniseed format of Ghost database entry from Satou Y et al., Development 2001; 128:2893-2904.

Original annotation:
Expression pattern: Brain Nerve cord.
Comment : brain and visceral ganglion.

Aniseed annotation : A staining has been detected in the nervous sytem but also in the visceral ganglion and the tail nerve cord.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	nervous system -

Dorsal view of an early tailbud stage wild type embryo probed for HNF6 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 :
HNF6 expression was expressed in A11.118, A11.117 and A10.57 cell pairs (posterior visceral ganglion and tail nerve cord).

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	tail nerve cord - visceral ganglion -

Dorso-lateral view (anterior is on the left) of an early tailbud probed for Onecut (HNF6) expression.
Original annotation : The Onecut gene is expressed in the more anterior precursors of the sensory vesicle (red arrowheads). Precursors of the visceral ganglion and more posterior nervous system are labeled.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Lateral view (anterior is on the left) of an early tailbud probed for Onecut (HNF6) expression.
Original annotation :At the early tailbud stage the Onecut gene is expressed in the more anterior precursors of the sensory vesicle (red arrowheads) and in the developping neural plate.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Vegetal view of an early tailbud probed for Onecut (HNF6) and neurogenin expression (respectively labelled in green and red).
Original annotation : At early tailbud stage, the merge images obtained by confocal microscopy demonstrated that these two genes are co-expressed in almost all the Onecut territories at level of the future sensory vesicle, visceral ganglion and caudal nerve cord, again with the exception of two small areas in the most anterior part of the nervous system corresponding to part of the future anterior sensory vesicle.
First image : Onecut ISH channel only.
Second image : Yellow color in the merged image represents co-expression territories.
Third image : Embryo used for imaging.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	caudal epidermal neurone (CEN) precursors - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Dorso-lateral view (anterior is on the left) of an early tailbud electroporated with Onecut/HNF6(-2590/-4)>LacZ (K construct in Pezzotti et al. 2014).
Original annotation : At early tailbud stage, precursors of the visceral ganglion and more posterior nervous system are labeled. At this stage a signal starts to appear in the cells of the future sensory vesicle. Comparing this result with the Onecut endogenous profile, these cells seems to correspond to the more posterior ones that express endogenous Onecut, while no signal is visible in the more anterior precursors of the sensory vesicle.
Aniseed annotation : expression is also observed in tail muscles.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail muscles - tail nerve cord - visceral ganglion -

Conversion to Aniseed format of Ghost database entry from Satou Y et al., Development 2001; 128:2893-2904.

Original annotation:
Expression pattern: brain, nerve cord.
Comment : brain and visceral ganglion.

Aniseed annotation : A staining has been detected in the nervous sytem but also in the tail nerve cord and the visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	nervous system -

Mid tailbud stage wild type embryo probed for HNF6 expression. No picture available in the article.

Original Annotation :
HNF6 expression in the visceral ganglion is localized in A12.238, A11.118 and A11.117 cell pairs.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	visceral ganglion -

Lateral and dorsal views (anterior is on the left) of mid tailbud embryos probed for Onecut (HNF6) expression.
Original annotation : At middle tailbud stage Onecut is expressed in the anterior part of the developing nervous system, at level of the future sensory vesicle (red arrow) and in four cells positioned in the central part and arranged along the neural folds, at level of the visceral ganglion precursors (black arrow). Prolonging staining time of the in situ hybridization experiments resulted in the detection of a signal in the posterior part of the tail (white arrow). This signal seems to be localized at level of the dorsal nerve cord.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Lateral view of a mid tailbud probed for Onecut (HNF6) and neurogenin expression (respectively labelled in red and green).
Original annotation : At middle tailbud stage it is possible to observe a full co-localization of the two genes not only in the visceral ganglion and dorsal nerve cord, but also in the anterior sensory vesicle precursors, where in the previous stages there was only Onecut (yellow arrowheads).
First image : Onecut ISH channel only.
Second image : Yellow color in the merged image represents co-expression territories, edge of the embryo is highlighted with a thin white line.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - visceral ganglion -

Lateral view of mid tailbud stage wild type embryo probed for Ci-HNF6 expression. Expression expended to the ventro-posterior and dorsal parts of sensory vesicle, while a few intermediate cells, in the ventral sensory vesicle remained unlabelled.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	dorsal midline posterior tail epidermis - tail nerve cord - visceral ganglion -

Lateral view of mid tailbud stage wild type embryo probed for Ci-HNF6 expression by fluorescent Fast red staining. The double labelling was performed on mid-tailbud embryos electroporated with the pCiTH-LacZ trasngene (expression in green).

Original Annotation : Expression of Ci-HNF6 was detected in anterior sensory vesicle.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle -

Dorso-lateral view (anterior is on the left) of a mid tailbud electroporated with Onecut/HNF6(-2590/-4)>LacZ (K construct in Pezzotti et al. 2014).
Original annotation : At this stage K construct continues to be active in the precursors of the visceral ganglion and a more posterior signal appears in the tail, at level of the epidermis which also seems to affect the neural tube. At middle tailbud stage the LacZ expression in the sensory vesicle becomes very similar to the endogenous Onecut gene, since the signal visible in the future sensory vesicle seems to correspond to the three cells in the anterior part of the nervous system expressing the endogenous Onecut.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - dorso lateral posterior tail epidermis - posterior sensory vesicle - tail nerve cord - visceral ganglion -

At tailbud stage, D construct (ie. Onecut/HNF6(-2590/-1740)>LacZ) is inactive (2nd line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	No expression -

At tailbud stage, E construct (ie. Onecut/HNF6(-1814/-1293)>LacZ) is active in ectopic mesenchyme (3rd line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At tailbud stage, A construct (ie. Onecut/HNF6(-1377/-866)>LacZ) is active ectopic mesenchyme (5th line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At tailbud stage, C construct (ie. Onecut/HNF6(-464/-4)>LacZ) is active ectopic mesenchyme (7th line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At tailbud stage, G construct (ie. Onecut/HNF6(-651/-444)>LacZ) is active in ectopic mesenchyme (9th line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At tailbud stage, H construct (ie. Onecut/HNF6(-464/-232)>LacZ) is active in ectopic mesenchyme (10th line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At tailbud stage, I construct (ie. Onecut/HNF6(-1307/-935)>LacZ) is active in ectopic mesenchyme (11th line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At tailbud stage, L construct (ie. Onecut/HNF6(-251/-4)>LacZ) is active in ectopic mesenchyme (11th line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

Dorso-lateral view (anterior is on the left) of a tailbud electroporated with Onecut/HNF6(-881/-444)>LacZ (B construct in Pezzotti et al. 2014).
Original annotation : 85% of embryos electroporated with the B construct showed LacZ expression in the sensory vesicle, visceral ganglion and ectopically in mesenchyme.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - dorso lateral posterior tail epidermis - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Dorso-lateral view (anterior is on the left) of a tailbud electroporated with Onecut/HNF6(-935/-625)>LacZ (F construct in Pezzotti et al. 2014).
Original annotation : The Onecut regulatory sequence contained in the F construct was the only one able to activate the expression of the LacZ gene in the same regions of the endogenous transcript.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Dorso-lateral view (anterior is on the left) of a tailbud electroporated with Onecut/HNF6(-935/-731)>LacZ (FA construct in Pezzotti et al. 2014).
Original annotation : 70% of embryos electroporated with the FA construct, lacking 58 bp at the 3', showed a partial expression of the LacZ reporter gene in the sensory vesicle around the ocellus, in the visceral ganglion and in the posterior part of the tail.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

At tailbud stage, FB construct (ie. Onecut/HNF6(-731/-625)>LacZ) is active in ectopic mesenchyme only (4th line of the diagram).
Original legend : Diagram of the transgenic constructs from F to FH.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At tailbud stage, FC construct (ie. Onecut/HNF6(-935/-834)>LacZ) is not active (5th line of the diagram).
Original legend : Diagram of the transgenic constructs from F to FH.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	No expression -

At tailbud stage, FD construct (ie. Onecut/HNF6(-833/-731)>LacZ) is not active (6th line of the diagram).
Original legend : Diagram of the transgenic constructs from F to FH.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	No expression -

Dorsal view of a WT Ciona robusta tailbud probed for Rx (KH.C12.152, red) and Onecut (green) expressions.
E: Onecut is expressed in the sensory vesicle and in the visceral ganglion.
D: DAPI staining for nuclei (blue).
F: Rx is expressed in the sensory vesicle.
G: Merge image of D, E, and F. Orange color in G indicates co-expression territory of Ci-Rx and Ci-Onecut.
Original comment: The most anterior cells expressing Ci-Rx consistently also co-express Ci-Onecut. Furthermore, we observed Ci-Onecut co-localization in the two more anterior groups of cells that express Ci-Rx. Unfortunately, it was not been possible to establish if Ci-Onecut is expressed in the more posterior cell marked by Ci-Rx, because Ci-Rx expression in this cell is too faint and transient to be detected with this technique.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - visceral ganglion -

Dorsal view of a WT Ciona robusta tailbud probed for Rx (KH.C12.152, red) and Onecut (green) expressions.
F: Rx is expressed in the sensory vesicle.
E: Onecut is expressed in the sensory vesicle and in the visceral ganglion.
D: DAPI staining for nuclei (blue).
G: Merge image of D, E, and F. Orange color in G indicates co-expression territory of Ci-Rx and Ci-Onecut.
Original comment: The most anterior cells expressing Ci-Rx consistently also co-express Ci-Onecut. Furthermore, we observed Ci-Onecut co-localization in the two more anterior groups of cells that express Ci-Rx. Unfortunately, it was not been possible to establish if Ci-Onecut is expressed in the more posterior cell marked by Ci-Rx, because Ci-Rx expression in this cell is too faint and transient to be detected with this technique.

Stained molecule	KH2012:KH.C12.152 (PAX6; RAX; RAX2)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle -

Dorso-lateral view (anterior is on the left) of a mid tailbud probed for Onecut (HNF6) expression.
Signal is detected in the anterior sensory vesicle and in the visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - visceral ganglion -

Vegetal view of a mid tailbud probed for Onecut (HNF6) and Arrestin expression (respectively labelled in red and green).
Original annotation : Onecut shows a clear co-localization with the Arrestin probe at larva stage thus demonstrating that at level of the sensory vesicle the Onecut gene is expressed in the photoreceptor cells.
First image : Onecut ISH channel only.
Second image : Yellow color in the merged image represents co-expression territories, edge of the embryo is highlighted with a thin white line.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - visceral ganglion -

Lateral view of a mid tailbud probed for Onecut (HNF6) expression.
Signal is detected in the sensory vesicle and in the visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - visceral ganglion -

Dorso-lateral view (anterior is on the left) of a mid tailbud electroporated with Onecut/HNF6(-1377/-4)>LacZ (V construct in Pezzotti et al. 2014).
Original annotation : the V construct was able to completely reproduce, in 95% of analyzed embryos, the Onecut expression pattern.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - dorso lateral posterior tail epidermis - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Dorso-lateral view (anterior is on the left) of a tailbud electroporated with Onecut/HNF6(-935/-674)>LacZ (FG construct in Pezzotti et al. 2014).
Original annotation : The FG construct is the only one able to reproduce the Onecut endogenous expression. The reporter gene expression was observed in the sensory vesicle, in particular in the area around the ocellus, in the visceral ganglion, in the tail and also ectopically in the mesenchyme
Red arrow indicates the more anterior signal at level of the sensory vesicle, black arrow the intermediate signal at level of the visceral ganglion and white arrow the more posterior one in the caudal nerve cord.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - dorso lateral posterior tail epidermis - posterior sensory vesicle - tail nerve cord - visceral ganglion -

At tailbud stage, 73% of transgenic embryos electroporated with the FH construct (ie. Onecut/HNF6(-833/-674)>LacZ) showed expression of the reporter gene only in ectopic mesenchyme while the remaining 27%, did not show any signal at all.
Original legend : Diagram of the transgenic constructs from F to FH.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

Conversion to Aniseed format of Ghost database entry from Satou Y et al., Development 2001; 128:2893-2904.

Original annotation:
Expression pattern: brain, nerve cord.
Comment : brain and visceral ganglion.

Aniseed annotation : A staining has been detected in the nervous sytem but also in the visceral ganglion and the tail nerve cord.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	nervous system -

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

Lateral and dorsal views of a WT Ciona robusta tailbud probed for Onecut expression. Onecut is expressed in the sensory vesicle and in the visceral ganglion (vg).
Original comment: Expression of Ci-Onecut is detected along the antero-posterior axis of the central nervous system (CNS).

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - visceral ganglion -

Conversion to aniseed format of DBTGR database entry from Kusakabe team.

Original annotation:
Brain vesicle; visceral ganglion; nerve cord.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Vegetal view of a tailbud probed for Onecut (HNF6) and neurogenin expression (respectively labelled in red and green).
Original annotation : It is possible to observe an almost complete co-localization of the two genes in the Onecut territories (ie. visceral ganglion, sensory vesicle and tail nerve chord).
First image : Onecut ISH channel only.
Second image : Yellow color in the merged image represents co-expression territories, edge of the embryo is highlighted with a thin white line.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

At late tailbud stage, FG(Ngn-OCa-OCbMut) construct (ie. Onecut/HNF6(-935/-674)Ngn-OCa-OCbMut>LacZ, the triple mutant) is not active.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	No expression -

At larva stage, FG(OCa-OCbMut) construct (ie. Onecut/HNF6(-935/-674)OCa-OCbMut>LacZ the double OC site mutant) showed weak activity in the sensory vesicle, the visceral ganglion and tail nerve chord in less than half of the embryos.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Lateral view of the visceral ganglion of a WT Ciona robusta late tailbud embryo co-electroporated with Onecut>unc76::GFP (green) and Islet>unc76::mCherry (red) reporter constructs.
Onecut enhancer drives GFP expression in differentiating motor neurons and nerve cord cells.
Original annotation:A −4.2 kb fragment immediately upstream of the transcription start site of the neuronal differentiation factor Onecut (also known as HNF6) drives reporter gene expression in differentiating neurons and nerve cord cells. This labels frondose endplates that contact the sides of the dorsal and anterior tail muscle cells.
Figure:
(B) Co-electroporation with Islet>mCherry reveals that these endplates do not belong to A10.64, which is strongly stained by this construct. (C) Magnification of dotted area in A. (C′) Magnification of dotted area in B. (C′′) Merged image of C and C′, showing that A10.64 might even be contacting and synapsing onto the frondose endplates (arrow).

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	tail nerve cord - visceral ganglion -

Lateral view of the visceral ganglion of a WT Ciona robusta late tailbud embryo co-electroporated with Onecut>unc76::GFP (green) and Islet>unc76::mCherry (red) reporter constructs.
Islet enhancer drives mCherry expression in A10.64 differentiating motor neuron 2.
Original annotation of the figure: (A) A −4.2 kb fragment immediately upstream of the transcription start site of the neuronal differentiation factor Onecut (also known as HNF6) drives reporter gene expression in differentiating neurons and nerve cord cells. This labels frondose endplates that contact the sides of the dorsal and anterior tail muscle cells.
(B) Co-electroporation with Islet>mCherry reveals that these endplates do not belong to A10.64, which is strongly stained by this construct. (C) Magnification of dotted area in A.
(C′) Magnification of dotted area in B.
(C′′) Merged image of C and C′, showing that A10.64 might even be contacting and synapsing onto the frondose endplates (arrow).

Stained molecule	KH2012:KH.L152.2 (ISL1; ISL2; LHX9)
Stained region(s)	visceral ganglion -

Lateral view of a WT Ciona robusta larva probed for Onecut expression. Onecut is expressed in the sensory vesicle (sv) and in the visceral ganglion (vg).

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - visceral ganglion -

Lateral view (anterior is on the left) of a larva probed for Onecut (HNF6) expression.
Original annotation : Red arrow indicates the signal at level of the sensory vesicle, black arrow the intermediate signal at level of the visceral ganglion and white arrow the more posterior signal in the caudal nerve cord.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Lateral view (anterior is on the left) of a larva probed for Onecut (HNF6) expression.
Original annotation : At larva stage, Onecut expression is restricted in the posterior-most part of the sensory vesicle, in an area that surrounds the ocellus (red arrow), at the level of the visceral ganglion (black arrow), and a faint signal is also detected in the tail nerve cord (white arrow).

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	epidermis - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Dorso-lateral view (anterior is on the left) of a larva electroporated with Onecut/HNF6(-2590/-4)>LacZ (K construct in Pezzotti et al. 2014).
Original annotation : In the larvae the LacZ gene is expressed at level of the sensory vesicle around the ocellus, in the visceral ganglion and in the tail. Red arrow indicates the signal at level of the sensory vesicle, black arrow the intermediate signal at level of the visceral ganglion and white arrow the more posterior signal in the caudal nerve cord.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

At larva stage, D construct (ie. Onecut/HNF6(-2590/-1740)>LacZ) is inactive (2nd line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	No expression -

At larva stage, E construct (ie. Onecut/HNF6(-1814/-1293)>LacZ) is active in ectopic mesenchyme (3rd line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

Dorso-lateral view (anterior is on the left) of a larva electroporated with Onecut/HNF6(-1377/-4)>LacZ (V construct in Pezzotti et al. 2014).
Original annotation : the V construct was able to completely reproduce, in 95% of analyzed embryos, the Onecut expression pattern.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - dorso lateral posterior tail epidermis - posterior sensory vesicle - tail nerve cord - visceral ganglion -

At larva stage, A construct (ie. Onecut/HNF6(-1377/-866)>LacZ) is active ectopic mesenchyme (5th line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At larva stage, C construct (ie. Onecut/HNF6(-464/-4)>LacZ) is active ectopic mesenchyme (7th line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At larva stage, G construct (ie. Onecut/HNF6(-651/-444)>LacZ) is active in ectopic mesenchyme (9th line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At larva stage, H construct (ie. Onecut/HNF6(-464/-232)>LacZ) is active in ectopic mesenchyme (10th line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At larva stage, I construct (ie. Onecut/HNF6(-1307/-935)>LacZ) is active in ectopic mesenchyme (11th line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At larva stage, L construct (ie. Onecut/HNF6(-251/-4)>LacZ) is active in ectopic mesenchyme (11th line of the diagram).
Original legend : Diagram of the transgenic constructs from K to L.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

Dorso-lateral view (anterior is on the left) of a larva electroporated with Onecut/HNF6(-881/-444)>LacZ (B construct in Pezzotti et al. 2014).
Original annotation : 85% of embryos electroporated with the B construct showed LacZ expression in the sensory vesicle, visceral ganglion and ectopically in mesenchyme.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - dorso lateral posterior tail epidermis - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Dorso-lateral view (anterior is on the left) of a larva electroporated with Onecut/HNF6(-935/-625)>LacZ (F construct in Pezzotti et al. 2014).
Original annotation : The Onecut regulatory sequence contained in the F construct was the only one able to activate the expression of the LacZ gene in the same regions of the endogenous transcript.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - dorso lateral posterior tail epidermis - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Dorso-lateral view (anterior is on the left) of a larva electroporated with Onecut/HNF6(-935/-674)>LacZ (FG construct in Pezzotti et al. 2014).
Original annotation : The FG construct is the only one able to reproduce the Onecut endogenous expression. The reporter gene expression was observed in the sensory vesicle, in particular in the area around the ocellus, in the visceral ganglion, in the tail and also ectopically in the mesenchyme
Red arrow indicates the more anterior signal at level of the sensory vesicle, black arrow the intermediate signal at level of the visceral ganglion and white arrow the more posterior one in the caudal nerve cord.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Dorso-lateral view (anterior is on the left) of a larva electroporated with Onecut/HNF6(-935/-731)>LacZ (FA construct in Pezzotti et al. 2014).
Original annotation : 70% of embryos electroporated with the FA construct, lacking 58 bp at the 3', showed a partial expression of the LacZ reporter gene in the sensory vesicle around the ocellus, in the visceral ganglion and in the posterior part of the tail.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

At larva stage, FB construct (ie. Onecut/HNF6(-731/-625)>LacZ) is active in ectopic mesenchyme only (4th line of the diagram).
Original legend : Diagram of the transgenic constructs from F to FH.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At larva stage, 73% of transgenic embryos electroporated with the FH construct (ie. Onecut/HNF6(-833/-674)>LacZ) showed expression of the reporter gene only in ectopic mesenchyme while the remaining 27%, did not show any signal at all.
Original legend : Diagram of the transgenic constructs from F to FH.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At larva stage, FC construct (ie. Onecut/HNF6(-935/-834)>LacZ) is not active (5th line of the diagram).
Original legend : Diagram of the transgenic constructs from F to FH.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	No expression -

At larva stage, FD construct (ie. Onecut/HNF6(-833/-731)>LacZ) is not active (6th line of the diagram).
Original legend : Diagram of the transgenic constructs from F to FH.
Left : Schematic representation of the regulatory sequences assayed by electroporation (black bars). LacZ reporter gene is represented by a blue box. Checkered box indicates the human β-globin basal promoter.
Right : Representation of the results observed in electroporated embryos at tailbud and larva stages. The number of positive embryos showing reporter gene expression is indicated by a percentage value. Empty squares indicate absence of any signal. Full colored squares indicate that all the positive embryos show LacZ expression in the corresponding territory. Squares with a colored dot indicate that less than 50% of the positive embryos show LacZ expression in the corresponding territory. e (green), ectopic mesenchyme; sv (red), sensory vesicle; t (yellow), tail; vg (violet), visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	No expression -

Dorso-lateral view of a larva electroporated with Onecut/HNF6(-935/-674)>LacZ (FG construct in Pezzotti et al. 2014).
Original annotation : The FG construct is the only one able to reproduce the Onecut endogenous expression. Red arrow indicates the signal at level of the sensory vesicle, black arrow the intermediate signal at level of the visceral ganglion and white arrow the more posterior one in the caudal nerve cord.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Dorso-lateral view of a larva electroporated with Onecut/HNF6(-935/-674)NgnMut>LacZ (FG(NgnMut) construct in Pezzotti et al. 2014).
Original annotation : At larva stage, about 40% of the transgenic embryos showed a positive signal in the sensory vesicle and the tail, but not in the visceral ganglion, in contrast to the 90% of positive larvae obtained with the wild type FG construct.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior anterior sensory vesicle -

Dorso-lateral view of a larva electroporated with Onecut/HNF6(-935/-674)OCaMut>LacZ (FG(OCaMut) construct in Pezzotti et al. 2014).
Original annotation : We electroporated the constructs mutated in the first (OCa) Onecut binding site and we observed that 70% of embryos show an expression of the reporter gene in the sensory vesicle, visceral ganglion and tail, but only less than 50% showed the reporter gene expression in the three territories at the same time, with less efficiency with respect to the wild type FG.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Dorso-lateral view of a larva electroporated with Onecut/HNF6(-935/-674)OCbMut>LacZ (FG(OCbMut) construct in Pezzotti et al. 2014).
Original annotation : we electroporated the constructs mutated in the second (OCb) Onecut binding site and we observed that 70% of embryos show an expression of the reporter gene in the sensory vesicle, visceral ganglion and tail, but only less than 50% showed the reporter gene expression in the three territories at the same time, with less efficiency with respect to the wild type FG.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Dorso-lateral view of a larva electroporated with Onecut/HNF6(-935/-674)Ngn-OCaMut>LacZ (FG(Ngn-OCaMut) construct in Pezzotti et al. 2014).
Original annotation : Transgenic embryos electroporated with the double mutant for Ngn and the first Onecut binding site (OCa), leaving wild type the second OC site (OCb), did not show any expression of the reporter gene in the territories of interest. Only 10% of embryos showed an ectopic mesenchymal expression.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

Dorso-lateral view of a larva electroporated with Onecut/HNF6(-935/-674)Ngn-OCbMut>LacZ (FG(Ngn-OCbMut) construct in Pezzotti et al. 2014).
Original annotation : Embryos electroporated with the double mutant in the sites for Ngn and for the second OC site (OCb) show a complete absence of the LacZ expression in the Onecut endogenous territories.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	No expression -

Dorso-lateral view of a larva electroporated with Onecut/HNF6(-935/-674)OCa-OCbMut>LacZ (FG(OCa-OCbMut) construct in Pezzotti et al. 2014).
Original annotation : The FG construct double mutant in both OC (Onecut) sites showed the same expression profile of the single OCa or OCb mutants in the sensory vesicle and tail.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -

Dorso-lateral view of a larva electroporated with Onecut/HNF6(-935/-674)Ngn-OCa-OCbMut>LacZ (FG(Ngn-OCa-OCbMut) construct in Pezzotti et al. 2014).
Original annotation : Larvae transgenic for the FG triple mutant in the Ngn and the two OC binding sites, the FG(Ngn–OCa–OCbMut) construct, showed a total absence of specific reporter gene expression.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)

At larva stage, FG(NgnMut) construct (ie. Onecut/HNF6(-935/-674)NgnMut>LacZ)is active in about 40% of the transgenic embryos : signal in the sensory vesicle and the tail, but not in the visceral ganglion.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord -

At tailbud stage, FG construct (ie. Onecut/HNF6(-935/-674)>LacZ) is active the sensory vesicle, in the visceral ganglion,in the tail nerve cord and in ectopic mesenchyme.

Stained molecule	KH2012:KH.C6.185 (ONECUT1; ONECUT2; ONECUT3)
Stained region(s)	anterior sensory vesicle - posterior sensory vesicle - tail nerve cord - visceral ganglion -