Ciona robusta adults were obtained from the National Bio-Resource Project for Ciona (NBRP, Japan). 50 unperturbed and Foxd-morphant embryos were collected at the 32-, 64-, and 112-cell stages. RNA was extracted using a Dynabeads mRNA DIRECT Purification Kit (Thermo Fischer Scientific) and libraries were made with an Ion Total RNA-Seq kit ver 2 (Thermo Fischer Scientific). The libraries were sequenced with an Ion PGM instrument (Thermo Fischer Scientific).

The purpose of this project is to identify genes positively and negatively regulated by signaling molecules that belongs to the TGFbeta superfamily.

Vegetal view of a 110-cell stage embryo stained for ETR expression. Staining is lost in the a-line neural plate retained in the A-line neural plate and extended to the A-line notochord precursors (A8.5/6/13/14).

Control: Vegetal view of a 110-cell stage embryo stained for ETR expression. Staining is detected in the a-line and A-line neural plate cells.

Vegetal view of a 110-cell stage embryo probed for Ci-ETR expression in which Nodal signalling has been blocked by injection of a nodal MO. The expression appeared similar to that of control embryos. Control sibling embryo shows the wild type pattern of the gene.

Vegetal view of a 110-cell stage embryo probed for Ci-ETR expression in which Nodal signalling has been blocked by overexpressing mRNA for tAlk4/5/7, a dominant negative type I nodal receptor. The expression appeared similar to that of control embryos. Control sibling embryo shows the wild type pattern of the gene.

Vegetal view of a 110-cell stage embryo probed for Ci-ETR expression in which Nodal signalling has been blocked by treatment with BS431542 inhibitor of Alk4/5/7 nodal type 1 receptor. The expression appeared similar to that of control embryos. Control sibling embryo shows the wild type pattern of the gene.

Vegetal view of a 110-cell stage embryo probed for Ci-ETR expression following ephrinAd RNA injection. Notochord precursors now expressed the neural marker Ci-ETR suggesting that both of the daughter cells of A6.2 and A6.4 cells had adopted a neural fate. Activation of ephrin signals can inhibit the FGF-Ras-MEK-ERK pathway in ascidian embryonic cells. Average of number cells positive : 13.5 for this marker, total number of samples analysed : 33.

Vegetal view of a 110-cell stage embryo probed for Ci-ETR expression following ephrinAd-MO injection. Ephrin-Ad-MO injection induced no expression of Ci-ETR. Average of number cells positive : 1.1 for this marker, total number of samples analyzed : 27. dnEph3-RNA injection gave similar results (1.2 cells, n = 22).

Vegetal view of a 110-cell stage wild type embryo probed for Ci-ETR expression. Embryos were traited with a pharmacological inhibitor U0126 from the 64 cell stage.

Vegetal view of a 110-cell stage wild type embryo probed for Ci-ETR expression. Embryos were traited with a pharmacological inhibitor U0126 from the 32 cell stage.

Original annotation: Ci-ETR was expressed in an average of 14 cells compared to 7.2 cells in control embryos. Judging from the position of the cells ectopically expressing Ci-ETR, it is most likely that these are cells in the notochord lineage, indicating that a cell fate transformation took place.

Vegetal view of a 110-cell stage wild type embryo probed for Ci-ETR expression. Embryos were traited with a pharmacological inhibitor U0126 from the 44 cell stage.

Vegetal view of a 64 cell stage embryo probed for Ci-ETR expression following egg micro-injection of a FGF9 MO. Ci-ETR was ectopically expressed in the primary notochord lineage and in the neural lineage.

Anterior view (animal to the top) of a Ciona robusta early gastrula stage embryo probed for Celf3 expression and injected with morpholino oligos against Prdm1-r.a, Prdm1-r.b and Hes.a.
Celf3 expression is observed in a8.17, a8.18, a8.19, a8.20, a8.25, a8.26, A8.15, A8.16, A8.7, A8.8 and in anterior epidermal cells.
Original annotation: The expression of the early neural marker Celf3 (formerly Etr) was not lost in the brain/palp lineage of Prdm1-r.a/b/Hes.a morphants. Its expression was expanded to the anterior epidermal cells of Prdm1-r.a/b/Hes.a morphants.
n, number of embryos examined. % : Percentages of embryos with Foxa.a expression in the neural lineage (cyan arrowheads).

Vegetal view of a Ciona robusta early gastrula stage embryo probed for Etr expression and injected with morpholino oligos against Eph-1.
Etr, a neural marker, is expressed in A8.7, A8.8, A8.15 and A8.16 blastomere pairs, and in most anterior a-line cells. The orientation of the embryo make observation of more animal cells impossible.

Neural plate and lateral views of a mid-gastrula stage embryo probed for Ci-ETR expression in which Nodal signalling has been blocked by incubation in the SB431542 inhibitor of Alk4/5/7 type I nodal receptor. The position of the neural plate was altered in SB431542-treated embryos,it remained "on top" of the embryo owing to defects in gastrulation movements associated with inhibition of Nodal signalling. Control sibling embryo shows the wild type pattern of the gene.

no major change in expression patternas compared to WT.

View of neurula stage animal cap explants, injected with FGF9/16/20 (AB086097) mRNA before fertilization, dissected at 8-cell stage, and stained by ISH for the neural marker ETR-1 (citb028e11). Injected FGF9/16/20 induces strong ETR-1 expression in part of the cap. The control picture demonstrate the absence of expression of ETR-1 in uninjected sibbling caps.

Dorsal view of a mid neurula stage embryo probed for RNA-binding protein BRUNOL4 (cluster ID 01087r1) (picture 1: DMSO-treated embryo, picture 2: Retinoic acid-treated embryo).

Original annotation:
Retinoic acid treatment caused reduction of mRNA in the anterior and posterior ends of the embryo. Expression in presumptive papilla was affected too. Brackets show the presumptive papilla region (precursors of palps at mid neurula stage are a9.36 and a9.40 cell pairs)

F: dorsal view of a late neurula treated with the MEK inhibitor PD184352 from the 8-cell satge showing ETR expression throughout the A-line neural plate and notochord precursors, but lost in the a-line neural plate.

G-H: Animal (G) and vegetal (H) views of a cleavage arrested PD-treated embryo (from 64-cell stage) showing Otx expression in all A-line neural (Yellow)and notochord (green)precursors. a-line neural plate (red) do not express any more.

Controls:

C: dorsal view of a late neurula showing ETR expression throughout the A-line neural plate, and part of the a-line neural plate (some lateral-most cells may not express).

D-E: Animal (D) and vegetal (E) views of a Cleavage arrested embryo (from 64-cell stage) showing Otx expression in all A-line (yellow) and a-line (red) precursors.

Dorsal view of an early tailbud stage embryo RA treated probed for RNA-binding protein BRUNOL4 (Cluster ID 01087r1) (picture 1: DMSO-treated embryo, picture 2: RA-treated embryo).

Original annotation:
Retinoic acid affected expression in the presumptive papilla region (arrowheads) and caused reduction of mRNA in the anterior and posterior ends of the embryo.

Mid tailbud embryo probed for Ci-ETR expression (no picture available in the article) following the injection of Ci-Rga-MO.

Ci-ETR was still expressed in the nervous system for 92% of embryos tested.

Lateral view of a mid tailbud stage embryo probed with ETR expression. Expression of tissue-specific differenciation markers in morpholino oligonucleotide-injected embryos at the stage corresponding to the mid tailbud stage. Embryos injected with MO against cieg007f18.

Original annotation: in the cieg007f18 knockdown embryos, whole-mount in situ hybridization showed that neural tissue was reduced.

Lateral view of a mid tailbud stage embryo probed with Ci-ETR (neural tissue) expression. Expression of tissue-specific differenciation markers in morpholino oligonucleotide-injected embryos at the stage corresponding to the mid tailbud stage.

Original annotation Embryos injected with MO against cilv006h21 as classified as " ball" phenotype: they developped into spherical-shaped cell masses, like balls. In these embryos, detection of the tissue specific markers indicated thaht almost the major tissues were lost or reduced.

Lateral view of a mid tailbud stage embryo probed with Ci-ETR (neural tissue) expression. Expression of tissue-specific differenciation markers in morpholino oligonucleotide-injected embryos at the stage corresponding to the mid tailbud stage.

Original annotation Embryos injected with MO against cieg028g04 as classified as " irregular" phenotype: embryos formed an irregular cellular mass. The a/p axis and trunk and tail could not be distinguished.

Lateral view of a mid tailbud stage embryo probed with Ci-ETR (neural tissue) expression. Expression of tissue-specific differenciation markers in morpholino oligonucleotide-injected embryos at the stage corresponding to the mid tailbud stage.

Original annotation: Embryos injected with MO against cieg009p15 as classified as " irregular" phenotype: embryos formed an irregular cellular mass. The a/p axis and trunk and tail could not be distinguished.

Lateral view of a mid tailbud stage embryo probed with Ci-ETR (neural tissue) expression. Expression of tissue-specific differenciation markers in morpholino oligonucleotide-injected embryos at the stage corresponding to the mid tailbud stage.

Original annotation: Embryos injected with MO against cieg039g16 is classified as " a/p axis" phenotype: embryos were extended along the anterior-posterior (a/p) axis, but did not form normal tadpoles, with head and tail being indistinguishable.

Lateral view of a mid tailbud stage embryo probed with Ci-ETR (neural tissue) expression. Expression of tissue-specific differenciation markers in morpholino oligonucleotide-injected embryos at the stage corresponding to the mid tailbud stage.

Original annotation: Embryos injected with MO against cicl017h21 is classified as " a/p axis" phenotype: embryos were extended along the anterior-posterior (a/p) axis, but did not form normal tadpoles, with head and tail being indistinguishable.

Lateral view of a mid tailbud stage embryo probed with Ci-ETR (eneural tissue) expression. Expression of tissue-specific differenciation markers in morpholino oligonucleotide-injected embryos at the stage corresponding to the mid tailbud stage.

Original annotation: Embryos injected with MO against ciad033i21 is classified as " flat" phenotype: eggs develop into flattened larvae and they lost the expression of markers for endoderm, notochord and mesenchyme.

Lateral view of a mid tailbud stage embryo probed for ETR expression in which FGF9/16/20 was overexpressed using electroporation with the pFoG driver. FGF9/16/20 is sufficient to induce ectopic midline and ESNs formation.

Lateral view of a mid tailbud stage embryo probed for ETR expression in which lefty was overexpressed using electroporation with the pFoG driver. Lefty prevents dorsal midline formation.

Lateral view of a mid tailbud stage embryo probed for ETR expression in which the ERK pathway has been blocked from the 16-cell stage. Dorsal ETR expression was abolished. Control sibling embryo shows the wild type pattern of the gene.

Lateral view of a mid tailbud stage embryo probed for ETR expression in which the ERK pathway has been blocked from the late 32-cell stage. Treatment at this stage has no effect on this neural marker. Control sibling embryo shows the wild type pattern of the gene.

Lateral view of mid tailbud stage embryo, injected with Ci-C10orf11 MO. Effects of functional suppression of Ci-C10orf11 on differenciation of neural cells. Differentiation of neural cells was assessed by expression of Ci-ETR.

Original annotation:
Ci-ETR expression was detected widely in Ci-C10orf11 knock-down embryos.

Lateral view of mid tailbud stage embryo, injected with Ci-PGAP1 MO. Effects of functional suppression of Ci-PGAP1 on differenciation of neural cells. Differentiation of neural cells was assessed by expression of Ci-ETR.

Lateral view of mid tailbud stage embryo, injected with Ci-ZF278 MO. Effects of functionnal suppression of Ci-ZF278 on differenciation of neural cells. Differenciation of neural cells was assessed by expression of Ci-ETR.

Original annotation:
The differentiation of neural cells was not completely inhibited in Ci-ZF278 knock-down embryos.

Lateral view of mid tailbud stage embryo, injected with Ci-Spatial MO. Effects of functional suppression of Ci-Spatial on differenciation of neural cells. Differentiation of neural cells was assessed by expression of Ci-ETR.

Original annotation:
The differentiation of neural cells was blocked in embryos with knock-down of Ci-spatial.

Lateral view of mid tailbud stage embryo, injected with Ci-FLJ10634 MO. Effects of functional suppression of Ci-FLJ10634 on differenciation of neural cells. Differentiation of neural cells was assessed by expression of Ci-ETR.

Original annotation:
The differentiation of neural cells was not completely inhibited in Ci-FLJ10634 knock-down embryos.

Lateral view of mid tailbud stage embryo, injected with Ci-Beta Catenin MO. Effects of functional suppression of Ci-Beta Catenin on differenciation of neural cells. Differentiation of neural cells was assessed by expression of Ci-ETR.

Original annotation:
Differentiation of neural cells occurred.

Double morphants of Ascl.b and Tox. Original annotation: expression almost completely lost in ESNs. Note that three spots seen near the tip of the tail are cells of the nerve cod, but not ESNs.

Tailbud stage embryo probed for Celf3.a expression, injected with Msx MO.
Original annotation : expression is downregulated in ESNs except in a few cells near the tip of the tail.

Overexpression of Msx in tailbud stage embryo probed for Celf3.a expression. Original annotation : Expression of Celf3.a, which marks ESNs is upregulated in embryos with Msx overexpression.
Aniseed annotation : There is ectopic expression in the tail epidermis. It seems that expression in tail nerve cord are downregulated.

Tailbud stage embryo probed for Celf3.a expression, injected with Tox MO. Original annotation : In some embryos, expression of Celf3.a is not completely lost, and several prospective ESNs express these genes.

Tailbud stage embryo probed for Celf3.a expression, injected with Tox MO. Original annotation :Expression is downregulated in morphant embryos.

Overexpression of Ascl.b in tailbud stage embryo probed for Celf3.a expression.
Original annotation :expression is seen in spots throughout the entire epidermal region of embryos

Lateral view of late a tailbud embryo showing continued expression after inhibition of MEK signalling by PD184352 treatment at the 8 cell stage. Expression is lost from the a line and maintained in the A line cells. Expression is probably found throughout both the A7.4 and A7.8 lineages as a result of the conversion of the visceral ganglion and nerve cord into a posterior sensory vesicle identity. The notochord precursors are also converted to the same post SV identity. The ventral midline b line cells retain expression in the CENs.

Expression of ETR at late tailbud stage, following the injection of FOG::Noggin construct.
The numbers show the total number of embryos analysed (n) and the percentage of embryos with staining in both dorsal and ventral epidermal sensory neurons.

Late tailbud stage embryo probed for Ci-ETR expression following egg micro-injection of a cieg003h01-MO.

Treatment with the gamma-secretase inhibitor DAPT, at early neurula stage results in varying degrees of midlines epiderman cell replacement by ETR-positive CESN precursors.

Treatment with the gamma-secretase inhibitor DAPT, at early tailbud stage results in varying degrees of midlines epiderman cell replacement by ETR-positive CESN precursors.

Treatment with the gamma-secretase inhibitor DAPT, at late neurula stage results in varying degrees of midlines epiderman cell replacement by ETR-positive CESN precursors.

Late tailbud stage embryo probed for Ci-ETR expression following egg micro-injection of a citb018a09-Mo.

"Overexpression of Klf1/2/4 using the pFog driver led to inhibition of Etr."
Embryos are in lateral views with anterior to the left and dorsal to the top.

« Overexpressing Msxb using the pFog driver led to Etr ectopic expression in lateral epidermis at late tailbud stages compared to controls. »
Embryos are in lateral views with anterior to the left and dorsal to the top.

"Dll-C overexpression inhibited Etr."
Embryos are in lateral views with anterior to the left and dorsal to the top.

Lateral view with anterior to the left of a late stage embryo probed for Ci-etr expression after DEAB treatment (150μM) from the early gastrula stage to the late neurula stage.
Original annotation: A decrease of anterior vCENs in the neurogenic midline region.

Lateral view with anterior to the left of a late stage embryo probed for Ci-etr expression after bFGF treatment (10ng/ml) at the early gastrula stage.
Original annotation: Expression was repressed in the anterior vCENs.

Lateral view with anterior to the left of a late stage embryo probed for Ci-etr expression after U0126 treatment (10μg/ml) at the early gastrula stage.
Original annotation: Expression was lost in the posterior vCENs.

Lateral view with anterior to the left of a late stage embryo probed for Ci-etr expression after LiCl treatment a Wnt pathway activator (300mM) at the early gastrula stage.
Original annotation: Expression was repressed in the anterior vCENs.

Lateral view with anterior to the left of a late stage embryo probed for Ci-etr expression after injection of an epidermally-targeted dominant-active form of b-catenin (pFT::∆N-b-catenin treatment) at the early gastrula stage.
Original annotation: Expression was repressed in the anterior vCENs.

Lateral view with anterior to the left of a late stage embryo probed for Ci-etr expression after retinoic acide treatment (1.5μM) from the early gastrula stage to the late neurula stage.
Original annotation: An increase of anterior vCENs in the neurogenic midline region and a loss of posterior vCENs was observed.

Lateral view of a larval stage wild type embryo probed for ETR expression. Overexpression of ADMP, using electroporation with the pFOG driver, suppressed lateral identity and induced midline identity and ESN formation.

Lateral view of a larval stage wild type embryo probed for ETR expression. NOGGIN overexpression abolishes ventral midline formation.