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	Figure 1. CESNs and Tail Epidermis Medio-Lateral Patterning(A–C) β-tubulin immunostaining showing CESN projections in the fin tunic. Confocal images projection with nuclei in red (propidium iodide) (C).(D and E) Acetylated α-tubulin showing the proximal part of the projections from paired CESNs (Acetylated α-tubulin in green, DAPI in blue, phalloidin in red).(F) 3-D projection extracted from a time lapse movie of an embryo injected with H2B-YFP mRNA and Fast Green (red). Trunk nuclei are green, while tail nuclei are colored to highlight the longitudinal rows made of single cell.(G and H) Schematic representation of the tail medio-lateral patterning (medial row, purple; medio-lateral row, yellow; lateral row, blue).(I–P) Expression patterns of tail epidermis markers at the mid-tailbud stage. Lateral view (I–L and P). Ventral view (M–O). Anterior is to the left. DAPI staining of nuclei in (M–P).
	Figure 2. Cell Lineage of the Tail Epidermis in Ciona (A) Color-coding illustrates the clonal base of dorso–ventral regionalization of tail epidermis. Pink, dorsal midline; light yellow, dorso–lateral domain; blue, lateral domain; yellow, ventro-lateral domain; purple, ventral midline. For a detailed description of each 110-cell stage b–line blastomere's contribution to the tail epidermis, see Figure S1. (B–E) composition of four representative clones derived from DiI-labelled epidermal precursors.(C) a lateral domain clone consists of 16 epidermal cells.(B and E) Dorsal (B) and ventral (E) midine clones consist of both epidermal cells and CESN pairs (green arrowheads). See Table S2 for a quantification of midline clone composition. (B) The progeny of a 110-cell stage dorsal precursor only contributes to the medial domain.(D) The progeny of a 110-cell stage ventral precursor contributes to both medial and medio-lateral domains.(E) A single-cell division segregates the ventral medial and medio-lateral domains.
	Figure 3. bFGF and BMP4 Induce Midline Fate and ESN Formation in Isolated b-Line Explantsb4.2 Blastomeres were isolated at the eight-cell stage and treated with proteins from the 16-cell stage.(A–D) Acetylated α-tubulin immunostaining at larval stage: CNS and PNS structures are labelled. Arrowheads indicate ESNs.(E–P) Molecular marker expression at early tailbud stages: midline marker KLF1/2/4 (E–H), lateral and medio-lateral marker citb003j16 (I–L), and tail nerve cord marker cilv038e16 (M–P). Top row, whole embryos (A,E,M) lateral view, (I) dorsal view, anterior to the left.Second row, control b4.2 blastomeres treated with BSA.Third row, b4.2 blastomeres treated with bFGF protein.Bottom row, b4.2 blastomeres treated with BMP4 protein.
	Figure 4. The FGF and Nodal Pathways Control Dorsal Midline and CESN Formation(A) bFGF-treated embryos show ectopic expression of the midline marker msxb at the late neurula stage when the treatment starts at the 16-cell or early 32-cell stages. Embryos do not respond to bFGF at the 64-cell stage. (B) Blocking Erk activity with the pharmacological inhibitor U0126 from the 16-cell stage abolishes dorsal KLF1/2/4 and ETR expression at tailbud stages, while treatment from the late 32-cell stage has no effect on these markers. FGF9/16/20 is sufficient to induce ectopic midline and ESNs formation (arrow), while Lefty prevents dorsal midline formation. (C) FGF9/16/20 MO–injected embryos show a loss of dorsal KLF1/2/4 expression while embryos injected with a control MO are not affected. (Lateral view, anterior to the left, dorsal to the top in all panels).
	Figure 5. The BMP Pathway Controls Ventral Midline and CESNs Formation(A) BMP4-treated embryos show ectopic expression of the midline marker KLF1/2/4 at the tailbud stage when the treatment starts at the 110-cell stage. Embryos do not respond to BMP4 at the early neurula stage. (B) ADMP overexpression transforms the entire tail epidermis into KLF1/2/4-positive tissue containing ETR-positive neurons. NOGGIN overexpression abolishes ventral midline formation. (C) The BMP ligand ADMP is expressed in B-line midline vegetal cells (blue on scheme) underlying the ventral midline precursors (pink on scheme) at the mid-gastrula stage. ADMP MO–injected embryos do not express KLF1/2/4 ventrally. (Lateral view, anterior to the left, dorsal to the top in all panels except in C for admp expression pattern: anterior to the top, vegetal view (first image), sagittal section (second image and scheme)).
	Figure 6. Delta/Notch Signalling Controls the Number of CESNs within Neurogenic Midlines(A,B,E,G) Electroporation of the construct pFOG::Venus (a bright YFP) has no effect on the midline domain and the CESNs, respectively, revealed by the KLF1/2/4 and β-thymosin-like probes. The expression levels of KLF1/2/4 at mid- to late tailbud stages (E) are lower than at mid-tailbud stages (A). (C and D) Electroporation of the dominant-negative construct pFOG::Venus-Su(H)DBM does not affect the expression of KLF1/2/4 but leads to an increase in the number of β-thymosin-like-positive cells. (F and H) Electroporation of pFOG::Delta2 results in a loss of β-thymosin-like-positive cells, without affecting KLF1/2/4 expression. (I and J) The construct p10.27::LacZ preferentially drives expression of β-galactosidase in the ventral tail epidermis (I), without affecting the development of CESNs (J).(K and L) Electroporation of p10.27::NICD, carrying an active form of Notch, does not affect the formation of the midline domain (K), but leads to a loss of CESNs in the ventral midline (arrow in L).(M–P) Treatment with the γ-secretase inhibitor, DAPT, at various developmental stages results in varying degrees of midline epidermal cell replacement by ETR-positive CESN precursors. e.neur., early neurula; l.neur., late neurula; and e.tbud, early tailbud.
	Figure 7. A Model for CESN FormationThe tail epidermis derives from the posterior animal b4.2 blastomeres. At the 32-cell stage, FGF9/16/20 induces Nodal and dorsal midline identity in the b6.5 blastomere. At the mid-gastrula stages, ADMP induces ventral midline fate in the overlying epidermis. These first inductive processes lead to a medio-laterally patterned epidermis comprising eight rows of cells in cross section. Finally, Notch signalling controls the number of CESNs in the midline through lateral inhibition.

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