Figure 1.
Cell lineages of the ascidian central nervous system.
At each developmental stage, cells contributing to the central nervous system are colored according to their origin in the 8-cell stage embryo. a-line CNS (red) originates from anterior animal blastomeres (a4.2 pair) and forms the anterior sensory vesicle. A-line CNS (orange) originates from anterior vegetal blastomeres (A4.1 pair) and forms the posterior sensory vesicle, the visceral ganglion and the tail nerve cord (only the ventral and lateral parts for the latter two regions). b-line CNS (purple) originates from posterior animal blastomeres (b4.2 pair) and forms the dorsal part of visceral ganglion and tail nerve cord. Drawings for 8-cell to early gastrula stages: lateral view with animal to the top and anterior to the left (top row) and animal view with anterior to the left (bottom row). Drawings for tailbuds are lateral views with dorsal to the top and anterior to the left and a cross-section through the tail showing the four cells originating from two distinct lineages (A- and b-line).
Figure 2.
FGF and Nodal signaling are required for posterior neural tissue formation.
A) Schematic representation of b6.5 lineage history with representation of embryos, cell lineage and gene expression at different stages. The different tissues and precursors are color coded: vegetal cells in grey, anterior (a-line) ectoderm in white, posterior (b-line) ectoderm in yellow, dorsal tail epidermis in green and dorsal tail nerve cord in purple. Embryos are in animal view (top row) or lateral view (bottom row) with anterior to the left. B) Expression of early and late b6.5 lineage markers when FGF-Erk and Nodal signaling pathways are disrupted. Msxb which is normally expressed in the four daughter cells of the b6.5 blastomere (Bi) is not expressed in U0126-treated (Biv) and SB431542-treated (Bvii) embryos. Animal views of Msxb at early gastrula stages (stages 10/11) (Bi, iv, vii). Schematic animal views of stage 10 embryos are depicted as insets in Msxb panels: anterior ectoderm in white, posterior ectoderm in yellow and gene expression in blue. Expression of Klf1/2/4 is lost in tail dorsal midline for both treatments (Bv and Bviii). The dorsal tail nerve cord marker KH.C7.391 is suppressed (Bvi and Bix). Lateral view with dorsal to the top and anterior to the left (Bii, iii, v, vi, viii and ix) at stage 19. Control DMSO-treated embryos (Bi-iii), U0126-treated embryos (Biv-vi) and SB431542-treated embryos (Bvii-ix). White arrows and arrowheads indicate sites with a loss of expression. C) Gene interactions revealed by loss-of-function data.
Figure 3.
Nodal acts downstream of FGF to posteriorize induced neural tissue.
Expression of posterior neural markers (Msxb (A), Delta2 (B), and Chordin (C)) and of the anterior neural marker Dmrt1 (D) in control embryos. FGF9/16/20 overexpression using the pFOG promoter via electroporation led to ectopic expression of Msxb (E) and Delta2 (F) throughout posterior ectoderm, of Chordin (G) through most of the ectoderm except the anterior-most part and of Dmrt1 (H) throughout anterior ectoderm at early gastrula stages (st. 10/11). These effects were suppressed by inhibition of Nodal signaling through Lefty overexpression (M-O) except for Dmrt1 (P). Overexpression of Lefty alone inhibited posterior marker expression (I-K) but did not affect expression of the anterior marker Dmrt1 (L). Overexpression of Nodal using the pFOG driver was sufficient to activate Msxb (Q) and Delta2 (R) in the neural plate, and Chordin (S) throughout the ectoderm. Ectopic Chordin expression was stronger in anterior ectoderm than in posterior ectoderm possibly reflecting the difference in expression levels between anterior and posterior expressing cells in control embryos. Dmrt1expression was downregulated (T). Combined overexpression of FGF9/16/20 and Nodal led to ectopic activation of Msxb (U) and Delta2 (V) in both anterior and posterior ectoderm. Under these conditions, Chordin was still expressed throughout the ectoderm but at weaker levels (W). Overexpression of Nodal downregulated ectopic activation in anterior ectoderm of Dmrt1 induced by FGF9/16/20 (X). Animal view with anterior to the top for all except insets in Q and R that show neural plate view with vegetal side to the left. For each panel a schematic animal view of stage 10 embryos depicts anterior ectoderm in white, posterior ectoderm in yellow and gene expression in blue.
Figure 4.
Otx is required for posterior neural tissue formation.
Overexpressing Otx in the ectoderm using the pFOG driver is sufficient to activate Msxb (B) and Delta2 (E) compared to control embryos (A, D) at stage 10. Upon injection of the Otx MO, Msxb (C) and Delta2 (F) expression is abolished at stage 10. The dorsal expression of the tail midline marker Klf1/2/4 is lost except in the posterior-most and ventral regions (I). The dorsal nerve cord marker KH.C7.391 expression is also suppressed (J). Control MO-injected embryos at stage 10 (A, D) and stage 19 (G, H). Animal view with anterior to the top (B, D-F). Vegetal view with anterior to the top (A and C). Lateral view with dorsal to the top, anterior to the left (G-J). White arrows and arrowheads indicate sites with a loss of expression. (K) Summary of gene interactions reported in this study and from previous studies [11], [29], [30], [35].
Figure 5.
The b6.5 line enhancers of Msxb and Delta2.
Schematic organization of tested enhancers depicting putative Otx (GATTA) (red bars), Fox (AAACA) (blue bars) binding sites and SBEs (AGAC) for Msxb (A) and Delta2 (F). Genomic browser view of gene loci with gene models (Msxb: KH.C2.957, Delta2: KH.L50.6), tested enhancers (grey bar), alignment profile of C. intestinalis and C. savignyi genomic sequences (pink) and ChiP-on-Chip data (green) [50] for Msxb (B) and Delta2 (G) (extracted from the Aniseed genome browser: http://www.aniseed.cnrs.fr/fgb2/gbrowse/ciona_intestinalis/ [70], and from the Ghost genome browser http://ghost.zool.kyoto-u.ac.jp/cgi-bin/gb2/gbrowse/kh/ [71]). Representative pictures for X-gal staining of electroporated embryos with the “msxb-b6.5 line” enhancer at stage 10 (C), stage 14 (D) and stage 16 (E), and with the “delta2-b6.5 line” enhancer at stage 10 (H), stage 14 (I) and stage 18 (J). Arrowheads indicate dorsal midline epidermis (blue), dorsal nerve cord (purple) and secondary muscle (black). Vegetal view, anterior to the top (C, H). Dorsal view, anterior to the left (D, I). Lateral view, dorsal to the top and anterior to the left (E, J). Additional staining was also observed in mesenchymal cells, a tissue highly permissive to transcriptional assays in Ciona [42].
Figure 6.
Otx, Fox and Smad putative binding sites control msxb-B enhancer activity.
A) Alignment of msxb-B sequences from C. intestinalis type A, C. intestinalis type B and C. savignyi. Putative transcription factor binding sites are in colored boxes as follows: canonical Fox (AAACA) in dark blue, canonical Otx (GATTA) in red, non-canonical Otx (GAATTA) in orange and SBE (AGAC) in yellow. B) The msxb-B enhancer is active in b6.5 derivatives as revealed by X-gal staining on late gastrula. Its activity is abolished upon injection of the Otx MO (C) or overexpression of Lefty (D). E) Schematic view of msxb-B enhancer and its mutated versions. Putative transcription factor binding sites position and orientation are represented by colored arrows with the same color code as in (A). Mutations are depicted by stars. The precise mutations are described in the main text and in figure S8. Transcriptional activity of the different enhancers was measured as the percentage of embryos with staining in the b6.5 derivatives at late gastrula stages (stage 14) (Table S1).
Figure 7.
A shared regulatory logic in Ciona intestinalis and Phallusia mammillata.
Schematic organization of tested enhancers with the same color code used in figures 4 and 5. Reporter gene activity is detected by X-gal staining after electroporation of “Ci-msxb-b6.5 line” enhancer (A, B), “Ci-delta2-b6.5 line” enhancer (C, D) and “Pm-msxb-b6.5 line” enhancer (E, F) into C. intestinalis (A, C, E) or P. mammillata (B, D, F) embryos. Transcriptional activity of the different enhancers, measured as the percentage of embryos with staining in the b6.5 derivatives, is detailed in Tables S1 and S2. In Phallusia mammillata embryos, Msxb (G) and Delta2 (J) are expressed in b6.5 derivatives. This expression is abolished upon inactivation of the FGF/MEK (H, K) or Nodal (I, L) signaling pathway. Dorsal view with anterior to the left (A-F). Animal view, anterior to the top (G-L).