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First published online July 11, 2006
doi: 10.1242/10.1242/dev.02466

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A signalling relay involving Nodal and Delta ligands acts during secondary notochord induction in Ciona embryos

Biologie du Développement, UMR 7009 CNRS/Universite Pierre et Marie Curie (Paris VI), Observatoire Océanologique, F-06230 Villefranche-sur-Mer, France.

View larger version (59K): [in a new window]   Fig. 1. Expression of notochord and endoderm markers following inhibition of ALK4/5/7 with SB431542. Embryo treatment is indicated at the top of the panels and the marker analysed is indicated at the left of the panels. Ci-Bra was analysed at the 64-cell stage, Ci-Titf at the early gastrula stage and Ci-Noto1 and Ci-Gataa at the early tailbud stage. For the top panel, the average number of cells expressing Ci-Bra is indicated; n=number of embryos analysed. For the rest of the panels, the numbers indicate the number of embryos expressing a given gene/total number of embryos analysed.

View larger version (50K): [in a new window]   Fig. 2. Nodal signalling is required for secondary notochord fate. (A-C) Embryo treatment is indicated above the panels. The marker analysed is shown on the left of the panels. For the graphs, embryo treatment is indicated on the x-axis and percentage of embryos on the y-axis. n=total number of embryos analysed. (A) Expression of Ci-Bra at the early gastrula stage and Ci-Noto1 at the early tailbud stage following inhibition of Nodal signalling. The schematic embryo, in a vegetal pole view, shows the positions of the primary and secondary notochord precursors (in blue) at the 110-cell stage. Arrowheads indicate the secondary notochord lineage. The insert in the control Ci-Noto1 panel is a cleaving embryo to indicate the stage at which the analysis was carried out. The graphs show the percentage of embryos showing expression of the gene indicated on the left, in one or two secondary notochord precursors. (B) Ablation of b5.3 on the right hand side (rb5.3) leads to an inhibition of Ci-Bra expression in the secondary notochord precursor on the ablated side. The schematic drawing of a 16-cell stage embryo, in animal pole view, shows the position of the b5.3 blastomere. The graph shows the percentage of secondary notochord precursors expressing strong or weak Ci-Bra on the left-(L) or right-(R) hand side of the embryo at the early gastrula stage. (C) Expression of Ci-Twist-like 1 at the early gastrula stage following inhibition of Nodal signalling. The graph shows the percentage of embryos expressing Ci-Twist-like 1 in one or both blastomeres for the lineages indicated in the colour scheme (see key). The schematic embryo on the left shows the positions of these blastomeres at the 110-cell stage, using the same colour scheme. The arrows indicate the secondary notochord precursors.

View larger version (41K): [in a new window]   Fig. 3. The temporal requirement of Nodal signalling for Ci-Bra expression in the secondary notochord precursors at the early gastrula stage. (A) Embryos were treated with SB431542 at different developmental time points, indicated along the x-axis of the graph. l32 corresponds to the late 32-cell stage. The percentage of embryos expressing Ci-Bra in one or two B8.6 blastomeres is indicated on the y-axis. A representative embryo for each time point is shown below the graph. (B) Schematic drawings of embryos from the late 32-cell stage to the 110-cell stage. The secondary notochord lineages are shown in blue. The name of the secondary notochord precursor at the different stages and the stage and orientation of the embryos in the drawings is indicated below each drawing. Nodal-expressing blastomeres are marked with a black dot for those with strong expression and a grey dot for those with weak expression. Black bars connecting blastomeres indicate their sister cell relationship. Below the drawings is shown a cell lineage tree of the B6.2 presumptive secondary notochord precursor from the 32-cell stage until the generation of the secondary notochord precursor at the 110-cell stage. Blastomeres containing notochord fate are outlined in blue.

View larger version (50K): [in a new window]   Fig. 4. Selective inhibition of the reception of either Nodal or Delta signals in one of the four founder cells of the eight-cell embryo. (A) Experimental design showing a vegetal pole view of the eight-cell stage embryo; eight-cell stage embryos were injected with tALK4/5/7 mRNA to block Nodal signal reception, Su(H)DBM mRNA to block Delta/Notch signal transduction or GFP mRNA as a control. mRNAs were selectively injected into either the A4.1 or the B4.1 blastomere on the right hand side and then analysed for Ci-Bra expression at the early gastrula stage. (B) A graph showing the percentage of embryos (y-axis) with strong or weak Ci-Bra expression in the B8.6 blastomere on the left-(L) or right-(R) hand side of the embryo. Injected RNAs and injected blastomere names are indicated on the x-axis. n=total number of embryos analysed. (C) A representative embryo for each of the treatments.

View larger version (45K): [in a new window]   Fig. 5. Ci-Delta2 expression at the 64-cell stage is a target of Nodal signalling. (A-C) Embryo treatment is indicated above the panels. n=total number of embryos analysed. (A) Expression of Ci-Delta2 at the 64-cell stage. The expression pattern is represented by schematic drawings on the right of each embryo panel. Ci-Delta2 is expressed in A7.8 (orange), A7.6 (mauve) and b7.10/b7.9 (green). On the schematic drawings, the secondary notochord precursor is marked by a blue dot. The graph shows the percentage of embryos (y-axis) showing expression in at least one blastomere for each of the different lineages (indicated by the colour scheme), following the treatment shown on the x-axis. (B) A graph showing the percentage of embryos (y-axis) in which Ci-Delta2 expression was detected in the different lineages on the left-(L) or right-(R) hand sides on the embryo following the treatments indicated on the x-axis. The colour scheme is the same as in A. Consistent with the observation that Ci-Bra and Ci-Nodal was sometimes recovered following b5.3 ablation, we observed expression of Ci-Delta2 in the trunk lateral cell precursor on the ablated side in 11% of embryos when analysed at the 76-cell stage (n=53). (C) A representative embryo for each of the treatments shown in B.

View larger version (66K): [in a new window]   Fig. 6. Ci-Delta2 encodes a Delta-like molecule. (A) Alignment of the putative DSL domain of Ci-Delta2 with DSLs from other Delta proteins. Ci, Ciona intestinalis; lv, Lytechinus variegatus (green sea urchin); Z, zebrafish (Danio rerio); C, chick; Dm, Drosophila melanogaster; spider, Cupiennius salei. The DSL domain of Ci-Delta2 was identified using the SMART programme, but gave a score less significant than the required threshold to be confidently predicted as a DSL domain using this programme (Letunic et al., 2004; Schultz et al., 1998). (B) Ci-Hes-b expression at the early gastrula stage is a target of Ci-Delta2. Expression of Ci-Hes-b is shown following the treatment indicated above the panels. Ectopic expression was sometimes observed in the mesenchyme lineages following injection of dnDel2 (far right). The graph shows the percentage of embryos (y-axis) showing expression in at least one cell of the A-line (A8.16, A8.8, in grey) and B-line (B8.6, in blue) following the treatments indicated on the x-axis. n=total number of embryos examined. The schematic drawing shows the positions of these blastomeres. Dots indicate the blastomere derivatives that were expressing Ci-Delta2 at the 64-cell stage. At the early gastrula stage, Ci-Delta expression is downregulated in A7.6, while that in A8.15/A8.16 (daughters of A7.8) becomes stronger (Hudson and Yasuo, 2005).

View larger version (49K): [in a new window]   Fig. 7. Delta2/Notch signalling is required for secondary notochord induction. (A-C) The embryonic treatment is indicated above the panels and the marker analysed on the left. n=total number of embryos examined. (A) Expression of Ci-Bra at the early gastrula stage and Ci-Noto1 at the early tailbud stage following inhibition of Delta2/Notch signalling. The graphs show the percentage of embryos (y-axis) expressing Ci-Bra or Ci-Noto1 in one or two B8.6 blastomeres following the treatments indicated on the x-axis. (B) Ablation of A6.3 on the left-hand side (lA6.3) leads to a loss of Ci-Bra expression in the secondary notochord precursor on the ablated side. The schematic drawing of a vegetal pole view 32-cell stage embryo shows the A6.3 blastomere (dark pink) relative to the presumptive secondary notochord precursor (blue). The graph shows the percentage of embryos (y-axis) expressing strong or weak Ci-Bra in B8.6 on the left-(L) or right-(R) hand side of the embryo, following ablation of A6.3 on the left-hand side. (C) Expression of Ci-Twist-like 1 at the early gastrula stage following inhibition of Delta signalling. The DAPT-treated embryo shown is tilted slightly (far right) in order to show more clearly the B8.6 blastomere (arrowhead), which is not expressing Ci-Twist-like 1. The graph shows the percentage of embryos (y-axis) expressing Ci-Twist-like 1 in at least one cell of the different lineages following the treatment indicated on the x-axis.

View larger version (27K): [in a new window]   Fig. 8. The role of Nodal and Delta2 during secondary notochord induction in Ciona embryos. The positions of blastomeres that are discussed in the text are indicated. Ci-Nodal signals are indicated by red arrows and Ci-Delta2 signals by green arrows. For each schematic drawing, the expression pattern of the gene indicated above it is shown by grey coloured blastomeres. Schematic drawings are vegetal pole views of an early 32-cell stage embryo, a 64-cell stage embryo and a 110-cell stage embryo, from left to right. Although Ci-Nodal expression is not detected until the late 32-cell stage, an early 32-cell stage embryo is shown so that the b6.5 blastomere can be seen on the vegetal pole view of the embryo (compare with Fig. 3B). The roles played by Ci-Nodal and Ci-Delta2 signals during secondary notochord induction during these stages is indicated below the drawings.

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