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First published online 29 August 2007
doi: 10.1242/dev.002352

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Sequential and combinatorial inputs from Nodal, Delta2/Notch and FGF/MEK/ERK signalling pathways establish a grid-like organisation of distinct cell identities in the ascidian neural plate

Developmental Biology Unit, Université Pierre et Marie Curie (Paris 6) and CNRS, Observatoire Océanologique, 06230 Villefranche-sur-Mer, France.

View larger version (35K): [in this window] [in a new window]   Fig. 1. The cell lineages and gene expression domains of the ascidian CNS. (A) Cell lineages of the CNS. Embryonic stages are indicated below the drawings. Blastomere names are indicated on the left half of the drawings. At the neural plate stage (neural plate shown only), blastomere names should be prefixed with an a9. for a-line and A9. for A-line, as indicated. Bars connecting blastomeres on the right half of the drawings indicate a sister cell relationship. A-line lineages are shown in yellow for medial precursors and tan for lateral precursors, with the secondary muscle precursor coloured in blue. a-line lineages are indicated in red (sensory vesicle) and pink (anterior epidermis and pharynx; because of the cell arrangement, these cells are considered part of the neural plate although they do not contribute to the CNS). In addition, cells from the b-line, positioned lateral to the A- and a-line neural plate, contribute to the dorsal part of the CNS (data not shown). The same colour code is used in all subsequent figures. At the neural plate stages, cells are organised into four columns (1-4) and six rows (I-VI), as indicated. (B) Schematic representation of the gene expression profiles in A-line neural lineages at early gastrula and neural plate stages. The right-hand-side A-line neural blastomeres of the bilaterally symmetrical embryo are represented; the position of the medial axis is indicated on the left. Bars above and below the schematics indicate gene expression for the markers used in this study (row II above, row I below). Position of the columns and rows are indicated (top and right). Sna, Snail; 07j15, cicl07j15; FGF8, FGF8/17/18; Chd, Chordin; FGF9, FGF9/16/20.

View larger version (77K): [in this window] [in a new window]   Fig. 2. Expression of various gene markers of the A-line neural cells following inhibition of Nodal signals. Embryo treatment is indicated above the panels and the marker analysed to the left of the panels. For control neural plate-stage embryos, nuclei are labelled with Hoechst (white) to allow the easy identification of individual cells. For this and subsequent figures, black dots on the schematics mark blastomeres expressing each given gene and, unless otherwise stated in the text, the vast majority of embryos exhibited bilateral staining. Expression of Ci-MRF and Ci-Tbx6b in A9.31 is indicated by an arrowhead on the right-hand side. For lateral markers, numbers indicate any expression over the total number of embryos analysed. For the medial marker cicl007j15, numbers indicate the mean number of cells (c) per embryo expressing a given gene. n, total number of embryos analysed.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Temporal dependency of early gastrula-stage A-neural markers to Nodal signal inhibition. Markers analysed are indicated on the left of the graphs. Graphs show the percentage of embryos with strong or weak expression in the lateral neural precursors (marked with arrowheads). Scoring was as follows: Ci-Snail, strong = at least two cells positive, weak = one cell positive or very weak expression in one to four cells; Ci-Ngn, strong = at least one cell positive, weak = at least one blastomere showing very weak expression; cicl007j15, strong = at least two lateral cells positive, weak = one lateral cell positive or weak expression in one to four lateral cells. For cicl007j15, only embryos with expression in one to four medial cells were included in the analysis. Numbers shown above the bars indicates the total number of embryos analysed. On the x-axis is shown the developmental time point when embryos were placed in inhibitor: C, control; 16, 32, 64, 76, 110 = cell stage.

View larger version (55K): [in this window] [in a new window]   Fig. 4. Expression of A-line neural markers at the early gastrula stage following Delta2/Notch inhibition or ectopic activation. (A) Expression of Ci-Delta2 at the 64- and 76-cell stages. Embryos and schematics are shown in lateral view with the vegetal pole to the left. Ci-Delta2-expressing blastomeres surround A7.8, the founder cell of columns 3 and 4, at the 64-cell stage, and surround A8.16, the precursor cell of column 4, at the 76-cell stage. Blastomere names are indicated. (B) Expression of markers, indicated on the left, at the early gastrula stage following the treatments indicated above the panels. For lateral markers, numbers indicate any expression seen in A-line neural cells over the total number of embryos analysed. The inserts for Ci-Delta2 expression are lateral views of embryos, showing that expression is slightly but consistently upregulated following Delta2/Notch inhibition, particularly in b-line cells. For cicl007j15, numbers indicate the mean number of positive cells (c) per embryo; n, total number of embryos analysed. Similar results were observed with dnDel2 injection (Ci-Snail, 35/35; Ci-Ngn, 5/48; cicl007j15, 3.2c; n=24). (C) Expression of markers, shown above the panels, following FOG::Delta2 electroporation. Numbers indicate: for Ci-Snail, the number of embryos showing expression represented by the panel over the total number of embryos analysed; for Ci-Hesb and Ci-Ngn, the number of embryos showing ectopic expression in A-line neural cells over the total number of embryos analysed. Ectopic expression of Ci-Hesb and Ci-Ngn was also sometimes observed in additional vegetal cells (data not shown). The insert on the bottom right of Ci-Hesb panel shows expression of Ci-Hesb in a control embryo.

View larger version (86K): [in this window] [in a new window]   Fig. 5. Expression of various neural plate markers following Delta2/Notch inhibition or ectopic expression. The marker analysed is indicated on the left and embryo treatment is indicated above the panels. Graphs show the percentage of embryos with ectopic, strong or weak expression according to the key indicated below. The same key is used for all subsequent figures. For Ci-Hesb and Ci-Chordin, `weak' means reduced expression compared with the majority of controls. For the remaining markers, `strong' means at least one A-line neural precursor cell with strong expression and `weak' means at least one cell with downregulated expression. Occasionally, following disruption of Delta2/Notch signalling, ectopic expression was seen for Ci-FGF8/17/18 and Ci-ephrinAb. This ectopic expression appeared in column 4. Following FOG::Delta2 electroporation (abbreviated to FOG::D2), Ci-FGF8/17/18 exhibited frequent unilateral expression. Of those scored positive, 49% expressed Ci-FGF8/17/18 on one side only (compared with 9% of control embryos from the same batches). Ectopic expression of Ci-COE following FOG::Delta2 electroporation appeared in column 3 (A9.30) in the majority of cases (see text for details) with 54% of these cases exhibiting unilateral A9.30 expression and 46% bilateral. Ectopic expression of Ci-MRF was observed only in column 3 (A9.29). Of those exhibiting ectopic expression, 66% had column 3 (A9.29) expression on one side only. In most cases, column 3 (A9.29) expression of Ci-MRF appeared slightly weaker than the expression in column 4 (A9.31). For Ci-FoxB, expression in both control and treated embryos was frequently (around half of those scored positive) observed on one side only. Arrowheads point to ectopic expression on the panels.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Analysis of neural plate markers following DAPT treatment at different embryological time points. Graphs show the percentage of embryos with strong, weak or ectopic expression. Scoring was carried out as indicated for Fig. 5. Numbers shown above the bars indicate the total number of embryos analysed. On the x-axis is shown the developmental time point when embryos were placed in DAPT inhibitor: C, control; 44, 76, 110 = cell stage; eG, early gastrula stage.

View larger version (81K): [in this window] [in a new window]   Fig. 7. FGF/MEK/ERK activity is required for row I versus row II fates. (A) Activation of ERK1/2 visualised by dpERK1/2 antibody staining. The schematics indicate the neural plate cells positive for ERK1/2 activation, with the rows of cells indicated. The cell outlines were estimated using the ERK staining image and a Hoechst-stained image of the same embryo (data not shown). The embryo on the left contains four rows of cells in the neural plate. At this stage, weak ERK1/2 activation can also be seen in the row III/IV precursors, which are not visible in the panel because of the orientation of the embryo. (B) Expression of row I markers following UO126 treatment from the early gastrula stage. Ci-Cdx is expressed in A9.31, A9.29 and A9.15 in control embryos (Imai et al., 2006). (C) Expression of row II markers following UO126 treatment from the early gastrula stage. Hoechst staining, shown on the right for Ci-COE and Ci-FGF8/17/18, confirms that ectopic expression in row I was seen in column 4 and column 3, respectively. (D) Injection of dnFGFRc mRNA into the A5.2 blastomere on the right-hand side. Descendants of A5.2 include those circled in red on the neural plate schematic. (Right) Numbers below the figures show the percentage of embryos that the panels represent and the total number of embryos analysed. Cont., control.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Expression of Ci-COE and Ci-MRF following UO126 treatment at different time points. Graphs show the percentage of embryos showing expression in A9.31 (row I/column 4). The key for the graph and scoring is the same as in Fig. 5. A sample of embryos was taken at the same time point that embryos were placed in inhibitor and analysed for Ci-Ngn expression to determine the percentage of embryos in each sample in which A8.16 had cleaved. These percentages are indicated on the graphs (arrows). On the x-axis: C, control; 110, 110-cell stage; eG, early gastrula stage; eG+30, 30 minutes after eG; eG+45, 45 minutes after eG. The minimum number of embryos analysed for each time point was 12 embryos for Ci-COE and 17 for Ci-MRF. This experiment was repeated two more times with similar results (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 9. Summary of the overlapping requirements of Nodal, Delta2 and FGF/MEK/ERK signalling pathways in the A-line neural plate.