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First published online 7 March 2007
doi: 10.1242/dev.003939


Development 134, 1491-1497 (2007)
Published by The Company of Biologists 2007


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Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos

Vincent Picco, Clare Hudson and Hitoyoshi Yasuo*

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


Figure 1
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Fig. 1. A binary choice between notochord and neural fates in ascidian embryos. All embryos are viewed from the vegetal pole with the anterior side up. Because ascidian embryos are bilaterally symmetrical, pairs of blastomeres will be referred to by their blastomere name (e.g. A6.4), rather than as `a pair of'. A4.1 (green), located in an anterior-vegetal position of the eight-cell-stage embryo, divides twice to generate four cells (outlined by green dotted line) in the 24-cell-stage embryo. These four cells consist of two mother cells of notochord/neural precursors, named A6.2 and A6.4 (red), one endoderm/mesenchyme mother cell and one endoderm precursor. From the 24- to 32-cell stages, no cell divisions take place in the A4.1-lineage. At the late 32-cell stage, the cells in the A4.1 lineage enter mitosis and each of the A6.2 and A6.4 mother cells divides along the anterior-posterior axis to give rise to one notochord (orange) and one neural (yellow) precursor at the 44-cell stage. Each of the precursors then divides in the medial-lateral direction to generate four notochord and four neural precursors in the 110-cell-stage embryo. Therefore, the A4.1 cell generates four notochord and four neural precursors at the 110-cell stage, whereby each of the A6.2 and A6.4 mother cells gives rise to two notochord and two neural precursors. The anterior-animal cell, a4.2, of the eight-cell-stage embryo, and its descendents, are coloured in blue.

 

Figure 2
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Fig. 2. Signals from the anterior-animal cells induce neural fate and suppress notochord fate. (A-J) Expression of a notochord-marker, Ci-Bra (A-E), and a neural marker, Ci-ETR (F-J), analysed at the 110-cell stage by in situ hybridisation in embryos and partial embryos derived from isolated cells, as indicated. (K,L) Histograms showing the average numbers of cells positive for Ci-Bra (magenta) and Ci-ETR (blue) in partial embryos as indicated on the left of the graphs. The data are expressed as mean±s.e.m. Significant differences compared to the results obtained by A4.1 isolation (for K) or by A6.2 or A6.4 isolation at the 24-cell stage (for L) were evaluated by the Student's t-test and indicated with asterisks (P<0.01). n, total number of samples analysed. Scale bars: 25 µm.

 

Figure 3
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Fig. 3. Spatial expression-profile of ephrin-Ad. (A-D) The expression pattern of the Ci-ephrin-Ad gene is shown in animal-pole (A and C; anterior up) and lateral (B and D; anterior to the left) views at the stages indicated. The position of the A6.2 blastomere is indicated by a red line in D.

 

Figure 4
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Fig. 4. Ephrin-Ad provides the notochord-suppressing signals from anterior-animal cells. Expression of Ci-Bra (A-D) and Ci-ETR (E-H) at the 64- and 110-cell stages, respectively, following the treatments indicated above the panels. The average number of cells positive for each marker gene is indicated in each panel, together with the total number of samples analysed. Embryos in A,C and D are slightly tilted in order to place the notochord and neural precursors in the same focal plane. Schematics in B and F represent vegetal-pole views of the 64- and 110-cell-stage embryos, respectively, with notochord in orange and neural cells in yellow. Black dots mark cells positive for Ci-Bra (in B) and Ci-ETR (in F) in the control embryo. dnEph3-RNA injection gave similar results to ephrin-Ad-MO injection (7.2 cells for Ci-Bra, n=34; 1.2 cells for Ci-ETR, n=22).

 

Figure 5
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Fig. 5. Forward ephrin-Ad-Eph signals attenuate activation of ERK1/2. (A) Western blot analysis to detect activated ERK1/2 using anti-dpERK1/2 (top panel) in 44-cell-stage embryos following the treatment indicated. Lower panel shows the total amount of ERK1/2 protein, as a loading control. (B-D) Activation of ERK1/2 was detected by immunohistochemistry with the same antibodies in 44-cell-stage embryos following the treatments indicated above the panels. Orange arrows point to notochord precursors and yellow arrows to neural precursors. Histograms below each panel show the number of embryos obtained exhibiting the pattern schematised in the grid below. The grid represents the four neural (upper) and four notochord (lower) precursors. ERK activation is represented by a black dot, with very weak detection indicated by a grey dot.

 

Figure 6
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Fig. 6. Ephrin-Ad acts upon the notochord/neural mother cells. (A) Experimental strategy. Eggs were injected with ephrin-Ad-MO, fertilised and cultured until the late 32-cell stage, when the notochord/neural mother cells were isolated. The isolated mother cells were further cultured until the equivalent of the early gastrula stage, when they were fixed for in situ hybridisation analyses for the expression of Ci-Bra and Ci-ETR. At this stage, the resultant partial embryos consist of four cells. (B) Histograms showing the average numbers of cells positive for Ci-Bra (magenta) and Ci-ETR (blue) in partial embryos as indicated on the left of the graphs. The data are expressed as mean±s.e.m. Significant differences compared to the results obtained from the control were evaluated by the Student's t-test and indicated with asterisks (P≤0.01). n, total number of samples analysed.

 





© The Company of Biologists Ltd 2007