Fig 1.
The anterior limit of Hox1 expression does not define a functional boundary between anterior FGF-sensitive and posterior FGF-insensitive somites.
Expression of Hox1 (lateral views, anterior to the left) and MLC (dorsal views, anterior to the left) at the L0 stage in control embryos (A, G), in RA treated embryos (B, H), in BMS009 treated embryos (C, I), in SU5402 treated embryos (D, J), in SU5402 and RA treated embryos (E, K) and in SU5402 and BMS009 treated embryos (F, L). All the treatments were performed at the blastula stage. The arrowheads indicate the anterior limit of Hox1 expression (A-F) or the anterior limit of the embryonic region with formed somites (G-L). (M) Graph presenting the percentage of the length of the embryo without Hox1 expression (dark grey) or without somites in the anterior region (light grey). The schematic embryos show how this percentage was calculated. (1) corresponds to the length of the anterior region without Hox1 expression, (2) corresponds to the total length of the embryo. (3) corresponds to the length of the anterior region without somite MLC expression. (4) corresponds to the total length of the embryo. Dark blue regions in the schematic embryos correspond to the territories expressing Hox1 (lateral view, anterior to the left) or MLC (dorsal view, anterior to the left) in SU5402 treated embryos. A one-way ANOVA analysis was undertaken and the result indicates that the means of the region without somites is not significantly different between the three treatments (SU5402, SU5402+RA and SU5402+BMS009) whereas the means of the region without Hox1 expression is significantly different between the three treatment conditions (SU5402, SU5402+RA and SU5402+BMS009). **P<0.003 (corrected p-value); Two samples Student t-test, n = 10 embryos. Error bars indicate s.e.m. Scale bars = 50μm.
Fig 2.
Interfering with RA signalling during posterior elongation does not affect somitogenesis.
Expression of MRF1 in L0 stage control embryos (A), and in embryos treated at the N3 stage with RA (B) or BMS009 (C) (dorsal views, anterior to the left). The expression shows that the number of formed somites is identical in treated and control embryos. Expression of Xlox and Tbx6/16 in L3 stage control embryos (D, G), and in embryos treated at the N3 stage with RA (E, H) or BMS009 (F, I) (side views, anterior to the left). The arrows indicate the position of the pigment spot. The double arrow lines indicate the size of the domain expressing Xlox or Tbx6/16. Enlargement of the photograph at the level of the pigment spot is presented for Tbx6/16 in situ hybridization on the top left of the panels. Scale bars = 50μm. Morphometric analysis of the expression domains of Xlox (J) and Tbx6/16 (K). Schematic larva with the domain of expression highlighted in blue-violet are presented (side view, anetrior to the left). (1) corresponds to the length of the embryo, posterior to the pigment spot, without Xlox expression. (2) corresponds to the length of the embryo with Xlox expression. (3) corresponds to the length of the posterior field of the embryo without Xlox expression. The percentage of the length of the field with Xlox expression was calculated as 2/(1+2+3)*100, the percentage of length of the anterior field without expression as 1/(1+2+3)*100 and the percentage of length of the posterior field without expression as 3/(1+2+3)*100 (J). One-way ANOVA analysis indicates that the the means of the percentage of length of the field with Xlox expression between the three conditions (control embryos, RA-treated embryos and BMS009-treated embryos) are significantly different as well as the means of the percentage of length of the posterior field without expression of Xlox (J). (4) corresponds to the length of the field posterior to the pigment spot showing Tbx6/16 expression. (5) correspond to the length of the posterior field of the embryo without Tbx6/16 expression. The percentage of length with Tbx6/16 expression was calculated as 4/(4+5)*100 (K). One-way ANOVA analysis indicates that the the means of the percentage of the length with Tbx6/16 expression are significantly different between the three conditions (control embryos, RA-treated embryos and BMS009-treated embryos). **P<0.005 (corrected p-value); *P<0,025 (corrected p-value); t-test, n = 3 embryos. Error bars indicate s.e.m.
Fig 3.
FGF and RA signals do not cross-talk during posterior elongation.
Expression of the FGF signalling pathway genes ER81/Erm/Pea3, Sprouty, FGFR, and of the ParaHox genes Cdx and Xlox in L1 control embryos (A, E, I, M, Q), and in embryos treated at the N3 stage with SU5402 (B, F, J, N, R), RA (C, G, K, O, S) or BMS (D, H, L, P, T) (lateral views, anterior to the left). For FGFR and Xlox, dorsal views of the tailbud region are also presented. Scale bars = 50μm.
Fig 4.
The asymmetry of somites depends on asymmetric Nodal expression and is not altered by RA signal pertubations.
Expression of Nodal in Omeprazole treated embryos at the N2 (A, B) and L0 (C-G) stages (dorsal views, anterior to the left). In wild type embryos expression of Nodal is similar to (A) and (C), and expression of Pitx is similar to (H) and (L). Expression of Pitx in Omeprazole treated embryos at the N2 (H-K) and L0 (L-O) stages (dorsal views, anterior to the left). The number of embryos showing these patterns of expression is indicated on each panel. (P) Graph presenting the number of embryos at the N2 and L0 stage presenting expression in the posterior mesoderm of Nodal or Pitx only on the left side, on both sides, or on neither side (l/r = left/right, n.e. = not expressed) (P). (Q-R) Texas Red X-Phalloidin (red) and anti-acetylated tubulin (green) labelled control L3 larvae (Q) and Omeprazole treated larvae showing symmetric somites (R). The number of larvae showing symmetric somites after Omeprazole treatment is indicated. Texas Red X-Phalloidin labelling in control (S), SB431552-treated (T), SB505124-treated (U), RA-treated (V) and BMS-treated (W) L3 stage larvae. The pictures represent dorsal views, with anterior to the left, focused on the central region of the larva. The dotted lines join the boundaries between somites on the right and left sides. Scale bars = 50μm.
Fig 5.
Evolutionary scenario for somitogenesis in chordates.
Evolutionary relationships among the three chordate clades are presented, as well as a schematic view of the morphology of embryos of the putative ancestor of chordates, cephalochordates, tunicates and vertebrates (all dorsal views except tunicates for which a lateral view is schematized) during posterior elongation. We propose that the ancestral chordate embryo was morphologically close to amphioxus and that the asymmetry of somite formation was under the control of Nodal. After the divergence of cephalochordates, an opposition between the RA and FGF pathways was acquired. In tunicates, the somitogenesis process was lost, probably as an adaptation to a reduced number of embryonic cells. In the vertebrate lineage, the opposition between RA and FGF gained importance in parallel to the acquisition of the PSM as an intermediate zone between both signals. The recruitment of RA in the control of somitogenesis permitted the acquisition of symmetry through the buffering of the left/right machinery controlled by Nodal. The Wnt pathway, indicated with a question mark, is a good candidate as a signal ancestrally controlling posterior elongation in chordates although up to now there are no functional data supporting this hypothesis.