Functions of the FGF signalling pathway in cephalochordates provide insight into the evolution of the prechordal plate

Abstract

The fibroblast growth factor (FGF) signalling pathway plays various roles during vertebrate embryogenesis, from mesoderm formation to brain patterning. This diversity of functions relies on the fact that vertebrates possess the largest FGF gene complement among metazoans. In the cephalochordate amphioxus, which belongs to the chordate clade together with vertebrates and tunicates, we have previously shown that the main role of FGF during early development is the control of rostral somite formation. Inhibition of this signalling pathway induces the loss of these structures, resulting in an embryo without anterior segmented mesoderm, as in the vertebrate head. Here, by combining several approaches, we show that the anterior presumptive paraxial mesoderm cells acquire an anterior axial fate when FGF signal is inhibited and that they are later incorporated in the anterior notochord. Our analysis of notochord formation in wild type and in embryos in which FGF signalling is inhibited also reveals that amphioxus anterior notochord presents transient prechordal plate features. Altogether, our results give insight into how changes in FGF functions during chordate evolution might have participated to the emergence of the complex vertebrate head.

Keywords: Amphioxus; Brachyury; Goosecoid; Head mesoderm; Notochord.

Fig. 1.

FGFR inhibition does not induce apoptosis. TUNEL assay (red) and DAPI (blue) staining in control, SU5402- and DNase I-treated embryos fixed at G6 (A-F), N1 (G-L), N2 (M-R), N4 (S-X), N5 (Y-D′) and T0 (E′-J′) stages. Dorsal views, except for T0 stage embryo pictures (lateral views). Anterior towards the left and dorsal towards the top in T0 stage embryo pictures. The inset shows that the TUNEL labelling does not coincide with the nuclei labelling. At least 10 embryos per stage were analysed and they all showed the same pattern. Scale bars: 50 µm.

Fig. 2.

Double in situ hybridization for Nodal with Hex, Netrin, Chordin, ADMP or Lhx2/9a. Double fluorescent in situ hybridization of Nodal and the endoderm marker Hex (A-H) and of Nodal and the dorsal axial markers Netrin (I-P), Chordin (Q-X), ADMP (Y-F′) and Lhx2/9a (G′-N′) in control and SU5402-treated embryos fixed at 15 hpf (G6) and 18 hpf (N1). No overlap is observed. Dorsal views and transverse views are shown for in situ hybridization data, with anterior towards the top and dorsal towards the top, respectively. Nuclei DAPI staining is in blue. The white arrowheads indicate the neural plate labelling observed for Netrin, Chordin and ADMP. At least five embryos per stage were analysed and they all showed the same pattern. Scale bar: 25 µm.

Fig. 3.

Nodal and Goosecoid are co-expressed in the anterior dorsal paraxial mesoderm, which also expresses Dmbx, after FGFR inhibition. Double in situ hybridization of Nodal and Goosecoid (A-J′) and of Nodal and Dmbx (K′-T″) in control and SU5402-treated embryos fixed at 15 hpf (G6) and 18 hpf (N1). Dorsal views and transverse views are shown for in situ hybridization data, with anterior towards the top and dorsal towards the top, respectively. Single-channel (DAPI in blue, Nodal in magenta, Goosecoid and Dmbx in green) and merged channel images are shown. For 18 hpf stage embryos, two transverse sections are presented: one at the level of the first somite pair (X,H′,H″,R″) and another one at the level of the second somite pair (Z,J′,J″,T″). The white arrowheads indicate the position of the first somite/presumptive somite pair in panels corresponding to 15 hpf embryos, and the position of the left first (C′-H′,C″-H″,M″-R″) or second somite region (I′,J′,I″,J″,S″,T″) in panels corresponding to 18 hpf embryos. At least five embryos per stage were analysed and they all showed the same pattern. Scale bar: 25 µm.

Fig. 4.

The presumptive anterior paraxial mesoderm cells are incorporated into the anterior notochord after FGFR inhibition. One representative embryo is presented for control (n=4/4) and SU5402-treated conditions (n=7/7). G4 stage control (A-C) and SU5402-treated (G-I) embryos after Kaede protein photoconversion in presumptive paraxial mesoderm and ventral epidermis. Images are blastopore views with dorsal towards the top. The fluorescence was later observed in the corresponding embryos at the T0 stage. (D-F,J-O) Orthogonal views are shown (xy below the main panel, yz on the right), and single channel as well as merged channel images are presented. For SU5402-treated embryo at T0, two orthogonal views are shown: one at the anterior level (J-L) and one at the posterior level (M-O). In controls (D-F), the red fluorescence (magenta) was observed in the ventral epidermis (arrowhead in F) and in the paraxial mesoderm (encircled by a white line in F) all along the antero-posterior axis. In SU5402-treated embryos (J-O), the red fluorescence was observed in the ventral epidermis (white arrowhead in L and O) as well as in the anterior notochord (encircled by a white line in L) and in the posterior somites (encircled by a white line in O). Scale bars: 50 µm.

Fig. 5.

Only the first somite pair integrates the anterior axial mesoderm after FGFR inhibition. (A) Confocal images after fluorescent in situ hybridization for Brachyury2 (green) and DAPI staining (white), and corresponding mesodermal nuclei segmentation images of control and SU5402-treated embryos at N2 (21 hpf), N3 (24 hpf) and N5 (30 hpf) stages. The notochord nuclei are in green and the somites and tailbud nuclei are in magenta. Dorsal views with anterior towards the top. Scale bar: 25 µm. (B,C) Graphs presenting the ratio of the number of nuclei of the regions coloured in the schemes presented below per the number of nuclei of the mesoderm (excluding the tailbud Brachyury2-expressing region) at N2 (B) and N3 (C) stages. One-way ANOVA and post-hoc Tukey's test analysis results are shown. N.S., non-significant, **P<0.01, ***P<0.001, ****P<0.0001, n=4 embryos. Data are median±s.e.m. with the box indicating interquartile range. Scale bar: 25 µm.

Fig. 6.

Two scenarios for the evolution of the anterior mesoderm in chordates. (A,B) Schematics of embryos at the early somitogenesis stage are presented as dorsal views with anterior towards the top. Arrows correspond to evolutionary steps. Colour coding of different mesodermal territories is shown in the key. PM, paraxial mesoderm; LPM, lateral plate mesoderm. (A) In this scenario, we considered that the chordate ancestor had a prechordal plate-like structure, as observed transiently in amphioxus. (B) In this scenario, we considered that the chordate ancestor had neither an anterior notochord nor a prechordal plate.