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First published online May 16, 2007
doi: 10.1242/10.1242/dev.000216

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Wnt signals provide a timing mechanism for the FGF-retinoid differentiation switch during vertebrate body axis extension

Division of Cell and Developmental Biology, College of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

View larger version (62K): [in this window] [in a new window]   Fig. 1. FGF signalling is required to maintain Wnt8c expression. (A-C') Expression of Fgf8 (A,A'), Wnt8c (B,B') and Raldh2 (C,C') in HH10 chicken embryos and corresponding transverse sections (at position of white lines): arrowheads indicate the last somite formed. Diagram on the left defines the different regions of the caudal end of the embryos at stage HH10. (D,E) Wnt8c is downregulated 6 hours after presomitic mesoderm removal. (F-I) FGF4 beads beneath caudal neural plate (F) ectopically maintain Wnt8c 6 hours (G) and 18 hours (H) later, whereas control PBS beads do not (I). (J,K) Caudal Wnt3a (J) or Wnt5a (K) expression is not maintained rostrally by FGF4 beads (9 hours later). (L) Origin of explants used in M,M'. (M,M') Wnt8c loss in preneural tube explants (pNT) cultured 24 hours (M) is prevented by FGF4 (M'). (O) Origin of caudal neural plate (CNP) explants used in N,N',P,P' and caudal presomitic mesoderm (CPM) explants used in Q,Q'. (N,N') Wnt8c is still expressed in CNP explants after 8 hours in DMSO control media (N) but is downregulated by SU5402 (N'). Fgf8 transcripts are still detected in CNP with control cells after 24 hours (P) but not in CPM explants (Q). Explants (P-Q') are derived from the same embryo.Wnt8c cells do not alter Fgf8 expression (P',Q'). Scale bars: 100 µm in A for A-C and in E for E,G-K; 50 µm in M for M-N' and in P for P-Q'.

View larger version (51K): [in this window] [in a new window]   Fig. 2. Somites and retinoic acid inhibit Wnt8c expression. (A) Origin of explants used in B-C'. (B) Caudal neural plate (CNP) expresses Wnt8c (purple) after 24 hours culture whereas its counterpart explant pair (B') loses Wnt8c next to the quail somites (QCPN, orange). CNP explants in DMSO (control) (C) express Wnt8c after 24 hours, whereas 9cisRA represses Wnt8c (C'). Wnt8c expression in control (D) and VAD quails (E,F). Wnt8c is ectopically expressed in VAD neural tube. Arrowheads indicate the last somite formed; r4, prospective rhombomere 4; s6, somite 6 which indicates the level of the prospective hindbrain/spinal cord boundary. (G) Origin of explants derived from VAD quails used in (H,H'). VAD neural tube (VAD NT) explants still express Wnt8c after 6 hours in DMSO (H), but this is lost with SU5402 (H'). r4, prospective rhombomere 4; s6, somite 6. Scale bars: 50 µm in B for B-C'; 100 µm in D for D-F; 25 µm in G for H,H'.

View larger version (68K): [in this window] [in a new window]   Fig. 3. Canonical Wnt signalling inhibits neuronal differentiation. (A,A') Electroporation of control IRES-GFP/PCINeo vector (A, Fast Red, GFP transcripts) has no effect on NeuroM (A'). (B,B') Electroporation of Wnt8c-IRES-GFP/PCINeo (B), reduces the amount of NeuroM-positive cells (B'). (C) Quantification of results shown in (A-B'): the percentage of NeuroM-positive cells on the electroporated side is significantly lower in presence of Wnt8c (four sectioned embryos), compared with the control (five sectioned embryos) (t-test, P=0.01). (D) Origin of neural tube (NT), preneural tube (pNT) and underlying presomitic mesoderm (PSM) explants used in (E-J'). (E-F') pNT explants either combined with control cells (E) or Wnt8c cells (E'), or cultured in control media (F) or media supplemented with LiCl (F'). Fewer NeuroM+ cells arise in pNT exposed for 24 hours to Wnt8c or to LiCl. (G-H') NT in control media (G-J), with FGF4 (G',I') or with LiCl (H',J'). Fewer Ngn1+ cells are present in NT exposed to FGF4 (I') or to LiCl (J'). Scale bars: 25 µm in A for A,A'; 20 µm in B for B,B'; 50 µm in E for E-F' and in G for G-H'.

View larger version (106K): [in this window] [in a new window]   Fig. 4. Wnt and FGF signalling inhibit neurogenesis via different mechanisms. (A-C') Neural tube (NT) explants in control media do not express Wnt8c at the time of excision or 24 hours later (A) but do express Wnt1 (B) and Wnt3a (C). FGF4 does not reactivate Wnt8c (A'), reduces strongly Wnt1 (B') and mildly Wnt3a (C'). (D-E') NT explants in control media with ethanol (D,E), CKI-7 (D') or FGF4 and CKI-7 (E'). Inhibiting Wnt signalling with CKI-7 does not alter NeuroM expression in NT (within 24 hours) and FGF4 in the presence of CKI-7 is still able to inhibit NeuroM (24 hours, compare E with E' and see Fig. 3G,G'). (F,F') RARß is expressed in the transition zone and the underlying rostral presomitic mesoderm at HH10 (F': section of F indicated by a white line). (G,G') NT explants in control media (G), with FGF4 (G'). pNT explants combined with control cells (H) or Wnt8c cells (H'). PSM explants combined with control cells (I) or Wnt8c cells (I'). RARß is dramatically inhibited by FGF4 in neural tube explants, but is not consistently affected by Wnt8c in pNT. RARß appears enhanced in underlying PSM by Wnt8c in 24 hours of culture. Scale bars: 50 µm in A for A-C',H-I', in D for D-E',G-G' and in F'; 100 µm in F.

View larger version (117K): [in this window] [in a new window]   Fig. 5. Onset of RA synthesis is promoted by canonical Wnt signalling once FGF signalling has declined. (A-B') Raldh2 is weakly expressed in caudal presomitic mesoderm (CPM) explants (drawn in A) cultured for 8 hours with control cells (B), whereas it is activated in presence of Wnt8c cells (B'). (C-F) Embryos cultured in control media (C,E) or media with LiCl (D,F). The Wnt responsive gene Lef1 is upregulated by LiCl, whereas Raldh2 expression is not consistently upregulated. (G-J) Comparison of embryos treated with DMSO (control media G,I) or SU5402 (H,J). SU5402 inhibits the expression of the FGF responsive gene Sprouty2 but Raldh2 is not affected by this treatment. (K,L) Compared with embryos in DMSO media (K), embryos cultured in presence of SU5402 and LiCl combined (L) show enhanced Raldh2 expression, and in some cases (L) this expands caudally. Arrow, node; arrowhead, last somite formed. Scale bars: 50 µm in B for B,B'; 100 µm in C for C-L.

View larger version (63K): [in this window] [in a new window]   Fig. 6. Loss of Wnt signalling delays Raldh2 onset. (A,B) Effects of control PBS beads (A) and beads soaked in sFRP2 (B) on Raldh2 onset. Asterisk in B indicates region of Raldh2 inhibition. (C-F) Embryos cultured in ethanol control media (C,E) or media supplemented with CKI-7 (D,F). Both Lef1 and Raldh2 are downregulated by CKI-7. (G,G') After 18 hours in culture, CPM explants come to express Raldh2 in control media (G), but this is prevented by DKK1 (G'). Arrow, node; arrowhead, last somite formed. Scale bars: 100 µm in A for A-F; 50 µm in G for G,G'.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Sequence of signalling events controlling maturation of the extending body axis. Model for the sequential action of Fgf8 (1), Wnt (2) and retinoid (3) signals in the extending body axis (see text for details). Grey lines indicate interactions shown elsewhere, and black lines those established in this work.

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