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First published online 10 August 2005
doi: 10.1242/dev.01964

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A balance of FGF, BMP and WNT signalling positions the future placode territory in the head

Department of Craniofacial Development, Guys Campus, Guys Tower, Floor 27, King's College London, London SE1 9RT, UK

View larger version (74K): [in a new window]   Fig. 1. The pre-placodal region is induced by converging signals from the heart mesoderm and the neural plate. In the extra-embryonic region, Hensen's node (A-C,A'-C'; brown) rapidly induces the pre-neural marker Sox3 (A,A'; blue), while the pre-placodal marker Six4 (B,B'; blue) and the epidermal marker Dlx5 (C,C'; blue) are induced only at the edge of the neural plate after 14-16 hours. Stage 5/6 neural plate grafts (D-H,D'-H'; brown) induce the expression of the pre-placodal markers Six1 (D,D'; blue) and Dach1 (E,E'; blue), as well as Dlx5 (F,F') and the crest markers Slug (G,G'; blue) and Sox9 (H,H'; blue). Head mesoderm ablation (I,J; I',J'; broken line in I,J) results in the loss of Six4 (I,I', arrow) and Eya2 (J,J', arrow). Grafts of head mesoderm (K-P,K'-P'; brown) induce the expression of Six1 (K,K'; blue), Six4 (L,L'; blue), Eya2 (M,M'; blue), Dach1 (N,N'; blue), Dlx5 (O,O'; blue) and low levels of Sox3 (P,P'; blue). Horizontal lines in A-P indicate the level of the sections shown in A'-P', respectively. Brown staining in A-H,A'-H',K-P and K'-P' represents QCPN immunoreactivity labelling quail tissue.

View larger version (104K): [in a new window]   Fig. 2. Inhibition of BMP signalling expands the expression of the pre-placodal genes Six4 and Eya2. Primitive streak stage embryos were electroporated with control GFP containing vector (A,D) or with SMAD6-IRES-GFP vector (B,C,E,F). Expression of Six4 (A-C; blue) and Eya2 (D-F, blue) was assessed by in situ hybridisation and the electroporated cells were visualised by staining with GFP antibody (brown in A,C,D,F). While Six4 and Eya2 expression is normal in control embryos (A,D), expression of both genes is expanded when BMP signalling is inhibited (B,C,E,F). Six4 expression is not observed in the trunk region (arrows in C) and ectopic expression of both Six4 (B,C, arrowheads) and Eya2 (E,F, arrowheads) is limited laterally.

View larger version (95K): [in a new window]   Fig. 3. WNT signalling antagonises the formation of the pre-placodal region (PPR). Primitive streak stage embryos were electroporated with GFP (A-E), ß-catenin (F-H,J,K), WNT8C (I) or Crescent (L-Q), and analysed for PPR and neural crest marker expression. Sections through control embryos (A-E) do not show any change in the expression of Six1 (A), Six4 (B), Eya2 (C), Pax7 (D) and Slug (E). Misexpression of ß-catenin (F-H,J,K; brown) or WNT8C (I, brown) narrows (brackets in F-I) Six1 (F), Six4 (G) and Eya2 (H,I) expression. By contrast, Pax7 (J) and Slug (K) expression expands into the placodal territory (arrowheads). Inhibition of WNT by Crescent (L-Q, brown) expands Six1 (L,M), Six4 (N) and Eya2 (O) expression into lateral (brackets in L,N,O) and trunk ectoderm (M, arrowhead), while Pax7 (P, arrowhead) and Slug (Q, arrowhead) expression is reduced.

View larger version (87K): [in a new window]   Fig. 4. Modulation of the WNT pathway does not change mesoderm or neural plate patterning. At head-fold stages, Wnt8c is expressed in the cranial lateral plate mesoderm (A,A'; lp) next to the pre-placodal region (ppr; bracket), in the trunk mesoderm (A,A''; mes) and the neural plate posterior to the pre-placodal region (A,A''; np). Lines in A indicate the positions of the sections shown in A' and A'', respectively. (B,C) Misexpression of Crescent (brown in C) does not result in expansion of the heart mesoderm marker Nkx2.5. (D-G) Misexpression of activated ß-catenin (brown in E and G) does not alter the expression of the regional neural markers Fgf3 (D,E) and Krox20 (F,G).

View larger version (99K): [in a new window]   Fig. 5. Segregation of pre-placodal region (PPR) and neural crest markers between head-fold (A-F') and early somite (G-J') stages. At stage 5-6 (A-F'), Six1 (A,A'), Six4 (B,B') and Eya2 (C,C') are expressed in the ectoderm encircling the anterior neural plate, while Dach1 (D,D') is found in the entire ectoderm. Pax7 (E,E') is expressed in a band of cells along the neural plate and partially overlaps with Six1, Six4 and Eya2 expression. PPR markers never colocalise with the neural plate marker Sox2 (F,F'). By stage 8, Six1 (G,G'), Six4 (H,H') and Eya2 (I,I') continue to surround the neural plate, but there is no overlap with Pax7 (K,K') or Slug (L,L'), which are confined to the neural folds. Dach1 remains expressed in most of the ectoderm (J,J'). Arrowheads in A'-C',G-I' label the medial limit of PPR gene expression; lines in A-L indicate the plane of the sections shown in A'-L', respectively.

View larger version (87K): [in a new window]   Fig. 6. Cooperation of FGF and BMP and WNT antagonists to induce the pre-placodal region. Misexpression of SMAD6 and Crescent (A-D, brown in B,D) leads to an expansion of Six4 (A,B; blue) and Eya2 (C,D; blue) expression into the extra-embryonic epiblast. (E-G) FGF8 induces ectopic Sox3 (E, arrowhead), Dlx5 (F, arrowhead) and Eya2 (H, arrowhead), but not Six4 (G, arrowhead). Misexpression of SMAD6, Crescent and FGF8 (I-P) results in ectopic induction of Six4 (I,J; blue) in the absence of the neural marker Sox2 (M,N) and of the heart mesoderm marker Nkx2.5 (O,P). When the FGF8 bead is replaced with an SU5402 bead after 5 hours (K,L), induction of Six4 expression is still observed. Brown staining in J,L,N,P represents immunostaining with anti-GFP antibody to visualise electroporated cells. (Q-T) Head mesoderm (brown in R,T) was grafted into the extra-embryonic region together with control (Q,R) or SU5402-coated beads (S,T). Six4 expression (blue) is induced in controls (Q,R), but not when FGF signalling is inhibited (S,T). Quail grafts are labelled using QCPN antibody (brown in R,T).

View larger version (30K): [in a new window]   Fig. 7. Model of the signalling interactions that position the pre-placodal region in the cranial ectoderm. (A) The section through an embryo viewed from anterior to posterior represents signalling within the ectoderm on the left and mesoderm-derived signals on the right. The pre-placodal region (dark blue) lies next to the neural crest (red) at the border of the anterior neural plate and is induced by FGF together with BMP and WNT antagonists derived from the underlying mesoderm (light blue). WNT signals from the lateral and posterior mesoderm (pink, right) cooperate with WNT from the trunk ectoderm (pink, left) to limit the PPR. BMP signals from the lateral ectoderm (grey) also prevent expansion of the PPR into more lateral regions. WNT proteins in the neural folds promote neural crest formation (red, left), but inhibit PPR gene expression. The neural plate expresses BMP and WNT inhibitors, which may account for its limited PPR inducing ability. (B) Model for neural crest and placode specification at the border of the neural plate; the terminology follows a recent molecular network for neural crest cell specification proposed by Meulemans and Bronner-Fraser (Meulemans and Bronner-Fraser, 2004), which classifies three categories of molecules: secreted inducers (orange), neural plate border specifiers (black) and neural crest specifiers (red). We propose that the Six/Eya/Dach network may act as pre-placode specifier (blue). Pax7 was classified as a neural plate border specifier (Meulemans and Bronner-Fraser, 2004) and is, like Msx1, expressed early; however, so far it is not clear which upstream signalling pathways induce Pax7. Our experiments reveal that at neural fold stages its expression is controlled by WNT. FGF signalling plays a dual role: it promotes border specifiers and later pre-placode specifiers, while levels of WNT and BMP activity control neural crest and placode fates in the border.