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First published online 14 December 2005
doi: 10.1242/dev.02196

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Convergence of Wnt and FGF signals in the genesis of posterior neural plate through activation of the Sox2 enhancer N-1

Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.

View larger version (48K): [in a new window]   Fig. 1. Assessment of activities of enhancer N-1 and its subfragments. (A) Scheme of assay using electroporation of chicken embryo at stage 4, and subsequent assessment of EGFP fluorescence. (B) Scheme of deletion analysis of the enhancer N-1. The sequences with enhancer activity are indicated in green, whereas those without activity are in purple. Each deletion construct was examined using more than 10 electroporated embryos, which gave identical results. (C) Enhancer activity of various constructs indicated by EGFP expression. (a-e) Stage 5 (a,c) and stage 9 (b,d,e) chicken embryos, 6 and 12 hours after electroporation, respectively, showing enhancer N-1 activity (c,d), compared with bright-field images (a,b) and expression of co-electroporated DsRed1-N1 (e). (f,g) Enhancer activity of trimerized N-1c, 6 and 12 hours after electroporation, respectively, emulating the activity of enhancer N-1 (compare c,d), in comparison with endogenous Sox2 expression of the same embryo at stage 9 (h) detected by in situ hybridization. (i,j) Loss of enhancer activity by deletion of N-1c sequence (N-1c). a to e, f to h, and i and j are data from the same embryos. Arrowheads indicate the node position.

View larger version (81K): [in a new window]   Fig. 2. Mutation analysis of the N-1c sequence. (A) Mutations introduced into the N-1c sequence and identified functional blocks. (B)(a-g) EGFP fluorescence indicating activity of wild-type and mutant enhancers in dorsal views of chicken embryos. Mutations are indicated in parentheses. Mut-A (b), Mut-B (c) and Mut-C (e) mutations resulted in a decrease in the enhancer activity and Mut-AB (d) and Mut-D (f) resulted in a large loss of the activity, whereas Mut-E (g) caused a widening of the area of tissue where the enhancer was active. (h,i) Cross section through the node of embryo, electroporated with wild-type N-1c and Mut-E constructs, respectively. Arrowheads indicate the node position. Each mutant enhancer was examined using more than 10 electroporated embryos, which all gave identical results.

View larger version (73K): [in a new window]   Fig. 3. Wnt signal-dependent regulation of enhancer N-1c. (A) Mutations and probe sequences for Blocks A and B, used for EMSA, and comparison with Lef1/Tcf-binding consensus. (B) EMSA performed using a sequence B probe, with recombinant cLef1 (left) or embryo nuclear extract (right). Arrows indicate cLef1-probe complex. (C) Effect of exogenous expression of stabilized ß-catenin (b), Wnt antagonists Dkk1 (c) or Sfrp1 (d) on enhancer N-1c activity. Successful electroporation of a broad embryonic area was confirmed by DsRed1 expression (insets). Anterior margin of the embryo and the node position are indicated by an arrow and an arrowhead, respectively. At least six electroporated embryos were used to examine the effects of these molecules, which all gave identical results.

View larger version (122K): [in a new window]   Fig. 4. Effect of ectopic administration of FGF8b on activity of trimeric N-1 enhancer in electroporated embryos. (A,B) A cFGF8b-expressing COS7 cell clump marked by DsRed1 expression was deposited in a distal location of an embryo immediately after electroporation. The same embryo 6 hours (A) and 12 hours (B) after electroporation, showing ectopic activation of the trimeric N-1c enhancer as indicated by the green fluorescence (arrowheads). (C)An embryo 12 hours after electroporation with deposition of normal COS7 cells. No ectopic activation of the enhancer was observed. (D) An FGF8b-soaked heparin bead (indicated by a red circle, soaked in 50 µg/ml FGF8b) deposited at a proximal site of area opaca of an electroporated embryo, activated the trimeric N-1c enhancer activity in the area opaca (arrowhead). The border between the area pellucida and area opaca is indicated by the white broken line. Six electroporated embryos were used for each experiment, which all gave essentially the same results.

View larger version (19K): [in a new window]   Fig. 5. Functional cooperation of Wnt and FGF signals demonstrated by transfection of 10T1/2 cells with firefly luciferase reporter bearing trimeric N-1c enhancer. (A) Activation of the trimerized N-1c enhancer by exogenous Wnt3 and cFGF8b. Wnt3 and cFGF8b were expressed by cotransfection of relevant expression vectors. Firefly luciferase expression was normalized using the expression of co-transfected Renilla luciferase. (B) Response of mutant forms of enhancer N-1c to FGF and Wnt signals. Luciferase expression level without stimulation with an effector was designated as 1. The unstimulated activity of trimeric N-1c enhancers compared with enhancer-less luciferase reporter were: N-1c (wild type), 5.0±0.4; Mut-AB, 6.4±0.3; Mut-C, 4.9±0.1; Mut-D, 2.8±0.2; Mut-E, 7.4±0.7 (mean ± s.e.m.). (a) Cotransfection with Wnt3 expression vector. (b Cotransfection with Fgf8b expression vector. (c) Cotransfection with Wnt3 plus Fgf8b expression vectors. (d) The same as c, but with addition of SU5402 immediately after transfection. (C) FGF-responsiveness of trimeric [D+E] subfragment of the enhancer N-1c. The activation levels with exogenous FGF8b derived from cotransfected expression vector are compared. The subfragment region of N-1c sequence is shown in Fig. 2A. Transfection was done at least in triplicate samples per experiment, and each panel shows a representative set of data derived from an experiment.

View larger version (90K): [in a new window]   Fig. 6. Effects of BMP signals on activity of enhancer N-1c trimer and Sox2 expression. (Top) Embryonic Sox2 expression (in situ hybridization) and (bottom) activity of enhancer N-1/N-1c (EGFP fluorescence) are compared using the same embryos. (A) Enhancer N-1. (B) Enhancer N-1c (trimer). (C) Effect of misexpression of constitutive-active (CA) Alk6, which wiped out Sox2 expression. (D,E) Effect of misexpression of BMP antagonists cNoggin (D) or dominant-negative (DN) Alk6 (E), posteriorly extending the Sox2 expression in a manner matching the activity of enhancer N-1c.

View larger version (15K): [in a new window]   Fig. 7. Model of organization of functional Blocks of enhancer N-1c. Enhancer N-1c is activated by Wnt and FGF signals and repressed in mesendodermal precursors, and subsequent steps plus inhibition of BMP activity leading to genesis of posterior neural plate.