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Fig. 5. Role of BMP inhibitors and FGF8 in placodal Six1 induction. Embryos shown in lateral views (anterior to the left). Transplantations involved hosts at early neural plate stage (stage 13) and were analysed at late neural fold or early tailbud stages (stage 186). (A,B) Implanting animal caps (pigmented) from BMP4-injected embryos (A) or beads (red asterisk; the bead itself is light blue) soaked with BMP4 (B) repress placodal Six1 expression (arrowheads) around grafts or beads, respectively. Insert in panel B shows control side (CS). (C,D) Animal caps from uninjected embryos grafted into the LPR strongly express Sox3 (C) but not Six1 despite normal Six1 expression in host ectoderm (D). (E) Animal caps (pigmented, green GFP label, arrows) from embryos injected with various constructs (E) or protein-soaked beads (K) were grafted into belly ectoderm. (E) noggin-injected grafts strongly express Sox3. (F) dnBMPR-injected grafts also express Sox3, but expression tends to be somewhat weaker and more patchy. (G) dnBMPR-injected grafts do not or only weakly affect placodal Six1 expression of host, whereas (H) noggin-injected grafts significantly broaden (arrowhead) placodal Six1 expression of host towards anterior graft border. (I) FGF8-injected grafts only weakly broaden (arrowhead) placodal Six1 expression in few cases. (J) noggin and FGF8 coinjected grafts broaden placodal Six1 expression and strongly induce Six1 (arrowhead) at anterior graft border. Beads (red asterisks) soaked with noggin (K) or FGF8 (L) alone do not, but beads soaked with noggin and FGF8 (M) do induce ectopic Six1 expression in ectoderm (beads enlarged in insert; Affi-Gel Blue beads used in panels K and M are light blue, while heparin acrylic beads used in panel L are colourless). (N) Anterior neural plate from stage 13 donors retains FGF8 expression after transplantation into belly ectoderm. Transverse sections (O,P) through embryo shown in panel N clearly show FGF8 expression (O) confined to GFP-labeled graft (P). (Q) In contrast, FGF8 expression is typically not maintained in LPR from stage 13 donors after transplanting into belly ectoderm.
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