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Fig. 1 . Induction of ectodermal markers at neural plate boundaries. Ectodermal marker expression in tailbud stage embryos (albino) that had received neural plate grafts (pigmented and/or mycGFP-labeled) into belly ectoderm at stage 13. (A-F′′) Grafts (arrows, outlined) are shown in overviews (A-F) and transverse sections in brightfield (A′-F′), green fluorescent channel (A′-F′), and overlay (A′′-F′′). Sox3 (A-A′′), Sox11 (B-B′′), Zic1 (C-C′′; inset shows higher magnification of expression in graft) and FoxD3 (D-D′′) are maintained or induced (asterisks) in graft. Six1 (F-F′′) is induced in host ectoderm (arrowheads), whereas Sox9 (E-E′′) is induced in the graft (asterisk) and sometimes in host ectoderm (arrowhead).
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Fig. 2. Distribution of competence for neural crest and panplacodal induction. (A-K′) Experimental overview. After grafting mycGFP-labeled or pigmented ectoderm from various locations and stages into the lateral neural plate border (NB) of stage 12-13 hosts (A,H), expression of panplacodal (Six1), neural crest (FoxD3) or neural (Sox3) markers was analyzed in tailbud stage embryos (B-K). (B′-K′) Sections through grafts. Arrows highlight graft borders. Asterisks indicate expression in graft, whereas arrowheads indicate expression adjacent to graft. (B-D′) After grafting stage 13 neural plate (NP) into the NB, FoxD3 but not Six1 is induced and Sox3 is maintained in the graft (inset in B, control side; inset in C′, magnified view of boxed area, arrows indicate pigment granules). (E-G′) After grafting stage 13 belly ectoderm (B) into the NB, Six1 but neither FoxD3 nor Sox3 is induced in the graft. (I-K′) After grafting stage 9 animal caps (AC) into the NB, FoxD3 and Sox3 but not Six1 are induced in the graft (inset in K, Sox3 is more widely induced in grafts placed into stage 13 NB; ab in J′, air bubble). (L,M) Comparison of FoxD3, Sox3 and Six1 induction after grafting belly (L) or neural plate ectoderm (M) into NBs of different stage hosts. Stage 12 and stage 13 NB are more conducive to FoxD3 and Six1 induction, respectively. (N) Decline of FoxD3 and Sox3 induction, and increase in Six1 induction with increasing age of belly ectoderm grafted into the NB. Data for Six1 at stage 13 taken from Ahrens and Schlosser (Ahrens and Schlosser, 2005). (O) Decline of Six1 induction with increasing age of neural plate ectoderm grafted into the NB.
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Fig. 4. Effects of Dlx3 or GATA2 gain or loss of function on neural ectodermal markers. (A-G′′) Neural plate stage embryos after unilateral injection (lower half; marked by light blue β-galactosidase or green mycGFP staining) of various constructs as indicated. Reductions (arrows), and broadening or ectopic expression domains (asterisks) in the neural (green) and non-neural ectoderm (orange) compared with the control side (arrowheads) are indicated. Double asterisks indicate lateral displacement owing to widening of the neural plate. For Msx1, additional examples of embryos are shown in the insets in C and C′.
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Fig. 5. Effects of Dlx3 or GATA2 gain or loss of function on non-neural ectodermal markers. (A-G′′) Neural plate stage embryos after unilateral injection (lower half; marked by light blue β-galactosidase or green mycGFP staining) of various constructs as indicated. Reductions (arrows) and broadening or ectopic expression domains (asterisks) in the neural (green) and non-neural (orange) ectoderm compared with the control side (arrowheads) are indicated. Insets depict additional embryos with ectopic expression of Six1 (A), Eya1 (B) and Dlx5 (D) in central neural plate.
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Fig. 6. Dlx3 and GATA2 are required cell-autonomously in the non-neural ectoderm for Six1 induction. (A-F′)Host embryos were co-injected with mycGFP mRNA and either Dlx3 MO (A-C′) or GATA2 MO (D-F′) at the two- to eight-blastomere stage and received a neural plate (NP) graft (pigmented) into their belly ectoderm (B-B′) at stage 13. After host embryos reached tailbud stage, grafts are shown in overview (A,D) and at higher magnifications (B-C′,E-F′) in brightfield (B-F), green fluorescent channel (B′-F′) and an overlay (B′-F′). Boxed areas in B and E are shown in detail in C and F, respectively. Six1 induction in non-neural host ectoderm around the graft (asterisks) is specifically suppressed in cells that received high levels of Dlx3 MO or GATA2 MO (arrows or outlined areas of green cells).
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Fig. 7. Dlx3 promotes different non-neural fates depending on the signaling environment. (A-B′) Keratin (A,A′) and Six1 (B,B′) expression in neural plate stage embryos after unilateral injection (lower half; marked by light-blue β-galactosidase staining) of Dlx3 mRNA and treatment with DMSO (A,B) or the BMP signaling antagonist dorsomorphin (A′,B′). Ectopic Keratin expression in the neural plate is strongly reduced, while ectopic Six1 expression is strongly enhanced by dorsomorphin treatment (inset in B′ shows another example). (C) Effects of signaling agonists or antagonists on ectopic expression of Keratin or Six1 in the neural plate (NP). After Dlx3 mRNA injection, the increase in ectopic Six1 expression by dorsomorphin was blocked by the Wnt agonist azakenpaullone and the FGF antagonist SU5402, whereas co-injection of FGF8 had no significant effect. After GATA2 mRNA injection, dorsomorphin did not increase ectopic Six1 expression, whereas Keratin (not shown) was never ectopically expressed (Fisher’s exact test; *P≤0.05, n.s., not significant). (D-E′′) Transverse sections through neural plate of embryos shown in A (D-D′′) and the inset of B′ (E-E′′). Sections are shown in brightfield (D,E) in the green fluorescent channel, showing mycGFP-positive Dlx3-injected cells (D′,E′); in the red fluorescent channel, showing Sox3 immunostaining (D′,E′); and in overlay (D′′,E′′). DAPI-stained nuclei are shown for orientation in all panels. Ectopic Keratin and Six1 expression is confined to Dlx3-injected cells (asterisks), whereas residual Sox3 staining on injected side is found only in cells that did not receive Dlx3 (arrows). not, notochord.
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Fig. 8. Model for regulation of ectodermal competence. (A) Genes that promote non-neural competence, including Dlx3 and GATA2 (orange), and those that promote non-neural competence, probably including Sox3 (green), cross-repress each others transcription (1, broken lines indicate indirect effects). Expression of non-neural competence genes is initially dependent on BMP signaling (2). In the presence of BMP, transcription of non-neural competence genes is therefore promoted over neural competence genes, whereas the reverse is true in the absence of BMP. However, persistent expression of these genes may lead to their autoactivation (3), thereby making their expression resilient to repression and BMP independent. Neural competence factors promote transcription of neural plate genes (4) or, in the presence of additional signals such as BMP, Wnt and FGF (5), neural crest genes. Non-neural competence factors promote transcription of epidermal genes (6) or, in the presence of additional signals such as BMP inhibitors, FGFs and Wnt inhibitors (7), panplacodal and placodal genes. (B) Owing to the dorsal secretion of BMP antagonists and crossrepressive interactions among competence genes, their initially overlapping expression domains will resolve into two distinct territories over time (t).
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Fig. S1. Expression of Pax genes is not induced in non-neural ectoderm. (A-C) After grafting neural plates (NP) from stage 13 pigmented donors into the belly ectoderm (B) of stage 13 albino hosts, Pax gene expression was analyzed in tailbud stage embryos. Grafts (outlined) are shown in high magnification views. Expression of Pax6, Pax3 and Pax2 is confined to grafts. (D-H) After grafting belly ectoderm (B) from stage 13 pigmented donors into the lateral neural plate border (NB) of stage 13 albino hosts, expression of Pax genes was analyzed in tailbud stage embryos. None of the Pax genes analyzed is induced in the graft (E is a transverse section through the embryo in D, showing absence of Pax6 in graft; insets in D,H show control sides).
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Fig. S2. Expression patterns of Dlx3, GATA2 and Sox3. (A) Dlx3 is expressed from stage 9 throughout the animal hemisphere but gradually becomes excluded from prospective neural ectoderm dorsally (asterisks) during gastrulation. (B) GATA2 is expressed from stage 10 in a pattern resembling Dlx3 expression. (C) Sox3 is expressed from stage 9 throughout the animal hemisphere but gradually becomes excluded from prospective non-neural ectoderm ventrally (asterisks) during gastrulation.
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Fig. S3. Effects of Dlx3 or GATA2 loss of function after injection of engrailed repressor constructs on neural and non-neural ectodermal markers. Neural plate stage embryos after unilateral injection (lower half; marked by light blue β-galactosidase or green mycGFP staining) of various constructs as indicated. Reductions (arrows), and broadening or ectopic expression domains (asterisks) in the neural (green) and non-neural ectoderm (orange) compared with the control side (arrowheads) are indicated. Double asterisks indicate lateral displacement owing to widening of the neural plate. See text for details.
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