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???displayArticle.abstract??? Cranial placodes, which give rise to sensory organs in the vertebrate head, are important embryonic structures whose development has not been well studied because of their transient nature and paucity of molecular markers. We have used markers of pre-placodal ectoderm (PPE) (six1, eya1) to determine that gradients of both neural inducers and anteroposterior signals are necessary to induce and appropriately position the PPE. Overexpression of six1 expands the PPE at the expense of neural crest and epidermis, whereas knock-down of Six1 results in reduction of the PPE domain and expansion of the neural plate, neural crest and epidermis. Using expression of activator and repressor constructs of six1 or co-expression of wild-type six1 with activating or repressing co-factors (eya1 and groucho, respectively), we demonstrate that Six1 inhibits neural crest and epidermal genes via transcriptional repression and enhances PPE genes via transcriptional activation. Ectopic expression of neural plate, neural crest and epidermal genes in the PPE demonstrates that these factors mutually influence each other to establish the appropriate boundaries between these ectodermal domains.
Fig. 1. (A) In the stage 16 Xenopus embryo there are four major ectodermal domains: neural plate, neural crest, pre-placodal ectoderm (PPE) and epidermis. (B) The expression pattern of six1 coincides with the PPE. (C) An idealized depiction of the ectodermal fate of the 32-cell embryo showing four dorsal blastomeres (blue) that contribute significantly to the neural plate, two ventrolateral blastomeres (green) that contribute significantly to the neural crest and PPE, and two ventral blastomeres (yellow) that contribute significantly to the ventralepidermis (from Moody, 1987).
Fig. 3. (A) Expression of bmp4 in the LNE on one side of embryo (right) reduced six1 placode expression compared with control, uninjected side (left). (B) Ventralepidermis containing chordin-expressing (red) cells in a dispersed pattern; there is no ectopic six1 expression. (C) Ventralepidermis containing a secondary axis (sox2, blue) after ectopic chordin expression (red). (D) Ventralepidermis containing a secondary axis/elongated clone (*) after ectopic chordin expression (red); ectopic six1 expression is at its anterior pole (stripe between arrows). (E) When co-injection of frzb-1+noggin mRNAs does not form a secondary axis (dispersed red cells), ectopic six1 is not induced. (F) When co-injection of frzb-1+noggin mRNAs forms a secondary axis (left), the ectopic six1 domain (arrow) extends further posterior (black bar) from the anterior tip of the secondary axis (*), compared with noggin alone embryos (right). (G) Co-injection of dnWnt8+noggin mRNAs expands the six1 expression domain (arrows) to encircle both the primary axis (*) and the induced secondary axis (red cells, inset). (H) Wnt8 expression in the LNE (left side) represses six1 (arrow). (I) cFGFR1 expression in the LNE (left side) represses six1 (arrow). (J) Explants were injected with either cfgfr1 or Wnt8 mRNA and cultured in 1 ng/ml Noggin. The high levels of six1 expression induced by this concentration of Noggin were significantly repressed by both factors. (K) Expression of frzb-1+noggin mRNAs either represses (left) or reduces (right) foxD3 expression on the treated side (arrows). (L) Wnt8 expression in the LNE (left side) expands foxD3 (arrow). (M) cFGFR1 expression in the LNE (left side) expands foxD3 (arrow).
Fig. 4. Neural plate stage control expression patterns (A,D,G,J). (B,E,H,K) Overexpression of six1-WT (B) represses keratin, (E) increases the placodal domain of sox11 (inset, control side), (H) represses foxD3, but (K) has no significant effect on sox2 expression. The asterisks indicate the injected sides. (C,F,I,L) Six1-MO knock-down (C) expands the keratin domain closer to the border of the neural plate [np; compare bars on injected (*) versus control sides], (F) reduces the placodal domain of sox11, (I) increases the width of foxD3 domain, and (L) expands sox2 (bars indicate distance from midline). Quantitation of changes is presented in Table 1. (M) Injection of six1-myc results in protein expression detected by Myc antibody (green). (N) No protein (green) is detected when six1-MO is co-injected. (O) Same section as in N showing presence of lysamine-tagged six1-MO. (P) Injection of six1-rescue mRNA restores normal foxD3 domain on six1-MO injected side (*).
Fig. 5. Constructs that cause transcriptional activation (six1VP16; six1+eya1) reduce keratin expression (A,B), expand the placodal domain of sox11 (E,F) and expand the foxD3 domain (I,J). Constructs that cause transcriptional repression (six1EnR; six1+groucho) reduce keratin expression (C,D), reduce sox11 placodal expression (G,H) and reduce the foxD3 domain (K,L).
Fig. 6. (A) Control zic2 expression; bracket indicates LNE domain. (B) Overexpression of six1-WT expands the zic2 LNE domain (bracket). (C) Six1 knock-down expands the zic2 LNE domain (bracket). (D) Control dlx6 expression. (E) Overexpression of six1-WT moves the dlx6 stripe laterally (arrow), and in some cases eliminates expression in places (arrowheads, inset). (F) Six1 knockdown reduces the lateraldlx6 stripe (arrow). (G) Overexpression of sox2 reduces six1 (arrow). (H,I) Overexpression of foxD3 (H) reduces six1 (arrow) and (I) expands zic2 LNE expression (arrow). (J,K) Over-expression of zic2 (J) reduces six1 (arrow) and (K) expands foxD3 (arrow). (L) Over-expression of dlx5 reduces six1 (arrow). mRNAs were injected on left-hand side; right-hand side is internal control.
In the stage 16 Xenopus embryo there are four major ectodermal domains: neural plate, neural crest, pre-placodal ectoderm (PPE) and epidermis.
In the stage 16 Xenopus embryo there are four major ectodermal domains: neural plate, neural crest, pre-placodal ectoderm (PPE) and epidermis.
The expression pattern of six1 coincides with the PPE.
In the stage 16 Xenopus embryo there are four major ectodermal domains: neural plate, neural crest, pre-placodal ectoderm (PPE) and epidermis.
In the stage 16 Xenopus embryo there are four major ectodermal domains: neural plate, neural crest, pre-placodal ectoderm (PPE) and epidermis.
