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First published online 3 November 2004
doi: 10.1242/dev.01516

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Six1 promotes a placodal fate within the lateral neurogenic ectoderm by functioning as both a transcriptional activator and repressor

Samantha A. Brugmann¹, Petra D. Pandur^1,*, Kristy L. Kenyon^2,, Francesca Pignoni² and Sally A. Moody^1,

¹ Department of Anatomy and Cell Biology, Institute for Biomedical Sciences, The George Washington University, Washington, DC 20037, USA
² Department of Ophthalmology, MEEI, Harvard Medical School, Boston, MA 02138, USA

View larger version (19K): [in a new window]   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 ventral epidermis (from Moody, 1987).

View larger version (25K): [in a new window]   Fig. 2. (A) RT-PCR analysis showing that explants injected with noggin or cerberus mRNAs express six1, whereas control and bmp4-injected explants do not. H4, loading control. (B) Explants were cultured in different concentrations of Noggin, and processed for six1 expression at stage 17. The largest percentage of explants stained at high levels was observed between 1 and 5 ng/ml of Noggin. (C) BMP4 antagonizes the 1 ng/ml Noggin-induction of six1. (D) Explants were cultured as in B, and the percentage of explants with high levels of marker gene expression were plotted.

View larger version (64K): [in a new window]   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) Ventral epidermis containing chordin-expressing (red) cells in a dispersed pattern; there is no ectopic six1 expression. (C) Ventral epidermis containing a secondary axis (sox2, blue) after ectopic chordin expression (red). (D) Ventral epidermis 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).

View larger version (56K): [in a new window]   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 (*).

View larger version (59K): [in a new window]   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).

View larger version (66K): [in a new window]   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 lateral dlx6 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.

View larger version (46K): [in a new window]   Fig. 7. Model of the three steps involved in PPE formation. (1) The embryonic ectoderm is separated into presumptive epidermis (E), pre-placodal (P), neural crest (NC) and neural plate (NP) domains in response to a combination of a dorsoventral gradient of BMP-antagonizing factors (Noggin) and a gradient of posteriorizing signals (Wnt, FGF). (2) six1 promotes the expression of placode genes at the expense of neural crest and epidermal genes. (3) A network of gene interactions further defines the ectodermal subdomains. Green arrows indicate that the expression of the downstream gene is enhanced, and red bars indicate that it is reduced. Lines are broken to indicate that interactions may be direct or include intermediary genes.