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Spemann organizer is a center of dorsal mesoderm and itself retains the mesoderm character, but it has a stimulatory role for neighboring ectoderm cells in becoming neuroectoderm in gastrula embryos. Goosecoid (Gsc) overexpression in ventral region promotes secondary axis formation including neural tissues, but the role of gsc in neural specification could be indirect. We examined the neural inhibitory and stimulatory roles of gsc in the same cell and neighboring cells contexts. In the animal cap explant system, Gsc overexpression inhibited expression of neural specific genes including foxd4l1.1, zic3, ncam, and neurod. Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and promoter analysis of early neural genes of foxd4l1.1 and zic3 were performed to show that the neural inhibitory mode of gsc was direct. Site-directed mutagenesis and serially deleted construct studies of foxd4l1.1 promoter revealed that Gsc directly binds within the foxd4l1.1 promoter to repress its expression. Conjugation assay of animal cap explants was also performed to demonstrate an indirect neural stimulatory role for gsc. The genes for secretory molecules, Chordin and Noggin, were up-regulated in gsc injected cells with the neural fate only achieved in gsc uninjected neighboring cells. These experiments suggested that gsc regulates neuroectoderm formation negatively when expressed in the same cell and positively in neighboring cells via soluble factors. One is a direct suppressive circuit of neural genes in gsc expressing mesoderm cells and the other is an indirect stimulatory circuit for neurogenesis in neighboring ectoderm cells via secreted BMP antagonizers.
Fig. 1. InhibitIon of neural gene expression either mediated by dnbr or chordin and noggin (Chrd/Nog) in AC explants of Xenopus.
3Flag-gsc (1 ng/embryo), dnbr (0.5 ng/embryo), chordin (0.5 ng/embryo) and noggin (0.5 ng/embryo) (Chrd/Nog) were injected at one-cell stage and the AC explants were dissected at stage 8 to grow until (A and B) stage 11 and (C and D) stage 24. The expression profiles of different germ layer specific marker genes were analyzed by RT-PCR. No RT (no reverse transcriptase added) served as a negative control while WE (whole embryos) served a positive control. (E) 3Flag-gsc (1 ng/embryo) mRNA was injected into one cell embryos, and the injected embryos and non-injected ones (control) were processed for whole mount in situ hybridization (WISH) with anti-sense foxd4l1.1 probe at stage 11.
Fig. 2. Identification of GREs within the neural target genes using ChIP-seq of 3Flag-gsc injected Xenopus embryos. (A and B) Coverage plot of 3Flag-gsc within the foxd4l1.1 and zic3 promoter regions. (C) Similarity within both (chr1s and chr1l) copies of foxd4l1.1 promoter region are shown. The active GRE (–213 to –218 bps) location is highlighted by an arrow. (D) Putative GREs revealed within the foxd4l1.1 promoter region by ChIP-sequencing. (E) Consensus binding motifs of Gsc within both (chr1s and chr1l) copies of foxd4l1.1 promoter region.
Fig. 3. Abolishment of the repressional activity of Gsc by site-directed mutagenesis of GRE within the foxd4l1.1(–1551) reporter construct. (A) foxd4l1.1(–1551) (40 pg/embryo), dnbr (1 ng/embryo), and 3Flag-gsc (1 ng/embryo) were injected at one-cell stage and reporter assay was performed at stage 11. RLU, relative reporter activity. (B) Schematic representation of serially-deleted foxd4l1.1(–1551) promoter constructs. (C) Serially-deleted foxd4l1.1 (40 pg/embryo) promoter constructs were injected with and without 3Flag-gsc (1 ng/embryo) at one-cell stage and relative promoter activity were measured at stage 11. (D) Site-directed mutagenesis scheme for foxd4l1.1(–1551) and foxd4l1.1(–301) promoter constructs, target sequences highlighted (red color and italic). (E and F) Relative reporter assay of foxd4l1.1(–1551), foxd4l1.1(–1551)mGRE, foxd4l1.1(–301), and foxd4l1.1(–301)mGRE with or with gsc were performed at stage 11. (G) 3Flag-gsc was injected at one-cell stage and ChIP-PCR was performed at stage 11. Specific primers of Foxd4l1.1 promoter region (containing GRE) were used for amplification, while ventx2.1 served as negative control. (H) zic3(–1805) reporter construct was injected (40 pg/embryo) with or without 3Flag-gsc (1 ng/embryo) and reporter assay was performed at stage 11. **P ≤ 0.01; ****P ≤ 0.0001; ns, non-significance.
Fig. 4. A dual role of Gsc in transcriptional regulation of foxd4l1.1. In all experiments, 3Flag-gsc (1 ng/embryo) and foxd4l1.1(-1551) (40 pg/embryo) were injected at one-cell stage and the ACs were dissected at stage 8 to grow until stage 11 and 24. (A) Conjugation scheme for AC explants used. (B) The relative promoter activity are measured. RLU, relative reporter activity. ****P ≤ 0.0001. (C) Schematic description of the conjugated AC explants of the embryos injected with 3Flag-gsc and NI. The expression profiles of (D) early (stage 11) and (E) late (stage 24) neural marker genes were analyzed by RT-PCR. No RT (no reverse transcriptase added) served as a negative control while WE (whole embryos) served as positive control. (F) Whole mount in situ hybridization (WISH) was performed with anti-sense foxd4l1.1 probe of at stage 11.
Fig. 5.
Schematic diagram depicting the dual role of Gsc in early neurogenesis. In mesoderm (dorsal organizer), Gsc inhibits the neural genes while strongly induces chordin and noggin expression. Newly translated Chordin and Noggin diffuse to neighboring cells (ectoderm) where they induce neurogenesis in a BMP inhibited manner.
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