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The POU domain gene, XlPOU 2, acts as a transcriptional activator during mid-gastrulation in Xenopus. Overexpression or misexpression of VP16-POU-GR, a fusion protein consisting of the strong activator domain of VP16 and the POU domain of XlPOU 2, results in ectopic expression of the neural-specific genes, nrp-1, en-2, and beta-tubulin. In contrast, overexpressing a dominant-inhibitory form of XlPOU 2 inhibits the chordin-induced neuralization of uncommitted ectoderm, and results in a loss of nrp-1 and en-2 expression in embryos. Furthermore, in uncommitted ectoderm, XlPOU 2 regulates the developmental neural program that includes a number of pre-pattern genes and at least one proneural gene, X-ngnr-1, thus playing a key role during neural determination.
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10559482
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Fig. 2. Induction of nrp-1 and b-tubulin by VP16-POU-GR.
(A-D') Coronal sections of embryos depicting the expression of nrp-1, and b-tubulin. VP16-POUGR mRNA was injected at the 8-16 cell stage into a dorsal animal blastomere (20-100 pg) (head lineage, including the nervous system, A, A', B, and B') or ventral animal blastomere (200 pg) (belly epidermis, C, C', D, and D'), and embryos were subsequently treated with dex at stage 11 or 14 and collected at stage 20-22. The embryos treated with dex at stage 11 show increased nrp-1 expression (A; black arrowhead in the inset shows the expanded region) and ectopic expression of b-tubulin (C) only in the injected sides (turquoise). However, embryos that were treated with dex at stage14 display a normal pattern of expression for both nrp-1 and b-tubulin (B and D, respectively). The non-dex-treated embryos also show a normal pattern of expression for both genes (A', B', C', and D'). White arrows depict the enlarged region shown in the insets. Abbreviations: nt, notochord.
Fig. 3. Repression of nrp-1 expression by ENG-POU-GR. (A and B)
Whole-mount in situ hybridization of stage 20-22 embryos depicting
nrp-1 expression following the injection of ENG-POU-GR (60-300 pg)
mRNA into a dorsal animal blastomere at 8-16 cell stage (see Section 4).
Dex-treated embryos display a loss of neural tissue (A; b-gal staining,
depicting the injected side is turquoise). Non-dex-treated embryos show
normal patterns of expression for nrp-1 (B). Black arrows indicate injected
side. The alkaline phosphatase reaction product is purple.
Fig. 5. Potential regulation of en-2 by XlPOU 2 in the MHB. (A) Expression pattern of XlPOU 2 by whole-mount in situ hybridization of a stage 24 embryo (dorsal view). XlPOU 2 is expressed predominantly in the MHB (black arrow) and in the pronephros (white arrow). (B) Expression pattern of en-2 by wholemount in situ hybridization of a stage 24 embryo (dorsal view). En-2 is expressed in the MHB. (C) Expression pattern of XlPOU 2 and en-2 by double wholemount in situ hybridization of a stage 24 embryo (dorsal view). XlPOU 2 expression (white arrows), is visualized as the turquoise reaction product of alkaline phosphatase (BCIP). (D) A high magnification view of XlPOU 2 and en-2 co-localized at the MHB (dark purple; magenta-phosphate 1 BCIP). (E,F) VP16-POU-GR injected and dex-treated embryo shows an expansion of the MHB (E; black arrows), while non-dex treated embryo shows normal expression pattern for en-2 (F; white arrows show b-gal staining and an arrow outlined in black indicates the normal boundary of en-2 expression). (G,H) ENG-POU-GR dramatically reduces en-2 expression in the injected and dex-treated embryo (compare G and H; white arrows indicate the b-gal staining and a black arrow show repression; H, an arrow outlined in black indicates the normal boundary of en-2 expression).
