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Mech Dev
2003 May 01;1205:607-16. doi: 10.1016/s0925-4773(03)00010-8.
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Depletion of the cell-cycle inhibitor p27(Xic1) impairs neuronal differentiation and increases the number of ElrC(+) progenitor cells in Xenopus tropicalis.
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The Xenopus p27(Xic1) gene encodes a cyclin dependent kinase (CDK) inhibitor of the Cip/Kip family. We have previously shown that p27(Xic1) is expressed in the cells of the neural plate as they become post-mitotic (Development 127 (2000) 1303). To investigate whether p27(Xic1) is necessary for cell cycle exit and/or neuronal differentiation, we used antisense morpholino oligos (MO) to knockdown the protein levels in vivo. For such knockdown studies, Xenopus tropicalis is a better model system than Xenopus laevis, since it has a diploid genome. Indeed, while X. laevis has two p27(Xic1) paralogs, p27(Xic1) and p28(Kix1), we have found only one ortholog in X. tropicalis, equidistant from the X. laevis genes. The X. tropicalis p27(Xic1) was expressed in a similar pattern to the X. laevis gene. Depletion of p27(Xic1) in X. tropicalis caused an increase in proliferation and a suppression of the neuronal differentiation marker, N-tubulin. At the same time, we found an increase in the expression of ElrC, a marker of cells as they undergo a transition from proliferation to differentiation. We conclude that p27(Xic1) is necessary for cells to exit the cell cycle and differentiate; in its absence, cells accumulate in a progenitor state. The expression of p27(Xic1) in the embryo is regionalised but the transcriptional regulation of p27(Xic1) is not well understood. We report the isolation of a p27(Xic1) genomic clone and we identify a 5' region capable of driving reporter gene expression specifically in the neural tube and the eye.
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Fig. 2. Expression of p27Xic1 in X. tropicalis and X. laevis. Xenopus tropicalis (A–C, F) and X. laevis (D, E). (A, D) At stage 13 both genes are strongly expressed in the notochord and somites. Expression in the ectoderm is present but weak. (B, E, F) At stage 16 expression in dorsal mesoderm persists but is also evident in the deep layer of the neural ectoderm (arrow, F). (C) By stage 27, p27Xic1 is expressed in the mesoderm of the tailbud, the neural tube and the eye. In the whole mounts, anterior is to the left.
Fig. 4. Comparison of N-tubulin and ElrC expression in the neural plate and neural tube. (A–D) Neural plate stage (stage 16) embryos hybridised with the markers indicated, some are additionally stained for proliferation by BrdU incorporation (brown nuclei; C, D). Arrows point to BrdU positive cells that express ElrC but not N-tubulin. Arrowhead points to an example of a BrdU positive cell that does not express ElrC. (E, F) Sections of tailbud neural tube (stage 23) at the level of the otic vesicle (ov), stained as indicated. Inset is a section demonstrating the location of BrdU in the neural tube. (G, H) Tadpole neural tube section (stage 34), stained as indicated. In all stages, the overlap between ElrC and N-tubulin expression is partial. At the neural tube stage, note that ElrC+ cells are located closer to the ventricle than N-tubulin+ cells.
Fig. 3. Effect of p27Xic1 MO on neuronal differentiation. (A) Western blot verifying p27Xic1 MO reduces the amount of endogenous p27Xic1 in X. tropicalis and the specific control MO has no affect, nPKC is a loading control. (B) X. tropicalis embryos were injected with p27Xic1 MO or control MO and were analysed at stage 18 for Sox3 or N-tubulin expression (purple) and BrdU incorporation (brown nuclei), as indicated. In the whole mounts, the injected side is towards the lower part of the panels and shown by detection of the FITC MO lineage tracer (light blue). Arrows point to the missing N-tubulin expression on the injected side
Fig. 5. Effect of p27Xic1 MO on ElrC expression and proliferation. (A�C) Neurula stage embryos (stage 18) injected with p27Xic1 and control MO, as indicated. Injected half is towards the lower part of the panels and is stained light blue by co-injection of FITC MO. (D�F) Sections that correspond to these embryos are shown immediately below each panel. Inset in F, shows ElrC expression at an earlier stage. An arrow indicates a gap in Elr-C expression in the control MO injected (F), which is present at an earlier stage (inset) but is missing from the p27Xic1 MO injected ectoderm (E). A broken line indicates the midline and the injected side is on the left. Brown nuclei represent BrdU incorporation (E�I). (G�I) Higher magnification images of p27Xic1 MO injected ectoderm (G), control MO injected ectoderm (H) and control uninjected side (I). Note that the BrdU stained nuclei in p27Xic1 MO injected ectoderm (G) are more numerous and smaller than the controls (H�I). (J) Quantification of ElrC+, BrdU+ cells in p27Xic1 MO injected ectoderm (n=45 sections from five embryos) versus control MO injected (n=39 sections from five embryos). In each case, a comparison is also made to the uninjected side. Although the increase of ElrC+, BrdU+ cells by the p27Xic1 MO was variable in no case did the p27Xic1 MO injected side have less such cells than the control side.
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Fig. 6. A p27Xic1 genomic clone drives expression in the neural tissue. (A) Genomic structure of the X. tropicalis p27Xic1. The coding sequence is boxed. An intron is located in the 3′ UTR, 7 nt from the stop codon. (B-D) Xenopus laevis transgenic embryos (stage 33), with a p27Xic1-GFP construct, show GFP expression in the neural tube and the eye. Inset in B is the bright field image. In C and D, GFP expression was detected by in situ hybridisation. (E, F) X. tropicalis endogenous p27Xic1 expression (stage 33), arrowheads indicate areas of expression absent from the transgenics.
Fig. 6. A p27Xic1 genomic clone drives expression in the neural tissue. (A) Genomic structure of the X. tropicalis p27Xic1. The coding sequence is boxed. An intron is located in the 30 UTR, 7 nt from the stop codon.
