XB-ART-7064Genes Cells May 1, 2002; 7 (5): 487-96.
BACKGROUND: The Wnt signal transduction pathway regulates various aspects of embryonal development and has been implicated in promoting cancer. Signalling by Wnts leads to the stabilization of cytosolic beta-catenin, which then associates with TCF transcription factors to regulate expression of Wnt-target genes. The Wnt pathway is further subject to cross-regulation at various levels by other components. RESULTS: Recent evidence suggests that a specific MAP kinase pathway involving the MAP kinase kinase kinase TAK1 and the MAP kinase NLK counteract Wnt signalling. In particular, it has been shown that TAK1 activates NLK, which phosphorylates TCFs bound to beta-catenin. This phosphorylation down-regulates the DNA-binding activity of a TCF-4/beta-catenin complex, and blocks activation of their target genes. To investigate the role of NLK in Xenopus development, we isolated xNLK, a Xenopus homologue of NLK. Our findings indicate that xNLK is expressed in neural tissues and induces the anterior-neural marker gene, Otx-2. Moreover, xSox11, which is induced by the expression of Chordin, co-operates with xNLK to induce neural development. These molecules also interact in mammalian cells, and expression of a mutant of xNLK lacking kinase activity was found to suppress the induction of neural marker gene expression by xSox11. CONCLUSIONS: Our findings indicate that xNLK may play a role in neural development together with xSox11 during early Xenopus embryogenesis.
PubMed ID: 12047350
Article link: Genes Cells
Species referenced: Xenopus laevis
Genes referenced: chrd.1 map3k7 nlk nlk.2 sia1 sox11 wnt8a
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|Figure 1 Primary structure of NLK. (A) The amino acid sequence of xNLK. The xNLK cDNA encodes a predicted protein of 447 amino acids. The GenBank accession number for xNLK is AB071285. (B) Structural comparison of Xenopus NLK, mouse NLK and Drosophila Nemo. The xNLK protein is 66% and 68% identical to mNLK and Nemo at the amino acid level, respectively.|
|Figure 2 Developmental expression of Xenopus NLK. (A) RT-PCR analysis shows that xNLK mRNA is maternal and expressed at all stages of early development. Lane numbers correspond to developmental stages: 0, egg; 7, 8, 9, blastula; 10.5, 12, gastrula; 20, neurula; 24, tailbud tadpole; 34, swimming tadpole. (B) Whole-mount in situ hybridization shows the localization of the xNLK transcript. xNLK transcript is distributed to the ectoderm in the early gastrula stages. Views of the vegetal pole ( left) and cross-section (right) are shown and dorsal is right (a). At the neurula ( b) and tailbud tadpole (c) stage, xNLK expression is high in the central nervous system and head. No signal is observed in a stage 32 embryo hybridized with a sense control probe (d). (C) Expression of xSox11 gene in the neural and tailbud tadpole stages. Whole-mount in situ hybridization shows that the xSox11 transcript is high in the central nervous system and head. Lateral views are shown.|
|Figure 3 Effects of xNLK on Wnt signalling. (A) Effect of xNLK on ectopic axis formation. mRNA encoding the indicated dorsalizing factor (β-catenin: 50 pg or Siamois: 1 pg) was injected into two ventral blastomeres at the four-cell stage with or without xNLK mRNA (1 ng). Embryos were examined for axial duplications at the tadpole stage, and examples are shown. (B) xNLK inhibits the expression of the Wnt target genes. Synthetic mRNAs were injected into the animal pole of two blastomeres at the two-cell stage, and total RNAs were immediately prepared. mRNA was analysed by RT-PCR for the expression of Wnt target genes, Xnr-3 and Siamois. 1: uninjected, 2: Xwnt8 (20 pg), 3: Xwnt8 + xNLK (100 pg), 4: Xwnt8 + xNLK (1 ng), 5: Xwnt8 + xNLK-KN (100 pg), 6: Xwnt8 + xNLK-KN (1 ng), 7: -RT. Histone, ubiquitously expressed, was used as a loading control.|
|Figure 4 RT-PCR analysis of neural marker gene expression by xNLK mRNA injection in animal caps. Synthetic mRNAs containing the xNLK sequence was injected into the animal pole at the two-cell stage. Animal caps injected with xNLK (2 ng) were cultured until the neurula stage 20 (early), and total RNA was harvested. RNA was analysed by RT-PCR for the presence of the indicated transcripts: 1, whole embryo; 2, uninjected; 3, xNLK; 4, -RT. xNLK induces the expression of the anterior- and panneural markers, Otx-2 and N-CAM, respectively, but not the other neural markers.|
|TTTACFigure 5 Effect of xNLK and xSox11 on Xenopus neural induction. (A) RT-PCR analysis of neural marker gene expression by xNLK and xSox11 mRNA injection in animal caps. Synthetic mRNAs containing xNLK or xSox11 sequences were injected into the animal pole at the two-cell stage. Animal caps injected with xNLK (1 ng) and xSox11 (200 pg), xNLK-KN (1 ng) and xSox11, xSox11, xNLK or xNLK-KN mRNA were cultured until gastrula stage 20, and total RNA was harvested. RNA was analysed by RT-PCR for the presence of the indicated transcripts: 1, whole embryo; 2, uninjected; 3, xSox11; 4, xSox11 + xNLK; 5, xSox11 + xNLK-KN; 6, xNLK; 7, NLK-KN; 8, -RT.(B) Chordin induces the expression of xSox11. Synthetic mRNA containing the Chordin sequence was injected into the animal pole of 2-cell stage. Animal caps injected with Chordin mRNA (2 ng) were cultured until gastrula stage 20, and total RNA was harvested. RNA was analysed by RT-PCR for the presence of the indicated transcripts: 1, whole embryo; 2, uninjected; 3, Chordin; 4, -RT. (C) xNLK-KN inhibits the expression of an anterior-marker gene induced by expression of Chordin. Synthetic mRNAs were injected into the animal pole of the two-cell stage, and total RNAs were prepared at neural stage 20. Otx-2 RNA, an anterior neural marker, was analysed by RT-PCR. 1: whole embryo, 2: uninjected, 3: Chordin (1 ng), 4: Chordin + xNLK (1 ng), 5: Chordin + xNLK-KN (1 ng), 6: -RT.|
|Figure 6 xSox11 associates with xNLK. Two hundred and ninety-three cells were transiently co-transfected with Flag-tagged xNLK and T7-tagged xSox11. Cell lysates were subjected to immunoprecipitation using the anti-Flag antibody and then immunoblotting using the anti-T7 antibody. Expression of xSox11 was measured by anti-T7 immunoblotting of samples from cell lysates.|
|nlk (nemo-like kinase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, vegetal view (left), horizontal view of bisected embryo (right).|
|nlk (nemo-like kinase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior left.|
|nlk (nemo-like kinase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.|