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Genes Cells
2002 May 01;75:487-96. doi: 10.1046/j.1365-2443.2002.00536.x.
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Involvement of NLK and Sox11 in neural induction in Xenopus development.
Hyodo-Miura J
,
Urushiyama S
,
Nagai S
,
Nishita M
,
Ueno N
,
Shibuya H
.
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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.
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, tailbudtadpole; 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 tailbudtadpole (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
tailbudtadpole 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, anteriorleft.
nlk (nemo-like kinase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anteriorleft, dorsal up.
