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With no lysine (K) (WNK) kinase family is conserved among many species and regulates SPAK/OSR1 and ion cotransporters. WNK is also involved in developmental and cellular processes, but the molecular mechanisms underlying its regulation in these processes remain unknown. In this study, we found that WNK4 is involved in fibroblast growth factor (FGF) signaling during Xenopus development. In Xenopus embryos, depletion of WNK4 by antisense morpholino oligonucleotides (MOs) results in a severe defect in anterior development and impaired expression of endogenous anterior markers. Defects in head formation or expression of anterior marker genes caused by suppression of endogenous WNK4 expression could be rescued by expression of wild-type WNK4, but not mutant WNK4 lacking its kinase activity. It is notable that morphants of Xenopus WNK4 inhibited the expression of anterior marker genes and the target genes induced by FGF signaling. Moreover, knockdown of Wnk4 significantly reduced the phosphorylation level of Osr1 induced by FGF. These results provide the first evidence that FGF signaling regulates WNK4 function required for anterior formation in Xenopus development.
Figure 1. Alignment of the Xenopus WNK4 (xWNK4) and human WNK4 (hWNK4). The conserved amino acids are represented by asterisks (*). The kinase domains have high identity (88%) and are shaded gray.
Figure 4. Xenopus WNK4 is involved in fibroblast growth factor (FGF) signaling. (A) RT-PCR analysis of neural marker genes, NCAM and BF-1, and FGF-target genes, FoxD5a, Spry2 and Zic3. Control-MO (10 ng) or xWNK4-MO (10 ng) was co-injected with FGFR1(CA) mRNA (250 pg) into four animal blastomeres of 8-cell embryos, and animal cap explants were dissected at stage 9 and cultured until stage 18. RNAs from animal cap explants were extracted at stage 18. WE: whole embryos. (B) Western blotting analysis. Control-MO (30 ng) or xWNK4-MO (30 ng) was co-injected with FGFR1(CA) mRNA(250 pg) and/or mOSR1 mRNA (50 pg) into four animal blastomeres of 8-cell embryos. Animal caps were dissected at the early gastrula stage (st.10) and were lysed.
Figure 2. Temporal and spatial expression of xWNK4. (A) RT-PCR was carried out using 1 μg of total RNA extracted from Xenopus embryos at different stages. 'U' indicates the unfertilized eggs, and numbers indicate the developmental stages. xWNK4 is maternally expressed, and its zygotic expression increased after neurula stage (st.14). (B-E) Localization of xWNK4 transcripts by whole-mount in situ hybridization. At the gastrula stage, xWNK4 is entirely expressed in ectodermal region (B, vegetal view, stage 10.5). Expression of xWNK4 is gradually restricted to anterior neural region (lateral view, C; stage 17, D; stage 23, E; stage 27).
Figure 3. xWNK4 functions in anterior neural development. (A) Morpholino (MO) (20 ng) and FLAG-tagged mRNAs (1000 pg) were co-injected with β-globin-FLAG mRNA (200 pg) as loading control into the animal poles of 4-cell stage embryos, and the injected animal caps were dissected at stage 10. Lysates from the animal caps were subjected to Western blotting with anti-FLAG antibody (M2, Sigma). (B-E) Phenotypes of injected embryos at stage 35. Control-MO (30 ng) or xWNK4-MO (30 ng) was co-injected with xWNK4 mRNA (1000 pg) or kinase negative xWNK4 mRNA (1000 pg) into two dorso-animal blastomeres of 8-cell embryos. (B) Control-MO. (C) xWNK4-MO. (D) xWNK4-MO with xWNK4 mRNA. (E) xWNK4-MO with kinase negative xWNK4 mRNA. (F) RT-PCR analysis of neural marker genes, NCAM (pan-neural), BF-1 (forebrain) and Rx1 (eye). RNAs from head regions of injected embryos were extracted at stage 25.
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