WNK4 is an essential effector of anterior formation in FGF signaling

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.


Introduction
With no lysine (K) (WNK) is a family of serine/threonine protein kinases that are characterized by an atypical location of the catalytic lysine and are conserved among many species, such as plants, nematode, fly, rat, mouse and human (Xu et al. 2000;Ver ıssimo & Jordan 2001;Moniz & Jordan 2010). There are four mammalian WNK family members, and positional cloning has identified two of them, WNK1 and WNK4, as genes linked to a hereditary form of human hypertension known as pseudohypoaldosteronism type II (PHAII) (Wilson et al. 2001). Several groups including our group previously discovered that WNK1 and WNK4 could phosphorylate and activate SPAK or OSR1 kinases, which in turn regulate various ion cotransporters, such as NKCC1, NKCC2 and NCC (Piechotta et al. 2002;Moriguchi et al. 2005;Vitari et al. 2005;Gagnon et al. 2006). Deletion in intron 1 of WNK1 gene in PHAII patients caused the increased expression of WNK1, which leads to hypertension by the misregulation of ion cotransporters (Wilson et al. 2001). WNK4 is known to be regulated by renin-angiotensin-aldosterone system. Angiotensin II induced WNK4 activation, which controlled NCC phosphorylation through SPAK/OSR1 kinases (San-Cristobal et al. 2009;Talati et al. 2010;van der Lubbe et al. 2011;Castañeda-Bueno et al. 2012). PHAII mutations in acidic region of WNK4 (D561A) disrupt this regulation (San-Cristobal et al. 2009). We also found that WNK4 D561A/+ knock-in mice, model mice of human PHAII patient, caused phenotypes similar to those of PHAII (Yang et al. 2007). Furthermore, serum-and glucocorticoid-induced protein kinase (SGK1), which was activated by aldosterone, regulated WNK4 activity (Bhargava et al. 2001). The phosphorylation by SGK1 inhibited the function of inhibitory domain of WNK4 (Ring et al. 2007). PHAII mutations in C-terminal region of WNK4 (R1185C) lost the regulation by SGK1 (Na et al. 2013). These results suggest that the dysregulation of WNK4 contributes to the pathogenesis of hypertension in PHAII patients.
The WNK-SPAK/OSR1 pathway is known to regulate various ion cotransporters and is widely conserved among many species (Piechotta et al. 2002;Moriguchi et al. 2005;Vitari et al. 2005;Gagnon et al. 2006;Hisamoto et al. 2008;Sato & Shibuya 2013). Wnk1 knockout mice die before embryonic day 13 (Zambrowicz et al. 2003;Sato & Shibuya 2013) and display defects in cardiac development (Xie et al. 2009). WNK1 is also required for cell division in cultured cells (Tu et al. 2011), and proliferation, migration and differentiation of neural progenitor cells (Sun et al. 2006). Furthermore, PHAII patients display a number of other clinical features, such as an intellectual impairment, dental abnormalities and impaired growth in addition to hypertension (Gordon 1986). Accordingly, the new role of the WNK signaling pathway described here may provide further insight into the development and pathogenesis of PHAII. Recently, we have identified Lhx8/Awh as a new downstream molecule in the WNK-SPAK/ OSR1 pathway and discovered a novel function for the WNK-Lhx8 pathway in neural development (Sato & Shibuya 2013).
In vertebrate, neural induction is established by inhibition of bone morphogenetic protein (BMP) signal in the ectoderm (Bier 1997;Sasai & De Robertis 1997;Wilson & Hemmati-Brivanlou 1997;Weinstein & Hemmati-Brivanlou 1999). Several inhibitors of BMP such as noggin and chordin are isolated from organizer region in Xenopus. However, inhibitory signals from organizer are not enough for neural induction, and other signals including fibroblast growth factor (FGF) are necessary for neural induction (Launay et al. 1996;Hongo et al. 1999;Powers et al. 2000). For examples, a kinase inhibitor of FGF receptor 1 (FGFR1) inhibits down-regulation of BMP in chick explants (Wilson et al. 2000). In Xenopus, FGF signals contribute to neural induction in animal cap cells (Kengaku & Okamoto 1995;Lamb & Harland 1995), and over-expression of dominant negative forms of FGFRs, which lack the intracellular domain, inhibits neural induction by noggin and chordin (Launay et al. 1996;Sasai et al. 1996).
FGF ligands contain more than 20 family members (Powers et al. 2000;Fukumoto 2008). Four FGFR genes, FGFR1 to FGFR4, are identified in vertebrates (Dailey et al. 2005;Eswarakumar et al. 2005). Each FGFR contains an FGF binding extracellular domain and a catalytic intracellular domain. When a ligand binds to a receptor, receptor dimerizes via the extracellular domain and its intracellular tyrosine kinase domain is autophosphorylated, causing its downstream signaling. The main downstream pathways include the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-3 kinase (PI3K) pathway and the phospholipase Cc (PLCc) pathway (Dailey et al. 2005;Eswarakumar et al. 2005).
In the present study, we report the novel finding that Xenopus WNK4 (xWNK4) is involved in FGF signaling. Depletion of xWNK4 resulted in defects in anterior neural development in Xenopus embryos, including the loss of eye and head structures. The phenotypes induced by depletion of endogenous xWNK4 were rescued by over-expression of wildtype xWNK4, but not by a kinase negative mutant of xWNK4. Moreover, the expression of FGF-inducible genes was inhibited by the depletion of xWNK4. These results reveal a new role of WNK4 function during anterior formation in Xenopus embryos.

Isolation of Xenopus WNK4 cDNA
To isolate Xenopus WNK4 (xWNK4) cDNA, we searched the Xenopus expressed sequence tag database with high homology to human WNK4 (hWNK4) and isolated the full-length cDNA from a Xenopus tailbud cDNA library by PCR method using primers based on the predicted WNK4 coding sequence (see Experimental Procedures). We found a single open reading frame of 1574 amino acids as a Xenopus homologous gene. Xenopus WNK4 contains a single kinase domain near the N-terminal region, which has remarkable homology (88% identity) with the human WNK4 one (Fig. 1A). This suggests that xWNK4 also regulates the downstream kinases, such as SPAK/ OSR1 (Moriguchi et al. 2005;Vitari et al. 2005;Gagnon et al. 2006).

Xenopus WNK4 is expressed maternally and throughout early embryogenesis
To assess the possible involvement of xWNK4 for early development, we first examined the temporal and spatial expression patterns of xWNK4 by RT-PCR analysis and whole-mount in situ hybridization. RT-PCR analysis of xWNK4 expression in early Xenopus embryos revealed that xWNK4 mRNA was expressed maternally, and its zygotic expression was increased after the neurula stage ( Fig. 2A). Expression of xWNK4 mRNA was detected entirely in ectoderm region at gastrula stage (Fig. 2B). After the neurula stage, transcripts of xWNK4 were gradually restricted to neural region, especially in anterior neural regions involving eye and brain at later stages ( Fig. 2C-E). These results suggest that xWNK4 might act in Xenopus early embryogenesis.

Xenopus WNK4 functions in anterior neural development
To examine the function of xWNK4 in early embryogenesis, we synthesized antisense morpholino oligonucleotide (MO) against xWNK4 (See Experimental procedures). By Western blotting analysis, we confirmed that injection of the xWNK4 -MO specifically reduced expression of xWNK4 protein (Fig. 3A). When xWNK4-MO was injected into two dorso-animal blastomeres at 8-cell stage, which develops mainly into neuroectodermal tissues and head structure, the resulting phenotype interfered with the head formation (Fig. 3B,C). We also confirmed that depletion of xWNK4 reduced expression of neural marker genes such as pan-neural marker, NCAM; forebrain marker, BF-1; eye marker, Rx1 (Fig. 3F). The anterior defects and the reduction of anterior neural marker expression by xWNK4-MO were rescued by co-injection of wild-type xWNK4 mRNA, but not by kinase-inactive xWNK4 mRNA (Fig. 3D-F). These results suggest that xWNK4 is specifically involved in anterior formation in Xenopus embryos.

Xenopus WNK4 is involved in FGF signaling
It is known that the FGF signaling pathway positively regulates Xenopus anterior neural development (Hongo et al. 1999). To examine whether xWNK4 would be involved in these signaling pathways in early embryogenesis, we analyzed the effects of xWNK4-MO on the expression of FGF-inducible neural marker genes in Xenopus animal cap cells. We found that co-injection of constitutively active FGF receptor 1 (FGFR1(CA)) mRNA with xWNK4-MO decreased the expression of neural marker genes, NCAM and BF-1, relative to animal cap cells coinjected with the control morpholino (Fig. 4A). Moreover, the expression of FGF-target genes, FoxD5a, Spry2 and Zic3, was also inhibited by depletion of endogenous xWNK4. We further attempted to examine the WNK-SPAK/OSR1 pathway in the FGF stimulation. A previous study has reported that WNKs are activated by hypertonic stimulation (Zag orska et al. 2007). To investigate whether WNK4 is activated by FGF stimulation, we carried out Western blotting analysis using antiphospho OSR1 antibody, which recognizes Ser325 of mouse OSR1 (mOSR1) phosphorylated by WNK kinases, with animal cap cells. We found that the phosphorylation level of mOSR1 was increased by the expression of FGFR1(CA) in animal cap cells (Fig. 4B). However, depletion of xWNK4 significantly reduced the phosphorylation level of mOsr1 (Fig. 4B). Taken together, these results indicate that xWNK4 may positively contribute to the FGF signaling pathway.

Discussion
In this report, we found a novel role of WNK4 gene in Xenopus embryos. Injection of xWNK4-MO demonstrated that xWNK4 is involved in the anterior formation during early embryogenesis. Moreover, in animal cap cells, xWNK4-MO inhibits the anterior neural marker, and FGF-target genes induced by FGFR1(CA). These results suggest that xWNK4 contributes to the FGF signaling in Xenopus embryos.
WNK has been shown to interact directly with and to phosphorylate SPAK/OSR1, which in turn regulates various ion cotransporters, such as NKCC1, NKCC2 and NCC (Piechotta et al. 2002;Moriguchi et al. 2005;Vitari et al. 2005;Gagnon et al. 2006). Moreover, we have recently shown that WNK regulates the expression of Lhx8 to play an essential role in neural development (Sato & Shibuya 2013). We also indicated the possibility that Drosophila WNK directly interacted with and phosphorylated Fray, fly homologue of SPAK/OSR1, which in turn regulated the expression of Arrowhead, fly homologue of Lhx8, and that this pathway is important for Drosophila abdominal and neural development (Sato & Shibuya 2013). Thus, WNK-SPAK/ OSR1 pathway may be able to be involved in multiple function, and possibly, its activities are modulated through the phosphorylation of SPAK/OSR1 by WNK. Our results showing that FGFR1(CA) can activate mOSR1 in animal cap cells (Fig. 4B) suggest that WNK activity is also regulated by FGF. These observations lead us to hypothesize that WNK, presumably activated by FGF, may interact with and regulate the expression of FGF-induced target genes. Actually, xWNK4-MO inhibited the expression of FGF-induced target genes in animal cap cells. However, it has not been directly shown whether WNK-SPAK/OSR1 pathway regulates the target genes following stimulation of the FGF signal-ing pathway. Additional experiments will be required to test this hypothesis, and further molecular analysis will be needed to assess the role of WNK-SPAK/OSR1 pathway in FGF signaling. To date, two regulatory pathways for head development have been proposed. One is a default state that is triggered by inhibiting growth factor signals required for trunk development, such as those induced by Wnt, BMP and Nodal (Harland 2000). The other includes growth factor-mediated active pathways, such as FGFs and IGFs (Hardcastle et al. 2000;Pera et al. 2001;Richard-Parpaillon et al. 2002). These pathways function in a parallel and/or cooperative manner to promote anteriorizing activity and to suppress the trunk mesoderm. Although the actual mediator(s) for anterior neural formation, which is regulated by WNK-SPAK/ OSR1 pathway in FGF signaling, is unclear at present, it is known that WNK activates the expression of Lhx8/Arrowhead gene and is involved in neural differentiation of mammalian cells and Drosophila neural development (Sato & Shibuya 2013). It is possible that WNK activates the expression of Lhx8 through the action of a yet-to-be-identified mediator(s) that regulates anterior tissue development, and further studies will be needed to identify the precise mediator(s) involved in this process. It will also be important to determine whether there is crosstalk between FGF-WNK-SPAK/OSR1 pathway and other signaling pathways to mediate anterior tissue development.

Plasmid construction and morpholino oligonucleotides
The Xenopus WNK4 (xWNK4) was amplified by PCR from a Xenopus tailbud cDNA library using the following primers: 5′-CGCCATCGATGGTACTTGTGTACATTGCTC-3′ and 5′-CGGCTCTAGATTACTTTACTTTTTCATCCTG-3′, and subcloned into pCS2+ vector. A kinase negative mutant of xWNK4 was constructed by replacing the lysine residue with methionine: K185M. A constitutive active FGFR1 was constructed by replacing the cysteine residue with tyrosine: C337Y. The MO (Gene Tools, LLC) used here were 5′-CCTCTTACCTCAGTTACAATTTATA-3′ (Control-MO), 5′-CAGGGACACTAACAGCAAACATCTC-3′ (xWNK4-MO). The specificity of xWNK4-MO was confirmed by its ability to inhibit the translation of FLAG-tagged mRNAs containing the targeted site with or without 5-mismatched sequences. MO (20 ng) and FLAG-tagged mRNAs (1000 pg) were co-injected with b-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).

Embryo handling
MOs and mRNAs were injected into four animal blastomeres at the 8-cell stage for dissection of animal caps or into two dorso-animal blastomeres at the 8-cell stage for RT-PCR analysis and observation of embryo phenotypes. Animal cap explants of the injected embryos were dissected at the late blastula stage (st.9), and total RNA was extracted at the neurula stage (st.18) for RT-PCR analysis. Head regions of embryos were dissected at the tailbud stage (st.25), and total RNA was extracted for RT-PCR analysis.

Whole-mount in situ hybridization
In situ hybridization was carried out using digoxigenin-labeled RNA probe and alkaline phosphatase substrate (NBT) (Roche) according to Harland (1991).