Endothelial-Specific Expression of WNK1 Kinase Is Essential for Angiogenesis and Heart Development in Mice
2009; Elsevier BV; Volume: 175; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2009.090094
ISSN1525-2191
AutoresJian Xie, Tao Wu, Ke Xu, Ivan Huang, Ondine Cleaver, Chou-Long Huang,
Tópico(s)Ion Transport and Channel Regulation
ResumoWNK1 [with-no-lysine (K)−1] is a ubiquitous serine/threonine kinase with a unique placement of the catalytic lysine residue. Increased WNK1 expression levels in humans causes a hypertension-hyperkalemia syndrome by altering renal Na+ and K+ transport. The function of WNK1 outside of the kidney remains elusive. In this study, we report that Wnk1 ablation causes cardiovascular developmental defects. The developing heart of null mutant embryos has smaller chambers and reduced myocardial trabeculation at E10.5. Yolk sac vessels in the E10.5 null mutant fail to remodel into a network of large and small vessels, and embryonic vessels show defective angiogenesis that involves both arteries and veins. The arterial marker neuropilin-1 and venous marker EphB4 are ectopically expressed in mutant veins and arteries, respectively. However, the orphan nuclear receptor COUP-TFII as well as the Notch signaling pathway, which are known to be critical for angiogenesis and artery-vein specification, are not significantly altered in Wnk1−/− mutants. Conditional deletion of Wnk1 in endothelial cells phenotypically copies defects caused by global Wnk1 ablation. Moreover, endothelial-specific expression of a Wnk1 transgene rescues cardiovascular developmental defects in Wnk1−/− mice. These findings identify a novel function of WNK1 in endothelial cells that is critical for angiogenesis and heart development, raising the possibility for a role of endothelial WNK1 in the control of blood pressure and postnatal angiogenesis and cardiac growth. WNK1 [with-no-lysine (K)−1] is a ubiquitous serine/threonine kinase with a unique placement of the catalytic lysine residue. Increased WNK1 expression levels in humans causes a hypertension-hyperkalemia syndrome by altering renal Na+ and K+ transport. The function of WNK1 outside of the kidney remains elusive. In this study, we report that Wnk1 ablation causes cardiovascular developmental defects. The developing heart of null mutant embryos has smaller chambers and reduced myocardial trabeculation at E10.5. Yolk sac vessels in the E10.5 null mutant fail to remodel into a network of large and small vessels, and embryonic vessels show defective angiogenesis that involves both arteries and veins. The arterial marker neuropilin-1 and venous marker EphB4 are ectopically expressed in mutant veins and arteries, respectively. However, the orphan nuclear receptor COUP-TFII as well as the Notch signaling pathway, which are known to be critical for angiogenesis and artery-vein specification, are not significantly altered in Wnk1−/− mutants. Conditional deletion of Wnk1 in endothelial cells phenotypically copies defects caused by global Wnk1 ablation. Moreover, endothelial-specific expression of a Wnk1 transgene rescues cardiovascular developmental defects in Wnk1−/− mice. These findings identify a novel function of WNK1 in endothelial cells that is critical for angiogenesis and heart development, raising the possibility for a role of endothelial WNK1 in the control of blood pressure and postnatal angiogenesis and cardiac growth. The cardiovascular system is the first organ system to develop during embryogenesis. In developing mouse embryos, blood circulation begins at about day 8.5 of gestation.1Risau W Flamme I Vasculogenesis.Annu Rev Cell Dev Biol. 1995; 11: 73-91Crossref PubMed Scopus (1352) Google Scholar, 2Folkman J D'Amore PA Blood vessel formation: what is its molecular basis?.Cell. 1996; 87: 1153-1155Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar The development of the vascular system occurs via two separate processes known as vasculogenesis and angiogenesis. Vasculogenesis involves in situ differentiation of endothelial cells from precursor angioblasts and assembly of these differentiating endothelial cells into tube-shaped blood vessels. This process results in the formation of the primordial heart tube and major axial vessels as well as a homogenous primary capillary plexus in the embryo proper and extraembryonic structures such as the yolk sac. Angiogenesis involves further growth, sprouting, branching from the primary vessels and remodeling of the vascular plexus to form a tree-like system of large and small vessels, eventually resulting in a mature vasculature.1Risau W Flamme I Vasculogenesis.Annu Rev Cell Dev Biol. 1995; 11: 73-91Crossref PubMed Scopus (1352) Google Scholar, 2Folkman J D'Amore PA Blood vessel formation: what is its molecular basis?.Cell. 1996; 87: 1153-1155Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar Angiogenesis is responsible for the formation of blood vessels in organs, such as the brain and kidney. Integral to the development of vasculature is the recruitment of mural cells (smooth muscle and/or pericytes) to form the external walls of mature vessels. The arterial and venous sides of the vascular system are anatomically, functionally and molecularly distinct. Specification of arteries and veins is an essential process of vascular development that requires reciprocal interactions between arterial and venous fated endothelial cells. Failure in artery/vein specification results in angiogenesis defects and almost always in embryonic lethality.3Wang HU Chen ZF Anderson DJ Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.Cell. 1998; 93: 741-753Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar, 4Gu C Rodriguez ER Reimert DV Shu T Fritzsch B Richards LJ Kolodkin AL Ginty DD Neuropilin-1 conveys Semaphorin and VEGF signaling during neural and cardiovascular development.Dev Cell. 2003; 5: 45-57Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar The molecular mechanisms controlling vascular development have been under intensive investigation, but much remains unknown. Dozens of genes have been identified that regulate the processes of vasculogenesis, angiogenesis, and artery-vein specification.5Coultas L Chawengsaksophak K Rossant J Endothelial cells and VEGF in vascular development.Nature. 2005; 438: 937-945Crossref PubMed Scopus (798) Google Scholar Vascular endothelial growth factor (VEGF) and its receptors VEGF-R1 (Flt1) and VEGF-R2 (Flk1), and transforming growth factor-β are essential regulators of the initial differentiation and assembly of endothelial cell into primary vessel plexus in vasculogenesis. Additional factors, including angiopoietins/Tie receptors, VEGFs/VEGF receptors, fibroblast growth factors (FGFs), Notch signaling pathway, transforming growth factor-β/Endoglin/Smad5, matrix metalloproteinases (MMPs), ephrins and receptors, neuropilins, are also involved in modulation of angiogenesis. Early hypotheses proposed that activation of the VEGF-Notch signaling pathway confers arterial fate and a lack of activation results in the formation of veins by default.6Adams RH Molecular control of arterial-venous blood vessel identity.J Anat. 2003; 202: 105-112Crossref PubMed Scopus (75) Google Scholar A more recent study, however, reports that the orphan nuclear receptor COUP-TFII is specifically expressed in venous-fated endothelial cells and actively confers venous fate by suppressing the VEGF-Notch signaling.7You LR Lin FJ Lee CT DeMayo FJ Tsai MJ Tsai SY Suppression of notch signaling by the Coup-TFII transcription factor regulates vein identity.Nature. 2005; 435: 98-104Crossref PubMed Scopus (480) Google Scholar Mice homozygous for deletion of COUP-TFII, however, do not display vascular defects in yolk sac,8Pereira FA Qiu Y Zhou G Tsai MJ Tsai SY The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development.Genes Dev. 1995; 13: 1037-1049Crossref Scopus (413) Google Scholar suggesting that yet additional factors are involved in the molecular determination of vein identity. WNK1 [with-no-lysine (K)−1] is a member of unique serine/threonine protein kinase family characterized by an atypical location of the catalytic lysine.9Xu B English JM Wilsbacher JL Stippec S Goldsmith EJ Cobb MH WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II.J Biol Chem. 2000; 275: 16795-16801Crossref PubMed Scopus (416) Google Scholar Despite the atypical kinase domain structure, WNK1 exhibits kinase activity and catalyzes phosphorylation of endogenous substrates.9Xu B English JM Wilsbacher JL Stippec S Goldsmith EJ Cobb MH WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II.J Biol Chem. 2000; 275: 16795-16801Crossref PubMed Scopus (416) Google Scholar Three additional members, WNK2, WNK3, and WNK4, are present in mammals.9Xu B English JM Wilsbacher JL Stippec S Goldsmith EJ Cobb MH WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II.J Biol Chem. 2000; 275: 16795-16801Crossref PubMed Scopus (416) Google Scholar, 10Wilson FH Disse-Nicodeme S Choate KA Ishikawa K Nelson-Williams C Desitter I Gunel M Milford DV Lipkin GW Achard JM Feely MP Dussol B Berland Y Unwin RJ Mayan H Simon DB Farfel Z Jeunemaitre X Lifton RP Human hypertension caused by mutations in WNK kinases.Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1221) Google Scholar, 11Verissimo F Jordan P WNK kinases, a novel protein kinase subfamily in multi-cellular organisms.Oncogene. 2001; 20: 5562-5569Crossref PubMed Scopus (226) Google Scholar Each member of the WNK family is encoded by a separate gene. WNK1 protein is over 2100 amino acids long, whereas WNK2, WNK3, and WNK4 range between 1200 and 1600 amino acids in length. The four WNK kinases share a conserved kinase domain, an autoinhibitory domain, 1–2 coiled-coil domains, and multiple proline-rich motifs for potential protein-protein interactions.9Xu B English JM Wilsbacher JL Stippec S Goldsmith EJ Cobb MH WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II.J Biol Chem. 2000; 275: 16795-16801Crossref PubMed Scopus (416) Google Scholar, 10Wilson FH Disse-Nicodeme S Choate KA Ishikawa K Nelson-Williams C Desitter I Gunel M Milford DV Lipkin GW Achard JM Feely MP Dussol B Berland Y Unwin RJ Mayan H Simon DB Farfel Z Jeunemaitre X Lifton RP Human hypertension caused by mutations in WNK kinases.Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1221) Google Scholar, 11Verissimo F Jordan P WNK kinases, a novel protein kinase subfamily in multi-cellular organisms.Oncogene. 2001; 20: 5562-5569Crossref PubMed Scopus (226) Google Scholar Few recognizable motifs exist beyond the aforementioned conserved domains/motifs. The human as well as mouse WNK1 gene each consists of 28 exons and undergoes tissue-specific alternative splicing.12Delaloy C Lu J Houot AM Disse-Nicodeme S Gasc JM Corvol P Jeunemaitre X Multiple promoters in the WNK1 gene: one controls expression of a kidney-specific kinase-defective isoform.Mol Cell Biol. 2003; 23: 9208-9221Crossref PubMed Scopus (141) Google Scholar, 13O'Reilly M Marshall E Speirs HJ Brown RW WNK1, a gene within a novel blood pressure control pathway, tissue-specifically generates radically different isoforms with and without a kinase domain.J Am Soc Nephrol. 2003; 14: 2447-2456Crossref PubMed Scopus (144) Google Scholar A full-length WNK1 encoded by all 28 exons is ubiquitously expressed.9Xu B English JM Wilsbacher JL Stippec S Goldsmith EJ Cobb MH WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II.J Biol Chem. 2000; 275: 16795-16801Crossref PubMed Scopus (416) Google Scholar, 12Delaloy C Lu J Houot AM Disse-Nicodeme S Gasc JM Corvol P Jeunemaitre X Multiple promoters in the WNK1 gene: one controls expression of a kidney-specific kinase-defective isoform.Mol Cell Biol. 2003; 23: 9208-9221Crossref PubMed Scopus (141) Google Scholar, 13O'Reilly M Marshall E Speirs HJ Brown RW WNK1, a gene within a novel blood pressure control pathway, tissue-specifically generates radically different isoforms with and without a kinase domain.J Am Soc Nephrol. 2003; 14: 2447-2456Crossref PubMed Scopus (144) Google Scholar A shorter splice variant of WNK1 that lacks the region encoded by the first four exons is predominantly expressed in the kidney.12Delaloy C Lu J Houot AM Disse-Nicodeme S Gasc JM Corvol P Jeunemaitre X Multiple promoters in the WNK1 gene: one controls expression of a kidney-specific kinase-defective isoform.Mol Cell Biol. 2003; 23: 9208-9221Crossref PubMed Scopus (141) Google Scholar WNK2 is predominantly expressed in heart, brain and colon.11Verissimo F Jordan P WNK kinases, a novel protein kinase subfamily in multi-cellular organisms.Oncogene. 2001; 20: 5562-5569Crossref PubMed Scopus (226) Google Scholar WNK3 and WNK4 are expressed in kidney, heart, and brain.11Verissimo F Jordan P WNK kinases, a novel protein kinase subfamily in multi-cellular organisms.Oncogene. 2001; 20: 5562-5569Crossref PubMed Scopus (226) Google Scholar, 14Holden S Cox J Raymond FL Cloning, genomic organization, alternative splicing and expression analysis of the human gene WNK3 (PRKWNK3).Gene. 2004; 335: 109-119Crossref PubMed Scopus (52) Google Scholar, 15Kahle KT Gimenez I Hassan H Wilson FH Wong RD Forbush B Aronson PS Lifton RP WNK4 regulates apical and basolateral Cl− flux in extrarenal epithelia.Proc Natl Acad Sci USA. 2004; 101: 2064-2069Crossref PubMed Scopus (150) Google Scholar In addition, WNK4 is expressed in many epithelial tissues.15Kahle KT Gimenez I Hassan H Wilson FH Wong RD Forbush B Aronson PS Lifton RP WNK4 regulates apical and basolateral Cl− flux in extrarenal epithelia.Proc Natl Acad Sci USA. 2004; 101: 2064-2069Crossref PubMed Scopus (150) Google Scholar WNK1 regulates sodium channel ENaC and the thiazide-sensitive sodium-chloride cotransporter by altering their cell surface abundance.16Yang CL Angell J Mitchell R Ellison DH WNK kinases regulate thiazide-sensitive Na-Cl cotransport.J Clin Invest. 2003; 111: 1039-1045Crossref PubMed Scopus (401) Google Scholar, 17Xu B Stippec S Chu PY Li XJ Lazrak A Ortega B Lee BH English JM Huang CL Cobb MH WNK1 activates SGK1 to regulate the epithelial sodium channel.Proc Natl Acad Sci USA. 2005; 102: 10315-10320Crossref PubMed Scopus (165) Google Scholar WNK1 has also been reported to interact with intersectin, a multimodular endocytic scaffold protein, to regulate internalization of a renal potassium channel ROMK1 from the cell surface.18He G Wang HR Huang SK Huang CL Intersectin links WNK kinases to endocytosis of ROMK.J Clin Invest. 2007; 117: 1078-1087Crossref PubMed Scopus (129) Google Scholar Large intronic deletions of WNK1 that lead to its overexpression cause pseudohypoaldosteronism type II (PHA II), an autosomal dominant disorder featuring hypertension and hyperkalemia (high blood potassium level).10Wilson FH Disse-Nicodeme S Choate KA Ishikawa K Nelson-Williams C Desitter I Gunel M Milford DV Lipkin GW Achard JM Feely MP Dussol B Berland Y Unwin RJ Mayan H Simon DB Farfel Z Jeunemaitre X Lifton RP Human hypertension caused by mutations in WNK kinases.Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1221) Google Scholar Alterations in WNK1 regulation of sodium and potassium transport in kidney contribute, at least partly, to the pathogenesis of hypertension and hyperkalemia in patients with WNK1 mutations. Mice heterozygous for Wnk1 mutation generated by a gene-trapping insertion within intron-1 have hypotension, supporting an important role of WNK1 in the control of blood pressure.19Zambrowicz BP Abuin A Ramirez-Solis R Richter LJ Piggott J BeltrandelRio H Buxton EC Edwards J Finch RA Friddle CJ Gupta A Hansen G Hu Y Huang Wenhu Jaing C Key Jr, BW Kipp P Kohlhauff B Ma Z-Q Markesich D Payne R Potter DG Quan N Shaw J Schrick J Shi Z-Z Sparks MJ Van Slightenhorst I Vogel P Walke W Xu N Zhu Q Person C Sands AT Wnk1 kinase deficiency lowers blood pressure in mice: A gene-trap screen to identify potential targets for therapeutic intervention.Proc Natl Acad Sci USA. 2003; 100: 14109-14114Crossref PubMed Scopus (301) Google ScholarWnk1 heterozygous mice, however, do not display apparent defects in renal Na+ transport, raising the possibility for additional role of WNK1 in other tissues in the control of blood pressure. Mice homozygous for Wnk1 mutation die before embryonic day 13 (E13), indicating an essential role of WNK1 in embryogenesis. However, the precise role of WNK1 in the embryonic development is not known. Here, we show that Wnk1-ablated mice display cardiac developmental and angiogenesis defects. The angiogenesis defect is evident in both yolk sac and embryos and is associated with an aberrant expression of arterial and venous markers. The defects in Wnk1-null embryos, however, are distinct from arterial-venous specification abnormalities observed in embryos with alterations of COUP-TFII and Notch signaling pathway. Conditional deletion of Wnk1 in endothelial cells phenocopies defects caused by global Wnk1 ablation. Endothelial-specific expression of a Wnk1 transgene rescues cardiovascular developmental defects in Wnk1−/− mice. These findings suggest that function of WNK1 in endothelial cells defines a novel mechanism for regulating angiogenesis and establishing or maintaining artery/vein identity and raise the possibility for a role of endothelial WNK1 in the control of blood pressure. Wnk1+/− mice produced by a gene-trap insertion in intron-1 have been described.19Zambrowicz BP Abuin A Ramirez-Solis R Richter LJ Piggott J BeltrandelRio H Buxton EC Edwards J Finch RA Friddle CJ Gupta A Hansen G Hu Y Huang Wenhu Jaing C Key Jr, BW Kipp P Kohlhauff B Ma Z-Q Markesich D Payne R Potter DG Quan N Shaw J Schrick J Shi Z-Z Sparks MJ Van Slightenhorst I Vogel P Walke W Xu N Zhu Q Person C Sands AT Wnk1 kinase deficiency lowers blood pressure in mice: A gene-trap screen to identify potential targets for therapeutic intervention.Proc Natl Acad Sci USA. 2003; 100: 14109-14114Crossref PubMed Scopus (301) Google ScholarTie2-Cre transgenic mice were from the Jackson Laboratories (Bar Harbor, Maine). Flk1-Cre/+ knockin mice were the gift of Dr. T. Sato.20Motoike T Markham DW Rossant J Sato TN Evidence for novel fate of Flk1+ progenitor: contribution to muscle lineage.Genesis. 2003; 35: 153-159Crossref PubMed Scopus (179) Google ScholarSox2-Cre transgenic mice were the gift of Dr. T. Carroll and Dr. A. McMahon.21Hayashi S Lewis P Pevny L McMahon AP Efficient gene modulation in mouse epiblast using a Sox2Cre transgenic mouse strain.Mech Dev. 2002; 119: S97-S101Crossref PubMed Scopus (316) Google Scholar All animal maintenance and experiments were conducted in accordance with the Guide for the Use and Care of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. For timed breeding, the appearance of vaginal plug was checked twice a day at 9 a.m. and 6 p.m. At 1 a.m. or 1 p.m. of the day in which a plug was found was designated as embryonic day 0. The embryonic stage was further confirmed by the number of somites at the time of embryo dissection. Mouse tail tips, ear punches, toe clips, or portions of yolk sacs of embryos were digested in Viagen DirectPCR reagents (Viagen Biotech, Los Angeles, CA) with 0.2 mg/ml proteinase K at 55°C overnight, heat-inactivated at 85°C for 45 minutes, and analyzed by polymerase chain reaction (PCR). All genotyping PCR reactions were performed with 35 cycles of 95°C, 20 seconds; 60°C, 30 seconds; and 72°C, 1 minute. The primers for genotyping of Wnk1 knockout were as described.19Zambrowicz BP Abuin A Ramirez-Solis R Richter LJ Piggott J BeltrandelRio H Buxton EC Edwards J Finch RA Friddle CJ Gupta A Hansen G Hu Y Huang Wenhu Jaing C Key Jr, BW Kipp P Kohlhauff B Ma Z-Q Markesich D Payne R Potter DG Quan N Shaw J Schrick J Shi Z-Z Sparks MJ Van Slightenhorst I Vogel P Walke W Xu N Zhu Q Person C Sands AT Wnk1 kinase deficiency lowers blood pressure in mice: A gene-trap screen to identify potential targets for therapeutic intervention.Proc Natl Acad Sci USA. 2003; 100: 14109-14114Crossref PubMed Scopus (301) Google Scholar Sequences of other primers for genotyping are in Table 1.Table 1List of Primers Used for Genotyping and Real-Time PCRPrimerSequencecF15′-TCTGTCTCTGCCTCCCAAGT-3′cF25′-TGACATCTGGAACACTTAAGACG-3′cR15′-CTGAGTTCAGCTTTCAGCATCCATGTC– TAG-3′cR25′-AGGGGATCGGCAATAAAAAG-3′F15′-GAGTTCTCTGCTGCCTCCTG-3′F25′-CCCCCTGAACCTGAAACATA-3′R15′-CTCCCACTCAGCACTGACAA-3′VEGF5′-GGCTTTACTGCTGTACCTCCA-3′,5′-ACAGGACGGCTTGAAGATGTA-3′Ang15′-TCCTGACTCAGCACCATGAC-3′,5′-GATGGCCTTGATGTTGCTCT-3′Ang25′-CGACTACGACGACTCAGTGC-3′,5′-TCTGGTTCTGCACCACATTC-3′Hey15′-GGTACCCAGTGCCTTTGAGA-3′,5′-ACCCCAAACTCCGATAGTCC-3′Hey25′-GGTCCAATTCACCGACAACT-3′,5′-TGGCAGATCCTTGTTTTTCA-3′Neuropilin15′-CGTCACACTCATGCACTGG-3′,5′-CCCTGAGAGAGCCACACACA-3′Neuropilin25′-TGTTCTGTCATTGGGGTTAGC-3′,5′-AACTGCAACTTTGATTTTCCG-3′Ephrin-B25′-GCGGGATCCAGGAGATCCCCAC– TTGGACT-3′,5′-GTGCGCAACCTTCTCCTAAG-3′EphB45′-GCGGGATTCCAGCGCTCTGGACAA– GATGAT-3′,5′-CATCTCAAAGGAGCCGAATC-3′COUP-TFII5′-AAGCACTACGGCCAGTTCAC-3′,5′-AGGCATCCTGCCTCTCTGTA-3′Flt-15′-GCTGCTTGGAGATCTCACTG-3′,5′-TCAGCAGCTCAAGTGTCACC-3′Flt-45′-TGCATGCTGGGTGGACTATCA-3′,5′-GCAGGAGGAGGAAGAGGAGC-3′Tie25′-GGACAGTGCTCCAACCAAATG-3′,5′-GACGGAAATGTTGAAAGGC-3′VCAM15′-GCTCAAATCGGTGACTCCAT-3′,5′-AACTGACAGGCTCCATGGTC-3′SM225′-ACGATGGAAACTACCGTGGA-3′,5′-GCTGGCCTTCCCTTTCTAAC-3′Sox185′-CTCCCTGTCACCAACGTCTC-3′,5′-GTGCAGATCCGGATTTTGTT-3′ Open table in a new tab A full-length rat WNK1 cDNA fragment was released from a pBluescript-WNK1 plasmid9Xu B English JM Wilsbacher JL Stippec S Goldsmith EJ Cobb MH WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II.J Biol Chem. 2000; 275: 16795-16801Crossref PubMed Scopus (416) Google Scholar by digestion with XbaI and SalI. The XbaI-SalI fragment was inserted between compatible NheI and XhoI sites in the multiple cloning sites of pBigT plasmid22Srinivas S Watanabe T Lin CS William CM Tanabe Y Jessell TM Costantini F Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.BMC Dev Biol. 2001; 1: 4Crossref PubMed Scopus (2320) Google Scholar downstream of the loxP-PGKneo-tpA-loxP cassette. The resulting pBigT-WNK1 construct was then digested with PacI and AscI into a ∼6-kb PacI-AscI fragment and a ∼5-kb PacI-PacI fragment. These two fragments were subsequently inserted into PacI-AscI sites of ROSA26PAS vector,22Srinivas S Watanabe T Lin CS William CM Tanabe Y Jessell TM Costantini F Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.BMC Dev Biol. 2001; 1: 4Crossref PubMed Scopus (2320) Google Scholar between the short arm and long arm of ROSA26 genomic sequence, to create the ROSA26-WNK1 targeting construct. Targeting construct was linearized by XhoI before electroporation. Culture and electroporation of ES cells and injection of ES cells into blastocysts were performed by the Transgenic Core Facility of University of Texas Southwestern Medical Center. For screening of ES cell clones positive for targeted ROSA-Wnk1 (RW) allele, electroporated G418-resistant ES cells were digested with Viagen DirectPCR reagent and 0.2 mg/ml proteinase K at 55°C for 4 hours, heat inactivation at 85°C for 40 minutes, and analyzed by PCR. PCR primers consisted of forward primer located upstream of the ROSA26 short arm (5′-AGGTAGGGGATCGGGACTCT-3′) and reverse primer in the 5′ loxP site of targeting vector (5′-GCAGGTCGAGGGACCTAATA-3′). Another reverse primer located in the ROSA26 short arm (5′-TAAGCCTGCCCAGAAGACTC-3′) was included in the reaction for internal PCR control. PCR was performed according to the manufacturer’s instructions (HotStarTaq, Qiagen, Valencia, CA) with 35 cycles of 95°C, 30 seconds; 60°C, 30 seconds; and 72°C, 2 minutes. PCR product for wild-type ROSA26 allele was about 1.2 kb; product for targeted ROSA26-Wnk1 allele was about 1.4 kb (data not shown). ES cell clones positive for the targeted RW allele were expanded and injected into C57BL/6 blastocysts to generate chimera founders. More than 30 male chimeras with 80% to 100% agouti coat color were backcrossed with C57BL/6 females. Germ-line transmission was determined by presence of agouti offspring and confirmed by genotyping PCR using primers specific for the targeting vector or rat WNK1 cDNA. The exon 2 of Wnk1 and two ∼3-kb genomic fragments flanking exon 2 (left and right arms) were amplified from mouse genomic DNA and subcloned into OsDupDel targeting vector (gift of Dr. L. James and Dr. O. Smithies)23Smithies O Kim HS Targeted gene duplication and disruption for analyzing quantitative genetic traits in mice.Proc Natl Acad Sci USA. 1994; 91: 3612-3615Crossref PubMed Scopus (162) Google Scholar, so that the exon 2 is in front of a reverse-oriented Neo cassette. The exon2-neo fragment was flanked at each end by single loxP site. This targeting construct was used to generate Wnk1flox mice as described above. For routine histological analysis, embryos were fixed with 4% paraformaldehyde at 4°C overnight. Thereafter, they were either embedded in OCT and frozen by dry ice/ethanol for cryosectioning or dehydrated (serial submersion in 50%, 70%, 95%, and 100% ethanol solution) and embedded in paraffin wax for sectioning. Hematoxylin and eosin staining was performed on paraffin-embedded sections. Immunostaining using polyclonal antibodies to Neuropilin-1 (R&D Systems, Minneapolis, MN; 1:200) and EphB4 (R&D Systems, Minneapolis, MN; 1:100) was performed on cryosections as described.7You LR Lin FJ Lee CT DeMayo FJ Tsai MJ Tsai SY Suppression of notch signaling by the Coup-TFII transcription factor regulates vein identity.Nature. 2005; 435: 98-104Crossref PubMed Scopus (480) Google Scholar Fluorescence-labeled second antibodies from the Jackson ImmunoResearch Laboratories (West Grove, PA) were used as instructed. Whole-mount immunostaining by anti-PECAM1 antibodies (at 1:300 dilution) was performed as described with modifications.7You LR Lin FJ Lee CT DeMayo FJ Tsai MJ Tsai SY Suppression of notch signaling by the Coup-TFII transcription factor regulates vein identity.Nature. 2005; 435: 98-104Crossref PubMed Scopus (480) Google Scholar Signal was detected using a biotinylated second antibody (at 1:500) and Vectastain Elite ABC kit (Vector Labs, Burlingame, CA). Exons 6-9 of Wnk1 was PCR-subcloned into pBluescript using the primers previously described.12Delaloy C Lu J Houot AM Disse-Nicodeme S Gasc JM Corvol P Jeunemaitre X Multiple promoters in the WNK1 gene: one controls expression of a kidney-specific kinase-defective isoform.Mol Cell Biol. 2003; 23: 9208-9221Crossref PubMed Scopus (141) Google Scholar Digoxigenin-labeled riboprobe was made from this fragment using digoxigenin-UTP (Roche, Indianapolis, IN) and MaxiScript in vitro transcription kit (Ambion, Austin, TX). For in situ hybridization on sections, the glass slides were washed three times for 3 minutes in phosphate-buffered saline, treated with 15 μg/ml proteinase K for 10 minutes, then rinsed in phosphate-buffered saline and fixed in 4% PFA for 5 minutes, and acetylated for 10 minutes (acetylation solution: 2.66 ml of triethanolamine, 350 μg of HCl, 750 μg of acetic anhydride, and 200 ml of water). Then the slides were transferred to a slide mailer containing hybridization buffer (50% formamide (Fisher, Pittsburgh, PA), 5X standard saline citrate (SSC), pH 4.5, 50 μg/ml ribonucleic acid from torula yeast, type VI (Sigma, St. Louis, MO), 1% sodium dodecyl sulfate, 50 μg/ml heparin (Sigma) for prehybridization at room temperature for 1 hour. Probe hybridization was performed with 100 μl of probe/slide (1 μg/ml Digoxigenin-labeled probe in hybridization solution, covered with coverslip) in a humidified chamber (humidified with 50% formamide/5X SSC) at 68°C overnight. After hybridization, the slides were immersed in 2X SSC at 72°C until coverslips separated, rinsed in 0.2X SSC at 72°C and room temperature for 1 minute each, rinsed briefly in MBST buffer (100 mmol/L maleic acid, 150 mmol/L NaCl, pH 7.5, 0.1% Tween 20), and then incubated in blocking solution (2% blocking reagent from Roche, 5% heat-inactivated sheep serum in MBST) for 1 hour at room temperature. Then the slides were applied with 250 μl of anti-Digoxigenin antibody (Roche), covered with parafilm, and incubated in a chamber humidified with MBST at 4°C overnight. The slides were then washed in MBST three times 30 minutes, and treated in NTMT (100 mmol/L NaCl, 100 mmol/L Tris, pH 9.5, 50 mmol/L MgCl2, 0.1% Tween 20) three times for 5 minutes. Color reaction was done in BM purple solution (Roche), and the slides were sealed using Permount (Fisher SP15-500). Total RNAs were extracted from tissues (embryo, yolk sac, etc) using TRIzol (Invitrogen, Carlsbad, CA). DNase-treated RNAs (1–2 μg) were reverse-transcribed using a TaqMan reverse-transcription kit (Applied Biosystems, Foster City, CA). The resulting cDNA samples (5 ng at 1–2 ng/μl dilution) were analyzed in real-time PCR reactions using SYBR PCR reagents (Bio-Rad, Hercules, CA) and the real-time PCR system from Applied Biosystems. Sequences of primers for genes analyzed by real time are in Table 1. Ablation of Wnk1 in mice causes death before embryonic day 13 (E13).19Zambrowicz BP Abuin A Ramirez-Solis R Richter LJ Piggott J BeltrandelRio H Buxton EC Edwards J Finch RA Friddle CJ Gupta A Hansen G Hu Y Huang Wenhu Jaing C Key Jr, BW Kipp P Kohlhauff B Ma Z-Q Markesich D Payne R Potter DG Quan N Shaw J Schrick J Shi Z-Z Sparks MJ Van Slightenhorst I Vogel P Walke W Xu N Zhu Q Person C Sands AT Wnk1 kinase deficiency lowers blood pressure in mice: A gene-trap screen to identify potential targets for therapeutic intervention.Proc Natl Acad Sci USA. 2003; 100: 14109-14114Crossref PubMed Scopus (301) Google Scholar The cause of death in Wnk1-ablated mice, however, is not known. To examine the role of WNK1 in embryogenesis, we
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