Cardiovascular Expression of the Mouse WNK1 Gene during Development and Adulthood Revealed by a BAC Reporter Assay
2006; Elsevier BV; Volume: 169; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.051290
ISSN1525-2191
AutoresCéline Delaloy, Juliette Hadchouel, Martine Imbert–Teboul, Maud Clemessy, Anne-Marie Houot, Xavier Jeunemaı̂tre,
Tópico(s)Pancreatic function and diabetes
ResumoLarge deletions in WNK1 are associated with inherited arterial hypertension. WNK1 encodes two types of protein: a kidney-specific isoform (KS-WNK1) lacking kinase activity and a ubiquitously expressed full-length isoform (L-WNK1) with serine threonine kinase activity. Disease is thought to result from hypermorphic mutations increasing the production of one or both isoforms. However, the pattern of L-WNK1 expression remains poorly characterized. We generated transgenic mice bearing a murine WNK1 BAC containing the nlacZ reporter gene for monitoring L-WNK1 expression during development and adulthood. We observed previously unsuspected early expression in the vessels and primitive heart during embryogenesis, consistent with the early death of WNK1−/− mice. The generalized cardiovascular expression observed in adulthood may also suggest a possible kidney-independent role in blood pressure regulation. The second unsuspected site of L-WNK1 expression was the granular layer and Purkinje cells of the cerebellum, suggesting a role in local ion balance or cell trafficking. In the kidney, discordance between endogenous L-WNK1 and transgene expression suggests that either cis-regulatory elements important for physiological renal expression lie outside the BAC sequence or that illegitimate interactions occur between promoters. Despite this limitation, this transgenic model is a potentially valuable tool for the analysis of spatial and temporal aspects of WNK1 expression and regulation. Large deletions in WNK1 are associated with inherited arterial hypertension. WNK1 encodes two types of protein: a kidney-specific isoform (KS-WNK1) lacking kinase activity and a ubiquitously expressed full-length isoform (L-WNK1) with serine threonine kinase activity. Disease is thought to result from hypermorphic mutations increasing the production of one or both isoforms. However, the pattern of L-WNK1 expression remains poorly characterized. We generated transgenic mice bearing a murine WNK1 BAC containing the nlacZ reporter gene for monitoring L-WNK1 expression during development and adulthood. We observed previously unsuspected early expression in the vessels and primitive heart during embryogenesis, consistent with the early death of WNK1−/− mice. The generalized cardiovascular expression observed in adulthood may also suggest a possible kidney-independent role in blood pressure regulation. The second unsuspected site of L-WNK1 expression was the granular layer and Purkinje cells of the cerebellum, suggesting a role in local ion balance or cell trafficking. In the kidney, discordance between endogenous L-WNK1 and transgene expression suggests that either cis-regulatory elements important for physiological renal expression lie outside the BAC sequence or that illegitimate interactions occur between promoters. Despite this limitation, this transgenic model is a potentially valuable tool for the analysis of spatial and temporal aspects of WNK1 expression and regulation. Familial hyperkalemic hypertension (FHHt, no. OMIM145260) is a hereditary form of hypertension caused by mutations affecting two members (WNK1 and WNK4) of a new family of serine threonine kinases, the “with no lysine (K),” or WNK, kinases.1Wilson F Disse-Nicodème S Choate K Ishikawa K Nelson-Williams C Desitter I Gunel M Milford D Lipkin G Achard J-M Feely M Dussol B Berland Y Unwin R Simon D Farfel Z Jeunemaitre X Lifton R Mutations in WNK kinases reveal a novel mechanism of human hypertension.Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1218) Google Scholar, 2Hadchouel J Delaloy C Faure S Achard JM Jeunemaitre X Familial hyperkalemic hypertension.J Am Soc Nephrol. 2006; 98: 421-428Google Scholar WNK4 is found primarily in the distal nephron, and a number of recent experiments have shown this kinase to be important in the regulation of ion handling.3Yamauchi K Rai T Kobayashi K Sohara E Suzuki T Itoh T Suda S Hayama A Sasaki S Uchida S Disease-causing mutant WNK4 increases paracellular chloride permeability and phosphorylates claudins.Proc Natl Acad Sci USA. 2004; 101: 4690-4694Crossref PubMed Scopus (228) Google Scholar, 4Kahle KT Wilson FH Lalioti M Toka H Qin H Lifton RP WNK kinases: molecular regulators of integrated epithelial ion transport.Curr Opin Nephrol Hypertens. 2004; 13: 557-562Crossref PubMed Scopus (63) Google Scholar, 5Wilson FH Kahle KT Sabath E Lalioti MD Rapson AK Hoover RS Hebert SC Gamba G Lifton RP Molecular pathogenesis of inherited hypertension with hyperkalemia: the Na-Cl cotransporter is inhibited by wild-type but not mutant WNK4.Proc Natl Acad Sci USA. 2003; 100: 680-684Crossref PubMed Scopus (359) Google Scholar However, the distribution of WNK1 is more complex due mostly to the production of different isoforms. A long ubiquitous isoform (L-WNK1) containing the kinase domain is produced under the control of proximal promoters whereas a shorter isoform is produced specifically in the distal nephron (KS-WNK1).6Xu 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 (414) Google Scholar, 7Delaloy 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 (139) Google Scholar This shorter isoform has no kinase activity and is generated from an alternative promoter upstream from exon 4a. It is unclear whether the FHHt-causing deletions in intron 1 of WNK1 affect the qualitative and quantitative production of these two isoforms. The typical renal symptoms of FHHt indicate a probable change in expression pattern, at least in the distal nephron. We previously observed an increase in WNK1 mRNA levels in leukocytes of affected patients, suggestive of hypermorphic mutations.1Wilson F Disse-Nicodème S Choate K Ishikawa K Nelson-Williams C Desitter I Gunel M Milford D Lipkin G Achard J-M Feely M Dussol B Berland Y Unwin R Simon D Farfel Z Jeunemaitre X Lifton R Mutations in WNK kinases reveal a novel mechanism of human hypertension.Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1218) Google Scholar This mechanism is consistent with recent in vitro findings that L-WNK1 inhibits WNK4, which itself inhibits the sodium co-transporter NCC, the potassium transporter ROMK1, and chloride transtubular transfer.8Kahle KT Wilson FH Lifton RP Regulation of diverse ion transport pathways by WNK4 kinase: a novel molecular switch.Trends Endocrinol Metab. 2005; 16: 98-103Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar It is also consistent with the inhibition of L-WNK1 by KS-WNK19Subramanya AR Yang CL Zhu X Ellison DH Dominant-negative regulation of WNK1 by its kidney-specific kinase-defective isoform.Am J Physiol Renal Physiol. 2006; 290: F619-F624Crossref PubMed Scopus (99) Google Scholar and with the effects of L-WNK1 on the epithelial sodium transporter ENaC.10Xu BE Stippec S Chu PY Lazrak A Li XJ Lee BH English JM Ortega B 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, 11Naray-Fejes-Toth A Snyder PM Fejes-Toth G The kidney-specific WNK1 isoform is induced by aldosterone and stimulates epithelial sodium channel-mediated Na+ transport.Proc Natl Acad Sci USA. 2004; 101: 17434-17439Crossref PubMed Scopus (115) Google Scholar The ubiquitous nature of WNK1 probably renders the whole situation much more complex. Indeed, L-WNK1 is produced in several epithelia, the heart, muscle, and brain. However, its distribution remains poorly characterized. Expression studies in adults have mostly involved multitissue Northern blots6Xu 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 (414) Google Scholar, 7Delaloy 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 (139) Google Scholar, 12Wilson 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 (685) Google Scholar, 13Verissimo F Jordan P WNK kinases, a novel protein kinase subfamily in multi-cellular organisms.Oncogene. 2001; 20: 5562-5569Crossref PubMed Scopus (226) Google Scholar, 14O'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 (143) Google Scholar, 15Choate KA Kahle KT Wilson FH Nelson-Williams C Lifton RP WNK1, a kinase mutated in inherited hypertension with hyperkalemia, localizes to diverse Cl−-transporting epithelia.Proc Natl Acad Sci USA. 2003; 100: 663-668Crossref PubMed Scopus (112) Google Scholar or the application of immunohistochemistry techniques to epithelia.15Choate KA Kahle KT Wilson FH Nelson-Williams C Lifton RP WNK1, a kinase mutated in inherited hypertension with hyperkalemia, localizes to diverse Cl−-transporting epithelia.Proc Natl Acad Sci USA. 2003; 100: 663-668Crossref PubMed Scopus (112) Google Scholar In other organs, such as the heart, it has proved difficult to obtain consistent results with techniques such as immunohistochemistry and Northern blotting due to alternative splicing of WNK1, hindering studies into the possible multiple functions of WNK1. Finally, WNK1 expression during development has not yet been studied despite its probable importance, as reflected by the early death of WNK1−/− embryos.16Zambrowicz 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 W Jaing C Key Jr, BW Kipp P Kohlhauff B Ma ZQ Markesich D Payne R Potter DG Qian N Shaw J Schrick J Shi ZZ Sparks MJ Van Sligtenhorst 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 (300) Google Scholar As a first step toward an analysis of the consequences of FHHt-causing WNK1 mutations, we designed an in vivo model for monitoring L-WNK1 expression during development and adulthood. We generated transgenic mice bearing the murine WNK1(mWNK1) locus modified to include a reporter gene. Species-specific variation in transcription factor binding sites may be responsible at some level for species-specific differences in gene expression and regulation. We constructed a mouse transgene to prevent these interferences between cis- and trans regulatory elements and, thus, potential discordance between the transgene and the endogenous gene expression. Moreover, we had previously shown that the organization, structure, and expression of the WNK1 gene are similar in mice and humans.7Delaloy 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 (139) Google Scholar The construct used was a bacterial artificial chromosome (BAC RP24-212e14) containing 47.4 kb upstream from the transcription start site and 11.2 kb downstream from the last mWNK1 exon, into which we incorporated in exon 2 the nuclear lacZ (nlacZ) gene as a reporter. Furthermore we introduced a stop codon in the KS-WNK1-specific exon 4a to prevent KS-WNK1 overexpression. We used this BAC reporter assay to analyze L-WNK1 expression in a physiological model, during embryogenesis and adulthood, with high sensitivity and single-cell resolution. This analysis provided unexpected findings, concerning WNK1 expression in the cardiovascular and central nervous systems in particular. This transgenic model is of potential value for the analysis of spatial and temporal aspects of WNK1 expression and regulation. BAC RP24-212e14 spanning the mWNK1 locus was identified on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/). The RPCI-24 library was constructed from C57BL/6 genomic DNA using the pTARBAC1 vector (CHORI). The chosen BAC included the complete mWNK1 gene sequence. It began at nucleotide −47440 relative to the mWNK1 transcription start site (sequenced BAC end: gi 13218338) and ended 11.4 kb after the last mWNK1 exon (sequenced BAC end: gi 13218335). Escherichia coli strains EL-250 and EL-350, electroporation conditions with BAC DNA and targeting cassettes, selection of recombinant clones, and excision of the selection cassette were described by Lee and colleagues17Lee EC Yu D Martinez de Velasco J Tessarollo L Swing DA Court DL Jenkins NA Copeland NG A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.Genomics. 2001; 73: 56-65Crossref PubMed Scopus (994) Google Scholar and Liu and colleagues.18Liu P Jenkins NA Copeland NG A highly efficient recombineering-based method for generating conditional knockout mutations.Genome Res. 2003; 13: 476-484Crossref PubMed Scopus (853) Google Scholar The BAC construct was checked at each step by DNA digestion and direct sequencing of the targeted regions, to ensure that the structure of the original BAC was conserved and that the clones included the homologous recombination events. We deleted the LoxP site from the backbone to prevent recombination between this site and the loxP sequences inserted in the BAC. We constructed a targeting cassette containing an ampicillin (amp) resistance gene replacing the LoxP sequence based on pTamp.17Lee EC Yu D Martinez de Velasco J Tessarollo L Swing DA Court DL Jenkins NA Copeland NG A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.Genomics. 2001; 73: 56-65Crossref PubMed Scopus (994) Google Scholar In the targeted BAC, the LoxP site was replaced by the amp gene. The exon 4a-targeting cassette was generated by inserting a translation stop codon just downstream from the start codon in exon 4a, followed by the SV40 polyadenylation signal (SV40pA). The kanamycin (kan) resistance gene was incorporated into the cassette to facilitate positive selection for homologous recombination in E. coli. LoxP sites flanking the kan gene made it possible to remove this gene by arabinose induction of the Cre recombinase carried in EL350 before the generation of transgenic mice, thereby excluding the possibility of promoter interference effects. Two DNA fragments cloned by polymerase chain reaction (PCR) were incorporated at the ends of the targeting cassette for bacterial homologous recombination at the exon 4a locus (Figure 1B). The 346-bp Ex4a-5′ sequence was cloned by PCR using forward primer 5′-CAAAAGTCAAGGAGGCAGAGC-3′ and reverse primer 5′-CATCAGAAAAGGCAACAGACTT-3′, and the 359-bp Ex4a-3′ sequence was cloned using forward primer 5′-TAAATTCTCATTGCTGCTGCTGT-3′ and reverse primer: 5′-GTTTGTTTGCCAACTAATCTGCT-3′. The exon 2-targeting cassette was generated by inserting the gene encoding β-galactosidase (lacZ) together with a nuclear localization signal (n) and the SV40pA signal downstream from an internal ribosome entry site sequence (IRES). Flp recognition target (FRT) sites flanking the kan gene made it possible to remove this gene by arabinose induction of Flp recombinase before the generation of transgenic mice. Two DNA fragments cloned by PCR were incorporated at the ends of the targeting cassette for bacterial homologous recombination at the exon 2 locus (Figure 1B). The 375-bp Ex2-5′ sequence was cloned by PCR using forward primer 5′-GTTT-TTCGAGACAGGGTTTCTCT-3′ and reverse primer 5′-TTCTTTGAATCTCTGCCTTTCAG-3′, and the 380-bp Ex2-3′ sequence was cloned using forward primer 5′-GGAATCCACAGTAAAAGGGAAAA-3′ and reverse primer 5′-CCATGTCTAGTGCTTCCTCTGTT-3′. In the targeted BAC, IRES-nlacZ was inserted 42 bases downstream from the first base of exon2. Transgenic mice were generated by microinjection of the circular BAC DNA into fertilized (C57BL/6JxSJL) F2 eggs at a concentration of 1 ng/μl using standard techniques.19Hogan B Beddington R Costantini F Lacy E Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1994Google Scholar Injected eggs were reimplanted the day after the injection into pseudopregnant CD1(ICR) foster mothers. Genomic DNA was prepared from mouse tails and analyzed by Southern blot or PCR analysis. For Southern blot analysis, 15 μg of DNA was digested by PstI and subjected to electrophoresis. The Ex4a 5′ sequence was used as a probe and allowed the detection of a 7.8-kb fragment in the endogenous mWNK1 gene and a 2.8-kb fragment in the BAC transgene. Transgene copy number for each transgenic line was determined by analysis of hybridization signals with the Molecular Imager FX and Quantity one program (Bio-Rad, Marnes la Coquette, France). For PCR analysis, the primers used were forward primer 5′-GTTTTTCGAGACAGGGTTTCTCT-3′ and reverse primer 5′-AACGCACACCGGCCTTATTCC-AAG-3′. Heterozygous males were crossed with nontransgenic females (C57BL/6N). Embryos were staged taking embryonic day (E) 0.5 as the day of the vaginal plug. Embryos and adult tissues were dissected in phosphate-buffered saline (PBS), fixed by incubation in 4% paraformaldehyde for 10 to 20 minutes at 4°C (depending on the embryonic stage or the size of the tissue), rinsed twice in PBS, and stained overnight at room temperature with 0.4 mg/ml 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside (X-Gal; Invitrogen, Cergy Pontoise, France) in PBS, 5 mmol/L K3Fe, 5 mmol/L K4Fe, 2 mmol/L MgCl2, 0.2% Nonidet P-40. They were then postfixed in paraformaldehyde. For cryostat sectioning, embryos and adult tissues were transferred to 15% sucrose in PBS at room temperature after paraformaldehyde fixation and then embedded in 7% gelatin and 15% sucrose in PBS before freezing by immersion in isopentane at −140°C. Cryosections (10 μm) were cut from these tissues and rinsed in PBS before X-gal staining overnight at room temperature, as previously described, with postfixing and counterstaining with eosin. We cut 200-μm kidney sections with a vibratome after perfusion of anesthetized transgenic mice with 4% paraformaldehyde via the right atrium. These sections were stained overnight in X-gal solution at room temperature before paraffin-embedding. Sections (8 to 10 μm) were deparaffinized, rehydrated, and endogenous peroxidase inactivated with 3% H2O2 in PBS. They were then blocked with the avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA) and 5% normal goat serum in PBS and incubated with a polyclonal anti-NCC (thiazide-sensitive Na-Cl co-transporter) antibody (dilution 1/2000),20Nijenhuis T Hoenderop JG Loffing J van der Kemp AW van Os CH Bindels RJ Thiazide-induced hypocalciuria is accompanied by a decreased expression of Ca2+ transport proteins in kidney.Kidney Int. 2003; 64: 555-564Crossref PubMed Scopus (103) Google Scholar anti-NKCC2 (BSC-1) (renal Na+, K+, 2Cl− co-transporter) antibody (dilution 1/400),21Kim GH Ecelbarger CA Mitchell C Packer RK Wade JB Knepper MA Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle's loop.Am J Physiol. 1999; 276: F96-F103PubMed Google Scholar or anti-β-ENaC (epithelial sodium channel β subunit) antibody (dilution 1/500)22Loffing J Pietri L Aregger F Bloch-Faure M Ziegler U Meneton P Rossier BC Kaissling B Differential subcellular localization of ENaC subunits in mouse kidney in response to high- and low-Na diets.Am J Physiol. 2000; 279: F252-F258PubMed Google Scholar for 2 hours in PBS, 1% bovine serum albumin, and 0.025% Tween. Sections were then washed twice in PBS and incubated for 30 minutes with biotinylated goat anti-rabbit IgG (Vector Laboratories). This secondary antibody was amplified and detected with the Vectastain ABC kit and the DAB peroxidase substrate kit (Vector Laboratories). The L- and KS-WNK1 isoforms were detected with RNA probes labeled with digoxigenin-labeled uridine triphosphate as recommended by the manufacturer (Roche Diagnostics, Meylan, France) and binding to exon 4a (KS-WNK1), exons 1 to 4 (L-WNK1) or exons 25 to 27 (L- and KS-WNK1). Whole-mount in situ hybridization on embryos (E9.5 to E13.5) and adult tissues was performed as previously described.23Eichmann A Yuan L Breant C Alitalo K Koskinen PJ Developmental expression of pim kinases suggests functions also outside of the hematopoietic system.Oncogene. 2000; 19: 1215-1224Crossref PubMed Scopus (95) Google Scholar In situ hybridization on microtome sections (10 μm) was performed as previously described.24Etchevers HC Vincent C Le Douarin NM Couly GF The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain.Development. 2001; 128: 1059-1068PubMed Google Scholar Total RNA from mouse tissues were reverse-transcribed by using the MMLV RT (Gibco BRL-Life Technologies, Invitrogen) and random hexamers (Roche Diagnostics). Exon 4b splice variant was detected with primers binding to exons 2 and 4b or to exons 4a and 4b. Adult mouse kidneys were microdissected at the plate-forme d'explorations fonctionnelles rénales in vivoand ex vivo, Institut des Cordeliers (Paris, France) as previously described.25Virlon B Cheval L Buhler JM Billon E Doucet A Elalouf JM Serial microanalysis of renal transcriptomes.Proc Natl Acad Sci USA. 1999; 96: 15286-15291Crossref PubMed Scopus (152) Google Scholar Real-time quantitative RT-PCR (QRT-PCR) assays were performed in a LightCycler (Roche Diagnostics) with the intercalation of SYBR Green (Roche Di-agnostics) as a fluorescence reporter. Primers were designed to discriminate between the long and kidney-specific isoforms. Forward primer from exon 1 (5′-GACAGTCTACAAAGGTCTGGACAC-3′) and reverse primer from exon 2 (5′-GACCCTTTAACATTTCAGCCTCTTC-3′) were used to amplify L-WNK1, and primers from exon 4a (5′-TTGTCATCATAAATTCTCATTGCTG-3′) and exon 5 (5′-AGGAATTGCTACTTTGTCAAAACTG-3′) were used to amplify KS-WNK1. Forward (5′-TTATGGAAGGTGCAGCCACTA-3′) and reverse (5′-CTGTTGCACCAGTAGCCATATCT-3′) primers were used to amplify exon 4b from both L- and KS-WNK1 transcripts. Forward primer from exon 4a (5′-TTATTGTAAATTCTCATTGCTGCTG-3′) and reverse primer from exon 4b (5′-ATATCTTGGCAGGCCAGTCTTTAT-3′) were used to amplify exon 4b from KS-WNK1 transcripts, and forward primer from exon 4 (5′-GAATATCCATACTCAGAATGCCAAA-3′) and reverse primer from exon 4b (5′-CCTGGTGATGACTCTTTTCCACTA-3′) were used to amplify exon 4b from L-WNK1 transcripts. Primers from the lacZ gene (lacZ forward 5′-TACACCAACGTAACCTATCCCATTA-3′ and lacZ reverse 5′-GAGTTAACGCCATCAAAAATAATTC-3′) were used to analyze the lacZ mRNA distribution along the nephron. For all of the samples, the amount of PCR product was calculated as a percentage of a RNA standard (whole kidney RNA) per mm of tubule length (arbitrary unit). Data were then corrected for PCR efficiency as previously described.26Nissant A Lourdel S Baillet S Paulais M Marvao P Teulon J Imbert-Teboul M Heterogeneous distribution of chloride channels along the distal convoluted tubule probed by single-cell RT-PCR and patch clamp.Am J Physiol. 2004; 287: F1233-F1243Google Scholar The nucleotide sequence of exon 4b of the mouse WNK1 gene has been deposited in the GenBank database under GenBank accession no. DQ402040. The ubiquitous kinase domain-containing isoform (L-WNK1) is produced under the control of proximal promoters upstream from and into exon 1 (pP), whereas the kidney-specific isoform with no kinase activity (KS-WNK1) is produced under the control of an alternative promoter (rP) in intron 4 (Figure 1A). We analyzed L-WNK1 expression by means of a genomic reporter assay, using a BAC bearing the mWNK1 locus (Figure 1B). We constructed two targeting cassettes—one to introduce the nlacZ reporter gene into mWNK1 exon 2 (Figure 1B) and the other to insert a stop codon into exon 4a, thereby preventing production of the KS-WNK1 isoform (Figure 1B). The homologous recombinant events were checked by means of several restriction enzyme digestions (Figure 1, C and D). Homologous recombination sites and selection cassette excision were checked by BAC sequencing. In the resulting construct, the nlacZ gene was fused to the exon 2 coding sequence, resulting in β-galactosidase expression under control of the proximal promoters of the BAC clone. Internal translation initiation from the IRES sequence located just upstream from the nlacZ coding sequence led to production of an efficient β-galactosidase enzyme rather than a WNK1-β-galactosidase fusion protein. We obtained four transgenic founders from this construct, and transmission of the BAC transgene to the F1 generation was confirmed by genomic Southern blotting (Figure 1E). Three of the four lines expressed the transgene. Transgenic lines 10 and 28 carried 2 and 12 copies of the transgene, respectively, at a single chromosomal location. Transgenic line 12 carried 6 and 10 copies at two chromosomal locations and was separated into two lines, each with transgene insertion at a single chromosomal location. The four independent transgenic lines had almost identical L-WNK1-nlacZ expression profiles, with only slight differences in expression intensities. The L-WNK1-nlacZ transgene was strongly expressed in the heart during early development. Whole-mount observation as early as day 7.5 of development revealed ubiquitous low-level nlacZ expression in the embryo. Marked changes in X-gal staining occurred during rapid organogenesis, between stages E8 and E9. During this period, reporter expression increased in the developing primitive heart (see Supplementary Figure 1 at http://ajp.amjpathol.org). The pattern of L-WNK1-nlacZ expression in E13 heart (Figure 2A) was similar to that of endogenous WNK1 (Figure 2B). We stained embryo cryosections to locate nlacZ expression more precisely in the primitive heart. At E12.5, expression was strongest in the outflow tract and atrioventricular canal (Figure 2C). At E14.5, expression was strongest in the trabeculations of the ventricular wall (Figure 2D). L-WNK1 cardiac expression was maintained in adults. In toto X. gal staining demonstrated sustained expression of the transgene (Figure 2E). Histological analysis showed that the transgene was expressed homogeneously and that its product was present in cardiomyocytes (Figure 2F). Endogenous cardiac L-WNK1 expression was confirmed by in situ hybridization (Figure 2G). At E10.5 the transgene was expressed throughout the developing vasculature (Figure 3A). β-Galactosidase activity was clearly detected in the intersegmental arteries and the capillaries of perineural tissues. At E13, β-gal activity was observed in the aorta and intersegmental arteries (Figure 3B), internal carotid arteries (Figure 3C), and vessels of the choroid plexus (Figure 3D). We stained cryosectioned E12.5 embryos to locate nlacZ expression precisely. The transgene was expressed ubiquitously at a low level, but was much more strongly expressed in the endothelial cells of arteries and veins (Figure 3E). The transgene was also expressed in the extraembryonic vasculature. It was detected in placenta (Figure 3F) and yolk sac vessels throughout development and throughout its capillary bed (Figure 3G). As development progressed, nlacZ expression was evident in many blood vessels throughout the body, in large and small arteries, capillaries, and veins. β-Galactosidase activity was detected in the adult abdominal aorta (Figure 4, A and B), vessels in the brain (Figure 4, D and E), the diaphragm, kidneys, skeletal muscle, and mesentery (Figure 4G). X-gal staining was detected in the small arteries and their connecting arterioles at the brain surface, and in the veins and venules (Figure 4, D and E). We stained cryosections of adult aorta with X-gal (Figure 4B) to identify the expressing cell types in the blood vessels. Both endothelial cells and smooth muscle cells were stained. The corresponding endogenous expression was assessed by in situ hybridization with an L-WNK1 RNA probe. Expression was confirmed in the aorta (Figure 4C), brain vessels (Figure 4F), and mesenteric vessels (Figure 4H). In the aorta, endogenous WNK1 expression was detected in the vascular endothelium and smooth muscle cell layers (Figure 4C). In embryos, almost no L-WNK1-nlacZ transgene expression was observed in the central nervous system, except in the capillaries surrounding the neural tube (Figure 5A) and some neurons of the floor plate (Figure 5B). The transgene was also expressed in epithelial cells of the optic layer (Figure 5C). In adult mice, the transgene was expressed in the cerebellum (Figure 5D). The most intense region of X-gal staining was the granular layer and cerebellar Purkinje cells, with only weak staining observed in the molecular layer and white matter (Figure 5, E and G). Expression levels in these layers appeared constant from 3 days after birth into adulthood (see Supplementary Figure 2 at http://ajp.amjpathol.org). In toto hybridization (Figure 5F) and in situ hybridization on cryosections (Figure 5H) gave similar results. During embryogenesis, β-galactosidase activity was detected in
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