Human Molecular Genetics, 2001, Vol. 10, No. 21 2385-2396
© 2001 Oxford University Press
Comparison of Pkd1-targeted mutants reveals that loss of polycystin-1 causes cystogenesis and bone defects
Renal Division, Department of Medicine, 1Department of Pathology, Brigham and Women’s Hospital, 2Department of Pathology, Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA, 3Division of Nephrology, Mayo Clinic, Rochester, MN, USA and 4Institute of Molecular Medicine, University of Oxford, Oxford, UK
Received June 8, 2001; Revised and accepted July 27, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A high level of polycystin-1 expression is detected in kidneys of all patients with autosomal dominant polycystic kidney disease (ADPKD). Mice that overexpress polycystin-1 also develop renal cysts. Whether overexpression of polycystin-1 is necessary for cyst formation is still unclear. Here, we report the generation of a targeted mouse mutant with a null mutation in Pkd1 and its phenotypic characterization in comparison with the del34 mutants that carry a ‘truncation mutation’ in Pkd1. We show that null homozygotes develop the same, but more aggressive, renal and pancreatic cystic disease as del34/del34. Moreover, we report that both homozygous mutants develop polyhydramnios, hydrops fetalis, spina bifida occulta and osteochondrodysplasia. Heterozygotes also develop adult-onset pancreatic disease. We show further that del34 homozygotes continue to produce mutant polycystin-1, thereby providing a possible explanation for increased immunoreactive polycystin-1 in ADPKD cyst epithelia in the context of the two-hit model. Our data demonstrate for the first time that loss of polycystin-1 leads to cyst formation and defective skeletogenesis, and indicate that polycystin-1 is critical in both epithelium and chondrocyte development.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic diseases, characterized by the progressive replacement of renal tissue by epithelial cysts. About 50% of ADPKD patients have liver cysts (1). Pancreatic cysts are also seen (2).
Polycystins are an expanding family of a novel class of transmembrane proteins. Two members of the polycystin family (polycystin-1 and -2) are mutated in human ADPKD. Polycystin-1 is a large (
460 kDa) membrane-associated glycoprotein with a number of adhesive domains in its extracellular N-terminal region, 7–11 transmembrane domains, and a small cytoplasmic tail (3–6). Polycystin-2 (110 kDa) is homologous to an 400-residue hydrophobic region of polycystin-1 (7) and to voltage-activated and transient receptor potential channel subunits, which suggest that polycystins are associated with ion transport. The C-terminal tail of polycystin-1 interacts with that of polycystin-2 (8,9). Polycystin-1 signaling may be mediated by G proteins (10,11) and its signaling pathway may intersect with that of Wnts, a family of secreted signaling molecules (11). Recently, three new polycystins, polycystin-L, -L2 and -REJ, have been identified (12–14). Polycystin-L, the first polycystin whose function was determined, forms a calcium-regulated, calcium-permeable cation channel when expressed in Xenopus oocytes, and was proposed to function as a transducer of Ca2+-mediated signaling in vivo (15). Very recently, it has been shown that co-assembly of polycystin-1 and -2 produces unique cation-permeable currents (16). Polycystin-2 alone also mediates cation currents and functions as a Ca2+-permeable non-selective cation channel (17–19).
Polycystin-1 is widely expressed in a number of tissues and cell types (20–23), and its expression is developmentally regulated. The highest level of polycystin-1 expression is found in fetal life, while a low level of expression is maintained throughout adulthood (21,22,24). It is thus highly likely that polycystin-1 function is not only important in organs that are affected in ADPKD, i.e. kidney, liver and pancreas, but also in the development of other organs and tissues. Homozygous mutant mice with a deletion of exon 34 of Pkd1 (del34, previously named Pkd1–) develop severe polycystic kidney and pancreatic disease and die during the perinatal period (25). Mice heterozygous for the del34 mutation develop late-onset polycystic kidney and liver disease (26). The del34 mutation is a truncation mutation that mimics many mutations seen in ADPKD patients (27–29). It is unclear, however, whether such a truncation mutation results in a form of polycystin-1 with gain of function or in a ‘truncated’ protein that has an effect on phenotype. In humans, polycystin-1 is overexpressed in renal cystic epithelia from ADPKD patients, as shown by a number of antibodies raised against various domains of the protein (20,21,24,30–33). The rare large deletions that create null mutations of PKD1 in humans are difficult to interpret (34,35) because they involve contiguous genes, including TSC2, a tuberous sclerosis gene that is independently associated with renal abnormalities such as cysts.
In the experiments described herein, we generated, by homologous recombination, a line of mice that are deficient in polycystin-1. Comparative analysis of the null and del34 mice revealed that mutations in Pkd1 result not only in epithelial defects (cyst formation) but also chondrocyte defects (osteochondrodysplasia and failure of neural arches to close). We show that polycystin-1 is present in del34 mutants and probably partially rescues its phenotype.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of Pkd1 null mutants
To study the effects of loss of polycystin-1 function in a model system, we targeted the 5' end of the murine Pkd1 gene by inserting a pgk-neomycin (neo) cassette into exon 4 by homologous recombination in embryonic stem (ES) cells (Fig. 1A). This insertion should result in a frame shift, generating a shortened peptide of 130 amino acids which lacks 97% of the polycystin-1 sequence. Eight recombinant ES clones were identified by Southern blot analysis with probes flanking the sequence in the targeting vector (Fig. 1B). Two independent clones (9c and h) were injected into C57BL/6 and BALB/c blastocysts, respectively. The resulting ‘null’ chimaeric mice, with >80% of the agouti coat, were crossed with C57BL/6 and BALB/c mice to produce (null/+) F1 progeny.
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We analyzed Pkd1 gene expression in null mutants by reverse-transcription polymerase chain reaction (RT–PCR) of a Pkd1 transcript, using two sets of primers located in exons 2 and 5 (Fig. 1A). Both sets of primers revealed a mutant transcript in mRNA from null homozygotes and heterozygotes (Fig. 1C). DNA sequencing of this mutant transcript revealed that part of the pgk-neo cassette was spliced into the mutant Pkd1 transcript, resulting in a stop codon 30 bp downstream of exon 3 (Fig. 1H).
Northern blot analysis of mRNA from embryonic day 12.5 (E12.5) null embryos with both 5' and 3' cDNA probes revealed a mutant transcript similar in size to that in wild-type (14 kb) Pkd1 mRNA. The mutant transcripts were expressed at a slightly lower level in homozygotes and heterozygotes after normalization with GAPDH expression (Fig. 1D). To determine whether these mutant transcripts produce stable Pkd1 protein, we performed western blot analysis with a monoclonal antibody 7e12 (36), directed against the extracellular N-terminal region of polycystin-1. A strong immunoreactive band of 460 kDa was detected in wild-type and heterozygous embryos and absent in homozygous null mutants (Fig. 1F).
Differential expression of Pkd1 gene in null and del34 mutants
To investigate whether a mutant polycystin-1 is present in the del34 mutants (25), expression of both Pkd1 transcript and protein was examined in del34 homozygotes and heterozygotes and compared with that of wild-type littermates. While del34 homozygotes and heterozygotes continue to express large (14 kb) mutant Pkd1 transcripts at a reduced intensity compared with wild-type (Fig. 1E), an 11 kb transcript was detected in del34 homozygotes and heterozygotes by a 5' Pkd1 probe (Fig. 1E). This transcript was not detected by the extreme 3' cDNA probe kg8 (data not shown).
While null homozygotes do not produce any detectable polycystin-1, antibody 7e12 detected a smaller band (400 kDa) and a nearly full-length protein in del34 heterozygotes and homozygotes by western analysis (Fig. 1G). While the nature of the nearly full length protein is unknown, the smaller protein is coincident in size to the predicted size of the truncated protein produced by the del34 mutants, which truncates the polycystin-1 peptide by 836 residues (25).
Rapid progression of polycystic kidney and pancreatic disease in homozygous null embryos
Timed pregnancies were generated to analyze null homozygous fetuses at various developmental stages. A total of 378 mouse embryos (218 in C57BL/6-129 and 160 in BALB/c-129 background) from E12.5 to the neonatal period were isolated (Table 1). Embryos older than E15.5 were grossly examined for their kidney size, and cystic lesions in their pancreas and liver, as well as the size of their heart and lungs. A total of 17 null homozygotes (from E12.5 onwards) were examined histologically for renal cystic lesions. To our surprise, kidney development in null mutants appears to proceed normally until E15.5, when cystic dilatation of renal tubules first becomes evident (Fig. 2A). This is the same stage at which cyst formation is observed in del34 embryos, thus indicating that polycystin-1 is required for tubular maturation but not for the initial stages of nephrogenesis, at least in the mouse strains investigated (Fig. 2B). As in del34 mutants, lectin-binding experiments in null animals revealed that proximal tubules dilated prior to collecting tubules.
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Null mutants seem to have larger and more renal cysts than del34 mutants at the same stage (Fig. 2E–F). Some strain dependence in the degree of cystic lesions in mice of a given mutation was noted. Kidney lesions in BALB/c-129 background appear to be milder than that in C57/BL-129 background at the same stage, and are similar between null mutants of newborn stage in BALB/c-129 background (Fig. 2E) and those at E17.5 in C57BL/6-129 background (Fig. 2C). This finding suggests a moderate strain dependence on renal disease severity. The rate of cyst development indicates a more aggressive disease in homozygous null kidneys than in del34 homozygotes of the same genetic background (Fig. 2A–F), thus providing the first evidence that the nature of mutation has an impact on disease progression.
The onset of pancreatic phenotype in null homozygous embryos was at E13.5 (i.e. at the same stage as in del34 mutants), but the dilatation of pancreatic ducts progressed much more rapidly in null mutants (Fig. 2G–J).
Kidney, liver and pancreatic cystic disease in Pkd1-targeted heterozygous mice
Among 40 null heterozygotes (2–24 months) examined, microscopic kidney cysts were found as early as 2.5 months; in contrast del34 heterozygotes develop scattered microscopic renal cysts at 9 months of age (26). Serial sections of kidneys from 10 null heterozygotes (2.5–14.5 months) revealed 2–30 cysts in seven (70%) mice (Table 2). Five out of 40 null heterozygotes developed visible renal cysts (at 13, 19.5, 21, 22.5 and 23 months old); three out of five had bilateral cysts.
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Liver cysts are the most common extra-renal manifestation of ADPKD, occurring in
75% of ADPKD patients aged over 60 (1). Despite the lack of discernible liver defects in null homozygotes, 12 out of 25 (48%) null heterozygotes (2.5–16 months) examined developed visible liver cysts, first observed at the age of 11 months (Table 2), similar in timing to that in del34 heterozygotes. All 15 (100%) older null heterozygous mice (18–24 months) had liver cysts. Six wild-type littermates (11.5–23 months) had no cystic lesions in the liver. Although we did not observe pancreatic cysts in 23 del34/+ heterozygotes (9–20 months), one out of 21 null heterozygotes >12 months (14.5 months) developed a single visible pancreatic cyst. Further study of a large population of old del34/+ mutants (>20 months) revealed that three out of 30 (10%) heterozygotes (at 22, 24 and 25 months, respectively) had macroscopic pancreatic cysts (Fig. 3A), whilst five aged-matched wild-type littermates showed no pancreatic cysts. There is no significant difference in terms of age of onset and severity of pancreatic cystic disease between theses two lines of Pkd1 mutants. Histology showed dilatation of pancreatic ducts (Fig. 3B) and multiple cystic structures lined by cuboidal cyst epithelium (Fig. 3C). Some cysts with small lumens also contained cuboidal epithelium, with a large portion of eosinophilic cytoplasm suggesting an acinar origin (Fig. 3D). Cystic lesions were surrounded by interstitial fibrosis with few atrophic acini and isolated islets of Langerhans (Fig. 3E). Pancreatic lipomatosis was noted in areas with extensive adipose tissue replacement (Fig. 3F). This constellation of findings, including cystic disease, fibrosis and lipomatosis, is identical to that observed in patients with cystic fibrosis (37).
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Pkd1 null mutation causes embryonic lethality, polyhydramnios and hydrops fetalis
Homozygous null embryos were found dead as early as E13.5, 2 days earlier than del34 homozygotes with a peak around E14.5 and E16.5. Only five out of 143 homozygotes survived to term but died immediately after birth. All five were of BALB/c-129 genetic background. In the C57BL/6-129 background, all null homozygous embryos succumbed by E17.5, 2 days earlier than their del34 counterparts.
Polyhydramnios was the first abnormality observed in the Pkd1 homozygous fetus, found in null/null embryos as early as E12.5 (Fig. 4A and B). It was followed by systemic edema of the whole embryo (hydrops fetalis) starting at E13.5 (Fig. 4C), consistent with a sequential association found in human fetuses (38,39). Polyhydramnios and hydrops fetalis were not obvious in del34 mutants until the later stages of fetal development (Fig. 4D). Histological examination of the null/null embryos revealed massive subcutaneous edema (Fig. 4E), which was also present in del34 embryos but to a lesser degree (data not shown). Anemia and congestive heart failure are major causes of non-immune hydrops fetalis in humans (40,41). However, examination of hemoglobin levels and erythrocyte morphologies as well as serial sections of four null and four del34 Pkd1 homozygous fetal hearts did not show any abnormality (data not shown). Another common cause of hydrops fetalis in humans is skeletal dysplasia (42). Organ-specific targeting of Pkd1 may elucidate whether the skeletal defects (see below) play a role in the development of hydrops and polyhydramnios in these Pkd1 mutants. Polyhydramnios may also be exacerbated by obstruction of the gastrointestinal tract due to enlarged cystic pancreas and kidneys at late embryonic stage.
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Spina bifida occulta and osteochondrodysplasia in Pkd1 homozygous mutants
Cartilage and bone develop from multiple embryological origins. The craniofacial skeleton in the first branchial arch and other regions of the developing head derive largely from neural crest cells. The ribs and vertebrae are from the sclerotomal part of the somites, and the appendicular skeleton is derived from the lateral mesoderm (43). Pkd1 is expressed at high levels in developing neural tube, neural crest derivatives and prechondrogenic tissue (44). Our in situ hybridization studies show that the strongest signals of Pkd1 were found in the perichondrium of developing vertebrae and long bone (Fig. 5A–G), but absent in the hypertrophic chondrocytes.
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To determine whether loss of polycystin-1 in bone has any effects in vivo, we looked for skeletal abnormalities in the mutants. Both null and del34 homozygotes developed spina bifida occulta at late embryonic or newborn stages (Fig. 6A). All five null homozygotes examined displayed severe defects in vertebral development (Table 3). Non-closure of neural arches started at cervical vertebrae and extended in some cases to sacral vertebrae, with the most severe defects in the lumbar region. The laminae of the lumbar vertebrae were extended; the intact arches remained open and failed to cover the dorsal part of the neural tube (Fig. 6B and E). Eight (73%) of 11 del34 homozygotes developed mild spina bifida occulta in the lumbar region (Fig. 6C and Table 3). At the newborn stage, the skeletal development in both del34 and null homozygotes was retarded compared with that of wild-type littermates (Fig. 6A and Table 3). There is a delay in bone mineralization of vertebrae, long bones and skull, most obviously in the null homozygotes (Fig. 6B–K), although ossification centers remained (Fig. 6E). The thyroid cartilage is malformed in the null homozygous mutants (Fig. 6L and Table 3). The long bones in null homozygotes were shorter and about a third thinner than those of wild-type animals (Fig. 6G and H). Histology of null homozygous long bones shows that the hypertrophic chondrocyte zone is reduced in length at E14.5, similar to what was seen in mice lacking Indian Hedgehog, indicating that loss of polycystin-1 can lead to defective chondrocyte differentiation and maturation. (Fig. 7A–D). At E17.5, bone marrow cavity is formed but shortened (Fig. 7E–H) in the null homozygotes. The defective overall ossification of skeletal elements of different origins makes a cell lineage defect as an unlikely cause. This condition is, in fact, reminiscent of human infantile osteodystrophy. These data suggest that polycystin-1 is required for the differentiation of chondrocytes. Its loss leads to defects in the development of the vertebral column, skeletal growth and ossification including intramembranous ossification.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Polycystin-1 is required in the final stages of kidney development
To our surprise we observed that some polycystin-1 null mutants were able to survive to late gestation, despite high levels of Pkd1 expression as early as the stem cell stage in development (Lu et al., unpublished data) and the wide distribution of the protein. Furthermore, the null kidneys were phenotypically normal until E15.5. Kidney development starts by formation of Wolffian-duct-derived ureteric buds that invade the metanephric mesenchyme at about E11 in the mouse. As a response to bud induction, the mesenchymal cells condense around the ureteric bud. During subsequent branching morphogenesis of the ureteric bud, the condensed mesenchymal cells further proliferate to form pretubular structures that undergo mesenchymal-epithelial transformation to form epithelium-lined tubules and fuse to the branching ureteric bud. These tubules undergo further functional and structural maturation to become fully differentiated tubules in permanent kidneys. This process can be classified into four stages: nephrogenic condensation, epithelialization, initial tubulogenesis and tubular maturation. Our data demonstrate that polycystin-1 is not required in the initial stages of kidney development, although Pkd1 transcript was strongly expressed in condensing mesenchyme at E12.5 (44). Our data also indicate that a truncated or partially functional polycystin-1 may reduce the rate of cyst formation but not delay the onset of cyst formation, a finding consistent with the previous observation that polycystin-1 expression in the kidney peaks between E15.5 and E18.5, coincident with renal tubule maturation (22–24,45). Thus, the data lend further support for a role of polycystin-1 in terminal differentiation of epithelial cells and maintenance of the structural integrity of renal tubules. Although glomerular expression of polycystin-1 has been reported (33), no obvious abnormality was seen in glomeruli. A similar phenotype has been found in mice with inactivation of the Pkd2 gene (46).
Loss of polycystin-1 function results in cyst formation in ADPKD
A two-hit hypothesis was proposed as the mechanism of cystogenesis (47) for the sporadic distribution of cysts observed in ADPKD. The hypothesis gained support from the finding of loss of heterozygosity in a small number of cysts (48–50). However, the two-hit theory is contradicted by the findings that polycystin-1 is overexpressed in epithelia from most cysts in kidneys from ADPKD patients (20,21,24,30–33), and that mice overexpressing normal human polycystin-1 develop renal cysts (51).
In this study, we show that homozygous and heterozygous null mutants both have phenotypes that are similar to those produced by polycystin-1-overexpressing del34 mutants (see Results). Two possible conclusions can be drawn from these results: (i) both overexpression and loss of polycystin-1 result in cyst formation and (ii) loss of polycystin-1 leads to cystogenesis. Although overexpression of normal human polycystin-1 in mice does result in cyst formation, these mice have normal fetal development and develop mild glomerular cysts only in late adulthood (51). Moreover, these mice also overexpress a full-length tuberous sclerosis 2 gene whose product is involved in growth control. While it is not clear at the present stage whether polycystin-1 overexpression contributes to cyst formation in ADPKD, the data from our null mutants clearly state that loss of polycystin-1 results in cyst formation in Pkd1 disease.
Although similar, the null and del34 phenotypes are not identical. In homozygotes, the renal tubule and pancreatic duct dilatation was much more rapidly progressive in null animals, and at a given age, the number of cysts in homozygotes was greater in null than in del34 mice with the same genetic background. These data provide the first evidence that the nature of the mutation affects disease progression. These data also suggest that a mutant polycystin-1 in del34 mice partially rescues the phenotype. Indeed, we did detect mutant proteins in del34 homozygotes by western blotting. The nature of the mutant proteins is currently unknown, but they may be the truncated and/or alternatively read-through or spliced forms of polycystin-1 (52). Although multiple RT–PCRs of the Pkd1 transcripts of the exon 32–37 region failed to identify alternative spliced transcripts, alternative splicing in other parts of the Pkd1 gene can not be excluded. On the other hand, the alternative readings of the genetic code, which include leftward and rightward ribosomal frameshifting, programmed termination read-through, and hopping (53,54), may allow the continued translation of mutant Pkd1 transcripts into near full-length mutant polycystin-1. Nevertheless, our results provide the first evidence of the concurrence of loss-of-function phenotype and increased polycystin-1 expression in Pkd1 homozygous mutants, thus providing a possible explanation for increased immunoreactive polycystin-1 in ADPKD cyst epithelia in the context of the two-hit model such that the overexpression of polycystin-1 in cysts in ADPKD is due to the detection of mutant polycystin-1 that is unable to function sufficiently. While the germline mutant allele generates increased levels of mutant (e.g. read-through) proteins with little or no function, gradual loss of the second allele due to somatic mutations initiates clonal cyst development in ADPKD.
Polycystin-1 is required for skeletogenesis
Both homozygous Pkd1-targeted mutants exhibit osteochondrodysplasia and delayed endochondral and intramembranous bone formation, demonstrating that polycystin-1 is required for normal mouse skeletogenesis. The function of polycystin-1 in the bone appears to mirror its function in the kidney, such that it is required for chondrocyte differentiation and maturation in the bone, as it is needed for epithelial differentiation and maturation in the kidney. These findings are consistent with polycystin-1 expression patterns in tissues originating from neural crest cells and sclerotic mesenchyme in which polycystin-1 is found at high levels between E12.5 and E17.5.
However, skeletal abnormality is not a general finding in ADPKD patients. The absence of skeletal abnormalities in ADPKD patients may be because of the lack of PKD1 homozygous patients to be assessed, as current study suggests that human PKD1 homozygotes likely die in uterus. In heterozygous patients, depending on the cell type undergoing a somatic hit, and the timing of the second hit, the skeletal phenotype described here may or may not be exhibited. It is noteworthy that several reports have suggested a link between human recessive and possibly dominant polycystic kidney disease and skeletal abnormality (55,56). Our study thus brings up an issue, that if one day we can correct the kidney disease so that homozygous PKD1 patients would survive, the skeletal defects in these children may require medical attention. No significant cardiovascular phenotype and intracranial aneurysm were detected in our mouse models. Whether the lack of these phenotypes is due to the specific mutations we introduced or other factors remains to be investigated.
While study of mouse homozygotes reveals the importance of polycystin-1 in a wider range of tissues than are clinically affected in ADPKD, the phenotype of heterozygous del34 and null mutants largely recapitulate human clinical patterns seen in ADPKD. Thus, our Pkd1 mutant mouse lines provide excellent models for studies of both the pathogenesis of ADPKD and polycystin functions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of Pkd1 null mutant mice and breeding scheme
To construct the targeting vector, a P1 clone (clone address, plate 243; control no. 13003, GenomeSystems) containing full-length Pkd1 genomic DNA was isolated from a 129Sv mouse genomic library. A 6.6 kb AvrII-NsiI fragment containing exons 2–6 was subcloned into vector litmus 28. A phosphoglycerate kinase (pgk) promoter-driven neo-resistant selection cassette was inserted into the PmlI site of exon 4 in the same orientation. The resulting targeting vector contained 1.1 kb of homologous sequence in the short arm and 5.5 kb in the long arm (Fig. 1A). The targeting vector was electroporated into 129Sv J1 ES cells (a gift from Dr. En Li) and selected according to standard procedures. Positive clones were injected into C57BL/6 and BALB/c blastocysts to generate chimeras.
The chimeras that were derived from C57BL/6 or BALB/c blastocysts were bred with the same inbred strain, respectively. The resulting F1 heterozygotes were bred with the respective C57BL/6 or BALB/c strain to produce more heterozygotes (N2). Homozygotes were generated by a cross between F1 heterozygotes or F1 and N2 heterozygotes. Homozygotes thus contain 129SvJ1 and C57BL/6 genetic components or 129SvJ1 and BALB/c genetic components and are defined as of either C57BL/6-129 or BALB/c-129 background in the text. Heterozygotes and homozygotes were determined by Southern blot of tail and yolk-sac genomic DNA.
Southern blot analysis to genotype ES cells and mice
To identify the targeted mutant ES cells and to genotype the mice, genomic DNA was digested with BamHI and analyzed by Southern blotting. A 1.1 kb NaeI fragment (probe 2), which detects a 3.7 kb fragment in wild-type DNA and a 5.4 kb fragment in targeted clones, was used (Fig. 1A and B). A 0.9 kb NsiI-AvrII genomic fragment (probe 1) located immediately upstream of the long arm AvrII site was used to confirm the homologous recombination in the 5' homology region. The wild-type SpeI fragment detected by this probe is 16 kb, and the mutant fragment is 14.8 kb (data not shown). The homologous recombination frequency was 20%.
Northern blot and RT–PCR analysis
For northern analysis, mRNA was isolated from E12.5 null and del34 whole embryos with a MicroPoly(A)Pure kit (Ambion), separated on 1% agarose/formaldehyde gels, and transferred to BrightStar-Plus positively charged nylon membranes (Ambion). A 1.1 kb mouse cDNA containing Pkd1 exons 2–6 was used as probes. Hybridization was performed with ExpressHyb hybridization solution (Clontech). Intensity analysis was performed on a Macintosh computer with the public domain NIH image program (developed at the US National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/).
RT–PCR was carried out with 5 µl total RNA from E16.5 embryos with the SuperScript preamplification system (Life Technologies). Two sets of primers were used (Fig. 1A): set 1, F27 in exon 2 (5'-CTT CAG ACG CTG GAC ATC G-3')/R37 in exon 5 (5'-GTT GCT TCT ACT TGC ACC TCT G-3'), amplified a 1500 bp fragment in homozygous (null/null) RNA and an 800 bp fragment in wild-type (+/+) RNA. Both fragments can be detected in heterozygous (null/+) RNA. Set 2, F27 in exon 2/R25 in exon 5 (5'-TAC TGC TGC CAC AGC ACC TG-3'), gives similar results (Fig. 1C). Each mutant transcript was cloned and sequenced by the dideoxy method.
In situ hybridization
Techniques for in situ analysis have been described in detail by Sassoon and Rosenthal (57). The probe used for analysis of Pkd1 was generated from a cDNA fragment corresponding to nucleotide positions 888–1281 (GenBank accession no. U70209) and subcloned into Bluescript SK. Antisense riboprobe was obtained by T7 polymerase with 35S-radiolabeled UTP (>1000 Ci/mmol), following linearization of the plasmid by EcoRI to yield a 400-base fragment, and used in a hybridization buffer with an 35 000 c.p.m./µl final probe concentration.
Antibodies and western blot analysis
Mouse monoclonal antibody 7e12 was raised against an epitope in the N-terminal flank-LRR-flank domain of human polycystin-1 (36).
For western blot analysis, total protein was measured by the Bio-Rad protein assay, 150 µg/lane of membrane fraction from E12.5 or E16.5 fetal tissue extracts was separated on 5% SDS-polyacrylamide gels at 50 V for 13 h followed by 100 V for 2 h at room temperature. Protein was transferred onto Immobilon-P PVDF membranes (Millipore) at 30 V for 17 h at 4°C in Tris–glycine transfer buffer (Tris 25 mM, glycine 190 mM). The membranes were blocked with 5% non-fat dry milk/TBST and incubated with a monoclonal antibody 7e12 to polycystin-1 at 1:1000. Bound protein was detected by enhanced chemiluminescence (Amersham-Pharmacia).
Histology and skeletal staining
For histological analysis, specimens were fixed in formalin, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin (H&E).
Bones and cartilages of completely skinned and freshly eviscerated newborn mice were stained in a mixture of 0.14% alcian blue and 0.12% alizarin red S in ethanol and glacial acetic acid (58) for 2.5 days. Specimens were then macerated in 1.8% KOH for 4 h, cleared in 0.3% KOH overnight and stored in pure glycerin.
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ACKNOWLEDGEMENTS |
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We thank Drs Stephen T.Reeders, Bjorn R.Olsen, Nuria Basora and Mr Eric Williams for discussion, Dr En Li for providing the ES cells, Dr Lin Geng for initial antibody work, Dr Xiaohong Fan for providing mice for skeleton staining, Dr Naomi Fukai for the protocol of skeletal staining and Mr Haidong You for technical assistance. This work was supported by research grants from the National Institutes of Health (NIDDK) to J.Z.
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FOOTNOTES |
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+ To whom correspondence ahould be addressed at: Harvard Institutes of Medicine, Room 522, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. Tel: +1 617 525 5860; Fax: +1 617 525 5861; Email: zhou@rics.bwh.harvard.eduPresent address:Weining Lu, Genetics Division, Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA
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REFERENCES |
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